PREVENTION OF ENVIRONMENTAL-TOXICANT TRIGGERED AUTOIMMUNITY BY CONSUMPTION OF THE OMEGA-3 POLYUNSATURATED FATTY ACID, DOCOSAHEXAENOIC ACID By Melissa A. Bates A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Food Science–Environmental Toxicology – Doctor of Philosophy 2017 ABSTRACT PREVENTION OF ENVIRONMENTAL-TOXICANT TRIGGERED AUTOIMMUNITY BY CONSUMPTION OF THE OMEGA-3 POLYUNSATURATED FATTY ACID, DOCOSAHEXAENOIC ACID By Melissa A. Bates While heredity is a primary predisposing factor for autoimmunity, cumulative exposures to environmental factors such as toxic stressors and diet greatly impact the latency and severity of autoimmune diseases such as lupus. The chapters within this dissertation test the hypothesis that dietary DHA prevents activation of autoreactive lymphocytes that establish the lung as a platform to exacerbate systemic autoimmunity after cSiO2 exposure in lupus-prone NZBWF1 mice. Herein, it is demonstrated that airway exposure to crystalline silica (cSiO 2) triggers early loss of selftolerance in the lung characterized by prolonged elevations in pro-inflammatory cytokines, and strikingly, early infiltration of autoreactive B and T cells into the lung which collectively drive production of autoantibodies that exacerbate systemic autoimmunity and glomerulonephritis in the lupus-prone female NZBWF1 mouse. Supplementing NZBWF1 mouse diets with docosahexaenoic acid (DHA), an ω-3 polyunsaturated fatty acid (PUFA), dose-dependently blocks this triggering in the lung, kidney, and systemic circulation. The anti-inflammatory effects of DHA were evident through ablation of lymphocytic infiltration and reduction of pro-inflammatory cytokines in the lungs that corresponded to reduced serum autoantibodies and cytokine concentrations and accordingly, reduced severity of cSiO2-triggered glomerulonephritis in the kidneys of NZBWF1 mice. To elaborate on these findings, we utilized an integrative approach with quantitative immunohistochemistry, transcriptomics, and targeted proteomics to identify relevant cell populations and putative molecular pathways responsible for cSiO 2-driven autoimmunity and targets for the ameliorative effects of DHA. Taken together, the findings in this dissertation establish a model that permits dissection of the countervailing roles of cSiO 2 (potentiation) and DHA (attenuation) in initiation and progression of autoimmunity. The results from these studies demonstrate how modification of cellular lipids with the ω-3 fatty acid, DHA, could be harnessed to attenuate lupus and related autoimmune diseases triggered by toxic environmental stressors. ACKNOWLEDGEMENTS To my mentor, Dr. Jim Pestka, I sincerely express my gratitude for your guidance. I couldn’t have chosen a better mentor. You must have seen some potential in me those years back, thank you for helping me see the potential in myself. To my guidance committee members, Dr. Jenifer Fenton, Dr. Jack Harkema, Dr. Wei Li and Dr. Cheryl Rockwell, I thank you for being my mentors and role models. I promise to give back as you’ve given to me. To Dr. Steve Bursian, thank you for that chance to volunteer in your lab those years ago. Dr. Hui-Ren Zhou, thank you for showing me how to work and think in a lab. This project would not have been successful without the contributions of Dr. Jack Harkema and the Experimental Toxicology Laboratory, especially Dr. Jim Wagner, Dr. Ning Li, Dr. Peyman Akbari, Dr. Devon Jackson-Humbles, Dr. Christina Brandenberger and Ryan Lewandowski. I cannot thank you enough for your knowledge and your thought-provoking questions. Without our collaborators and co-authors, especially Dr. Andrij Holian, this research could not have been completed. Thank you for showing me the beauty of a strong collaborative team. To all the support staff I’ve had the pleasure to work, especially Kathy Joseph and Amy Porter of MSU Histopathology, thank you for your expertise. I’ll be challenged to find a team of technicians that is as fun to work with and can produce better results than you. To all the friends I’ve made in graduate school, especially Josephine Wee, Austin Pickens, David Duriancik and Jason Wiesinger, you’ve influenced my life beyond the confines of this dissertation. I look forward to collaborating one day. To Guy, I couldn’t have done it without you. iv TABLE OF CONTENTS LIST OF TABLES ix LIST OF FIGURES xii KEY TO ABBREVIATIONS xvii CHAPTER 1: INTRODUCTION, CHAPTER SUMMARIES, AND LITERATURE REVIEW Introduction Chapter Summaries Literature Review The Immune System Autoimmunity Murine Models of SLE The Role of the Exposome in Autoimmunity Ectopic Lymphoid Tissue (ELT) in Autoimmunity Anti-Inflammatory Effects of Dietary Lipids REFERENCES 1 1 2 5 5 8 11 14 18 24 36 CHAPTER 2: SILICA TRIGGERS INFLAMMATION AND ECTOPIC LYMPHOID NEOGENESIS IN THE LUNGS IN PARALLEL WITH ACCELERATED ONSET OF SYSTEMIC AUTOIMMUNITY AND GLOMERULONEPHRITIS IN THE LUPUS-PRONE NZBWF1 MOUSE Abstract Introduction Materials and Methods  Mice  cSiO2 exposure  Necropsy and tissue collection  Kidney histopathology  Lung histopathology  BALF processing and differential staining  IgG and autoantibody measurement  Cytokine analysis  Statistics Results  cSiO2 elicits early onset of proteinuria and glomerulonephritis  cSiO2 induces marked lymphoplasmacytic infiltration in lung parenchyma cSiO2 elicits IgG producing plasma cells in lung CD45R+ and CD3+ infiltrating lymphocytes in lung organize into ectopic lymphoid tissue after cSiO2 exposure in NZBWF1 mice cSiO2 induces leukocyte infiltration and elevates IgG, IgA, and IgM in v 61 61 62 64 64 66 66 67 67 69 69 69 70 70 70 72 77 77 BALF cSiO2 induces elevation of MCP-1, TNF-α, and IL-6 in BALF cSiO2 triggers systemic autoantibody and proinflammatory cytokine responses Discussion Conclusions APPENDIX REFERENCES 81 81 84 84 92 94 96 CHAPTER 3: SILICA-TRIGGERED AUTOIMMUNITY IN LUPUS-PRONE MICE BLOCKED BY DOCOSAHEXAENOIC ACID CONUMPTION 104 Abstract 104 Introduction 105 Materials and Methods 108  Animals and diets 108  Experimental design 109  Necropsy and tissue collection 113  Fatty acid analyses 114  Kidney histopathology 114  Lung histopathology 115  Lung immunohistochemistry 115  Lung morphometry for B and T cells 116  BALF cell quantitation and identification 116  IgG and autoantibody measurement 117  Cytokine analyses 117  Statistics 118 Results 118  Consumption of DHA-rich microalgal oil dose-dependently increases tissue DHA 118  DHA consumption suppresses cSiO2-triggered proteinuria and lupus nephritis 120  DHA consumption abrogates cSiO2-induced inflammatory cell response in lungs 124  Dietary DHA prevents cSiO2-induced ELT neogenesis and autoimmunity in the lung 127  DHA consumption suppresses cSiO2-triggered secretion of proinflammatory and B-cell stimulatory cytokines in BALF 132  Dietary DHA attenuates cSiO2-induced systemic autoantibody and cytokine elevation 136 Discussion 136 Conclusions 144 APPENDIX 145 REFERENCES 152 vi CHAPTER 4: DYNAMIC MRNA AND PROTEIN SIGNATURES OF SILICATRIGGERED AUTOIMMUNITY SUPPRESSED BY DOCOSAHEXAENOIC ACID IN LUPUS-PRONE NZBWF1 MICE 160 Abstract 160 Introduction 161 Materials and Methods 166  Animals and diets 166  Experimental design 168  Necropsy and tissue collection 170  Lung histopathology 170  CD45R immunohistochemistry for B-cells 171  CD3 immunohistochemistry for T-cells 172  CD21/CD35 for follicular dendritic cells 172  Kidney histopathology 173  Morphometric quantitation of immunohistochemistry 173  RNA preparation 174 TM  Gene expression analysis with nCounter 175  Targeted proteome microarray 176  Autoantibody microarray 177  Statistics 177 Results: Histopathology 179  Dietary DHA blocks cSiO2-induced inflammation and cell infiltration  in lung 179  Dietary DHA delays formation of ELT over time in lung of cSiO2-treated NZBWF1 mice 187  Dietary DHA diminishes protein deposition and CD45R+ infiltration in kidney of cSiO2-treated NZBWF1 mice at 13 wks post final exposure 192  Kidney histopathology at 13 wk post final exposure correlates with lung inflammation 194 Discussion: Histopathology 197 Conclusions: Histopathology 203 Results: Targeted transcriptomics 204  mRNA expression reveals putative mechanisms for suppressive effect of dietary DHA on cSiO2-triggered inflammation in lung 204  DHA suppresses cSiO2-triggered mRNA gene expression in lung at 1 wk post final exposure 205  DHA suppresses cSiO2-triggered mRNA gene expression in lung at 5 wk post final exposure 211  DHA suppresses cSiO2-triggered mRNA gene expression in lung at 9 wk post final exposure 220  DHA suppresses cSiO2-triggered mRNA gene expression in lung at 13 wk post final exposure 230  DHA suppresses cSiO2-triggered mRNA gene expression in spleen at 13 wk post final exposure 230 vii  DHA suppresses cSiO2-triggered mRNA gene expression in kidney at 13 wk post final exposure Discussion: Targeted transcriptomics Conclusions: Targeted transcriptomics Results: Targeted proteome microarray  Targeted proteome microarray analysis identifies novel biomarkers of cSiO2-triggered pulmonary and systemic inflammation decreased by DHA Targeted proteome microarray identifies protein signatures of cSiO2-triggered inflammation decreased by DHA in BALF over time  Targeted proteome microarray identifies protein signatures of cSiO2-triggered inflammation decreased by DHA in plasma over time Targeted proteome microarray identifies protein signatures in plasma of cSiO2-treated mice that parallel with those in BALF Discussion: Targeted proteome microarray Conclusions: Targeted proteome microarray Results: Autoantibody array  Dietary DHA inhibited cSiO2-elicited repertoire of autoreactive IgM and IgG autoantibodies in BALF and plasma of lupus-prone NZBWF1 mice Discussion: Autoantibody array Conclusions: Autoantibody array Summary: Chapter 4 APPENDIX REFERENCES CHAPTER 5: CONCLUSION AND PROPOSED FUTURE DIRECTIONS Conclusion Future Directions Dietary DHA as an intervention after cSiO2 exposure Early events after cSiO2 exposure that exacerbate systemic autoimmunity in lupus-prone mice Susceptibility of male NZBWF1 mice to autoimmune triggering by cSiO2 exposure Effect of cSiO2 on other AD in murine models of autoimmunity Additional environmental factors that trigger autoimmunity Utility of the plasma lipidome to identify individuals at risk for SLE APPENDICES APPENDIX A: Short-term in vivo experiments with cSiO2 in NZBWF1 mice APPENDIX B: In vitro models of cSiO2 exposure REFERENCES viii 246 259 273 273 273 276 291 300 301 317 318 318 323 326 327 330 564 588 588 589 589 590 592 592 593 594 596 597 683 691 LIST OF TABLES Table 1. Characteristics of select murine models of human SLE. 13 Table 2. Summary of the anti-inflammatory mechanisms of ω-3 PUFAs. 28 Table 3. Summary of human clinical trials employing ω-3 PUFA supplementation in SLE. 32 Table 4. cSiO2 exposure increases severity of lymphocytic cell infiltration in lungs of NZBWF1 mice relative to C57Bl/6 mice. 76 Table 5. Compositions of experimental diets. 110 Table 6. Fatty acid content of experimental diets based on GLC analysis. 111 Table 7. Histopathological assessment of cSiO2-triggered pulmonary lung inflammation in lupus-prone NZBWF1 and NZW/LacJ mice fed DHA. 126 Table 8. Fatty acid composition of kidney. 146 Table 9. Fatty acid composition of lung. 148 Table 10. Fatty acid composition of RBCs. 150 Table 11. Compositions of experimental diets. 167 Table 12. Histopathological assessment of cSiO2-triggered pulmonary inflammation in lupus-prone NZBWF1 mice fed CON or DHA-enriched diets at 1 wk post cSiO2 exposure. 181 Table 13. Histopathological assessment of cSiO2-triggered pulmonary inflammation in lupus-prone NZBWF1 mice fed CON or DHA-enriched diets at 5 wk post cSiO2 exposure. 183 Table 14. Histopathological assessment of cSiO2-triggered pulmonary inflammation in lupus-prone NZBWF1 mice fed CON or DHA-enriched diets at 9 wk post cSiO2 exposure. 185 Table 15. Histopathological assessment of cSiO2-triggered pulmonary inflammation in lupus-prone NZBWF1 mice fed CON or DHA-enriched diets at 13 wk post cSiO2 exposure. 189 Table 16. cSiO2-induced lung inflammation correlates with kidney histopathology at 13 wks post final exposure to cSiO2. 198 ix Table 17. mRNA signatures in lung at 1 wk post final exposure in cSiO2-exposed NZBWF1 mice. 208 Table 18. mRNA signatures in lung at 5 wk post final exposure in cSiO2-exposed NZBWF1 mice. 214 Table 19. mRNA signatures in lung at 9 wk post final exposure in cSiO2-exposed NZBWF1 mice. 223 Table 20. mRNA signatures in lung at 13 wk post final exposure in cSiO2-exposed NZBWF1 mice. 233 Table 21. mRNA signatures in spleen at 13 wk post final exposure in cSiO 2-exposed NZBWF1 mice. 243 Table 22. mRNA signatures in kidney at 13 wk post final exposure in cSiO2exposed NZBWF1 mice. 249 Table 23. Identification of a DHA-attenuated mRNA IFN-signature induced in cSiO2-treated NZBWF1 mice fed CON diet. 266 Table 24. Targeted proteome analysis of BALF at 1 wk post final exposure to cSiO 2. 277 Table 25. Targeted proteome analysis of BALF at 5 wk post final exposure to cSiO 2. 282 Table 26. Targeted proteome analysis of BALF at 9 wk post final exposure to cSiO2. 286 Table 27. Targeted proteome analysis of BALF at 13 wk post final exposure to cSiO2. 292 Table 28. Targeted proteome analysis of plasma at 5, 9, and 13 wk post final exposure to cSiO2. 297 Table 29. Summary of proteins identified in targeted proteome analysis of BALF following cSiO2 exposure in NZBWF1 mice fed CON or 1.0% DHAenriched diet. 302 Table 30. Summary of proteins identified in targeted proteome analysis of plasma following cSiO2 exposure in NZBWF1 mice fed CON or 1.0% DHAenriched diet. 307 Table 31. Complete mRNA transcript profile in lung at 1 wk post final exposure in cSiO2-exposed NZBWF1 mice. 333 x Table 32. Complete mRNA transcript profile in lung at 5 wk post final exposure in cSiO2-exposed NZBWF1 mice. 372 Table 33. Complete mRNA transcript profile in lung at 9 wk post final exposure in cSiO2-exposed NZBWF1 mice. 411 Table 34. Complete mRNA transcript profile in lung at 13 wk post final exposure in cSiO2-exposed NZBWF1 mice. 451 Table 35. Complete mRNA transcript profile in spleen at 13 wk post final exposure in cSiO2-exposed NZBWF1 mice. 491 Table 36. Complete mRNA transcript profile in kidney at 13 wk post final exposure in cSiO2-exposed NZBWF1 mice. 531 Table 37. mRNA signatures upregulated in lung of cSiO2-exposed NZBWF1 mice compared to VEH + CON mice 24 hrs post final exposure. 600 Table 38. Complete mRNA transcript profile in lung 24 hr post single exposure to cSiO2 in NZBWF1 mice. 604 Table 39. Complete mRNA transcript profile in lung 24 hr post final exposure to cSiO2 in NZBWF1 mice. 644 xi LIST OF FIGURES Figure 1. General overview of management and treatment approaches to SLE. 10 Figure 2. Examples of the exposome. 15 Figure 3. Summary of biological elongation of long-chain ω-3 PUFAs from essential dietary precursors. 26 Figure 4. Experimental design for intranasal cSiO2 exposure. 65 Figure 5. cSiO2 exposure accelerates development of proteinuria in NZBWF1 mice. 71 Figure 6. cSiO2 exposure increases severity of lupus nephritis in kidneys of NZBWF1 mice. 72 Figure 7. cSiO2 exposure in C57Bl/6 mice induced kidney lesions resembling lupus nephritis. 74 Figure 8. cSiO2 elicits perivascular and peribronchiolar lymphocytic infiltration in lungs of NZBWF1 and C57Bl/6 mice. 75 Figure 9. Marked accumulation of IgG producing plasma cells occurs in lungs of cSiO2-exposed NZBWF1 mice. 78 Figure 10. Intranasal cSiO2 exposure induces infiltration of CD45R+ and CD3+ lymphocytes in lungs that resemble ectopic lymphoid tissue. 79 Figure 11. Intranasal cSiO2 exposure induces infiltration of lymphocytes and PMN leukocytes in BALF of NZBWF1 and C57Bl/6 mice. 82 Figure 12. cSiO2 exposure elevates IgG (A), IgA (B), and IgM (C) concentrations in BALF of NZBWF1 and C57Bl/6 mice. 83 Figure 13. cSiO2 induces elevation of MCP-1 (A, D), TNF-α (B, E), and IL-6 (C, F) in BALF and plasma of NZBWF1 mice. 85 Figure 14. cSiO2 exposure increases anti-dsDNA antibodies (A) and anti-nuclear antibodies (B) in plasma of NZBWF1 mice. 86 Figure 15. cSiO2 does not significant alter anti-dsDNA autoantibodies in plasma of C57Bl/6 mice. 95 Figure 16. Experimental design. 112 xii Figure 17. Consumption of microalgal oil dose-dependently increases DHA in kidney, lung and RBC. 119 Figure 18. Dietary supplementation with DHA attenuates cSiO2-induced proteinuria in NZBWF1 mice. 121 Figure 19. DHA consumption suppresses cSiO2-induced glomerulonephritis in NZBWF1 mice. 122 Figure 20. DHA dose-dependently reduces severity of lupus nephritis in cSiO2exposed NZBWF1 mice. 123 Figure 21. DHA supplementation prevents cSiO2-induced pneumonitis. 125 Figure 22. DHA consumption abrogates cSiO2-induced macrophage, lymphocyte, and polymorphonuclear leukocyte accumulation in BALF. 128 Figure 23. B and T cell infiltration in lungs of NZBWF1 mice following cSiO 2 exposure is prevented by dietary supplementation with DHA. 129 Figure 24. cSiO2-triggered B and T cell infiltration in lungs of NZBWF1 mice is dose-dependently prevented by DHA consumption. 131 Figure 25. Dietary DHA suppresses cSiO2-induced elevation of total IgG and antidsDNA Ig in BALF of NZBWF1 mice. 133 Figure 26. cSiO2-induced elevations of proinflammatory cytokines MCP-1, TNF-α and IL-6 in BALF and plasma are decreased by DHA consumption in NZBWF1 mice. 134 Figure 27. cSiO2-induced elevation of B cell stimulating cytokines BAFF and osteopontin are decreased in BALF and plasma in NZBWF1 mice fed DHA. 135 Figure 28. Dietary DHA attenuates cSiO2-induced elevation of total IgG and antidsDNA Ig in plasma of NZBWF1 and NZW/LacJ mice. 137 Figure 29. Putative mechanisms for DHA-mediated suppression of cSiO2-induced autoimmunity. 140 Figure 30. Experimental design. 169 Figure 31. DHA consumption prevented cSiO2-triggered inflammation in lungs at 1 wk post final exposure. 180 xiii Figure 32. DHA consumption prevented cSiO2-triggered inflammation in lungs at 5 wk post final exposure. 182 Figure 33. DHA consumption prevented cSiO2-triggered inflammation in lungs at 9 wk post final exposure. 184 Figure 34. DHA consumption prevented cSiO2-triggered inflammation in lungs at 13 wk post final exposure. 188 Figure 35. Dietary DHA dose-dependently attenuates development of ELT in lungs of cSiO2-treated NZBWF1 mice over time as indicated by immunohistochemistry for CD45R+ (B-cells) (A) and CD3+ (T-cells) (B). 191 Figure 36. fDCs are induced in ELT of cSiO2-treated NZBWF1 mice at 13 wks post final exposure to cSiO2 as indicated by immunohistochemistry for CD21/CD35. 193 Figure 37. Dietary DHA trended towards decreased cSiO2-accelerated protein accumulation in renal tubules at 13 wks post final exposure to cSiO 2. 195 Figure 38. Dietary DHA dose-dependently attenuated cSiO2-elicited B-cell accumulation in kidney at 13 wks post final exposure to cSiO2 as indicated by immunohistochemistry for CD45R+ cells. 196 Figure 39. Network visualization of immune genes impacted by cSiO2 identified 1 wk post final exposure in lung of cSiO2-exposed NZBWF1 mice fed CON diet. 206 Figure 40. Volcano plots depicting immune-related mRNA gene expression data at 1 wk post final exposure in lung of cSiO2-treated NZBWF1 mice. 207 Figure 41. Network visualization of immune genes impacted by cSiO2 identified 5 wk post final exposure in lung of cSiO2-exposed NZBWF1 mice fed CON diet. 212 Figure 42. Volcano plots depicting immune-related mRNA gene expression data at 5 wk post final exposure in lung of cSiO2-treated NZBWF1 mice. 213 Figure 43. Network visualization of immune genes impacted by cSiO2 identified 9 wk post final exposure in lung of cSiO2-exposed NZBWF1 mice fed CON diet. 221 Figure 44. Volcano plots depicting immune-related mRNA gene expression data at 9 wk post final exposure in lung of cSiO2-treated NZBWF1 mice. 222 xiv Figure 45. Network visualization of immune genes impacted by cSiO2 identified at 13 wk post final exposure in lung of cSiO2-exposed NZBWF1 mice fed CON diet. 231 Figure 46. Volcano plots depicting immune-related mRNA gene expression data at 13 wk post final exposure in lung of cSiO2-treated NZBWF1 mice. 232 Figure 47. Summary of immune genes impacted by cSiO2 identified at 1, 5, 9, and 13 wk post final exposure in lung of cSiO2-exposed NZBWF1 mice fed CON diet. 240 Figure 48. Network visualization of immune genes impacted by cSiO2 identified at 241 13 wk post final exposure in spleen of cSiO2-exposed NZBWF1 mice fed CON diet. Figure 49. Volcano plots depicting immune-related mRNA gene expression data at13 wk post final exposure in spleen of cSiO2-treated NZBWF1 mice. 242 Figure 50. Network visualization of immune genes impacted by cSiO2 identified at 13 wk post final exposure in kidney of cSiO2-exposed NZBWF1 mice fed CON diet. 247 Figure 51. Volcano plots depicting immune-related mRNA gene expression data at 13 wk post final exposure in kidney of cSiO2-treated NZBWF1 mice. 248 Figure 52. Summary of proteins altered in cSiO2-treated mice fed CON and cSiO2treated fed 1.0% DHA in BALF (A) and plasma (B) over time. 275 Figure 53. Summary of antigen-specific IgG autoantibodies in BALF at 13 wks post final exposure to cSiO2 in lupus-prone NZBWF1 mice. 319 Figure 54. Summary of antigen-specific IgM autoantibodies in BALF at 13 wks post final exposure to cSiO2 in lupus-prone NZBWF1 mice. 320 Figure 55. Summary of antigen-specific IgG autoantibodies in plasma at 13 wks post final exposure to cSiO2 in lupus-prone NZBWF1 mice. 321 Figure 56. Summary of antigen-specific IgM autoantibodies in plasma at 13 wks post final exposure to cSiO2 in lupus-prone NZBWF1 mice. 322 Figure 57. Certificate of analysis for DHASCOTM algal oil in DHA-enriched AIN93G diets. 331 Figure 58. Volcano plots depicting immune-related mRNA gene expression data at 24 hr post single exposure (A) or 24 hr post final exposure (B) to cSiO2 in lung of NZBWF1 mice. 599 xv Figure 59. Phagocytosis of cSiO2 particles is not affected in vitro by DHA. 684 Figure 60. IL-1β dose-response to cSiO2 in vitro in RAW 264.7 cells stably transfected with ASC. 686 Figure 61. IL-1β response to cSiO2 in relation to time in vitro with MPI cells. 687 Figure 62. Development of an in vitro model of efferocytosis. 689 xvi KEY TO ABBREVIATIONS AD Autoimmune disease AIN American Institute of Nutrition ALA Alpha linolenic acid ANOVA Analysis of variance ARA Arachidonic acid BAFF B-cell activating factor BALF Bronchoalveolar lavage fluid BH Benjamani-Hochberg BILAG British Isles Lupus Assessment Group CI Confidence interval CON Control COPD Chronic obstructive pulmonary disease cSiO2 Crystalline silica DAMP Damage associated molecular pattern DHA Docosahexaenoic acid DHASCO Microalgal oil containing 40% DHA EFSA European Food Safety Authority ELT Ectopic lymphoid tissue EPA Eicosapentaenoic acid FAME Fatty acid methyl esters FC Fold change FDA United States Food and Drug Administration fDC Follicular dendritic cells FSS Fatigue severity score xvii GLC Gas liquid chromatography GPR G-protein coupled receptor H&E Hematoxylin and eosin IBD Inflammatory bowel disease IFN Interferon LA Linoleic acid Lupus Systemic lupus erythematosus MFI Mean fluorescent intensity MUFA Monounsaturated fatty acid NIH National Institutes of Health NIEHS National Institute for Environmental Health Sciences NHANES National Health and Nutrition Examination Survey NSAID Non-steroid anti-inflammatory drugs NZBWF1 New Zealand Black White F1 NZM New Zealand Mixed OPN Osteopontin PAMP Pathogen-associated molecular pattern PASH Periodic-Acid Schiff and hematoxylin PGA Physician's global assessment PI Post-instillation PPAR peroxisome proliferator-activated receptor PUFA Polyunsaturated fatty acid RAND-SF36 RAND corporation short form-36 questions SLAM-R Systemic Lupus Activity Measure - Revised SLICC Systemic Lupus International Collaborating Clinics xviii SLE Systemic lupus erythematosus SLEDAI SLE Disease Activity Index SLO Secondary lymphoid organ SNR Signal to noise ratio SPM Specialized pro-resolving metabolites VEH Vehicle xix CHAPTER 1: INTRODUCTION, CHAPTER SUMMARIES, AND LITERATURE REVIEW Introduction Autoimmune diseases (AD) are a heterogenous collection of over 80 chronic debilitating illnesses afflicting some 25 million Americans. Systemic lupus erythematous (SLE or lupus) is a prototypical AD characterized by loss of immunological tolerance to the body’s own tissues. All AD, including lupus, are generally regarded as incurable and as a result, inflict tremendous individual suffering and social burdens. Genetically inherited susceptibility (genome) is the primary predisposing factor for autoimmunity, however, lifetime exposures to environmental factors (exposome) may have an equal influence on the onset of autoimmunity. National Institutes of Health (NIH) expert committees have identified two exposome components critical to AD onset and progression: 1) toxic stressors and 2) dietary factors [1]. Regarding toxic stressors, crystalline silica (cSiO 2) is an environmental toxicant etiologically linked to lupus and other AD. An estimated 1.7 million Americans are exposed to respirable cSiO 2 from manufacturing, mining, construction, agriculture practices, and military deployment [2]. Experimental animal and epidemiological studies have linked exposure to the respiratory toxicant, cSiO2 to lupus and other autoimmune diseases [3–5]. In contrast, numerous animal and clinical studies have demonstrated that ω-3 polyunsaturated fatty acids (PUFAs) can suppress chronic inflammation, thus making them attractive candidates for the prevention and treatment of chronic inflammatory diseases such as lupus [6]. However, little is known about how environmental factors such cSiO2 trigger lupus or how DHA supplementation could be harnessed to block environmental triggering. 1 For several years, the focus of Dr. Pestka’s lab has been the study of immunological aberrations induced by foodborne mycotoxins. Early investigations by our lab demonstrated that chronic dietary exposure to the mycotoxin, deoxynivalenol, increased IgA accumulation in the kidney, thus resembling IgA nephropathy, a type of autoimmune glomerulonephritis [7]. Later studies demonstrated that feeding ω-3 PUFAs in parallel with dietary exposure to deoxynivalenol suppressed IgA nephropathy [8]. Over time, the individual effects of deoxynivalenol and ω-3 PUFAs on autoimmunity were extended to lupus-prone mouse strains. While deoxynivalenol could induce IgA nephropathy in non-autoimmune mouse strains, it was not shown to exacerbate lupus in genetically-predisposed mice [9]. A recent study compared dietary lipids containing either ω-3 or ω-6 PUFAs compared to ω-9 monounsaturated fatty acid (MUFA) on spontaneous development of autoimmunity in female, lupus-prone mice, NZBWF1 mice. Only the ω-3 PUFA enriched diet could preferentially prevent spontaneous onset of autoimmunity as indicated by suppression of systemic autoantibodies and glomerulonephritis [10]. Taken together, there is a crucial need for further study on the interplay between the exposome and genetic factors that contribute to autoimmunity. The results presented in this dissertation address this gap in knowledge. Herein, cSiO2, a known toxic trigger of lupus, is employed to understand how AD triggering by environmental toxicants can be attenuated by selective dietary modulation of cellular lipids. Chapter Summaries Chapter 1 provides an in-depth evaluation of the current understanding of the complexity of lupus with a focus on environmental factors, namely, respiratory toxicant exposure and diet, that influence the onset of autoimmunity. Despite this current understanding, it is evident that there 2 is ambiguity in how toxic stressors, such as cSiO2, might trigger initiation of AD, and how DHA supplementation could be harnessed to block such environmental triggering. In Chapter 2, prior findings by others were expanded to develop a model of cSiO2triggered autoimmunity in female NZBWF1 mice, a widely used model of human lupus. These findings demonstrate that intranasal cSiO2 exposure (0.25 or 1.0 mg) dose-dependently reduced latency and increased intensity of glomerulonephritis. Furthermore, it was found that cSiO2 elicited robust development of ectopic lymphoid tissue (ELT) in the lungs that closely paralleled heightened systemic inflammation and distal (kidney) manifestations of SLE in these mice. Taken together, these findings suggest that the lung serves as a platform to initiate the early onset of systemic autoimmunity and glomerulonephritis in mice genetically prone to lupus. This chapter has been published in PLoS ONE (2015) and can be accessed online at https://www.ncbi.nlm.nih.gov/pubmed/25978333. In Chapter 3, cSiO2-triggered lupus was compared in NZBWF1 mice fed DHA-enriched diets calorically equivalent to human consumption of 0, 2, 6 and 12 g DHA per day. Dietary DHA dose-dependently suppressed cSiO2-triggered proteinuria and glomerulonephritis in parallel with decreased plasma proinflammatory cytokines and anti-dsDNA immunoglobulins. Striking effects of dietary DHA were further evident in the lung. DHA dose-dependently suppressed IgG and antidsDNA immunoglobulins that occurred in parallel with dramatic suppression of ELT in the lungs of cSiO2-exposed, lupus-prone mice. Taken together, the protective effect of DHA-supplemented diets on were not only reflected in the kidney, but also evident systemically and in the lung. Building on the conclusion from Chapter 2, these results indicate that the predominate effect of DHA on attenuation of glomerulonephritis resides upstream of the kidney, at the level of the lung. 3 This chapter has been published in PLoS ONE (2016) and can be accessed online at https://www.ncbi.nlm.nih.gov/pubmed/27513935. Given that blockade of cSiO2-triggered ELT neogenesis by DHA may be intimately related to suppressed systemic autoimmunity and glomerulonephritis, the focus of Chapter 4 is eludciation of how DHA impacts pathogenic sequalae of cSiO2-triggered ELT neogenesis in the lung over time using histopathology in parallel with an integrative profiling approach to relate lung ELT neogenesis to immune system activation by way of mRNA and protein signatures. Findings in Chapters 2 and 3 are built upon to reveal a more detailed understanding of the complexity of cSiO2-triggered lupus in NZBWF1 mice which overlap considerably with those known in humans. It is demonstrated that DHA suppresses multiple inflammatory pathways induced by cSiO2 throughout the pathogenesis of ELT in the lung. Taken together, cSiO2 induces early and sustained inflammation indicative of the collective efforts of several activated cell types and pathways in the lung that catalyze systemic autoimmunity. Results from this chapter will be published as multiple, first-authored manuscripts upon additional analyses to further associate DHA-suppression of ELT in the lung to attenuated systemic and kidney autoimmune responses. In Chapter 5, conclusions drawn from the collective findings presented in Chapters 2-4 are summarized and related to practical applications with dietary DHA to reduce the risk of lupus and other autoimmune diseases in genetically predisposed individuals and/or those exposed to respiratory toxicants. New research directions are briefly proposed that will enhance our understanding of how manipulating dietary lipids can be harnessed to block AD initiation and progression by toxic environmental stressors. Supplementary information in Chapters 2, 3, and 4 are presented as appendices. In Appendix A in Chapter 5, preliminary data is presented from short-term in vivo studies that will 4 guide future studies in elucidation of early inflammatory events induced by cSiO 2 in the lung that are targeted by DHA. In Appendix B, preliminary data from in vitro models of macrophages are presented that support the utility of these cell models to explore specifically how DHA attenuates cSiO2-induced toxicity in these cells and possibly as a link to how DHA ultimately suppresses systemic autoimmunity triggered by cSiO2-exposure. Literature Review The Immune System The immune system is an exquisitely balanced network of cells (broadly referred to as leukocytes) programmed to 1) identify foreign substances which could cause harm to the body and 2) protect the host from perceived threats by initiating an appropriately timed and sufficiently defensive immune response. The immune system has innate and adaptive branches, which coordinate to survey the body for potentially harmful foreign microbes, injurious substances and remove them, and thereby protect the health of the individual. The innate immune system rapidly responds to foreign antigens The innate immune system is so termed because it does not require any previous exposure to the harmful agent to be able to detect it and respond accordingly. As a result, the actions of the innate immune system are less specific but respond rapidly to foreign molecules. Since these innate immune cells are the ‘first-responders’ of the immune system, they are found extensively in locations of the body that interface between internal tissues and the outside environment (e.g. skin, the linings of the nasal cavities, lungs and intestines). These sites are the most likely entry points for potentially threatening foreign substances [11]. Cells of the innate immune system distinguish foreign substances from the host by recognizing surface epitopes. This is accomplished through a 5 diverse collection of receptors (termed pattern recognition receptors) that sense specific sequences on the surface of an invading substance (e.g. lipopolysaccharide on gram negative bacteria) which are termed pathogen associated molecular patterns (PAMPs) [12]. Alternatively, a variety of receptors on innate immune cells can detect cellular products that are released from damaged cells as a result of toxic injury. These products are called damage associated molecular patterns (DAMPs) [13,14]. Macrophages are key effector cells of innate immunity One of the most important cells of the innate immune system is the resident tissue macrophage. Macrophages are poised at the interface between the external environmental and internal tissues and regulate immune function at these sites [15,16]. Macrophages are “scavenging” cells whose hallmark function is phagocytosis – the internalization of particles to be degraded through enzymatic processes within the cytoplasm of the macrophage. Phagocytosis results in the destruction and clearance of pathogens and other harmful substances from the host. Phagocytosis also ensures removal of dying and dead cells – a process known as efferocytosis. Resident tissue macrophages also function as “tissue immune guards” and elicit an inflammatory response upon recognition of foreign antigens [17]. Depending on the amount of the foreign substance, macrophages may either eliminate the threat on their own, or recruit immune cells from the systemic circulation to assist in elimination of the foreign substance through inflammation. Inflammation protects the body Inflammation is the localized coordination of several defensive responses towards harmful stimuli at the site of tissue injury. During the acute phase of an inflammatory response, local epithelial cells and/or leukocytes secrete a variety of chemical messengers that direct recruited cells to the site of inflammation and dictate their function [18]. For example, chemokines direct 6 recruited cells to the site of injury in a concentration gradient-dependent manner, whereas cytokines direct the function of the recruited cells towards a certain effect tailored to eliminate the perceived threat. An important quality of infiltrating cells is the timely resolution of inflammation and later, tissue repair [19]. Unresolved inflammation persists to a state of chronic inflammation, which results in cell damage and tissue destruction. Importantly, phagocytosis in a negative feedback loop, promotes an anti-inflammatory macrophage phenotype that resolves inflammation [20]. The adaptive immune system coordinates antigen-specific responses In contrast to the generalized specificities of the innate immune system, the adaptive immune system targets a foreign substance in a highly specific manner. While the initiation of an adaptive immune response is less rapid then that of the innate immune system, the adaptive immune system is long-lasting. Cells of the adaptive immune system (termed lymphocytes) identify foreign substances by recognizing antigens displayed in the context of antigen-presenting cells (i.e. dendritic cells, macrophages, and B-cells) This recognition drives the activation of Band T-cells of the adaptive immune system. These two cell types eliminate foreign invaders by two predominate mechanisms: 1) “antibody-mediated” resulting from the action of B-cells which decorate foreign microbes, cells, or other substances with antibodies to target their removal and 2) “cell-mediated” resulting from the direct action of T-cells on the foreign microbes, cells, or substance [21]. Given the powerful destructive capacity of the immune system on foreign antigens, perhaps its most important feature of the host immune system is the ability to discern foreign agents from itself. Numerous checkpoints exist during maturation of the immune system to maintain this capacity, however, failure to distinguish self-antigens from foreign antigens results in autoimmune 7 diseases (AD) [22–24]. In individuals afflicted with AD, the immune system senses itself as ‘nonself’ and unleashes harmful, powerful responses that damage normal healthy tissue. Autoimmunity Systemic lupus erythematosus is a prototypical AD AD are an enigmatic mosaic of over 80 chronic debilitating illnesses afflicting some 25 million Americans. Systemic lupus erythematosus (SLE) is a hereditary, prototypical AD that affects approximately 300,000 Americans, predominately women of child-bearing age [25]. SLE is characterized by loss of tolerance to nuclear self-antigens, resulting in excessive production of autoreactive antibodies by antibody-producing plasma cells. In systemic circulation, these autoantibodies bind their cognizant self-antigen, leading to formation of circulating immunecomplexes that are subsequently deposited in target organs, which promote tissue injury by recruiting inflammatory leukocytes [26,27]. SLE can have damaging effects on nearly every organ system in the body, manifesting as renal complications, pulmonary difficulties, cardiovascular events, musculoskeletal effects and loss of central nervous function. As a result, individuals with SLE incur tremendous financial costs and diminished quality of life [28–31]. The damage caused by SLE itself and side-effects of treatments are cumulative and in severe cases often progress to organ failure and death. While advances in medical treatment and supportive therapy has decreased the mortality associated with SLE [32], the most common causes of death in individuals with SLE are cardiovascular disease, infection, and severe organ damage, usually manifesting in the central nervous system or kidneys [33,34]. SLE treatments have several limitations SLE, like most AD, is generally regarded as incurable. Owing to the heterogeneity of SLE, its treatments are widely variable and must be assessed case-by-case depending on individual 8 background, and disease phenotype and severity. Organ injury predicts mortality in SLE [35,36], and early onset of organ damage is correlated with accelerated death [37]. Therefore, therapeutic regimes for SLE patients focus on preventing further tissue injury. Treatment of severe disease typically involves two phases: 1) an induction phase during severe disease activity consisting of high doses of powerful immunosuppressant agents and 2) a maintenance phase during disease remission that was achieved following induction therapy (Figure 1) [38,39]. Antimalarial medications (e.g. hydroxychloroquine) and non-steroid anti-inflammatory drugs (NSAIDs) (e.g. naproxen and diclofenac) are effective for mild disease activity and prevention of lupus flares [39,40]. However, aggressive disease requires powerful immunosuppressive agents such as the alkylating agent cyclophosphamide and high-doses of glucocorticoids. These medications have severe side effects. Because of its toxicity, cyclophosphamide is reserved for patients with severe organ injury [41]. Long-term use of glucocorticoids is problematic; side effects include loss of bone density and increased susceptibility to infection [42–45]. Some conventional lupus therapeutics have teratogenic properties (e.g. methotrexate and mycophenolate mofetil) and/or reproductive organ toxicity (e.g. cyclophosphamide) [40]. Considering the predominant population of individuals afflicted with SLE are women of child-bearing age, this represents a significant hurdle in management of SLE. Breakthrough progress in SLE treatment: belilumab In cases of SLE that do not respond to conventional therapeutics, emerging biologics are in development that neutralize specific immune targets such as cytokines and cell receptors [46– 48]. Belimumab (Benlysta), a monoclonal antibody that halts B-cell proliferation by neutralization of B-cell activating factor (BAFF) [49–51], was approved for treatment of SLE by the United States Food and Drug Administration (FDA) and European Medicine Evaluation Agency in 2011 9 Figure 1. General overview of management and treatment approaches to SLE. Modified from [39]. 10 [52]. It was the first SLE-specific drug to be approved by the FDA in 52 years, indicating that new and emerging pharmaceuticals are relatively unsuccessful. Importantly, belimumab is currently only recommended for those patients with subcutaneous and musculoskeletal manifestations of SLE [53,54]; currently, clinical trials with belimumab in lupus nephritis are recruiting participants [55]. Murine Models of SLE There is a clear need for developing novel approaches and pharmaceuticals to treat and control SLE and other AD. Testing these strategies requires animal models. Several induced and spontaneous murine models of SLE have been developed to delineate genetic contributions and molecular mechanisms that underlie the pathogenesis of this AD [56–60]. According to the Systemic Lupus International Collaborating Clinics (SLICC) for lupus diagnosis, individuals must present with either 1) at least 4 out of 17 criteria that consists of both clinical (e.g. arthritis, rash) and immunological (e.g. elevated autoantibodies) manifestations of lupus or 2) biopsy indicated lupus nephritis and the presence of autoantibodies characteristic of SLE [61,62]. Accordingly, SLE murine models should exhibit some of these features and the selected model chosen that best matches the disease phenotype being studied. Induced murine models of SLE Pristane, a component of mineral oil, diesel exhaust, and a byproduct of petroleum processing, induces lupus-like disease in several mouse strains [63–65]. It is the only murine model of SLE that develops the “IFN signature”, an important feature SLE in some patients [66]. Observations of non-autoimmune mouse strains that transgenically over- or underexpress certain genes, or are administered biological agents (e.g. cytokines) have also provided key insight on how these immune components influence the course of autoimmunity [67–74]. BAFF is a key cytokine 11 involved in the proliferation of autoreactive B-cells. Accordingly, murine models that transgenically overexpress BAFF develop SLE-like disease, which consists of heightened autoantibodies and neuropsychiatric manifestations consistent with that in SLE [74]. Spontaneous murine models of human SLE Amongst the most widely used spontaneous models of SLE are the New Zealand Black White F1 (NZBWF1) mice and related New Zealand Mixed (NZM) strains (e.g. NZM2410) and MRL/lpr mouse strain (Table 1) [75]. NZBWF1 mice develop autoimmune responses that closely mirrors human SLE. Lupus in NZBWF1 mice predominately affects female mice that develop severe renal injury between 26 and 34wks of age; mice succumb to glomerulonephritis by 1 yr of age [76]. Importantly, susceptibility loci that contribute to disease in NZBWF1 have human homologues [77–79]. Therefore, studies in this strain are likely translatable to humans. As a result, NZBWF1 mice have been widely used as a preclinical model for currently approved (e.g. BAFF and cyclophosphamide) and other potential therapies for SLE [57]. In addition to classic manifestations of SLE (e.g. anti-dsDNA IgG and lupus nephritis), NZBWF1 mice develop other physiological conditions (e.g. hypertension, metabolic syndrome) that model human SLE and its co-morbidities [80–82]. The closely related NZM2410 strain develop aggressive SLE that equally effects both male and female mice and is characterized by accelerated onset of proteinuria compared to NZBWF1 mice [60,83]. Owing to their homozygous genome, NZM2410 mice have been particularly useful in delineating genetic susceptibility to lupus [60,84]. However, NZM2410 mice quickly succumb to lupus nephritis, which may mask subtle pathogenic changes that drive SLE. MRL/lpr mice develop features resembling numerous AD, including the central nervous system and arthritic effects of SLE and rheumatoid arthritis, respectively, and lacrimal and salivary 12 Table 1. Characteristics of select murine models of human SLE. Modified from [75]. Characteristics of Selected Murine Models of Human SLE Sex Bias 50% mortality Lupus Nephritis Clinical Features Pristane No Strain dependent Yes ANA, Anti-dsDNA, anti-RNA, serositis, arthritis BAFF-Tg No 42 wks Yes ANA, Anti-dsDNA Animal Induced models Spontaneous models NZBWF1 Female 36 wks Yes ANA, anti-dsDNA NZM2410 No 26 wks Yes ANA, anti-dsDNA BXSB/yaa Male 22 wks Yes ANA, anti-dsDNA, neuropathy MRL/lpr No 21 wks Yes ANA, anti-dsDNA, anti-RNA, arthritis, neuropathy 13 gland pathologies associated with Sjӧgren’s syndrome [85–87]. Importantly, MRL/lpr mice carry a fas gene mutation that contributes substantially to SLE in this strain [88,89]; however, mutations of this gene have not been associated with human SLE [90], and as such, may not be an appropriate model for disease pathogenesis. Taken together, factors that specifically drive SLE are difficult to determine with this model. The Role of the Exposome in Autoimmunity Environmental factors contribute to human disease AD is widely acknowledged to be dependent not only on genetic factors, but environmental factors that play a complex role in disease onset, flaring and progression. This is exemplified in studies of SLE in monozygotic twins whereby in only ~25% of cases do both twins develop the disease [91–93]. This discrepancy implies that genetics alone is insufficient to explain AD pathogenesis. Many AD, including SLE, display widely heterogenous phenotypes with disparate tissue manifestations and severity of disease. This suggests that 1) several molecular pathways contribute to the pathogenesis of autoimmunity and 2) other factors (i.e. environmental) influence their development. The concept of the exposome was outlined to identify factors aside from genome-driven mechanisms that influence the course of human disease (Figure 2)[94]. First described in 2005 by Christopher Wild [95], the exposome is defined as “the measure of cumulative environmental influences and biological responses throughout the lifespan, including exposures from environment, diet, behavior and endogenous processes” [96]. Exposure to cSiO2 is associated with SLE Many environmental factors suspected to contribute to autoimmunity are based on anecdotal observations [97]. However, there is a lack of highly powered epidemiological studies and mechanistic studies in murine models. This precludes confident conclusions on the relevance 14 Figure 2. Examples of the exposome. Clockwise from top: microbial infections, medications and access to health care, exercise, industry and air pollution, geographical location, cigarette smoke exposure, work place hazards, wood smoke exposure, and diet. 15 of environmental influences to human incidence of AD. To resolve this uncertainty, a National Institute of Environmental Health Science (NIEHS) Expert Committee was convened in 2012 to clarify the role of suspected environmental exposures in human incidence of AD. They concluded that a clear positive relationship exists between occupational exposure to cSiO2 and SLE, as well as several other AD [98]. An estimated 1.7 million Americans are exposed to respirable cSiO2 from mining, construction, manufacturing, farming, hydraulic fracturing, and military deployment [99]. Consistent with epidemiological studies [20,100–102], airway exposure of cSiO2 to lupus-prone mice markedly decreased onset time and enhanced disease severity [3–5,103,104]. cSiO2 evokes toxicity in the lung by several mechanisms Following airway exposure, respirable cSiO2 particles are deposited in the alveoli where they are engulfed by alveolar macrophages. Prior work indicates that following cSiO2 phagocytosis by alveolar macrophages, lysosomal membrane permeabilization triggers inflammasome activation and resultant cytokine release and cell death and release of cSiO2 particles: these events culminate in recurrent pulmonary exposure to cSiO2 and sustained inflammation [103,105–113]. cSiO2 evokes cell death in alveolar macrophages in vitro and in vivo by two principal mechanisms – pyroptosis [114,115] and apoptosis [109,113,116]. Pyroptosis is a lytic mode of cell death that occurs in macrophages following inflammasome-mediated activation of caspase-1 and results in secretion of proinflammatory mediators including cytokines (e.g IL-1β) and DAMPs that trigger inflammation [117,118]. In contrast, apoptosis following cSiO2 exposure results from lysosomal membrane permeabilization-driven release of proteases that does not result in lysis of the cell membrane or inflammation [119], provided that cell corpses are rapidly phagocytized through the process of efferocytosis [120–122]. 16 Efferocytosis is a homeostatic process that serves to promote resolution of inflammation and maintenance of self-tolerance through phagocytosis of apoptotic cells [20,100,101]. Apoptotic cells not removed by efferocytosis proceed to the state of secondary necrosis, in which the cell membrane containing apoptotic cells disintegrates, resulting in the release of nuclear components (including DAMPs and cytokines). These normally sequestered epitopes or self-proteins are no longer distinguished as ‘self’ [123]. These can be presented by antigen-presenting cells to B-cells. In the context of inflammation (such as that elicited by cSiO2), the net result is activation of autoreactive B- and T-cells [124,125]. Importantly, aberrant apoptosis and defective efferocytosis have both been implicated in human SLE [126–128]. Accumulation of apoptotic cells have been observed in tissues and lymph nodes during SLE [129–131], which may result from accelerated rates of apoptosis [131–133] in parallel with deficient efferocytosis [102,128]. Observations in cSiO2-treated lupus prone mice support this contention [5,134]. Remnants of apoptotic cells following cSiO2 exposure have been detected in lung draining lymph nodes in Balb/c mice [135]. Autoantibodies isolated from sera of cSiO2 exposed, lupus-prone NZM2410 mice were capable of binding apoptotic alveolar macrophages, suggesting the immunoreactivity of these self-derived antigens [134]. Additionally, treatment with rottlerin, an inhibitor of apoptosis, prior to and after cSiO2 exposure in NZM2410 mice was associated with decreased systemic autoantibodies and attenuated proteinuria and immune complex deposition in the kidney [5]. cSiO2 induces an innate immune response cSiO2 and SLE share in common a state of heightened, chronic inflammation. Many of the immune processes that drive the toxicity of cSiO2 in the lung overlap with early events in the pathogenesis of SLE [136], which might explain in part, the increased incidence of SLE in cSiO2- 17 exposed individuals. The initial inflammatory response to cSiO2 is driven by injured alveolar macrophages and epithelial cells that release TNF-α, IL-1α, IL-1β and HMGB-1 and inflammatory chemokines MCP-1 and MIP-2 [115,137–142]. Indeed, autoantibodies towards alarmins, including HMGB-1, have been isolated in sera from individuals with SLE; these positively correlated with disease scores [143]. Furthermore, individuals with active SLE have increased systemic TNF-α and IL-1β [72,144,145] and elevated expression of these cytokines in tissuespecific pathologies [146,147]. Both Type I interferon (IFN) (e.g. IFN-α, IFN-β) and type II IFN (e.g. IFN-γ) have been implicated in the pathogenesis of SLE [148–152]. Type I IFNs released predominately by plasmacytoid dendritic cells have been widely implicated in driving the disease [148–150]. In recent years, emerging evidence indicates that IFN-γ is a major contributor to autoimmunity. In the early phases of SLE, elevated IFN-γ in human sera precedes elevation of autoantibodies and type I IFN [153], and may predict lupus flares [151,154]. IFN-γ is known to mediate, in part, cSiO2-driven inflammation, followed later by Type I IFNs and related downstream gene expression [136,152]. Of critical relevance, both type I and II IFN stimulate expression of BAFF [155,156], which is strongly associated with AD. Although speculative, cSiO2-induced IFNs may mediate elevated BAFF and serve as an additional mechanism by which cSiO2 can trigger AD. In support of this contention, humans exposed to cSiO2 develop B-cell driven production of autoantibodies and kidney disease, thus closely mirroring lupus [157–159]. Ectopic Lymphoid Tissue (ELT) in Autoimmunity ELT occur in chronically inflamed tissues In AD, dysregulation of one or many tolerogenic mechanisms result in the activation and proliferation of autoreactive B and T lymphocytes which drive antibody and cell-mediated tissue 18 destruction. Numerous checkpoints exist to maintain immune tolerance to self-antigens including: deletion of autoreactive B and T lymphocytes, costimulatory molecule expression, cytokine stimulation, and specialized cell types, including T reg cells, that suppress lymphocyte activation. The importance of secondary lymphoid organs (SLO) (e.g. spleen, lymph nodes) as sites of tolerance, antigen presentation and activation/proliferation of B and T lymphocytes is wellestablished. However, the de novo formation of lymphoid tissue in localized sites of chronic inflammation is recognized as a critical source of antigen-specific B and T lymphocytes, and therefore, could contribute to proliferation of autoreactive lymphocytes in AD. Ectopic lymphoid tissues (ELT) are induced during chronic inflammation to remediate high concentrations of locally-present antigens in inflamed tissue [160]. ELT function much like SLOs; ELT structurally and functionally serve as a site for antigen accumulation, presentation, and proliferation/activation of B and T lymphocytes [161,162]. However, the greatest difference is that while SLOs are programmed to develop in specific body sites, ELT neogenesis occurs in chronically inflamed tissue. Thus, ELT neogenesis is initiated by an inflammatory trigger in a localized tissue that do not normally harbor SLOs. ELT structurally and functionally resemble SLOs Due to their common features, much of our current understanding of ELT is derived from study of lymphoid organogenesis [163]. Both ELT and SLO rely on lymphatic vessels and high endothelial vessels to transport antigen and naïve B and T cells, respectively, into the lymphoid tissue [164,165] – a process that requires several key chemokines, cytokines and surface molecules that traffic these cells into lymphoid tissues and promote their interaction with antigen-presenting cells. Importantly, these cells work in a positive feedback loop, such that continual recruitment and activation of lymphocytes further amplifies the adaptive immune response and maintains the 19 lymphoid tissue (reviewed in [166]). Like secondary lymphoid organs, ELT consist of distinct B cell aggregates spatially segregated from dispersed T cell zones. These B cell aggregates are built upon a scaffold of follicular dendritic cells (fDCs) that supply signals to developing B cells that determine their fate (i.e. survival vs. apoptosis) in the germinal center [167] (reviewed in [168]), which is the site of clonal expansion, somatic hypermutation, and affinity maturation of B cells to immunoglobulin secreting plasma cells [169]. Importantly, fDCs have the ability to retain and present antigen long after inflammation has resolved [170,171], which may mediate, in part, the sustained formation of ELT. ELT are pathogenic in autoimmunity ELT have the capacity to critically influence disease progression because of their proximity to sites of inflammation and generation of antigen-specific responses. As an extension of the defensive role of secondary lymphoid organs in adaptive immunity, ELT are induced as a protective mechanism to resolve inflammation induced by foreign antigens. In microbial infections and cancer, ELT are generally associated with desirable outcomes [172–178]. However, in AD, which are defined by loss of self-tolerance, ELT have been implicated in AD progression and severity. ELT are detectable in several AD with tissue-specific pathologies, including synovial joints in rheumatoid arthritis [179–181], salivary glands in Sjӧgren’s syndrome [182–184], meninges in multiple sclerosis [185,186], and kidneys in SLE [187–190]. In patients with Sjӧgren’s syndrome or rheumatoid arthritis, it is estimated that up to 40% of individuals have identifiable ELT [191]. In AD, presence of ELT positively correlates with disease severity and production of autoantibodies [169,192], therefore, ELT likely contribute to the progression of AD. The prevalence of ELT in AD is likely to be underestimated because of the difficulty in obtaining 20 biopsies – the only method to detect ELT in situ. Therefore, reliable biomarkers of their presence are urgently awaited. Components of the exposome initiate ectopic lymphoid tissue – a potential link to autoimmunity? ELT are commonly associated with mucosal tissues (e.g. gut and lungs) [141,177,193– 195], likely resulting from environmental factors that interact with these tissues. However, it is important to note that not all tissues undergoing chronic inflammation develop ELT. This suggests that specific signals, either as a direct result of the inflammatory stimuli or indirectly by its effects on resident and stromal cells at the site of injury, mediate in part, ELT neogenesis. Environmental agents known to initiate ectopic lymphoid tissues include microbes, chemicals, and particles. Microbial infections are suspected to be a trigger of autoimmunity, but clear causative relationships remain to be elucidated. However, it is well established that microbial populations have a role in normal and induced lymphoid tissue. Indeed, commensal microbiota is required for proper formation of Peyer’s patches in the gut [196,197] and regulates intestinal microbiota homeostasis [198]. Microbial dysbiosis has been associated with ELT neogenesis in the gut and the development of inflammatory bowel disease (IBD) [193,194,199], and in the lungs as a possible link to chronic obstructive pulmonary diseases (COPD) that feature ELT [200–203]. Of relevance, both IBD and COPD have pathogenic origins characteristic of autoimmunity; the contribution of ELT in generation of these diseases has not been fully elucidated. Chemical exposures are also known to initiate ELT neogenesis. A prime example of this is tobacco smoke, a complex mixture of > 4,000 chemicals. ELTs have been identified in the lungs of patients with moderate to severe chronic obstructive pulmonary disease [200–203], for which the greatest risk factor is cigarette smoke exposure. Without question, smoking cessation is key to slowing COPD progression. However, even with cessation, pulmonary ELT and cigarette-smoke 21 induced anti-nuclear antibodies persist [203], which is believed to in part, promote emphysema and COPD progression. Furthermore, cigarette smoke is widely accepted to be a risk factor for autoimmunity – a clear association has been demonstrated for rheumatoid arthritis [98], and probable correlations have been extended to SLE [204]. Other associations with chemicallyinduced ELT and incidence of autoimmune-like features have been described. Nacionales and colleagues demonstrated that ELT initiated by intraperitoneal pristane exposure produced type I IFN [205], which likely contributed to initiation and progression of AD in this model. Particle exposures may also initiate ELT and ultimately, influence autoimmunity. Because of the persistence of these environmental particles in the lung after exposure, ELTs are likely to be sustained following exposure. Bates et al. 2015 [3] showed dramatic induction of ELT in lungs of cSiO2-exposed lupus-prone NZBWF1 mice. Lymphocyte infiltrates in the lungs of cSiO2treated NZBWF1 mice consisted of B- and T-cell aggregates and importantly, dramatic induction of IgG-producing cells which were absent in vehicle-treated mice. The presence of these pulmonary ELT coincided with accelerated onset of and enhanced severity of systemic SLE and lupus nephritis in NZBWF1 mice. Taken together, the findings of Bates et al. 2015 [93] are highly consistent with the role of ELT in AD and provide a crucial mechanistic link as to how ELT induced by environmental particles can trigger systemic AD. ELT are resistant to conventional therapeutics While the basic biology of ectopic lymphoid tissue neogenesis is moderately wellunderstood, how environmental factors, inflamed tissue, and/or host defense responses drive pathogenic or protective roles of ELT are currently study of intense research [163]. B and T cells are critical towards the maintenance of ELT, therefore, it is logical that treatments targeting this disruption should be effective in ameliorating pathological influences of ELT in the context of 22 autoimmunity. However, cell-specific therapies have had limited success. Rituximab, an antiCD20 B-cell depleting monoclonal antibody and abatacept, a T-cell neutralizing monoclonal antibody, have had a minimal effect on ELT in rheumatoid arthritis [206,207] and SLE [208], indicating that ELT are resistant to otherwise effective pharmaceuticals. This effect likely owes to the ability of ELT to be ‘self-sufficient’, and support B cell and T-cell maturation within these niches. In this case, one approach that could be effective in disrupting formation of ELT is neutralization of BAFF, which is the target of belimumab. Kang and colleagues (2017) exquisitely demonstrated that BAFF mediated ELT neogenesis in kidneys of lupus-prone mice [190]. In this model, neutralization of BAFF prevented the organization of ELT which was associated with decreased lupus nephritis [190]. Other novel pharmaceuticals that target known pathways in ELT neogenesis and maintenance are currently undergoing clinical trials [163]. However, these studies do not consider disruption of ELT as primary or secondary endpoints, therefore animal models that develop ELT will be crucial in determining the effect of novel drug entities on AD. Considering that: 1) ELT are initiated in tissues with chronic inflammation 2) ELT can persist for many months after resolution of inflammation, and 3) are resistant to lymphocyte depleting therapies, alternative strategies that ameliorate chronic inflammation could prove beneficial in diseases where ELT have been associated with poor disease outcome. One such approach that could be harnessed is with dietary ω-3 polyunsaturated fatty acids (PUFAs). Findings from Bates et al. 2016 support this contention [209]. In this study, lupus-prone NZBWF1 were fed diets enriched with the ω-3 PUFA, docosahexaenoic acid (DHA) prior to and after cSiO2 exposure until sacrifice. DHA dose-dependently reduced lung ELT, which occurred with suppressed autoantibodies and cytokines in bronchoalveolar lavage fluid (BALF) and plasma. Importantly, DHA-fed mice were also protected from cSiO2 accelerated onset of and severity of 23 lupus nephritis. Taken together, these findings support a countervailing effect of dietary ω-3 PUFAs on environmentally-triggered AD. Anti-Inflammatory Effects of Dietary Lipids Lipids are macronutrients that support the structure and function of the body. Lipids may be utilized as a source of energy, incorporated into the lipid bilayer of the plasma membrane of cells, or serve as signaling molecules that influence cellular responses in the body [210]. Cells use lipids for several purposes. They can be used by a cell as a source of energy, or can be incorporated into the lipid bilayer of the plasma membrane [211]. The plasma membrane has several important functions, including phagocytosis and roles in cellular migration and communication [212]. Alterations in lipids that compose the plasma membrane impacts the physiological function of the cell [213]. Fatty acids (FA) are one important type of lipid in the body that are incorporated into phospholipids which form the plasma membrane. FA consist of a carboxyl group (referred to as the α-terminus) and an aliphatic chain of variable length; the last carbon in the aliphatic chain is referred to as the ω-terminus. FA can be further classified based on the length of the aliphatic chain and the number of double bonds in the aliphatic chain. Saturated fatty acids (SFA) contain no double bonds, monounsaturated fatty acids (MUFA) contain one double bond, and polyunsaturated fatty acids (PUFA) contain two or more double bonds in the aliphatic chain. Humans have the necessary enzymes to synthesize and elongate SFA and MUFA from endogenous precursors [214]. Unlike plants, humans do not have the enzyme to convert the MUFA oleic acid (C18:1 ω-9) to linoleic acid (C18:2 ω-6) (LA); humans also lack the enzyme necessary to convert LA to alphalinolenic acid (C18:2 ω-3) (ALA) [215]. Taken together, humans cannot synthesize ω-6 or ω-3 PUFAs, and they cannot convert between classes [216]. As a result, LA and ALA are essential 24 FAs and must be consumed through diet, which then serve as precursors for longer chain PUFAs within their respective origin. LA (found in plant oils, such as corn and soybean) is the precursor for longer chain ω-6 PUFAs such as arachidonic acid (C20:4 ω-6) (ARA). ALA (found in seeds and nuts, such as flax and walnuts) is the precursor for longer chain ω-3 PUFAs such as eicosapentaenoic acid (C20:5 ω-3) (EPA) and docosahexaenoic acid (C22:6 ω-3) (DHA). However, the low efficiency of endogenous enzymes that catalyze reactions for longer and further desaturated PUFAs limits these conversions in mammals [217]. Therefore, dietary consumption of pre-formed ARA (predominately from eggs and red meat), EPA, and DHA (from algal and fish oils) is the predominate source of these FA in the body (Figure 3). Cell membranes incorporate FA into their lipid bilayer such that they model dietary intake of FAs. Therefore, the type of FA consumed influences the composition of the lipid bilayer and ultimately, cellular function. By replacing the predominant constitutive ω-6 PUFAs, LA and ARA, which are readily consumed in Western-style diets [218], DHA and EPA are readily integrated into plasma membrane phospholipids in cells throughout the body, including those critical to innate and adaptive immunity [6,219,220]. Wide acceptance yet low dietary intake of ω-3 PUFAs In 2012, fish oil was consumed by 20 million Americans [221]; the top consumer motivation for their use was “health benefits” [222]. ω-3 PUFA’s are widely touted as antiinflammatory and are attractive candidates for prophylactic and therapeutic approaches in chronic inflammatory conditions, including AD [223,224]. However, most individuals consume far fewer ω-3 PUFAs than recommended; many regions of the world are shifting towards a Western-style 25 Figure 3. Summary of biological elongation of long-chain ω-3 PUFAs from essential dietary precursors. (LA and ALA, ω-6 and ω-3 PUFAs, respectively) and dietary sources of pre-formed ω-3 PUFAs (indicated in left margins). Modified from [6]. 26 diet, which is grossly deficient in ω-3 PUFAs [225]. Indeed, recent National Health and Nutrition Examination Survey (NHANES) data collected in the United States has estimated that on average, Americans only consume 0.072 mg/d EPA+DHA, far less than recommended amounts [226]. Importantly, dietary intake alone is unlikely to achieve amounts of ω-3 PUFAs necessary to provide significant anti-inflammatory effects in chronic inflammatory diseases; Calder (2013) suggests that intakes of >2.0 g/d of ω-3 PUFAs are required to achieve this effect [227], which can optimally be achieved through dietary supplementation or pharmaceutical preparations. The most recognized benefit of ω-3 PUFAs is in cardiovascular disease [228,229]; prescription preparations of EPA and DHA (e.g. Lovaza and Epanova) are prescribed up to 4.0 g/d for treatment of high triglycerides, and importantly, are well tolerated [230]. In support of this feasibility of this approach, the European Food Safety Authority (EFSA) has declared that up to 5.0 g/d of ω-3 PUFAs is safe [231]. Anti-inflammatory effects of ω-3 PUFAs influence innate and adaptive immunity Anti-inflammatory effects of ω-3 PUFAs are attributed to alterations in: 1) altered production of eicosanoids, 2) production of bioactive lipid mediators, 3) intracellular signaling, transcription factor activity and gene expression, and 4) membrane structure/function to alter receptor:ligand interactions (reviewed in [232]) (summarized in Table 2). Eicosanoids are a diverse category of lipid mediators derived from ARA and EPA that collectively include lipoxins, prostaglandins, and thromboxanes that influence progression and resolution of inflammation. Eicosanoids are derived from metabolism of free fatty acids that are liberated from the plasma membrane by activated phospholipase A2. The free fatty acids serve as substrates for cyclo-oxygenase, lipoxygenase or cytochrome P450 enzymes. Competition for metabolism of liberated PUFAs by these enzymes results in different profiles of eicosanoids. 27 Table 2. Summary of the anti-inflammatory mechanisms of ω-3 PUFAs. Table modified from [223]. Summary of the Anti-Inflammatory Effects of ω-3 PUFAs Anti-inflammatory effect Likely mechanism Decreased generation of ARA-eicosanoids Decreased ARA in cell membranes; inhibition of ARA metabolism Increased generation of eicosanoids (EPA) and docosanoids (DHA) Increased EPA and DHA in phospholipids of cell membranes Increased generation of EPA and DHA specialized pro-resolving metabolites Increased EPA and DHA in phospholipids of cell membranes Decreased generation of inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) Decreased activation of NF-κB; activation of PPARγ; altered activity of other transcription factors Decreased expression of adhesion molecules Decreased activation of NF-κB; altered activity of other transcription factors Decreased leukocyte chemotaxis Decreased expression of receptors for chemoattractants Decreased receptor:ligand interactions Altered expression of co-stimulatory molecules 28 Generally, eicosanoids derived from ω-6 PUFAs (i.e. ARA) are pro-inflammatory, whereas eicosanoids derived from EPA and docosanoids from DHA are regarded as anti-inflammatory [19]. Importantly, ω-3 PUFAs can also be metabolized into lipid mediators known as specialized proresolving metabolites (SPMs) that enhance resolution of tissue inflammation [233]. These SPMs include classes of DHA-derived lipid metabolites such as the D-series resolvins, protectins, and maresins and have several functions that promote resolution of inflammation, including enhanced phagocytosis of apoptotic cells and pathogenic bacteria, suppression of cytokine responses, and inhibition of leukocyte activation [19,234,235]. The anti-inflammatory effects of free FA and their metabolites are attributed to their ability to act in an autocrine or paracrine fashion through their receptors to blunt inflammatory responses. One mechanism of action for DHA’s anti-inflammatory effects is activation of the peroxisome proliferator-activated receptor gamma (PPARγ), which represses transcription of NF-κB and subsequent cytokine responses [236,237]. In addition, DHA binding G-protein receptor (GPR) 120/40 inhibits activation of TLR and TNF-α pro-inflammatory pathways, and promotes M2, antiinflammatory macrophage phenotypes [238]. Similarly, DHA metabolites act via the lipoxin A4 receptor (ALX4) and GPR32 to promote inflammation resolving phenotypes [239,240]. Lipids in cell membranes can also blunt inflammation by influencing receptor-ligand interactions. ω-3 PUFAs suppress adhesion molecule expression on endothelial cells and circulating leukocytes, thereby limiting leukoocyte chemotaxis into inflamed tissues [241,242]. In addition, incorporation of ω-3 PUFAs in the lipid bilayer alters the structure of the plasma membrane which influences leukocyte activation. Cell-cell communication is critical for leukocyte activation, especially in lymphocytes, where expression of co-stimulatory molecules by B- and Tcells is necessary for activation following antigen presentation. Importantly, ω-3 PUFAs, including 29 DHA, have been shown to not only decrease expression of these co-stimulatory molecules [243], but also reduce antigen presentation [244]. Ultimately, these effects result in decreased lymphocyte proliferation [245,246]. Taken together, ω-3 PUFAs have pleiotropic anti-inflammatory effects on multiple innate and adaptive immune functions and are likely beneficial in complex, chronic inflammatory conditions such as AD. SLE not only causes inflammation itself, resulting in significant organ injury, but can also manifest because of inflammatory conditions, such as those elicited by environmental factors. Therefore, a “catch-all” approach to dampen inflammation through ω-3 PUFAs may be an effective means to blunt initiation of and progression of SLE. Association of ω-3 PUFAs in lupus – evidence from human studies Human studies suggest that there is a relationship between PUFAs and lupus. Women with SLE were found to have lower circulating ω-3 PUFAs [247]; Elkan et al. [248] extended this finding by validating self-reported ω-3 PUFA dietary intake with tissue ω-3 PUFA concentrations and concluded that: 1) individuals with SLE consumed fewer ω-3 PUFAs which was reflected in lower ω-3 PUFA incorporation in tissues compared to healthy controls 2) individuals with higher tissue ω-3 PUFA had decreased disease activity whereas those with higher ω-6 PUFA had worse disease. These findings are in accord with those from Bengtsson et al. [249] and Wu et al. [250] who independently demonstrated that differences in fatty acid metabolism distinguish SLE patients from healthy control individuals. Conclusions on the effect of ω-3 PUFAs in human lupus are hindered by experimental design While there are limited investigations on the utility of ω-3 PUFA supplementation in individuals afflicted with SLE [251], these studies have significant limitations stemming from common hurdles in clinical trials. These include small sample sizes resulting in underpowered 30 studies, confounding effects of background treatments, inadequate patient stratification at baseline and/or inclusion criteria, and a lack of consensus on how to assess improvement. Several lupus activity indices have been defined for determining SLE disease activity and are effective at assessing individual disease activity [252]. However, these methods vary widely in their criteria, were not designed to be implemented in clinical trials, and there is no consensus on which indices to use [253]. These issues are reflected in clinical trials assessing the utility of ω-3 PUFAs in SLE. In addition to the variability introduced by design of clinical trials, ω-3 PUFAs supplements are themselves widely heterogenous and contain varying proportions of EPA and DHA which could be oxidized and/or inaccurately labeled [254–256]. What few clinical trials have been conducted use varying sources and amounts of ω-3 PUFA supplements with a small number of participants. Importantly, Nording et al. identified that following ω-3 PUFA supplementation in healthy individuals, expectedly, plasma and tissue ω-3 PUFA levels increased [257]. However, highly variable levels of some lipid metabolites were identified between subjects, which may indicate individual differences in fatty acid incorporation and metabolism that further contribute to the ambiguity of ω-3 PUFAs in SLE [251]. Results of ω-3 PUFAs on SLE in human trials are inconsistent Application of complex mixtures (i.e. ω-3 PUFAs supplements) to a disease with considerable individual variability such as SLE expectedly leads to heterogenous outcomes. This is reflected in clinical trials that have found conflicting results regarding the benefit of ω-3 PUFAs in this disease, even by the same investigator [258,259] (summarized in Table 3). For example, Arriens et al. [260] and Bello et al. [261] used the SLE Disease Activity Index (SLEDAI) criteria for disease activity and found no effect of ω-3 PUFA supplements on disease activity, but found contrasting results on inflammatory endpoints. 31 Table 3. Summary of human clinical trials employing ω-3 PUFA supplementation in SLE. Table modified from [251]. Clinical Trials Employing ω-3 PUFA Supplements in SLE selected studies with n > 20 subjects Study Subjects grams ω-3 PUFA/day Disease Activity Index Effect on Disease Activity Clark et al. 1993 n = 21 with SLE double blind, crossover, placebo controlled 2.7g EPA SLEDAI No improvement on disease activity or kidney function with ω-3 PUFA n = 52 with SLE double blind, placebo controlled 1.6g EPA SLAM-R Reduced disease activity with ω-3 PUFA n = 60 with SLE double blind, placebo controlled 1.8g EPA SLAM-R 1.2g DHA BILAG Reduced disease activity with ω-3 PUFA on both disease indices n = 85 with SLE double blind, placebo controlled 1.4g EPA PGA 1.1g DHA SLEDAI n = 32 with SLE single blind, placebo controlled 2.25g EPA PGA 2.25g DHA SLEDAI Σ = 4.5g RAND-SF36 Duffy et al. 2004 Wright et al. 2009 Bello et al. 2013 Arriens et al. 2015 1.7g DHA Σ = 4.4g 1.1g DHA Σ = 2.7g Σ = 3.0g Σ = 2.5g FSS No improvement on disease activity with ω-3 PUFA by either disease index Improvement on disease activity with ω-3 PUFA by PGA No improvement on disease activity with ω-3 PUFA by other disease indices Systemic Lupus Erythematosus Disease Activity Index (SLEDAI); Systemic Lupus Activity Measure Revised (SLAM-R); British Isles Lupus Assessment Group (BILAG); Physician's Global Assessment (PGA); RAND Corporation Short Form-36 Questions (RAND-SF36); Fatigue Severity Score (FSS) 32 Wright et al. [262] and Duffy et al. [263] used Systemic Lupus Activity Measure-Revised (SLAM-R) criteria for disease activity and found a significant difference between treatments regarding disease activity, but contrasting results on inflammatory markers. From this example, it is evident that conclusions on the benefits of ω-3 PUFAs in SLE are less than convincing and require further investigations with consistent criteria for disease activity and serological parameters that corroborate disease activity. A clear beneficial effect of ω-3 PUFAs in murine models of SLE As a result of the inherent difficulty of conducting robust, highly controlled clinical trials, ω-3 PUFA supplementation in murine models that spontaneously develop SLE have been particularly useful [10,264–271]. Under these controlled conditions, ω-3 PUFA supplementation has apparent beneficial effects on SLE. In studies by the Fernandes laboratory, NZBWF1 were fed diet supplemented with either EPA- or DHA- enriched fish oil throughout life and monitored for disease progression. DHA was superior to EPA at extending the lifespan of NZBWF1 mice, effects that were related to decreased activation of transcription factors PI3K, Akt, and NF-κB [268]. The Fernandes group extended their findings by using Lovaza, a pharmaceutical preparation of EPA+DHA, which showed a dose-response effect at extending the lifespan of NZBWF1 mice [269]. In our laboratory, NZBWF1 mice fed DHA-enriched fish oil did not develop significantly elevated auto-antibodies in serum up to and including 34 wks of age, effects that were reflected by undetectable proteinuria. Additionally, we observed down-regulation of several genes in the spleen and kidney that were associated with inflammatory responses, B cell activation/differentiation, leukocyte recruitment, antigen presentation and T cell activation [10]. In comparison, high oleic safflower oil (ω-9 MUFA control oil) and corn oil (ω-6 PUFA control oil) enriched diets did not effectively reduce expression of these genes, indicating that the preventative effects of DHA- 33 enriched diets are attributable to DHA, rather than substitution of ARA with another unsaturated fatty acid (i.e. oleic acid) [266]. Not only do ω-3 PUFAs delay onset of SLE and delay progression of lupus nephritis in female NZBWF1 mice, but have beneficial effects evident in other murine models of SLE. Comparable results on the effects of ω-3 PUFAs in the kidney of autoimmune-prone male BXSB mice and MRL/lpr mice have been identified [272–274]. Several relevant findings have been found in MRL/lpr mice. Dietary substitution with ω-3 PUFA in these mice resulted in decreased production of ω-6 leukotrienes, which was associated with protection from renal disease [275]. In addition to lupus nephritis, MRL/lpr mice develop several other autoimmune characteristics, including rheumatoid arthritis, which was decreased by feeding ω-3 PUFAs to this strain [276]. Finally, another manifestation of SLE is neurological complications. BAFF-Tg mice, which overexpress BAFF, develop an SLE-like disease, including neuropsychiatric features consistent with SLE. Mice of this strain fed ω-3 PUFA enriched diet had decreased neuroinflammation [277], consistent with a protective role of ω-3 PUFAs in the brain [278]. Suggestions for future clinical trials with ω-3 PUFAs in SLE Taken together, under well-designed and controlled environments, ω-3 PUFA supplementation has shown a beneficial effect in the context of AD. Murine models that develop SLE-like disease have consistently demonstrated that dietary ω-3 PUFAs have a clear beneficial effect on several markers of inflammation, including immune activation and autoantibodies, as well as tissue-specific pathologies that occur in human SLE, including lupus nephritis, arthritis, and neurological manifestations. To better understand how ω-3 PUFAs may be utilized to control human SLE will require stringent design of clinical trials. One approach to consider in future clinical trials is application of lupus responder indices. These differ from disease activity indices 34 (e.g. SLEDAI, SLAM-R) in that they were designed to be more sensitive towards identifying individual response to treatment, and it will be of interest to see if these indices resolve the discrepancies in previous studies of ω-3 PUFAs in SLE. In a separate approach, future trials could also consider using laboratory analyses in lieu of defined activity indices, and consider individual disease activity before participant enrollment [253]. Regardless of the chosen endpoints for future clinical studies, future investigations should incorporate baseline and endpoint ω-3 PUFA content in red blood cells, a validated index of tissue ω-3 PUFA status [279], and collect data on dietary intake of ω-3 and ω-6 PUFAs over the study period. These steps will allow investigators to dissect individual responses toward ω-3 PUFA supplementation and identify confounding effects of varying dietary intake of ω-3 and ω-6 PUFAs that may resolve the discrepancies in findings. In addition, studies should limit supplements to those containing either DHA or EPA to attribute putative benefits to a particular ω-3 PUFA on a given outcome in SLE. Conclusion: While hereditary is the primary predisposing factor for autoimmunity, cumulative exposure to environmental factors such as toxic stressors and diet greatly impact latency and severity of autoimmune diseases such as lupus [95,280]. However, it remains unclear how respiratory toxicants, such as cSiO2, might trigger initiation and flaring of ADs such as lupus and how DHA supplementation could be harnessed to block such environmental triggering. Within this dissertation, using lupus-prone NZBWF1 mice, we have established a model that permits dissection of the countervailing roles of cSiO2 (potentiation) and dietary DHA (attenuation) on the initiation and progression of autoimmunity which may be similarly applied to other environmental factors. 35 REFERENCES 36 REFERENCES 1. NIEHS. Autoimmune diseases [Internet]. 2012 [cited 16 Feb 2016]. Available: https://www.niehs.nih.gov/health/materials/autoimmune_diseases_508.pdf 2. NIOSH. Silica [Internet]. 2016 [cited 16 Feb 2016]. 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Available: https://www.niaid.nih.gov/topics/autoimmune/Documents/adccfinal.pdf 60 CHAPTER 2: SILICA TRIGGERS INFLAMMATION AND ECTOPIC LYMPHOID NEOGENESIS IN THE LUNGS IN PARALLEL WITH ACCELERATED ONSET OF SYSTEMIC AUTOIMMUNITY AND GLOMERULONEPHRITIS IN THE LUPUSPRONE NZBWF1 MOUSE This chapter has been published in Bates et al. (2015) PLoS ONE 10(5): e0125481. Abstract Genetic predisposition and environmental factors influence the development of human autoimmune disease. Occupational exposure to crystalline silica (cSiO2) has been etiologically linked to increased incidence of autoimmunity, including systemic lupus erythematosus (SLE), but the underlying mechanisms are poorly understood. The purpose of this study was to test the hypothesis that early repeated short-term cSiO2 exposure will modulate both latency and severity of autoimmunity in the lupus-prone female NZBWF1 mouse. Weekly intranasal exposure to cSiO2 (0.25 and 1.0 mg) for 4 wk beginning at 9 wk of age both reduced latency and increased intensity of glomerulonephritis. cSiO2 elicited robust inflammatory responses in the lungs as evidenced by extensive perivascular and peribronchial lymphoplasmacytic infiltration consisting of IgGproducing plasma cells, and CD45R+ and CD3+ lymphocytes that were highly suggestive of ectopic lymphoid tissue (ELT). In addition, there were elevated concentrations of immunoglobulins and the cytokines MCP-1, TNF-α and IL-6 in bronchoalveolar lavage fluid. cSiO2-associated kidney and lung effects paralleled dose-dependent elevations of autoantibodies and proinflammatory cytokines in plasma. Taken together, cSiO2-induced pulmonary inflammation and ectopic lymphoid neogenesis in the NZBWF1 mouse corresponded closely to 61 systemic inflammatory and autoimmune responses as well as the early initiation of pathological outcomes in the kidney. These findings suggest that following airway exposure to cSiO2, in mice genetically prone to SLE, the lung serves as a platform for triggering systemic autoimmunity and glomerulonephritis. Introduction Development of autoimmunity is widely acknowledged to be impacted not only by genetic predisposition, but by environmental factors that potentially accelerate initiation of autoimmune responses and increase severity of organ-specific pathologies [1]. Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease that affects 300,000 Americans, most of whom are women, and is often associated with glomerulonephritis and kidney failure [2]. In the United States, it is estimated that at least 1.7 million workers are employed in occupations (e.g. mining, construction, manufacturing and custodial industries) with potential exposure to crystalline silica (cSiO2) [3]. Human epidemiological studies have linked occupational exposure to cSiO2 to increased SLE incidence [4-6]. For example, a population-based, case-control study conducted with participants from the Carolina Lupus Cohort reported that development of SLE was more prevalent in individuals with occupations associated with cSiO2 exposure [5]. Similarly, in a cohort of 15,000 uranium miners heavily exposed to cSiO2, 28 men were diagnosed with SLE, whereas the predicted SLE prevalence for a group of this size was ≤ 1.5 individuals [7]. Finckh and coworkers [6] found that occupational exposure to cSiO2, but not organic solvents, contributed to increased SLE in urban women. Other studies report SLE-like symptoms in individuals with silicosis, including lymphocyte activation and elevated antinuclear antibodies, immunoglobulins, and immune complexes [8-11]. Both in vivo and in vitro studies have yielded several lines of evidence that cSiO2-induced 62 cell death of alveolar macrophages is a critical event in cSiO2-induced exacerbation of autoimmunity [12-17]. Phagocytosis of cSiO2 particles by alveolar macrophages induces NF-κB activation [18,19]. This results in production of proinflammatory cytokines, including TNF- [20], which are correlated with disease activity in individuals with SLE [21,22]. Aberrant apoptosis and/or necrosis of cSiO2-laden alveolar macrophages not only releases these particles, enabling continued pulmonary re-exposure to cSiO2 and sustained inflammation, but could also expose normally sequestered nuclear antigens (e.g. dsDNA). Both excessive apoptosis and defective clearance of apoptotic debris contribute to aberrant self-antigen presentation, driving consequent autoimmune sequealae [12,23]. Mouse strains that spontaneously develop SLE have been used to explore how toxicant exposure affects the pathogenesis of autoimmunity [24-27]. One such strain, NZM2410, bred from the widely used SLE model, NZBWF1 [24], exhibits rapid onset of lupus nephritis with most mice succumbing to glomerulonephritis by 25 wk of age [28]. When intranasally instilled with cSiO2, both male and female NZM2410 mice exhibit accelerated onset and increased severity of SLErelated responses including elevated autoreactive antibodies, proteinuria, and glomerulonephritis [29]. It was further determined that concentrations of TNF- in bronchoalveolar lavage fluid of cSiO2-exposed NZM2410 mice were significantly higher than saline-treated control mice [30]. Autoantibodies isolated from sera of cSiO2-exposed NZM2410 mice were found to bind to apoptotic debris derived from alveolar macrophages [31]. Importantly, the effects of cSiO2 on autoimmunity could be attenuated in this strain in vivo by concurrent instillation with rottlerin, an inhibitor of apoptosis [32]. There are several issues that limit the use of the NZM2410 phenotype to uncover mechanisms by which cSiO2 triggers SLE. Not only does this strain develop rapid onset and 63 progression of lupus nephritis compared to other lupus-prone mouse models [24], but the majority of NZM2410 mice succumb to glomerulonephritis within 2 wk of proteinuria appearance [33]. Furthermore, contrary to SLE prevalence in human population and other murine models [24], NZM2410 mice do not display female sex bias. Finally, while NZM2410 mice have been useful for delineating genetic contributions to SLE, most preclinical studies of putative therapeutic agents employ the NZBWF1 mouse, which displays slower development of autoimmunity [25]. Taken together, investigation of subtle underlying mechanisms and preventative measures to counter toxicant-triggered SLE might be enhanced by employing the less-penetrant NZBWF1 phenotype. The purpose of this study was to test the hypothesis that short-term repeated intranasal cSiO2 exposure will both decrease latency to onset and increase severity of autoimmunity in female NZBWF1 mice. The results presented herein suggest that cSiO2 triggered robust autoimmune and inflammatory responses in the lung, including the formation of ectopic lymphoid tissue, that closely paralleled early onset of glomerulonephritis and exacerbated systemic autoimmune responses. Materials and Methods Mice All experiments were approved by the Institutional Animal Care and Use Committee at Michigan State University in accordance with the National Institutes of Health guidelines for animal use. Female 7 wk old lupus-prone NZBWF1 and control C57Bl/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME), randomized into treatment groups, and allowed to acclimate for 2 wk prior to cSiO2 exposure. Upon arrival and throughout the study, mice were fed semi-purified American Institute of Nutrition (AIN)-93G diet containing high oleic safflower oil [34]. Mice were housed 4 per cage with free access to food and water and maintained at constant 64 Figure 4. Experimental design for intranasal cSiO2 exposure. Beginning at 9 wk of age, NZBWF1 and C57Bl/6 mice were dosed intranasally with 25μl PBS containing 0, 0.25 mg, or 1.0 mg cSiO2 once per wk, for 4 wk. Proteinuria was monitored over the course of the experiment and all animals sacrificed at 24 wk of age. 65 temperature and humidity (21°C–24°C and 40–55%, respectively) under a 12-h light/dark cycle. cSiO2 exposure The experimental design for cSiO2 exposure, summarized in Fig. 4, was based on previous protocols [29-32,35]. cSiO2 (Min-U-Sil-5, average particle diameter 1.5-2.0 μm) was originally obtained from Pennsylvania Sand Glass Corporation (Pittsburgh, PA). Stock suspensions of each dose were prepared fresh in sterile phosphate buffered saline (PBS) just prior to intranasal exposure and sonnicated for 1 min before use. At 9 wk of age, mice were anesthetized with 4% isoflurane and instilled intranasally with 0, 0.25 and 1.0 mg of cSiO2 in 25 μl PBS. To mimic intense occupational exposure to cSiO2 over a short time period, mice were instilled once per wk for 4 wk. Urine was collected weekly and evaluated for proteinuria by 2P urine reagent strips (Cortez Diagnostics, Inc., Calabasas, CA). Mice were sacrificed and tissues collected 12 wk after the final exposure to cSiO2. At this time point, more than 50% of animals exposed to the high cSiO2 dose exhibited moderate proteinuria, defined as ≥ 300 mg/dl. In addition to NZBWF1, female C57Bl/6 mice were used as a control to discern histopathologic effects of high cSiO2 exposure in kidney and lung. C57Bl/6 mice were dosed with either 25 μl PBS or 1.0 mg cSiO2 in 25 μl PBS following the above protocol and terminated 12 wk after the final exposure. Necropsy and tissue collection Animals were euthanized by intraperitoneal injection with 56 mg/kg BW sodium pentobarbital and exsanguination via the abdominal aorta. Blood was collected using heparinized syringes and centrifuged at 3500 x g for 10 min at 4°C for separation of plasma. The collected plasma was stored at -80°C until future analysis. Following BALF collection as described previously [36], lungs were intratracheally fixed with 10% (v/v) neutral buffered formalin at constant pressure (30 cm H2O) for a minimum 1 h and stored in fixative until further processing 66 for histology. The right kidney was excised and the cranial portion fixed for 24 h in 10% neutral buffered formalin. Kidney histopathology Formalin-fixed kidneys were paraffin-embedded, sectioned and stained with either hematoxylin and eosin (H&E) or Periodic acid-Schiff and hematoxylin (PASH). Stained sections were evaluated for lupus nephritis in a blinded fashion by a board-certified veterinary pathologist using a modified International Society of Nephrology/Renal Pathology (ISN/RPS) Lupus Nephritis Classification system [37]. Slide sections were graded as follows: (0) no tubular proteinosis and normal glomeruli; (1) mild tubular proteinosis with multifocal segmental proliferative glomerulonephritis and occasional early glomerular sclerosis and crescent formation; (2) moderate tubular proteinosis with diffuse segmental proliferative glomerulonephritis, early glomerular sclerosis and crescent formation; and (3) marked tubular proteinosis with diffuse global proliferative and sclerosing glomerulonephritis. Lung histopathology Randomly orientated, serial sections of the formalin-fixed left lung lobe were processed routinely and embedded in paraffin. Tissue sections (5 μm) were deparaffinized and stained with H&E for histopathology. Tissues were semi-quantitatively scored by a board-certified veterinary pathologist for the following lung lesions: (a) presence of lymphocytic cell infiltration within perivascular and peribronchial regions, (b) alveolitis defined as the presence of alveolar infiltration of vacuolated macrophages, neutrophils, and lymphocytes, granuloma formation in the alveolus, type II epithelial cell hyperplasia, and thickened alveolar wall, and (c) presence of alveolar proteinosis. Individual lungs were graded for these lesions using the following criteria (% of total pulmonary tissue examined): (0) no changes compared to control mice; (1) minimal (<10%); (2) 67 slight (10-25%); moderate (26-50%); (4) severe (51-75%); or (5) very severe (>75%) of total area affected. Immunohistochemistry for IgG was performed on paraffin-embedded lungs. Tissue sections (5 μm) were deparaffinized and subjected to heat-induced epitope retrieval with citrate buffer (pH 6.0) for 30 min at 100ºC. Endogenous peroxidase was blocked with 3% (v/v) H2O2/methanol for 30 min at room temperature followed by blocking non-specific protein binding with normal horse serum (Vector Laboratories Inc., Burlingame, CA) for 30 min. Tissue endogenous avidin and biotin binding sites were blocked by incubation with Avidin D (Vector Laboratories) followed by d-Biotin (Sigma Aldrich, St. Louis, MO), each for 15 min. Goat antimouse IgG γ-chain specific (Alpha Diagnostic Inc., San Antonio, TX) was diluted 1:4000 in normal antibody diluent (ScyTek Laboratories Inc., Logan, UT) and incubated for 60 min followed by incubation with 11µg/ml biotinylated horse anti-goat IgG H+L (Vector Laboratories, Inc.) in normal antibody diluent for 30 min. Peroxidase enzyme was incubated on slides for 30 min with Ready-to-Use Elite Peroxidase Reagent (Vector Laboratories, Inc.). Finally, slides were incubated for 15 min with Nova Red substrate (Vector Laboratories, Inc.) and counterstained with hematoxylin. B and T lymphocytes were identified using rat anti-mouse CD45R (BD Biosciences, San Jose, CA) (1:300) and rabbit anti-mouse CD3 (1:100), respectively on deparaffinized tissue sections (4 μm) that were subjected to heat-induced epitope retrieval with citrate buffer (pH 6.0) for 30 min at 100ºC (CD45R) or 125ºC for 15 min (CD3). Following pretreatments, formation of avidin – biotin complex and primary antibody staining were performed as described above. Bound CD45R and CD3 antibodies were detected by incubation with biotinylated rat and goat anti-rabbit in conjunction with Peroxidase Reagent and Nova Red substrate. 68 BALF processing and differential staining Cytological slides from 150 μl BALF were prepared by centrifugation at 400 x g for 10 min using a Shandon Cytospin 3 (Shandon Scientific, Pittsburgh, PA), allowed to air dry, and stained with Diff-Quick (Fisher Scientific). A total of 200 cells were counted and cells identified as monocytes/macrophages, lymphocytes, and polymorphonuclear (PMN) leukocytes using morphological criteria. Remaining BALF was centrifuged at 2400 x g for 15 min and supernatant collected and stored at -80°C for IgG and cytokine analysis. IgG and autoantibody measurement Total IgG, IgA and IgM were quantitated in BALF using an ELISA method described previously [38]. Briefly, 96-well plates were coated with 50µL of 10 µg/ml goat anti-mouse IgA, IgM, or IgG, respectively (Alpha Diagnostics, Inc.). The standard curve was generated using mouse reference serum (Bethyl Laboratories, Inc., Montgomery, TX). Plates were read on an ELISA reader (Molecular Devices, Menlo Park, CA) at 650 nm and Ig concentrations calculated from the standard curves using Softmax software (Molecular Devices). Total Ig antibodies for nuclear antigens and double-stranded DNA (dsDNA) in plasma were measured according to manufacturer’s instructions using commercial ELISA kits (Alpha Diagnostics, Inc.). Cytokine analysis BALF and plasma were diluted two-fold and analyzed for the inflammatory cytokines IL6, IL-10, MCP-1, IFN-γ, TNF, and IL-12p70 using a Mouse Inflammation Cytometric Bead Array (BD Biosciences). Data was acquired using a FACSCalibur flow cytometer (BD Biosciences) and cytokine concentrations were calculated from standard curves using FCAP Array Software (BD Biosciences). 69 Statistics All treatment groups consisted of 8 mice and data presented as group mean ± SEM. Data were plotted and analyzed using SigmaPlot 11.0 for Windows (Jandel Scientific; San Rafael, CA). Grubb’s test was performed to identify and exclude outliers from statistical analysis. Differences between cSiO2- and vehicle-treated NZBWF1 mice were analyzed by one-way ANOVA on Ranks with Dunn’s method. Differences between cSiO2- and vehicle-treated C57Bl/6 mice, and NZBWF1 and C57Bl/6 histopathological lung grades were analyzed by Mann-Whitney Rank Sum Test. The Spearman rank-order correlation coefficient was used to correlate cSiO2 dose to experimental endpoint for NZBWF1. A P value of < 0.05 was considered statistically different for all study outcomes. Results cSiO2 elicits early onset of proteinuria and glomerulonephritis The effects of weekly intranasal exposure to 0, 0.25 mg, or 1.0 mg cSiO2 from 9 to 12 wk of age on renal function were monitored by measuring urinary protein at weekly intervals. Proteinuria (≥ 300 mg/dl) was observed in 13% and 62% of NZBWF1 mice treated with 1.0 mg cSiO2 at 10 and 12 wk post-dosing, respectively (Fig 5). In contrast, proteinuria was not evident in NZBWF1 mice dosed with 0.25 mg cSiO2 or vehicle up to 12 wk post-treatment. Proteinuria was not observed in control C57Bl/6 mice treated with vehicle or 1.0 mg cSiO2 up to 12 wk postdosing. To confirm nephritogenic effects of cSiO2, kidney sections from NZBWF1 mice at 12 wk post-treatment were assessed histopathologically following H&E and PASH staining (Figure 6 A, B). Kidneys from NZBWF1 mice treated with 1.0 mg cSiO2 exhibited frequent tubular proteinosis and proliferative glomerulonephritis (Fig. 6B). Lymphocytic infiltration at the renal pelvis as well 70 Figure 5. cSiO2 exposure accelerates development of proteinuria in NZBWF1 mice. Proteinuria was monitored weekly until sacrifice 12 wk after the final cSiO2 exposure when most mice exposed to 1.0 mg cSiO2 were over threshold (≥ 300 mg/dl). Proteinuria was not detected in NZBWF1 mice dosed with 0.25 mg cSiO2 or vehicle. C57Bl/6 mice exposed to cSiO2 or vehicle did not develop detectable proteinuria over the course of the experiment. 71 Figure 6. cSiO2 exposure increases severity of lupus nephritis in kidneys of NZBWF1 mice. Representative light photomicrographs of PASH stained kidney sections in NZBWF1 mice at 24 wks of age exposed to vehicle (A) and 1.0mg cSiO2 (B). NZBWF1 mice exposed to 1.0mg cSiO2 developed extensive glomerulonephritis (black arrow) and tubular proteinosis (asterisk). NZBWF1 mice were individually graded for lupus nephritis following the modified ISN/RPS classification system as described in the Materials and Methods (C). Animals exposed to cSiO2 developed more severe lesions characteristic of lupus nephritis than vehicle-exposed mice. cSiO2 dose significantly correlated with lupus nephritis (Spearman rank-order coefficient = 0.64, p < 0.05). 72 as sclerosis and crescent formation were additional common findings in animals treated at this dose. These responses were largely absent in NZBWF1 mice treated with vehicle (Fig. 6A) or 0.25 mg cSiO2 as well as control C57Bl/6 mice treated with vehicle or 1.0 mg cSiO2 (Fig. 6A, B). When mice were graded individually for glomerulonephritis severity, only 13% (1/8) of NZBWF1 mice treated with vehicle were ranked 1 due to the occurrence of minimal tubular proteinosis and rare glomeruli with segmental mesangial hypercellularity, whereas the remaining vehicle-treated mice were ranked 0 (Fig. 6C). In contrast, cSiO2 elicited renal lesions characteristic of lupus nephritis, with 38% (3/8) and 63% (5/8) of NZBWF1 mice treated with 0.25 mg and 1.0 mg of the agent, respectively, being ranked 1. Additionally, 25% (2/8) of NZBWF1 mice treated with 1.0 mg cSiO2 had renal lesions ranked 2 or 3, whereas no mice treated with vehicle or 0.25 mg cSiO2 developed such severe lesions. Interestingly, while all C57Bl/6 mice exposed to vehicle were ranked 0, indicating no renal lesions consistent with glomerulonephritis, 38% of mice dosed with 1.0 mg cSiO2 developed renal lesions ranked 1 or 2 (Fig. 7C). Thus, cSiO2 appeared to induce modest glomerulonephritis in some of the C57Bl/6 control mice without onset of evident proteinuria. cSiO2 induces marked lymphoplasmacytic infiltration in lung parenchyma Histologically, conspicuous lymphoplasmacytic infiltrates were present in interstitial tissues surrounding bronchioles and associated blood vessels throughout the lung in NZBWF1 mice exposed to 1.0 mg cSiO2 (Fig. 8C). This response was less severe in NZBWF1 mice treated with 0.25 mg cSiO2 and minimal in NZBWF1 mice treated with vehicle (Fig. 8A, B; Table 4). C57Bl/6 mice exposed to 1.0 mg cSiO2 had minimal perivascular and peribronchial lymphoplasmacytic infiltrates as compared to vehicle controls (Fig. 8D, E) and NZBWF1 mice treated with cSiO2 (Table 4). 73 Figure 7. cSiO2 exposure in C57Bl/6 mice induced kidney lesions resembling lupus nephritis. Representative light photomicrographs of PASH stained kidney sections in C57Bl/6 mice at 24 wks of age exposed to vehicle (A) and 1.0mg cSiO2 (B). Some renal histopathological lesions were observed in cSiO2-exposed C57Bl/6 mice (B) with notable renal tubular proteinosis (asterisk) and several glomeruli with global mesangial hypercellularity (white arrows). C57Bl/6 mice were individually graded for lupus nephritis as described in the Materials and Methods (C). Animals exposed to cSiO2 developed more severe lesions characteristic of lupus nephritis than vehicle exposed mice. 74 Figure 8. cSiO2 elicits perivascular and peribronchiolar lymphocytic infiltration in lungs of NZBWF1 and C57Bl/6 mice. Representative light photomicrographs of H&E stained lung sections from NZBWF1 mice exposed to vehicle (A), 0.25 mg cSiO2 (B), or 1.0 mg cSiO2 (C) and C57Bl/6 mice given vehicle (D), or 1.0mg cSiO2 (E). Br = bronchiole, bv = bronchial vasculature, ap = alveolar parenchyma. Black arrows indicate lymphocytic infiltration in perivascular and peribronchial regions. Lymphocytic infiltration was semi-quantitatively graded as described in the Materials and Methods (Table 4). 75 Table 4. cSiO2 exposure increases severity of lymphocytic cell infiltration in lungs of NZBWF1 mice relative to C57Bl/6 mice. 76 In addition to the pronounced inflammatory cell infiltration observed in the interstitial tissue of the lung, diffuse alveolar lesions were histologically evident after cSiO2 exposure in NZBWF1 and C57Bl/6 mice. In contrast to the aforementioned lymphocytic response in the lung tissues, the severity of alveolar lesions in NZBWF1 mice was comparable to C57Bl/6 mice given the same amount of cSiO2 (Table 4). Dosing of NZBWF1 mice with 0.25 mg cSiO2 also elicited the responses observed with 1.0 mg cSiO2 in this strain, albeit to a more modest extent. cSiO2 elicits IgG producing plasma cells in lung Based on the prominent lymphoplasmacytic response in the lungs of cSiO2-treated NZBWF1 mice, the effects of intranasal cSiO2 on IgG production in these tissues was assessed immunohistochemically. Numerous IgG-laden lymphocytes were found present in the perivascular and peribronchiolar lymphoplasmacytic aggregates in cSiO2-treated but not vehicle-treated NZBWF1 mice (Fig. 9B, D). In addition, H&E staining of lung sections from NZBWF1 mice treated with cSiO2 revealed occasional plasma cells containing Russell bodies (conspicuous cytoplasmic accumulation of hyaline material) that were immunohistochemically identified as containing IgG globulin. Taken together, these results suggest that both production and secretion of IgG by infiltrating plasma cells is induced in both lungs and airways after intranasal cSiO2 exposure in NZBWF1 mice. CD45R+ and CD3+ infiltrating lymphocytes in lung organize into ectopic lymphoid tissue after cSiO2 exposure in NZBWF1 mice Infiltrating lymphocytes induced by cSiO2 in lungs of NZBWF1 mice were further characterized by immunohistochemistry for B and T lymphocytes. Distinct aggregates of CD45R+ cells (B lymphocytes) (Fig. 10B, F, and J) interspersed with CD3+ cells (T lymphocytes) (Fig. 10C, G, K) were observed peripheral to both bronchiolar airways and blood vessels in lung 77 Figure 9. Marked accumulation of IgG producing plasma cells occurs in lungs of cSiO2exposed NZBWF1 mice. Representative immunohistochemical photomicrographs of IgG in lungs of NZBWF1 mice. Photomicrographs taken at low magnification are shown in A and B whereas images at high magnification are shown in C and D. Vehicle-exposed mice did not indicate positive IgG staining (A,C). cSiO2 induced marked infiltration of IgG-laden lymphocytes peripheral to both blood vessels (bv) and bronchiole airways (BA) (black arrows in B, white arrows in D). IgG was also detected extracellularly within alveolar parenchyma (ap) (stippled arrows in B, black arrows in D) 78 Figure 10. Intranasal cSiO2 exposure induces infiltration of CD45R+ and CD3+ lymphocytes in lungs that resemble ectopic lymphoid tissue. Representative light photomicrographs of lung tissue sections from mice treated with 0.0 mg cSiO2 (vehicle controls; A, D, E, H, I and L) or 1.0 mg cSiO2 (B, C, F, G, J and K). 79 Figure 10 (cont’d). Some lung sections were immunohistochemically stained to identify B lymphocytes (CD45R+) (A, B, E, F, I, and J), while others (C, D, G, H, K, and L) were immunohistochemically stained to identify T lymphocytes (CD3+). Photomicrographs taken at high magnifications of blood vessels (v) and bronchioles (b) are illustrated in E, F, G, H and I, J, K, L, respectively. In cSiO2-treated mice, both bronchiolar airways and blood vessels were fully or partially circumscribed by thick interstitial infiltrates of mononuclear cells (arrows in B and C). These infiltrates were primarily comprised of B lymphocytes (solid arrows in F and J) and T lymphocytes (stippled arrows in G and K). B cells tended to form distinct focal aggregates (solid arrows in F and J) and T cells (stippled arrows in G and K) were more diffusely distributed throughout the peribronchiolar and perivascular lymphoid infiltrates. Control mice had only a few widely scattered B (solid arrow in E, I) and T (stippled arrow in H, L) cells present in the alveolar parenchyma (a), but no distinct lymphoid cell cuffing around the bronchioles or blood vessels. All tissues were counterstained with hematoxylin. e, airway epithelium. 80 interstitial tissue of NZBWF1 mice exposed to 1.0 mg cSiO2. The distinct localization of CD45R+ aggregated cells in conjunction with infiltrating CD3+ lymphocytes in the lungs following cSiO2 exposure in NZBWF1 mice structurally resembled the spatial architecture of ectopic lymphoid tissue (ELT). The identification of ELT in parallel with the observation of IgG-producing plasma cells suggest that these structures functionally contribute to initiation of antigen-specific adaptive immune responses in lungs of NZBWF1 mice following intranasal cSiO2 exposure. cSiO2 induces leukocyte infiltration and elevates IgG, IgA, and IgM in BALF Effects of enhanced lymphocytic infiltration following intranasal cSiO2 exposure were also evident in the airways. In the recovered BALF, treatment with 0.25 mg and 1.0 mg cSiO2 in NZBWF1 mice elicited increased recruitment of lymphocytes (33% and 43%, respectively) relative to vehicle control where the majority of cells were monocytes/macrophages in both NZBWF1 and C57Bl/6 mice (Fig. 11). Relative to NZBWF1 mice, 1.0 mg cSiO2 in C57Bl/6 mice elicited a modest increase of lymphocytes in BALF (23%). Similar PMN leukocyte responses were observed after 1.0mg cSiO2 exposure in both NZBWF1 (19%) and C57Bl/6 mice (17%). Based on the observed increase in lymphocytes following intranasal cSiO2 exposure, we performed ELISA to quantitate IgG, IgA and IgM in BALF of NZBWF1 and C57Bl/6 mice. IgG in BALF of NZBWF1 was substantially elevated in a dose-dependent manner following cSiO2 treatment (Fig. 12A). cSiO2 also increased IgA and IgM in BALF of both NZBWF1 and C57Bl/6 mice, albeit to a lesser extent than IgG (Fig. 12B, C). cSiO2 induces elevation of MCP-1, TNF-α, and IL-6 in BALF BALF of NZBWF1 mice was further analyzed for the cytokines IL-6, IL-10, MCP-1, IFNγ, TNF-α, and IL-12p70. While effects on IFN-γ, IL-10 or IL-12p70 were not detectable, cSiO2 treatment dose-dependently induced elevation of the proinflammatory cytokines MCP-1, TNF-α, 81 Figure 11. Intranasal cSiO2 exposure induces infiltration of lymphocytes and PMN leukocytes in BALF of NZBWF1 and C57Bl/6 mice. Cytometric slides prepared from BALF were stained with Diff-Quick and 200 cells per slide identified as monocytes/macrophages (Mo), lymphocytes (Lymph) or polymorphonuclear (PMN) cells. In NZBWF1 mice, group mean ± SEM at 0.0 mg cSiO2were 97.2 ± 0.4%, 2.4 ± 0.3%, and 0.5 ± 0.1% for Mo, Lymph and PMN, respectively. Group mean ± SEM for 0.25 mg cSiO2 group were 50.8 ± 2.9%, 32.8 ± 3.5%, and 14.0 ± 1.8%, respectively and for the 1.0 mg cSiO2 group were 39.3 ± 1.7%, 43.0 ± 2.5%, and 18.7 ± 1.8%, respectively. In C57Bl/6 mice, group mean ± SEM at 0.0 mg cSiO2 were 95.9 ± 0.9%, 3.1 ± 0.7% and 1.0 ± 0.2% for Mo, Lymph, and PMN, respectively. Group mean ± SEM for 1.0mg cSiO2 were 59.9 ± 3.7%, 22.6 ± 3.7, and 17.3 ± 2.1% for Mo, Lymph, and PMN, respectively. 82 Figure 12. cSiO2 exposure elevates IgG (A), IgA (B), and IgM (C) concentrations in BALF of NZBWF1 and C57Bl/6 mice. Total immunoglobulins in BALF were measured by ELISA. Data are group mean ± SEM (n = 7–8/gp) and were analyzed by one-way ANOVA on Ranks with Dunn’s method (NZBWF1) or Mann-Whitney Rank Sum Test (C57Bl/6). Asterisk indicates a statistically significant difference between cSiO2 treatment and vehicle control (p < 0.05). In BALF of NZBWF1 mice, cSiO2 dose correlated significantly (p < 0.05) with IgG (Spearman rankorder correlation coefficient = 0.80), IgA (Spearman rank-order correlation coefficient = 0.72), and IgM (Spearman rank-order correlation coefficient = 0.85). 83 and IL-6 (Fig. 13 A-C). cSiO2 triggers systemic autoantibody and proinflammatory cytokine responses Marked elevations of total Ig anti-dsDNA antibodies (Fig. 14A) and anti-nuclear antibodies (Fig. 14B) were detected in plasma of cSiO2-dosed NZBWF1 mice compared to vehicle-treated mice. No changes in plasma anti-dsDNA antibodies were detected in C57Bl/6 mice exposed to cSiO2 or vehicle (Fig. 60). cSiO2 dose-dependently induced elevation of the proinflammatory cytokines MCP-1, TNF-α, and IL-6 (Fig. 13 D-F) in NZBWF1 mice whereas effects on plasma cytokines IFN-γ, IL-10 or IL-12p70 were undetectable. Discussion Human epidemiological findings support the contention that airway exposure to cSiO2 is etiologically linked to development of autoimmunity [4-6,39-43]. However, such studies shed little light on the mechanisms that underlie this putative association. Towards that end, we investigated the effects of short-term repeated cSiO2 exposure on autoimmune disease development in the NZBWF1 female mouse, a widely used experimental animal model for SLE. Several novel observations regarding this lupus-prone mouse strain were made 12 wk after the final cSiO2 exposure. First, cSiO2 triggered the onset of and exacerbated glomerulonephritis. Second, concurrent with kidney effects, cSiO2 induced marked lymphocyte infiltration in the lungs as well as dramatic increases in total immunoglobulins (IgG, IgA and IgM), and proinflammatory cytokine concentrations. Third, a striking observation in the lungs of NZBWF1 mice was the identification of extensive ELT that likely contributes to early triggering of SLE after cSiO2 exposure. Finally, in plasma, we found that cSiO2 exposure elicited marked elevations in autoreactive antibodies and proinflammatory cytokine concentrations. Collectively, these findings suggest intensive cSiO2 exposure via the airways over a discrete period evoked long-lasting injurious inflammatory and in 84 Figure 13. cSiO2 induces elevation of MCP-1 (A, D), TNF-α (B, E), and IL-6 (C, F) in BALF and plasma of NZBWF1 mice. Proinflammatory cytokine concentrations in BALF and plasma were determined by cytometric bead array. Data are group mean ± SEM (n = 5–8/gp) and were analyzed by one-way ANOVA on Ranks with Dunn’s method. Asterisk indicates a statistically significant difference in analyte between cSiO2 treatment and vehicle control (p < 0.05). cSiO2 dose correlated significantly (p < 0.05) with BALF MCP-1 (Spearman rank-order correlation coefficient = 0.90), BALF TNF-α (Spearman rank-order correlation coefficient = 0.89), and BALF IL-6 (Spearman rank-order correlation coefficient = 0.82). cSiO2 dose also correlated significantly (p < 0.05) with plasma TNF-α (Spearman rank-order correlation coefficient = 0.60), and IL-6 (Spearman rank-order correlation coefficient = 0.49). n.d. indicates not detected. 85 Figure 14. cSiO2 exposure increases anti-dsDNA antibodies (A) and anti-nuclear antibodies (B) in plasma of NZBWF1 mice. 86 autoimmune responses that were evident at the site of exposure (lung), systemically, and at a distal tissue site (kidney). Elevated plasma titers of autoreactive antibodies are an immunological hallmark for human SLE. Systemic autoantibodies form immune complexes with circulating self-antigens (e.g. dsDNA and nucleosome fractions). When deposited in the kidney [29,44-46], these immune complexes promote proinflammatory cytokine and chemokine production that mediate infiltration by mononuclear cells and consequent tissue injury [47]. Congruent with these mechanisms, heightened severity of glomerulonephritis was observed here in cSiO2-treated NZBWF1 mice concurrently with early onset of proteinuria. These findings are consistent with previous observations in cSiO2-exposed NZM2410 mice [29]. Several epidemiological studies have identified an association between occupational cSiO2 exposure and development of end-stage renal disease [39,48,49], particularly glomerulonephritis [50,51]. In a previous study by our laboratory, NZBWF1 mice fed diet identical to that employed here (safflower oil-based AIN-93G) spontaneously developed proteinuria at 30 wk of age [34]. Herein, NZBWF1 dosed with 1.0 mg cSiO2 exhibited proteinuria beginning at 22 wk of age. Thus, intranasal exposure to cSiO2 likely decreased the time to onset of lupus nephritis by 8 wk. Interestingly, kidney lesions were also increased in cSiO2-treated C57Bl/6 mice, albeit to a more modest extent. The observation of renal pathology consistent with lupus nephritis in C57Bl/6 mice supports the notion of an association between cSiO2 exposure and kidney disease. Consistent with these findings, autoimmune-like responses in kidneys including IgG deposition have been previously described after intratracheal administration of fibrogenic particles in C57Bl/6 mice [52,53]. While overt proteinuria (≥ 300 mg/dl) did not develop over the 12 wk duration of our study, it might be speculated that given sufficient time, short-term repeated airway exposure to 87 cSiO2 might induce autoimmune nephritis in C57Bl/6 and other mice without evident genetic predisposition to SLE. Although a prior study of cSiO2-induced pulmonary inflammation in NZM2410 autoimmune mice qualitatively described inflammatory cell infiltration in the lung [29], this report is the first to semi-quantitatively grade this response. A critical finding in this study was that NZBWF1 mice exposed to cSiO2 exhibited extensive lymphoplasmacytic infiltration in peribronchial and perivasculature regions of the lungs. Similar but more modest effects were observed in C57Bl/6 mice. This observation is consistent with the findings of Singh et al. [54] who suggested that the magnitude of perivasculature and peribronchial inflammation induced by pulmonary toxicant exposure is dependent on genetic background. A critical question relates to the relevance of the doses employed in this study to actual human exposure. Using the NIOSH limit for respirable cSiO2 established at 0.05mg/m3/d and the assumption that human ventilation rate is 6.0L/min, we calculate that humans could be exposed to 1433mg cSiO2 in 40 years of work (8h/d for 5 d/wk) with respirable cSiO2. Based on the assumption that mice ventilation rate is 0.03L/min, we estimate that an equivalent lifetime exposure to cSiO2 would be 8.28 mg in mouse. Therefore, the two cumulative doses of cSiO2 used in this study, 1 and 4 mg, represent approximately one eighth and one half, respectively, of a human lifetime exposure to cSiO2 at the recommended NIOSH exposure limit. We opted here to employ intranasal instillation of cSiO2 based on its successful use in previous studies with NZM2410 mice [29,30,32,35]. Other routes of cSiO2 administration in murine species have been described including inhalation, intratracheal and trans-oral instillation (oropharyngeal aspiration) [55-57]. Development of silicosis in four in-bred mouse strains, including lupus-prone MRL/MpJ mice, was assessed 16 wks after inhalation exposure to 70 mg 88 m-3 cSiO2 for 5 h for 12 consecutive days [58]. MRL/MpJ mice developed extensive pulmonary lymphocytic infiltrates, similar to those observed herein with lupus-prone NZBWF1 mice, whereas Balb/c mice given the same exposure exhibited only mild pulmonary histopathological lesions. Although inhalation exposure of rodent species is the gold standard for modeling human airway exposure to respirable toxicants such as cSiO2, the need for specialized inhalation exposure chambers, rigorous technical considerations and high operational costs limit these experiments to only a few laboratory facilities. A comparative study of intratracheal, trans-oral, and intranasal instillation in Balb/c mice receiving a single 1.0 mg dose of cSiO2 revealed that each method was capable of eliciting significant pulmonary inflammation 4 h after exposure [59]. Intranasal instillation resulted in comparatively less inflammation relative to both trans-oral and intratracheal instillation. This could be attributed to trapping of cSiO2 particles in the nasal cavity, resulting in delayed passage to the lower airways [59]. However, this effect is not likely to be an issue in longterm studies of intranasal exposure to cSiO2, as mice treated in this manner eventually develop pulmonary fibrosis, a hallmark of cSiO2 exposure [35,60,61]. These latter studies support the contention that the high-dose, short-term repeated intranasal exposure employed here is a suitable model for investigations of environmentally triggered onset of autoimmunity in lupus-prone mice. Nevertheless, in the future it will be of interest to compare the effects of other routes of cSiO2 exposure, especially inhalation, on development of autoimmunity in NZBWF1 mice. An unexpected observation in this study was extensive ectopic lymphoid neogenesis in the lung after cSiO2 exposure in NZBWF1 mice. ELT morphologically and functionally resemble secondary lymphoid tissues (e.g. lymph nodes) by supporting maturation and differentiation of B cells into plasma cells that produce class-switched antibodies [62]. ELT have been previously identified in target tissues of several autoimmune diseases, including the kidneys of aged 89 NZWBF1 mice [63], synovial joints in rheumatoid arthritis [64,65], and salivary glands in Sjögren’s syndrome [66-68], where they contribute to local manifestations of autoimmunity. Although events that drive formation of ELT remain poorly defined, neogenesis of ectopic lymphoid structures is indicative of tissue-specific chronic inflammation [69] and importantly, may influence systemic autoimmunity [70]. Taken together, cSiO2-triggered formation of pulmonary ELT likely contributed to the exacerbated systemic autoimmunity observed in NZBWF1 mice following cSiO2 exposure. Elevated BALF concentrations of TNF-, MCP-1, and IL-6 observed here in cSiO2exposed NZBWF1 mice are key observations because these cytokines play critical roles in both initiating innate and amplifying adaptive immune responses. The sustained elevation of these cytokines in BALF up to 12 wk after cSiO2 exposure suggests that the initial innate immune response was insufficient to contain the inflammation, supporting a previously described model for continuous alveolar macrophage apoptosis after cSiO2 exposure with ensuing self-antigen presentation [12]. Robust elevation of TNF- in BALF has been similarly reported in cSiO2exposed NZM2410 mice [30]. TNF- induces apoptosis in alveolar macrophages and therefore could contribute to increased apoptotic debris containing autoantigens [12,31]. This cytokine can also stimulate production of additional proinflammatory cytokines, which could further amplify the immune response to cSiO2 [71-74]. Released predominately by monocytes/macrophages, MCP-1 recruits monocytes from systemic circulation to local sites of inflammation after tissue injury in attempt to contain or resolve further inflammation. The heightened elevation of this chemokine in BALF 12 wks following cSiO2 exposure further supports the notion that cSiO2 induces a continuous cycle of alveolar macrophage apoptosis, as sustained recruitment of circulating monocytes to the lungs by MCP-1 is likely an innate attempt to repopulate lost alveolar 90 macrophages and resolve inflammation. We observed robust increases in the percent of lymphocytes in BALF recovered from cSiO2-treated NZBWF1 mice. Previous studies in cSiO2 exposed NZM2410 mice have indicated that both B and T cells contribute to increased lymphocytes in BALF [30]. IL-6 has a well-established role in both CD4+ T cell survival and Bcell differentiation to Ig-secreting plasma cells [75]. Taken together, the increase of lymphocytes in the presence of IL-6 in BALF likely promotes antigen-specific effector responses in the lungs. Indeed, in the lung, cSiO2-induced elevation of IL-6 corresponded to significant increases of IgG in BALF of NZBWF1 mice. Mechanistic studies have indicated that alveolar macrophages are key to not only cSiO2induced pulmonary pathogenesis [12], but are also essential for development of IgG immune complex-mediated inflammation in the lungs [76,77]. Following exposure to chrysotile asbestos, in vitro production of superoxide anion by alveolar macrophages was enhanced by stimulation with IgG [78]. Proinflammatory cytokines in lungs were markedly reduced in C57Bl/6 mice deficient in FcγRIII, which mediates activation of mononuclear phagocytes by binding the Fc region of the IgG molecule [79]. The notion that IgG both initiates and enhances lung inflammatory responses through the alveolar macrophage is therefore of possible significance to cSiO2 triggering of SLE in autoimmune-prone mice. Extensive deposition/production of IgG in lungs of cSiO2exposed NZBWF1 mice could act synergistically with defective clearance of apoptotic macrophages, further contributing to acceleration of autoimmunity. Airway exposure to cSiO2 stimulates alveolar macrophages, epithelial cells, and fibroblasts that mediate recruitment of circulating monocytes, neutrophils, and lymphocytes by releasing an array of inflammatory mediators including cytokines [57,80-82]. The close proximity of these inflammatory cells to both the airways and vasculature in this study suggest that these cells are not 91 only capable of mediating production of proinflammatory mediators that impact the lung, but that may also be secreted into systemic circulation, thereby exacerbating development of systemic autoimmunity. It is notable that cSiO2-induced plasma increases of TNF- and IL-6 mirrored elevations of these cytokines in BALF. Importantly, systemic concentrations of TNF- and IL-6 correlate with SLE disease activity in humans [83] and treatment with exogenous IL-6 exacerbates glomerulonephritis in NZBWF1 mice [84]. There was also a trend towards elevated MCP-1 in plasma of NZBWF1 mice exposed to cSiO2. Urinary MCP-1 concentration has been identified as a biomarker of disease activity in lupus nephritis [85,86], and one study indicated that renal expression of MCP-1 correlates with NF-ΚB activation in kidney [87]. Overall, these results suggest that elevated plasma proinflammatory cytokines induced after cSiO2 exposure might further contribute to production of plasma autoantibodies as well as exacerbated renal pathology. Interestingly, cytokine array analysis of cSiO2-exposed NZM2410 mice failed to reveal any significant difference in plasma cytokines IL-4, IFN-γ, IL-10, IL-12, and TNF-α [30] suggesting some inherent differences in the response to intranasal cSiO2 might exist between that strain and the NZBWF1 employed here. Conclusions To summarize, the results presented here suggest that following airway exposure to cSiO2, the lung serves as a platform for the early triggering and exacerbation of systemic autoimmunity and glomerulonephritis in the NZBWF1 mouse. This model can serve as a starting point for further studies to gain insight into toxicant-triggered autoimmunity. First, it will be essential to characterize antigen-presenting cell and lymphocyte subpopulations recruited to and migrating out of the lung after cSiO2 exposure. These cells have the potential to drive subsequent tissue-specific homing of effector cell populations that mediate pathological outcomes in the lung and kidney. 92 Second, while it is apparent that cSiO2 induces plasma elevation of proinflammatory cytokines, further studies are warranted to ascertain if these originate from lung ELT, inflamed kidneys, and/or immune tissues such as spleen. Third, this model of cSiO2-accelerated lupus can be used to study potential approaches for prevention and intervention in occupationally exposed human populations. A particularly attractive approach is the consumption of n-3 polyunsaturated fatty acids in fish oil which have been shown to delay onset and severity of autoimmune nephritis in NZBWF1 and other models [34,88]. 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Prost Leukotriene Essent Fatty Acids 82: 251-258. 103 CHAPTER 3: SILICA-TRIGGERED AUTOIMMUNITY IN LUPUS-PRONE MICE BLOCKED BY DOCOSAHEXAENOIC ACID CONSUMPTION This chapter has been published in Bates et al. (2016) PLoS ONE 12(2): e0171877. Abstract Occupational exposure to respirable crystalline silica (cSiO2, quartz) is etiologically linked to systemic lupus erythematosus (lupus) and other human autoimmune diseases (ADs). In the female NZBWF1 mouse, a widely used animal model that is genetically prone to lupus, short-term repeated intranasal exposure to cSiO2 triggers premature initiation of autoimmune responses in the lungs and kidneys. In contrast to cSiO2’s triggering action, consumption of the ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) prevents spontaneous onset of autoimmunity in this mouse strain. The aim of this study was to test the hypothesis that consumption of DHA will prevent cSiO2-triggered autoimmunity in the female NZBWF1 mouse. Mice (6 wk old) were fed isocaloric AIN-93G diets containing 0.0, 0.4, 1.2 or 2.4 % DHA. Two wk after initiating feeding, mice were intranasally instilled with 1 mg cSiO2 once per wk for 4 wk and maintained on experimental diets for an additional 12 wk. Mice were then sacrificed and the lung, blood and kidney assessed for markers of inflammation and autoimmunity. DHA was incorporated into lung, red blood cells and kidney from diet in a concentration-dependent fashion. Dietary DHA dose-dependently suppressed cSiO2-triggered perivascular leukocyte infiltration and ectopic lymphoid tissue neogenesis in the lung. DHA consumption concurrently inhibited cSiO2–driven elevation of proinflammatory cytokines, B-cell proliferation factors, IgG and anti-dsDNA Ig in both bronchoalveolar lavage fluid and plasma. DHA’s prophylactic effects were further mirrored in reduced proteinuria and 104 glomerulonephritis in cSiO2-treated mice. Taken together, these results reveal that DHA consumption suppresses cSiO2 triggering of autoimmunity in female NZBWF1 mice as manifested in the lung, blood and kidney. Our findings provide novel insight into how dietary modulation of the lipidome might be used to prevent or delay triggering of AD by cSiO2. Such knowledge opens the possibility of developing practical, low-cost preventative strategies to reduce the risk of initiating AD and subsequent flaring in cSiO2-exposed individuals. Additional research in this model is required to establish the mechanisms by which DHA suppresses cSiO2-induced autoimmunity and to ascertain unique lipidome signatures predictive of susceptibility to cSiO2triggered AD. Introduction Autoimmune diseases (ADs) affect 24 million individuals in the U.S., inflicting tremendous individual suffering and societal costs [1]. The genome is universally recognized to be a primary predisposing factor for AD; however, the exposome, an individual’s lifetime environmental exposures, is a contributor of considerable and underappreciated importance. One component of the exposome likely to be critical to AD onset and progression is exposure to respirable particles such as crystalline silica (cSiO2, quartz) [2]. An estimated 2.3 million Americans employed in construction, manufacturing, mining, hydraulic fracturing, farming and custodial service are exposed to high concentrations of cSiO2 [3]. These individuals are at increased risk of developing ADs that include systemic lupus erythematosus (lupus), rheumatoid arthritis, scleroderma and antineutrophil cytoplasmic antibody-associated vasculitis [4-7]. Lupus, a prototypical AD, is characterized by loss of tolerance to self-antigens, activation of autoreactive B and T cells and consequential production of pathogenic autoantibodies [8]. These antibodies complex with self-antigens (e.g. nuclear components, including dsDNA) and deposit in 105 tissues facilitating mononuclear cell infiltration. In the kidney, these events drive onset and progression of glomerulonephritis - a burdensome and potentially fatal complication of lupus. Mouse strains that spontaneously develop lupus have been widely employed to understand how toxic stressors impact AD pathogenesis [9]. One example is the NZM2410 mouse, which develops lupus nephritis early in life causing them to succumb by 25 wk of age [10]. Intranasal cSiO2 instillation of NZM2410 accelerates the onset of lupus-related responses comprising of elevated autoreactive antibodies, proteinuria, and glomerulonephritis [11-14]. Accordingly, both experimental animal and human epidemiological studies suggest that cSiO2 could be a significant environmental trigger for lupus and potentially other ADs. The female New Zealand Black White (F1) mouse (NZBWF1), another widely used murine model of human lupus [9], spontaneously shows autoantibody production early in life. Glomerulonephritis onset is observed at approximately 34 wk of age, resulting in very high mortality by 12 months of age. Our laboratory recently demonstrated that weekly intranasal exposure to cSiO2 for 4 wk beginning at 9 wk of age of NZBWF1 mice dramatically shortened glomerulonephritis onset time to 22 wk of age [15]. Concurrently, cSiO2 elicited intense inflammatory responses in the lungs as evidenced by robust perivascular and peribronchial lymphocytic cell infiltration. These immune cells consisted of large numbers of B (CD45R +) cells, T (CD3+) cells and IgG-secreting plasma cells indicative of ectopic lymphoid tissue (ELT) neogenesis. Consistent with pulmonary inflammation and ELT development, bronchiolar lavage fluid (BALF) from cSiO2-exposed mice displayed elevated concentrations of IgG and the cytokines MCP-1, TNF-α and IL-6. cSiO2 exposure also evoked increases in plasma autoantibodies and proinflammatory cytokines. The discovery that cSiO2-induced inflammation and ELT neogenesis within the pulmonary compartment of NZBWF1 mice occurs in parallel with systemic 106 inflammatory and autoimmune responses strongly suggests the lung functions as a platform for AD triggering by this respirable particle. Another component of an individual’s exposome that potentially influences susceptibility to autoimmunity is diet [16]. Fat is a macronutrient that prominently influences immune function, with dietary polyunsaturated fatty acids (PUFA) being of particular relevance [17]. PUFA that contain more than one double bond in their backbone are classified as ω-3 or ω-6 according to the position of the first double bond relative to the terminal carbon in the aliphatic chain. Generally, ω-6 PUFA are regarded as proinflammatory in nature, whereas ω-3 PUFA such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are recognized to be antiinflammatory. Linoleic acid (C18:2 ω-6; LA), the major PUFA found in food oils derived from plants (e.g. corn and soybean), is the primary PUFA found in Western diets. Following consumption by humans, LA is enzymatically elongated and desaturated to arachidonic acid (C20:4 ω-6; ARA). Both LA and ARA become major components of cell membranes throughout the body. Although the ω-3 PUFA α-linolenic acid (C18:3 ω-3; ALA) is also found in plant oils, humans and other mammals have limited capacity to elongate it to eicosapentaenoic acid (C20:5 ω-3; EPA) and docosahexaenoic acid (22:6 ω-3; DHA) and afterwards incorporate these metabolites into cell membranes [18]. Thus, humans require exogenous sources for ω-3 PUFA other than plants or terrestrial animals. Certain marine microalgae efficiently catalyze the formation of EPA and DHA and cold-water fish consuming such algae readily bioconcentrate these ω-3 PUFA into their tissue [19]. Consequently, fish, fish oil and, more recently, microalgal oil are important sources of EPA and DHA for humans [20]. Animal and clinical studies over the past 30 years indicate that ingestion of ω-3 PUFA both prevents and resolves inflammation and, hence, might help those individuals who have a genetic predisposition to AD [21]. With the exception of 107 multivitamins, ω-3 PUFA are the most widely used nutritional supplement [22, 23]. An estimated 30 million American adults regularly consume ω-3 PUFA suggesting both wide usage and consumer acceptance; however, the impact of dose on potential health effects remains unclear. In a prior study, we compared latency and severity of autoimmunity in NZBWF1 mice consuming 1) an ω-3 PUFA- diet containing DHA-enriched fish oil, 2) an ω-6 PUFA-rich Western-type diet containing corn oil or 3) an ω-9 monounsaturated fatty acid (MUFA)-rich Mediterranean-type diet containing high oleic safflower oil [24]. Spontaneous elevations of plasma autoantibodies, proteinuria and glomerulonephritis occurred in mice at 34 wk of age fed ω-6 PUFA or ω-9 MUFA diets; however, these endpoints were dramatically diminished or even blocked in mice fed the DHA-rich diet. Inhibition of autoimmunity in ω-3 PUFA-fed NZBWF1 mice was associated with generalized downregulation of genes in both the kidney and spleen that contribute to inflammatory responses, antigen presentation, T cell activation, B cell activation/differentiation and leukocyte recruitment. Notably, many of these genes are currently under consideration as biomarkers and/or biotherapeutic targets for lupus. Nothing is known about the interactive effects cSiO2 exposure and ω-3 consumption on the development of AD. To address this knowledge gap, we tested the hypothesis that consumption of DHA-rich microalgal oil will prevent cSiO2-triggered autoimmunity in the female NZBWF1 mouse. The results show that DHA supplementation dose-dependently suppressed most indicators of cSiO2-accelerated autoimmune pathogenesis in lung, blood and kidney in this lupus-prone mouse model. Materials and Methods Animals and diets The Institutional Animal Care and Use Committee at Michigan State University reviewed 108 and approved all animal procedures in accordance with National Institutes of Health guidelines (AUF# 01/12-002-00). Female 6 wk old NZBWF1 and control NZW/LacJ mice were obtained from Jackson Laboratories (Bar Harbor, ME). Upon arrival, mice were randomized into experimental groups. They were then housed four per cage with free access to food and water. Animals were maintained at constant temperature and humidity (21°C–24°C and 40–55%, respectively) under a 12 h light/dark cycle. Formulations of experimental diets are summarized in Table 5. Briefly, modified American Institute of Nutrition (AIN)-93G diet containing 70 g/kg fat was prepared as described previously [24, 25]. All diets contained 10 g/kg food-grade corn oil to provide basal essential fatty acids. Control diet (CON) was formulated with 60 g/kg food-grade high-oleic safflower oil (Hain Pure Foods, Boulder, CO). High-oleic safflower oil was replaced with 10, 30, or 60 g/kg microalgal oil containing 40% DHA (DHASCOTM, provided by Dr. Kevin Hadley, Martek Biosciences Corporation Columbia, MD) yielding experimental diets that corresponded to 0.4, 1.2, or 2.4% (w/w) DHA, respectively. Based on energy percentages, these concentrations are estimated to mimic human consumption of 2, 6, and 12 g/d of DHA, respectively. Fatty acid compositions based on total lipids in each diet were analyzed as described below and are summarized in Table 6. Experimental design The overall study design is depicted in Fig. 16. Upon arrival, NZBWF1 mice (6 wk old) were fed CON diet or 0.4, 1.2, or 2.4% DHA-containing diets and maintained on assigned diet until experiment termination. To limit formation of lipid oxidation products, diets were prepared biweekly, stored at -20°C until use, and provided fresh to mice every other day. Beginning at 8 wk of age, mice were anesthetized with 4% isoflurane and intranasally instilled with1.0 mg cSiO2 109 Table 5. Compositions of experimental diets. Experimental Group CON 0.4% DHA Ingredient 1.2% DHA 2.4% DHA (g/kg ) Casein 200 200 200 200 Dyetrose 132 132 132 132 Cornstarch 397.5 397.5 397.5 397.5 Sucrose 100 100 100 100 Cellulose 50 50 50 50 t-Butylhydroquinone (TBHQ) 0.01 0.01 0.01 0.01 AIN 93G Salt Mix 35 35 35 35 AIN 93G Vitamin Mix (with vitamin E) 10 10 10 10 L-cysteine 3 3 3 3 Choline Bitartrate 2.5 2.5 2.5 2.5 Corn Oila,b 10 10 10 10 High-Oleic Safflower Oila,c 60 50 30 0 DHA-Enriched Algal Oila,d - 10 30 60 a As reported by the manufacturer b Corn oil contained 612 g/kg linoleic acid and 26 g/kg oleic acid c High oleic safflower oil contained 750 g/kg oleic acid and 140 g/kg linoleic acid d Algal oil contained 395 g/kg DHA and 215 g/kg oleic acid 110 Table 6. Fatty acid content of experimental diets based on GLC analysis. Experimental Group CON 0.4% DHA 1.2% DHA 2.4% DHA (% of fatty acid of total lipid in the diet) Fatty Acid 16:0 8.31 ± 0.05 8.94 ± 0.14 10.1 ± 0.04 12.1 ± 0.06 16:1 0.12 ± 0.00 0.21 ± 0.01 0.37 ± 0.005 0.65 ± 0.01 18:0 2.61 ± 0.02 2.58 ± 0.03 2.47 ± 0.01 2.25 ± 0.02 18:1 trans 0.05 ± 0.00 0.05 ± 0.00 0.06 ± 0.00 0.06 ± 0.00 18:1 cis 46.4 ± 0.35 42.6 ± 0.90 35.6 ± 0.43 22.3 ± 0.10 18:2 (ω-6) 36.6 ± 0.29 36.1 ± 0.46 34.5 ± 0.29 32.2 ± 0.47 18:3 (ω-3) 3.15 ± 0.03 3.25 ± 0.06 3.26 ± 0.04 3.22 ± 0.04 20:4 (ω-6) 0.02 ± 0.00 0.02 ± 0.00 0.02 ± 0.00 0.02 ± 0.00 20:5 (ω-3) 0.0 ± 0.00 0.0 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 22:5 (ω-3) 0.0 ± 0.00 0.03 ± 0.00 0.09 ± 0.00 0.19 ± 0.00 22:6 (ω-3) 0.0 ± 0.00 2.47 ± 0.30 7.74 ± 0.17 17.6 ± 0.52 ∑ SFA 12.1 ± 0.07 13.7 ± 0.26 16.8 ± 0.09 22.4 ± 0.12 ∑ MUFA 47.8 ± 0.36 44.0 ± 0.90 37.1 ± 0.43 24.0 ± 0.10 ∑ PUFA (ω-3) 3.15 ± 0.03 5.75 ± 0.34 11.1± 0.18 21.0 ± 0.50 ∑ PUFA (ω-6) 36.6 ± 0.29 36.1 ± 0.46 34.6 ± 0.29 32.3 ± 0.47 (ω-6):(ω-3) 11.6:1 6.3:1 3.1:1 1.5:1 111 Figure 16. Experimental design. Beginning at age 6 wk, NZBWF1 were assigned CON diet or 0.4, 1.2, and 2.4% DHA diet. NZW/LacJ mice were assigned either CON diet or 2.4% DHAcontaining diet. Then, starting at age 8 wk, mice were dosed intranasally with 25 μl PBS VEH or 25 μl PBS containing 1.0 mg cSiO2 weekly for 4 wk. Proteinuria was monitored over the course of the experiment and all animals euthanized at 23 wk of age (i.e. 12 wk PI) 112 (Min-U-Sil-5, 1.5-2.0 μm average particle size, Pennsylvania Sand Glass Corporation, Pittsburgh, PA) in 25 μl PBS or PBS vehicle (VEH) once per wk for 4 wk [15]. The total dose of cSiO2 (4 mg) in mice approximates one half of a human lifetime exposure at the recommended occupational health guideline established by the Occupational Safety and Health Administration [3]. Urine was collected weekly and evaluated for protein using reagent dipsticks (Cortez Diagnostics, Inc., Calabasas, CA). Mice were euthanized and tissues collected 12 wk after the final cSiO2 exposure. At this time point, 3 out of 8 mice fed CON diet and exposed to cSiO2 displayed proteinuria (defined as > 300 mg/dL). Female NZW/LacJ mice were used as controls for selected endpoints; these are a parental line for the NZBWF1 hybrid that do not spontaneously develop lupus [10, 26]. Age-matched female NZW/LacJ mice were fed either CON or 2.4% DHA-enriched AIN-93G for 2 wk prior to vehicle or cSiO2 exposure and then maintained on experimental diets as described above. Necropsy and tissue collection Mice were euthanized by intraperitoneal injection with 56 mg/kg BW sodium pentobarbital. Blood was collected from the abdominal aorta using heparinized syringes and centrifuged at 3500 x g for 10 min at 4°C. Resultant plasma was stored at -80°C. Red blood cell pellets were immediately frozen in liquid nitrogen and stored at -80°C for fatty acid analysis. Bronchoalveolar lavage fluid (BALF) was collected from whole lungs as described previously [27]. After lavage, the right lung lobes were removed, frozen in liquid nitrogen and stored at -80°C for fatty acid analysis. The left lung lobe was fixed with 10% neutral buffered formalin (Fisher Scientific, Pittsburgh, PA) at constant pressure (30 cm H2O) for minimum of 1 h and stored in fixative until further processing for histology. The right kidney was excised and the cranial portion fixed in 10% neutral buffered formalin for 24 h. The left kidney was removed and stored at -80°C 113 for fatty acid analysis. Fatty acid analyses Fatty acid concentrations of diets were determined using a modification of a direct transesterification method [28] as reported previously [29]. Total lipids were extracted from lung and kidney tissue according to the method of Bligh and Dyer [30] using a mixture of methanol:chloroform:water (2:2:1.8, by vol). Red blood cells were isolated and purified from whole blood according to Burton and coworkers [31] and lipids extracted as described for lung and kidney. Fatty acid methyl esters (FAME) were prepared from lipid extracts using KOH solution and boron trifluoride in methanol [32]. Gas liquid chromatography (GLC) analysis of FAME extracts were performed on a GC-2010 gas chromatograph (Shimadzu) equipped with a flame ionization detector using a CP-Sil 88 capillary column (100m × 0.25mm I.D. × 0.25 µm film thickness, Varian). Gas chromatographic conditions were as detailed previously [29]. Individual FAME were identified by comparison of retention times with known FAME standards (GLC reference standard 63-A and GLC reference standard 455 from Nu-Chek Prep Inc.). Fatty acid results are expressed as percentages (w/w) of fatty acids detected with a chain length between 10 and 24 carbon atoms. The lower limit of detection was <0.001 g/100 g fatty acids. Kidney histopathology Formalin-fixed, paraffin-embedded kidneys were sectioned to 5 μm and stained with either hematoxylin and eosin (H&E) or Periodic Acid Schiff and hematoxylin (PASH). Slide sections were individually graded by a board-certified veterinary pathologist graded using a modified International Society of Nephrology-Renal Pathology Society Lupus Nephritis Classification [33] as follows: (0) no tubular proteinosis and normal glomeruli; (1) mild tubular proteinosis with multifocal segmental proliferative glomerulonephritis and occasional early glomerular sclerosis 114 and crescent formation; (2) moderate tubular proteinosis with diffuse segmental proliferative glomerulonephritis, early glomerular sclerosis and crescent formation; and (3) marked tubular proteinosis with diffuse global proliferative and sclerosing glomerulonephritis. Lung histopathology The formalin-fixed inflated left lung lobe was cut in randomly oriented 2 mm thick serial sections. All sections (4-6 per left lung lobe) were embedded in paraffin for histopathological and morphometric analysis. Tissue sections (5 μm) were routinely stained with H&E for cellular morphometry and histopathology. Sections were semi-quantitatively scored by a board-certified veterinary pathologist for the following lung lesions: (a) presence of lymphocytic cell infiltration within perivascular and peribronchial regions, (b) alveolitis defined as the presence of alveolar infiltration of vacuolated macrophages, neutrophils, and lymphocytes, granuloma formation in the alveolus, type II epithelial cell hyperplasia, and thickened alveolar wall, and (c) presence of alveolar proteinosis. Individual lungs were graded for these lesions using the following criteria (% of total pulmonary tissue examined): (0) no changes compared to control mice; (1) minimal (<10%); (2) slight (10-25%); (3) moderate (26-50%); (4) severe (51-75%); or (5) very severe (>75%) of total area affected. Lung immunohistochemistry Immunohistochemistry was performed on formalin-fixed, paraffin embedded lung sections for identification of B and T lymphocytes in lung parenchyma. Briefly, B and T cells were identified using rat monoclonal anti-mouse CD45R (BD Biosciences, San Jose, CA) (1:300) and rabbit polyclonal anti-mouse CD3 (Abcam, Cambridge, MA) (1:100), respectively. Deparaffinized tissue sections (4 μm) were first subjected to heat-induced epitope retrieval with citrate buffer (pH 6.0) at 100ºC for 30 min (CD45R) or 125ºC for 15 min (CD3). Following pretreatments to block 115 non-specific binding and endogenous biotin, primary antibody staining was performed. Bound CD45R and CD3 antibodies were detected by incubation with biotinylated secondary antibodies followed by Peroxidase Reagent and Nova Red substrate (Vector Laboratories, Inc., Burlingame, CA) prior to being counterstained with hematoxylin. Lung morphometry for B and T cells To quantify B and T cell infiltration, slides stained with antibodies to CD45R and CD3, respectively, were scanned and digitized with a VS110 (Olympus, Hicksville, NY) virtual slide system. The lung tissues on scanned slides were selected as region of interest and 10% of the lung section was captured at 20X magnification by systematic random sampling with NewCast software (Visiopharm, Hoersholm, Denmark). The volume density of CD45R+ or CD3+ cells in the bronchial and perivascular regions of the lungs was estimated by projecting a point grid over the randomly sampled images with the STEPanizer 1.8 Stereology Tool (http://www.stepanizer.com/)[34]. The numbers of points hitting the CD45R+ or CD3+ cells as well as the whole lung tissues (i.e. lung parenchyma and interstitium) were counted. Finally, the volume density or percentage of CD45R+ and CD3+ per reference area was calculated. BALF cell quantitation and identification Total cell numbers in BALF were determined by counting intact cells using a hemocytometer without Trypan Blue exclusion. Cytological slides from BALF were prepared by centrifugation at 400 x g for 10 min using a Shandon Cytospin 3 (Shandon Scientific, PA), allowed to air dry, and stained with Diff-Quick (Fisher Scientific). Remaining BALF was centrifuged at 2400 x g for 15 min and supernatant collected and stored at -80°C. Differential cell counts for macrophages/monocytes, lymphocytes, neutrophils, and eosinophils in BALF were determined using morphological criteria from 200 total cells on cytological slides. 116 IgG and autoantibody measurement Total IgG in BALF and plasma was determined by ELISA [35]. Briefly, 96-well plates were coated with 50 µL per well of 5 µg/ml goat anti-mouse IgG (γ-chain specific, Alpha Diagnostics, Inc., cat # 40120) in carbonate coating buffer. Afterwards, BALF and plasma samples as well as standards were loaded on the plate, followed by another incubation step with horseradish peroxidase-labeled goat anti-mouse IgG (γ-chain specific) (Alpha Diagnostics, Inc., cat # 40121). The standard curve was generated with mouse reference serum (Bethyl Laboratories, Inc., Montgomery, TX). Plates were read on an ELISA reader (Molecular Devices, Menlo Park, CA) and IgG calculated from the standard curve using Softmax software (Molecular Devices). To quantify anti-dsDNA Ig [36], 96-well plates were coated with 5 µg/ml dsDNA purified from calf thymus (Alpha Diagnostics, Inc. cat # DNAD25-N-1) in borate buffered saline. BALF, plasma samples or standards (mouse anti-dsDNA, Abcam clone HYB331/01) were loaded on the plate. Bound antibody was measured using horseradish peroxidase-labeled goat anti-mouse IgG (Fab2specific) (Cappel Labs, cat # 3211021). Since this detector antibody reacts with all immunoglobulin classes (IgG, IgA, IgM, IgD and IgE), data were reported as anti-dsDNA Ig (ng/ml). Anti-dsDNA Ig in samples was determined from standard curve. Cytokine analyses BALF supernatant and plasma were analyzed for the proinflammatory cytokines MCP-1, TNF-α, IFN-γ, IL-6, IL-1β, IL-10, IL-12p70, and IL-17a by flow cytometric bead array using Flex Set reagents (BD Biosciences, San Jose, CA). Sample data were acquired using a FACSCalibur flow cytometer (BD Biosciences) and cytokine concentrations were calculated from standard curves using FCAP Array Software (BD Biosciences). In addition to the Flex Set array, B cell stimulating cytokines in BALF supernatant and plasma were quantitated using mouse B-cell 117 Activating Factor (BAFF) and Osteopontin DuoSets (R&D Systems, Minneapolis, MN) according to manufacturer’s instructions. Statistics All treatments consisted of 8 mice per group and data presented as x̄ ± SEM. A p value of < 0.05 was considered statistically different for all study outcomes. Grubb’s outlier test was performed and any identified outliers were excluded from statistical analysis. Data were analyzed using SigmaPlot 11.0 for Windows (Jandel Scientific; San Rafael, CA). Student’s t test was used to determine differences in CON-fed mice instilled with VEH or cSiO2. The effects of consuming DHA diets on cSiO2-triggered responses were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons. If the assumption of normality or equal variance failed, Kruskal-Wallis one-way ANOVA on Ranks followed by Dunn’s test was used. The Spearman rank-order correlation coefficient was used to correlate DHA concentrations in diet to experimental endpoints in cSiO2-exposed NZBWF1 mice. Comparisons between VEHexposed NZW/LacJ mice and cSiO2-exposed counterparts fed CON or DHA-enriched diets were conducted using Student’s t-test. If the assumption of normality or equal variance failed, differences were analyzed by Mann Whitney Rank Sum test. Results Consumption of DHA-rich microalgal oil dose-dependently increases tissue DHA Substitution of high oleic safflower oil in AIN-93G diets with increasing amounts of DHA oil (Tables 5, 6) dose-dependently increased DHA in lung (Fig. 17A Table 9, Appendix B), red blood cells (Fig. 17B, Table 10, Appendix B) and kidney (Fig. 17C, Table 8, Appendix B) in NZBWF1 mice 12 wk after the last cSiO2 instillation. These effects co-occurred with reductions of the ω-6 PUFA ARA, suggesting that DHA effectively replaced ARA from cell membrane phospholipids 118 Figure 17. Consumption of microalgal oil dose-dependently increases DHA in kidney, lung and RBC. DHA incorporation in tissues increased in a dose-dependent manner that coincided with a reduction in ARA. (A) Kidney (r2 = 0.725, P<0.05) (B) lung (r2 = 0.961, p < 0.001), and (C) red blood cell (r2 = 0.876, p < 0.001). 119 in these three tissues. There was very little EPA detected in the tissues indicating that there was no retroconversion of DHA. Finally, cSiO2 exposure did not significantly affect DHA content in tissues of CON-fed mice. DHA consumption suppresses cSiO2-triggered proteinuria and lupus nephritis Consistent with our prior findings [15], short-term repeated intranasal exposure to cSiO2 triggered the premature onset of proteinuria in CON-fed NZBWF1 mice as compared to VEHinstilled CON-fed mice (Fig 18). Proteinuria in cSiO2-exposed animals was apparent beginning at 10 wk post-instillation (PI) with 38% of this group responding by 12 wk PI. In contrast, cSiO2exposed NZBWF1 mice fed 0.4% DHA showed delayed proteinuria onset, first detectable 11 wk PI; by 12 wk PI, 25% of this group was affected. None of the cSiO2-exposed NZBWF1 mice fed either 1.2% or 2.4% DHA exhibited proteinuria upon termination at 12 wk PI. Proteinuria was not evident at 12 wk PI in VEH- or cSiO2-instilled NZW/LacJ mice, which were controls without genetic susceptibility to autoimmunity. DHA’s preventative effects on cSiO2-triggered renal injury in NZBW1 mice were confirmed histologically. CON-fed mice instilled with cSiO2 exhibited moderate to severe diffuse glomerular hypercellularity, mesangial matrix expansion, lymphocytic infiltration, and tubular proteinosis characteristic of glomerulonephritis as compared to CON diet-fed mice instilled with VEH (Fig 19A, B). In contrast, NZBWF1 mice fed diets containing 2.4% DHA exhibited marked reduction of such lesions (Fig 18C). Individual NZBWF1 mice were graded for severity of lupus nephritis (Fig 20). CON-fed mice that received cSiO2 showed significantly higher lupus nephritis scores than CON-fed mice instilled with VEH. In contrast, consumption of diets containing 1.2 or 2.4% DHA markedly reduced severity of cSiO2-induced nephritis. Consumption of diets containing 0.4% DHA did not affect cSiO2-induced nephritis. Hence, DHA consumption 120 Figure 18. Dietary supplementation with DHA attenuates cSiO2-induced proteinuria in NZBWF1 mice. Proteinuria (>300 mg/dl) was monitored weekly until sacrifice after the final cSiO2 instillation. VEH-instilled NZBWF1 mice fed CON diet did not develop proteinuria. Proteinuria was undetectable in NZW/LacJ exposed to VEH or cSiO2 over the duration of the experiment. 121 Figure 19. DHA consumption suppresses cSiO2-induced glomerulonephritis in NZBWF1 mice. Representative photomicrographs of H&E stained kidney section in NZBWF1 (A-C) and NZW/LacJ (D-F). Letters indicate CON-fed, VEH-exposed mice (A, D), CON-fed, cSiO2-exposed mice (B, E) and 2.4% DHA-fed, cSiO2-exposed mice(C, F). CON-fed NZBWF1 mice instilled with cSiO2 (B) developed extensive glomerulonephritis (black arrows) and tubular proteinosis (*). Mild histopathological lesions were also observed in some NZW/LacJ mice exposed to cSiO2 (E). Dietary supplementation with 2.4% DHA decreased severity of lesions in cSiO2-exposed NZBWF1(C), and NZW/LacJ mice (F). 122 Figure 20. DHA-dose dependently reduces severity of lupus nephritis in cSiO 2-exposed NZBWF1 mice. NZBWF1 and NZW/LacJ mice were individually graded following the modified ISN/RPS lupus nephritis classification system as described in Materials and Methods. Slide sections from kidneys were graded as follows: (0) no tubular proteinosis; (1) mild tubular proteinosis, early sclerosis, and mild crescent formation; (2) moderate tubular proteinosis, early sclerosis, and crescent formation; (3) marked tubular proteinosis with diffuse global proliferative and sclerosing glomerulonephritis. Data are ± SEM (n = 8). Symbols: * indicates significant difference from CON-fed mice instilled with VEH (p < 0.05); # indicates significant difference from CON-fed mice instilled with cSiO2 (p < 0.05). DHA dose-dependently reduced cSiO2triggered lupus nephritis in NZBWF1 mice (r2 = -0.414, p < 0.05). 123 suppressed cSiO2-triggered kidney injury in the female NZBWF1 mouse. cSiO2 instillation also induced renal lesions indicative of mild nephritis in NZW/LacJ control mice PI (Fig 19E). These results indicate that intranasal exposure to cSiO2 per se might be sufficient to induce nephritis even in the absence of strong genetic predisposition to autoimmunity. Consistent with effects of DHA observed in cSiO2-exposed NZBWF1 mice, supplementation with 2.4% DHA appeared to reduce the severity of kidney lesions in NZW/LacJ mice exposed to cSiO2 (Fig 19F). DHA consumption abrogates cSiO2-induced inflammatory cell response in lungs CON-fed NZBWF1 mice instilled with cSiO2 exhibited extensive inflammation in the lungs consisting of significant perivascular and peribronchiolar mononuclear cell infiltration compared to those instilled with VEH (Fig 21A, B). While diet supplemented with 0.4% DHA modestly reduced perivascular mononuclear infiltration, consumption of the 1.2 and 2.4% DHA diets dramatically suppressed cSiO2 induced mononuclear cell infiltration (Fig 21C). NZW/LacJ mice fed CON diet and intranasally instilled with cSiO2 also developed pulmonary mononuclear cell infiltration (Fig 21D, E), but this response was much less severe than in NZBWF1 mice. Nonetheless, consumption of 2.4% DHA reduced cSiO2-triggered mononuclear cell infiltration in this mouse strain (Fig 21F). Observations in H&E stained serial lung sections from NZBWF1 and NZW/LacJ mice were confirmed by semi-quantitatively grading severity of pulmonary inflammation (Table 7). cSiO2 induced lymphocytic cell infiltration in both CON diet-fed NZBWF1 and NZW/LacJ mice; however, the response induced in autoimmune-prone NZBWF1 mice was considerably more severe. DHA diets were effective at reducing this infiltration in NZBWF1 mice in a dosedependent manner. Infiltration of mononuclear cells in cSiO2-treated NZW/LacJ mice was also 124 Figure 21. DHA supplementation prevents cSiO2-induced pneumonitis. Representative photomicrographs of H&E stained lung sections from NZBWF1 (A-C) and NZW/LacJ (D-F) mice exposed to VEH (A, D), cSiO2 fed CON diet (B, E), and cSiO2 fed 2.4% DHA (C, F). Black arrows in light photomicrographs denote marked leukocyte infiltration that circumvented both the vasculature and airways in the lung following cSiO2 exposure (B). Dietary DHA dramatically reduced cSiO2-induced pulmonary inflammation as evident by the absence of cellular accumulation in (C, F). Lymphocytic cell infiltration was semi-quantitatively graded as indicated in Table 3). Abbreviations: ba = bronchiolar airway, v = blood vessel, tb = terminal bronchiole, a = alveolus. 125 Table 7. Histopathological assessment of cSiO 2-triggered pulmonary lung inflammation in lupus-prone NZBWF1 and NZW/LacJ mice fed DHA. Mice were graded individually for severity of lung inflammation (% of total pulmonary tissue examined) as follows: 0, no changes; 1, minimal (<10%); 2, slight (10-25%); 3, moderate (26-50%); 4, severe (51-75%) 5; very severe (>75%) of total area affected. Data are mean ± SEM (n=8/gp). Symbols: * indicates significant difference from CON-fed mice instilled with VEH (p < 0.05); # indicates significant difference from CON-fed mice instilled with cSiO2 (p < 0.05). DHA dose-dependently decreased lymphocytic cell infiltration (r2 = -0.845, p < 0.001) and alveolitis (r2 = -0.622, p < 0.001) in NZBWF1 mice. 126 suppressed by feeding diets containing 2.4% DHA. Dietary supplementation with 2.4% DHA reduced alveolitis in NZBWF1 mice as compared to CON diet-fed mice, whereas consumption of 0.4 and 1.2% DHA diets had no effect. These effects were not observed in NZW/LacJ mice. Finally, DHA consumption did not influence cSiO2-triggered alveolar proteinosis in NZBWF1 or NZW/LacJ mice. Consequently, DHA supplementation primarily targeted lymphocytic cell infiltration in the lung. Macrophages, lymphocytes, and neutrophils were dramatically increased in the BALF of CON-fed NZBWF1 mice instilled with cSiO2, while NZW/LacJ mice exhibited slighter increases (Fig 22A-C). Total cell counts in BALF of cSiO2-exposed NZBWF1 mice were approximately 3-fold higher relative to NZW/LacJ mice. Consumption of 1.2 and 2.4% DHA markedly impaired macrophage, lymphocyte, and neutrophil cellular infiltration in cSiO2-exposed NZBWF1 mice. Consumption of the 0.4% DHA diet reduced lymphocyte counts in BALF but did not affect macrophage or neutrophil infiltration. cSiO2-exposed NZW/LacJ mice fed 2.4% DHA also showed a trend towards decreased lymphocyte and neutrophil infiltration compared to CONfed mice. Dietary DHA prevents cSiO2-induced ELT neogenesis and autoimmunity in the lung Lymphocyte populations in lungs of NZBWF1 mice were characterized immunohistochemically employing antibodies to the pan B and T lymphocyte markers CD45R and CD3, respectively. Both CD45R+ (Fig 23A, B) and CD3+ cells (Fig 23D, E) indicative of cSiO2-induced ELT neogenesis were detected in lung parenchyma in CON-fed mice. Morphometry revealed that CD45R+ cells (Fig 24A) were more abundant in lung parenchyma than CD3+ cells (Fig 24B). These findings confirmed previous observations that intranasal cSiO2 exposure induces ELT development in NZBWF1 mice [15]. Dietary supplementation with 0.4, 1.2, and 2.4% DHA dose-dependently suppressed cSiO2-triggered B and T cell expansion (Fig 127 Figure 22. DHA consumption abrogates cSiO2-induced macrophage, lymphocyte, and polymorphonuclear leukocyte accumulation in BALF. Differential counts of macrophages (A), lymphocytes (B), and neutrophils (C) in BALF of NZBWF1 and NZW/LacJ mice. Data are x̄ ± SEM (n=8). Symbols: * indicates significant difference from CON-fed mice instilled with VEH (p < 0.05); # indicates significant difference from CON-fed mice instilled with cSiO2 (p < 0.05). DHA dose-dependently decreased macrophages (r2 = -0.545, p < 0.05), lymphocytes (r2 = -0.599, p < 0.001), and neutrophils (r2 = -0.448, p < 0.05) in NZBWF1 mice. 128 Figure 23. B and T cell infiltration in lungs of NZBWF1 mice following cSiO2 exposure is prevented by dietary supplementation with DHA. Representative light photomicrographs of lung tissue sections from CON-fed NZBWF1 mice treated with VEH (A, D), CON-fed NZBWF1 mice treated with cSiO2 (B, E), 2.4% DHA-fed mice treated with cSiO2 (C, F). 129 Figure 23 (cont’d). Lung sections were stained with either CD45R to identify B-lymphocytes (AC) or CD3 to identify T cells (D-F) and counterstained with hematoxylin. Inflammatory cell infiltrates in peribronchiolar and perivascular interstitium induced by cSiO2 (asterisk in B and E) consisted of both B and T lymphocytes as indicated by positive immunohistochemical staining (black arrows in B and E, respectively). B-lymphocytes tended to form distinct aggregates whereas T lymphocytes, which were more diffusely, scattered throughout lymphoid cells aggregates. Dietary DHA blocked B and T cell accumulation in lungs of cSiO2-treated NZBWF1 mice as evident by marked reduction in CD45R+ and CD3+ cells. Abbreviations: ba = bronchiolar airway, e = airway epithelium, a = alveolus, v = blood vessel, *= interstitium. 130 Figure 24. cSiO2-triggered B and T cell infiltration in lungs of NZBWF1 mice is dosedependently prevented by DHA consumption. Morphometric quantitation of B cell (A) and T cell (B) cellular infiltration in lung parenchyma in CON- and DHA-fed mice exposed to VEH or cSiO2. Data are x̄ ± SEM (n=8). Symbols: * indicates significant difference from CON-fed mice instilled with VEH (p < 0.05); # indicates significant difference from CON-fed mice instilled with cSiO2 (p < 0.05). DHA consumption dose-dependently decreased CD45R+ (r2= -0.707, p<0.001) and CD3+ (r2 = -0.728, p<0.001) cellular infiltration. 131 23C, F). The CD45R+ cell area in lung parenchyma was reduced by DHA consumption 80, 98, and 96%, respectively, relative to cSiO2-exposed NZBWF1 fed CON diet (Fig 24A). In analogous fashion, DHA supplementation reduced CD3+ cell area in lung parenchyma by 41, 79, and 83%, respectively (Fig 24B). Consistent with immunohistochemical findings, cSiO2 triggered robust elevations in total IgG and anti-dsDNA Ig concentrations, clinical signs of active lupus, in the BALF of CON diet-fed NZBWF1 mice (Fig 25A, B). cSiO2-triggered elevation in total IgG was suppressed in mice fed diets containing 1.2 and 2.4% DHA. Likewise, DHA feeding at 0.4, 1.2 and 2.4% decreased anti-dsDNA Ig in BALF by 82, 97, and 95% respectively, relative to CONfed cSiO2-exposed NZBWF. Accordingly, dietary DHA consumption dramatically diminished cSiO2-induced ELT neogenesis and production of total and autoreactive IgG production in the lung. DHA consumption suppresses cSiO2–triggered secretion of proinflammatory and B-cell stimulatory cytokines into BALF cSiO2 instillation of CON-fed NZBWF1 mice elicited increased concentrations of MCP-1, TNF-α, and IL-6 in BALF compared to CON-fed mice instilled with VEH (Fig 26A-C). DHA consumption dose-dependently attenuated these responses, with the 1.2 and 2.4% DHA diets being more efficacious than the 0.4% DHA diet. Other cytokines in the flow cytometric bead panel IFNγ, IL-1β, IL-10, IL-12p70, and IL-17a fell below the limit of detection. The effects of DHA on two B-cell stimulating cytokines, BAFF and OPN, were also assessed in BALF of NZBWF1 mice. Both cytokines were significantly induced in CON-fed mice instilled with cSiO2 (Fig 27A, B). Dietary supplementation with DHA dose-dependently decreased ciSO2-induced BAFF and a similar trend was observed for ciSO2-induced OPN. 132 Figure 25. Dietary DHA suppresses cSiO2-induced elevation of total IgG and anti-dsDNA Ig in BALF of NZBWF1 mice. Total IgG (A) and anti-dsDNA Ig (B) in BALF of NZBWF1 mice was quantitated by ELISA. Data are x̄ ± SEM (n=8). Symbols: * indicates significant difference from CON-fed mice instilled with VEH (p < 0.05); # indicates significant difference from CONfed mice instilled with cSiO2 (p < 0.05). Dietary DHA dose-dependently decreased total IgG (r2= -0.574, p < 0.001) and anti-dsDNA Ig (r2 = -0.546, p < 0.05) in BALF. 133 Figure 26. cSiO2-induced elevations of proinflammatory cytokines MCP-1, TNF-α and IL-6 in BALF and plasma are decreased by DHA consumption in NZBWF1 mice. MCP-1 (A, D), TNF-α, (B, E) and IL-6 (C, F)) were quantitated in BALF (left panel) and plasma (right panel) by flow cytometric bead array. Data are x̄ ± SEM (n=8). The designation n.d. indicates below the limit of detection. Bars without same letter are significantly different (p<0.05). Symbols: * indicates significant difference from CON-fed mice instilled with VEH (p < 0.05); # indicates significant difference from CON-fed mice instilled with cSiO2 (p < 0.05). DHA dose-dependently decreased BALF concentrations of MCP-1 (r2 = -0.791, p < 0.001), TNF- α (r2 = -0.577 p < 0.001), and IL-6 (r2= -0.810, p < 0.001). DHA dose-dependently decreased plasma MCP-1 (r2 = -0.871, p < 0.001) and TNF-α (r2= -0.527, p < 0.05). 134 Figure 27. cSiO2-induced elevation of B cell stimulating cytokines BAFF and osteopontin are decreased in BALF and plasma in NZBWF1 mice fed DHA. B cell stimulating cytokines B cell activating factor (BAFF) (A, C) and osteopontin (OPN) (B, D) were quantitated by ELISA in BALF (left panel) and plasma (right panel). Data are x̄ ± SEM (n=8). Symbols: * indicates significant difference from CON-fed mice instilled with VEH (p < 0.05); # indicates significant difference from CON-fed mice instilled with cSiO2 (p < 0.05). DHA dose-dependently decreased BAFF in BALF (r2 = -0.507, p < 0.05) and plasma (r2 = -0.539, p < 0.05). DHA dose-dependently decreased OPN in BALF (r2= -0.330, p = 0.06) and in plasma (r2 = -0.493, p < 0.05). 135 Dietary DHA attenuates cSiO2-induced systemic autoantibody and cytokine elevation As observed in BALF, CON-fed NZBWF1 mice instilled with cSiO2 exhibited plasma elevations of total IgG and anti-dsDNA Ig relative to their VEH-exposed counterparts (Fig 28 A, B). Consumption of diets containing 1.2 and 2.4 % DHA suppressed cSiO2-induced IgG elevation. Dietary DHA also dose-dependently decreased anti-dsDNA Ig concentrations in plasma of NZBWF1 mice. Differences in total IgG were not evident between CON-fed NZW/LacJ mice exposed to VEH or cSiO2. Interestingly, cSiO2-exposed NZW/LacJ mice fed DHA had significantly decreased total IgG in plasma compared to cSiO2-exposed mice fed CON diet (Fig 28A). Differences in anti-dsDNA Ig were not detected among any NZW/LacJ treatment groups (Fig 28B). Plasma was also analyzed for cytokines to evaluate systemic effects of cSiO2 and DHA. In CON-fed NZBWF1 mice, cSiO2 instillation caused an elevation in systemic MCP-1 and TNF-α. Dose-dependent reductions in MCP-1 and TNF-in plasma occurred because of dietary treatment with DHA (Fig 26D, E). cSiO2 treatment did not increase IL-6 in plasma but there was a trend toward decreased levels of this cytokine in DHA-fed mice (Fig 26F). Other cytokines IFN-γ, IL1β, IL-10, IL-12p70, and IL-17a fell below the limit of detection. cSiO2 exposure also induced plasma BAFF in CON-fed NZBWF1 mice (Fig 27C). Consumption of 1.2, and 2.4% DHA diets significantly reduced cSiO2-induced systemic BAFF responses. Similar trends were observed for OPN with 1.2% DHA diet significantly suppressing this B cell factor (Fig 27D). Discussion Heredity is widely viewed to be critical in the manifestation of autoimmunity in an individual; however, the exposome can impact both latency and severity of an AD [37]. This investigation is unique because it is the first to employ an animal model genetically predisposed 136 Figure 28. Dietary DHA attenuates cSiO2-induced elevation of total IgG and anti-dsDNA Ig in plasma of NZBWF1 and NZW/LacJ mice. Total IgG (A) and anti-dsDNA Ig (B) in plasma of NZBWF1 mice was quantitated by ELISA. Data are x̄ ± SEM (n=8). Symbols: * indicates significant difference from CON-fed mice instilled with VEH (p < 0.05); # indicates significant difference from CON-fed mice instilled with cSiO2 (p < 0.05). DHA dose-dependently decreased plasma total IgG in NZBWF1 (r2= -0.493, p < 0.05) and NZW/LacJ mice (r2 = -0.814, p = < 0.001). DHA dose-dependently decreased plasma anti-dsDNA Ig in NZBWF1 (r2= -0.567, p < 0.001). 137 to lupus, a prototypical AD, to identify how two highly relevant and countervailing exposome, cSiO2 exposure and ω-3 PUFA content of the diet, influence latency, and progression of autoimmunity. Importantly, we demonstrate that dietary DHA dose-dependently inhibited cSiO2triggered inflammation, ELT neogenesis and autoantibody production in the lung. Moreover, these effects were recapitulated systemically as evidenced by concurrent DHA-mediated suppression of cSiO2-induced elevations in cytokines and autoantibodies in plasma. Lastly, DHA consumption acted further downstream at the kidney by preventing glomerulonephritis and resultant proteinuria. Collectively, these data support the contention that triggering of autoimmunity by cSiO2 might be prevented or delayed by modulating dietary lipid composition. Our findings confirm recent observations that weekly intranasal exposure of female NZBWF1 mice to 1 mg cSiO2 for 4 wk triggers autoimmune responses in the lung, blood and kidney [15]. Notably, there was robust development of ELT in the lung as evidenced by extensive perivascular and peribronchial lymphoplasmacytic infiltration consisting of T cells, B cells, and IgG-producing plasma cells. We further detected elevated concentrations of autoantibodies, cytokines (TNF-α and IL-6), the chemokine MCP-1 and B cell stimulatory factors (BAFF, OPN) in BALF and plasma. Both reduced latency and increased intensity of glomerulonephritis paralleled inflammatory events in the lung and plasma. Hence, our current and previous findings strongly suggest that, following airway exposure to cSiO2, the lung might serve as a staging point for triggering systemic autoimmunity and lupus nephritis. Initiation of lupus involves, in part, impaired phagocytosis of dead or dying cells leading to aberrant autoantigen presentation and production of autoantibodies, an immunological hallmark for this AD [38-43]. These autoantibodies form complexes with self-antigens (e.g. dsDNA and nucleosome fractions) that subsequently deposit widely in tissues. In the kidney, such immune 138 complexes promote proinflammatory cytokine and chemokine production that mediate infiltration by mononuclear cells and consequent tissue injury [44-47]. Lupus morbidity and mortality often correspond with severity of autoimmune glomerulonephritis [48]. It has been convincingly established that airway exposure to cSiO2 or other particles unleashes a vicious, repetitive cycle in alveolar macrophages of phagocytosis → lysosomal membrane permeabilization → inflammasome activation → cell death → release of free cSiO2 particles → phagocytosis that drives chronic inflammation in the lung [49, 50]. This cycle may deplete the alveolar macrophage population, thereby promoting cell debris accumulation, secondary necrosis, alarmin release, and self-antigen presentation that culminate in early loss of tolerance with consequent production of pathogenic autoantibodies. Intriguingly, many of the processes that drive toxicity of cSiO2 particles in the lung overlap with well-established early events in lupus pathogenesis [51]. Consistent with this model, our findings, summarized in Fig 29, suggest that DHA consumption attenuates cSiO2-induced i) cytokine and chemokine release, ii)lymphocyte activation, proliferation and homing, iii) B cell differentiation to plasma cells and iv)IgG secretion by plasma cells. Several points are noteworthy regarding the cSiO2 dosing regimen employed here. Because the mouse lifetime is approximately 30 to 40 times less than that of a person, a contracted period of cSiO2 dosing (4 wk) was used to mimic long-term chronic human exposure. The total cSiO2 dose in this study approximates one half of a human lifetime occupational exposure at the current OSHA exposure limit [3]. Although other routes of cSiO2 administration in mice have been reported such as intratracheal and trans-oral instillation (oropharyngeal aspiration), we chose intranasal instillation based on its successful prior use in NZBWF1 and NZM2410 mice [12, 13, 52, 15]. In further support of our choice, an investigation comparing these approaches reported in 139 Figure 29. Putative mechanisms for DHA-mediated suppression of cSiO2-induced autoimmunity. The data presented here suggest that cSiO2-triggered pulmonary inflammation and ectopic lymphoid neogenesis drive systemic autoimmunity and glomerulonephritis in the female NZBWF1 mouse. Red downward arrows indicate potential action sites for suppressive effects of DHA that can be further predicted from these data. 140 mice receiving a single 1 mg dose of cSiO2 by each technique elicited similarly robust pulmonary inflammation [53]. Little is known regarding the distribution of cSiO2 particles outside of the lung following exposure and how such distribution might impact chronic toxicity to cSiO2. Sparse case reports from individuals occupationally exposed to cSiO2 have reported not only the development of typical cSiO2-induced pulmonary diseases (i.e. silicosis), but also observations of cSiO2 particles in peripheral organs such the lymph node, spleen, and kidney [54]. However, computational models have predicted that distribution of cSiO2 particles to these compartments is negligible[55], suggesting that pathological changes observed in these distal tissues are more likely a result of immune system dysfunction rather than direct effects of particle toxicity. An alternative possibility for the pronounced suppressive effect of DHA we observed in both the lung and distal organs of cSiO2-exposed NZBWF1 mice is dependent on restoration of the function of the mucociliary escalator, which mediates clearance of particle-laden alveolar macrophages from the respiratory tract. In a process termed "particle overload", high particle lung burdens (such as those encountered by chronic exposure in occupational settings and doses of cSiO2 utilized in this study), result in alveolar macrophages saturated with engulfed cSiO2 that cease translocation to the mucociliary escalator, culminating in inhibition of this clearance mechanism and contributing to chronic toxicity [56, 57]. It is tempting to speculate that DHA could facilitate enhanced phagocytosis of cSiO2 particles, possibly by increasing the fluidity of macrophage cellular membranes [58], which may facilitate pulmonary clearance of cSiO2-laden alveolar macrophages in lieu of the high concentrations of cSiO2. In support of this possibility, previous studies have demonstrated that DHA and/or its metabolites enhanced phagocytosis of zymosan beads [59, 60]; however no studies have specifically addressed their effect on phagocytosis or clearance of environmental particulates. 141 The NZW/LacJ strain was selected as a control for this study because it is a parental strain for NZBWF1 that does not spontaneously develop glomerulonephritis [10, 26]. It was therefore notable that NZW/LacJ mice also exhibited modest pneumonitis and nephritis after cSiO2 exposure and that this could be prevented by consumption of DHA. Interestingly, we similarly found that cSiO2 induces similar effects in C57Bl6 mice, which are also not prone to autoimmunity [15]. Numerous preclinical/clinical studies have demonstrated that dietary ω-3 PUFA supplementation suppresses and sometimes even reverses innate immune cell-driven inflammation thus making these dietary lipids attractive candidates for the prevention/treatment of chronic inflammatory diseases [21]. For example, ω-3 PUFAs suppress proinflammatory cytokine production, lymphocyte proliferation, cytotoxic T cell activity, natural killer cell activity, macrophage-mediated cytotoxicity, neutrophil/monocyte chemotaxis, MHCII expression and antigen presentation. Mechanistically, the anti-inflammatory effects of ω-3 PUFAs have been linked to alterations in: 1) production of bioactive lipid mediators, 2) intracellular signaling, transcription factor activity, and gene expression; and 3) membrane structure/function [61]. The potential of fish oil or semi-purified ω-3 PUFAs to specifically delay, prevent, and ameliorate autoimmunity has been investigated extensively in lupus-prone mouse models. Early studies with NZBWF1 mice demonstrated that initiating feeding of menhaden oil, DHA ethyl ester or EPA ethyl ester early in life markedly reduce severity and incidence of renal disease as well as extend the lifespan compared to mice fed beef tallow [62-64]. These findings coincide with reductions of autoantibodies and circulating immune complexes. Elegant studies from the Fernandes laboratory have related the delayed onset and decreased severity of renal disease exhibited in fish oil-fed NZBWF1 mice to reduced IL-1β, TNF-α, TGFβ1, ICAM-1 and fibronectin expression and increased expression of antioxidant enzymes [65-69]. The ameliorative effects of 142 ω-3 PUFAs have been similarly replicated in two other murine lupus models, BXSB/MpJ and MRL-1pr/1pr, as evidenced by decreased plasma proinflammatory cytokines, proteinuria, and glomerular injury as well as increased lifespan [70, 63, 71-73]. In a recent study comparing the effects of consuming ω-3 PUFA-, ω-6 PUFA- and ω-9 MUFA-rich diets on spontaneous AD development in NZBWF1 mice, we observed elevated plasma autoantibodies, proteinuria and glomerulonephritis in mice fed the latter two diets [24]. In contrast, all three endpoints were markedly attenuated in mice consuming the ω-3 PUFA diet, which contained primarily DHA. Current Western diets predominantly supply ω-6 PUFA but are deficient in ω-3 PUFA [61]. LA, the primary ω-6 PUFA in plant-derived oils, and its metabolite ARA are major PUFA in phospholipids of cell membranes. By replacing these, DHA is readily integrated into plasma membrane phospholipids in cells throughout the body, including those that mediate immune function [17]. Consistent with this paradigm, we observed dose-dependent increases in DHA and decreases in ARA in lung, kidney and RBCs. DHA was selected for this study over other ω-3 PUFA such as EPA because it has been shown to be superior in preventing spontaneous autoimmunity in NZBWF1 mice [74]. The DHA-rich microalgal oil employed in this study is free of heavy metals and organic pollutants that might be encountered in oils produced from farmed and wild cold-water fish [19]. As this product is formulated to be a source of DHA, the ω-3 PUFA EPA is not present. Accordingly, the microalgal oil employed here is preferable to fish oil-derived formulations because experimental results are attributable to DHA and not confounded by the presence of EPA or other ω-3 PUFAs. Representative ω-3 PUFA intake recommendations for healthy people range from 0.5 to 2 g/d, but higher levels of consumption up to 20 g/d have been used in clinical therapy trials for established lupus [75-77]. The 0.4, 1.2, and 2.4 % dietary DHA concentrations used in this study 143 account for 0.9, 2.7, and 5.4% of total energy intake. Upon extrapolation, a human eating 2000 kcal/d (8.368 MJ/d) would require 2, 6, and 12 g/d to correlate with the amounts consumed in this study. Consequently, in terms of energy percentage in a typical human diet, the concentrations employed here span a range attainable through diet, supplement consumption or by prescription. Therefore, given that some beneficial effects occurred at all three DHA doses, our findings have physiologic relevance to humans. During the course of this study, the European Food Safety Authority deemed that human consumption of supplements containing up to 5 g/d ω-3 PUFA derived from microalgal oil is considered safe [78]. Future perspectives of this model should therefore focus on effects of consuming 5 g/d DHA or lower human equivalents (i.e. < 2.4% of total energy intake) and consider effects of DHA consumption by lupus-prone mice during early life-stages on long-term susceptibility to environmental AD triggers. Conclusions Our findings reveal for the first time that DHA consumption dose-dependently suppresses cSiO2 triggering of autoimmunity in female NZBWF1 mice as manifested in the lung, blood and kidney (Fig 16). These observations provide a foundation for further mechanistic exploration of how modulation of the lipidome may be used to prevent or delay triggering of AD by cSiO2 and potentially other respirable toxicants. Such knowledge possibly could lead to development of practical, low-cost preventative strategies to reduce the risk of developing AD in cSiO2-exposed individuals or even slow progression of existing autoimmunity [79]. Future studies will focus on understanding the mechanisms by which DHA and/or its metabolites suppress cSiO2-triggered autoimmunity in this model emphasizing the alveolar macrophage. It will be particularly interesting to determine if unique lipidome signatures are predictive of protective effects of ω-3 PUFA on populations exposed to environmental AD triggers. 144 APPENDIX 145 APPENDIX Chapter 3 Supplementary Information Table 8. Fatty acid composition of kidney. This table was submitted in Bates et al. 2016 as Supplementary Table 1. Table 8 (cont’d) Treatment VEH CON cSiO2 cSiO2 cSiO2 cSiO2 CON 0.4% DHA 1.2% DHA 2.4% DHA Fatty Acid % of fatty acid in kidney 16:0 18.79 ± 0.10 18.89 ± 0.31 20.32 ± 0.36 21.99 ± 0.67 24.49 ± 1.16 16:1 (ω-7) trans 0.02 ± 0.001 0.02 ± 0.001 0.02 ± 0.001 0.02 ± 0.001 0.03 ± 0.001 16:1 (ω-7) cis 2.98 ± 0.710 2.82 ± 0.620 2.07 ± 0.690 2.02 ± 0.410 3.12 ± 0.800 18:0 12.88 ± 1.29 13.64 ± 0.99 14.28 ± 1.06 12.18 ± 1.03 12.90 ± 1.13 18:1 trans 0.20 ± 0.010 0.20 ± 0.010 0.21 ± 0.020 0.18 ± 0.020 0.16 ± 0.010 18:1 cis 24.64 ± 3.48 21.83 ± 2.42 15.15 ± 1.13 20.74 ± 2.53 15.97 ± 1.94 18:2 (ω-6) 9.12 ± 0.220 9.19 ± 0.390 10.27 ± 0.66 10.44 ± 0.25 7.32 ± 0.490 20:0 0.11 ± 0.001 0.10 ± 0.010 0.11 ± 0.001 0.09 ± 0.010 0.10 ± 0.010 18:3 (ω-6) 0.05 ± 0.001 0.05 ± 0.001 0.03 ± 0.001 0.02 ± 0.001 0.01 ± 0.001 20:1 (ω-9) 0.31 ± 0.010 0.30 ± 0.010 0.25 ± 0.010 0.20 ± 0.010 0.14 ± 0.010 18:3 (ω-3) 0.12 ± 0.020 0.11 ± 0.020 0.07 ± 0.001 0.09 ± 0.010 0.07 ± 0.020 20:2 (ω-6) 0.18 ± 0.020 0.19 ± 0.010 0.20 ± 0.010 0.13 ± 0.010 0.08 ± 0.010 22:0 0.05 ± 0.010 0.05 ± 0.001 0.06 ± 0.010 0.04 ± 0.001 0.05 ± 0.001 20:3 (ω-6) 0.59 ± 0.060 0.61 ± 0.040 0.91 ± 0.130 0.72 ± 0.100 0.30 ± 0.040 20:4 (ω-6) 18.10 ± 2.03 17.96 ± 1.64 11.34 ± 2.32 3.44 ± 0.440 1.22 ± 0.230 24:0 0.07 ± 0.010 0.08 ± 0.010 0.09 ± 0.010 0.08 ± 0.010 0.09 ± 0.010 20:5 (ω-3) 0.09 ± 0.010 0.10 ± 0.010 2.11 ± 0.340 5.37 ± 0.710 9.42 ± 1.080 24:1 (ω-9) 0.06 ± 0.001 0.06 ± 0.001 0.06 ± 0.010 0.05 ± 0.001 0.03 ± 0.001 22:4 (ω-6) 0.47 ± 0.050 0.51 ± 0.080 0.05 ± 0.010 0.01 ± 0.001 0.00 ± 0.000 146 Table 8 (cont’d) Treatment VEH CON cSiO2 cSiO2 cSiO2 cSiO2 CON 0.4% DHA 1.2% DHA 2.4% DHA Fatty Acid % of fatty acid in kidney 22:5 (ω-6) 0.47 ± 0.050 0.55 ± 0.100 0.01 ± 0.001 0.00 ± 0.000 0.00 ± 0.000 22:5 (ω-3) 0.29 ± 0.030 0.31 ± 0.030 0.41 ± 0.020 0.46 ± 0.040 0.55 ± 0.040 22:6 (ω-3) 6.50 ± 0.740 6.50 ± 0.460 14.13 ± 1.20 15.50 ± 1.41 18.99 ± 1.59 ∑ SFA 32.62 ± 1.17 33.40 ± 0.93 36.69 ± 0.93 35.46 ± 0.31 39.37 ± 0.81 ∑ MUFA 29.00 ± 4.13 26.10 ± 2.99 20.30 ± 2.49 24.85 ± 3.17 20.22 ± 2.72 ∑ PUFA (ω-3) 7.11 ± 0.740 8.78 ± 1.700 17.54 ± 1.82 21.51 ± 2.06 29.15 ± 2.65 ∑ PUFA (ω-6) 28.97 ± 2.01 29.14 ± 1.52 22.94 ± 2.74 15.87 ± 1.44 8.92 ± 0.740 147 Table 9. Fatty acid composition of lung. This table was submitted in Bates et al. 2016 as Supplementary Table 2. Table 9 (cont’d) Treatment VEH CON cSiO2 cSiO2 cSiO2 cSiO2 CON 0.4% DHA 1.2% DHA 2.4% DHA Fatty Acid % of fatty acid in lung 16:0 27.50 ± 0.43 40.77 ± 1.14 44.32 ± 1.21 42.96 ± 0.43 45.22 ± 1.31 16:1 (ω-7) trans 0.04 ± 0.001 0.04 ± 0.001 0.04 ± 0.001 0.04 ± 0.001 0.05 ± 0.003 16:1 (ω-7) cis 7.70 ± 0.260 8.42 ± 0.350 8.96 ± 0.440 9.73 ± 0.290 9.24 ± 0.800 18:0 6.40 ± 0.200 5.13 ± 0.200 4.28 ± 0.130 4.12 ± 0.090 4.52 ± 0.150 18:1 trans 0.18 ± 0.010 0.11 ± 0.010 0.09 ± 0.010 0.09 ± 0.001 0.13 ± 0.050 18:1 cis 35.87 ± 0.77 23.35 ± 1.03 20.90 ± 1.03 21.60 ± 0.55 19.96 ± 1.14 18:2 (ω-6) 6.37 ± 0.190 4.97 ± 0.160 4.84 ± 0.230 4.72 ± 0.070 3.15 ± 0.120 20:0 0.07 ± 0.002 0.04 ± 0.004 0.03 ± 0.001 0.03 ± 0.002 0.03 ± 0.001 18:3 (ω-6) 0.11 ± 0.004 0.09 ± 0.005 0.06 ± 0.002 0.04 ± 0.001 0.02 ± 0.001 20:1 (ω-9) 0.38 ± 0.008 0.24 ± 0.018 0.18 ± 0.009 0.14 ± 0.007 0.09 ± 0.005 18:3 (ω-3) 0.11 ± 0.008 0.08 ± 0.003 0.07 ± 0.006 0.07 ± 0.003 0.04 ± 0.003 20:2 (ω-6) 0.14 ± 0.002 0.12 ± 0.005 0.09 ± 0.006 0.07 ± 0.002 0.04 ± 0.002 22:0 0.04 ± 0.001 0.03 ± 0.002 0.03 ± 0.002 0.03 ± 0.001 0.02 ± 0.003 20:3 (ω-6) 0.37 ± 0.011 0.31 ± 0.020 0.36 ± 0.020 0.25 ± 0.006 0.13 ± 0.005 20:4 (ω-6) 4.83 ± 0.150 5.27 ± 0.498 1.97 ± 0.130 0.59 ± 0.016 0.22 ± 0.006 24:0 0.06 ± 0.002 0.05 ± 0.002 0.05 ± 0.003 0.05 ± 0.002 0.06 ± 0.002 20:5 (ω-3) 0.03 ± 0.002 0.04 ± 0.001 0.41 ± 0.030 0.69 ± 0.018 1.06 ± 0.089 24:1 (ω-9) 0.09 ± 0.003 0.10 ± 0.003 0.09 ± 0.005 0.08 ± 0.002 0.07 ± 0.008 22:4 (ω-6) 1.66 ± 0.071 1.52 ± 0.064 0.17 ± 0.016 0.03 ± 0.002 0.01 ± 0.002 22:5 (ω-6) 0.29 ± 0.018 0.43 ± 0.031 0.01 ± 0.001 0.01 ± 0.001 0.01 ± 0.001 22:5 (ω-3) 0.24 ± 0.008 0.23 ± 0.011 0.52 ± 0.019 0.57 ± 0.021 0.59 ± 0.012 22:6 (ω-3) 1.39 ± 0.043 1.44 ± 0.030 5.89 ± 0.210 7.09 ± 0.123 9.65 ± 0.217 ∑ SFA 35.67 ± 0.54 48.26 ± 1.10 51.21 ± 1.08 50.16 ± 0.75 53.62 ± 1.22 148 Table 9 (cont’d) Treatment VEH CON cSiO2 cSiO2 cSiO2 cSiO2 CON 0.4% DHA 1.2% DHA 2.4% DHA Fatty Acid % of fatty acid in lung ∑ MUFA 44.92 ± 0.79 32.71 ± 0.97 30.64 ± 1.03 32.05 ± 0.84 26.86 ± 1.46 ∑ PUFA (ω-3) 2.12 ± 0.040 2.76 ± 0.210 7.07 ± 0.200 8.69 ± 0.130 11.67 ± 0.27 ∑ PUFA (ω-6) 13.78 ± 0.23 12.51 ± 0.67 7.52 ± 0.300 5.71 ± 0.080 3.58 ± 0.140 149 Table 10. Fatty acid composition of RBCs. This table was submitted in Bates et al. 2016 as Supplementary Table 3. Table 10 (cont’d) Treatment VEH CON cSiO2 cSiO2 cSiO2 cSiO2 CON 0.4% DHA 1.2% DHA 2.4% DHA Fatty Acid % of fatty acid in RBC 16:0 43.69 ± 1.90 42.20 ± 2.26 40.26 ± 3.06 40.50 ± 2.94 45.02 ± 2.10 16:1 (ω-7) trans 0.08 ± 0.010 0.08 ± 0.010 0.06 ± 0.010 0.06 ± 0.004 0.05 ± 0.003 16:1 (ω-7) cis 1.58 ± 0.370 2.11 ± 0.440 2.45 ± 0.670 4.16 ± 0.930 3.01 ± 0.960 18:0 21.99 ± 2.30 20.54 ± 2.50 16.64 ± 2.73 12.60 ± 2.12 13.78 ± 1.66 18:1 trans 1.05 ± 0.150 1.02 ± 0.230 0.99 ± 0.270 0.74 ± 0.190 0.65 ± 0.140 18:1 cis 17.56 ± 3.44 21.05 ± 3.99 24.48 ± 5.23 24.41 ± 3.91 18.01 ± 3.10 18:2 (ω-6) 4.25 ± 1.060 4.02 ± 0.650 5.88 ± 1.000 5.59 ± 0.560 4.67 ± 0.320 20:0 0.36 ± 0.020 0.33 ± 0.030 0.28 ± 0.040 0.16 ± 0.020 0.17 ± 0.020 18:3 (ω-6) 0.00 ± 0.000 0.00 ± 0.000 0.00 ± 0.000 0.00 ± 0.000 0.00 ± 0.000 20:1 (ω-9) 0.32 ± 0.080 0.32 ± 0.050 0.34 ± 0.070 0.24 ± 0.040 0.17 ± 0.020 18:3 (ω-3) 0.00 ± 0.000 0.00 ± 0.000 0.00 ± 0.000 0.00 ± 0.000 0.00 ± 0.000 20:2 (ω-6) 0.00 ± 0.000 0.02 ± 0.010 0.00 ± 0.000 0.00 ± 0.000 0.00 ± 0.000 22:0 0.45 ± 0.050 0.20 ± 0.050 0.10 ± 0.020 0.06 ± 0.010 0.07 ± 0.010 20:3 (ω-6) 0.14 ± 0.040 0.20 ± 0.030 0.35 ± 0.050 0.25 ± 0.030 0.16 ± 0.020 20:4 (ω-6) 1.63 ± 0.350 2.36 ± 0.320 1.68 ± 0.250 0.73 ± 0.090 0.38 ± 0.040 24:0 0.69 ± 0.090 0.29 ± 0.060 0.17 ± 0.030 0.13 ± 0.020 0.15 ± 0.030 20:5 (ω-3) 0.00 ± 0.000 0.00 ± 0.000 0.18 ± 0.050 0.72 ± 0.110 1.40 ± 0.170 24:1 (ω-9) 0.41 ± 0.080 0.25 ± 0.110 0.05 ± 0.010 0.03 ± 0.005 0.01 ± 0.004 22:4 (ω-6) 0.24 ± 0.050 0.31 ± 0.050 0.07 ± 0.010 0.03 ± 0.010 0.01 ± 0.004 22:5 (ω-6) 0.09 ± 0.020 0.13 ± 0.010 0.00 ± 0.000 0.00 ± 0.000 0.01 ± 0.004 22:5 (ω-3) 0.00 ± 0.000 0.02 ± 0.010 0.11 ± 0.020 0.19 ± 0.020 0.28 ± 0.030 22:6 (ω-3) 0.15 ± 0.030 0.26 ± 0.030 1.36 ± 0.270 2.35 ± 0.330 5.08 ± 0.620 ∑ SFA 68.59 ± 4.13 65.09 ± 4.74 58.95 ± 5.76 55.55 ± 4.94 61.57 ± 3.50 150 Table 10 (cont’d) Treatment VEH CON cSiO2 cSiO2 cSiO2 cSiO2 CON 0.4% DHA 1.2% DHA 2.4% DHA Fatty Acid % of fatty acid in RBC ∑ MUFA 21.23 ± 3.60 25.09 ± 4.14 28.79 ± 5.40 30.00 ± 4.55 22.22 ± 3.84 ∑ PUFA (ω-3) 0.15 ± 0.030 0.33 ± 0.050 1.65 ± 0.340 5.31 ± 0.500 7.93 ± 0.740 ∑ PUFA (ω-6) 6.37 ± 1.210 7.04 ± 1.030 8.06 ± 1.180 6.72 ± 0.590 5.23 ± 0.340 151 REFERENCES 152 REFERENCES 1. 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J Rheumatol. 2004;31(8):1551-1556. 77. Wright SA, O'Prey FM, McHenry MT, Leahey WJ, Devine AB, Duffy EM et al. A randomised interventional trial of omega-3-polyunsaturated fatty acids on endothelial function and disease activity in systemic lupus erythematosus. Ann Rheum Dis. 2008;67(6):841-848.doi: 10.1136/ard.2007.077156. 78. EFSA. Scientific opinion on the extension of use for DHA and EPA-rich algal oil from Schizochytrium sp. as an novel food ingredient. EFSA Journal. 2014;12(10):3843-3860. 79. Pollard KM, Hultman P, Kono DH. Toxicology of autoimmune diseases. Chem Res Toxicol. 2010;23(3):455-466. doi: 10.1021/tx9003787. CHAPTER 4: DYNAMIC MRNA AND PROTEIN SIGNATURES OF SILICATRIGGERED AUTOIMMUNITY SUPPRESSED BY DOCOSAHEXAENOIC ACID IN 159 LUPUS-PRONE NZBWF1 MICE Bates, M.A., Akbari, P., Wee, J., Langohr, I.M., Parsey, G., Li, Q.Z., Harkema, J.R., Holian, A., and Pestka, J.J. Portions of this chapter will be submitted as multiple manuscripts (2018) Abstract Heredity is a primary predisposing factor for autoimmunity, however, environmental factors can critically influence the latency and severity of autoimmune diseases such as systemic lupus erythematosus (SLE). Dietary supplementation with the ω-3 polyunsaturated fatty acid (PUFA), docosahexaenoic acid (DHA), abrogates cSiO2-induced pulmonary ectopic lymphoid neogenesis, systemic autoimmunity, and nephritis at 24 wk of age. The purpose of this study was to test the hypothesis that cSiO2-triggered inflammation in the lung precedes systemic autoimmunity and nephritis in female NZBWF1 mice and is a primary target for the prophylactic action of DHA. Beginning at 6 wk of age, female NZBWF1 mice were fed an isocaloric AIN-93G diet containing 0.0, 0.4, or 1.0% DHA. Two wks later, mice were intranasally instilled with 1.0 mg cSiO2 once per week for 4 wk and maintained on experimental diets until sacrifice. At 1, 5, 9, and 13 wk post final exposure to cSiO2, cohorts of mice were sacrificed and tissues from the lung, spleen, and kidney were collected for histology. A focused mRNA expression array was used to relate lung ELT to immunological alterations prior to and at the onset of glomerulonephritis. BALF and plasma were analyzed for inflammatory proteins and autoantibodies by protein microarrays to temporally associate lung, spleen and kidney pathology to systemic autoimmunity. Using this integrative approach, we found that DHA blocked cSiO2-mediated infiltration of B- and T- cells in the lung in parallel to suppression of inflammatory gene and protein expression over time. The 160 prophylactic effect of DHA on cSiO2-triggered inflammation was further reflected in downregulated gene expression in the kidney and to a lesser extent, the spleen, at 13 wk post final exposure to cSiO2. Effects of cSiO2 on autoimmunity was recapitulated by a diverse profile of autoantibodies in BALF and plasma that were strikingly attenuated by DHA. Taken together, these results elucidate novel mechanistic insight into the countervailing effect of dietary DHA on environmental triggers on autoimmune disease. Introduction Systemic lupus erythematosus (SLE or lupus) is a devastating autoimmune disease with complex etiology and widely variable disease manifestation. It is widely acknowledged that lupus and other ADs are driven predominately by genetics. However, an underappreciated contribution to the heterogeneity of lupus may be the role of environmental factors, such as toxic stressors and diet, that influence lupus onset and disease severity [1,3]. The initiating event in lupus is loss of tolerance to self-antigens which elicits production of autoreactive antibodies and formation of circulating immune complexes (reviewed in [4,5]). These deposit in tissues, most notably the kidney, where they promote infiltration and activation of circulating mononuclear cells, mediating advanced glomerulonephritis that can culminate in end-stage renal failure (reviewed in [6]). Taken together, progression of lupus is coordinated by a dynamic network of aberrant activation of innate and adaptive immunity. The identification of specific factors that drive lupus in humans are difficult to dissect owing to the heterogeneity of lupus and inherent variance in human populations. Therefore, murine models of lupus such as the female NZBWF1 mouse offer a reductionist approach to dissect countervailing factors of autoimmunity. Elucidation into causal relationships between complex diseases and dietary influences has been expedited by technological advancements in high-throughput, comprehensive biological 161 profiling (“-omics”) approaches [7,8]. Untargeted approaches that capture global, unbiased alterations in RNA, protein, and beyond are powerful tools for discovery-based experiments [9– 11]. These studies have contributed to identification of underlying mechanisms that predicate autoimmunity, diagnostic indices and protein biomarkers for disease susceptibility, tissue injury, predictive outcomes for treatment effects, and elucidation of novel pharmaceutical targets [12– 16]. Capturing wide-scale biological changes over time is particularly valuable as it enables a dynamic view of the pathogenesis of chronic diseases such as lupus. However, these approaches are expensive and require extensive pre- and post-data processing and statistical analysis to derive meaningful interpretation [17]. Furthermore, computational algorithms to analyze time-course data using global RNA profiling methods are lacking [18,19], and mass-spectrometry based proteomic approaches are notoriously variable [20–23]. Coincident with the growth of global profiling methods is the advancement of targeted analysis with target microarrays, which have been expanded to include hundreds of analytes and no longer exclusively rely on target amplification, thus reducing limitations of traditional PCR approaches [24,25]. Thus, RNA and protein microarrays are an acceptable compromise between cost and effort to identify putative mechanisms of action that collectively delineate the pathogenesis of autoimmune diseases. Experimental animal and epidemiological studies have linked airway exposure of the respiratory toxicant, cSiO2, to kidney disease, lupus and other autoimmune diseases (rheumatoid arthritis, Sjӧgren’s syndrome, scleroderma, systemic vasculitis) [26–30]. 2.3 million Americas are employed in industries with high exposure to cSiO2; the majority (2.0 million) are employed in construction, with other high-exposure industries including stone cutting, foundries, and hydraulic fracturing [31]. To address the known health effects of respirable cSiO 2, in 2016, the U.S. Occupational Safety Health Administration decreased the permissible exposure limit to 25μg/m 3 162 to protect workers in industries with high exposure to cSiO 2 [31]. With these new regulations, OSHA estimates that up to 40% of workers are exposed to respirable cSiO 2 concentrations above the permissible exposure limit [32]. Thus, a significant population of Americans are exposed to the health hazards of respirable cSiO2 despite current protective measures. Respiratory cSiO2 exposure elicits overt and chronic inflammation that affects not only the lung, but distal organs by way of systemic inflammation [33–37]. Chronic inflammation is a defining feature of autoimmune diseases, and is both a trigger of and consequence of autoimmunity. Inflammatory agents, such as foreign antigens or environmental toxicants such as cSiO2, elicit cell-mediated secretion of cytokines, lipid metabolites, and alarmins coincident with upregulation of chemokine and adhesion molecule expression [33]. Collectively, this results in recruitment and activation of infiltrating immune cells, which in context of autoimmunity, predicate rampant leukocyte responses towards the body’s own tissues, inciting further inflammation. Ultimately, unresolved inflammation escalates tissue injury and resultant disease severity. A hallmark pathological feature in autoimmunity that reflects the association between unresolved inflammation and loss of self-tolerance is ectopic lymphoid tissue (ELT). ELT share similar structure and function to secondary lymphoid organs owing to common cell types that both initiate their development and orchestrate an adaptive immune response (reviewed in [38,39]). However, ELT are induced at sites of unresolved inflammation and thus, do not occur in preprogrammed locations in the body. The de novo formation of ELT facilitate accelerated initiation of an adaptive immune response by promoting antigen presentation and rapid activation of naïve B- and T-cells to remediate the offending agent at the site of inflammation [40–42]. 163 ELT contribute to autoimmune disease severity and progression. Target tissues of autoimmune diseases such kidneys (systemic lupus erythematosus), synovial joints (rheumatoid arthritis), and salivary glands (Sjӧgren’s syndrome) have been observed to contain B- and T-cell aggregates characteristic of ELT that correlate with disease severity [41,43–48]. Pharmaceuticals that deplete B-lymphocytes have little ability to disrupt established ELT in afflicted target organs in autoimmune disease and thus likely contribute to local and systemic injury by countering the effectiveness of conventional therapeutics (reviewed in [49]). ELT have been associated with accelerated systemic B-cell repopulation following successful treatment with the B-cell depleting therapy, rituximab [50]. This finding suggests that ELT in autoimmunity contribute to the systemic pool of circulating B-cells and may influence onset of active autoimmunity. Indeed, several studies have indicated that ELT function as a “survival niche”, thus shielding autoreactive plasma cells from immunosuppressive therapies [50–53], such as rituximab, which does not deplete long-lived plasma cells [54]. Therefore, alternative measures are warranted that block the initiation and/or formation of ELT, thus limiting their pathological contribution to AD progression and severity. One alternative approach to mitigate autoimmune disease and other chronic health conditions is by dietary fatty acids, particularly, ω-3 PUFAs [55]. Two important ω-3 PUFAs are eicosapentaenoic acid (EPA) (C20:5ω-3) and docosahexaenoic acid (DHA) (C22:6ω-3). EPA and DHA can be endogenously synthesized from dietary intake of the precursor essential fatty acid, alpha-linolenic acid (ALA) (C18:2ω-3). However, the low efficiency of endogenous enzymes involved in the elongation and desaturation of these products to EPA and DHA limits these conversions in mammals [56]. Therefore, dietary supplementation with ω-3 PUFAs provides sufficient quantities of pre-formed ω-3 PUFAs that are readily incorporated into phospholipids of the lipid bilayer of cellular membranes by replacing the predominate constitutive ω-6 PUFAs, 164 linoleic acid (LA) (C18:2ω-6) and arachidonic acid (ARA) (C20:4ω-6). Incorporation of ω-3 PUFAs occurs in numerous cells and tissues in the body, including immune cells [57,58]. In general, ω-6 PUFAs are considered pro-inflammatory whereas ω-3 PUFAs are considered antiinflammatory and inflammation resolving. The net effect of modification of cellular lipids with ω3 PUFAs is altered function of immune cells towards less inflammatory phenotypes, and thus could be applied to dampen the immune system in chronic inflammatory conditions [59,60]. Prior work by our laboratory suggests that the ω-3 PUFA, DHA, is a practical and effective way to prevent environmental triggering of lupus [61–63]. Previously, we revealed that airway exposure to cSiO2 triggers early autoimmunity and severe glomerulonephritis in female NZBWF1 mice, a widely used genetic model of lupus (Chapter 2). cSiO 2 elicited in the lung proinflammatory cytokine production, leukocyte homing, B-cell differentiation and profuse development of ELT containing B-, T-, and plasma cells [64]. Furthermore, supplementation of diets with the ω-3 PUFA, DHA, dose-dependently suppressed cSiO2-triggered inflammation, ELT development, autoimmunity and glomerulonephritis (Chapter 3) [62]. These conclusions were limited to analyses conducted after glomerulonephritis onset – 3 months following cSiO2 exposure. Not knowing early in vivo events that initiate autoimmunity precludes expansion of how modulation of dietary lipids can be exploited to counter environmental triggers of lupus and other autoimmune diseases. Towards this end, we tested the hypothesis that cSiO2-triggered ELT in the lung 1) precedes systemic autoimmunity and nephritis in lupus-prone NZBWF1 and 2) is a primary target for the prophylactic action of DHA. Herein, we utilized an integrative profiling approach to elucidate cellular, molecular, and protein signatures that catalyze autoimmunity and lupus nephritis following cSiO2 exposure that are markedly attenuated by dietary DHA. Taken together, the results 165 presented herein provide novel mechanistic avenues as to how DHA can be harnessed to block environmental triggers of autoimmunity. Materials and Methods Animals and diets All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Michigan State University in accordance with the National Institutes of Health guidelines (AUF #01/15-021-00). Female 6 wk old lupus-prone NZBWF1 mice were obtained from Jackson Laboratories (Bar Harbor, ME) and randomized into treatment groups. Mice were housed four per cage with free access to food and water. Mice were kept at constant temperature (21℃-24℃) and humidity (40-55%) under a 12/h light/dark cycle. Composition of experimental diets are summarized in Table 11. Upon arrival and for the duration of the study, mice were fed semi-purified American Institute of Nutrition (AIN)-93G containing 70g/kg fat [1]. To provide basal essential fatty acids, all diets were formulated with 10g/kg food-grade corn oil. Control diet (CON) was formulated with 60 g/kg food-grade higholeic safflower oil (Hain Pure Food, Boulder, CO). To prepare DHA-enriched diets, high-oleic safflower oil was substituted with 10 or 25 g/kg microalgal oil containing 40% DHA (DHASCO, provided by Dr. Kevin Hadley, Martek, Columbia MD). This yielded experimental diets that contained 0.4 or 1.0% (w/w) DHA, respectively. These dietary modifications were selected to model, on a caloric basis, two patterns of human DHA consumption: 1) 0.4% DHA, which is approximately equivalent to human consumption of 2 g per day and could feasibly achieved through dietary intake alone g and 2) 1.0% DHA, which is approximately equivalent to human 166 Table 11. Compositions of experimental diets. Experimental Group CON Ingredient 0.4% DHA 1.0% DHA (g/kg) Casein 200 200 200 Dyetrose 132 132 132 Cornstarch 397.5 397.5 397.5 Sucrose 100 100 100 Cellulose 50 50 50 t-Butylhydroquinone (TBHQ) 0.01 0.01 0.01 AIN 93G Salt Mix 35 35 35 AIN 93G Vitamin Mix (with vitamin E) 10 10 10 L-cysteine 3 3 3 Choline Bitartrate 2.5 2.5 2.5 Corn Oila,b 10 10 10 High-Oleic Safflower Oila,c 60 50 35 DHA-Enriched Algal Oila,d - 10 25 a As reported by the manufacturer b Corn oil contained 612 g/kg linoleic acid and 26 g/kg oleic acid c High oleic safflower oil contained 750 g/kg oleic acid and 140 g/kg linoleic acid d Algal oil contained 395 g/kg DHA and 215 g/kg oleic acid 167 consumption of 5 g per day and can be achieved through supplementation with DHA. This level of DHA consumption has been recognized as safe by the European Food Safety Authority [2]. Experimental design The experimental design for this study is depicted in Fig. 30. Upon arrival, at 6 wks of age, cohorts of mice (n = 8) were assigned either CON, 0.4% and 1% DHA-enriched diets and maintained on that diet until sacrifice. To prevent the formation of lipid oxidation products, diets were prepared weekly and stored at -20℃ until use. Fresh feed was provided to mice every day. cSiO2 (Min-U-Sil-5, 1.5-2.0 μm average particle size, Pennsylvania Sand Glass Corporation, Pittsburgh, PA) was acid washed and dried prior to preparation of stock suspensions of cSiO2 in PBS vehicle (VEH). Stock suspensions were prepared fresh in VEH prior to exposure and suspensions sonicated and vortexed rapidly for 1 min prior to intranasal instillation (25μl volume). Mice were exposed to cSiO2 as described previously [3,4]. At 8 wks of age, mice were anesthetized with 4% isoflurane and intranasally instilled with 1.0 mg cSiO2 in 25μl PBS or VEH. Exposures were repeated once per week for 4 wk. All mice in this experiment were exposed to cSiO2 at the same time. Thereafter, cohorts of mice were sacrificed over time at 1, 5, 9, and 13 wks post cSiO2 exposure. The mice were age 12, 16, 20 and 24 wks of age, respectively, at each of these timepoints. These time periods were selected to capture pathological changes in lupusprone, NZBWF1 mice following cSiO2 exposure prior to and during onset of glomerulonephritis as observed in prior studies (Chapters 2 and 3) [3,4]. 168 Figure 30. Experimental design. Beginning at 6 wks of age, female, lupus-prone NZBWF1 mice were assigned either control, 0.4%, or 1.0% DHA-enriched AIN-93G. Mice were maintained on assigned diet until sacrifice at designated time point. Beginning at 8 wks of age, mice were intranasally instilled with either PBS (VEH) or 1.0 mg cSiO2. Exposures were repeated once per wk, for 4 wks. Cohorts of mice (n = 8/gp) were sacrificed at 1, 5, 9, and 13 wks post exposure, corresponding to 12, 16, 20, or 24 wks of age respectively. 169 Necropsy and tissue collection For each timepoint, experimental cohorts of mice (VEH + CON, cSiO2 + CON, cSiO2 + 0.4% DHA, cSiO2 + 1.0% DHA) were sacrificed and tissues collected. Animals were euthanized by intraperitoneal injection with 56 mg/kg BW sodium pentobarbital and exsanguinated via the abdominal aorta. Blood was collected with heparin-coated syringes and centrifuged at 3500 x g for 10 min at 4℃ for separation of plasma which was stored in single-use aliquots at -80℃ until future analysis. BALF was centrifuged at 2400 x g for 15 mins and supernatant collected and stored at -80℃ in single-use aliquots for targeted proteome and autoantibody analysis. Following total lung lavage, the right lungs lobes were ligated and excised. The accessory lobe was stored in RNAlater (Ambion, Inc., Calabras, CA) overnight at 4℃ and held at -80℃ until RNA extraction. The middle and caudal lobes were frozen on dry ice, and the cranial lobe was snap frozen in liquid nitrogen prior to being stored at -80℃. The left lung lobe was fixed with 10% (v/v) neutral buffered formalin (NBF) at constant pressure for at least 1 h and stored in fixative until further processing for histology. The spleen and left kidney were placed in RNAlater (Ambion) overnight at 4℃ and then stored at -80℃ until further processing for RNA extraction. Finally, the right kidney was excised and fixed in NBF until further processing for histology. Lung histopathology Randomly orientated, serial sections of the formalin-fixed left lung lobe were processed routinely and embedded in paraffin. Tissue sections (5 μm) were deparaffinized and stained with H&E for histopathology. Tissues were scored semi-quantitatively in a blinded fashion by a boardcertified veterinary pathologist for the following lung lesions: (a) presence of lymphoid aggregates 170 within perivascular and peribronchial regions, (b) histologically evident ectopic lymphoid tissue, (c) presence of alveolar proteinosis, (d) alveolitis defined as the presence of alveolar infiltration of vacuolated macrophages, neutrophils, and lymphocytes, granuloma formation in the alveolus, (d) type II epithelial cell hyperplasia and thickened alveolar wall, and (e) mucous cell metaplasia. Individual lungs were graded for these lesions using the following criteria (% of total pulmonary tissue examined): (0) no changes compared to control mice; (1) minimal (<10%); (2) slight (10– 25%); moderate (26–50%); (4) severe (51–75%); or (5) very severe (>75%) of total area affected. CD45R immunohistochemistry for B-cells Immunohistochemistry was performed on formalin-fixed, paraffin embedded, left lung lobe and right kidney for identification of CD45R+ (B-cells). Sections were cut at 7μm with a Thermo Electron Cryotome FSE (Thermo Fisher Scientific, Inc.) and placed on slides coated with 2% 3-aminopropyltriethoxysilane and dried at 56℃ overnight. The paraffin slides were subsequently deparaffinized in xylene and hydrated through descending grades of ethyl alcohol to distilled water followed by Tris Buffered Saline (TBS) (pH 7.4) without surfactant (Scytek Labs, Logan, UT) for 5 min for pH adjustment. Following TBS, paraffin embedded tissue section underwent heat induced epitope with Citrate Plus Retrieval pH 6.0 (Scytek Labs) for 30 mins at 125℃ followed by a 30 min incubation at room temperature (RT). Endogenous peroxidase was blocked with 3% hydrogen peroxide and methanol (1:4) for 30 mins followed by tap and distilled water rinses. After pretreatments, standard micro-polymer complex staining was performed at RT on a IntelliPath Flex Autostainer (Biocare Medical, Concord, CA). All staining steps were followed by rinses in TBS autowash buffer (Biocare Medical). After blocking for nonspecific protein with Rodent Block M (Biocare) for 20 min, sections were incubated with 1:600 rat anti-CD45R 171 monoclonal antibody (catalog # 550286) (Becton Dickson, Franklin Lakes, NJ) in normal antibody diluent (Scytek Labs) for 1 hr to identify B cells. Bound CD45R antibody was detected with ProMark Rat on Mouse HRP Polymer by 30 min incubations with probe and polymer, respectively, and reaction developed with Romulin 3-amino-9-ethylcarbazole (AEC) (Scytek) chromagen prior to being counterstained with 1:10 hematoxylin for 1 min followed by distilled water. Slides were air dried before cover slips were applied with permanent synthetic mounting media. CD3 immunohistochemistry for T-cells Immunohistochemistry was performed on formalin-fixed, paraffin embedded, left lung lobe and for identification CD3+ (T-cells) in lung parenchyma. Sections were cut and processed as described above with some modifications. Following deparaffinization and rehydration, tissue sections underwent heat induced epitope with Citrate Plus Retrieval pH 6.0 (Scytek Labs) for 30 mins at 100℃ followed by a 10 min incubation at RT. Endogenous peroxidase was blocked as described above, and non-specific binding was blocked with Rodent Block M for 10 min. After pretreatments, 1:250 rabbit anti-CD3 polyclonal antibody (catalog #ab5690) (Abcam, Cambridge, MA) was incubated for 1 hr to identify T cells. Bound CD3 antibody was detected with ProMark Rat on Mouse HRP polymer (Biocare) with 15 min incubation for probe and polymer, respectively and reaction developed with AEC for 5 min prior to being counterstained with hematoxylin. CD21/CD35 for follicular dendritic cells Immunohistochemistry for CD21/CD35 was performed on snap frozen cranial lobe of the right lung. Tissues were sectioned at 7μm and sections air dried overnight on 2% 3aminopropyltriethoxysilane coated slides. Sections were fixed with acetate and formalin for 10 min at RT, and endogenous peroxidase blocked using 3% hydrogen peroxide in TBS for 30 mins.. 172 Blocking for non-specific binding was performed with Rodent Block M (Biocare) for 5 min. Follow pretreatments, 1:500 rat anti-CD21/35 monoclonal antibody (catalog # 553817) (Becton Dickson) was incubated for 1 hr to identify follicular dendritic cells. Bound CD21/CD35 antibody was detected with ProMark Rat on Mouse HRP polymer (Biocare) with 15 min incubation for probe and polymer, respectively and reaction developed with AEC for 5 min prior to being counterstained with hematoxylin. Kidney histopathology Formalin-fixed kidneys were randomly sectioned, paraffin-embedded, cut and stained with either hematoxylin and eosin (H&E) or Periodic acid-Schiff and hematoxylin (PASH). Stained sections were evaluated for lupus nephritis in a blinded fashion by a board-certified veterinary pathologist using a modified International Society of Nephrology/Renal Pathology (ISN/RPS) Lupus Nephritis Classification system. Slide sections were graded as follows: (0) no tubular proteinosis and normal glomeruli; (1) mild tubular proteinosis with multifocal segmental proliferative glomerulonephritis and occasional early glomerular sclerosis and crescent formation; (2) moderate tubular proteinosis with diffuse segmental proliferative glomerulonephritis, early glomerular sclerosis and crescent formation; and (3) marked tubular proteinosis with diffuse global proliferative and sclerosing glomerulonephritis. Morphometric quantitation of immunohistochemistry B, T, and fDCs cells were identified with antibodies to CD45R, CD3, and CD21/CD35, respectively. To quantitate cell infiltration in lungs (CD45R, CD3, and CD21/CD35) and kidneys (CD45R) indicative of ELT, tissue morphometry was performed as described by us previously. Briefly, slides were digitized with a VS110 (Olympus, Hicksville, NY) virtual slide system. The 173 entire tissue was selected as the region of interest and 20% of the sections were captured by systematic random sampling with NewCast software (Visiopharm, Hoersholm, Denmark) and a virtual magnification of 20X. The percentage of CD45R+, CD3+, or CD21/CD35+ cells was calculated by projecting a point grid over randomly sampled images with the STEPanizer 1.8 stereology tool and tallying the number of points falling onto positive staining or reference tissue. RNA preparation Extraction of total RNA from lung, spleen, and kidney were performed on samples stored in RNAlater. Tissues were homogenized with a high-speed rotor mechanical homogenizer in 0.5 ml of TriReagent (Sigma Aldrich, St. Louis, MO) and total volume adjusted to 1.0 ml with TriReagent after homogenization. RNA extraction with TriReagent was performed according to manufacturers instructions. Briefly, after homogenization in TriReagent, 100μl 1-bromo-3chloropropane was added to facilitate phase separation. After centrifugation 12,000 x g for 10 min at 4℃, 500μl of the aqueous layer (containing solubilized RNA) was transferred to 500μl isopropanol and shaken vigorously to precipitate RNA. After centrifugation, precipitated RNA was washed with 1.0 ml 75% ethanol, allowed to air dry, then solubilized in nuclease-free water (Ambion). Extracted RNA was further purified and genomic DNA removed by RNeasy Mini Kit with DNase treatment (Qiagen, Valencia, CA). Total RNA was dissolved in nuclease-free water and quantified using a NanoDrop-1000 (Thermo Fisher Scientific, Wilmington, DE). Samples were then analyzed for RNA integrity using a LabChip Gx Analyzer (Caliper Life Sciences, Waltham, MA). Samples with RIN values > 7.0 were included for gene expression analysis. 174 Gene expression analysis with nCounterTM To assess the effects of dietary DHA on cSiO2-induced changes in immune gene expression, RNA (n = 7-8/gp) were analyzed using the nCounter Mouse PanCancer Immune Profiling Panel (catalog # 115000142) (Nanostring Technologies, Inc., Seattle WA), which includes 770 immune-related genes, 40 housekeeping genes and 6 positive controls. Assays were performed and quantitated on the nCounter MAX system, sample preparation station, and digital analyzer (Nanostring Technologies) according to manufacturers instructions. Briefly, reporter and capture probes were hybridized to target analytes for 16 h at 65℃. After hybridization, samples were washed to remove excess probes, then purified target-probe complexes immobilized, aligned, and counted to obtain gene expression data. Raw data were analyzed using Nanostring’s proprietary software nSolver 3.0.22 and the Advanced Analysis Module. Background subtraction was performed using the 8 included negative controls and was calculated as the mean ± 2σ. Genes that had counts below this threshold were excluded from subsequent analysis. Data normalization was performed on background subtracted samples using internal positive controls and selected reference genes that were identified with the geNorm algorithm. Normalized data were then log2 transformed for downstream analysis. Network analyses for gene interactions in the statistically significant set of genes from each timepoint were performed using STRING database version 10.5 (http://string-db.org/) which curates both experimental and predicted gene interactions. Only interactions between genes identified in the nSolver data analysis were considered for gene interactions by STRING and confidence level for associations was set at 0.7. Networks generated by STRING were visualized with Cytoscape 3.0. 175 Targeted proteome microarray BALF and plasma were analyzed for the expression of 200 immune-related proteins using the Quantibody Mouse Cytokine Array 4000 (catalog #QAM-CAA-4000) (RayBiotech, Inc., Norcross, GA), a multiplexed sandwich ELISA based array. Due to cost constraints, n = 5/gp from VEH-treated mice fed CON, cSiO2-treated mice fed CON, and cSiO2-treated mice fed 1% DHA at 1, 5, 9, and 13 wks post exposure (BALF) and 5, 9 and 13 wks post exposure (plasma) were analyzed. Samples were randomly selected from each group, packed on dry ice and sent overnight to RayBiotech where all sample preparation, reagent preparation, and data acquisition was performed. Briefly, BALF and plasma were diluted 3-fold and added onto glass slides containing immobilized analyte-specific, capture antibodies and allowed to incubate for 2 h. Following incubation and washing, a cocktail of analyte-specific, biotinylated detection antibodies was added, incubated for 2 h, and finally incubated with Cy3-labeled streptavidin. Fluorescent images were acquired using a GenePix 4000B Microarray Scanner (Molecular Devices, Sunnyvale, CA) and then converted into digits using GenePix software (Molecular Devices). Quadruplicate readings for fluorescent intensity were acquired for each sample and averaged to derive the mean fluorescent intensity (MFI). Several analytes across submitted samples fell above or below the limits of the included standard curves. Per manufacturers’ suggestion (personal communication), data for all samples were calculated using MFI for a given analyte. Signal to noise ratios (SNR) were calculated as MFI compared to background signal from a given analyte. SNR > 2.0 were considered positive for analyte signal above background. Data for cSiO2-treated mice fed CON or DHA are expressed as log2fold change of SNR relative to SNR of VEH-treated mice fed CON. 176 Autoantibody microarray Autoantibody profiling for IgG and IgM in plasma and BALF at 13 wks post exposure (n = 7-8/gp) was performed using an antigen-coated microarray at the University of Texas Southwestern Genomics and Microarray Core. Samples were first treated with DNAse, then diluted 1:50, and added to microarray plates coated with 126 antigens known to elicit autoreactive antibodies in murine and human lupus. Then, Cy3 conjugated anti-IgG and Cy5 conjugated antiIgM were added and fluorescent images acquired with a GenePix 4400A Microarray Scanner (Molecular Devices). Fluorescent images were converted to signal intensity values using GenePix 7.0, background subtracted, and normalized to internal controls for IgG or IgM, respectively. Statistics Histopathology All treatments consisted of 7-8 mice per group and data presented as mean ± SEM. A p value of < 0.05 was considered statistically different for all study outcomes. Grubb’s outlier test was performed and any identified outliers were excluded from statistical analysis. Data were analyzed using SigmaPlot 11.0 for Windows (Jandel Scientific; San Rafael, CA). Student’s t test was used to determine differences in CON-fed mice instilled with VEH or cSiO2. The effects of consuming DHA diets on cSiO2-triggered responses were analyzed by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls (SNK) post hoc test for multiple comparisons. If the assumption of normality or equal variance failed, Kruskal-Wallis one-way ANOVA on ranks followed by Dunn’s test was used. The Spearman rank-order correlation coefficient was used to correlate DHA concentrations in diet and histopathological endpoints in the lung and kidney. 177 mRNA analysis Gene expression analysis was performed using multivariate linear regression models in nSolver 3.0. p-values were corrected using the Benjamini-Hochberg (BH) false discovery rate. Statistically significant, differentially expressed genes were defined as those with expression levels corresponding to log2(fold change) > ± 1.2 and BH p-value < 0.05, relative to the corresponding control group. Targeted proteome SNR > 2.0 for protein and autoantibody analytes were used for statistical analyses. If analytes had SNR < 2.0, these analytes were transformed to 2.0 to enable statistical comparisons between treatment groups. All data transformations, statistical analysis, and heat map generation were performed with Python 2.7.13 (https://www.python.org/downloads/release/python-2713/) in conjunction with NumPy (https://www.numpy.org), SciPy (https://www.scipy.org) and Pandas (https://pandas.pydata.org) libraries. SNR’s for targeted proteome data in BALF and plasma were analyzed using Student’s t-test for statistically significant differences between 1) cSiO2-treated mice fed CON or DHA vs VEH treated mice fed CON and 2) cSiO2 treated mice fed DHA vs cSiO2 treated mice fed CON. Autoantibody arrays At the time of this writing, statistical analyses for autoantibody array in BALF and plasma are pending future sample analyses being processed by collaborators at the University of Texas Southwest (UTSW). SNRs > 2.0 will be analyzed using Student’s t-test to determine statistically significant differences between cSiO2 treated mice fed CON vs VEH treated mice fed CON. Oneway ANOVA followed by SNK post hoc test will be utilized to assess statistically significant 178 differences between cSiO2 treated mice fed CON vs cSiO2 treated mice fed either 0.4% or 1.0% DHA-enriched diet and p-values < 0.05 considered statistically significant. Results: Histopathology Dietary DHA blocks cSiO2-induced inflammation and cell infiltration in lung A primary objective for this study was to test the hypothesis that following cSiO2 exposure in female, lupus-prone NZBWF1 mice that 1) ELT neogenesis in the lungs occurs prior to the development of systemic autoimmunity and downstream glomerulonephritis and 2) the lung is a primary target for the prophylactic effect of DHA. Towards this end, we first investigated the progression of histopathological lesions induced by cSiO2 in the lung over time. Randomly sectioned lungs at each timepoint were stained with H&E and assessed in a blinded fashion by a board-certified veterinary pathologist using a semi-quantitative scale as we have described previously [3,4]. Results from these interpretations are depicted in tables 9, 10, 11, and 12 for 1, 5, 9, and 13 wks post exposure, respectively. At 1 wk (Fig. 31, Table 12) and 5 wk (Fig. 32, Table 13) post final exposure to cSiO2, lesions were minimal but still evident in cSiO2-treated mice. Whereas ectopic lymphoid tissue were not overtly evident at these earlier time points, lymphoid aggregates circumventing the airways and blood vessels were present to a slight degree in some cSiO2 treated mice. Similarly, mild alveolar proteinosis and alveolitis were present following cSiO2 exposure to lupus-prone NZBWF1 mice. The effects of dietary DHA were to some extent apparent at these time points on some endpoints, particularly with regarding its suppressive effect on alveolitis (1 wk post final exposure, Table 12) and lymphoid aggregates (5 wk post final exposure, Table 13). The relatively minor suppressive effect of DHA at these early time points is likely attributable to comparatively 179 Figure 31. DHA consumption prevented cSiO2-triggered inflammation in lungs at 1 wk post final exposure. Representative H&E photomicrographs from NZBWF1 exposed to VEH + CON (A); cSiO2 + CON (B); cSiO2 + 0.4% DHA (C); cSiO2 + 1.0% DHA (D) at 1 wk post final exposure. Black arrows in photomicrographs indicate periairway and perivasculature lymphocytic infiltration. Dietary DHA suppressed lymphocytic infiltration as evident by the sparsity of infiltrating cells and was semi-quantitatively graded as shown in table 9. Abbreviations: a = airway; v = blood vessel; p = alveolar parenchyma. 180 Table 12. Histopathological assessment of cSiO2-triggered pulmonary inflammation in lupus-prone NZBWF1 mice fed CON or DHA-enriched diets at 1 wk post cSiO2 exposure. Pulmonary Lesions - 1 wk post final cSiO2 exposure Alveolitis Type 2 alveolar epithelial cell hyperplasia Mucous cell metaplasia 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.2 1.5 ± 0.3* 1.0 ± 0.0* 0.0 ± 0.0 0.0 ± 0.0 0.9 ± 0.1 0.0 ± 0.0 1.1 ± 0.1 1.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.8 ± 0.2 0.0 ± 0.0 1.0 ± 0.0 0.4 ± 0.1# 0.0 ± 0.0 0.0 ± 0.0 Group Lymphoid aggregates Ectopic lymphoid tissue Alveolar proteinosis VEH + CON 0.6 ± 0.2 0.0 ± 0.0 cSiO2 + CON 1.1 ± 0.1* cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA NZBWF1 mice (n = 7-8/gp) were graded individually for severity of lung inflammation (% of total pulmonary tissue) as follows: 0, no changes; 1, minimal (<10%); 2, slight (10-25%); 3, moderate (26-50%); 4, severe (51-75%); 5, very severe (> 76%) of total area affected. Data are mean ± SEM. * indicates significant difference between cSiO2 + CON vs VEH + CON (p < 0.05) † indicates significant difference between cSiO2 + 0.4% DHA vs cSiO2 + CON (p < 0.05) # indicates significant difference between cSiO2 + 1.0% DHA vs cSiO2 + CON (p < 0.05) 181 Figure 32. DHA consumption prevented cSiO2-triggered inflammation in lungs at 5 wk post final exposure. Representative H&E photomicrographs from NZBWF1 exposed to VEH + CON (A); cSiO2 + CON (B); cSiO2 + 0.4% DHA (C); cSiO2 + 1.0% DHA (D) at 5 wk post final exposure. Black arrows in photomicrographs indicate periairway and perivasculature lymphocytic infiltration. Dietary DHA suppressed lymphocytic infiltration as evident by the sparsity of infiltrating cells and was semi-quantitatively graded as shown in table 10. Abbreviations: a = airway; v = blood vessel; p = alveolar parenchyma. 182 Table 13. Histopathological assessment of cSiO2-triggered pulmonary inflammation in lupus-prone NZBWF1 mice fed CON or DHA-enriched diets at 5 wk post cSiO2 exposure. Pulmonary Lesions - 5 wk post final cSiO2 exposure Alveolitis Type 2 alveolar epithelial cell hyperplasia Mucous cell metaplasia 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.1 ± 0.1 2.2 ± 0.2* 1.2 ± 0.2* 0.0 ± 0.0 0.0 ± 0.0 1.1 ± 0.1 0.0 ± 0.0 2.2 ± 0.2 1.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.8 ± 0.1# 0.0 ± 0.0 2.1 ± 0.1 1.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 Group Lymphoid aggregates Ectopic lymphoid tissue Alveolar proteinosis VEH + CON 0.6 ± 0.2 0.0 ± 0.0 cSiO2 + CON 1.5 ± 0.2* cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA NZBWF1 mice (n = 7-8/gp) were graded individually for severity of lung inflammation (% of total pulmonary tissue) as follows: 0, no changes; 1, minimal (<10%); 2, slight (10-25%); 3, moderate (26-50%); 4, severe (51-75%); 5, very severe (> 76%) of total area affected. Data are mean ± SEM. * indicates significant difference between cSiO2 + CON vs VEH + CON (p < 0.05) † indicates significant difference between cSiO2 + 0.4% DHA vs cSiO2 + CON (p < 0.05) # indicates significant difference between cSiO2 + 1.0% DHA vs cSiO2 + CON (p < 0.05) 183 Figure 33. DHA consumption prevented cSiO2-triggered inflammation in lungs at 9 wk post final exposure. Representative H&E photomicrographs from NZBWF1 exposed to VEH + CON (A); cSiO2 + CON (B); cSiO2 + 0.4% DHA (C); cSiO2 + 1.0% DHA (D) at 9 wk post final exposure. Black arrows in photomicrographs indicate periairway and perivasculature lymphocytic infiltration. Dietary DHA suppressed lymphocytic infiltration as evident by the sparsity of infiltrating cells and was semi-quantitatively graded as shown in table 11. Abbreviations: a = airway; v = blood vessel; p = alveolar parenchyma. 184 Table 14. Histopathological assessment of cSiO2-triggered pulmonary inflammation in lupus-prone NZBWF1 mice fed CON or DHA-enriched diets at 9 wk post cSiO2 exposure. Pulmonary Lesions - 9 wk post final cSiO2 exposure Group Lymphoid aggregates Ectopic lymphoid tissue Alveolar proteinosis Alveolitis Type 2 alveolar epithelial cell hyperplasia Mucous cell metaplasia VEH + CON 1.0 ± 0.0 0.4 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 cSiO2 + CON 3.1 ± 0.2* 2.4 ± 0.2* 4.0 ± 0.2* 2.6 ± 0.3* 0.6 ± 0.2* 0.0 ± 0.0 cSiO2 + 0.4% DHA 2.2 ± 0.1† 1.2 ± 0.2† 4.0 ± 0.0 1.9 ± 0.2 0.6 ± 0.2 0.0 ± 0.0 cSiO2 + 1.0% DHA 1.1 ± 0.2# 0.6 ± 0.2# 4.0 ± 0.0 1.1 ± 0.2# 0.6 ± 0.2 0.0 ± 0.0 NZBWF1 mice (n = 7-8/gp) were graded individually for severity of lung inflammation (% of total pulmonary tissue) as follows: 0, no changes; 1, minimal (<10%); 2, slight (10-25%); 3, moderate (26-50%); 4, severe (51-75%); 5, very severe (> 76%) of total area affected. Data are mean ± SEM. * indicates significant difference between cSiO2 + CON vs VEH + CON (p < 0.05) † indicates significant difference between cSiO2 + 0.4% DHA vs cSiO2 + CON (p < 0.05) # indicates significant difference between cSiO2 + 1.0% DHA vs cSiO2 + CON (p < 0.05) 185 mild severity of lesions induced by cSiO2 at these time points, which developed into dramatic lesions at later time points. 9 wk post final exposure marked the time when dramatic appearance of cSiO2-induced pulmonary lesions were evident in the lung of NZBWF1 mice, which were markedly more severe than these features in earlier time points (Fig. 33, Table 14). The formation of ELT in cSiO2-treated mice fed CON diet was apparent and similarly paralleled by an increase in lymphoid aggregates at this time. These features were found to be predominately present as lymphocytic infiltrates that extensively encompassed the airways and blood vessels in the lungs of cSiO2 treated mice. The suppressive effect of DHA on these lymphocytic lesions at 9 wk post final exposure was pronounced in comparison to its relatively slight effect at 1 and 5 wk post final exposure to cSiO2. In contrast to the strong suppressive effect of DHA on lesions consisting of lymphocytes, DHA did not appreciably impact the development of alveolar proteinosis or type II alveolar epithelial cell hyperplasia. There was no statistically significant difference in the severity of these lymphocyte-independent lesions by feeding cSiO2-treated mice DHA at the amounts of DHA used in this study. Consistent with our previous findings, at 13 wk post final exposure (Fig. 34, Table 15) when mice were 24 wks of age, histologically evident inflammation and ELT induced by cSiO2 were most severe (Fig. 34). In addition to amplification of the severity of previously described lesions evident in cSiO2-treated mice, we also observed mucous cell metaplasia of these cells around the airways, which was previously not identified at earlier time points. The impact of DHA on suppression of lymphocytic-dependent lesions (i.e. lymphoid aggregates and ectopic lymphoid tissue) were evident with both concentrations of DHA employed in this study. 1.0% DHA 186 statistically reduced cSiO2-induced alveolitis at this time point, however, this response was not evident in mice fed diet enriched with 0.4% DHA. Consistent with what was observed in earlier time points following cSiO2 exposure, DHA did not have a marked impact on histopathological lesions induced by cSiO2 which are lymphocyte-independent, including alveolar proteinosis, type II alveolar epithelial cell hyperplasia, and mucous cell metaplasia. To summarize, the progression of cSiO2-induced lesions in the lung were time dependent and attributable to cSiO2 exposure, as these histopathological lesions were not evident in VEH treated mice fed CON diet. These findings are consistent with our model that dietary DHA attenuates an unresolved cycle of inflammation induced by cSiO2 in the lungs that escalates over time and precedes the development of systemic inflammation and glomerulonephritis in kidneys of lupus-prone NZBWF1 mice. Dietary DHA delays formation of ELT over time in lung of cSiO2-treated NZBWF1 mice Immunohistochemistry was used with morphometry to 1) characterize the cellular phenotype of leukocytes present in cSiO2-induced pulmonary histopathological lesions and 2) follow DHA attenuation of cSiO2-induced ELT formation over time on FFPE lung section. Specifically, CD45R+ and CD3+ cells, were labeled to reflect B- and T-cells, respectively. Results for quantitation of CD45R+ and CD3+ cells in lung over time are displayed in Fig. 35. cSiO2 induced early and sustained B- and T-cells in lungs of NZBWF1 mice. Both B- and T-cells were markedly elevated within 1wk post final cSiO2 instillation in lungs of NZBWF1 mice fed CON diet. The B and T cell infiltrates in the lung of cSiO2-treated NZBWF1 mice fed CON dramatically increased over time and was drastically diminished by feeding DHA. At 1 and 5 wks post exposure to cSiO2, B- and T-cells in lung of cSiO2-treated, CON-fed mice were mostly diffuse infiltrates and did not appear to markedly organize into distinct ELT at these time points. However, by 9 wks post exposure, cSiO2 elicited pronounced formation of focal 187 Figure 34. DHA consumption prevented cSiO2-triggered inflammation in lungs at 13 wk post final exposure. Representative H&E photomicrographs from NZBWF1 exposed to VEH + CON (A); cSiO2 + CON (B); cSiO2 + 0.4% DHA (C); cSiO2 + 1.0% DHA (D) at 13 wk post final exposure. Black arrows in photomicrographs indicate periairway and perivasculature lymphocytic infiltration. Dietary DHA suppressed lymphocytic infiltration as evident by the sparsity of infiltrating cells and was semi-quantitatively graded as shown in table 12. Abbreviations: a = airway; v = blood vessel; p = alveolar parenchyma. 188 Table 15. Histopathological assessment of cSiO2-triggered pulmonary inflammation in lupus-prone NZBWF1 mice fed CON or DHA-enriched diets at 13 wk post cSiO2 exposure. Pulmonary Lesions - 13 wk post final cSiO2 exposure Group Lymphoid aggregates Ectopic lymphoid tissue Alveolar proteinosis Alveolitis Type 2 alveolar epithelial cell hyperplasia Mucous cell metaplasia VEH + CON 0.9 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 cSiO2 + CON 3.6 ± 0.2* 3.4 ± 0.2* 4.0 ± 0.1* 2.8 ± 0.2* 2.2 ± 0.2* 0.2 ± 0.2 cSiO2 + 0.4% DHA 2.0 ± 0.2† 1.6 ± 0.3† 4.1 ± 0.1 2.2 ± 0.2 1.6 ± 0.3 0.4 ± 0.2 cSiO2 + 1.0% DHA 1.6 ± 0.2# 1.1 ± 0.3# 4.1 ± 0.1 2.0 ± 0.2# 1.6 ± 0.3 0.1 ± 0.1 NZBWF1 mice (n = 7-8/gp) were graded individually for severity of lung inflammation (% of total pulmonary tissue) as follows: 0, no changes; 1, minimal (<10%); 2, slight (10-25%); 3, moderate (26-50%); 4, severe (51-75%); 5, very severe (> 76%) of total area affected. Data are mean ± SEM. * indicates significant difference between cSiO2 + CON vs VEH + CON (p < 0.05) † indicates significant difference between cSiO2 + 0.4% DHA vs cSiO2 + CON (p < 0.05) # indicates significant difference between cSiO2 + 1.0% DHA vs cSiO2 + CON (p < 0.05) 189 B-cell aggregates interspersed with more diffuse T-cells in CON-fed mice, thus resembling the structure of ELT described in Chapters 2 and 3. This continued to be enhanced in magnitude by 13 wks following the final exposure to cSiO2. Importantly, both 0.4% and 1.0% DHA markedly decreased B- and T-cells at all timepoints, thus limiting the formation of ELT following cSiO2 exposure in DHA-fed NZBWF1 mice. These results are consistent with those observed in H&E observations. B- and T-cells are the effector cells of ELT that mediate the adaptive immune response evoked by ELT. The cell type that is the initiator cell involved in the formation of and maintenance of ELT, and arguably the definitive marker for these structures follicular dendritic cells (fDCs). In Chapters 2 and 3, we previously did not identify fDCs in our cSiO2-treated NZBWF1 mice, namely resulting from the unavailability of commercially available antibodies for fDCs that are compatible with formalin-fixed tissue. To determine if fDCs were present in ELT of cSiO2-treated NZBWF1, we investigated this possibility at 13 wks post final exposure to cSiO2 with immunohistochemistry for CD21/CD35 on frozen lung sections. Marked infiltration of fDCs at 13 wk post final exposure was evident in NZBWF1 exposed to cSiO2 which were identified predominately in the perivascular and peribronchial regions, which is consistent with the location of CD45R+ and CD3+ cell infiltrate. In contrast to the marked attenuation by DHA on B-cells and T-cells, the effect of DHA was less dramatic on fDCs and did not notably impact cSiO2-elicited fDCs at 13 wks post final exposure to cSiO2 (Fig. 36). However, this may be explained by the large variation in cSiO2treated mice fed CON diet, thereby obscuring more subtle effects of DHA on fDC populations in ELT in lung of cSiO2-treated NZBWF1 mice. 190 Figure 35. Dietary DHA dose-dependently attenuates development of ELT in lungs of cSiO 2treated NZBWF1 mice over time as indicated by immunohistochemistry for CD45R+ (Bcells) (A) and CD3+ (T-cells) (B). * indicates p < 0.05 between cSiO2 + CON vs VEH + CON; † indicates p < 0.05 between cSiO2 + 0.4% DHA vs cSiO2 + CON; # indicates p < 0.05 between cSiO2 + 1.0% DHA vs cSiO2 + CON. Spearman’s rank order correlation (r2) for dietary DHA concentration and CD45R+ = -0.66 (p < 0.0001); r2 for CD45R+ cells and CD3+ cells = 0.75 (p = < 0.0001). 191 In summary, cellular infiltration consisting of B and T cell infiltrates in lung are induced early during the progression of cSiO2-triggered pulmonary inflammation and mark early initiation of ELT neogenesis in the lung following cSiO2 exposure. In addition, we identified fDCs present as constituent members of ELT in the lungs of cSiO2-treated mice. Interestingly, B- and T-cells were strongly attenuated by feeding DHA. However, fDCs were not dramatically affected by DHA. This could indicate that the prophylactic effect of DHA on attenuation of cSiO2-triggered lung ELT neogenesis occurs independent of the presence of fDCs. Dietary DHA diminishes protein deposition and CD45R+ infiltration in kidney of cSiO 2treated NZBWF1 mice at 13 wks post final exposure At the time of this writing, interpretation of H&E and PASH for severity of lupus nephritis lesions in kidneys of NZBWF1 is ongoing. However, we utilized H&E stained kidney sections to morphometrically quantitate protein present in the tubules of cSiO2-treated mice at 13 wks post final exposure (Fig. 37). Consistent with previous studies in Chapters 2 and 3, which assessed this end point through semi-quantitative 2-parameter reagent sticks for proteinuria, we found that cSiO2-treated mice fed CON diet had accumulated protein in the renal tubules (Fig. 37) In contrast, cSiO2-treated mice fed DHA tended to have reduced protein present in the renal tubules, however, this difference was not statistically significant (p = 0.12). In previous studies, we preliminarily observed CD45R+ staining in kidneys of cSiO2treated mice that qualitatively appeared to be decreased by DHA (unpublished observations). However, the sectioning method for kidneys in these studies did not allow for full assessment of these lesions by qualitative observation or morphometry. To address this issue, in this study, kidneys were randomly sectioned kidneys to capture a global, unbiased assessment of cellular infiltration in this study. We rationalized that since CD45R+ cell infiltration peaked at 13 wks post 192 Figure 36. fDCs are induced in ELT of cSiO2-treated NZBWF1 mice at 13 wks post final exposure to cSiO2 as indicated by immunohistochemistry for CD21/CD35. * indicates p < 0.05 between cSiO2 + CON vs VEH + CON. r2 for dietary DHA concentration and fDCs = -0.288 (p = 0.17). r2 for CD21/35+ and CD3+ cells = 0.54 (p = 0.006); r2 for CD21/35+ and CD45R+ cells = 0.363 (p = 0.09). 193 final exposure cSiO2 in the lung, that time point would also capture these cells if they were present in the kidney. Much to our surprise, similarly to the lung, CD45R+ cells seemed to localize into distinct, focal aggregates, resembling the ELT we observed in the lung of cSiO2-treated NZBWF1 mice. In the kidney, CD45R+ aggregates induced by cSiO2 were found to occur predominately in the interstitum between renal arteries and veins and the renal pelvis. Kidney histopathology at 13 wk post final exposure correlates with lung inflammation In contrast to the strong attenuation observed with DHA on CD45R+ infiltrates in the lung, cSiO2-treated NZBWF1 mice fed CON diet tended to have increased B-cells compared to VEHtreated mice fed CON diet, however, this difference was not statistically significant (p = 0.12). Similarly, dietary DHA tended to decreased CD45R+ cells in the kidney, however this difference was also not statistically significant (p = 0.12) (Fig. 38). We speculated that the lack of statistical significance in the kidney could be attributed to the high variation observed within cSiO2-treated mice fed CON, which obscured the effect of DHA on cSiO2-induced protein in renal tubules and CD45R+ infiltrates in kidney. To address this possibility, we utilized Spearman’s rank order correlation coefficient to determine if dietary DHA had a dose-response effect on these endpoints which is indicated in figure legends for Fig. 37 and Fig. 38 for tubule protein and CD45R+ infiltrates, respectively. With this approach, we determined that dietary DHA decreased cSiO2induced CD45R+ infiltrates compared to NZBWF1 mice fed CON diet. However, this approach did not identify an effect of DHA on protein in tubules. Another explanation for the discrepancy in these results is that the large variation observed in cSiO2-treated mice is attributable to a responder vs non-responder index. To test this possibility, Spearman’s rank order correlation coefficient was calculated at 13 wks post final exposure to cSiO2 194 Figure 37. Dietary DHA trended towards decreased cSiO2-accelerated protein accumulation in renal tubules at 13 wks post final exposure to cSiO2. * indicates p < 0.05 between cSiO2 + CON vs VEH + CON. r2 for dietary DHA concentration and tubular protein density = -0.29 (p = 0.17). 195 Figure 38. Dietary DHA dose-dependently attenuated cSiO2-elicited B-cell accumulation in kidney at 13 wks post final exposure to cSiO2 as indicated by immunohistochemistry for CD45R+ cells. r2 for dietary DHA concentration and CD45R+ cell density = -0.419 (p = 0.04). 196 between the lung histopathological endpoints described in previous sections of this chapter (i.e. histopathological lesions; B-, T-, and fDC infiltrates) and observed kidney effects. Several interesting correlations between lung inflammation and kidney histopathology were elucidated with this comparison (Table 16). Most strikingly, tubular protein (indicative of glomerulonephritis) was strongly correlated (p < 0.05) with both pulmonary lymphoid aggregates and ectopic lymphoid tissue. In addition, CD45R+ infiltrates in kidney correlated with pulmonary lymphoid aggregates ELT, alveolitis, type II cell hyperplasia, and CD45R+ cells in lung. These findings were particularly valuable as we conclude that suppression of lung ELT by DHA in cSiO2treated lupus-prone confers a protective effect on cSiO2-accelerated onset of kidney pathology and glomerulonephritis. Discussion: Histopathology In Chapters 2 and 3, we demonstrated that dietary DHA dose-dependently decreased cSiO2induced ELT in female, lupus-prone NZBWF1 at 24 wks of age which occurred in parallel with decreased autoantibodies in BALF and plasma. In this study, we utilized pan immunohistochemistry markers for CD45R, CD3, and CD21/CD35 to characterize the infiltration of these cell types, respectively, over time. At study termination (13 wk post final cSiO2 exposure), we confirmed the histopathological findings in previously reported results and expanded them by finding that: 1) fDCs occur in ELT of cSiO2-treated NZBWF1 mice, 2) B-cell infiltrates in kidneys of cSiO2-treated mice were consistent with ELT induced by cSiO2 in lungs, and 3) dietary DHA dose-dependently suppressed B- and T- cell infiltration over time in the lungs and B-cell infiltration in kidneys. ELT require persistent, unresolved inflammation and three principal cell types to initiate an adaptive immune response: B-cells, T-cells, and fDCs. Histopathological interpretation of the lung sections over time of cSiO2-treated NZBWF1 mice revealed several 197 Table 16. cSiO2-induced lung inflammation correlates with kidney histopathology at 13 wks post final exposure to cSiO2. Spearman’s rank order correlation coefficient (r2) and the corresponding p-value for each endpoint between lung and kidney are given below. * indicates pvalue < 0.05. Correlations Between Lung and Kidney at 13 Wks Post Final Exposure to cSiO2 Kidney Histopathology Tubular protein CD45R+ (B-cells) Lung Histopathology r2 p-value r2 p-value Lymphoid aggregates 0.42 0.04* 0.57 0.004* ELT 0.46 0.02* 0.59 0.002* Proteinosis -0.26 0.22 0.02 0.91 Alveolitis -0.08 0.69 0.63 0.001* Type II cell hyperplasia 0.33 0.12 0.62 0.001* Mucous cell metaplasia 0.24 0.25 0.32 0.12 CD45R+ (B-cells) 0.35 0.1 0.61 0.002* CD3+ (T-cells) 0.35 0.1 0.37 0.07 CD21/CD35+ (fDCs) 0.28 0.18 0.13 0.54 198 prominent features of our model of environmentally-triggered autoimmune disease. Evident within 1 wk post final exposure to cSiO2 in CON-fed mice were hallmark features of persistent lung inflammation (e.g. lymphocytic aggregates, alveolitis, and alveolar proteinosis) that amplified over time. Taken together, these findings indicate that there is no resolution of inflammation following cSiO2 exposure. When pathogenesis of ELT was assessed over time, it was found that dietary DHA decreased cSiO2-elevated B- and T-cells within 1 wk post final exposure to cSiO2 and dramatically diminished B- and T-cell infiltration in the lungs over time. Collectively, these features indicate that 1) cSiO2-triggered inflammation occurring in the lung prior to 1 wk post final exposure is sufficient to incite persistent cell infiltration and 2) DHA has early and sustained ability to block infiltration of B- and T-cells that promote the formation of ELT. Taken together, future studies should incorporate short-term experiments with single or multiple exposures to cSiO2 to identify early inflammatory events that are targeted by DHA. Importantly, 9 wk post final exposure to cSiO2 marked the formation of ELT in the lung of CON-fed mice. Given that ELT contribute to autoimmunity and are resistant to therapeutics (reviewed in [48]), this finding has practical implications. If ELT induced after cSiO2 exposure are the primary driving force behind cSiO2-triggered autoimmunity, then the period preceding their appearance in the lung may mark a window of opportunity to initiate therapeutic interventions, including DHA, following heavy particle burdens to environmental toxicants such as cSiO2. Indeed, preliminary observations of the utility of dietary DHA as an interventional approach on cSiO2-triggered autoimmunity support this contention (Akbari, P., unpublished observations). It is interesting to note that both cSiO2 and DHA appear to have a more pronounced on Bcells compared to T-cells in the lung. This finding indicates that B-cells have a considerable impact on the overall phenotype induced by intranasal cSiO2 exposure and attenuated by DHA. One 199 tempting possibility is that DHA is incorporated to a greater degree in B-cells compared to T-cells, thus attributing a stronger effect of DHA on B-cells. Poggi et al. 2015 found no difference in the amount of DHA between CD4+ or CD8+ T-cells, CD19+ B-cells, CD14+ monocytes isolated from healthy human volunteers. However, this study, nor any others, have compared the fatty acid composition between classes of lymphocytes following supplementation with DHA in humans or mice, thus representing a possible direction for future studies. It might be further speculated that DHA may selectively increase certain subtypes of CD3+ T-cells, such as Treg cells [66–68], thereby promoting a tolerogenic effect in ELT of cSiO2-treated mice. This hypothetical increase in T reg cells mediated by DHA would be captured by CD3 staining. Brown et al. 2004 [69] previously demonstrated that cSiO2 exposure to lupus-prone NZM2410 mice did not alter the absolute number of Treg cells, but did increase the total number of CD4+ cells, thus relatively decreasing the number of Treg cells. It is possible that such skewing in cSiO2-treated NZBWF1 mice can be blocked by feeding DHA. Future studies should therefore consider alterations in the balance of lymphocyte subsets in addition to absolute number. In addition to the well-established role of B- and T- cells in ELT and secondary lymphoid organs, fDCs are an additional cell type in germinal centers that is key to initiation of the adaptive immune response. fDCs predominately dictate the fate of B-cells in germinal centers by serving as a platform for B-cell activation and survival [70]. Unlike conventional and plasmacytoid dendritic cells, fDCs express low levels of MHC II molecules and PPRs, and are instead identified by their surface expression of FcγRIIb, complement receptor (CR) 1 (CD35), and CR2 (CD21) (reviewed in [71]). Due to this receptor expression, fDCs preferentially present immune complexes of antibody or complement-opsonized antigens. In addition to their antigen presentation function, fDCs also secrete B-cell chemotactic (e.g. CXCL13) [72,73] and survival factors (e.g. BAFF) 200 (reviewed in [74]). Taken together, these features of fDCs contribute to activation, differentiation and survival of B-cells in germinal centers such as those found in ELT. fDCs have tremendous potential to influence to the pathogenesis of SLE and other autoimmune diseases. Aside from their role in recruitment and activation of B-cells in ELT, fDCS regulate immunological tolerance to self-antigens [75–79]. We identified a trend, albeit it not statistically significant, for dietary DHA to attenuate fDCs elicited by cSiO2 exposure at 13 wks post final exposure to cSiO2. Since fDCs are critical for B-cell survival, this may indicate further evidence for a preferential effect of dietary DHA on B-cell activation and survival as opposed to another cell type as discussed above. Whether this occurs by actions on B-cells as opposed to survival factors secreted by fDCs, such as BAFF, will require future investigations. Alternatively, it is possible that the effect of dietary DHA is more pronounced at earlier time points. At the time of this writing, we did not perform staining for fDCs in lung tissue at earlier time points. It is conceivable that since B- and T-cell infiltrates appeared to structurally organize into ELT at 9 wks post final exposure to cSiO2 and beyond, it seems plausible that 9 wks post final exposure to cSiO2 or earlier would mark the appearance of fDCs in lung tissue. This possibility can be resolved by performing future staining for fDCs at earlier time points. Overall, elucidation of the role of ω-3 PUFAs, including DHA, on fDC function is completely underexplored, and could potentially uncover novel applications for ω-3 PUFAs on fDC-mediated pathologies, including ELT. While dietary DHA was effective at decreasing lymphocyte histopathological lesions induced in the lung by cSiO2 exposure, one must also consider that DHA appears to have little to no effect on alveolar proteinosis, and type II cell hyperplasia in the lung. These two pathological features are not mutually exclusive: type II cells produce pulmonary surfactant and alveolar proteinosis is a result of defective macrophage clearance of excess surfactant [80]. Defective 201 macrophage function (and consequently, alveolar proteinosis) could result from the phenomenon of “overloaded macrophages” resulting in cessation of phagocytic function following excessive particle burdens [81,82]. In parallel, the ability of DHA to attenuate cellular emigration into inflamed tissues may actually promote alveolar proteinosis, as infiltrating monocytes that could compensate the lost function of particle-laden alveolar macrophages are prevented from migrating into the lung [83,84]. Taken together, dietary DHA did not restore alveolar macrophage function of clearance of pulmonary surfactant. Nevertheless, future studies should address if supplementary DHA alters other phagocytic functions of alveolar macrophages, such as efferocytosis or particle clearance, following cSiO2 exposure that may contribute to the ameliorative effect of DHA on autoimmunity. A goal in this study was to determine if cSiO2-induced appearance of ELT exposure in NZBWF1 mice precede systemic autoimmunity and ultimately, glomerulonephritis. At the time of this writing, grading for severity of lupus nephritis by a board-certified veterinary pathologist in mice treated with cSiO2 fed CON or DHA enriched diets over time are under interpretation. We correlated tubular protein deposition and CD45R+ cell infiltration in the kidney to cSiO2-induced lung inflammation at 13 wks post final exposure. Indeed, pulmonary ELT correlated with renal tubular protein density and CD45R+ infiltration in kidney which was dosedependently attenuated by dietary DHA. However, there is a need for further confirmation by staining for CD3 and/or CD21/CD35 in the kidney. Nevertheless, the identification of B-cell infiltrates in the kidney is strongly indicative of ELT, which likely enhanced lupus nephritis. Indeed, renal ELT have been identified individuals with SLE and severity of lupus nephritis correlated with B- and T-cell aggregates in kidney [85,86]. 202 This is the first report of ELT induction in multiple tissue compartments following localized toxicant exposures. Given that cSiO2 particles in this study are too large to diffuse into systemic circulation via the lung and are insoluble in water, it seems unlikely that these particles could be deposited in tissues distal to the lung. We did not observe cSiO2 particles in kidney by birefringent microscopy (unpublished observations). Therefore, it is unlikely that deposition of cSiO2 particles in the kidney directly promotes CD45R+ B-cell infiltration. Future studies should explore the possibility of autoreactive lymphocyte migration from ELT in the lung to the kidney and/or accelerated systemic immune complex or complement deposition to the kidney to clarify the role of lung ELT on kidney pathology. The gold-standard for characterization of ELT in afflicted tissues is by immunohistochemistry or immunofluorescence. However, microscopy-based approaches have inherent limitations. The small number of fluorophores available for antibody conjugation and substrates employed to develop staining by immunohistochemistry limits characterization in a given sample to a select few markers. In this study, we utilized pan markers for CD45R+, CD3+, and CD21/35+ cells indicative of B-, T-, and fDCs. However, this approach does not distinguish between subclasses of lymphocytes or their activation status, thus limiting our conclusions to generalized statements about the effect of cSiO2 and DHA on a given cell population. Nevertheless, the cell populations identified herein are candidates for more thorough characterization in follow-up studies that employ flow cytometry. Conclusions: Histopathology Taken together, dietary DHA attenuated the pathological formation of ELT in the lung and kidney following airway cSiO2 exposure over time. Given that heightened B- and T-cell infiltration in the lung occurred within 1 wk post final exposure to cSiO2, future studies should employ 203 experiments with acute and subacute cSiO2 exposure to explore early targets of DHA that dampen the development of lung ELT and systemic autoimmunity following cSiO2 exposure in NZBWF1 mice (Appendix A). Results: Targeted transcriptomics mRNA expression reveals putative mechanisms for suppressive effect of dietary DHA on cSiO2-triggered inflammation in the lung Having established with histopathology that in our model, cSiO2 induced marked inflammation in the lungs of NZBWF1 mice shortly after the last exposure to cSiO2, we utilized a targeted transcriptome approach to 1) identify patterns of gene expression that may predicate and/or delineate mechanisms of cSiO2-triggered systemic autoimmunity in NZBWF1 mice and 2) determine the primary effect of DHA attributable to its suppressive effect on cSiO2-triggered autoimmunity. For this analysis, we utilized the nCounter Mouse PanCancer Immune Profiling panel, which consists of 770 genes broadly encompassing innate, adaptive, humoral and cellmediated inflammatory pathways. Due to cost constraints, we elected to only use this approach on the lung at 1, 5, 9, and 13 wks post exposure, and the spleen and kidney at 13 wks post exposure to cSiO2. As this is the first time that a multiplexed gene expression analysis has been conducted in cSiO2-treated, lupus-prone mice, we elected to use a less-conservative approach to define statistically significant, differentially expressed genes. Statistically significant, differentially expressed genes in cSiO2-treated mice fed either CON or DHA-enriched diets were defined as those with expression levels corresponding to log2 fold change > ± 1.2 and BH p-value < 0.05, relative to VEH-treated mice fed CON diet. Log2(FC), 95% confidence interval (CI), and BH pvalue are summarized in tabular form for each time point within a given tissue and sorted by log2(FC) according to cSiO2-treated mice fed CON diet. To identify gene interactions present 204 between the mRNA transcripts identified within a given tissue at a given time point, the genes upregulated by cSiO2 were input into String database to identify physical interactions within this identified set. mRNA transcripts were manually binned into categories according to similar function and colored as indicated in the networks that were output from the String database. DHA suppresses cSiO2-triggered mRNA gene expression in lung at 1 wk post final exposure At 1 wk post final exposure in the lung, 48 genes were upregulated in cSiO2 treated NZBWF1 mice fed CON diet, 5 genes were upregulated in cSiO2 fed 0.4% DHA, and remarkably, 0 genes were upregulated in cSiO2 fed 1.0% DHA (Fig. 39, Fig. 40). In general, at 1 wk post final exposure, mRNA transcripts that were significantly upregulated in cSiO2-treated mice fed CON diet fell predominately into chemokine and IFN categories, with fewer members being identified that were classified into cytokines, including the IL-1 superfamily, lymphocyte activation, and macrophage functions. Expectedly, chemokines were strongly upregulated by cSiO2 exposure in the lung in mice fed CON diet, and to a lesser extent in the mice fed DHA. This is consistent with our model of leukocyte infiltration occurring in the lungs following cSiO2 exposure and suppression of this response by dietary DHA. To determine the predominate effect of DHA, BH p-values and 95% CI were considered. Since all data are expressed relative to VEH treated mice fed CON diet, it is valuable to identify what genes in the cSiO2-treated mice fed DHA have 1) CI that include or are close to zero and 2) have BH p-values > 0.05. In this regard, this indicates genes that are not statistically different from VEH treated mice fed CON diet. With this criteria, mRNA transcripts classified as IFN genes were those predominately identified as matching these characteristics (Table 17). This indicates that one of the principal mechanisms through which DHA could be mediating it suppressive effects on lung inflammation following cSiO2 exposure is by decreasing IFN production and IFN stimulated 205 Figure 39. Network visualization of immune genes impacted by cSiO 2 identified 1 wk post final exposure in lung of cSiO2-exposed NZBWF1 mice fed CON diet. Nodes with similar function were colored as depicted. Disconnected nodes were removed for ease of visualization. 206 Figure 40. Volcano plots depicting immune-related mRNA gene expression data at 1 wk post final exposure in lung of cSiO 2treated NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for individual genes within each treatment group as log2(fold change) relative to VEH + CON mice and corresponding BH p-value for that gene. Vertical lines indicate log2(fold change) = 1.2; horizontal line indicates BH p-value = 0.05. (A) indicates cSiO2 + CON relative to VEH + CON treated mice; (B) indicates effect of DHA on cSiO2-induced gene expression expressed relative to VEH + CON treated mice. 207 Table 17. mRNA signatures in lung at 1 wk post final exposure in cSiO2-exposed NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log 2(fold change) relative to VEH + CON mice. Data are included for all treatment groups for those genes which were log2(fold change) ± > 1.2 and BH p-value < 0.05 in cSiO2 + CON treated NZBWF1 mice. Table 17 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl5 3.33 [2.36 , 4.31] 1.4E-04 1.06 [0.74 , 1.37] 8.1E-05 0.49 [0.35 , 0.63] 6.8E-05 Cxcl3 2.91 [2.12 , 3.69] 7.8E-05 1.31 [0.94 , 1.69] 5.7E-05 0.46 [0.32 , 0.61] 2.0E-04 Lcn2 2.78 [2.36 , 3.21] 8.1E-07 1.55 [1.36 , 1.74] 2.0E-08 0.60 [0.53 , 0.67] 1.8E-08 Chil3 2.69 [1.70 , 3.69] 6.4E-04 1.19 [0.53 , 1.84] 8.2E-03 0.49 [0.32 , 0.67] 4.9E-04 Ctla4 2.43 [2.08 , 2.78] 6.1E-07 1.18 [0.94 , 1.43] 2.7E-06 0.41 [0.33 , 0.48] 1.9E-06 Mmp9 2.14 [1.51 , 2.77] 1.4E-04 1.04 [0.70 , 1.38] 1.6E-04 0.45 [0.32 , 0.58] 6.9E-05 Pdcd1 2.14 [1.47 , 2.82] 2.0E-04 1.19 [0.84 , 1.54] 7.1E-05 0.39 [0.25 , 0.53] 5.7E-04 Fcer2a 2.03 [1.60 , 2.46] 9.4E-06 1.23 [0.98 , 1.47] 2.0E-06 0.40 [0.33 , 0.47] 1.2E-06 Marco 2.02 [1.47 , 2.57] 8.0E-05 1.25 [1.01 , 1.49] 1.2E-06 0.50 [0.41 , 0.58] 8.4E-07 Foxp3 1.93 [1.60 , 2.25] 1.7E-06 1.06 [0.88 , 1.23] 2.8E-07 0.36 [0.29 , 0.44] 4.3E-06 Il12b 1.93 [1.42 , 2.44] 6.5E-05 1.05 [0.81 , 1.29] 6.5E-06 0.35 [0.25 , 0.45] 6.9E-05 Irf7 1.85 [1.15 , 2.55] 7.4E-04 0.78 [0.33 , 1.22] 1.0E-02 0.08 [0.01 , 0.14] 6.5E-02 208 Table 17 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cfb 1.79 [1.55 , 2.04] 6.1E-07 0.76 [0.66 , 0.86] 3.2E-08 0.31 [0.28 , 0.34] 3.9E-09 Ccl8 1.79 [1.13 , 2.45] 6.1E-04 0.90 [0.52 , 1.27] 1.3E-03 0.25 [0.13 , 0.38] 6.1E-03 Slc11a1 1.78 [1.11 , 2.45] 7.0E-04 1.28 [1.11 , 1.44] 3.5E-08 0.28 [0.22 , 0.33] 2.0E-06 Il1rn 1.77 [1.48 , 2.06] 1.7E-06 0.96 [0.84 , 1.09] 3.2E-08 0.32 [0.28 , 0.37] 1.6E-07 Ccl3 1.74 [1.19 , 2.29] 2.0E-04 1.01 [0.74 , 1.29] 3.6E-05 0.38 [0.28 , 0.47] 3.5E-05 Il1r2 1.65 [1.24 , 2.07] 4.7E-05 0.77 [0.56 , 0.99] 4.7E-05 0.32 [0.23 , 0.41] 8.7E-05 Birc5 1.60 [1.08 , 2.12] 2.5E-04 0.89 [0.65 , 1.13] 3.5E-05 0.29 [0.20 , 0.39] 2.1E-04 Clec5a 1.57 [1.28 , 1.85] 2.8E-06 0.94 [0.79 , 1.09] 2.2E-07 0.28 [0.24 , 0.33] 6.4E-07 Trem2 1.55 [1.24 , 1.85] 4.6E-06 0.87 [0.70 , 1.03] 1.2E-06 0.30 [0.27 , 0.34] 1.8E-08 C3ar1 1.54 [1.26 , 1.82] 2.8E-06 0.76 [0.59 , 0.92] 4.1E-06 0.24 [0.19 , 0.28] 2.0E-06 Mx1 1.54 [0.98 , 2.09] 5.7E-04 0.67 [0.29 , 1.04] 8.9E-03 0.28 [0.19 , 0.36] 1.8E-04 Tigit 1.52 [0.96 , 2.09] 6.5E-04 0.69 [0.36 , 1.01] 2.9E-03 0.26 [0.15 , 0.37] 1.6E-03 Cxcl2 1.52 [0.83 , 2.21] 2.6E-03 0.68 [0.34 , 1.01] 3.9E-03 0.28 [0.14 , 0.42] 6.1E-03 Msr1 1.46 [1.01 , 1.91] 1.7E-04 0.85 [0.60 , 1.10] 6.3E-05 0.28 [0.20 , 0.37] 1.1E-04 Oas2 1.42 [0.97 , 1.87] 2.1E-04 0.35 [0.04 , 0.66] 7.3E-02 0.01 [-0.04 , 0.06] 7.9E-01 Oasl1 1.40 [0.73 , 2.08] 4.2E-03 0.68 [0.36 , 0.99] 2.8E-03 0.04 [-0.04 , 0.13] 4.8E-01 Ccl9 1.39 [1.06 , 1.72] 3.1E-05 0.82 [0.70 , 0.95] 1.4E-07 0.27 [0.22 , 0.32] 1.9E-06 Cxcr1 1.37 [1.02 , 1.73] 6.1E-05 0.64 [0.46 , 0.82] 5.1E-05 0.24 [0.17 , 0.30] 8.6E-05 209 Table 17 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value C1qa 1.35 [1.10 , 1.61] 3.6E-06 0.70 [0.59 , 0.82] 2.9E-07 0.23 [0.20 , 0.27] 2.0E-07 Tnfrsf9 1.35 [0.63 , 2.08] 7.6E-03 0.70 [0.35 , 1.04] 3.8E-03 0.32 [0.18 , 0.46] 2.1E-03 Mx2 1.34 [0.80 , 1.88] 1.1E-03 0.50 [0.21 , 0.79] 1.1E-02 0.06 [0.00 , 0.13] 1.4E-01 Isg15 1.34 [0.75 , 1.93] 2.1E-03 0.47 [0.05 , 0.89] 7.2E-02 0.08 [0.04 , 0.13] 8.6E-03 Csf2 1.32 [1.05 , 1.59] 7.5E-06 0.66 [0.50 , 0.81] 8.3E-06 0.32 [0.26 , 0.38] 1.7E-06 Siglec1 1.32 [0.85 , 1.79] 4.8E-04 0.76 [0.54 , 0.99] 7.0E-05 0.08 [0.01 , 0.16] 8.6E-02 Ccl6 1.29 [1.02 , 1.55] 8.0E-06 0.78 [0.67 , 0.90] 1.1E-07 0.27 [0.23 , 0.31] 9.7E-08 Il23r 1.29 [0.91 , 1.67] 1.4E-04 0.73 [0.55 , 0.91] 1.7E-05 n.d n.d n.d C1qb 1.28 [1.04 , 1.53] 4.4E-06 0.64 [0.51 , 0.76] 1.7E-06 0.19 [0.16 , 0.22] 6.4E-07 Ifi44 1.26 [0.72 , 1.81] 1.8E-03 0.41 [0.07 , 0.74] 5.4E-02 -0.01 [-0.07 , 0.05] 7.9E-01 Ccl7 1.25 [0.68 , 1.81] 2.7E-03 0.92 [0.66 , 1.19] 5.6E-05 0.07 [-0.05 , 0.19] 3.8E-01 Slc7a11 1.24 [0.77 , 1.72] 7.5E-04 0.48 [0.26 , 0.70] 2.6E-03 0.19 [0.10 , 0.28] 3.4E-03 Ccl4 1.21 [0.66 , 1.77] 2.6E-03 0.72 [0.43 , 1.00] 8.4E-04 0.25 [0.17 , 0.34] 4.4E-04 Ifit1 1.20 [0.70 , 1.70] 1.5E-03 0.44 [0.18 , 0.71] 1.2E-02 0.05 [-0.01 , 0.10] 1.9E-01 Tnfsf11 1.20 [0.60 , 1.80] 5.1E-03 0.68 [0.37 , 0.98] 2.0E-03 0.25 [0.15 , 0.35] 1.5E-03 210 genes. Importantly, this occurred in a concentration dependent manner, whereby the highest concentration of DHA had a more suppressive effect than the lower concentration of DHA employed in this study. DHA suppresses cSiO2-triggered mRNA gene expression in lung at 5 wk post final exposure This trend was similarly evident at 5 wk post final exposure in the lung. In total, 90 genes were upregulated and 2 genes were downregulated in cSiO2 treated mice fed CON diet, 7 geneswere upregulated in cSiO2 treated mice fed 0.4% DHA, and 0 genes were upregulated in cSiO2 treated NZBWF1 mice fed 1.0% DHA (Fig. 41, Fig. 42). Chemokines and IFN remained as distinct populations of mRNA transcripts that were upregulated by cSiO2 exposure at this time and to level of expression greater than that observed at 1 wk post final exposure (Table 18). Importantly, mRNA transcripts classified as being indicative of macrophage function, cytokines, including those in the IL-1 superfamily, were increased in number. Both the observed increase in expression level and the increase in mRNA transcripts support a role for the amplification of inflammation occurring over time following cSiO2 exposure. However, emergence of mRNA signatures that were not present at 1wk post final exposure but were evident at 5 wk post final exposure to cSiO2 are notable. This time point marks the appearance of two additional families of mRNA transcripts that were not present at 1 wk post final exposure to cSiO2: lymphocyte activation, including antigen processing, and immunoglobulin receptors. Of critical relevance to the formation of ELT in the lungs, the TNF superfamily cytokine, lta, was detected at this time point. Taken together, 5 wks post final exposure to cSiO2 may mark the initiation of the pathological sequalae of events that ultimately lead to the formation of ELT in the lungs following cSiO2 exposure. Interestingly, here the predominate effect of DHA could once again be attributed to suppression of IFN, and also antigen processing and lymphocyte activation. 211 Figure 41. Network visualization of immune genes impacted by cSiO 2 identified 5 wk post final exposure in lung of cSiO2-exposed NZBWF1 mice fed CON diet. Nodes with similar function were colored as depicted. Disconnected nodes were removed for ease of visualization. 212 Figure 42. Volcano plots depicting immune-related mRNA gene expression data at 5 wk post final exposure in lung of cSiO2treated NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for individual genes within each treatment group as log2(fold change) relative to VEH + CON mice and corresponding BH p-value for that gene. Vertical lines indicate log2(fold change) = 1.2; horizontal line indicates BH p-value = 0.05. (A) indicates cSiO2 + CON relative to VEH + CON treated mice; (B) indicates effect of DHA on cSiO2-induced gene expression expressed relative to VEH + CON treated mice. 213 Table 18. mRNA signatures in lung at 5 wk post final exposure in cSiO 2-exposed NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log 2(fold change) relative to VEH + CON mice. Data are included for all treatment groups for those genes which were log2(fold change) ± > 1.2 and BH p-value < 0.05 in cSiO2 + CON treated NZBWF1 mice. Table 18 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 5 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl5 4.76 [4.23 , 5.30] 8.1E-08 2.06 [1.66 , 2.47] 3.0E-06 0.87 [0.77 , 0.97] 2.7E-08 Cxcl3 4.03 [3.50 , 4.56] 1.3E-07 1.63 [1.35 , 1.90] 8.3E-07 0.62 [0.52 , 0.72] 7.7E-07 Lcn2 3.49 [3.04 , 3.95] 1.3E-07 1.70 [1.53 , 1.88] 2.5E-08 0.67 [0.60 , 0.74] 2.4E-08 Ccl3 3.40 [2.99 , 3.80] 9.0E-08 1.62 [1.47 , 1.77] 1.3E-08 0.69 [0.64 , 0.75] 1.6E-09 Ccl8 2.99 [2.21 , 3.77] 3.0E-05 1.27 [0.89 , 1.65] 1.2E-04 0.33 [0.19 , 0.48] 1.7E-03 Ccl2 2.92 [2.22 , 3.62] 1.5E-05 1.04 [0.77 , 1.31] 3.0E-05 0.41 [0.32 , 0.50] 6.6E-06 Cxcl2 2.89 [2.46 , 3.32] 4.6E-07 1.46 [1.29 , 1.63] 6.0E-08 0.53 [0.45 , 0.60] 2.6E-07 Il1rn 2.88 [2.28 , 3.48] 4.9E-06 1.24 [0.96 , 1.53] 1.2E-05 0.48 [0.38 , 0.59] 7.1E-06 Cxcl10 2.85 [1.80 , 3.91] 5.0E-04 0.63 [0.23 , 1.04] 2.2E-02 0.20 [0.05 , 0.34] 3.6E-02 Cxcl1 2.71 [2.21 , 3.20] 1.9E-06 0.97 [0.62 , 1.31] 4.9E-04 0.44 [0.36 , 0.52] 9.5E-07 Cxcl9 2.70 [1.59 , 3.82] 1.1E-03 0.71 [0.24 , 1.17] 2.4E-02 0.17 [-0.01 , 0.34] 1.5E-01 Fcer2a 2.65 [2.15 , 3.15] 2.4E-06 1.19 [1.00 , 1.38] 5.6E-07 0.44 [0.37 , 0.52] 9.1E-07 214 Table 18 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 5 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Clec5a 2.49 [2.09 , 2.90] 9.3E-07 1.11 [0.93 , 1.30] 7.8E-07 0.47 [0.40 , 0.55] 3.4E-07 Msr1 2.36 [1.85 , 2.86] 6.1E-06 0.98 [0.77 , 1.19] 6.9E-06 0.40 [0.33 , 0.47] 1.1E-06 Ccl4 2.34 [1.69 , 2.99] 5.4E-05 n.d [n.d , n.d] n.d 0.40 [0.32 , 0.48] 3.2E-06 Pdcd1 2.33 [1.86 , 2.80] 4.6E-06 1.11 [0.85 , 1.37] 1.3E-05 0.42 [0.32 , 0.52] 1.4E-05 Mx1 2.25 [1.60 , 2.91] 7.7E-05 0.56 [0.40 , 0.72] 6.5E-05 0.16 [0.11 , 0.22] 3.1E-04 Marco 2.24 [1.76 , 2.71] 5.7E-06 1.24 [1.06 , 1.42] 3.5E-07 0.56 [0.48 , 0.64] 2.6E-07 Ccl7 2.24 [1.45 , 3.02] 3.4E-04 0.57 [0.28 , 0.87] 6.2E-03 0.22 [0.12 , 0.31] 2.9E-03 C3ar1 2.23 [1.74 , 2.73] 7.8E-06 0.85 [0.72 , 0.98] 4.0E-07 0.35 [0.32 , 0.38] 7.6E-09 Spink5 2.20 [1.85 , 2.55] 8.4E-07 1.07 [0.89 , 1.25] 8.3E-07 0.40 [0.33 , 0.47] 9.4E-07 Tnfrsf9 2.16 [1.65 , 2.66] 1.2E-05 1.01 [0.79 , 1.24] 9.3E-06 0.35 [0.25 , 0.45] 7.5E-05 Irf7 2.16 [1.33 , 3.00] 7.1E-04 0.40 [0.19 , 0.60] 6.7E-03 0.05 [-0.01 , 0.11] 1.9E-01 Cfb 2.15 [1.80 , 2.50] 9.6E-07 0.80 [0.68 , 0.93] 5.0E-07 0.28 [0.22 , 0.33] 1.9E-06 Clec4n 2.11 [1.78 , 2.45] 8.4E-07 0.90 [0.75 , 1.06] 1.1E-06 0.36 [0.29 , 0.42] 1.3E-06 Ctla4 2.10 [1.74 , 2.47] 1.3E-06 0.88 [0.72 , 1.05] 2.2E-06 0.37 [0.30 , 0.44] 1.9E-06 Ifi44 2.06 [1.38 , 2.74] 2.3E-04 0.28 [0.09 , 0.47] 3.0E-02 -0.03 [-0.08 , 0.03] 4.6E-01 Oas2 2.04 [1.46 , 2.62] 6.3E-05 0.25 [0.08 , 0.43] 3.3E-02 0.00 [-0.07 , 0.06] 9.0E-01 Oasl1 2.02 [1.20 , 2.83] 1.0E-03 0.47 [0.18 , 0.76] 1.7E-02 0.03 [-0.06 , 0.12] 6.6E-01 Chil3 2.01 [1.21 , 2.81] 8.7E-04 1.49 [1.11 , 1.87] 2.8E-05 0.79 [0.62 , 0.97] 6.6E-06 215 Table 18 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 5 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il12b 2.00 [1.48 , 2.52] 3.1E-05 0.94 [0.72 , 1.17] 1.7E-05 0.35 [0.26 , 0.43] 1.9E-05 Ccl9 1.98 [1.68 , 2.28] 6.5E-07 0.91 [0.75 , 1.07] 1.1E-06 0.32 [0.27 , 0.38] 1.0E-06 Tnfsf11 1.98 [1.55 , 2.40] 6.7E-06 0.79 [0.64 , 0.93] 1.9E-06 0.29 [0.24 , 0.34] 9.4E-07 Ccl12 1.97 [1.28 , 2.65] 3.2E-04 0.39 [0.10 , 0.69] 4.3E-02 0.18 [0.08 , 0.28] 8.3E-03 Mmp9 1.96 [1.71 , 2.22] 1.3E-07 0.98 [0.86 , 1.10] 6.4E-08 0.42 [0.38 , 0.46] 7.6E-09 Ambp 1.95 [0.58 , 3.32] 2.6E-02 0.64 [-0.04 , 1.31] 1.5E-01 0.29 [0.03 , 0.55] 8.6E-02 Trem2 1.93 [1.61 , 2.25] 1.0E-06 0.98 [0.87 , 1.10] 6.0E-08 0.46 [0.41 , 0.51] 2.0E-08 Csf2 1.92 [1.57 , 2.26] 1.7E-06 0.81 [0.61 , 1.01] 2.0E-05 0.38 [0.32 , 0.44] 8.4E-07 Zbp1 1.85 [1.21 , 2.49] 3.1E-04 0.25 [0.08 , 0.42] 2.9E-02 0.00 [-0.05 , 0.05] 9.6E-01 Spp1 1.83 [1.40 , 2.27] 1.3E-05 0.86 [0.63 , 1.08] 4.2E-05 0.37 [0.29 , 0.45] 7.0E-06 Clec7a 1.83 [1.23 , 2.43] 2.1E-04 0.72 [0.41 , 1.02] 2.0E-03 0.29 [0.17 , 0.42] 1.4E-03 Isg15 1.78 [1.06 , 2.50] 9.5E-04 0.31 [0.14 , 0.48] 8.0E-03 0.04 [-0.01 , 0.08] 1.9E-01 Foxp3 1.77 [1.34 , 2.20] 1.6E-05 0.80 [0.61 , 0.98] 1.2E-05 0.33 [0.25 , 0.41] 2.0E-05 Ccl6 1.77 [1.32 , 2.23] 2.7E-05 0.97 [0.73 , 1.21] 2.3E-05 0.41 [0.32 , 0.50] 7.7E-06 Ccl20 1.73 [0.69 , 2.77] 1.2E-02 0.39 [-0.13 , 0.91] 2.5E-01 0.01 [-0.19 , 0.21] 9.5E-01 Tigit 1.71 [1.25 , 2.16] 3.6E-05 0.60 [0.40 , 0.80] 2.7E-04 0.19 [0.12 , 0.25] 2.5E-04 Oas3 1.71 [0.97 , 2.45] 1.6E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d C1qa 1.69 [1.37 , 2.01] 2.4E-06 0.71 [0.62 , 0.80] 1.8E-07 0.23 [0.19 , 0.26] 4.9E-07 216 Table 18 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 5 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value S100a8 1.65 [1.29 , 2.01] 7.1E-06 0.68 [0.50 , 0.86] 4.3E-05 0.30 [0.23 , 0.38] 1.5E-05 Ccr6 1.64 [1.06 , 2.23] 3.5E-04 0.43 [0.27 , 0.59] 7.8E-04 0.15 [0.10 , 0.19] 1.3E-04 Il1a 1.61 [1.33 , 1.90] 1.7E-06 0.59 [0.48 , 0.69] 1.1E-06 0.26 [0.21 , 0.30] 6.8E-07 Osm 1.60 [1.18 , 2.02] 3.4E-05 0.69 [0.54 , 0.84] 6.9E-06 0.23 [0.17 , 0.29] 4.5E-05 Fcgr1 1.59 [1.07 , 2.11] 2.1E-04 0.32 [0.18 , 0.46] 2.1E-03 0.08 [0.03 , 0.13] 1.6E-02 Ifit1 1.59 [1.00 , 2.18] 5.1E-04 0.23 [0.09 , 0.37] 1.5E-02 0.02 [-0.03 , 0.07] 5.6E-01 Birc5 1.58 [1.10 , 2.05] 9.9E-05 0.67 [0.50 , 0.84] 2.7E-05 0.26 [0.20 , 0.33] 2.4E-05 Ctss 1.57 [1.14 , 2.00] 4.2E-05 0.58 [0.40 , 0.76] 1.7E-04 0.24 [0.17 , 0.32] 1.1E-04 C1qb 1.56 [1.24 , 1.88] 4.7E-06 0.59 [0.50 , 0.68] 4.0E-07 0.18 [0.15 , 0.21] 1.4E-06 Ccr1 1.55 [1.23 , 1.87] 4.8E-06 0.77 [0.60 , 0.94] 8.9E-06 0.30 [0.23 , 0.37] 8.5E-06 Usp18 1.52 [0.97 , 2.06] 4.3E-04 0.30 [0.17 , 0.44] 2.8E-03 0.05 [0.00 , 0.09] 8.5E-02 Blnk 1.51 [1.00 , 2.02] 2.5E-04 0.45 [0.32 , 0.58] 1.1E-04 0.19 [0.15 , 0.22] 3.7E-06 Ccl22 1.50 [0.98 , 2.03] 3.2E-04 0.92 [0.70 , 1.14] 2.0E-05 0.34 [0.25 , 0.43] 2.4E-05 Cd14 1.49 [1.20 , 1.78] 3.2E-06 0.76 [0.61 , 0.91] 3.3E-06 0.27 [0.22 , 0.31] 7.7E-07 Fcgr2b 1.47 [1.19 , 1.75] 2.4E-06 0.67 [0.56 , 0.79] 1.1E-06 0.25 [0.20 , 0.29] 1.0E-06 Mx2 1.45 [0.76 , 2.14] 2.9E-03 0.33 [0.14 , 0.52] 1.3E-02 0.03 [-0.04 , 0.10] 5.0E-01 Cfi 1.43 [1.06 , 1.80] 2.8E-05 0.49 [0.29 , 0.70] 1.5E-03 0.17 [0.09 , 0.25] 2.4E-03 Fcgr3 1.40 [0.95 , 1.86] 1.9E-04 0.62 [0.39 , 0.85] 7.4E-04 0.24 [0.15 , 0.33] 7.1E-04 217 Table 18 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 5 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd83 1.39 [1.13 , 1.65] 2.4E-06 0.48 [0.38 , 0.58] 5.5E-06 0.21 [0.18 , 0.23] 3.0E-08 Slc11a1 1.37 [1.09 , 1.66] 4.8E-06 0.67 [0.53 , 0.80] 5.0E-06 0.28 [0.22 , 0.34] 4.5E-06 Tnfrsf4 1.36 [0.97 , 1.74] 6.3E-05 0.61 [0.42 , 0.81] 1.8E-04 0.24 [0.18 , 0.30] 2.6E-05 Ccr5 1.36 [0.85 , 1.87] 5.3E-04 0.41 [0.24 , 0.57] 1.1E-03 0.15 [0.08 , 0.21] 3.1E-03 Arg2 1.35 [1.01 , 1.68] 2.3E-05 0.88 [0.66 , 1.10] 2.6E-05 0.33 [0.26 , 0.40] 6.5E-06 Il23r 1.35 [0.94 , 1.76] 1.1E-04 0.68 [0.48 , 0.87] 7.3E-05 0.22 [0.13 , 0.31] 1.4E-03 Tlr2 1.34 [1.11 , 1.58] 1.7E-06 0.50 [0.38 , 0.63] 1.7E-05 0.23 [0.19 , 0.27] 1.5E-06 Cxcr1 1.34 [1.01 , 1.67] 1.9E-05 0.73 [0.52 , 0.93] 6.8E-05 0.34 [0.29 , 0.39] 3.4E-07 Icos 1.34 [0.99 , 1.70] 3.6E-05 0.37 [0.25 , 0.50] 3.2E-04 0.15 [0.10 , 0.19] 2.5E-04 H2-Q2 1.34 [0.86 , 1.83] 4.3E-04 0.27 [0.00 , 0.54] 1.3E-01 0.06 [-0.03 , 0.15] 3.0E-01 Emr1 1.33 [0.90 , 1.77] 2.1E-04 0.50 [0.28 , 0.71] 1.9E-03 0.19 [0.11 , 0.27] 2.2E-03 Il1b 1.31 [1.04 , 1.58] 4.8E-06 0.42 [0.26 , 0.58] 9.0E-04 0.20 [0.15 , 0.26] 3.7E-05 Ly86 1.30 [0.97 , 1.63] 2.8E-05 0.35 [0.20 , 0.49] 1.5E-03 0.13 [0.08 , 0.18] 5.4E-04 H2-DMb2 1.29 [0.84 , 1.74] 3.4E-04 0.39 [0.24 , 0.53] 7.4E-04 0.14 [0.09 , 0.18] 5.3E-04 Il1r2 1.27 [0.86 , 1.69] 2.0E-04 0.91 [0.64 , 1.18] 1.1E-04 0.28 [0.20 , 0.37] 8.7E-05 Lta 1.27 [0.83 , 1.71] 3.2E-04 0.41 [0.25 , 0.57] 8.6E-04 0.18 [0.13 , 0.22] 4.5E-05 Cx3cr1 1.25 [0.84 , 1.66] 2.1E-04 0.31 [0.07 , 0.55] 4.9E-02 0.13 [0.05 , 0.21] 1.4E-02 Slamf7 1.25 [0.77 , 1.74] 7.0E-04 0.34 [0.23 , 0.45] 2.5E-04 0.14 [0.10 , 0.18] 2.6E-05 218 Table 18 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 5 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd68 1.24 [1.04 , 1.44] 8.7E-07 0.60 [0.52 , 0.69] 3.5E-07 0.27 [0.24 , 0.30] 5.8E-08 Pou2af1 1.23 [0.63 , 1.82] 3.5E-03 0.23 [0.04 , 0.43] 6.6E-02 0.09 [0.02 , 0.17] 6.0E-02 Cd86 1.21 [0.93 , 1.49] 1.1E-05 0.56 [0.44 , 0.68] 5.9E-06 0.18 [0.14 , 0.23] 1.3E-05 Cd80 1.21 [0.85 , 1.57] 8.8E-05 0.57 [0.43 , 0.70] 1.6E-05 0.20 [0.16 , 0.25] 4.0E-06 Rsad2 1.21 [0.55 , 1.86] 6.6E-03 0.16 [-0.01 , 0.34] 1.5E-01 0.07 [0.02 , 0.12] 3.3E-02 Ncf4 1.20 [0.98 , 1.42] 1.7E-06 0.40 [0.30 , 0.50] 2.0E-05 0.17 [0.14 , 0.20] 1.7E-06 Cdkn1a -1.49 [-1.82 , -1.16] 7.9E-06 -0.27 [-0.67 , 0.13] 2.9E-01 -0.14 [-0.21 , -0.06] 8.6E-03 Thbs1 -1.54 [-1.90 , -1.18] 1.2E-05 -0.79 [-0.94 , -0.64] 2.3E-06 -0.30 [-0.37 , -0.23] 1.0E-05 219 DHA suppresses cSiO2-triggered mRNA gene expression in lung at 9 wk post final exposure At 9 wk post final exposure to cSiO2 in the lung, 106 genes were upregulated and 4 genes were downregulated in cSiO2 treated mice fed CON diet, 13 genes were upregulated in cSiO2treated mice fed 0.4% DHA, and 0 genes were upregulated in cSiO2 treated mice fed 1.0% DHA (Fig. 43, Fig. 44). At this time point, the families representing the mRNA signatures (Table 19) appeared similar to those at 5 wk post final exposure. This indicates that by this time point, the dynamics of the shifting cellular response to cSiO2 have stabilized, and as reflected in the expression data, continue to amplify over time. As consistent in earlier time points, IFN was a predominate effect that mediated both the inflammatory response to cSiO2 and the suppressive effect of DHA (Table 19). There are several additional noteworthy mRNA transcripts present at this time point that were decreased by dietary DHA. Activation-induced cytidine deaminase (aicda) was dramatically induced in mice treated with cSiO2 fed CON diet, but was undetectable in mice that were given cSiO2 and either concentration of DHA. This enzyme is crucial for B cell differentiation, including Ig class switching and somatic hypermutation, which likely crucially contributes to the formation of autoreactive antibodies in the lung following cSiO2 exposure to NZBWF1 mice. Furthermore, two cytokines known to contribute to development of ELT were upregulated at this time point: IL21 and IL-6. These findings are in accordance with histopathological findings that identified 9 wk post final exposure to cSiO2 to mark the emergence of ELT in lungs of cSiO2 treated mice fed CON diet. 220 Figure 43. Network visualization of immune genes impacted by cSiO 2 identified 9 wk post final exposure in lung of cSiO2-exposed NZBWF1 mice fed CON diet. Nodes with similar function were colored as depicted. Disconnected nodes were removed for ease of visualization. 221 Figure 44. Volcano plots depicting immune-related mRNA gene expression data at 9 wk post final exposure in lung of cSiO2treated NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for individual genes within each treatment group as log2(fold change) relative to VEH + CON mice and corresponding BH p-value for that gene. Vertical lines indicate log2(fold change) = 1.2; horizontal line indicates BH p-value = 0.05. (A) indicates cSiO2 + CON relative to VEH + CON treated mice; (B) indicates effect of DHA on cSiO2-induced gene expression expressed relative to VEH + CON treated mice. 222 Table 19. mRNA signatures in lung at 9 wk post final exposure in cSiO 2-exposed NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log 2(fold change) relative to VEH + CON mice. Data are included for all treatment groups for those genes which were log2(fold change) ± > 1.2 and BH p-value < 0.05 in cSiO2 + CON treated NZBWF1 mice. Table 19 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl5 4.87 [4.30 , 5.45] 6.4E-09 2.49 [2.28 , 2.70] 2.8E-10 0.93 [0.84 , 1.03] 3.6E-09 Ccl2 4.07 [3.46 , 4.67] 5.3E-08 2.07 [1.77 , 2.37] 3.8E-08 0.66 [0.53 , 0.78] 8.9E-07 Lcn2 3.82 [3.43 , 4.22] 3.9E-09 2.00 [1.87 , 2.13] 2.5E-11 0.74 [0.69 , 0.79] 9.6E-11 Cxcl3 3.68 [2.93 , 4.42] 1.0E-06 1.87 [1.51 , 2.23] 7.1E-07 0.66 [0.52 , 0.80] 2.9E-06 Cxcl10 3.43 [2.97 , 3.89] 2.1E-08 1.27 [0.89 , 1.65] 5.2E-05 0.23 [0.07 , 0.38] 2.2E-02 Cxcl1 3.40 [3.00 , 3.80] 6.4E-09 1.67 [1.50 , 1.84] 1.9E-09 0.63 [0.55 , 0.71] 1.7E-08 Ccl7 3.37 [2.77 , 3.97] 3.1E-07 1.67 [1.35 , 1.98] 6.3E-07 0.48 [0.35 , 0.61] 3.3E-05 Cxcl9 3.24 [2.52 , 3.95] 2.2E-06 1.10 [0.68 , 1.52] 3.9E-04 0.21 [0.02 , 0.41] 8.2E-02 Ccl3 3.15 [2.77 , 3.54] 8.9E-09 1.63 [1.46 , 1.81] 3.1E-09 0.58 [0.49 , 0.67] 1.9E-07 Ccl8 3.11 [2.39 , 3.83] 3.2E-06 1.39 [0.99 , 1.79] 3.5E-05 0.39 [0.21 , 0.57] 2.2E-03 Il1rn 2.97 [2.63 , 3.31] 6.4E-09 1.50 [1.34 , 1.65] 2.4E-09 0.53 [0.46 , 0.59] 1.7E-08 Oasl1 2.87 [2.36 , 3.38] 3.1E-07 1.10 [0.69 , 1.50] 3.3E-04 0.12 [-0.03 , 0.28] 2.0E-01 223 Table 19 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value C3ar1 2.83 [2.48 , 3.19] 1.2E-08 1.39 [1.18 , 1.60] 5.5E-08 0.41 [0.32 , 0.50] 4.2E-06 Msr1 2.72 [2.31 , 3.13] 6.3E-08 1.40 [1.22 , 1.59] 1.4E-08 0.46 [0.38 , 0.54] 3.1E-07 Irf7 2.63 [2.04 , 3.22] 2.5E-06 1.07 [0.59 , 1.55] 1.5E-03 0.11 [-0.06 , 0.27] 2.9E-01 Pdcd1 2.61 [2.15 , 3.08] 3.2E-07 1.20 [0.95 , 1.44] 1.3E-06 0.37 [0.25 , 0.48] 9.2E-05 Ccl12 2.55 [2.16 , 2.94] 6.7E-08 0.97 [0.71 , 1.24] 2.1E-05 0.32 [0.22 , 0.41] 8.8E-05 Isg15 2.54 [2.10 , 2.98] 2.5E-07 0.99 [0.59 , 1.38] 6.0E-04 0.12 [-0.01 , 0.26] 1.3E-01 Ccl4 2.54 [2.08 , 3.01] 3.6E-07 0.98 [0.65 , 1.30] 1.2E-04 0.26 [0.12 , 0.40] 6.0E-03 Trem2 2.47 [2.26 , 2.68] 9.7E-10 1.34 [1.22 , 1.47] 7.0E-10 0.47 [0.42 , 0.52] 3.6E-09 Ifi44 2.46 [1.99 , 2.93] 6.1E-07 0.93 [0.51 , 1.34] 1.5E-03 0.08 [-0.07 , 0.23] 4.0E-01 Fcer2a 2.46 [1.88 , 3.03] 3.9E-06 1.21 [0.98 , 1.43] 5.8E-07 0.43 [0.35 , 0.52] 1.6E-06 Clec5a 2.42 [2.15 , 2.68] 5.2E-09 1.28 [1.16 , 1.39] 6.4E-10 0.47 [0.42 , 0.53] 1.2E-08 Cfb 2.42 [2.05 , 2.80] 7.9E-08 1.12 [0.89 , 1.36] 1.7E-06 0.35 [0.28 , 0.43] 2.4E-06 Oas2 2.41 [1.89 , 2.93] 1.8E-06 0.96 [0.63 , 1.29] 1.7E-04 0.11 [-0.02 , 0.23] 1.6E-01 Zbp1 2.37 [1.99 , 2.75] 1.2E-07 0.89 [0.53 , 1.24] 6.0E-04 0.14 [0.02 , 0.27] 7.1E-02 Cxcl2 2.37 [1.97 , 2.78] 2.2E-07 1.12 [0.89 , 1.34] 1.0E-06 0.40 [0.32 , 0.47] 5.0E-07 Mx1 2.24 [1.80 , 2.67] 6.8E-07 0.82 [0.46 , 1.18] 1.2E-03 0.14 [0.02 , 0.26] 5.6E-02 Cxcl13 2.24 [1.57 , 2.92] 3.6E-05 0.52 [0.07 , 0.98] 5.9E-02 0.14 [-0.07 , 0.34] 2.9E-01 Tnfrsf9 2.20 [1.81 , 2.58] 2.7E-07 1.15 [1.00 , 1.30] 1.3E-08 0.36 [0.31 , 0.40] 3.7E-08 224 Table 19 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Spp1 2.19 [1.94 , 2.45] 6.4E-09 1.19 [1.05 , 1.33] 4.6E-09 0.41 [0.36 , 0.47] 3.7E-08 Spink5 2.16 [1.68 , 2.64] 2.2E-06 n.d n.d n.d n.d n.d n.d Ccr5 2.13 [1.82 , 2.44] 4.5E-08 0.83 [0.64 , 1.01] 3.5E-06 0.22 [0.15 , 0.30] 2.4E-04 Oas3 2.12 [1.46 , 2.79] 5.1E-05 0.83 [0.44 , 1.22] 1.9E-03 0.08 [-0.07 , 0.23] 4.0E-01 Il12b 2.10 [1.65 , 2.56] 1.8E-06 0.99 [0.77 , 1.21] 3.3E-06 0.37 [0.28 , 0.46] 9.9E-06 C1qa 2.07 [1.79 , 2.34] 1.9E-08 0.85 [0.71 , 0.98] 1.2E-07 0.25 [0.19 , 0.32] 1.9E-05 Clec4n 2.05 [1.85 , 2.24] 3.3E-09 1.05 [0.96 , 1.13] 2.5E-10 0.37 [0.33 , 0.42] 1.2E-08 Ifit1 2.01 [1.65 , 2.37] 3.2E-07 0.73 [0.39 , 1.07] 1.8E-03 0.08 [-0.03 , 0.19] 2.4E-01 Fcgr1 2.01 [1.65 , 2.37] 3.2E-07 0.76 [0.46 , 1.05] 5.0E-04 0.15 [0.06 , 0.24] 1.2E-02 C1qb 1.99 [1.69 , 2.29] 6.2E-08 0.78 [0.64 , 0.92] 3.1E-07 0.24 [0.17 , 0.31] 6.6E-05 Mx2 1.92 [1.56 , 2.28] 4.6E-07 0.76 [0.45 , 1.06] 6.4E-04 -0.02 [-0.17 , 0.14] 8.7E-01 Marco 1.85 [1.38 , 2.32] 8.2E-06 1.14 [0.88 , 1.40] 4.5E-06 0.57 [0.49 , 0.65] 6.7E-08 Fcgr2b 1.84 [1.57 , 2.11] 5.2E-08 0.98 [0.88 , 1.09] 3.1E-09 0.32 [0.28 , 0.37] 1.0E-07 Tigit 1.83 [1.32 , 2.34] 1.7E-05 0.84 [0.55 , 1.12] 1.8E-04 0.19 [0.07 , 0.31] 1.6E-02 Ccl9 1.77 [1.56 , 1.98] 6.4E-09 0.88 [0.77 , 0.99] 7.9E-09 0.30 [0.25 , 0.35] 2.7E-07 Csf2 1.77 [1.48 , 2.07] 1.5E-07 0.94 [0.81 , 1.06] 1.4E-08 0.36 [0.32 , 0.40] 9.5E-09 Il21 1.72 [1.03 , 2.42] 4.8E-04 n.d n.d n.d n.d n.d n.d Il1a 1.71 [1.49 , 1.93] 1.5E-08 0.89 [0.78 , 1.00] 1.1E-08 0.31 [0.26 , 0.35] 7.0E-08 225 Table 19 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfsf11 1.70 [1.40 , 2.01] 3.3E-07 0.87 [0.76 , 0.97] 8.7E-09 0.26 [0.20 , 0.32] 4.1E-06 Foxp3 1.70 [1.35 , 2.06] 1.4E-06 0.98 [0.78 , 1.19] 1.6E-06 0.27 [0.20 , 0.34] 2.3E-05 Slamf7 1.70 [1.31 , 2.09] 3.1E-06 0.74 [0.49 , 0.99] 1.6E-04 0.17 [0.07 , 0.28] 1.3E-02 Ctss 1.67 [1.46 , 1.89] 1.4E-08 0.80 [0.65 , 0.96] 1.0E-06 0.24 [0.17 , 0.30] 3.1E-05 Cd14 1.67 [1.38 , 1.97] 3.0E-07 0.84 [0.74 , 0.95] 7.9E-09 0.28 [0.23 , 0.34] 5.2E-07 Siglec1 1.67 [1.27 , 2.07] 4.5E-06 0.83 [0.63 , 1.03] 6.8E-06 0.19 [0.14 , 0.23] 9.7E-06 Aicda 1.66 [0.81 , 2.52] 3.1E-03 n.d n.d n.d n.d n.d n.d Cd68 1.65 [1.48 , 1.82] 3.9E-09 0.91 [0.82 , 1.00] 1.3E-09 0.31 [0.27 , 0.35] 1.3E-08 Slc11a1 1.62 [1.41 , 1.83] 1.5E-08 0.74 [0.60 , 0.88] 6.2E-07 0.25 [0.19 , 0.31] 1.2E-05 Ccl6 1.62 [1.39 , 1.84] 3.4E-08 0.94 [0.81 , 1.07] 2.6E-08 0.35 [0.32 , 0.39] 3.6E-09 Cxcr1 1.62 [1.18 , 2.06] 1.5E-05 1.00 [0.79 , 1.21] 1.8E-06 0.39 [0.30 , 0.47] 3.9E-06 Fcgr4 1.61 [1.36 , 1.86] 7.6E-08 0.37 [0.28 , 0.46] 1.0E-05 0.15 [0.07 , 0.22] 3.7E-03 Gzmk 1.61 [1.10 , 2.12] 6.3E-05 0.42 [0.10 , 0.74] 3.5E-02 0.09 [-0.06 , 0.24] 3.2E-01 Birc5 1.61 [1.08 , 2.14] 7.9E-05 0.64 [0.37 , 0.92] 9.9E-04 0.19 [0.07 , 0.30] 1.4E-02 Usp18 1.59 [1.19 , 1.99] 6.5E-06 0.61 [0.30 , 0.92] 3.5E-03 0.04 [-0.07 , 0.14] 5.9E-01 Ifit3 1.58 [1.27 , 1.89] 7.2E-07 0.62 [0.33 , 0.90] 2.0E-03 0.07 [-0.01 , 0.16] 1.4E-01 Tlr2 1.51 [1.30 , 1.72] 3.2E-08 0.73 [0.60 , 0.86] 3.2E-07 0.25 [0.20 , 0.29] 3.7E-07 Arg2 1.51 [1.23 , 1.80] 5.0E-07 0.82 [0.72 , 0.92] 8.0E-09 0.35 [0.29 , 0.41] 2.3E-07 226 Table 19 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Pou2af1 1.50 [1.13 , 1.88] 5.9E-06 0.48 [0.27 , 0.70] 1.4E-03 0.13 [0.03 , 0.23] 4.4E-02 Ccl20 1.50 [0.49 , 2.52] 1.7E-02 0.39 [-0.09 , 0.87] 1.7E-01 0.08 [-0.12 , 0.28] 5.4E-01 Blnk 1.49 [1.13 , 1.85] 4.7E-06 0.59 [0.39 , 0.80] 1.6E-04 0.15 [0.07 , 0.24] 9.5E-03 Plau 1.48 [1.27 , 1.69] 3.7E-08 0.79 [0.68 , 0.91] 4.5E-08 0.25 [0.20 , 0.30] 1.3E-06 Ccr1 1.44 [1.15 , 1.74] 1.2E-06 0.83 [0.71 , 0.95] 2.9E-08 0.28 [0.23 , 0.32] 4.5E-07 Il6 1.43 [0.78 , 2.08] 1.2E-03 0.72 [0.39 , 1.05] 1.6E-03 0.13 [0.00 , 0.26] 1.2E-01 Ccr6 1.42 [0.88 , 1.97] 3.3E-04 0.48 [0.22 , 0.74] 4.7E-03 0.11 [0.00 , 0.22] 9.7E-02 Irf4 1.40 [0.97 , 1.82] 4.2E-05 0.57 [0.32 , 0.83] 1.3E-03 0.12 [0.01 , 0.22] 8.6E-02 Tnfrsf17 1.40 [0.89 , 1.92] 2.2E-04 0.67 [0.42 , 0.93] 3.9E-04 n.d n.d n.d Ctla4 1.39 [0.96 , 1.81] 4.2E-05 0.80 [0.59 , 1.01] 1.4E-05 0.21 [0.13 , 0.28] 2.8E-04 Cd200r1 1.38 [1.22 , 1.54] 6.4E-09 0.76 [0.68 , 0.84] 2.4E-09 0.28 [0.24 , 0.31] 1.7E-08 Cx3cr1 1.38 [1.14 , 1.62] 2.7E-07 0.38 [0.27 , 0.50] 6.1E-05 0.17 [0.09 , 0.24] 1.5E-03 Cfi 1.37 [1.08 , 1.66] 1.5E-06 0.68 [0.51 , 0.85] 9.0E-06 0.25 [0.19 , 0.31] 9.9E-06 Rsad2 1.37 [0.87 , 1.87] 2.1E-04 0.47 [0.13 , 0.82] 2.8E-02 0.03 [-0.09 , 0.14] 7.1E-01 Tnf 1.36 [1.00 , 1.72] 1.1E-05 0.65 [0.47 , 0.83] 3.0E-05 0.23 [0.15 , 0.31] 2.8E-04 Tnfrsf4 1.36 [0.99 , 1.72] 1.5E-05 0.65 [0.48 , 0.82] 1.3E-05 0.20 [0.13 , 0.27] 2.1E-04 Lag3 1.36 [0.93 , 1.80] 6.3E-05 0.50 [0.27 , 0.73] 1.7E-03 0.07 [-0.03 , 0.18] 2.7E-01 Lgals3 1.35 [1.15 , 1.55] 5.3E-08 0.83 [0.73 , 0.93] 4.6E-09 0.29 [0.25 , 0.33] 3.7E-08 227 Table 19 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Fcer1g 1.33 [1.18 , 1.48] 6.4E-09 0.50 [0.40 , 0.60] 1.1E-06 0.19 [0.14 , 0.24] 1.2E-05 Clec7a 1.33 [1.10 , 1.56] 2.2E-07 0.59 [0.48 , 0.70] 4.5E-07 0.21 [0.18 , 0.25] 4.4E-07 Osm 1.33 [0.93 , 1.73] 3.9E-05 0.67 [0.47 , 0.86] 5.4E-05 0.17 [0.10 , 0.24] 8.8E-04 Csf2rb 1.31 [1.13 , 1.50] 3.4E-08 0.68 [0.57 , 0.80] 2.3E-07 0.25 [0.22 , 0.29] 1.6E-08 Itgam 1.30 [1.05 , 1.54] 5.0E-07 0.55 [0.41 , 0.70] 1.3E-05 0.16 [0.10 , 0.21] 4.9E-04 Il2ra 1.30 [0.97 , 1.63] 7.0E-06 0.70 [0.54 , 0.87] 4.7E-06 0.20 [0.15 , 0.26] 3.0E-05 Il21r 1.29 [0.92 , 1.66] 2.6E-05 0.44 [0.21 , 0.68] 4.8E-03 0.09 [0.00 , 0.19] 1.2E-01 Bst2 1.26 [0.98 , 1.54] 2.5E-06 0.53 [0.30 , 0.76] 1.2E-03 0.07 [0.01 , 0.14] 7.6E-02 Slc7a11 1.25 [0.92 , 1.58] 1.1E-05 0.86 [0.70 , 1.03] 6.7E-07 0.35 [0.28 , 0.41] 1.2E-06 Cxcr3 1.25 [0.84 , 1.66] 7.6E-05 0.48 [0.20 , 0.75] 8.4E-03 0.05 [-0.06 , 0.16] 4.6E-01 Emr1 1.22 [1.03 , 1.40] 6.7E-08 0.49 [0.38 , 0.59] 2.5E-06 0.15 [0.10 , 0.20] 1.2E-04 Cd274 1.21 [1.02 , 1.40] 1.0E-07 0.59 [0.47 , 0.72] 2.1E-06 0.20 [0.15 , 0.24] 3.9E-06 Tnfrsf11a 1.21 [1.00 , 1.42] 2.6E-07 0.65 [0.57 , 0.73] 7.9E-09 0.21 [0.16 , 0.25] 2.9E-06 Cd180 1.21 [0.99 , 1.43] 4.0E-07 0.43 [0.27 , 0.58] 3.0E-04 0.11 [0.04 , 0.18] 1.4E-02 Cd83 1.21 [0.97 , 1.46] 9.5E-07 0.56 [0.48 , 0.64] 2.7E-08 0.18 [0.13 , 0.22] 9.5E-06 Il1r2 1.21 [0.77 , 1.66] 2.3E-04 0.66 [0.47 , 0.85] 4.2E-05 0.25 [0.15 , 0.36] 1.0E-03 Ly9 1.20 [0.94 , 1.46] 1.8E-06 0.63 [0.49 , 0.76] 2.0E-06 0.15 [0.08 , 0.21] 1.7E-03 Tap1 1.20 [0.94 , 1.46] 1.9E-06 0.51 [0.33 , 0.70] 3.0E-04 0.10 [0.04 , 0.16] 1.7E-02 228 Table 19 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Icos 1.20 [0.88 , 1.51] 1.0E-05 0.54 [0.34 , 0.74] 3.0E-04 0.12 [0.05 , 0.18] 8.5E-03 Camp -1.20 [0.37 , -1.93] 1.0E-05 -0.41 [-0.70 , -0.12] 2.5E-02 -0.05 [-0.20 , 0.10] 6.0E-01 Ccr3 -1.25 [0.36 , -1.96] 1.0E-05 -0.64 [-0.99 , -0.29] 5.4E-03 -0.18 [-0.32 , -0.04] 4.4E-02 Ppbp -1.40 [0.31 , -2.01] 1.0E-05 -0.64 [-0.89 , -0.39] 5.2E-04 -0.15 [-0.24 , -0.06] 1.4E-02 Hamp -1.51 [0.24 , -1.98] 1.0E-05 -0.65 [-0.90 , -0.39] 6.0E-04 -0.28 [-0.41 , -0.15] 2.1E-03 Cfd -2.53 [0.58 , -3.67] 1.0E-05 -0.74 [-1.40 , -0.09] 6.3E-02 -0.54 [-0.81 , -0.27] 4.2E-03 229 DHA suppresses cSiO2-triggered mRNA gene expression in lung at 13 wk post final exposure At 13 wk post final exposure to cSiO2 in the lung, 99 genes were upregulated and 9 genes were downregulated in cSiO2-treated mice fed CON diet, 22 genes were upregulated in cSiO2treated mice fed 0.4% DHA, and 0 genes were upregulated in cSiO2 treated mice fed 1.0% DHA (Fig. 45, Fig. 46). Overall, the same mRNA signatures present at 9 wk post final exposure were also present at 13 wks post final exposure to cSiO2 in the lung (Table 20). Again, at this time point, there was no alteration in the classification of mRNA transcripts upregulated by cSiO2. Overall, the level of mRNA expression increased at 13 wk compared to 9 wk post final exposure to cSiO2. An important note is that levels of mRNA expression in the cSiO2 treated mice fed DHA increased compared to mice given the same treatment at 9 wks post final exposure. However, this change in expression was comparatively minor relative to that of cSiO2-treated mice fed CON diet (Table 20). Taken together, mice fed 1.0% DHA had global and sustained immunosuppressive effects in the lung throughout the pathogenesis of cSiO2-triggered ELT (Fig 47). DHA suppresses cSiO2-triggered mRNA gene expression in spleen at 13 wk post final exposure In the spleen, cSiO2 induced mRNA signatures that were subtle compared to those observed in the lungs. In total, 23 genes were upregulated and 9 genes were downregulated in cSiO2 treated mice fed CON diet, 2 genes upregulated and 1 gene downregulated in cSiO2 treated mice fed 0.4% DHA, and 0 genes were upregulated in cSiO2 treated mice fed 1.0% DHA (Fig. 48, Fig. 49). In spleen, chemokines were most represented in upregulated genes, and to a lesser extent, IFN and IL-1 superfamily (Table 21). A explanation for the low expression of mRNA in the spleen at this time is that the spleen is a lymphoid organ, and basal expression levels is expectedly high. 230 Figure 45. Network visualization of immune genes impacted by cSiO2 identified 13 wk post final exposure in lung of cSiO2-exposed NZBWF1 mice fed CON diet. Nodes with similar function were colored as depicted. Disconnected nodes were removed for ease of visualization. 231 Figure 46. Volcano plots depicting immune-related mRNA gene expression data at 13 wk post final exposure in lung of cSiO 2treated NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for individual genes within each treatment group as log2(fold change) relative to VEH + CON mice and corresponding BH p-value for that gene. Vertical lines indicate log2(fold change) = 1.2; horizontal line indicates BH p-value = 0.05. (A) indicates cSiO2 + CON relative to VEH + CON treated mice; (B) indicates effect of DHA on cSiO2-induced gene expression expressed relative to VEH + CON treated mice. 232 Table 20. mRNA signatures in lung at 13 wk post final exposure in cSiO 2-exposed NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log2(fold change) relative to VEH + CON mice. Data are included for all treatment groups for those genes which were log2(fold change) ± > 1.2 and BH p-value < 0.05 in cSiO2 + CON treated NZBWF1 mice. Table 20 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl5 4.99 [3.95 , 6.03] 1.4E-05 2.94 [2.60 , 3.27] 5.9E-08 0.97 [0.81 , 1.14] 1.6E-06 Cxcl3 4.46 [3.43 , 5.49] 2.6E-05 2.22 [1.78 , 2.67] 6.1E-06 0.83 [0.67 , 0.99] 4.9E-06 Lcn2 3.99 [3.55 , 4.43] 1.1E-07 2.38 [2.15 , 2.60] 9.5E-09 0.93 [0.84 , 1.01] 1.4E-08 Ccl2 3.87 [3.38 , 4.35] 3.0E-07 2.33 [1.97 , 2.69] 8.1E-07 0.91 [0.79 , 1.03] 2.1E-07 Ccl3 3.46 [2.87 , 4.05] 3.9E-06 2.11 [1.84 , 2.38] 1.8E-07 0.82 [0.70 , 0.95] 6.6E-07 Ccl8 3.41 [2.19 , 4.62] 7.6E-04 1.57 [0.96 , 2.18] 9.9E-04 0.64 [0.40 , 0.87] 7.1E-04 Cxcl13 3.24 [2.11 , 4.37] 6.8E-04 1.20 [0.58 , 1.82] 6.2E-03 0.49 [0.26 , 0.72] 3.6E-03 C3ar1 3.17 [2.47 , 3.88] 2.1E-05 1.57 [1.33 , 1.81] 8.1E-07 0.53 [0.42 , 0.64] 6.6E-06 Ccl7 3.15 [2.58 , 3.73] 4.8E-06 1.87 [1.52 , 2.22] 3.1E-06 0.70 [0.57 , 0.83] 2.8E-06 Cxcl9 3.15 [1.84 , 4.45] 2.1E-03 1.17 [0.62 , 1.72] 3.6E-03 0.40 [0.19 , 0.60] 6.9E-03 Cxcl1 2.89 [2.30 , 3.48] 1.1E-05 1.61 [1.38 , 1.84] 4.9E-07 0.57 [0.47 , 0.67] 1.8E-06 Cxcl10 2.86 [1.88 , 3.84] 6.2E-04 1.39 [0.79 , 2.00] 2.2E-03 0.47 [0.23 , 0.72] 6.6E-03 233 Table 20 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccl12 2.85 [2.19 , 3.52] 2.8E-05 1.46 [1.13 , 1.79] 1.7E-05 0.52 [0.39 , 0.65] 3.0E-05 Ccl4 2.81 [1.82 , 3.79] 7.0E-04 1.58 [1.10 , 2.06] 1.7E-04 0.52 [0.32 , 0.72] 9.4E-04 Pdcd1 2.81 [1.70 , 3.93] 1.5E-03 1.34 [1.00 , 1.68] 4.1E-05 0.46 [0.31 , 0.61] 2.6E-04 C1qb 2.70 [1.96 , 3.45] 1.2E-04 1.06 [0.74 , 1.39] 1.7E-04 0.36 [0.25 , 0.48] 2.0E-04 Trem2 2.56 [2.11 , 3.02] 4.3E-06 1.34 [1.15 , 1.53] 4.6E-07 0.53 [0.45 , 0.61] 6.6E-07 Tigit 2.53 [1.57 , 3.48] 1.1E-03 1.01 [0.68 , 1.35] 3.3E-04 0.36 [0.23 , 0.50] 6.6E-04 Gzmk 2.49 [1.45 , 3.53] 2.1E-03 0.94 [0.45 , 1.43] 6.4E-03 0.29 [0.12 , 0.47] 1.4E-02 Il1rn 2.48 [2.10 , 2.86] 2.2E-06 1.51 [1.24 , 1.78] 2.6E-06 0.59 [0.49 , 0.69] 1.7E-06 Cfb 2.48 [1.99 , 2.96] 8.6E-06 1.32 [1.08 , 1.56] 2.5E-06 0.49 [0.37 , 0.61] 2.9E-05 Ccr5 2.47 [1.60 , 3.35] 7.2E-04 0.97 [0.61 , 1.32] 7.1E-04 0.29 [0.17 , 0.40] 1.5E-03 Spp1 2.46 [1.83 , 3.10] 6.6E-05 1.54 [1.26 , 1.82] 2.8E-06 0.60 [0.51 , 0.69] 5.1E-07 Cxcl2 2.45 [1.53 , 3.37] 1.1E-03 1.54 [1.11 , 1.97] 8.8E-05 0.53 [0.35 , 0.72] 4.6E-04 Fcgr4 2.44 [1.74 , 3.15] 1.8E-04 0.79 [0.33 , 1.25] 1.3E-02 0.22 [0.09 , 0.35] 1.5E-02 C1qa 2.44 [1.63 , 3.25] 5.0E-04 0.98 [0.66 , 1.29] 2.8E-04 0.35 [0.23 , 0.46] 3.4E-04 Oasl1 2.39 [1.43 , 3.35] 1.7E-03 1.14 [0.67 , 1.61] 1.6E-03 0.30 [0.02 , 0.58] 9.4E-02 Tnfrsf17 2.39 [1.38 , 3.41] 2.3E-03 0.98 [0.46 , 1.50] 7.4E-03 0.33 [0.20 , 0.47] 1.5E-03 Chil3 2.33 [0.32 , 4.35] 6.9E-02 1.96 [0.91 , 3.01] 7.7E-03 0.45 [0.22 , 0.69] 7.2E-03 Msr1 2.26 [1.83 , 2.69] 7.3E-06 1.29 [1.07 , 1.52] 2.2E-06 0.50 [0.41 , 0.59] 1.7E-06 234 Table 20 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Aicda 2.23 [0.42 , 4.03] 5.5E-02 0.57 [-0.25 , 1.39] 2.7E-01 0.30 [0.01 , 0.59] 1.1E-01 Prg2 2.17 [1.26 , 3.08] 2.3E-03 0.93 [0.37 , 1.48] 1.4E-02 0.25 [0.12 , 0.38] 7.9E-03 Tnfsf11 2.12 [1.50 , 2.74] 1.8E-04 1.05 [0.82 , 1.28] 1.3E-05 0.38 [0.28 , 0.48] 5.2E-05 Isg15 2.09 [1.29 , 2.89] 1.3E-03 0.95 [0.49 , 1.41] 4.3E-03 0.26 [0.01 , 0.50] 9.9E-02 Cfi 2.05 [1.22 , 2.89] 1.8E-03 1.13 [0.93 , 1.34] 2.6E-06 0.36 [0.30 , 0.42] 1.6E-06 Slamf7 2.05 [1.20 , 2.90] 2.1E-03 0.84 [0.56 , 1.12] 3.3E-04 0.30 [0.19 , 0.41] 6.5E-04 Oas3 2.01 [1.15 , 2.87] 2.5E-03 0.85 [0.45 , 1.24] 3.3E-03 0.23 [0.02 , 0.43] 9.3E-02 Oas2 2.00 [1.22 , 2.78] 1.4E-03 0.83 [0.46 , 1.19] 2.4E-03 0.17 [0.00 , 0.35] 1.1E-01 Irf7 2.00 [1.04 , 2.96] 4.9E-03 0.92 [0.48 , 1.36] 4.0E-03 0.24 [-0.01 , 0.50] 1.3E-01 Pou2af1 1.97 [0.97 , 2.98] 6.8E-03 0.57 [0.16 , 0.99] 3.4E-02 0.21 [0.07 , 0.34] 2.7E-02 Ccr6 1.96 [0.96 , 2.95] 6.8E-03 0.50 [0.13 , 0.86] 3.9E-02 0.17 [0.05 , 0.28] 3.1E-02 Birc5 1.95 [1.31 , 2.59] 4.7E-04 0.69 [0.43 , 0.95] 8.0E-04 0.28 [0.19 , 0.36] 1.8E-04 Clec4n 1.91 [1.50 , 2.33] 1.8E-05 1.21 [1.09 , 1.32] 9.5E-09 0.47 [0.40 , 0.54] 4.5E-07 Lag3 1.88 [0.94 , 2.83] 6.2E-03 0.53 [0.18 , 0.89] 2.5E-02 0.20 [0.09 , 0.30] 8.3E-03 Il10 1.88 [0.93 , 2.82] 6.2E-03 0.83 [0.44 , 1.21] 3.4E-03 0.33 [0.20 , 0.46] 1.1E-03 Zbp1 1.79 [1.14 , 2.44] 8.0E-04 0.76 [0.41 , 1.11] 3.2E-03 0.21 [0.06 , 0.37] 3.5E-02 Clec5a 1.73 [1.36 , 2.10] 1.7E-05 1.17 [0.98 , 1.36] 9.5E-07 0.48 [0.42 , 0.55] 2.1E-07 Blnk 1.73 [0.94 , 2.53] 3.8E-03 0.57 [0.27 , 0.88] 7.9E-03 0.21 [0.12 , 0.30] 2.5E-03 235 Table 20 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Fcgr1 1.71 [0.99 , 2.43] 2.3E-03 0.67 [0.37 , 0.97] 2.5E-03 0.16 [0.02 , 0.30] 7.0E-02 Ifi44 1.68 [0.88 , 2.47] 4.4E-03 0.79 [0.37 , 1.22] 7.9E-03 0.20 [-0.02 , 0.42] 1.6E-01 Foxp3 1.64 [1.00 , 2.27] 1.3E-03 0.87 [0.58 , 1.16] 3.6E-04 0.31 [0.19 , 0.43] 1.1E-03 Ctss 1.63 [1.04 , 2.22] 8.2E-04 0.81 [0.62 , 1.00] 2.1E-05 0.28 [0.20 , 0.35] 7.9E-05 Pdcd1lg2 1.63 [1.01 , 2.25] 1.1E-03 0.65 [0.39 , 0.91] 1.3E-03 0.24 [0.14 , 0.33] 1.4E-03 Ifit1 1.60 [0.90 , 2.29] 2.7E-03 0.80 [0.37 , 1.23] 7.7E-03 0.21 [0.01 , 0.40] 9.5E-02 Irf4 1.60 [0.85 , 2.35] 4.3E-03 0.43 [0.08 , 0.78] 5.9E-02 0.16 [0.06 , 0.26] 2.0E-02 Clec4a2 1.58 [1.06 , 2.10] 4.5E-04 0.57 [0.24 , 0.90] 1.3E-02 0.14 [0.03 , 0.24] 5.4E-02 Fcgr2b 1.57 [1.31 , 1.83] 3.7E-06 1.06 [0.88 , 1.24] 1.6E-06 0.40 [0.32 , 0.48] 4.7E-06 Il6 1.57 [1.07 , 2.06] 3.3E-04 0.85 [0.55 , 1.15] 6.0E-04 0.27 [0.16 , 0.38] 1.2E-03 Ccr1 1.54 [1.26 , 1.81] 4.5E-06 0.95 [0.72 , 1.17] 2.5E-05 0.35 [0.29 , 0.41] 1.6E-06 Ccl9 1.54 [1.18 , 1.89] 2.6E-05 0.87 [0.68 , 1.07] 1.5E-05 0.33 [0.23 , 0.42] 1.3E-04 Arg2 1.53 [1.17 , 1.89] 2.9E-05 1.08 [0.82 , 1.34] 2.6E-05 0.45 [0.38 , 0.52] 8.2E-07 Il21r 1.53 [0.66 , 2.41] 1.2E-02 0.39 [0.02 , 0.76] 1.0E-01 0.16 [0.03 , 0.28] 5.3E-02 Cd14 1.50 [1.22 , 1.77] 4.8E-06 0.92 [0.73 , 1.12] 9.7E-06 0.33 [0.25 , 0.41] 2.5E-05 Slc11a1 1.50 [0.95 , 2.06] 9.6E-04 0.78 [0.54 , 1.01] 1.7E-04 0.29 [0.20 , 0.38] 2.0E-04 Cd68 1.46 [1.21 , 1.72] 4.3E-06 0.85 [0.70 , 0.99] 1.6E-06 0.32 [0.27 , 0.36] 6.4E-07 Tnfrsf4 1.46 [0.97 , 1.95] 5.1E-04 0.65 [0.44 , 0.87] 3.0E-04 0.26 [0.19 , 0.32] 4.5E-05 236 Table 20 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnf 1.45 [0.92 , 1.99] 8.9E-04 0.65 [0.31 , 0.99] 6.6E-03 0.32 [0.21 , 0.42] 2.7E-04 Emr1 1.44 [1.09 , 1.79] 3.7E-05 0.60 [0.38 , 0.81] 6.1E-04 0.17 [0.09 , 0.25] 3.0E-03 Cxcr3 1.44 [0.75 , 2.13] 4.6E-03 0.37 [-0.01 , 0.74] 1.2E-01 0.15 [0.05 , 0.25] 2.5E-02 Il1b 1.41 [0.95 , 1.87] 4.4E-04 0.55 [0.35 , 0.75] 6.4E-04 0.10 [-0.01 , 0.21] 1.5E-01 Pik3cg 1.40 [0.96 , 1.83] 3.1E-04 0.54 [0.32 , 0.76] 1.6E-03 0.19 [0.12 , 0.26] 6.9E-04 Itgam 1.39 [0.87 , 1.92] 1.1E-03 0.66 [0.42 , 0.90] 7.1E-04 0.21 [0.12 , 0.30] 1.5E-03 Ccr8 1.37 [0.63 , 2.10] 9.0E-03 0.83 [0.53 , 1.13] 6.0E-04 0.26 [0.13 , 0.39] 6.4E-03 Il1r2 1.34 [0.76 , 1.93] 2.7E-03 0.77 [0.44 , 1.11] 2.2E-03 0.28 [0.15 , 0.40] 2.5E-03 Ada 1.34 [0.73 , 1.96] 3.7E-03 0.39 [0.09 , 0.68] 4.4E-02 0.15 [0.06 , 0.24] 1.7E-02 Slamf6 1.32 [0.53 , 2.12] 1.6E-02 0.38 [0.11 , 0.65] 3.4E-02 0.17 [0.07 , 0.26] 1.4E-02 Ccl6 1.31 [0.95 , 1.68] 1.4E-04 0.97 [0.82 , 1.12] 8.8E-07 0.40 [0.34 , 0.46] 6.4E-07 Tlr2 1.30 [1.02 , 1.57] 1.7E-05 0.74 [0.56 , 0.91] 2.5E-05 0.27 [0.19 , 0.34] 7.5E-05 Fcer2a 1.30 [0.45 , 2.15] 2.4E-02 1.00 [0.61 , 1.40] 1.2E-03 0.36 [0.21 , 0.51] 1.9E-03 Fcgr3 1.29 [1.07 , 1.51] 3.9E-06 0.71 [0.57 , 0.84] 4.2E-06 0.23 [0.17 , 0.29] 6.6E-05 Cd200r1 1.29 [0.99 , 1.58] 2.6E-05 0.79 [0.68 , 0.90] 4.9E-07 0.29 [0.24 , 0.33] 6.6E-07 Fpr2 1.28 [1.02 , 1.53] 9.5E-06 0.45 [0.13 , 0.77] 3.3E-02 0.14 [-0.01 , 0.29] 1.5E-01 Cx3cr1 1.28 [0.84 , 1.72] 6.5E-04 0.38 [0.07 , 0.69] 6.1E-02 0.10 [-0.03 , 0.23] 2.4E-01 Itgax 1.27 [0.99 , 1.56] 2.2E-05 0.56 [0.33 , 0.79] 1.6E-03 0.17 [0.11 , 0.22] 4.8E-04 237 Table 20 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Rsad2 1.27 [0.46 , 2.09] 2.1E-02 0.73 [0.28 , 1.18] 1.6E-02 0.18 [-0.06 , 0.43] 2.4E-01 Il1a 1.26 [0.86 , 1.67] 4.1E-04 0.80 [0.62 , 0.98] 1.3E-05 0.30 [0.24 , 0.37] 1.2E-05 Marco 1.26 [0.63 , 1.90] 6.1E-03 1.02 [0.77 , 1.27] 3.4E-05 0.46 [0.34 , 0.58] 6.6E-05 Sh2d1b1 1.25 [0.76 , 1.75] 1.6E-03 0.25 [-0.15 , 0.64] 3.2E-01 0.00 [-0.08 , 0.09] 9.4E-01 Il2ra 1.25 [0.75 , 1.75] 1.7E-03 0.77 [0.53 , 1.01] 1.8E-04 0.29 [0.20 , 0.38] 2.3E-04 Ulbp1 1.25 [0.72 , 1.77] 2.3E-03 0.61 [0.39 , 0.82] 5.7E-04 0.22 [0.14 , 0.30] 5.1E-04 Ctla4 1.25 [0.51 , 1.99] 1.5E-02 0.51 [0.22 , 0.80] 1.2E-02 0.23 [0.14 , 0.32] 1.1E-03 Cd83 1.24 [0.86 , 1.62] 2.8E-04 0.53 [0.35 , 0.71] 4.1E-04 0.16 [0.09 , 0.23] 2.2E-03 Usp18 1.24 [0.65 , 1.82] 4.4E-03 0.61 [0.32 , 0.89] 3.8E-03 0.17 [0.03 , 0.31] 7.0E-02 Cfp 1.23 [0.65 , 1.81] 4.3E-03 0.46 [0.23 , 0.68] 4.6E-03 0.17 [0.08 , 0.26] 6.0E-03 Lgals3 1.21 [0.98 , 1.44] 7.3E-06 0.85 [0.67 , 1.04] 1.3E-05 0.34 [0.30 , 0.38] 1.8E-07 Csf2rb 1.21 [0.94 , 1.48] 2.2E-05 0.71 [0.60 , 0.82] 8.1E-07 0.27 [0.22 , 0.31] 1.6E-06 Fcer1g 1.21 [0.88 , 1.54] 1.1E-04 0.51 [0.31 , 0.70] 1.0E-03 0.16 [0.09 , 0.23] 2.0E-03 Sh2b2 1.21 [0.73 , 1.69] 1.5E-03 0.58 [0.34 , 0.83] 2.0E-03 0.20 [0.15 , 0.25] 3.9E-05 Plau 1.20 [0.83 , 1.57] 2.6E-04 0.79 [0.62 , 0.95] 9.7E-06 0.29 [0.23 , 0.34] 4.2E-06 Lyve1 -1.23 [-1.76 , -0.69] 2.8E-03 -0.57 [-0.78 , -0.36] 7.1E-04 -0.13 [-0.22 , -0.04] 2.6E-02 Il11ra1 -1.23 [-2.00 , -0.46] 1.9E-02 -0.54 [-0.78 , -0.31] 2.2E-03 -0.20 [-0.26 , -0.14] 1.3E-04 Angpt1 -1.25 [-1.93 , -0.57] 1.0E-02 -0.49 [-0.70 , -0.29] 1.7E-03 -0.16 [-0.24 , -0.09] 3.4E-03 238 Table 20 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Kdr -1.26 [-1.86 , -0.67] 4.4E-03 -0.57 [-0.69 , -0.44] 1.4E-05 -0.23 [-0.30 , -0.16] 1.3E-04 Dll4 -1.33 [-1.78 , -0.88] 5.8E-04 -0.64 [-0.84 , -0.43] 2.5E-04 -0.23 [-0.29 , -0.17] 6.8E-05 Cd8b1 -1.41 [-2.16 , -0.66] 8.6E-03 -0.82 [-1.19 , -0.44] 3.4E-03 -0.29 [-0.42 , -0.16] 2.9E-03 Angpt2 -1.60 [-2.18 , -1.02] 8.0E-04 -0.59 [-0.70 , -0.49] 2.8E-06 -0.22 [-0.27 , -0.17] 1.9E-05 Hamp -2.28 [-3.99 , -0.56] 4.2E-02 -0.88 [-1.82 , 0.06] 1.4E-01 -0.16 [-0.34 , 0.03] 1.8E-01 Cfd -3.00 [-5.20 , -0.80] 3.8E-02 -1.03 [-2.17 , 0.12] 1.6E-01 -0.19 [-0.47 , 0.09] 2.9E-01 239 Figure 47. Summary of immune genes impacted by cSiO2 identified at 1, 5, 9, and 13 wk post final exposure in lung of cSiO2-exposed NZBWF1 mice fed CON diet. Nodes with similar function were colored as depicted. Disconnected nodes were removed for ease of visualization. 240 Figure 48. Network visualization of immune genes impacted by cSiO 2 identified 13 wk post final exposure in spleen of cSiO2-exposed NZBWF1 mice fed CON diet. with similar function were colored as depicted. Disconnected nodes were removed for ease of visualization. 241 Figure 49. Volcano plots depicting immune-related mRNA gene expression data at 13 wk post final exposure in spleen of cSiO 2treated NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for individual genes within each treatment group as log2(fold change) relative to VEH + CON mice and corresponding BH p-value for that gene. Vertical lines indicate log2(fold change) = 1.2; horizontal line indicates BH p-value = 0.05. (A) indicates cSiO2 + CON relative to VEH + CON treated mice; (B) indicates effect of DHA on cSiO2-induced gene expression expressed relative to VEH + CON treated mice. 242 Table 21. mRNA signatures in spleen at 13 wk post final exposure in cSiO2-exposed NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log 2(fold change) relative to VEH + CON mice. Data are included for all treatment groups for those genes which were log2(fold change) ± > 1.2 and BH p-value < 0.05 in cSiO2 + CON treated NZBWF1 mice. Table 21 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl14 3.87 [2.47 , 5.27] 6.6E-04 1.71 [1.10 , 2.32] 2.5E-03 0.41 [0.18 , 0.64] 7.4E-02 Ccl8 3.32 [2.72 , 3.91] 2.2E-05 1.36 [1.03 , 1.70] 3.4E-04 0.40 [0.22 , 0.59] 3.4E-02 Fcgr4 2.93 [2.11 , 3.76] 1.2E-04 1.19 [0.77 , 1.61] 2.4E-03 0.26 [0.11 , 0.41] 7.8E-02 Ccl12 2.24 [1.66 , 2.83] 8.3E-05 0.95 [0.64 , 1.25] 1.7E-03 0.24 [0.11 , 0.38] 7.4E-02 Klra2 2.06 [1.54 , 2.57] 6.2E-05 0.80 [0.48 , 1.12] 4.7E-03 0.12 [0.01 , 0.22] 2.1E-01 C3ar1 1.98 [1.30 , 2.65] 4.4E-04 0.84 [0.50 , 1.18] 5.0E-03 0.21 [0.09 , 0.33] 7.4E-02 Il1b 1.92 [1.37 , 2.48] 1.5E-04 0.62 [0.33 , 0.92] 1.1E-02 0.06 [-0.07 , 0.19] 6.8E-01 Ccl4 1.66 [1.12 , 2.20] 3.1E-04 0.63 [0.32 , 0.94] 1.3E-02 0.14 [0.04 , 0.24] 1.2E-01 Gzmk 1.63 [1.10 , 2.16] 2.9E-04 0.65 [0.37 , 0.94] 7.1E-03 0.16 [0.01 , 0.31] 2.5E-01 Il12b 1.50 [0.99 , 2.01] 4.2E-04 0.54 [0.26 , 0.81] 1.4E-02 0.17 [0.07 , 0.26] 7.8E-02 Il21 1.49 [1.02 , 1.96] 2.4E-04 0.48 [0.21 , 0.75] 2.3E-02 0.10 [-0.04 , 0.25] 4.5E-01 Il1rn 1.45 [1.14 , 1.76] 3.0E-05 0.51 [0.33 , 0.69] 2.4E-03 0.08 [-0.01 , 0.17] 3.1E-01 243 Table 21 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccl2 1.45 [1.02 , 1.88] 1.7E-04 0.42 [0.15 , 0.68] 3.6E-02 0.09 [-0.02 , 0.20] 3.9E-01 Itgax 1.45 [0.87 , 2.04] 1.3E-03 0.54 [0.22 , 0.86] 2.6E-02 0.10 [0.00 , 0.20] 2.5E-01 Fpr2 1.44 [0.89 , 2.00] 1.0E-03 0.62 [0.37 , 0.87] 4.7E-03 0.15 [0.05 , 0.25] 9.9E-02 Ccl3 1.42 [1.12 , 1.72] 3.0E-05 0.61 [0.32 , 0.90] 1.1E-02 0.09 [-0.01 , 0.19] 3.1E-01 Lag3 1.40 [1.10 , 1.69] 3.0E-05 0.47 [0.33 , 0.61] 1.4E-03 0.15 [0.08 , 0.22] 4.5E-02 Msr1 1.37 [0.84 , 1.90] 1.0E-03 0.48 [0.26 , 0.70] 9.3E-03 0.12 [0.04 , 0.19] 9.6E-02 Tnfrsf8 1.35 [1.06 , 1.63] 3.0E-05 0.47 [0.33 , 0.61] 1.4E-03 0.17 [0.12 , 0.22] 4.3E-03 Oas2 1.34 [1.02 , 1.67] 6.2E-05 0.44 [0.27 , 0.62] 4.7E-03 0.09 [0.01 , 0.16] 2.1E-01 Socs3 1.30 [0.92 , 1.68] 1.7E-04 0.46 [0.26 , 0.66] 7.5E-03 0.13 [0.05 , 0.21] 7.8E-02 Cx3cr1 1.28 [0.90 , 1.66] 1.7E-04 0.50 [0.29 , 0.72] 7.1E-03 0.10 [0.02 , 0.17] 1.7E-01 Il10 1.27 [0.84 , 1.70] 4.3E-04 0.43 [0.23 , 0.63] 9.9E-03 0.09 [0.02 , 0.16] 1.6E-01 Ltf -1.20 [-1.81 , -0.59] 5.6E-03 -0.14 [-0.60 , 0.31] 6.9E-01 -0.04 [-0.15 , 0.07] 7.8E-01 Ctsg -1.22 [-2.10 , -0.35] 3.6E-02 -0.07 [-0.63 , 0.49] 8.9E-01 0.04 [-0.13 , 0.21] 8.5E-01 Il1rl1 -1.33 [-1.97 , -0.68] 4.2E-03 -0.32 [-0.57 , -0.07] 8.1E-02 -0.08 [-0.19 , 0.03] 4.4E-01 Tal1 -1.40 [-2.04 , -0.75] 3.1E-03 -0.22 [-0.49 , 0.05] 2.5E-01 0.02 [-0.08 , 0.12] 8.9E-01 Ccl24 -1.52 [-2.13 , -0.91] 1.3E-03 -0.51 [-0.83 , -0.18] 3.8E-02 -0.11 [-0.23 , 0.02] 3.6E-01 Tfrc -1.57 [-2.33 , -0.81] 4.3E-03 -0.26 [-0.54 , 0.02] 2.0E-01 0.01 [-0.10 , 0.12] 9.4E-01 Rsad2 -1.69 [-2.57 , -0.81] 6.7E-03 -0.05 [-0.33 , 0.22] 8.2E-01 0.10 [-0.01 , 0.20] 3.1E-01 244 Table 21 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Icam4 -2.14 [-3.12 , -1.16] 3.0E-03 -0.30 [-0.63 , 0.03] 2.0E-01 0.02 [-0.10 , 0.14] 9.1E-01 Cxcl15 -2.66 [-3.78 , -1.54] 1.7E-03 -1.29 [-1.76 , -0.81] 2.8E-03 -0.60 [-0.80 , -0.41] 6.1E-03 245 DHA suppresses cSiO2-triggered mRNA gene expression in kidney at 13 wk post final exposure Dramatic induction of gene expression was evident in the kidney 13 wks post final exposure to cSiO2. In total, 168 genes were upregulated in cSiO2 treated mice fed CON diet, 1 gene was upregulated in cSiO2 treated mice fed 0.4% DHA, and 0 genes were upregulated in cSiO2 treated mice fed 1.0% DHA (Fig. 50, Fig. 51). The expression profile in the kidney paralleled in lung. IFN and chemokine families of mRNA transcripts were prominently induced by intranasal cSiO2 exposure in the kidney at 13 wks post final exposure, as were mRNA signatures indicative of complement, lymphocyte activation and antigen processing (Table 22). In addition, the emergence of two additional families of mRNA transcripts, adhesion molecules and collagen deposition, were present in the kidneys but not identified in the lungs following cSiO2 exposure. In addition to the dramatic effects of cSiO2 in the kidney, the suppressive effects of DHA on cSiO2 -induced mRNA signatures were also apparent. Similarly to the results in the spleen at 13 wks post final exposure to cSiO2, the suppressive effects of DHA in the kidney were so dramatic that it is difficult to attribute any specific family of mRNA signatures as being the predominant factor responsible for mediating the suppressive effects of DHA (Table 22); nearly all transcripts identified in cSiO2 treated mice fed 1% DHA were not significantly different from VEH treated mice fed CON. These findings illustrate the impressive protective role of DHA on kidney inflammation and glomerulonephritis following airway exposure to cSiO2 in lupus prone NZBWF1 mice. Overall, the gene expression analyses performed at select time points in the lung, spleen, and kidney of cSiO2 -exposed NZBWF1 mice fed CON or DHA-enriched diets support our model that cSiO2 induces a repetitive cycle of unresolved inflammation in the lung that predicates the 246 Figure 50. Network visualization of immune genes impacted by cSiO2 identified 13 wk post final exposure in kidney of cSiO2-exposed NZBWF1 mice fed CON diet. Nodes with similar function were colored as depicted. Disconnected nodes were removed for ease of visualization. 247 Figure 51. Volcano plots depicting immune-related mRNA gene expression data at 13 wk post final exposure in kidney of cSiO 2treated NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for individual genes within each treatment group as log2(fold change) relative to VEH + CON mice and corresponding BH p-value for that gene. Vertical lines indicate log2(fold change) = 1.2; horizontal line indicates BH p-value = 0.05. (A) indicates cSiO2 + CON relative to VEH + CON treated mice; (B) indicates effect of DHA on cSiO2-induced gene expression expressed relative to VEH + CON treated mice. 248 Table 22. mRNA signatures in kidney at 13 wk post final exposure in cSiO 2-exposed NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log2(fold change) relative to VEH + CON mice. Data are included for all treatment groups for those genes which were log 2(fold change) ± > 1.2 and BH p-value < 0.05 in cSiO2 + CON treated NZBWF1 mice. Table 22 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Fpr2 3.76 [2.83 , 4.68] 5.1E-04 1.36 [0.89 , 1.84] 1.5E-02 0.40 [0.17 , 0.64] 7.6E-02 Itgam 3.22 [2.13 , 4.31] 9.4E-04 0.97 [0.37 , 1.57] 4.5E-02 0.20 [0.01 , 0.39] 2.7E-01 Fcgr4 3.13 [2.16 , 4.09] 9.4E-04 0.94 [0.44 , 1.44] 3.3E-02 0.22 [0.06 , 0.38] 1.5E-01 Lcn2 3.04 [1.07 , 5.02] 1.9E-02 0.79 [-0.09 , 1.67] 2.1E-01 0.00 [-0.08 , 0.09] 9.5E-01 Ccl8 3.01 [1.03 , 4.99] 2.0E-02 0.08 [-0.87 , 1.03] 9.1E-01 -0.16 [-0.56 , 0.23] 6.6E-01 Ccl12 2.97 [2.05 , 3.88] 9.4E-04 0.87 [0.43 , 1.31] 3.3E-02 0.10 [-0.07 , 0.28] 5.2E-01 Clec4a2 2.95 [1.99 , 3.91] 9.4E-04 0.92 [0.40 , 1.43] 3.5E-02 0.17 [0.04 , 0.31] 1.8E-01 Fcgr1 2.78 [1.91 , 3.64] 9.4E-04 0.81 [0.40 , 1.21] 3.3E-02 0.12 [0.03 , 0.20] 1.4E-01 C3ar1 2.78 [1.89 , 3.66] 9.4E-04 0.89 [0.49 , 1.30] 3.0E-02 0.19 [0.11 , 0.28] 2.5E-02 Ccr5 2.77 [1.47 , 4.08] 4.2E-03 0.94 [0.35 , 1.53] 4.7E-02 0.15 [-0.07 , 0.38] 4.6E-01 Tlr8 2.63 [1.73 , 3.54] 9.4E-04 0.80 [0.39 , 1.22] 3.3E-02 0.15 [0.04 , 0.25] 1.4E-01 Ccl2 2.60 [1.74 , 3.45] 9.4E-04 0.88 [0.48 , 1.28] 3.0E-02 0.15 [0.00 , 0.31] 2.8E-01 249 Table 22 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il1b 2.59 [1.93 , 3.26] 5.1E-04 0.62 [0.31 , 0.93] 3.3E-02 0.19 [0.07 , 0.32] 1.2E-01 Sele 2.57 [1.94 , 3.20] 5.1E-04 n.d n.d n.d 0.43 [0.24 , 0.62] 2.6E-02 Klra2 2.57 [1.75 , 3.38] 9.4E-04 0.69 [0.29 , 1.08] 3.6E-02 0.14 [0.04 , 0.23] 1.3E-01 Ccl9 2.53 [1.66 , 3.41] 9.4E-04 0.80 [0.34 , 1.26] 3.6E-02 0.19 [0.06 , 0.32] 1.4E-01 Ccl5 2.49 [1.36 , 3.62] 3.5E-03 0.53 [0.01 , 1.04] 1.6E-01 0.04 [-0.09 , 0.16] 7.7E-01 Irf7 2.47 [1.58 , 3.36] 1.1E-03 0.89 [0.47 , 1.31] 3.0E-02 0.17 [0.03 , 0.30] 1.9E-01 Ccl3 2.45 [1.60 , 3.29] 9.4E-04 0.82 [0.39 , 1.24] 3.3E-02 0.27 [0.13 , 0.40] 4.9E-02 Ctss 2.44 [1.55 , 3.33] 1.1E-03 0.64 [0.24 , 1.05] 4.7E-02 0.08 [-0.01 , 0.17] 3.5E-01 Hck 2.43 [1.78 , 3.07] 5.1E-04 0.70 [0.33 , 1.06] 3.3E-02 0.20 [0.04 , 0.36] 1.9E-01 C1qb 2.41 [1.57 , 3.25] 9.4E-04 0.68 [0.29 , 1.08] 3.6E-02 0.10 [-0.01 , 0.20] 3.2E-01 C1qa 2.33 [1.53 , 3.14] 9.4E-04 0.64 [0.26 , 1.02] 4.1E-02 0.08 [-0.01 , 0.18] 3.3E-01 Oas2 2.31 [1.60 , 3.03] 9.4E-04 0.69 [0.41 , 0.97] 2.1E-02 0.09 [0.02 , 0.16] 1.8E-01 Emr1 2.27 [1.38 , 3.16] 1.9E-03 0.64 [0.22 , 1.06] 5.4E-02 0.08 [-0.03 , 0.19] 4.1E-01 Zbp1 2.25 [1.53 , 2.97] 9.4E-04 0.49 [0.16 , 0.83] 6.1E-02 0.04 [-0.09 , 0.18] 7.5E-01 Cx3cr1 2.24 [1.48 , 3.01] 9.4E-04 0.60 [0.23 , 0.96] 4.5E-02 0.08 [0.00 , 0.16] 2.8E-01 Tnfsf13b 2.22 [1.46 , 2.99] 9.4E-04 0.62 [0.23 , 1.01] 4.7E-02 0.14 [0.00 , 0.28] 2.9E-01 Ncf4 2.21 [1.54 , 2.87] 9.4E-04 0.58 [0.23 , 0.92] 4.2E-02 0.10 [-0.04 , 0.24] 4.4E-01 Col1a1 2.21 [0.93 , 3.50] 1.2E-02 0.42 [-0.29 , 1.13] 4.0E-01 0.01 [-0.09 , 0.12] 9.1E-01 250 Table 22 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Pou2f2 2.19 [1.44 , 2.94] 9.4E-04 0.59 [0.20 , 0.97] 5.4E-02 0.13 [0.00 , 0.26] 2.8E-01 Fcer1g 2.18 [1.42 , 2.94] 9.8E-04 0.67 [0.35 , 1.00] 3.1E-02 0.12 [0.02 , 0.21] 1.9E-01 Cxcl10 2.18 [1.40 , 2.96] 1.1E-03 0.74 [0.36 , 1.12] 3.3E-02 0.18 [0.01 , 0.34] 2.4E-01 Isg15 2.18 [1.40 , 2.96] 1.1E-03 0.68 [0.36 , 1.00] 3.0E-02 0.12 [-0.02 , 0.25] 3.7E-01 Cxcl9 2.18 [1.26 , 3.11] 2.6E-03 0.37 [-0.09 , 0.83] 2.6E-01 0.04 [-0.14 , 0.22] 8.1E-01 Lif 2.18 [0.88 , 3.48] 1.3E-02 0.58 [0.09 , 1.07] 1.1E-01 0.10 [0.02 , 0.18] 1.9E-01 Oasl1 2.17 [1.17 , 3.17] 3.8E-03 0.81 [0.35 , 1.27] 3.5E-02 0.15 [-0.01 , 0.31] 3.0E-01 Ifit1 2.16 [1.38 , 2.93] 1.1E-03 0.78 [0.39 , 1.17] 3.3E-02 0.14 [-0.02 , 0.30] 3.5E-01 Fcgr3 2.15 [1.38 , 2.92] 1.1E-03 0.58 [0.13 , 1.02] 8.6E-02 0.07 [-0.02 , 0.16] 3.7E-01 Mx2 2.12 [1.36 , 2.87] 1.0E-03 0.79 [0.49 , 1.10] 1.7E-02 0.12 [-0.05 , 0.29] 4.4E-01 Slc11a1 2.08 [1.34 , 2.82] 1.0E-03 0.58 [0.26 , 0.90] 3.4E-02 0.10 [-0.03 , 0.24] 4.0E-01 Mrc1 2.05 [1.12 , 2.97] 3.4E-03 0.54 [0.12 , 0.95] 8.5E-02 0.07 [0.02 , 0.12] 1.3E-01 Socs3 2.05 [1.01 , 3.10] 6.1E-03 0.50 [0.08 , 0.92] 1.1E-01 0.03 [-0.04 , 0.10] 6.5E-01 C5ar1 2.01 [1.38 , 2.64] 9.4E-04 0.67 [0.37 , 0.97] 3.0E-02 0.15 [0.04 , 0.26] 1.7E-01 Cybb 2.01 [1.37 , 2.64] 9.4E-04 0.56 [0.27 , 0.84] 3.3E-02 0.14 [0.04 , 0.24] 1.4E-01 Ccl28 2.01 [0.68 , 3.35] 2.0E-02 0.38 [-0.16 , 0.92] 3.2E-01 0.13 [0.01 , 0.24] 2.2E-01 Itgal 1.99 [1.46 , 2.51] 5.1E-04 0.45 [0.15 , 0.74] 5.6E-02 0.11 [-0.03 , 0.26] 4.0E-01 Lyz2 1.99 [1.16 , 2.82] 2.4E-03 0.53 [0.13 , 0.93] 8.1E-02 0.05 [-0.09 , 0.19] 7.2E-01 251 Table 22 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il21r 1.97 [1.13 , 2.82] 2.7E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Tlr9 1.95 [1.32 , 2.57] 9.4E-04 0.51 [0.27 , 0.75] 3.0E-02 0.05 [-0.05 , 0.14] 6.2E-01 C7 1.95 [0.97 , 2.93] 5.8E-03 0.48 [0.05 , 0.90] 1.3E-01 0.06 [-0.01 , 0.13] 3.8E-01 Ccr2 1.94 [1.05 , 2.83] 3.7E-03 0.52 [0.10 , 0.93] 9.9E-02 0.05 [-0.01 , 0.12] 3.8E-01 Cd68 1.93 [1.28 , 2.58] 9.4E-04 0.46 [0.12 , 0.80] 7.9E-02 0.06 [-0.01 , 0.13] 3.6E-01 Cd48 1.93 [1.21 , 2.65] 1.3E-03 0.46 [0.13 , 0.79] 7.2E-02 0.02 [-0.09 , 0.13] 8.8E-01 Ly86 1.92 [1.18 , 2.65] 1.6E-03 0.44 [0.06 , 0.83] 1.2E-01 0.02 [-0.07 , 0.11] 8.2E-01 Cd84 1.91 [1.10 , 2.73] 2.6E-03 0.34 [-0.06 , 0.74] 2.3E-01 0.04 [-0.06 , 0.14] 6.6E-01 Msr1 1.89 [0.99 , 2.78] 4.3E-03 0.55 [0.09 , 1.00] 1.1E-01 0.06 [-0.04 , 0.15] 5.1E-01 Col3a1 1.87 [0.76 , 2.97] 1.2E-02 0.36 [-0.27 , 0.98] 4.2E-01 -0.03 [-0.11 , 0.04] 6.6E-01 Ccl20 1.87 [0.34 , 3.41] 4.9E-02 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Cxcl1 1.86 [0.57 , 3.15] 2.5E-02 0.53 [-0.05 , 1.11] 2.0E-01 0.05 [-0.08 , 0.17] 6.8E-01 Cxcl13 1.84 [0.62 , 3.06] 2.1E-02 0.18 [-0.28 , 0.64] 5.9E-01 -0.01 [-0.19 , 0.16] 9.3E-01 Ticam2 1.83 [1.22 , 2.44] 9.4E-04 0.50 [0.21 , 0.80] 3.8E-02 0.09 [-0.04 , 0.21] 4.5E-01 Itgb2 1.83 [1.20 , 2.46] 9.4E-04 0.41 [0.11 , 0.72] 7.9E-02 0.08 [-0.05 , 0.20] 5.0E-01 Vcam1 1.83 [0.88 , 2.79] 6.8E-03 0.53 [0.07 , 0.98] 1.2E-01 0.05 [-0.01 , 0.12] 3.7E-01 Spn 1.82 [1.18 , 2.46] 1.0E-03 0.47 [0.18 , 0.77] 4.7E-02 0.10 [-0.04 , 0.24] 4.2E-01 Ptprc 1.80 [1.12 , 2.48] 1.5E-03 0.39 [0.09 , 0.69] 8.3E-02 0.05 [-0.05 , 0.15] 6.3E-01 252 Table 22 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Runx1 1.80 [0.93 , 2.67] 4.6E-03 0.38 [-0.12 , 0.88] 3.0E-01 0.00 [-0.08 , 0.08] 9.9E-01 Il10ra 1.79 [1.08 , 2.50] 2.0E-03 0.47 [0.22 , 0.73] 3.3E-02 0.06 [-0.03 , 0.15] 4.9E-01 Cd14 1.79 [0.87 , 2.71] 6.5E-03 0.45 [-0.02 , 0.92] 1.9E-01 0.05 [0.01 , 0.08] 1.9E-01 Csf3r 1.77 [1.00 , 2.53] 2.8E-03 0.47 [0.11 , 0.82] 8.4E-02 0.07 [-0.04 , 0.18] 4.8E-01 Nlrc5 1.77 [1.00 , 2.55] 2.9E-03 0.32 [0.00 , 0.64] 1.7E-01 0.01 [-0.11 , 0.14] 9.1E-01 Selplg 1.74 [1.03 , 2.45] 2.2E-03 0.28 [-0.02 , 0.58] 1.9E-01 0.01 [-0.08 , 0.11] 8.9E-01 Syk 1.73 [1.04 , 2.43] 2.0E-03 0.32 [-0.05 , 0.70] 2.2E-01 0.03 [-0.07 , 0.13] 7.7E-01 Cd4 1.73 [0.79 , 2.66] 8.3E-03 0.19 [-0.24 , 0.61] 5.6E-01 -0.15 [-0.28 , -0.02] 2.0E-01 H2-DMb2 1.71 [0.26 , 3.16] 5.5E-02 0.32 [-0.24 , 0.88] 4.2E-01 -0.06 [-0.20 , 0.08] 6.4E-01 Klra17 1.70 [0.77 , 2.64] 9.0E-03 0.37 [-0.05 , 0.80] 2.2E-01 -0.01 [-0.08 , 0.07] 9.2E-01 Ccl4 1.69 [0.90 , 2.49] 4.2E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Tlr7 1.66 [0.95 , 2.37] 2.7E-03 0.39 [0.09 , 0.69] 8.4E-02 0.05 [-0.04 , 0.14] 5.2E-01 Ifit3 1.66 [0.89 , 2.43] 3.8E-03 0.65 [0.31 , 0.99] 3.3E-02 0.11 [-0.05 , 0.26] 4.8E-01 Ly9 1.65 [0.94 , 2.35] 2.7E-03 0.20 [-0.16 , 0.55] 4.3E-01 -0.04 [-0.16 , 0.08] 7.2E-01 Ddx60 1.64 [0.97 , 2.30] 2.2E-03 0.50 [0.31 , 0.68] 1.5E-02 0.08 [0.00 , 0.17] 2.9E-01 Havcr2 1.63 [1.19 , 2.08] 5.5E-04 0.45 [0.28 , 0.62] 1.5E-02 n.d [n.d , n.d] n.d Sh2d1b1 1.61 [1.01 , 2.20] 1.3E-03 0.45 [0.17 , 0.73] 4.7E-02 0.03 [-0.06 , 0.13] 7.3E-01 Tap1 1.61 [1.00 , 2.23] 1.6E-03 0.41 [0.17 , 0.64] 3.8E-02 0.07 [0.00 , 0.13] 2.8E-01 253 Table 22 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd74 1.61 [0.86 , 2.37] 4.1E-03 0.31 [0.01 , 0.61] 1.6E-01 0.00 [-0.06 , 0.07] 9.5E-01 C4b 1.60 [0.80 , 2.39] 5.3E-03 0.48 [0.24 , 0.72] 3.3E-02 0.08 [0.01 , 0.14] 2.1E-01 Il2rg 1.59 [1.07 , 2.11] 9.4E-04 0.18 [-0.04 , 0.41] 2.6E-01 0.06 [-0.05 , 0.16] 5.6E-01 Psmb8 1.59 [0.89 , 2.28] 2.9E-03 0.36 [0.06 , 0.65] 1.1E-01 0.01 [-0.07 , 0.09] 9.1E-01 Btk 1.58 [0.92 , 2.24] 2.4E-03 0.35 [0.02 , 0.69] 1.5E-01 0.01 [-0.10 , 0.12] 9.2E-01 Itga4 1.56 [1.03 , 2.09] 9.4E-04 0.32 [0.04 , 0.60] 1.2E-01 0.09 [-0.02 , 0.20] 3.6E-01 Lilra5 1.56 [1.02 , 2.11] 9.8E-04 0.45 [0.21 , 0.69] 3.3E-02 0.08 [0.02 , 0.14] 1.8E-01 Cd53 1.55 [0.87 , 2.23] 2.9E-03 0.33 [0.00 , 0.65] 1.7E-01 0.03 [-0.09 , 0.14] 8.1E-01 Csf2rb 1.54 [0.88 , 2.19] 2.6E-03 0.34 [-0.03 , 0.71] 2.0E-01 0.03 [-0.09 , 0.15] 8.2E-01 Cd180 1.54 [0.85 , 2.24] 3.4E-03 0.32 [0.05 , 0.60] 1.1E-01 -0.01 [-0.11 , 0.09] 9.3E-01 H2-Eb1 1.54 [0.82 , 2.26] 4.1E-03 0.25 [0.00 , 0.50] 1.7E-01 -0.01 [-0.08 , 0.06] 8.8E-01 Tmem173 1.54 [0.74 , 2.35] 7.0E-03 0.33 [-0.01 , 0.68] 1.9E-01 0.07 [0.01 , 0.14] 2.0E-01 H2-Aa 1.53 [0.79 , 2.27] 4.5E-03 0.22 [-0.04 , 0.49] 2.3E-01 0.00 [-0.07 , 0.06] 9.5E-01 Cd5 1.53 [0.63 , 2.43] 1.2E-02 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Bst2 1.52 [0.96 , 2.09] 1.3E-03 0.49 [0.23 , 0.75] 3.3E-02 0.07 [0.00 , 0.15] 2.9E-01 Itgax 1.52 [0.91 , 2.13] 2.0E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Usp18 1.51 [0.87 , 2.14] 2.5E-03 0.45 [0.21 , 0.69] 3.3E-02 0.05 [-0.06 , 0.16] 6.2E-01 H2-Ab1 1.50 [0.80 , 2.21] 4.2E-03 0.28 [-0.02 , 0.58] 1.9E-01 0.01 [-0.04 , 0.06] 8.6E-01 254 Table 22 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Lcp1 1.49 [0.95 , 2.04] 1.1E-03 0.30 [0.05 , 0.55] 1.0E-01 0.07 [-0.02 , 0.15] 3.8E-01 Xaf1 1.49 [0.95 , 2.03] 1.1E-03 0.42 [0.22 , 0.61] 3.0E-02 0.06 [-0.02 , 0.13] 4.0E-01 Pik3cd 1.49 [0.88 , 2.10] 2.2E-03 0.39 [0.11 , 0.67] 7.2E-02 0.06 [-0.02 , 0.14] 4.1E-01 H2-Ea-ps 1.49 [0.78 , 2.19] 4.3E-03 0.24 [-0.02 , 0.50] 2.0E-01 0.00 [-0.06 , 0.05] 9.9E-01 H2-K1 1.48 [0.80 , 2.17] 3.8E-03 0.41 [0.12 , 0.70] 6.6E-02 0.03 [-0.03 , 0.08] 6.0E-01 Slamf7 1.48 [0.63 , 2.32] 1.1E-02 0.08 [-0.26 , 0.42] 7.6E-01 -0.02 [-0.13 , 0.09] 8.8E-01 Psmb9 1.47 [0.87 , 2.07] 2.2E-03 0.33 [0.10 , 0.56] 6.5E-02 0.03 [-0.05 , 0.11] 7.4E-01 Ccl6 1.47 [0.72 , 2.21] 6.0E-03 0.46 [0.13 , 0.79] 7.2E-02 0.10 [0.04 , 0.16] 7.6E-02 Gbp5 1.46 [0.86 , 2.05] 2.2E-03 0.37 [0.13 , 0.61] 5.3E-02 0.04 [-0.05 , 0.12] 6.6E-01 Cd200r1 1.46 [0.54 , 2.39] 1.7E-02 0.32 [-0.11 , 0.75] 3.1E-01 0.01 [-0.09 , 0.11] 9.1E-01 Tnfrsf1b 1.45 [0.99 , 1.91] 9.4E-04 0.27 [0.04 , 0.50] 1.1E-01 0.06 [-0.03 , 0.16] 4.5E-01 Ebi3 1.45 [0.87 , 2.04] 2.0E-03 0.41 [0.13 , 0.70] 6.3E-02 0.07 [-0.04 , 0.17] 4.9E-01 Cd3g 1.45 [0.45 , 2.45] 2.4E-02 -0.08 [-0.48 , 0.32] 8.0E-01 -0.12 [-0.25 , 0.02] 3.5E-01 Cxcr6 1.44 [0.63 , 2.24] 9.7E-03 0.13 [-0.11 , 0.38] 4.4E-01 -0.05 [-0.14 , 0.03] 4.9E-01 Clec5a 1.42 [0.78 , 2.06] 3.4E-03 0.35 [0.05 , 0.65] 1.1E-01 0.04 [-0.03 , 0.11] 6.0E-01 Itgae 1.42 [0.75 , 2.10] 4.3E-03 0.19 [-0.09 , 0.47] 3.3E-01 -0.03 [-0.09 , 0.03] 6.6E-01 Irf4 1.42 [0.68 , 2.17] 7.0E-03 0.25 [-0.10 , 0.60] 3.1E-01 n.d. [n.d, n.d.) n.d. Cd86 1.41 [0.81 , 2.01] 2.6E-03 0.27 [0.09 , 0.45] 5.9E-02 0.04 [-0.03 , 0.11] 5.4E-01 255 Table 22 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value H2-DMa 1.40 [0.73 , 2.06] 4.3E-03 0.30 [-0.02 , 0.62] 1.9E-01 0.01 [-0.04 , 0.06] 8.8E-01 Il2rb 1.38 [0.63 , 2.12] 8.2E-03 0.14 [-0.08 , 0.36] 3.8E-01 -0.01 [-0.10 , 0.08] 9.1E-01 Ltb 1.38 [0.32 , 2.45] 3.8E-02 0.02 [-0.43 , 0.47] 9.5E-01 -0.13 [-0.30 , 0.05] 4.2E-01 Spp1 1.38 [0.30 , 2.47] 4.0E-02 0.31 [-0.20 , 0.83] 3.9E-01 -0.01 [-0.03 , 0.01] 6.7E-01 Cfp 1.37 [0.76 , 1.98] 3.1E-03 0.22 [-0.18 , 0.62] 4.4E-01 -0.01 [-0.10 , 0.09] 9.5E-01 Lag3 1.37 [0.70 , 2.04] 5.1E-03 0.31 [0.09 , 0.52] 6.9E-02 0.02 [-0.07 , 0.12] 8.1E-01 Pik3cg 1.36 [0.61 , 2.10] 9.0E-03 0.31 [0.01 , 0.61] 1.5E-01 0.04 [-0.08 , 0.15] 7.3E-01 Cxcr3 1.36 [0.61 , 2.11] 9.0E-03 0.13 [-0.15 , 0.40] 5.2E-01 -0.06 [-0.17 , 0.04] 5.0E-01 Clec7a 1.35 [0.66 , 2.04] 6.1E-03 0.37 [0.12 , 0.63] 6.5E-02 0.07 [0.03 , 0.12] 7.7E-02 C3 1.35 [0.37 , 2.33] 3.0E-02 0.52 [0.12 , 0.93] 8.7E-02 0.09 [0.01 , 0.17] 2.0E-01 Cfd 1.35 [-0.86 , 3.56] 2.9E-01 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Irak3 1.34 [0.92 , 1.77] 9.4E-04 0.36 [0.16 , 0.57] 3.6E-02 0.10 [0.03 , 0.16] 1.3E-01 Cdkn1a 1.34 [0.57 , 2.12] 1.1E-02 0.39 [0.09 , 0.69] 8.7E-02 0.11 [0.00 , 0.22] 2.9E-01 Casp1 1.33 [0.78 , 1.88] 2.4E-03 0.18 [-0.08 , 0.44] 3.3E-01 0.03 [-0.04 , 0.10] 6.6E-01 Ikzf1 1.33 [0.69 , 1.97] 4.5E-03 0.19 [-0.09 , 0.47] 3.4E-01 -0.02 [-0.11 , 0.06] 7.9E-01 Socs1 1.33 [0.66 , 2.00] 5.8E-03 0.28 [0.01 , 0.55] 1.5E-01 -0.01 [-0.13 , 0.11] 9.5E-01 Cxcl11 1.33 [0.65 , 2.01] 6.4E-03 0.41 [0.14 , 0.69] 5.8E-02 0.05 [-0.02 , 0.13] 4.3E-01 Cd33 1.33 [0.56 , 2.11] 1.2E-02 0.34 [0.05 , 0.63] 1.1E-01 0.07 [0.01 , 0.13] 2.1E-01 256 Table 22 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd83 1.31 [0.88 , 1.75] 9.4E-04 0.26 [0.04 , 0.47] 1.1E-01 0.03 [-0.03 , 0.10] 6.1E-01 Gzma 1.31 [0.79 , 1.84] 2.0E-03 0.29 [0.08 , 0.49] 7.1E-02 0.02 [-0.06 , 0.11] 8.1E-01 Gpr183 1.31 [0.59 , 2.04] 9.2E-03 0.32 [-0.02 , 0.66] 2.0E-01 0.02 [-0.04 , 0.09] 7.4E-01 Icam1 1.30 [0.78 , 1.82] 2.0E-03 0.42 [0.20 , 0.64] 3.3E-02 0.09 [0.04 , 0.15] 9.6E-02 Batf 1.29 [0.79 , 1.79] 1.7E-03 0.24 [-0.02 , 0.50] 2.1E-01 -0.03 [-0.10 , 0.04] 6.6E-01 H2-Ob 1.29 [0.56 , 2.01] 9.7E-03 0.16 [-0.19 , 0.50] 5.5E-01 -0.02 [-0.10 , 0.06] 8.1E-01 Card11 1.29 [0.48 , 2.09] 1.6E-02 0.02 [-0.31 , 0.36] 9.3E-01 -0.03 [-0.16 , 0.10] 8.1E-01 Fn1 1.29 [0.36 , 2.22] 2.9E-02 0.38 [-0.14 , 0.89] 3.1E-01 0.00 [-0.06 , 0.06] 9.7E-01 Tnfrsf12a 1.29 [0.32 , 2.25] 3.4E-02 0.27 [-0.14 , 0.69] 3.5E-01 0.03 [-0.01 , 0.07] 4.2E-01 Amica1 1.28 [0.67 , 1.89] 4.3E-03 0.17 [-0.09 , 0.44] 3.5E-01 -0.01 [-0.06 , 0.03] 7.5E-01 Ctsw 1.28 [0.48 , 2.09] 1.6E-02 -0.07 [-0.37 , 0.23] 7.5E-01 -0.13 [-0.23 , -0.03] 1.7E-01 Cd276 1.28 [0.46 , 2.11] 1.8E-02 0.42 [0.05 , 0.80] 1.3E-01 0.06 [-0.01 , 0.13] 3.8E-01 Sh2b2 1.27 [0.85 , 1.69] 9.4E-04 n.d [n.d , n.d] n.d -0.02 [-0.10 , 0.06] 8.1E-01 Vim 1.27 [0.67 , 1.87] 4.3E-03 0.30 [-0.04 , 0.64] 2.2E-01 0.02 [-0.03 , 0.08] 6.6E-01 Irf1 1.26 [0.75 , 1.77] 2.0E-03 0.31 [0.11 , 0.52] 5.4E-02 0.05 [-0.01 , 0.11] 3.4E-01 Tnfrsf14 1.25 [0.75 , 1.75] 2.0E-03 0.25 [0.07 , 0.43] 7.4E-02 0.02 [-0.03 , 0.08] 6.0E-01 Il1rl2 1.25 [0.64 , 1.85] 4.7E-03 0.48 [0.18 , 0.78] 4.7E-02 0.10 [0.03 , 0.16] 1.3E-01 H2-DMb1 1.25 [0.59 , 1.92] 7.4E-03 0.21 [-0.07 , 0.50] 3.1E-01 0.01 [-0.04 , 0.07] 8.1E-01 257 Table 22 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Zap70 1.25 [0.32 , 2.18] 3.3E-02 n.d [n.d , n.d] n.d -0.02 [-0.12 , 0.07] 8.1E-01 Csf1 1.24 [0.81 , 1.67] 9.4E-04 0.34 [0.10 , 0.58] 6.7E-02 0.05 [-0.01 , 0.11] 3.6E-01 Herc6 1.24 [0.60 , 1.88] 6.5E-03 0.38 [0.18 , 0.59] 3.3E-02 0.07 [-0.03 , 0.17] 4.5E-01 Ciita 1.24 [0.51 , 1.98] 1.2E-02 0.18 [-0.15 , 0.50] 4.4E-01 -0.02 [-0.11 , 0.07] 8.1E-01 Irgm2 1.21 [0.81 , 1.61] 9.4E-04 0.39 [0.21 , 0.57] 3.0E-02 0.05 [-0.04 , 0.14] 5.3E-01 Inpp5d 1.21 [0.67 , 1.74] 3.1E-03 0.28 [0.04 , 0.51] 1.1E-01 0.04 [-0.01 , 0.09] 3.9E-01 Xcr1 1.21 [0.60 , 1.83] 6.0E-03 0.21 [-0.03 , 0.46] 2.3E-01 -0.07 [-0.15 , 0.00] 2.9E-01 Stat1 1.20 [0.74 , 1.66] 1.6E-03 0.31 [0.14 , 0.49] 3.5E-02 0.03 [-0.04 , 0.11] 6.6E-01 Cd8a 1.20 [0.56 , 1.84] 7.4E-03 0.08 [-0.16 , 0.32] 6.5E-01 -0.03 [-0.13 , 0.07] 7.5E-01 Tnfrsf13b 1.20 [0.53 , 1.87] 9.7E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Icos 1.20 [0.31 , 2.09] 3.2E-02 -0.02 [-0.32 , 0.28] 9.3E-01 -0.07 [-0.19 , 0.04] 5.0E-01 Cd3d 1.20 [0.30 , 2.09] 3.4E-02 0.04 [-0.34 , 0.42] 9.0E-01 -0.10 [-0.25 , 0.06] 4.9E-01 258 development of glomerulonephritis in the kidney. The mRNA signatures support the histopathological observations that the severity of lesions in the lung increase over time, which are also mirrored by the increase in the level of gene expression observed in the lung over time. In addition, by classifying mRNA transcripts into families with similar function, putative mechanisms can be elucidated that delineate the inflammatory sequalae of cSiO2 in the lung that predicate development of systemic autoimmunity, and are primary targets for the suppressive effect of DHA in this context. Remarkably, dietary DHA diminished cSiO2-induced gene expression in the lung, spleen, and most notably, the kidney at all time points investigated in this study, thus illustrating the impressive ability of dietary DHA to suppress inflammation via multiple pathways and prevent the onset of accelerated systemic autoimmunity following cSiO2 exposure to lupus-prone NZBWF1 mice. Discussion: Targeted transcriptomics Genetic predisposition is the primary driving factor of human incidence of lupus and other AD. Murine models that spontaneously develop lupus, including the female NZBWF1 mouse, have contributed extensively to identification of mechanisms that mediate the development of lupus in genetically predisposed individuals. Technological advancements in high-thoroughput transcriptomics approaches have facilitated the exploration of global (e.g. RNASeq [85]) and targeted (e.g. Affymetrix [86] and Nanostring [23]) alterations in gene expression that contribute to disease pathogenesis and potentially identify novel therapeutic targets [87,88]. Many commercially-available platforms exist to perform gene expression analysis. Here, we utilized the nCounter Mouse PanCancer Immune Profiling panel, which consists of 770 genes that induce and regulate inflammation in both innate and adaptive immunity. We selected this technology over other approaches because there is minimal sample preparation required, does not 259 require cDNA conversion and does not rely on target amplification [23], which can be a major sources of variation in traditional qRT-PCR approaches (reviewed in [89]). Using this approach, we determined that airway exposure to cSiO2 in lupus-prone, NZBWF1 mice fed CON diet induced robust inflammation consistent with phenotypes expressed by neutrophils, macrophages, and lymphocytes that amplified over time and was globally suppressed by feeding DHA. Furthermore, mRNA gene expression analysis revealed mechanistic insight into the pathogenesis of cSiO2-triggered autoimmunity. In the lung, rampant chemokine and cytokine production, persistent complement activation, and sustained IFN activity together contribute to the unresolved inflammation induced by cSiO2 exposure. Dietary DHA markedly attenuated these responses in a concentration-dependent manner. Chemokines were consistently upregulated genes induced by cSiO2 exposure. Chemokines are released from injured cells to recruit resident and circulating leukocytes to sites of inflammation by way of concentration gradients. In general, C-X-C motif chemokines are chemotactic for neutrophils, which predominately express CXCR2 and to a lesser extent, CXCR1 receptors. The exception to this rule are C-X-C motif chemokines that lack the amino acid sequence, E-L-R, on the first cysteine residue of the C-X-C motif (i.e. CXCL4, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, and CXCL16) [90]. These chemokines are generally chemotactic for lymphocytes. On the contrary, C-C motif chemokines are generally chemotactic for monocytes and to some extent, lymphocytes that express CCR receptors. Herein, we identified both C-X-C chemokines and C-C chemokines in lung, spleen and kidney, thus implicating neutrophils, macrophages, and lymphocytes in the progression of cSiO2-triggered autoimmunity. Interestingly, CXCL5 and CXCL3 were consistently among the most highly upregulated genes in cSiO2-treated NZBWF1 mice, which predominately recruit neutrophils to sites of inflammation by way of 260 CXCR2. Our working model of cSiO2-triggered autoimmunity proposed in Chapter 3 has focused on the toxicity of cSiO2 towards alveolar macrophages. Chemokines identified in this study were consistent with monocyte recruitment into inflamed tissues. These include CCL2, CCL7, CCL8, and CCL12, a.k.a monocyte chemoattract protein (MCP)-1, MCP-2, MCP-3, and MCP-5, respectively. Neutrophils may also contribute to cSiO2-triggered autoimmunity. Degranulation of recruited neutrophils may release proteolytic enzymes into the extracellular space, thus resulting in bystander tissue injury and promoting lung inflammation [91–93]. In addition, neutrophils at sites of inflammation can die by NETosis, a form of ‘cell suicide’ that releases neutrophil extracellular traps (NET) consisting of nuclear material (e.g.DNA) that could be a source of autoantigen. One study has reported production of NETs following cSiO2 exposure to human peripheral neutrophils in vitro [94]. Whether this occurs in vivo and is a direct consequence of neutrophil phagocytosis of cSiO2 particles remains to be elucidated. Nevertheless, NETs have been implicated in autoimmunity. NETs are persistent in sera of individuals with SLE and interestingly, correlate with glomerulonephritis (reviewed in [95]). Taken together, neutrophils should be further explored as a possible mechanism of cSiO2-triggered autoimmunity. C-X-C motif chemokines that are chemoattractant for B and T lymphocytes were also identified to be persistently upregulated by cSiO2 in lung, spleen, and kidney. These were attenuated by feeding DHA. Of interest here, CXCL13, otherwise known as B-lymphocyte chemoattractant (BLC) was highly expressed in the lung and kidney of cSiO2 treated mice fed CON diet. CXCL13 is preferentially produced by fDCs in B-cell follicles of lymphoid organs [72], and to some extent, by T follicular helper cells [96,97] and TH17 cells [98]. In recent years, experimental anti-CXCL13 antibodies have shown promising results on murine models of 261 rheumatoid arthritis and multiple sclerosis [99], and thus, clinical trials that block CXCL13 are urgently awaited. In addition, chemokines that recruit and direct the position of activated T-cells, namely CXCL9, CXCL10, CXCL11, and CXCL16, and receptors for these chemokines, CXCR3 and CXCR6, were identified in the lungs of cSiO2-exposed NZBWF1 mice and attenuated by dietary DHA. Taken together, the identification of these chemokines and their receptors support our model of continual and persistent lymphocyte recruitment following airway exposure to cSiO2 that can be attenuated by dietary DHA. Many of the upregulated genes induced by intranasal cSiO2 exposure are molecules present on activated lymphocytes and by antigen presenting cells, which include dendritic cells, B-cells, and monocytes. In a previous study conducted by our laboratory [60], we demonstrated that feeding DHA-enriched fish oil attenuated spontaneous development of autoimmunity. This was associated with downregulated expression of CD4 T-cell related genes in the kidney at 34 wks of age in female, NZBWF1 mice. Here, we show that the effects of DHA are not solely limited to the kidney, but can be expanded to include effects on B- and T- cells in the lung following cSiO2 exposure that attenuate downstream kidney inflammation. Upregulation of adhesion molecules that facilitate cellular infiltration are commonly observed in renal biopsies from individuals with lupus nephritis [100–102]. Interestingly, these were upregulated in the kidney by airway cSiO2 exposure in lupus-prone mice fed CON diet. Several cell adhesion molecules expressed by infiltrating lymphocytes (ITGA4, ITGAL) as well as endothelial cells (e.g. SELE, SELPLG, VCAM-1, ICAM-1) that collectively promote cellular infiltration were decreased by feeding DHA. The ability of DHA to prevent upregulation of adhesion molecules in the kidney induced by cSiO2 exposure is a probable explanation for the suppression of cellular infiltration observed with histopathological observations in the kidney. 262 The expression of several cytokines that are of critical relevance to the formation and maintenance of lymphoid tissues were upregulated in lungs, spleen and kidney of cSiO2-treated mice fed CON diet and blunted by dietary DHA. Cytokines and their receptors identified here include lymphotoxin-α, IL-21, IL-21R, TNFSF11 (RANKL), TNFRSF11A (RANK or TRANCE), and IL-6. Lymphotoxin-α, otherwise known as TNF-β, and RANKL, which is induced by IL-21 [103], is critical to formation of lymphoid tissues. Mice deficient in lymphotoxin alpha or RANKL fail to properly develop lymph nodes and Peyer’s patches [104,105]. IL-21/IL-21R blockade has shown positive effects in preclinical trials in murine models of rheumatoid arthritis and SLE and was associated with decreased IL-6, which likely contributed to decreased autoantibodies observed in these studies [106,107]. In support of this contention, in murine models of rheumatoid arthritis, a combination of IL-6 and IL-21 blockade was more effect than either therapy alone on attenuation of Th17-drived autoimmunity [108]. Taken together, dietary DHA attenuated several key factors that are implicated in ELT formation that were upregulated by cSiO2 and importantly, could be applied as ‘catch-all’ approach to attenuate many aberrant pathways that collectively influence the pathogenesis of SLE. The IL-1 superfamily is a key component of the innate immune system and functions to rapidly initiate an inflammatory response following toxic stimuli via several mechanisms. IL-1α, IL-1β, the decoy receptor IL1R2, and the receptor antagonist, IL1RN, were upregulated by exposure to cSiO2 in lungs of NZBWF1 mice. Both IL-1α and IL-1β can be released by dying cells. Cells dying by necrosis release IL-1α, which serves as an alarmin to alert the immune system of an inflammatory stimulus. Cells dying by pyroptosis in an NLRP3 inflammasome-dependent manner release IL-1β by way of caspase-1 activation. Caspase-1, IL-1 β and IRAK3 were upregulated in the kidneys of cSiO2-treated NZBWF1 mice in parallel with IL-1β in this study. 263 Aberrant IL-1 signaling and inflammasome activation is suspected to contribute to the pathogenesis of autoimmune diseases such as SLE (reviewed in [109]). Importantly, DHA and its bioactive metabolites have been previously demonstrated to attenuate inflammasome activation [110,111], and thus may represent a central mechanism by which DHA alleviates cSiO2-triggered inflammation and systemic autoimmunity in lupus-prone NZBWF1 mice. The contribution of complement towards pathogenesis of SLE is complex; simple models of deficiency or excess are insufficient to explain all aspects of complement that are dysregulated in SLE. In this study, we identified upregulation of the classical complement pathway (e.g. C1qb and C1qa) and alternative complement pathway (e.g. Cfb and Cfp). Homozygous deficiency of C1q, albeit rare, is the number one predictive factor towards development of SLE [112]; upwards of 85-90%% of individuals with homozygous C1q deficiency develop SLE or SLE-like symptoms [113]. In this regard, upregulation of C1q by cSiO2 would seem contradictory to the contention that complement deficiency mediates SLE. However, one proposed explanation for the increased susceptibility of individuals with C1q deficiency to SLE is attributable to defective efferocytosis [114]. C1q can opsonize apoptotic cells, thus targeting them for removal by phagocytes that express CR1/CR2. Others have demonstrated that C1q-mediated efferocytosis contributes towards a pro-resolving, M2 macrophage phenotype [115]. Indeed, herein, we identified macrophage markers consistent with those of M2 phenotype, including Chil3 and Arg2 [116,117]. While speculative, one explanation for the upregulation of C1q in this study is that this is an artifact of heavy particle burden in lungs of cSiO2 exposed mice, thereby resulting in excessive apoptosis and a compensatory increase in C1q to promote efferocytosis of cell corpses. On the other hand, C1q upregulation following cSiO2 exposure could be attributable to unresolved inflammation; the 264 observation that DHA decreased the expression of complement system components upregulated by cSiO2 in parallel with the evident suppression of inflammation, support this idea. Type I and type II IFN constitute the “IFN signature”; the IFN signature is strongly associated with SLE in up to 65% of patients with SLE and correlates with disease severity [11,118]. The panel of genes used to define the IFN signature varies by laboratory, however, we observed striking overlap in the IFN signature with Li et al. 2010 (Table 23), who further correlated the IFN signature with autoantibody responses in the same patients [14]. Much to our excitement, members of this laboratory are our collaborators in this study, so further statistical analysis on these putative correlations between our mRNA data here and autoantibody profiles in figures X and Y are anxiously awaited. Type I IFNs (i.e. IFN-α and IFN-β) have been established to be pathogenic in SLE (reviewed in [119]). In addition, emerging evidence in recent years has implicated type II IFN (i.e. IFN-γ) in SLE [120–122]. The main cellular source of Type I IFN are plasmacytoid dendritic cells which is dependent on IRF4 activation of TLR7/9 [123,124]; these genes were upregulated in lung (IRF4) and kidney (TLR7/9) by cSiO2 exposure in CON fed mice. They have been widely implicated in autoimmunity [124] and of particular relevance here, in ELT of pristane-induced of autoimmunity [125]. Furthermore, exogenous administration of IFN-α to NZBWF1 mice accelerated disease onset [126,127] and decreased the efficacy of pharmacological interventions [128]. Type II IFN, consisting of the sole family member IFN-γ, is secreted predominately by NK and T-cells. IFN-γ heightens adaptive immunity by increased antigen presentation through enhanced expression of MHC II molecules [129] and immunoglobulin production [122]. Exogenous administration of IFN-γ to lupus-prone NZBWF1 mice accelerates glomerulonephritis [130]; IFN-γ receptor deletion attenuated renal injury and autoantibody [126]. 265 Table 23. Identification of a DHA-attenuated mRNA IFN-signature induced in cSiO2-treated NZBWF1 mice fed CON diet. mRNA was measured using the nCounter Mouse PanCancer Immune Panel in this study were compared to IFN signatures previously identified in individuals with SLE by Li et al. 2010. mRNA expression data was considered for all time points in all tissues analyzed. Criteria for inclusion was log2(fold change) ≥ ± 1.2 and p < 0.05. N/a indicates gene is not applicable as it was not included in the mRNA analysis of the present study; yes/no indicates if gene was expressed. Table 23 (cont’d) IFN-signature in cSiO2-exposed NZBWF1 mice fed CON or 1.0% DHA Identified in human IFN signature by Li et al. 2010a Expressed in cSiO2-treated NZBWF1 mice fed CONc Expressed in cSiO2-treated NZBWF1 mice fed 1.0% DHAc TOR1B N/a N/a FAM3B N/a N/a USP18 Yes No SIGLEC-1 Yes No IFIT5 N/a N/a IFRG28 N/a N/a PRKR N/a N/a OASL Yes No LGALS3 Yes No IFIHI N/a N/a C1QB Yes No 266 Table 23 (cont’d) IFN-signature in cSiO2-exposed NZBWF1 mice fed CON or 1.0% DHA Identified in human IFN signature by Li et al. 2010a Expressed in cSiO2-treated NZBWF1 mice fed CONc Expressed in cSiO2-treated NZBWF1 mice fed 1.0% DHAc GBP1 N/a N/a IFI44 Yes No GBP2 No No CARD15 N/a N/a SOCS1 Yes No XAF1 Yes No OAS2 Yes No ABCA1 N/a N/a PLSCR1 N/a N/a ISG95 N/a N/a RIGI N/a N/a LGP2 N/a N/a GADD45B N/a N/a PARP9 N/a N/a NT5C3 No No STAT1 Yes No TRIM22 N/a N/a SERPING1 No No 267 Table 23 (cont’d) IFN-signature in cSiO2-exposed NZBWF1 mice fed CON or 1.0% DHA Identified in human IFN signature by Li et al. 2010a Expressed in cSiO2-treated NZBWF1 mice fed CONc Expressed in cSiO2-treated NZBWF1 mice fed 1.0% DHAc NUB1 No No CLORF29 N/a N/a OAS3 Yes No GBP5 Yes No IFIT2 No No IFIT1 Yes No IL1RN Yes No OAS1 N/a N/a OASL1 Yes No ZBP1 Yes No CEB1 No No BST2 Yes No IFI27 No No ESPTI1 N/a N/a IFI35 No No TAP1 Yes No SP110 N/a N/a STAT1 Yes No 268 Table 23 (cont’d) IFN-signature in cSiO2-exposed NZBWF1 mice fed CON or 1.0% DHA Identified in human IFN signature by Li et al. 2010a Expressed in cSiO2-treated NZBWF1 mice fed CONc Expressed in cSiO2-treated NZBWF1 mice fed 1.0% DHAc IFIT4 N/a N/a CIG5 N/a N/a STAT2 No No MX2 Yes No IRF7 Yes No SCOTIN N/a N/a MT2A N/a N/a MX1 Yes No ADAR N/a N/a GIP2 N/a N/a ISG20 No No LY6E N/a N/a G1P3 N/a N/a IFITM1 No No Percentage: 66.60% 0.00% a citation: [14] b indicates gene was included in nCounter Mouse PanCancer Immune Profiling Panel 269 Table 23 (cont’d) IFN-signature in cSiO2-exposed NZBWF1 mice fed CON or 1.0% DHA Identified in human IFN signature by Li et al. 2010a c Expressed in cSiO2-treated NZBWF1 mice fed CONc indicates expression in mRNA gene expression analysis in present study 270 Expressed in cSiO2-treated NZBWF1 mice fed 1.0% DHAc Collectively, our findings implicate a pathogenic role for type I and/or type II IFN in cSiO2triggered SLE in NZBWF1 mice. However, defining the predominate IFN class that was affected by cSiO2 is difficult owing to the promiscuous nature of cytokines that induce IFN and IFNstimulated genes. The capacity of cytokine stimulation to induce either type I or type II IFN in vitro is dependent on cell type [131,132], and in many instances, both type I and type II IFN stimulate the same genes, albeit with varying potency [11,133]. Nevertheless, cSiO2 upregulated expression of IFN and IFN-stimulated gene expression were dramatically suppressed by DHA, and future studies can be conducted that address the limitation described above. DHA induced modification of IFN signaling is poorly described in the literature relative to other immunological alterations induced by DHA. Scarce studies on the effect of feeding DHA on IFN-γ are inconsistent regarding the pattern of expression by DHA [134–137], and thus may indicate that the effect of DHA on IFN-γ or its receptor is cell-type specific. No studies have investigated the effect of DHA on plasmacytoid dendritic cells or IFN-α in the context of immune-related outcomes. One explanation for the lack of studies on IFN and DHA could lie in potentially negative outcomes associated with DHA related to decreased IFN in the context of viral infection. This represents a problem that must be considered for all immunosuppressive agents, including DHA: a careful balance must exist between the necessity for host immunity and control of systemic inflammation caused by other conditions. Nevertheless, future investigations should exploit the immense potential for the utility of DHA on inflammatory diseases elicited in part by excess IFN, including autoimmune diseases. This is the first time that nCounter technology has been applied in lupus-prone mice. We employed this targeted transcriptomic approach to elucidate immunological changes following cSiO2 exposure that are attenuated by dietary DHA. nCounter has been repeatedly demonstrated 271 to correlate well other microarray platforms [138,139]. Nevertheless, qRT-PCR remains the goldstandard approach, therefore validation of select targets by qRT-PCR are currently being conducted at the time of this writing. Irrespective of the approach, gene expression profiling is not without its limitations. Targeted transcriptome approaches such as nCounter are inherently biased as they do not globally profile mRNA changes and instead focus on a subset of genes, usually identified by whole RNA transcriptome profiling methods [138]. As a result, analytical approaches designed for discovery, such as gene ontology, are limited in application as identification of enriched genes is difficult considering that the targeted approach is itself enriched for specific genes. However, global RNA profiling methods, such as RNASeq are cost-prohibitive. To decrease cost, investigators often limit the number of samples profiled, thus decreasing statistical power and potentially obscuring more subtle gene changes that influence the observed phenotype. Also, transcriptomic profiling on cSiO2-triggered inflammation in whole tissue homogenates limits the interpretation of results for several reasons: 1) it does not allow for the discrimination that the observed suppression by DHA on cSiO2-triggered inflammation is not simply attributable to the decreased infiltration of lymphocytes as opposed to DHA-mediated attenuation of transcription and 2) cannot attribute a mRNA signature to a given cell type. Nevertheless, the targets identified herein can be employed as endpoints in future studies that address these limitations by several approaches such as normalizing the number of cells to experimental endpoints with reductionist in vitro models or flow cytometry, or performing mRNA analyses on isolated populations obtained by cell separation columns or by laser dissection of lesions of interest from FFPE histopathological samples [23]. 272 Conclusions: Targeted transcriptomic In summary, our findings reveal novel mRNA signatures targeted by DHA that ablate the progression of cSiO2-triggered autoimmunity in NZBWF1 mice over time. Given the marked upregulation of inflammatory gene expression in kidney of cSiO2-exposed NZBWF1 mice fed CON diet at 13 wk post final exposure, it will be critical to assess gene expression in the kidney at earlier time points to identify cSiO2-triggered pathways to nephritis. Of particular interest and relevance is the discovery of early and sustained IFN signaling in cSiO2-exposed NZBWF1 mice that was 1) closely related to the IFN signature in individuals with SLE [66] and 2) completely suppressed by dietary DHA. These observations warrant future studies to investigate the unexplored potential of DHA to block aberrant IFN networks in lupus and other autoimmune diseases. Results: Targeted proteome microarray Targeted proteome microarray analysis identifies novel biomarkers of cSiO 2-triggered pulmonary and systemic inflammation decreased by DHA mRNA expression analysis revealed heightened immune activation by cSiO2 in the lungs, spleen, and kidney of NZBWF1 mice that was attenuated by dietary DHA at the transcription level. We next sought to investigate the effect of cSiO2 and DHA at the translation level of immunerelated proteins by using a targeted proteome microarray in BALF and plasma of NZBWF1 mice. Due to cost constraints, BALF was analyzed at 1, 5, 9, and 13 wks post final exposure to cSiO2 in VEH-treated mice fed CON, cSiO2-treated mice fed CON, and cSiO2-treated mice fed 1.0% DHA (n = 5/gp). Plasma was analyzed at 5, 9, and 13 wks post final exposure to cSiO2. 273 To facilitate interpretation of experimental results, the 200 proteins included in RayBiotech Quantibody Mouse Cytokine Array 4000 were manually assigned a functional category that best described the protein function. Statistically significant (p < 0.05) proteins elicited by cSiO2 exposure in BALF of NZBWF1 mice 1, 5, 9, and 13 wks post final exposure are summarized in Tables 24, 25, 26, and 27 respectively, and plasma at 5, 9, and 13 wks post final exposure is summarized in Table 28. In general, the total number of proteins significantly altered by cSiO2 exposure in mice fed CON diet increased in BALF over time: 65 proteins at 1 wk post final exposure, 60 proteins at 5 wk post final exposure, 82 proteins at 9 wk post final exposure and 82 proteins at 13 wk post final exposure when compared to VEH-treated mice fed CON diet. In comparison, mice given cSiO2 fed 1.0% DHA had fewer total proteins significantly altered: 57 proteins at 1 wk post final exposure, 49 proteins at 5 wk post final exposure, 57 proteins at 9 wk post final exposure, and 68 proteins at 13 wk post final exposure when compared to VEH-treated mice fed CON diet. These findings are summarized graphically in Fig. 52. Overall, cSiO2 increased protein expression relative to VEH treated mice, and mice fed DHA given cSiO2 had decreased protein expression compared to cSiO2 -treated mice fed CON diet. To determine the individual contribution of protein functional categories towards the observed phenotype in cSiO2 \-treated mice, the following formula was used: % effect of cSiO2 on functional category = ( (# of proteins identified within a group) ) × 100 (total # of proteins within a group) To determine the effect of dietary DHA on cSiO2-induced protein expression, proteins identified within cSiO2-treated mice fed 1.0% DHA that were not statistically different from expression leels expression levels in VEH-treated mice fed CON diet were considered. Here, the 274 Figure 52. Summary of proteins altered in cSiO2-treated mice fed CON and cSiO2-treated fed 1.0% DHA in BALF (A) and plasma (B) over time. n.d. indicates not determined. 275 following formula was used to classify and categorize protein functional groups induced by cSiO2 that were also impacted by feeding dietary DHA in female, lupus-prone NZBWF1 mice: % effect of DHA on functional category = ( (# of proteins not significantly different within a group) ) ×100 (total # of proteins upregulated by cSiO2 within a group) Targeted proteome microarray identifies protein signatures of cSiO 2-triggered inflammation decreased by DHA in BALF over time At 1 wk post final exposure to cSiO2 (Table 24) in CON fed mice, the rank order for protein functional categories in BALF is as follows and individual percentages indicated in parentheses: adhesion molecules (72.7%) > enzymes (50.0%) > chemokines (43.3%) > signal transduction (38.7%) > growth factors (31.7%) > co-stimulatory molecules (28.6%) > TNF superfamily (22.7%) > cytokines (5.3%). In BALF at 1 wk post final exposure to cSiO2, the rank order for protein functional categories effected by DHA is as follows and individual percentages indicated in parentheses: growth factors (30.7%) > chemokines (15.4%) > adhesion molecules (12.5%) > signal transduction (8.3%) > cytokines (0%) = enzymes (0%) = TNF superfamily (0%). At 5 wk post final exposure to cSiO2 (Table 25), the rank order for protein functional categories in the BALF induced by cSiO2 in CON fed mice is as follows: adhesion molecules (72.7%) > enzymes (45.0%) > chemokines (43.3%) > co-stimulatory molecules (42.9%) > growth factors (34.1%) > signal transduction (29.0%) > TNF superfamily (13.6%) > cytokines (3.3%). For DHA at this time point, co-stimulatory molecules were most effected (33.0%) > growth factors (29.0%) > chemokines (23%) > enzymes (22%) > adhesion molecules (12.5%) > cytokines (0%) = TNF superfamily (0%). At 9 wk post final exposure to cSiO2 (Table 26), the rank order for protein functional categories induced by cSiO2 in the BALF is as follows: adhesion molecules (82.8%) > chemokines 276 Table 24. Targeted proteome analysis of BALF at 1 wk post final exposure to cSiO 2. Protein expression data were measured by RayBiotech Quantibody Mouse Cytokine Array 4000. Data are expressed as log2(FC) of cSiO2-treated mice fed CON or 1.0% DHA relative to VEH-treated mice fed CON. P-values < 0.05 were considered statistically significant. FC indicates fold change. Underlined target names and p-value indicate protein is not statistically different between cSiO2-treated mice fed 1.0% DHA and VEH-treated mice fed CON diet. Table 24 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 1 wk post final exposure in BALF cSiO2 + CON Functional Category Target name Protein name Adhesion molecule L-Selectin Chemokine cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value L-selectin 2.96 2.0E-04 2.29 1.0E-04 VCAM-1 Vascular cell adhesion protein 1 2.77 1.0E-04 1.81 1.0E-04 E-Selectin E-selectin 1.79 1.0E-04 1.64 1.0E-04 Periostin Periostin 1.51 6.0E-04 0.70 8.0E-04 ICAM-1 Intercellular adhesion molecule 1 0.69 1.7E-02 0.96 1.0E-04 JAM-A Junctional adhesion molecule A 0.56 1.1E-02 0.81 5.0E-04 E-Cadherin Cadherin-1 0.42 3.5E-03 0.15 1.6E-01 P-Selectin P-selectin 0.34 1.5E-02 0.19 3.2E-02 KC Growth-regulated alpha protein 3.07 1.0E-04 2.92 6.0E-04 LIX C-X-C motif chemokine 5 2.68 1.0E-04 3.20 1.0E-04 277 Table 24 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 1 wk post final exposure in BALF cSiO2 + CON Functional Category Co-stimulatory molecule Cytokine Enzyme Target name Protein name TARC cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value C-C motif chemokine 17 2.54 1.0E-04 3.67 1.0E-04 MDC C-C motif chemokine 22 2.41 1.0E-04 3.07 1.0E-04 CCL6 C-C motif chemokine 6 1.79 4.0E-04 2.20 1.0E-04 Lungkine C-X-C motif chemokine 15 1.68 3.8E-03 1.52 6.0E-04 MIP-1G C-C motif chemokine 9 1.67 1.0E-04 1.87 1.0E-04 MIP-1B C-C motif chemokine 4 1.49 9.0E-04 0.41 5.7E-03 C5a Complement C5 1.30 1.2E-03 1.52 1.0E-04 MCP-5 C-C motif chemokine 12 0.92 1.9E-02 0.00 n.d BLC C-X-C motif chemokine 13 0.77 3.3E-02 0.46 6.0E-04 CXCL16 C-X-C motif chemokine 16 0.60 2.9E-03 0.88 1.0E-04 MIP-3B C-C motif chemokine 19 0.28 9.7E-03 0.23 1.5E-01 CD6 T-cell differentiation antigen CD6 2.68 1.0E-04 1.88 1.0E-04 CD48 CD48 antigen 1.46 1.0E-04 1.07 1.0E-04 IL-1RA Interleukin-1 receptor antagonist protein 4.04 1.0E-04 3.46 1.0E-04 OPN Osteopontin -0.34 3.0E-03 -0.17 3.1E-02 Pro-MMP-9 Matrix metalloproteinase-9 7.17 1.0E-04 7.04 1.0E-04 MMP-3 Stromelysin-1 4.84 2.0E-04 3.59 1.0E-04 278 Table 24 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 1 wk post final exposure in BALF cSiO2 + CON Functional Category Growth factor Target name Protein name AXL cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value Tyrosine-protein kinase receptor UFO 2.88 1.0E-04 1.88 1.0E-04 ACE Angiotensin-converting enzyme 1.71 1.0E-04 1.41 1.0E-04 MMP-2 72 kDa type IV collagenase 1.32 8.0E-04 0.88 2.0E-04 RAGE MAPK/MAK/MRK overlapping kinase 0.86 5.8E-03 1.42 1.5E-03 MMP-10 Stromelysin-2 0.77 1.6E-03 0.64 5.4E-03 Cystatin C Cystatin-C 0.62 1.7E-03 0.75 1.0E-04 Renin 1 Renin-1 0.53 2.0E-02 0.52 9.0E-04 Neprilysin Neprilysin 0.20 2.1E-02 0.53 2.2E-02 Progranulin Granulins 3.85 2.8E-03 2.76 1.0E-04 IGF-1 Insulin-like growth factor I 2.64 1.9E-03 0.86 2.8E-03 PDGF-AA Platelet-derived growth factor subunit A 2.08 1.0E-04 2.24 1.0E-04 IGFBP-3 Insulin-like growth factor-binding protein 3 1.93 1.0E-04 1.34 1.0E-04 PLGF-2 Placenta growth factor 1.83 5.0E-04 1.23 3.1E-03 Decorin Decorin 1.64 7.8E-03 0.46 4.5E-01 Fetuin A Alpha-2-HS-glycoprotein 1.53 4.1E-02 0.82 9.8E-02 VEGF Vascular endothelial growth factor A 0.97 2.9E-03 1.44 1.0E-04 ADIPOQ Adiponectin 0.92 1.0E-04 0.98 1.0E-04 Gas 6 Growth arrest-specific protein 6 0.88 6.6E-03 0.96 3.3E-03 279 Table 24 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 1 wk post final exposure in BALF cSiO2 + CON Functional Category Signal transduction TNF superfamily cSiO2 + 1.0% DHA Target name Protein name log2(FC) p-value log2(FC) p-value Epiregulin Proepiregulin 0.76 3.1E-02 0.10 6.6E-02 IGFBP-2 Insulin-like growth factor-binding protein 2 0.73 3.2E-02 1.05 1.0E-04 VEGF R1 Vascular endothelial growth factor receptor 1 0.34 3.5E-02 0.10 2.0E-01 NOV Protein NOV homolog 5.20 1.0E-04 5.19 1.0E-04 Osteoactivin Transmembrane glycoprotein NMB 2.53 1.0E-04 2.06 1.0E-04 FCG RIIB Low affinity immunoglobulin gamma Fc region receptor II 2.48 1.4E-03 2.10 1.0E-04 Galectin-1 Galectin-1 2.45 1.0E-04 1.70 1.0E-04 Resistin Resistin 2.21 8.0E-04 1.63 1.0E-04 Clusterin Clusterin 1.04 1.1E-03 0.99 6.0E-04 MBL-2 Mannose-binding protein C 1.02 6.7E-03 0.58 1.5E-03 CD36 Platelet glycoprotein 4 0.97 8.9E-03 0.90 1.3E-03 MFG-E8 Lactadherin 0.93 5.3E-03 1.30 2.3E-03 Nope Immunoglobulin superfamily DCC subclass member 4 0.56 2.0E-04 0.62 2.0E-04 CRP C-reactive protein 0.43 4.2E-02 0.32 1.0E-03 Testican 3 Testican-3 0.37 9.0E-04 0.44 1.6E-01 TNF RII Tumor necrosis factor receptor superfamily member 1B 3.24 1.0E-04 2.97 1.0E-04 OPG Tumor necrosis factor receptor superfamily member 11B 3.08 9.9E-03 2.16 1.0E-04 TNF RI Tumor necrosis factor receptor superfamily member 1A 1.88 1.0E-04 1.75 1.0E-04 280 Table 24 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 1 wk post final exposure in BALF cSiO2 + CON Functional Category Target name Protein name EDAR TRAIL cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value Tumor necrosis factor receptor superfamily member EDAR 0.99 1.0E-04 1.01 3.0E-04 Tumor necrosis factor ligand superfamily member 10 0.67 1.4E-02 0.63 2.4E-02 281 Table 25. Targeted proteome analysis of BALF at 5 wk post final exposure to cSiO 2. Protein expression data were measured by RayBiotech Quantibody Mouse Cytokine Array 4000. Data are expressed as log2(FC) of cSiO2-treated mice fed CON or 1.0% DHA relative to VEH-treated mice fed CON. P-values < 0.05 were considered statistically significant. FC indicates fold change. Underlined target names and p-value indicate protein is not statistically different between cSiO2-treated mice fed 1.0% DHA and VEH-treated mice fed CON diet. Table 25 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 5 wk post final exposure in BALF cSiO2 + CON Functional Category Target name Protein name Adhesion molecule VCAM-1 Chemokine cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value Vascular cell adhesion protein 1 4.00 2.0E-04 3.36 1.0E-04 L-Selectin L-selectin 3.72 1.1E-02 2.84 1.0E-04 Periostin Periostin 2.85 5.4E-03 2.04 1.0E-04 E-Selectin E-selectin 2.43 2.6E-03 2.02 3.0E-04 P-Selectin P-selectin 1.61 6.0E-04 1.17 1.6E-02 ICAM-1 Intercellular adhesion molecule 1 1.51 1.0E-04 1.32 1.0E-04 P-Cadherin Cadherin-3 0.89 1.0E-04 0.58 2.2E-01 E-Cadherin Cadherin-1 0.43 1.4E-02 0.49 2.0E-04 KC Growth-regulated alpha protein 4.46 8.0E-04 4.28 1.0E-04 LIX C-X-C motif chemokine 5 3.09 7.0E-04 3.35 1.0E-04 282 Table 25 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 5 wk post final exposure in BALF cSiO2 + CON Functional Category log2(FC) p-value log2(FC) p-value C-C motif chemokine 22 2.63 6.7E-03 3.18 1.0E-04 MIP-1B C-C motif chemokine 4 2.51 2.3E-02 1.06 1.0E-04 MIP-1G C-C motif chemokine 9 2.38 1.0E-04 2.20 1.0E-04 TARC C-C motif chemokine 17 2.27 9.0E-04 3.81 1.0E-04 CCL6 C-C motif chemokine 6 2.18 1.8E-03 2.22 1.0E-04 C5a Complement C5 2.04 7.0E-04 2.10 1.0E-04 Lungkine C-X-C motif chemokine 15 1.82 8.3E-03 1.63 1.7E-03 MIP-3A C-C motif chemokine 20 0.88 4.8E-02 0.70 1.4E-01 MIP-3B C-C motif chemokine 19 0.67 1.0E-03 0.21 1.4E-02 MIP-1A C-C motif chemokine 3 0.49 4.6E-02 0.50 6.6E-02 Eotaxin Eotaxin 0.39 4.0E-02 0.08 2.5E-01 CD48 CD48 antigen 2.24 4.0E-04 1.31 1.0E-04 CD6 T-cell differentiation antigen CD6 1.65 7.6E-03 0.52 2.1E-01 CD40L CD40 ligand 0.81 1.0E-02 0.37 3.9E-02 Cytokine IL-1RA Interleukin-1 receptor antagonist protein 4.74 9.0E-04 4.32 1.0E-04 Enzyme Pro-MMP-9 Matrix metalloproteinase-9 7.45 4.0E-04 7.57 1.0E-04 MMP-3 Stromelysin-1 5.08 3.0E-04 3.82 1.0E-04 AXL Tyrosine-protein kinase receptor UFO 3.22 8.6E-03 2.50 4.0E-04 Co-stimulatory molecule Target name Protein name MDC cSiO2 + 1.0% DHA 283 Table 25 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 5 wk post final exposure in BALF cSiO2 + CON Functional Category Growth factor Target name Protein name ACE cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value Angiotensin-converting enzyme 1.93 4.8E-02 1.41 1.0E-04 MMP-2 72 kDa type IV collagenase 1.31 5.5E-03 0.65 5.0E-03 RAGE MAPK/MAK/MRK overlapping kinase 1.25 1.5E-03 1.74 2.5E-03 MMP-10 Stromelysin-2 1.02 2.3E-02 0.01 9.3E-01 Renin 1 Renin-1 0.70 7.3E-03 0.53 2.8E-02 Granzyme B Granzyme B 0.34 2.1E-02 0.00 1.0E+00 IGF-1 Insulin-like growth factor I 4.42 6.0E-03 2.79 8.0E-04 Progranulin Granulins 3.71 6.0E-03 2.68 5.9E-03 IGFBP-3 Insulin-like growth factor-binding protein 3 2.61 2.6E-03 1.97 1.0E-04 Fetuin A Alpha-2-HS-glycoprotein 2.30 1.1E-02 1.05 6.1E-02 Decorin Decorin 1.97 2.2E-02 0.54 3.1E-01 PLGF-2 Placenta growth factor 1.64 2.3E-03 0.86 1.0E-02 PDGF-AA Platelet-derived growth factor subunit A 1.51 4.9E-03 2.10 1.0E-04 Epiregulin Proepiregulin 1.50 3.4E-02 0.00 1.0E+00 VEGF Vascular endothelial growth factor A 1.14 4.5E-02 2.18 5.5E-03 IGFBP-2 Insulin-like growth factor-binding protein 2 0.87 4.2E-02 1.56 1.0E-04 ADIPOQ Adiponectin 0.74 2.0E-04 0.74 9.0E-04 Gas 1 Growth arrest-specific protein 1 0.53 3.7E-02 0.07 2.2E-01 284 Table 25 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 5 wk post final exposure in BALF cSiO2 + CON Functional Category Signal transduction TNF superfamily Target name Protein name IGFBP-5 cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value Insulin-like growth factor-binding protein 5 0.48 1.5E-03 0.12 1.3E-02 G-CSF Granulocyte colony-stimulating factor 0.46 3.2E-02 0.03 3.5E-01 NOV Protein NOV homolog 4.23 5.2E-03 5.00 3.0E-04 FCG RIIB Low affinity immunoglobulin gamma Fc region receptor II 3.09 1.7E-03 2.46 3.0E-04 LGALS1 Gaectin-1 2.95 3.0E-04 2.02 1.0E-04 Osteoactivin Transmembrane glycoprotein NMB 2.80 4.0E-03 2.30 1.0E-04 Resistin Resistin 2.41 3.0E-04 1.76 6.0E-04 MFG-E8 Lactadherin 1.98 6.7E-03 2.27 1.0E-04 MBL-2 Mannose-binding protein C 1.85 1.0E-04 1.66 1.5E-03 Clusterin Clusterin 1.35 2.0E-04 0.88 3.9E-03 CRP C-reactive protein 0.75 1.0E-04 0.71 1.0E-04 TNF RII Tumor necrosis factor receptor superfamily member 1B 3.51 2.0E-04 3.27 1.0E-04 OPG Tumor necrosis factor receptor superfamily member 11B 3.29 9.7E-03 3.12 3.1E-03 TNF RI Tumor necrosis factor receptor superfamily member 1A 2.03 7.0E-04 2.05 1.0E-04 285 Table 26. Targeted proteome analysis of BALF at 9 wk post final exposure to cSiO 2. Protein expression data were measured by RayBiotech Quantibody Mouse Cytokine Array 4000. Data are expressed as log2(FC) of cSiO2-treated mice fed CON or 1.0% DHA relative to VEH-treated mice fed CON. P-values < 0.05 were considered statistically significant. FC indicates fold change. Underlined target names and p-value indicate protein is not statistically different between cSiO2-treated mice fed 1.0% DHA and VEH-treated mice fed CON diet. Table 26 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 9 wk post final exposure in BALF cSiO2 + CON Functional Category Target name Protein name Adhesion molecule L-Selectin Chemokine cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value L-selectin 4.23 1.0E-04 3.45 5.0E-04 VCAM-1 Vascular cell adhesion protein 1 4.05 1.0E-04 3.25 2.0E-04 Periostin Periostin 3.63 2.0E-04 2.57 3.1E-03 E-Selectin E-selectin 3.13 1.1E-03 2.16 1.0E-04 P-Selectin P-selectin 2.36 4.1E-02 1.80 3.7E-02 P-Cadherin Cadherin-3 1.96 1.5E-02 0.90 1.3E-03 ICAM-1 Intercellular adhesion molecule 1 0.86 1.8E-02 0.40 1.0E-04 E-Cadherin Cadherin-1 0.40 3.1E-03 0.25 4.4E-01 JAM-A Junctional adhesion molecule A 0.32 1.1E-02 0.18 5.3E-01 KC Growth-regulated alpha protein 4.08 1.0E-04 3.98 1.0E-01 286 Table 26 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 9 wk post final exposure in BALF cSiO2 + CON Functional Category Co-stimulatory molecule Target name Protein name LIX cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value C-X-C motif chemokine 5 3.84 2.0E-04 3.43 1.4E-02 BLC C-X-C motif chemokine 13 3.62 1.0E-02 1.03 2.0E-02 RANTES C-C motif chemokine 5 3.32 6.9E-03 1.64 3.5E-02 MIP-1B C-C motif chemokine 4 3.07 6.0E-04 2.12 1.7E-02 MCP-1 C-C motif chemokine 2 2.93 2.7E-02 2.26 4.5E-02 CCL6 C-C motif chemokine 6 2.85 1.0E-04 2.70 5.5E-03 MCP-5 C-C motif chemokine 12 2.70 5.0E-04 1.21 4.4E-02 TARC C-C motif chemokine 17 2.55 7.2E-03 3.10 2.5E-02 MIP-3A C-C motif chemokine 20 2.49 2.7E-03 1.01 6.1E-02 MIP-1G C-C motif chemokine 9 2.29 1.0E-04 1.97 3.0E-04 MDC C-C motif chemokine 22 2.24 2.5E-03 2.63 1.3E-02 C5a Complement C5 2.19 1.0E-04 2.15 9.9E-03 Lungkine C-X-C motif chemokine 15 1.36 6.0E-04 0.58 3.0E-01 Eotaxin Eotaxin 1.23 2.0E-04 0.41 5.3E-02 MIP-3B C-C motif chemokine 19 0.76 1.0E-04 0.33 9.3E-02 MIP-1A C-C motif chemokine 3 0.70 1.4E-02 0.82 3.7E-02 MIG C-X-C motif chemokine 9 0.52 1.1E-02 0.14 3.5E-01 CD48 CD48 antigen 2.90 1.0E-04 1.89 4.2E-03 287 Table 26 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 9 wk post final exposure in BALF cSiO2 + CON Functional Category Cytokine Enzyme Growth factor Target name Protein name CD6 cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value T-cell differentiation antigen CD6 2.77 2.4E-03 1.96 2.6E-03 B7-1 T-lymphocyte activation antigen CD80 1.10 7.8E-03 0.54 2.1E-01 CD40L CD40 ligand 0.90 3.2E-03 0.32 2.1E-01 IL-1RA Interleukin-1 receptor antagonist protein 5.23 1.0E-04 4.37 5.2E-03 IL-28 IFN lambda-2 0.65 4.8E-02 0.07 3.5E-01 gp130 Interleukin-6 receptor subunit beta 0.61 3.1E-02 0.19 8.2E-02 Pro-MMP-9 Matrix metalloproteinase-9 6.80 4.7E-03 7.23 8.7E-03 MMP-3 Stromelysin-1 5.92 1.0E-04 4.91 9.4E-03 AXL Tyrosine-protein kinase receptor UFO 3.42 2.0E-04 1.86 2.9E-02 ACE Angiotensin-converting enzyme 2.58 2.0E-04 1.69 3.2E-02 MMP-2 72 kDa type IV collagenase 2.44 1.0E-04 1.29 1.5E-03 MMP-10 Stromelysin-2 2.03 1.3E-03 0.97 4.9E-02 Renin 1 Renin-1 1.72 3.1E-03 1.25 3.0E-04 Granzyme B Granzyme B 0.70 1.1E-02 0.18 1.8E-02 FLT-3L Fms-related tyrosine kinase 3 ligand 0.66 9.3E-03 -0.03 9.0E-01 Neprilysin Neprilysin 0.58 1.4E-02 -0.07 2.0E-01 Progranulin Granulins 5.55 1.9E-03 4.48 1.2E-02 IGF-1 Insulin-like growth factor I 4.68 5.0E-04 3.05 6.2E-03 288 Table 26 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 9 wk post final exposure in BALF cSiO2 + CON Functional Category Signal transduction Target name Protein name IGFBP-3 cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value Insulin-like growth factor-binding protein 3 3.17 1.0E-04 2.14 5.0E-03 Decorin Decorin 3.11 2.0E-03 1.60 1.8E-01 Fetuin A Alpha-2-HS-glycoprotein 2.97 3.0E-04 1.78 1.3E-03 PLGF-2 Placenta growth factor 2.81 7.0E-04 1.93 4.0E-03 Epiregulin Proepiregulin 2.57 3.6E-03 0.66 2.9E-02 PDGF-AA Platelet-derived growth factor subunit A 1.48 1.1E-03 1.43 2.8E-02 VEGF Vascular endothelial growth factor A 1.34 2.1E-02 1.83 3.3E-02 ADPNQ Adiponectin 1.30 1.0E-04 1.10 1.0E-04 IGFBP-5 Insulin-like growth factor-binding protein 5 0.95 3.6E-03 0.58 2.0E-01 Gas 1 Growth arrest-specific protein 1 0.77 4.1E-03 0.41 1.5E-01 VEGF R1 Tumor necrosis factor receptor superfamily member EDAR 0.66 1.8E-02 0.03 3.5E-01 Gas 6 Growth arrest-specific protein 6 0.64 3.5E-02 0.62 1.5E-01 G-CSF Granulocyte colony-stimulating factor 0.58 1.4E-02 0.33 1.9E-01 IGFBP-2 Insulin-like growth factor-binding protein 2 0.58 3.2E-02 0.63 1.5E-01 HGF Hepatocyte growth factor 0.51 4.7E-02 0.03 1.9E-01 NOV Protein NOV homolog 4.38 1.3E-03 4.50 1.2E-02 Osteoactivin Transmembrane glycoprotein NMB 4.13 1.0E-04 3.53 5.9E-03 Resistin Resistin 3.61 1.2E-02 2.45 8.4E-03 289 Table 26 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 9 wk post final exposure in BALF cSiO2 + CON Functional Category TNF superfamily Target name Protein name FCG RIIB cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value Low affinity immunoglobulin gamma Fc region receptor II 3.23 1.0E-04 2.71 2.1E-02 Galectin-1 Galectin-1 2.97 1.0E-04 2.75 2.0E-04 MBL-2 Mannose-binding protein C 1.89 1.0E-04 2.10 5.0E-03 Clusterin Clusterin 1.82 1.0E-04 1.73 1.0E-04 Leptin Leptin 1.78 1.0E-04 0.50 1.8E-02 MFG-E8 Lactadherin 1.73 4.0E-04 2.02 1.7E-02 Nope Immunoglobulin superfamily DCC subclass member 4 1.66 3.0E-04 1.09 1.4E-03 Kremen-1 Kremen protein 1 1.35 1.3E-02 0.70 6.3E-02 DLL4 Delta-like protein 4 1.29 1.9E-02 1.06 1.8E-02 CRP C-reactive protein 0.96 1.0E-04 0.71 4.0E-04 Chemerin Retinoic acid receptor responder protein 2 0.82 8.8E-03 0.75 1.0E-04 Lipocalin-2 Neutrophil gelatinase-associated lipocalin 0.40 2.3E-02 0.51 1.3E-01 TNF RII Tumor necrosis factor receptor superfamily member 1B 4.02 2.0E-04 3.28 8.0E-03 OPG Tumor necrosis factor receptor superfamily member 11B 3.50 1.4E-03 2.84 5.3E-03 TNF RI Tumor necrosis factor receptor superfamily member 1A 2.73 1.0E-04 2.24 1.2E-02 TRAIL Tumor necrosis factor ligand superfamily member 10 1.05 4.2E-02 0.87 4.8E-02 CD40 Tumor necrosis factor receptor superfamily member 5 1.01 5.4E-03 0.38 3.7E-02 TWEAK R Tumor necrosis factor receptor superfamily member 12A 0.51 3.2E-02 -0.10 7.4E-01 290 chemokines (60.0%) > co-stimulatory molecules (57.7%) > enzymes (50.0%) > signal transduction (48.4%) > growth factors (42.5%) > TNF superfamily (27.3%) > cytokines (10.0%). For DHA at this time point, the rank order is as follows: cytokines (66.6%) > co-stimulatory molecules (50.0%) > growth factors (47.0%) > adhesion molecules (22.2%) > TNF superfamily (16.6%) > enzymes (10%) > signal transduction (6.6%). At 13 wk post final exposure to cSiO2 (Table 24), the rank order for protein functional categories induced by cSiO2 in BALF is as follows: adhesion molecules (72.7%) > chemokines (63.3%) > co-stimulatory molecules (57.0%) > enzymes (50.0%) > signal transduction (48.4%) > growth factors (36.6%) > TNF superfamily (31.8%) > cytokines (13.3%). For DHA at 13 wks post final exposure, cytokines were most effected (50.0%) > TNF superfamily (29.0%) > chemokines (26.0%) > signal transduction (20.0%) > enzymes (10.0%) > growth factors (6.6%) > adhesion molecules (0%) = co-stimulatory molecules (0%) (Table 27). Targeted proteome microarray identifies protein signatures of cSiO2-triggered inflammation decreased by DHA in plasma over time Effects of cSiO2 treatment on inflammatory protein expression in the plasma were expectedly more subtle: 9 proteins were altered at 5 wk post final exposure, 7 proteins at 9 wk post final exposure, and 15 proteins at 13 wk post final exposure when compared to VEH-treated mice fed CON diet. In comparison, mice given cSiO2 fed 1.0% DHA had dramatic alterations: 0 proteins were altered at 5 wk post final exposure, 1 protein at 9 wk post final exposure, and 2 proteins at 13 wk post final exposure when compared to VEH-treated mice fed CON diet. These findings are summarized in Table 28. 291 Table 27. Targeted proteome analysis of BALF at 13 wk post final exposure to cSiO 2. Protein expression data were measured by RayBiotech Quantibody Mouse Cytokine Array 4000. Data are expressed as log2(FC) of cSiO2-treated mice fed CON or 1.0% DHA relative to VEH-treated mice fed CON. P-values < 0.05 were considered statistically significant. FC indicates fold change. Underlined target names and p-value indicate protein is not statistically different between cSiO2-treated mice fed 1.0% DHA and VEH-treated mice fed CON diet. Table 27 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 13 wk post final exposure in BALF cSiO2 + CON Functional Category Target name Protein name Adhesion molecule L-Selectin Chemokine cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value L-selectin 4.69 1.1E-02 4.22 6.0E-04 VCAM-1 Vascular cell adhesion protein 1 4.43 1.0E-04 4.15 1.0E-04 Periostin Periostin 4.31 1.0E-04 3.55 1.0E-04 E-Selectin E-selectin 3.42 1.0E-04 2.55 4.6E-03 P-Selectin P-selectin 2.21 3.0E-04 1.70 2.9E-03 P-Cadherin Cadherin-3 1.77 1.1E-03 1.32 1.9E-02 JAM-A Junctional adhesion molecule A 0.48 2.1E-02 0.59 1.0E-04 E-Cadherin Cadherin-1 0.23 6.3E-03 0.25 1.9E-02 KC Growth-regulated alpha protein 5.10 1.0E-04 5.18 6.0E-04 LIX C-X-C motif chemokine 5 4.56 1.0E-04 4.39 1.0E-04 292 Table 27 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 13 wk post final exposure in BALF cSiO2 + CON Functional Category Co-stimulatory molecule Target name Protein name RANTES cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value C-C motif chemokine 5 4.24 2.2E-02 1.41 1.4E-01 BLC C-X-C motif chemokine 13 3.98 1.0E-04 2.20 9.6E-02 MCP-5 C-C motif chemokine 12 3.68 1.3E-02 1.93 5.4E-02 MIP-1B C-C motif chemokine 4 3.00 1.9E-03 3.16 4.4E-03 CCL6 C-C motif chemokine 6 2.91 3.0E-04 2.91 2.0E-04 MIP-3A C-C motif chemokine 20 2.83 3.7E-03 1.48 1.4E-02 TARC C-C motif chemokine 17 2.79 1.0E-04 3.55 1.0E-04 MIP-1G C-C motif chemokine 9 2.63 1.0E-04 2.58 1.0E-04 MCP-1 C-C motif chemokine 2 2.36 2.2E-02 3.04 1.0E-04 Lungkine C-X-C motif chemokine 15 2.20 2.0E-04 1.88 6.0E-04 MDC C-C motif chemokine 22 2.07 4.0E-04 2.79 1.0E-04 C5a Complement C5 1.74 1.0E-04 1.90 1.0E-04 Eotaxin Eotaxin 1.22 2.6E-03 0.48 5.5E-03 MIP-3B C-C motif chemokine 19 0.90 2.0E-04 0.38 4.2E-02 MIP-1A C-C motif chemokine 3 0.51 2.0E-04 1.51 5.8E-03 I-TAC C-X-C motif chemokine 11 0.40 1.0E-02 0.00 1.0E+00 TECK C-C motif chemokine 12 0.39 1.9E-02 0.30 6.0E-02 CD6 T-cell differentiation antigen CD6 3.02 9.5E-03 1.81 9.8E-03 293 Table 27 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 13 wk post final exposure in BALF cSiO2 + CON Functional Category Cytokine Enzyme Growth factor cSiO2 + 1.0% DHA Target name Protein name log2(FC) p-value log2(FC) p-value CD48 CD48 antigen 2.95 1.0E-04 2.20 6.0E-04 B7-1 T-lymphocyte activation antigen CD80 1.26 4.5E-03 0.62 1.2E-01 CD40L CD40 ligand 0.54 3.4E-03 0.68 1.6E-02 IL-1RA Interleukin-1 receptor antagonist protein 5.39 4.0E-04 5.06 5.0E-04 IL-28 IFN lambda-2 0.49 6.0E-04 0.16 2.9E-01 IL-7 RA Interleukin-7 receptor subunit alpha -0.30 2.0E-02 0.08 7.0E-01 OPN Osteopontin -0.30 2.0E-02 -0.49 1.0E-04 MMP-3 Stromelysin-1 6.14 1.0E-04 5.61 3.0E-04 Pro-MMP-9 Matrix metalloproteinase-9 6.06 1.0E-04 7.18 1.8E-03 AXL Tyrosine-protein kinase receptor UFO 4.21 1.0E-04 2.88 4.1E-03 ACE Angiotensin-converting enzyme 3.57 1.0E-04 3.37 2.0E-04 MMP-10 Stromelysin-2 2.60 2.7E-02 1.88 1.0E-04 MMP-2 72 kDa type IV collagenase 2.43 2.0E-04 1.70 5.0E-04 Renin 1 Renin-1 1.54 3.0E-04 1.05 1.4E-03 RAGE MAPK/MAK/MRK overlapping kinase 1.32 2.2E-03 1.46 7.4E-03 FLT-3L Fms-related tyrosine kinase 3 ligand 0.70 4.4E-03 0.39 8.6E-03 Granzyme B Granzyme B 0.58 1.7E-02 0.45 5.2E-02 IGFBP-1 Insulin-like growth factor I 6.39 1.0E-04 4.99 1.4E-02 294 Table 27 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 13 wk post final exposure in BALF cSiO2 + CON Functional Category Signal transduction Target name Protein name Progranulin cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value Granulins 5.30 2.0E-04 4.34 2.0E-04 Decorin Decorin 3.55 1.3E-03 1.97 2.9E-02 Fetuin A Alpha-2-HS-glycoprotein 3.48 1.0E-04 2.43 5.8E-03 IGFBP-3 Insulin-like growth factor-binding protein 3 3.43 1.0E-04 2.84 1.0E-04 Epiregulin Proepiregulin 3.12 1.0E-04 1.86 1.7E-02 PLGF-2 Placenta growth factor 2.45 1.1E-03 1.68 1.0E-03 IGFBP-2 Insulin-like growth factor-binding protein 2 1.88 1.0E-04 1.84 1.0E-04 ADIPOQ Adiponectin 1.29 2.0E-04 1.33 1.6E-03 Meteorin Meteorin 1.21 4.4E-03 0.46 2.9E-02 IGFBP-5 Insulin-like growth factor-binding protein 5 1.09 1.0E-04 0.53 2.2E-02 PDGF-AA Platelet-derived growth factor subunit A 1.08 2.0E-04 1.69 1.0E-04 G-CSF Granulocyte colony-stimulating factor 0.89 2.0E-04 0.49 4.6E-02 Gas 1 Growth arrest-specific protein 1 0.81 1.1E-02 0.23 1.7E-01 VEGF Vascular endothelial growth factor A 0.77 2.6E-03 2.14 1.0E-04 NOV Protein NOV homolog 4.03 1.0E-04 5.27 1.5E-02 Osteoactivin Transmembrane glycoprotein NMB 3.64 1.0E-04 3.37 1.0E-04 Resistin Resistin 3.46 1.8E-02 2.70 1.0E-04 Galectin-1 Galectin-1 3.41 1.0E-04 3.24 3.0E-04 295 Table 27 (cont’d) Protein expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice at 13 wk post final exposure in BALF cSiO2 + CON Functional Category TNF superfamily Target name Protein name FCG RiiB cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value Low affinity immunoglobulin gamma Fc region receptor II 3.17 1.0E-04 2.96 4.0E-04 MBL-2 Mannose-binding protein C 2.75 1.0E-04 2.84 1.7E-03 Leptin Leptin 2.15 6.0E-04 1.50 1.3E-03 Clusterin Clusterin 1.70 1.0E-04 1.47 1.0E-04 DLL4 Delta-like protein 4 1.29 1.0E-04 2.08 2.0E-01 MFG-E8 Lactadherin 1.24 1.0E-04 2.40 1.0E-04 CRP C-reactive protein 1.17 1.0E-04 1.25 1.0E-04 Nope Immunoglobulin superfamily DCC subclass member 4 0.82 3.8E-03 0.55 7.2E-03 CD36 Tumor necrosis factor receptor superfamily member EDAR 0.67 2.7E-03 0.74 1.0E-04 Chemerin Retinoic acid receptor responder protein 2 0.65 1.1E-02 0.24 3.9E-01 LOX-1 Oxidized low-density lipoprotein receptor 1 0.49 3.1E-02 0.12 5.1E-01 TNF RII Tumor necrosis factor receptor superfamily member 1B 3.90 1.0E-04 3.65 1.0E-04 OPG Tumor necrosis factor receptor superfamily member 11B 3.43 3.0E-04 3.49 6.0E-04 TNF RI Tumor necrosis factor receptor superfamily member 1A 3.22 1.0E-04 2.95 1.0E-04 TRAIL Tumor necrosis factor ligand superfamily member 10 1.35 2.9E-02 -0.36 3.5E-01 TWEAK R Tumor necrosis factor receptor superfamily member 12A 1.25 2.0E-04 1.04 2.0E-04 CD40 Tumor necrosis factor receptor superfamily member 5 0.81 1.0E-02 0.42 1.9E-02 BAFF R Tumor necrosis factor receptor superfamily member 13C 0.55 4.8E-02 -0.03 3.7E-01 296 Table 28. Targeted proteome analysis of plasma at 5, 9, and 13 wk post final exposure to cSiO 2. Protein expression data were measured by RayBiotech Quantibody Mouse Cytokine Array 4000. Data are expressed as log 2(FC) of cSiO2-treated mice fed CON or 1.0% DHA relative to VEH-treated mice fed CON. P-values < 0.05 were considered statistically significant. FC indicates fold change. Underlined target names and p-value indicate protein is not statistically different between cSiO2-treated mice fed 1.0% DHA and VEHtreated mice fed CON diet. Table 28 (cont’d) Protein expression of cSiO2-treated NZBWF1 relative to VEH+CON mice in plasma 5 Wk Post Final Exposure cSiO2 + CON Functional Category Target name Protein name Adhesion molecule E-selectin Chemokine cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value E-selectin 0.38 1.3E-02 0.13 5.8E-02 CXCL16 C-X-C motif chemokine 16 0.77 5.6E-03 -0.07 2.1E-02 KC Growth-regulated alpha protein 0.70 4.7E-02 -0.32 3.8E-01 MCP-5 C-C motif chemokine 12 0.63 7.3E-03 -0.71 8.0E-01 CRP C reactive protein -0.23 7.8E-03 0.22 9.6E-01 Cytokine Gremlin Gremlin-1 1.21 2.4E-02 -0.02 9.2E-02 Growth factor IGFBP-6 Insulin-like growth factor-binding protein 6 0.12 4.1E-02 -0.13 4.4E-01 ADIPOQ Adiponectin -0.30 4.0E-02 0.33 8.7E-01 TNF RII Tumor necrosis factor receptor superfamily member 1B 0.28 2.2E-02 -0.48 6.4E-02 TNF superfamily 297 Table 28 (cont’d) Protein expression of cSiO2-treated NZBWF1 relative to VEH+CON mice in plasma 9 Wk Post Final Exposure cSiO2 + CON Functional Category Target name Protein name Adhesion molecule P-selectin cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value P-selectin 0.39 7.5E-03 0.27 5.8E-02 E-selectin E-selectin 0.21 3.7E-02 0.24 2.4E-01 Chemokine CCL6 C-C motif chemokine 6 0.32 4.3E-02 0.11 6.7E-01 Co-stimulatory molecule CD6 T-cell differentiation antigen CD6 1.03 3.9E-02 -0.13 8.5E-01 Cytokine MCP-5 C-C motif chemokine 12 0.88 9.6E-03 0.04 8.4E-01 IL-28 IFN lambda 2 0.66 3.0E-04 0.54 4.0E-02 OPG Tumor necrosis factor receptor superfamily member 11B 0.22 9.7E-03 0.09 6.7E-01 TNF superfamily 13 Wk Post Final Exposure cSiO2 + CON Functional Category Target name Protein name Adhesion molecule E-selectin Chemokine Cytokine cSiO2 + 1.0% DHA log2(FC) p-value log2(FC) p-value E-selectin 0.30 2.5E-02 -0.06 8.3E-02 BLC C-X-C motif chemokine 13 1.81 2.2E-03 -0.17 1.9E-02 TARC C-C motif chemokine 17 0.94 3.5E-02 -1.04 8.2E-01 LIX C-X-C motif chemokine 5 -1.76 7.0E-04 1.36 4.6E-01 IL-17E Interleukin-17E 0.40 2.1E-02 -0.33 6.6E-01 IL-28 IFN lambda 2 0.19 5.0E-02 0.28 3.1E-02 gp130 Interleukin-6 receptor subunit beta -0.99 1.6E-02 0.73 1.4E-01 298 Table 28 (cont’d) Protein expression of cSiO2-treated NZBWF1 relative to VEH+CON mice in plasma Functional Category Growth factor TNF superfamily Target name Protein name log2(FC) p-value log2(FC) p-value RAGE MAPK/MAK/MRK overlapping kinase -0.27 2.5E-02 -0.13 1.3E-02 Pro-MMP-9 Matrix metalloproteinase-9 -0.53 3.6E-03 0.17 5.0E-04 IGFBP-2 Insulin-like growth factor-binding protein 2 0.73 2.2E-02 -1.03 5.5E-02 Shh-N Sonic hedgehog protein -0.15 3.2E-02 0.23 4.6E-01 Persephin Persephin -0.82 1.4E-02 0.47 2.2E-01 PDGF-AA Platelet-derived growth factor subunit A -1.55 2.0E-04 1.51 9.2E-01 bFGF Fibroblast growth factor 2 -1.76 3.6E-03 0.95 2.2E-01 OX40 Ligand Tumor necrosis factor receptor superfamily member 4 1.18 1.4E-02 -0.61 6.6E-01 CD40 Tumor necrosis factor receptor superfamily member 5 0.41 3.9E-02 0.05 2.8E-01 299 To determine the functional categories predominately driving the phenotype in the plasma, the same strategy was applied as in the BALF. At 5 wk post final exposure, the contribution of cSiO2 was determined to be: chemokines (13.3%) > adhesion molecules (9.1%) > growth factors (4.8%) > cytokines (4.5%) > co-stimulatory molecules (n/a) = enzymes (n/a) = signal transduction (n/a). Impressively, at 5 wks post final exposure to cSiO2, DHA suppressed all of these effects equally; chemokines (100%) = adhesion molecules (100%) = growth factors (100%) = cytokines (100%). At 9 wk post final exposure to cSiO2, the rank order for proteins in the plasma were as follows: adhesion molecules (18.2%) > co-stimulatory molecules (14.3%) > cytokines (6.6%) > TNF superfamily (4.5%) > chemokines (3.3%) > enzymes (n/a) = growth factors (n/a) = signal transduction (n/a). Similarly to 5 wk post final exposure, at 9 wk post final exposure, feeding DHA had a dramatic impact on cSiO2-altered proteins: adhesion molecules (100%) = chemokines (100%) = co-stimulatory molecules (100%) = TNF superfamily (100%) > cytokines (50%) > enzymes (n/a) = growth factors (n/a) = signal transduction (n/a). At 13 wk post final exposure to cSiO2, the protein rank order in the plasma is as follows: growth factors (23.8%) > enzymes (15.0%) > chemokines (10%) = cytokines (10%) > adhesion molecules (9.1%) = TNF superfamily (9.1%) > signal transduction (n/a). For DHA, growth factors (100%) = adhesion molecules (100%) = TNF superfamily (100%) > chemokines (66.0%) = cytokines (66.0%) > enzymes (33%) > signal transduction (n/a). Targeted proteome microarray identifies protein signatures in plasma of cSiO 2-treated mice that parallel with those in BALF Much to our delight, the effects of cSiO2 on protein expression in the BALF were reflected in the plasma. Impressively, 22/29 (76%) proteins identified to be altered by cSiO2 in the plasma 300 were also identified in the BALF (Table 29, Table 30). DHA had a pronounced effect in the plasma compared to the BALF, whereby 28/31 total proteins identified were not statistically different from VEH-treated mice fed CON diet. Taken together, these systemic proteins may be further explored as potential biomarkers for 1) pulmonary injury that may predicate the development of systemic autoimmunity, 2) the suppressive effect of DHA in the lung following airway toxicant exposure and 3) as potentially novel biomarkers for spontaneous SLE and the effects of therapeutics. At the time of this writing, experimental validation of these proteins by ELISA is incomplete. To circumvent this issue and yet validate the findings herein, a Pubmed search was conducted for each protein identified in BALF and plasma with the term “systemic lupus erythematosus”. The number of Pubmed IDs matching this search criteria are reported in Table 29 and Table 30 for BALF and plasma, respectively. Of the 94 proteins identified in BALF, 70 proteins had previously been described in this context, whereas 24 proteins had not. Of the 29 proteins identified in plasma, 19 had been previously identified, whereas 10 had not. Discussion: Targeted proteome microarray Many of the same proteins identified in the targeted proteome analysis were also identified as mRNA transcripts identified by nCounter gene expression. These observations further support our working model of cSiO2-triggered autoimmunity described in Chapter 3. It is well known that cSiO2 exposure to the lung induces secretion of prototypical proinflammatory cytokines, (i.e. IL1β, IL-6, and TNF-α) from alveolar macrophages and epithelial cells, [140–143] the first cells to encounter these particles in the lung. While we did not observe elevations of these cytokines, we did identify surrogate markers for these cytokines in BALF (i.e. IL1RA, gp130, and TNFRI/RII, respectively). Among the pleotropic effects of these cytokines are increased adhesion molecule upregulation on epithelial and endothelial cells that facilitate leukocyte infiltration into inflamed 301 Table 29. Summary of proteins identified in targeted proteome analysis of BALF following cSiO 2 exposure in NZBWF1 mice fed CON or 1.0% DHA-enriched diet. Data are included for 1, 5, 9, and 13 wk post exposure to cSiO2. Proteins are summarized by function, principal cell type expressing the protein, and principal target cell of protein action (indicated by +). Underlined target names and p-value indicate protein is not statistically different between cSiO2-treated mice fed 1.0% DHA and VEH-treated mice fed CON diet. # of PMIDs were determined by entering “target name” + “systemic lupus erythematosus” into PubMed (search performed on 09/09/2017). Abbreviations: Mφ/Mo (macrophage, monocyte); PMN (polymorphonuclear); DC (dendritic cells); Endo. (endothelial cells); Epi. (epithelial cells). Table 29 (cont’d) Intercellular adhesion molecule 1 JAM-A Junctional adhesion molecule A L-Selectin L-selectin P-Cadherin Cadherin-3 Periostin Periostin + P-Selectin P-selectin + VCAM-1 Vascular cell adhesion protein 1 BLC C-X-C motif chemokine 13 C5a Complement C5 + + CCL6 C-C motif chemokine 6 + + Chemokine + + + 1 + + + + + Epi. ICAM-1 + Endo. E-selectin T cell Cadherin-1 E-Selectin B cell E-Cadherin DC Adhesion molecule PMN Protein name Mφ/Mo Target name Epi. Functional Category + # of PMIDs Target Cell Endo. T cell B cell DC PMN Mφ/Mo Source Cell + + + + + + 36 + 56 0 + 9 + 15 + + + + 302 + + + + + + + + + + + + + + + + + + + 3 + 18 + 57 28 57 0 Table 29 (cont’d) Co-stimulatory molecule Cytokine C-X-C motif chemokine 15 MCP-1 C-C motif chemokine 2 + MCP-5 C-C motif chemokine 12 + MDC C-C motif chemokine 22 + MIG C-X-C motif chemokine 9 MIP-1A C-C motif chemokine 3 MIP-1B C-C motif chemokine 4 MIP-1G C-C motif chemokine 9 MIP-3A C-C motif chemokine 20 MIP-3B C-C motif chemokine 19 RANTES C-C motif chemokine 5 TARC C-C motif chemokine 17 TECK C-C motif chemokine 25 B7-1 T-lymphocyte activation antigen CD80 CD40L CD40 ligand CD48 CD48 antigen CD6 T-cell differentiation antigen CD6 gp130 Interleukin-6 receptor subunit beta IL-1RA Interleukin-1 receptor antagonist protein IL-28 IFN lambda-2 + Epi. C-X-C motif chemokine 5 Lungkine + Endo. LIX T cell Growth-regulated alpha protein B cell KC DC C-X-C motif chemokine 11 PMN Eotaxin I-TAC + Mφ/Mo Eotaxin + Epi. + # of PMIDs Target Cell Endo. C-X-C motif chemokine 16 T cell CXCL16 B cell Protein name DC Target name PMN Functional Category Mφ/Mo Source Cell 3 0 + + + + + + + + + + + + + + + + + + + + + + + + + + + + 22 + + + + + + + + + + + + + + + + + + + + + + + + 22 18 0 + + + + + + + + + 17 + + + + + + + + + + + + + + + + 0 21 4 2 + + 5 14 2 0 + + 6 11 67 + + 10 + 2 + 5 43 + 0 + + + + 0 + 10 + + + 303 + + + + 66 + + + 2 Table 29 (cont’d) + + Angiotensin-converting enzyme + + AXL Tyrosine-protein kinase receptor UFO + + Cystatin C Cystatin-C + + FLT-3L Granzyme B MMP-10 Fms-related tyrosine kinase 3 ligand MMP-2 72 kDa type IV collagenase MMP-3 Stromelysin-1 Neprilysin Neprilysin Pro-MMP-9 Matrix metalloproteinase-9 + + RAGE MAPK/MAK/MRK overlapping kinase + + Renin 1 Renin-1 ADIPOQ Adiponectin Decorin + + + Stromelysin-2 + + + + + + + + + + + 5 + 3 + + + + + + + + Decorin + + Epiregulin Proepiregulin + + Fetuin A Alpha-2-HS-glycoprotein Gas 1 Growth arrest-specific protein 1 + + Gas 6 Growth arrest-specific protein 6 + + G-CSF Granulocyte colony-stimulating factor HGF Hepatocyte growth factor IGF-1 Insulin-like growth factor I + + + + + + IGFBP-2 Insulin-like growth factor-binding protein 2 + + + + + IGFBP-5 Insulin-like growth factor-binding protein 5 + + + + + Meteorin Meteorin + + + + 1 + + 24 + + 24 + + 3 + 56 + + 7 + + 0 + + 2 + + 0 + + + + 83 14 + + + + 8 + + 31 + + + 10 + + + Granzyme B + Epi. Osteopontin ACE + Endo. OPN T cell + B cell + DC + PMN + Mφ/Mo Interleukin-7 receptor subunit alpha Epi. T cell IL-7 RA # of PMIDs Target Cell Endo. B cell Growth factor Protein name DC Enzyme Target name PMN Functional Category Mφ/Mo Source Cell + + 7 + + + + 8 + 0 + + + + + 12 15 9 12 + + + + + + + + 19 + + + + + + + + + 3 + + + + + + + + + 0 + + + + + 0 304 Table 29 (cont’d) Signal transduction Endo. Epi. T cell + B cell + DC + PMN Epi. + Mφ/Mo Endo. T cell B cell DC # of PMIDs Target Cell + + 2 + + 0 Target name Protein name PDGF-AA Platelet-derived growth factor subunit A PLGF-2 Placenta growth factor Progranulin Granulins + VEGF Vascular endothelial growth factor A + VEGF R1 Vascular endothelial growth factor receptor 1 CD36 Platelet glycoprotein 4 Chemerin Retinoic acid receptor responder protein 2 + + + Clusterin Clusterin + + + CRP C-reactive protein + + + DLL4 Galectin-1 Delta-like protein 4 Low affinity immunoglobulin gamma FC region receptor II Galectin-1 Kremen-1 Kremen protein 1 Leptin Leptin Lipocalin-2 Neutrophil gelatinase-associated lipocalin LOX-1 Oxidized low-density lipoprotein receptor 1 MBL-2 Mannose-binding protein C MFG-E8 Lactadherin Nope Immunoglobulin superfamily DCC subclass member 4 + + + + 0 NOV Protein NOV homolog + + + + 0 + + + + 0 + + FCG RIIB Osteoactivin Resistin TNF superfamily PMN Functional Category Mφ/Mo Source Cell + + + + + + + + + + + + + + + + + + + + + + Testican-3 BAFF R Tumor necrosis factor receptor superfamily member 13C CD40 Tumor necrosis factor receptor superfamily member 5 305 + 0 9 7 23 6 0 + + + + + + + + + 6 + 2 + + + + + 0 + + 57 13 + + + + + + 1 + + + + 5 + 0 7 + + + 21 14 + + + 1 + + + + + + + + + + + 38 + + + Resistin + + + Testican 3 + + + + + + + Transmembrane glycoprotein NMB + 4 + + + + + 53 Table 29 (cont’d) # of PMIDs Epi. Endo. T cell B cell DC PMN Mφ/Mo Epi. Target Cell Endo. T cell B cell Protein name DC Target name PMN Functional Category Mφ/Mo Source Cell OPG Tumor necrosis factor receptor superfamily member EDAR Tumor necrosis factor receptor superfamily member 11B + TNF RI Tumor necrosis factor receptor superfamily member 1A + + + + + + + + + + + + 1 TNF RII Tumor necrosis factor receptor superfamily member 1B + + + + + + + + + + + + 1 TRAIL Tumor necrosis factor ligand superfamily member 10 + TWEAK R Tumor necrosis factor receptor superfamily member 12A + + 49 EDAR 306 1 + + + + + 17 6 + + + + Table 30. Summary of proteins identified in targeted proteome analysis of plasma following cSiO 2 exposure in NZBWF1 mice fed CON or 1.0% DHA-enriched diet. Data are included for 5, 9, and 13 wk post exposure to cSiO2. Proteins are summarized by function, principal cell type expressing the protein, and principal target cell of protein action (indicated by +). # of PMIDs were determined by entering target name + systemic lupus erythematosus into PubMed (search performed on 09/09/2017). Underlined target names indicate protein is not statistically different between cSiO2-treated mice fed 1.0% DHA and VEH-treated mice fed CON diet. Italicized target names indicate that protein was also identified in BALF (see table 28). Abbreviations: Mφ/Mo (macrophage, monocyte); PMN (polymorphonuclear); DC (dendritic cells); Endo. (endothelial cells); Epi. (epithelial cells). Table 30 (cont’d) # of PMID s BLC C-X-C motif chemokine 13 + + + + 28 CCL6 C-C-motif chemokine 6 + CXCL16 C-X-C motif chemokine 16 + KC Growth-regulated alpha protein LIX C-X-C motif chemokine 5 + MCP-5 C-C motif chemokine 12 + TARC C-C-motif chemokine 17 CD6 T-cell differentiation antigen CD6 gp130 Interleukin-6 receptor subunit beta Gremlin Gremlin-1 Co-stimulatory molecule Cytokine Epi. 18 Endo. 36 + T cell + + B cell + + DC PMN + + Epi. + P-selectin Endo. E-selectin P-selectin T cell E-selectin B cell Adhesion molecule DC Protein name PMN Target name Mφ/Mo Target Cell Functional Category Chemokine Mφ/Mo Source Cell + 0 + + + + + + + + + + + 3 14 2 0 + + + + 2 10 + + + + 0 10 + 307 + + 0 Table 30 (cont’d) Enzyme Growth factor Signal transduction TNF superfamily Stromelysin-2 + Pro-MMP-9 Matrix metalloproteinase-9 + + RAGE MAPK/MAK/MRK overlapping kinase + + ADIPOQ Adiponectin bFGF Fibroblast growth factor 2 IGFBP-2 Insulin-like growth factor-binding protein 2 + + IGFBP-6 Insulin-like growth factor-binding protein 6 + + PDGF-AA Platelet-derived growth factor subunit A Persephin Persephin Shh-N Sonic hedgehog protein + + CRP C reactive protein + + + CD40 Tumor necrosis factor receptor superfamily member 5 Tumor necrosis factor receptor superfamily member 11B + + + + + Epi. IFN lambda-2 MMP-10 Endo. IL-28 T cell Interleukin-17E B cell IL-17E DC Protein name PMN Mφ/Mo Epi. Target name OPG # of PMID s Target Cell Endo. T cell B cell DC PMN Functional Category Mφ/Mo Source Cell 0 + + + + + + + + + + + + 2 + + 1 + 56 + 7 + 7 + + + + + + + + + + 12 + + + + + + + + + + + + 4 + + + + + + + + + + + + 0 + + + + 0 + + + + + 0 OX40 Ligand Tumor necrosis factor receptor superfamily member 4 TNF RII Tumor necrosis factor receptor superfamily member 1B 308 + + + + + + + + + + + + + + + 0 23 6 53 + 17 + + + + + + + + + + + + + 3 + 1 tissues [144,145]. The direction of infiltrating leukocytes is mediated by chemokines, which were induced shortly after cSiO2 exposure and sustained throughout the duration of this study. The chemokines identified here exert their function on cells of the innate and adaptive immune systems. Chemokines that influence the infiltration of neutrophils (i.e. CXCL5, KC, and CXCL15) and monocytes (i.e. CCL2, CCL12) were identified here in addition to chemokines that have been associated with secondary lymphoid organ lymphocyte trafficking and ELT neogenesis. MDC, CCL19, and IL-7R-expressing cells (lymphoid tissue inducer cells) are key initiators of lymphoid tissue neogenesis and are accordingly implicated in ELT neogenesis [37,38,146]. Furthermore, chemotactic proteins that recruit T-cells and organize their spatial localization within lymphoid tissues were identified in BALF of cSiO2-treated NZBWF1 mice. These included TARC (thymus activated and regulated chemokine), TECK (thymus expressed chemokine), I-TAC (IFN-inducible T-cell alpha chemoattractant), and RANTES (regulated on activation, normal T-cell expressed and secreted). Regarding B-cells, CXCL13 is critical for localization of B-cells into lymphoid follicles, and has been previously been observed to be overexpressed in ELT of several autoimmune diseases [99], including COPD [147], rheumatoid arthritis [148,149], and type I diabetes [150]. The sustained, heightened expression of chemokines in BALF is supported by a coincident increase of co-stimulatory molecules (CD80, CD40L, CD48, and CD6) in cSiO2-treated NZBWF1. Protein constituents in BALF of humans have been used clinically as biomarkers of tissue injury and as diagnostic tools for pulmonary diseases [151,152]. However, this procedure requires sedation, a high-level of skill, and is invasive, therefore, proteins identified in the plasma that reflect tissue injury are more suitable biomarkers. Herein, we identified 22 proteins altered by cSiO2 in both the plasma and BALF that could potentially be employed as biomarkers. Excitingly, the majority of these proteins were suppressed by feeding DHA. Thus, they could be utilized as 309 biomarkers for the response of DHA and other therapeutics on pulmonary and systemic autoimmune diseases. Upregulated soluble selectins (e.g. E- and P-selectin) in sera of cSiO2-treated mice was decreased by feeding DHA. Elevated soluble adhesion molecules are frequently observed in individuals with lupus (reviewed in [153]). However, the clinical utility of these are markers is difficult to delineate as studies report mixed results pertaining to their association with disease. Increased sE- and sP-selectin appear to positively correlate with vascular damage [153–155], yet are protective in glomerulonephritis [156–158]. Nephritic kidneys have enhanced expression of membrane-bound selectins [156,159] which are ligands that circulating leukocytes bind to infiltrate inflamed tissues [108]. On the other hand, soluble selectins function as receptor antagonists, thus occupying selectin receptors on circulating leukocytes and preventing cellular infiltration [155,158,160]. Despite this contradiction on cSiO2-accelerated glomerulonephritis, increased soluble selectins induced by cSiO2 in the present study could be indicative of potential cardiovascular effects, which are completely unknown. Cardiovascular damage in SLE, either directly due to disease activity or as a side effect of conventional therapies, has surpassed glomerulonephritis as the leading cause of death in SLE [161,162]. Furthermore, cardiovascular disease in SLE is associated with low levels of ω-3 PUFAs [163,164] and supplementation of SLE patients with ω-3 PUFAs had improved indicators of cardiovascular health [165–167]. Taken together, further investigation into the cardiovascular effects of cSiO2 and DHA in NZBWF1 mice could be pursued in future studies. Remarkably, many chemokines involved in ELT neogenesis, most notably CXCL13, were identified in the BALF and plasma. Serum CXCL13 has been utilized as a biomarker of kidney ELT in individuals with lupus nephritis [168]. Furthermore, sCXCL13 has been utilized for early 310 detection of rheumatoid arthritis and is a biomarker of treatment effect [148,149,169]. Individuals with circulating CXCL13 had replenished systemic antibodies sooner than patients with lower levels of CXCL13 following treatment with rituximab, a B-cell depleting monoclonal antibody [169]. Accordingly, CXCL13 blockade by pharmacological preparations is an attractive approach for autoimmune disease treatment (reviewed in [170]). Strikingly, CXCL13 was increased by cSiO2 exposure and drastically decreased by DHA; levels of CXCL13 in cSiO2-treated mice fed 1.0% DHA were statistically significantly lower than in VEH-treated mice fed CON diet. The suppressive effect of DHA were also observed on other chemokines that have been associated with biomarkers of SLE. TARC and CXCL16, while understudied, have also shown promise as a biomarker of lupus nephritis [171–173]. Regarding CXCL5 (LIX), a neutrophil chemoattractant, only one study to our knowledge has implicated tissue-expression of this chemokine in autoimmune glomerulonephritis [174]. While promising in this study, an inherent limitation is the translatable application of mouse to human protein expression. MCP-5, KC, and CCL6 are chemokines expressed in mouse that were identified in this study, but are not expressed in not humans. MCP-1, the human homolog to MCP-5 (which was identified herein), has a well-established pathogenic role in SLE and lupus nephritis was not affected by cSiO2 or DHA in this study, but was previously identified in chapters 2 and 3 [61,63]. Urinary concentrations of MCP-1 correlate well with impending renal flares, in contrast to IL-8, which showed poor correlation with renal flares in the same study [175]. IL-8, the human homolog to murine KC, is a neutrophil chemoattractant that may have limited application as a urinary marker of SLE, but has been identified as a plasma biomarker of kidney complications in SLE [176]. No studies have previously identified a role for CCL6 or its human homologs, CCL14 or CCL15, in lupus or identified any effect of ω-3 PUFAs on this 311 chemokine. Here, CCL6 was upregulated by cSiO2 and decreased by DHA in plasma of NZBWF1 mice. Therefore, this is the first report to identify a potential role for this chemokine in pathogenesis of SLE. CCL6 has pleiotropic chemoattractant properties for by way of its receptor, CCR1, which is preferentially expressed on CD4+ T-cells and monocytes [177]. Administration of CCR1 antagonists to lupus-prone NZBWF1 reduced lupus nephritis which was accordingly, associated with decreased T-cell and macrophage infiltration [178]. Importantly, we have previously demonstrated that CD4+ T-cell infiltration into the kidneys of NZBWF1 mice was markedly attenuated by feeding DHA-enriched fish oil [60]. While the role of CCL6 in lupus is unclear at this time, this chemokine should be further explored relative to its role in autoimmunity and as a target for DHA. The presence of soluble lymphocyte markers in sera that are usually expressed as membrane proteins have also been exploited as biomarkers of autoimmunity. Surface expression of CD6 is a marker of activated T-cells. One report has previously indicated that sCD6 is elevated in individuals with Sjӧgren’s syndrome [179], although this is the first time sCD6 has been identified in a murine SLE model. Carrasco et al. 2017 [180], detected sCD6 in cell culture supernatant following in vitro activation of peripheral blood leukocytes, suggesting sCD6 is a product of increased T-cell activation. Furthermore, the authors observed that sCD6 was attenuated by addition of metalloproteinase inhibitors, thus suggesting enzyme cleave as a putative mechanism for the generation of sCD6 in sera. CD40 and OX40L are expressed on antigen-presenting cells and bind their cognate ligand, CD40L and OX40, respectively, which are expressed by activated T-cells [181,182]. The mechanisms that drive sCD40 and sOX40L are less understood, however, both sCD40 and sOX40L have been previously utilized as biomarkers for lupus nephritis in SLE 312 and other autoimmune disease manifestations [183–186]. Taken together, attributes of these soluble proteins are consistent with the role of activated T-cells in progression of lupus nephritis. It is difficult to discern the attenuative effect of DHA on sCD6, sCD40, and sOX40L in cSiO2treated NZBWF1, as this could reflect a generalized downregulation of T-cell activation. Nevertheless, enzyme-mediated cleave of sCD6, sCD40, and sOX40L should be explored in future studies as a biomarker for human and murine autoimmune diseases mediated by excessive T-cell activation. Intriguingly, we observed elevated serum enzymes in cSiO2-treated NZBWF1 mice that were attenuated by feeding DHA. These included matrix metalloproteinases, MMP-10 and proMMP-9. These enzymes are suggested to contribute to lupus nephritis through tissue matrix remodeling thereby orchestrating binding of immune complexes [187]. The role of MMP-10 in progression of autoimmunity is not well characterized but has been identified in cutaneous lesions associated with SLE [188]. Wu et al. 2016 [189] employed the same targeted proteome array utilized herein and demonstrated increased serum MMP-10 in patients with SLE compared to healthy controls. There was no correlation with serum MMP-10 and active lupus nephritis, however, endogenous expression of MMP-10 is low in kidneys [190]. (Pro)-MMP-9 has a role in progression of lupus nephritis [187,191], however its utility as a serum biomarker for SLE and/or lupus nephritis is confounded by conflicting results [192–195]. A likely explanation for this discrepancy could be explained by the findings of Robak et al. 2006 [196]. In this study, total MMP-9 was upregulated in sera of SLE-patients, yet active MMP-9 was decreased. All MMPs are secreted as zymogens, thus failure to distinguish between these two forms could explain the lack of consensus on (pro)-MMP-9 and SLE. 313 While MMPs may have limited application as biomarkers at this time, the finding of decreased sRAGE in sera of cSiO2-treated mice is intriguing. RAGE (receptor for advanced glycation endproducts) is normally expressed as a membrane-bound receptor that activates NF-κB to induce pro-inflammatory gene transcription [197]. While seemingly contradictory, levels of sRAGE appear to inversely correlate with disease activity [198–200]. This could be attributable to a decoy receptor function of sRAGE, which binds RAGE ligands (interestingly, which include endogenous alarmins such as HMGB-1 and S100 proteins) in sera, thus preventing them from binding membrane-bound RAGE and inducing inflammation (reviewed in [200]). Taken together, decreased sRAGE induced by cSiO2 in NZBWF1 mice may increase alarmin-mediated RAGE activation, thus exacerbating inflammation. However, DHA did not have an appreciable effect on sera concentrations of sRAGE, therefore, its contribution the anti-inflammatory effect of DHA in cSiO2-triggered autoimmunity is likely minimal. Interestingly, metabolism-linked proteins that are associated with SLE were altered by cSiO2 exposure in NZBWF1 mice fed CON diet that were ablated by feeding DHA. cSiO2 decreased adiponectin expression in mice fed CON diet but was elevated in cSiO2-treated mice fed DHA. Adiponectin is a hormone secreted by adipocytes that is generally regarded as antiinflammatory, particularly in obesity-related conditions [201]. Of significant relevance, increased adiponectin is also observed to be protective in lupus nephritis, as indicated through by the observation of more severe disease in MRL/lpr mice deficient in adiponectin [202]. Activation of PPAR-γ with synthetic ligands (e.g. troglitazone and rosiglitazone) increase plasma adiponectin [203,204] and ameliorated autoimmunity in MRL/lpr mice [205]. Interestingly, natural PPAR-γ ligands include DHA and its oxidized metabolites (reviewed in [206]). Therefore, DHA and/or 314 DHA metabolite-mediated activation of PPAR-γ could contribute to the protective effects of DHA on cSiO2-triggered autoimmunity in NZBWF1 mice. Related to adiponectin function is IGFBP2, which is nearly ubiquitously expressed on all cells due to their requirement for insulin-like growth factors, the endogenous ligands of IGFBP2. Herein, cSiO2-increased expression of IGFBP2. Emerging evidence indicates that IGFBP-2 is positively correlated with lupus nephritis in human cohorts [189,207–209]. While no mechanistic action has been proposed for IGFBP2, it can be inferred that this results from aberrant metabolic signaling, which has previously been implicated in lupus pathogenesis [210]. However, the related protein, IGFBP6, was recently identified to have chemotactic properties for T-cells preferentially to other leukocytes isolated from patients with rheumatoid arthritis [211]. Our findings herein of DHA-mediated suppression of systemic IGFBP2 are consistent with those reported previously [212], but this is the first report to associate DHA with attenuation IGFBP2 in autoimmunity. C-reactive protein, an acute phase protein that is typically associated with the early stages of infection, was decreased by cSiO2 exposure in CON fed mice. The role of CRP as a biomarker of human lupus is unclear; diagnostic evaluations of individuals with lupus often display widely varying concentrations of plasma CRP, and studies report contrasting results on levels of systemic CRP and association with disease activity (reviewed in [213]). These findings might be explained by the heterogenous nature of SLE in humans. While the utility of CRP as a practical biomarker for human SLE may be limited, treatment of NZBWF1 mice with exogenous CRP ameliorated disease progression and was attributed at least in part, by enhancing phagocytosis of chromatin, a nuclear antigen that elicits autoantibodies in this strain [214,215]. The finding that DHA increased CRP expression in cSiO2-exposed mice warrants future investigation into the role of CRP in cSiO2triggered autoimmunity. 315 Of the proteins identified herein, no studies have implicated sonic hedgehog (Shh-N) or persephin in lupus, both of which are neurotrophic proteins implicated in neurological diseases. Overexpression of Shh-N has been implicated in pathogenesis synovial joints of rheumatoid arthritis [216,217] yet deficiency contributed to multiple sclerosis [218,219]. Persephin has been demonstrated to be protective in neurodegenerative disorders, such as Parkinson’s disease [220,221]. The observation that cSiO2 decreased sHH and persephin in sera could indicate loss of neurological function, which is a severe complication of SLE observed in humans and NZBWF1 mice [222,223]. Feeding DHA to cSiO2 exposed lupus-prone mice in this study reversed cSiO2induced deficiencies of neurotrophic factors. Of particular interest herein, one well-characterized attribute of DHA is neuroprotection (reviewed in [224]). Though strictly hypothetical, this possibility warrants future studies that investigate 1) cSiO2-induced neurological manifestations of SLE in NZBWF1 mice and 2) the application of DHA towards this condition and other autoimmune diseases with neurological complications. The proteins identified here that parallel BALF and plasma in cSiO2-exposed NZBWF1 mice warrant further study into the utility as these proteins as biomarkers of human SLE. However, mechanistic contributions of these proteins are difficult to delineate for several reasons. It is impossible to determine whether these proteins are released passively by dying cells or are actively released by cells to promote inflammation. It is also difficult to attribute observed effects of cSiO2 in BALF and plasma proteins to a response that specifically contributes towards acceleration of autoimmunity as opposed to features that overlap with a generalized inflammatory response induced by cSiO2. To address this issue, future studies should consider running parallel experiments with control strains, such as the non-autoimmune parent strain, NZW/LacJ as employed in Chapter 3. 316 We encountered several limitations in this analysis related to sample dilution that limit this application. As this was a discovery-based approach, we elected to utilize standard dilutions conducted at RayBiotech. BALF is inherently dilute, and many analytes were below the limit of detection, particularly in VEH-treated mice fed CON. On the contrary, standard dilutions for plasma are likely determined based on what would be expected in non-autoimmune mouse strains. Therefore, lupus-prone mice could be expected to have higher baseline concentrations of these proteins present in the circulation, and a proinflammatory stimulus such as cSiO2 would expectedly increase this concentration further. Indeed, many analytes in plasma were above the maximum limit imposed by the standard curve, and thus limiting the absolute quantitation of inflammatory proteins identified herein. Nevertheless, proteins of further interest identified herein can be quantitated by ELISA in future studies. Conclusions: Targeted proteome microarray The identification of plasma proteins herein in our model of respiratory-toxicant triggered autoimmunity opens the door for future studies to potentially employ these proteins as biomarkers for 1) triggers of autoimmunity, 2) impending onset of autoimmunity and 3) the counter effect of immunosuppressive agents, including DHA. Finally, we identified proteins in sera of cSiO2-treated NZBWF1 mice that could reflect lupus-mediated tissue injury beyond the kidney. Thus, future investigations into the effect of airway cSiO2 exposure and dietary DHA on additional target tissues, notably, the cardiovascular and neurological systems are warranted. 317 Results: Autoantibody array Dietary DHA inhibited cSiO2-elicited repertoire of autoreactive IgM and IgG autoantibodies in BALF and plasma of lupus-prone NZBWF1 mice Having established that dietary DHA attenuated rampant and progressive inflammation at both the transcription and translational level following cSiO2 exposure in NZBWF1 mice, we finally investigated the effect of cSiO2 and DHA on IgG and IgM autoantibody profiles in BALF and plasma at 13 wk post final exposure to cSiO2. At the time of this writing, statistical analyses of these analyses, and profiling of autoantibodies in BALF and plasma at earlier time points is under completion by collaborators at UTSW. Nevertheless, illustrative descriptions of these results are presented here to illustrate the dramatic and diverse repertoire of autoantibodies elicited by cSiO2 exposure in CON fed mice and the impressive attenuation of DHA on IgG and IgM autoantibodies in BALF and plasma of cSiO2 -induced autoantibodies. The autoantibody profile we performed in the BALF of cSiO2-treated mice is the first time this autoantibody array has been used on BALF. We expected to identify a subset of antigens that were reactive with the autoantibodies present in our sample, and identify putative sources of these antigens. We did not expect to observe the dramatic and diverse autoantibody profile elicited in cSiO2-treated NZBWF1 mice fed CON diet as shown in Fig 53-56. Interestingly, both IgG (Fig. 53, Fig. 55) and IgM autoantibodies (Fig. 54, Fig. 56) were elevated at 13 wks post final exposure. Visually, DHA impressively reduced these responses in the BALF. However, complete statistical analyses are pending analysis, therefore, no firm conclusions can be made on 1) to what extent dietary DHA decreased cSiO2-elicited autoantibodies and 2) which autoantibodies were not effected by feeding DHA. An additional observation from these data at 13 wk post final exposure to cSiO2 was that both IgG and IgM autoantibodies in BALF (Fig. 53, Fig. 54) visually appeared 318 Figure 53. Summary of antigen-specific IgG autoantibodies in BALF at 13 wks post final exposure to cSiO2 in lupus-prone NZBWF1 mice. Values indicated are log2(signal) of VEHtreated mice fed CON, and cSiO2-exposed mice fed CON, 0.4%, or 1.0% DHA-enriched diet. 319 Figure 54. Summary of antigen-specific IgM autoantibodies in BALF at 13 wks post final exposure to cSiO2 in lupus-prone NZBWF1 mice. Values indicated are log2(signal) of VEHtreated mice fed CON, and cSiO2-exposed mice fed CON, 0.4%, or 1.0% DHA-enriched diet. 320 Figure 55. Summary of antigen-specific IgG autoantibodies in plasma at 13 wks post final exposure to cSiO2 in lupus-prone NZBWF1 mice. Values indicated are log2(signal) of VEHtreated mice fed CON, and cSiO2-exposed mice fed CON, 0.4%, or 1.0% DHA-enriched diet. 321 Figure 56. Summary of antigen-specific IgM autoantibodies in plasma at 13 wks post exposure to cSiO2 in lupus-prone NZBWF1 mice. Values indicated are log2(signal) of VEHtreated mice fed CON, and cSiO2-exposed mice fed CON, 0.4%, or 1.0% DHA-enriched diet. 322 to be higher than these same autoantibodies in the plasma (Fig. 55, Fig. 56). Again, DHA appeared to attenuate cSiO2-induced IgG and IgM autoantibodies in plasma, however, this conclusion requires further statistical analysis to be warranted. In summary, although statistical analysis is incomplete at the time of this writing, the dramatic induction of autoantibodies in BALF and plasma following cSiO2 exposure in CON fed mice is visually evident. The observation that IgG and IgM autoantibodies appear to higher in the BALF than plasma is further evidence to strengthen our hypothesis that cSiO2 exposure in the lung of lupus-prone NZBWF1 induces ELT that produce a diverse array of autoreactive antibodies that accelerate systemic autoimmunity and are a primary target for the prophylactic effect of DHA on cSiO2 -triggered autoimmunity in lupus-prone NZBWF1 mice. Discussion: Autoantibody array Autoantibodies are an invaluable diagnostic tool for lupus and other autoimmune diseases. Most notably is the presence of anti-nuclear antibodies [225], which are present in up to 95% of individuals with SLE [226,227]. Autoantibody profiling of serum from patients and murine models of autoimmunity have been previously employed to: 1) identify patients with characteristics of autoimmunity prior to clinical manifestation [228–231], 2) correlate gene and protein expression to disease phenotype and severity [232–234], and 3) identify novel antigens that react with autoantibodies [235,236]. Microarrays can be further applied to identify antigens that trigger loss of tolerance, thus elucidating mechanistic understanding of the progression of SLE [236], as exclusion and/or stratification criteria for participants in clinical trials that may resolve the failure of previous candidate therapeutics [237–239], and enhance precision medicine by identifying individuals who may respond to a given therapeutic [240,241]. 323 Previous studies by us [60,61] and others [242–245] have shown the marked suppressive effect of DHA on autoantibodies in SLE. However, these efforts have predominately focused on the effect of DHA on a select few autoantibodies. Up to 180 antigens that elicit autoantibodies have been identified in SLE, which far exceeds that of other autoimmune diseases [246], and may explain in part, the relative lack of success of SLE-specific therapeutics compared to those in rheumatoid arthritis, which have generally been successful [239]. Classifications of autoantibody reactivity include those toward nuclear antigens (e.g. ss and dsDNA, histones, nucleosomes), intracellular (e.g. ribosomal proteins) and extracellular cellular components (e.g. phospholipids, matrix proteins and complement). All immunoglobulin isotypes (i.e. IgM, IgG, IgA, and IgE) have been identified as autoantibodies, however, not all autoantibodies in SLE are pathogenic. In general, IgM autoantibodies seem to have a protective role [247–249], whereas IgG autoantibodies are regarded as pathogenic, particularly in the context of lupus nephritis [250]. Indeed, a protective role for IgM autoantibodies on glomerulonephritis has been elucidated with lupus-prone mouse strains. Delayed lupus nephritis was observed in NZBWF1 mice treated with IgM anti-dsDNA antibodies [251] and lupus-prone MRL/lpr mice deficient in IgM had accelerated onset of glomerulonephritis in parallel with increased IgG [250]. Li et al. 2010 suggested that class-switching from IgM to IgG autoantibodies, a process that may be driven in part, by IFN, could predicate the development of full SLE [14]. In support of this contention, a potentially valuable diagnostic criterion is the ratio of IgG:IgM autoantibodies; a shift towards increased IgG compared to IgM has been utilized as a biomarker lupus nephritis [252]. Visually, IgG and IgM were detected in the BALF and plasma of cSiO2-treated NZBWF1 mice in this study. A shift of IgG:IgM autoantibodies that may indicate impending onset of active SLE caused by cSiO2 exposure in these mice cannot be declared without 324 data on a time-dependent autoantibody profile, which are being completed at the time of this writing. Synovial joint fluid and cerebrospinal fluid from tissue-specific autoimmune diseases, rheumatoid arthritis and neurological manifestations of multiple sclerosis and SLE, respectively have been invaluable towards identification of autoantibodies that mediate tissue injury in these autoimmune diseases [253–256]. Our study is the first to use autoantibody profiling on BALF. IgM and IgG autoantibodies were profiled in this study. IgM is the first antibody produced by antibody-secreting plasma cells and is secreted as a pentamer. It is the ‘default’ isotype produced by plasma cells and thus, does not require B-cell class-switching to be secreted [257]. As a result, IgM has a large capacity to bind antigens, but at comparatively low affinity. For these reasons, it is interesting to note the IgM response observed in the BALF at 13 wk post final exposure to cSiO2. This may indicate several features of cSiO2-triggered autoimmunity. First, since IgM is the first antibody produced upon antigen encounter, it is usually indicative of an early inflammatory response. In that vein, the observation of IgM autoantibodies in BALF at time this could indicate the active production of neo-antigens in the lung. Alternatively, long-lived IgM-secreting plasma cells could be retained and expanded in the inflamed lungs of cSiO2-treated mice, thus serving as a ‘survival niche’ as has previously been identified in kidneys of NZBWF1 mice [258] and hypothesized to be a function of ELT in autoimmunity. However, these are speculations until IgM in BALF over time and further statistical analyses can be conducted on cSiO2-treated mice. Nevertheless, IgM is a potent activator of the classical complement pathway [257], and the IgM identified herein likely contributed to the persistent activation of complement we observed in lungs of cSiO2-treated NZBWF1 mice. We also observed activation of the alternative complement pathway in the lung, which can be induced 325 by IgA antibodies [259]. Therefore, it would be of future interest to profile IgA autoantibodies in BALF and plasma of cSiO2-treated NZBWF1 mice. IgG is produced following class-switch recombination of B-cells and is secreted as a monomer. There are four subtypes of IgG, each with varying effector function. The general functions of IgG can be summarized as complement activation, pathogen opsonization, and toxin neutralization [90]. Because of these potent features, IgG autoantibodies are largely regarded as pathogenic in SLE. The striking IgG response in BALF and plasma of cSiO2-treated NZBWF1 mice illustrates the potent effect of cSiO2 on autoimmunity in NZBWF1 mice and demonstrates dramatic attenuation attributed to feeding DHA. Also noteworthy is comparison between the IgG response in the BALF and plasma; visually, the IgG in BALF appears to be greater than that in the plasma. This is further evidence for the role of lung as a platform that triggers systemic autoimmunity following airway cSiO2exposure to lupus-prone mice. Conclusions: Autoantibody array Herein, we characterize an elaborate profile of autoantibodies induced by cSiO2 that could ultimately contribute towards mechanistic understanding of the pathogenesis of toxicant-triggered autoimmunity. Not only are the findings of DHA important in the context of toxicant-triggered autoimmunity, but it is also possible to expand the effect of DHA to autoimmune diseases outside of SLE. In this regard, autoimmune diseases with autoantibody profiles that overlap those identified herein could also potentially harness a dietary approach with DHA to control the progression of these diseases. 326 Summary: Chapter 4 We employed an integrative profiling approach to characterize cellular, molecular, and protein signatures that catalyze autoimmunity and lupus nephritis following cSiO2 exposure that are markedly attenuated by dietary DHA. Collectively, the findings reported herein indicate that rampant and unresolved inflammation occurs early in the pathogenesis of cSiO2-triggered autoimmunity that is amplified over time. Furthermore, the protective effect of DHA is evident throughout the course of pathological events that ultimately predicate exacerbated systemic autoimmunity by cSiO2 exposure. Taken together, the findings herein offer a much larger appreciation for the complexity of cSiO2 -triggered lupus and the multiple pathways targeted by DHA that collectively influence the progression of autoimmunity in lupus-prone NZBWF1 mice. Cost constraints limited the number of experimental controls included in the present study, which limits further conclusions on the data reported herein. One limitation is that nonautoimmune control mouse strains were not included in treatment groups. Therefore, it is difficult to dissect features of cSiO2 -induced inflammation that contribute to autoimmunity as opposed to those indicative of a general inflammatory response to cSiO2. Future studies should consider employing Balb/c mice, a commonly employed strain with close genetic phylogeny to NZBWF1 mice [260], the non-autoimmune parent strain to NZBWF1 mice, the NZW/LacJ mouse (employed in Chapter 3), or the C57Bl6 mouse (employed in Chapter 2), a murine model for cSiO2 exposure [261–264]. An additional limitation of this study is that mice fed DHA without cSiO2 exposure were not included, which was not economically feasible in this study considering the large number of animals required and the numerous analyses employed. Therefore, we cannot discern if attenuation by dietary DHA is specifically directed towards cSiO2-induced upregulation, or simply attributable to global suppression by DHA. Nevertheless, the data herein suggest that future 327 mechanistic studies should focus on short-term experiments conducted in vivo and in vitro, therefore, this negative control can be feasibly included in those studies. The beneficial effects of dietary DHA in the context of cSiO2 -triggered autoimmunity in lupus-prone mice are evident. However, one must also consider that global immunosuppression by DHA and other broadly immunosuppressive agents can be deleterious, particularly in the context of infection [67,265–267]. Nevertheless, the European Food Safety Authority (EFSA) considered potential adverse outcomes of DHA and other ω-3 PUFAs, including those of immune function, and concluded that human intake up to 5 g/d DHA is safe [65]. In this study, we fed NZBWF1 mice concentrations of DHA equivalent to human consumption of 2 g/d (0.4% DHA; achievable by dietary intake) and 5 g/d (1.0% DHA; based on EFSA recommendation) and demonstrated beneficial effects of DHA on toxicant-triggered autoimmunity at both concentrations employed. Nevertheless, studies that directly compare both the effectiveness and safety between DHA and other immunosuppressive agents used as the standard of care for individuals with SLE are warranted. Despite these limitations, the present study elucidated several features of our model of cSiO2 -triggered autoimmunity that are targets of DHA. The findings reported herein serve as endpoints for ongoing research efforts to dissect the complexity of cSiO2 -triggered autoimmunity. Furthermore, the identification of numerous dysregulated pathways reported herein are observed in humans with SLE. Signatures indicative of persistent recruitment of neutrophils, monocytes/macrophages, lymphocytes, and antigen-presenting cells, including dendritic cells, were identified herein, implying that both innate and adaptive immune systems influence the progression of cSiO2 -triggered autoimmunity. It is unlikely that a single population is solely responsible for driving cSiO2 -accelerated autoimmunity. A more probable scenario requires the 328 collective coordination of these cells. Towards this end, reductionist in vitro approaches can be harnessed to identify what cell population triggers the cascade to cSiO2 -triggered autoimmunity. Another additional key finding of this study was that cellular and protein signatures are established within 1 wk post final exposure to cSiO2 and attenuated by DHA at this time. This suggests that DHA prevents early cSiO2 toxicity (within the 4 wk cSiO2 exposure period) in the lung that critically impact the later establishment of the lung as a platform to trigger systemic autoimmunity. 329 APPENDIX 330 APPENDIX Chapter 4 Supplementary Information Figure 57. Certificate of analysis for DHASCOTM algal oil in DHA-enriched AIN-93G diets. 331 Figure 57 (cont’d). 332 Table 31. Complete mRNA transcript profile in lung at 1 wk post final exposure in cSiO 2-exposed NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log2(fold change) relative to VEH + CON mice. Expression data are included for all mRNA transcripts that were above background threshold. Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl5 3.33 [2.36 , 4.31] 1.4E-04 1.37 [0.74 , 1.37] 8.1E-05 0.63 [0.35 , 0.63] 6.8E-05 Cxcl3 2.91 [2.12 , 3.69] 7.8E-05 1.69 [0.94 , 1.69] 5.7E-05 0.61 [0.32 , 0.61] 2.0E-04 Lcn2 2.78 [2.36 , 3.21] 8.1E-07 1.74 [1.36 , 1.74] 2.0E-08 0.67 [0.53 , 0.67] 1.8E-08 Chil3 2.69 [1.70 , 3.69] 6.4E-04 1.84 [0.53 , 1.84] 8.2E-03 0.67 [0.32 , 0.67] 4.9E-04 Ctla4 2.43 [2.08 , 2.78] 6.1E-07 1.43 [0.94 , 1.43] 2.7E-06 0.48 [0.33 , 0.48] 1.9E-06 Mmp9 2.14 [1.51 , 2.77] 1.4E-04 1.38 [0.70 , 1.38] 1.6E-04 0.58 [0.32 , 0.58] 6.9E-05 Pdcd1 2.14 [1.47 , 2.82] 2.0E-04 1.54 [0.84 , 1.54] 7.1E-05 0.53 [0.25 , 0.53] 5.7E-04 Fcer2a 2.03 [1.60 , 2.46] 9.4E-06 1.47 [0.98 , 1.47] 2.0E-06 0.47 [0.33 , 0.47] 1.2E-06 Marco 2.02 [1.47 , 2.57] 8.0E-05 1.49 [1.01 , 1.49] 1.2E-06 0.58 [0.41 , 0.58] 8.4E-07 Foxp3 1.93 [1.60 , 2.25] 1.7E-06 1.23 [0.88 , 1.23] 2.8E-07 0.44 [0.29 , 0.44] 4.3E-06 Il12b 1.93 [1.42 , 2.44] 6.5E-05 1.29 [0.81 , 1.29] 6.5E-06 0.45 [0.25 , 0.45] 6.9E-05 Irf7 1.85 [1.15 , 2.55] 7.4E-04 1.22 [0.33 , 1.22] 1.0E-02 0.14 [0.01 , 0.14] 6.5E-02 Cfb 1.79 [1.55 , 2.04] 6.1E-07 0.86 [0.66 , 0.86] 3.2E-08 0.34 [0.28 , 0.34] 3.9E-09 333 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccl8 1.79 [1.13 , 2.45] 6.1E-04 1.27 [0.52 , 1.27] 1.3E-03 0.38 [0.13 , 0.38] 6.1E-03 Slc11a1 1.78 [1.11 , 2.45] 7.0E-04 1.44 [1.11 , 1.44] 3.5E-08 0.33 [0.22 , 0.33] 2.0E-06 Il1rn 1.77 [1.48 , 2.06] 1.7E-06 1.09 [0.84 , 1.09] 3.2E-08 0.37 [0.28 , 0.37] 1.6E-07 Ccl3 1.74 [1.19 , 2.29] 2.0E-04 1.29 [0.74 , 1.29] 3.6E-05 0.47 [0.28 , 0.47] 3.5E-05 Il1r2 1.65 [1.24 , 2.07] 4.7E-05 0.99 [0.56 , 0.99] 4.7E-05 0.41 [0.23 , 0.41] 8.7E-05 Birc5 1.60 [1.08 , 2.12] 2.5E-04 1.13 [0.65 , 1.13] 3.5E-05 0.39 [0.20 , 0.39] 2.1E-04 Clec5a 1.57 [1.28 , 1.85] 2.8E-06 1.09 [0.79 , 1.09] 2.2E-07 0.33 [0.24 , 0.33] 6.4E-07 Trem2 1.55 [1.24 , 1.85] 4.6E-06 1.03 [0.70 , 1.03] 1.2E-06 0.34 [0.27 , 0.34] 1.8E-08 C3ar1 1.54 [1.26 , 1.82] 2.8E-06 0.92 [0.59 , 0.92] 4.1E-06 0.28 [0.19 , 0.28] 2.0E-06 Mx1 1.54 [0.98 , 2.09] 5.7E-04 1.04 [0.29 , 1.04] 8.9E-03 0.36 [0.19 , 0.36] 1.8E-04 Tigit 1.52 [0.96 , 2.09] 6.5E-04 1.01 [0.36 , 1.01] 2.9E-03 0.37 [0.15 , 0.37] 1.6E-03 Cxcl2 1.52 [0.83 , 2.21] 2.6E-03 1.01 [0.34 , 1.01] 3.9E-03 0.42 [0.14 , 0.42] 6.1E-03 Msr1 1.46 [1.01 , 1.91] 1.7E-04 1.10 [0.60 , 1.10] 6.3E-05 0.37 [0.20 , 0.37] 1.1E-04 Oas2 1.42 [0.97 , 1.87] 2.1E-04 0.66 [0.04 , 0.66] 7.3E-02 0.06 [-0.04 , 0.06] 7.9E-01 Oasl1 1.40 [0.73 , 2.08] 4.2E-03 0.99 [0.36 , 0.99] 2.8E-03 0.13 [-0.04 , 0.13] 4.8E-01 Ccl9 1.39 [1.06 , 1.72] 3.1E-05 0.95 [0.70 , 0.95] 1.4E-07 0.32 [0.22 , 0.32] 1.9E-06 Cxcr1 1.37 [1.02 , 1.73] 6.1E-05 0.82 [0.46 , 0.82] 5.1E-05 0.30 [0.17 , 0.30] 8.6E-05 334 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value C1qa 1.35 [1.10 , 1.61] 3.6E-06 0.82 [0.59 , 0.82] 2.9E-07 0.27 [0.20 , 0.27] 2.0E-07 Tnfrsf9 1.35 [0.63 , 2.08] 7.6E-03 1.04 [0.35 , 1.04] 3.8E-03 0.46 [0.18 , 0.46] 2.1E-03 Mx2 1.34 [0.80 , 1.88] 1.1E-03 0.79 [0.21 , 0.79] 1.1E-02 0.13 [0.00 , 0.13] 1.4E-01 Isg15 1.34 [0.75 , 1.93] 2.1E-03 0.89 [0.05 , 0.89] 7.2E-02 0.13 [0.04 , 0.13] 8.6E-03 Csf2 1.32 [1.05 , 1.59] 7.5E-06 0.81 [0.50 , 0.81] 8.3E-06 0.38 [0.26 , 0.38] 1.7E-06 Siglec1 1.32 [0.85 , 1.79] 4.8E-04 0.99 [0.54 , 0.99] 7.0E-05 0.16 [0.01 , 0.16] 8.6E-02 Ccl6 1.29 [1.02 , 1.55] 8.0E-06 0.90 [0.67 , 0.90] 1.1E-07 0.31 [0.23 , 0.31] 9.7E-08 Il23r 1.29 [0.91 , 1.67] 1.4E-04 0.91 [0.55 , 0.91] 1.7E-05 n.d [n.d , n.d] n.d C1qb 1.28 [1.04 , 1.53] 4.4E-06 0.76 [0.51 , 0.76] 1.7E-06 0.22 [0.16 , 0.22] 6.4E-07 Ifi44 1.26 [0.72 , 1.81] 1.8E-03 0.74 [0.07 , 0.74] 5.4E-02 0.05 [-0.07 , 0.05] 7.9E-01 Ccl7 1.25 [0.68 , 1.81] 2.7E-03 1.19 [0.66 , 1.19] 5.6E-05 0.19 [-0.05 , 0.19] 3.8E-01 Slc7a11 1.24 [0.77 , 1.72] 7.5E-04 0.70 [0.26 , 0.70] 2.6E-03 0.28 [0.10 , 0.28] 3.4E-03 Ccl4 1.21 [0.66 , 1.77] 2.6E-03 1.00 [0.43 , 1.00] 8.4E-04 0.34 [0.17 , 0.34] 4.4E-04 Ifit1 1.20 [0.70 , 1.70] 1.5E-03 0.71 [0.18 , 0.71] 1.2E-02 0.10 [-0.01 , 0.10] 1.9E-01 Tnfsf11 1.20 [0.60 , 1.80] 5.1E-03 0.98 [0.37 , 0.98] 2.0E-03 0.35 [0.15 , 0.35] 1.5E-03 Clec4n 1.19 [0.89 , 1.49] 1.0E+00 0.81 [0.38 , 0.81] 3.8E-04 0.28 [0.18 , 0.28] 5.6E-06 Cx3cr1 1.19 [0.85 , 1.53] 2.0E+00 0.73 [0.39 , 0.73] 9.0E-05 0.22 [0.06 , 0.22] 1.2E-02 335 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ctss 1.17 [0.89 , 1.45] 3.0E+00 0.84 [0.54 , 0.84] 4.1E-06 0.20 [0.13 , 0.20] 1.7E-06 Cxcl13 1.15 [0.43 , 1.86] 4.0E+00 0.54 [-0.01 , 0.54] 1.2E-01 0.22 [-0.01 , 0.22] 1.8E-01 Cd14 1.14 [0.86 , 1.41] 5.0E+00 0.67 [0.43 , 0.67] 4.8E-06 0.27 [0.16 , 0.27] 3.2E-05 C3 1.13 [0.92 , 1.34] 6.0E+00 0.72 [0.56 , 0.72] 3.2E-08 0.22 [0.15 , 0.22] 1.9E-06 Ccr1 1.12 [0.72 , 1.52] 7.0E+00 0.82 [0.46 , 0.82] 4.7E-05 0.25 [0.10 , 0.25] 1.4E-03 Zbp1 1.11 [0.62 , 1.61] 8.0E+00 0.61 [-0.03 , 0.61] 1.5E-01 0.09 [-0.03 , 0.09] 5.0E-01 Cd74 1.09 [0.73 , 1.44] 9.0E+00 0.83 [0.66 , 0.83] 1.7E-08 0.19 [0.13 , 0.19] 1.5E-06 Fcgr1 1.08 [0.73 , 1.43] 1.0E+01 0.57 [0.16 , 0.57] 8.7E-03 0.18 [0.05 , 0.18] 1.5E-02 Tnf 1.08 [0.70 , 1.45] 1.1E+01 0.89 [0.39 , 0.89] 8.4E-04 0.21 [0.10 , 0.21] 2.6E-04 Ifitm1 1.07 [0.89 , 1.25] 1.2E+01 0.52 [0.35 , 0.52] 1.8E-06 0.28 [0.20 , 0.28] 8.0E-07 Cd68 1.07 [0.81 , 1.34] 1.3E+01 0.75 [0.51 , 0.75] 1.3E-06 0.23 [0.14 , 0.23] 1.3E-05 Csf2rb 1.06 [0.74 , 1.39] 1.4E+01 0.83 [0.40 , 0.83] 3.1E-04 0.22 [0.12 , 0.22] 9.1E-05 Cxcr2 1.05 [0.24 , 1.86] 1.5E+01 0.84 [0.10 , 0.84] 4.7E-02 0.37 [0.06 , 0.37] 4.0E-02 Ifit3 1.04 [0.54 , 1.54] 1.6E+01 0.64 [0.07 , 0.64] 5.2E-02 0.07 [-0.07 , 0.07] 9.9E-01 S100a8 1.04 [0.28 , 1.79] 1.7E+01 0.77 [0.09 , 0.77] 4.9E-02 0.32 [0.04 , 0.32] 6.0E-02 Card9 1.03 [0.60 , 1.46] 1.8E+01 0.62 [0.21 , 0.62] 4.5E-03 0.27 [0.08 , 0.27] 8.9E-03 Icos 1.02 [0.84 , 1.21] 1.9E+01 0.62 [0.35 , 0.62] 5.0E-05 0.19 [0.12 , 0.19] 1.7E-05 336 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd33 1.02 [0.79 , 1.25] 2.0E+01 0.54 [0.38 , 0.54] 7.5E-07 0.18 [0.12 , 0.18] 5.6E-06 Tlr9 1.02 [0.56 , 1.48] 2.1E+01 0.73 [0.29 , 0.73] 1.4E-03 0.15 [0.00 , 0.15] 1.1E-01 Slamf7 1.00 [0.64 , 1.37] 2.2E+01 0.88 [0.59 , 0.88] 1.6E-06 0.19 [0.09 , 0.19] 3.7E-04 Lta 1.00 [0.57 , 1.44] 2.3E+01 0.68 [0.23 , 0.68] 4.3E-03 0.22 [0.06 , 0.22] 1.3E-02 Cxcl10 1.00 [0.10 , 1.90] 2.4E+01 0.93 [0.15 , 0.93] 3.2E-02 0.18 [-0.13 , 0.18] 8.0E-01 Il2ra 1.00 [0.72 , 1.28] 2.5E+01 0.67 [0.28 , 0.67] 1.1E-03 0.19 [0.09 , 0.19] 7.4E-04 Ccl2 0.99 [0.40 , 1.57] 2.6E+01 1.01 [0.33 , 1.01] 4.6E-03 0.32 [0.07 , 0.32] 2.0E-02 H2-Q10 0.98 [0.41 , 1.56] 2.7E+01 0.70 [0.18 , 0.70] 1.3E-02 0.21 [-0.01 , 0.21] 1.9E-01 Cd27 0.96 [0.39 , 1.54] 2.8E+01 0.86 [0.40 , 0.86] 4.5E-04 0.20 [0.04 , 0.20] 2.9E-02 Itgax 0.95 [0.74 , 1.17] 2.9E+01 0.63 [0.47 , 0.63] 1.0E-07 0.21 [0.14 , 0.21] 1.2E-06 Fcgr2b 0.94 [0.69 , 1.19] 3.0E+01 0.62 [0.39 , 0.62] 7.7E-06 0.23 [0.15 , 0.23] 3.2E-06 Cd6 0.92 [0.64 , 1.19] 3.1E+01 0.59 [0.31 , 0.59] 1.4E-04 0.17 [0.06 , 0.17] 5.3E-03 Trem1 0.91 [0.50 , 1.32] 3.2E+01 0.68 [0.28 , 0.68] 1.3E-03 0.24 [0.08 , 0.24] 6.3E-03 Cd46 0.90 [0.18 , 1.63] 3.3E+01 0.83 [0.18 , 0.83] 1.9E-02 n.d [n.d , n.d] n.d Ncf4 0.88 [0.61 , 1.15] 3.4E+01 0.61 [0.41 , 0.61] 1.8E-06 0.18 [0.11 , 0.18] 3.6E-05 Usp18 0.88 [0.44 , 1.31] 3.5E+01 0.63 [0.16 , 0.63] 1.3E-02 0.07 [-0.02 , 0.07] 4.2E-01 Arg2 0.87 [0.58 , 1.17] 3.6E+01 0.69 [0.29 , 0.69] 9.4E-04 0.25 [0.10 , 0.25] 2.1E-03 337 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Klrg1 0.87 [0.51 , 1.24] 3.7E+01 0.71 [0.33 , 0.71] 4.9E-04 0.23 [0.09 , 0.23] 1.6E-03 Tap1 0.87 [0.58 , 1.17] 3.8E+01 0.50 [0.19 , 0.50] 2.1E-03 0.07 [-0.02 , 0.07] 4.5E-01 Ccl12 0.87 [0.39 , 1.34] 3.9E+01 0.70 [0.12 , 0.70] 2.9E-02 0.21 [-0.01 , 0.21] 1.9E-01 Cdk1 0.87 [0.38 , 1.35] 4.0E+01 0.71 [0.16 , 0.71] 1.6E-02 0.24 [0.06 , 0.24] 2.0E-02 C1ra 0.86 [0.68 , 1.04] 4.1E+01 0.59 [0.43 , 0.59] 1.7E-07 0.16 [0.12 , 0.16] 8.0E-07 Clec4a2 0.86 [0.62 , 1.10] 4.2E+01 0.53 [0.30 , 0.53] 4.0E-05 0.21 [0.11 , 0.21] 1.3E-04 Psmb10 0.85 [0.52 , 1.19] 4.3E+01 0.68 [0.53 , 0.68] 1.7E-08 0.02 [-0.03 , 0.02] 8.4E-01 Cxcl1 0.84 [0.17 , 1.52] 4.4E+01 0.26 [-0.25 , 0.26] 9.8E-01 0.26 [0.03 , 0.26] 5.3E-02 Ccr6 0.84 [0.36 , 1.32] 4.5E+01 0.58 [0.07 , 0.58] 4.7E-02 0.15 [-0.02 , 0.15] 2.7E-01 Tnfrsf18 0.84 [0.42 , 1.26] 4.6E+01 0.61 [0.10 , 0.61] 3.0E-02 0.27 [0.10 , 0.27] 3.6E-03 Tnfrsf4 0.83 [0.54 , 1.12] 4.7E+01 0.65 [0.34 , 0.65] 1.2E-04 0.21 [0.11 , 0.21] 2.0E-04 Ccr5 0.82 [0.57 , 1.07] 4.8E+01 0.50 [0.24 , 0.50] 3.1E-04 0.17 [0.07 , 0.17] 1.4E-03 Blnk 0.82 [0.41 , 1.22] 4.9E+01 0.60 [0.22 , 0.60] 2.2E-03 0.18 [0.05 , 0.18] 1.5E-02 Clec7a 0.81 [0.56 , 1.05] 5.0E+01 0.46 [0.25 , 0.46] 1.1E-04 0.19 [0.08 , 0.19] 1.7E-03 Fcgr3 0.81 [0.56 , 1.05] 5.1E+01 0.44 [0.27 , 0.44] 1.2E-05 0.22 [0.18 , 0.22] 3.9E-09 Serpinb2 0.81 [0.22 , 1.39] 5.2E+01 0.35 [-0.29 , 0.35] 8.9E-01 0.01 [-0.19 , 0.01] 1.6E-01 Il10ra 0.80 [0.56 , 1.03] 5.3E+01 0.46 [0.11 , 0.46] 1.4E-02 0.15 [0.07 , 0.15] 1.2E-03 338 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccr8 0.79 [0.50 , 1.08] 5.4E+01 0.76 [0.26 , 0.76] 4.1E-03 0.27 [0.11 , 0.27] 2.0E-03 Ebi3 0.79 [0.56 , 1.02] 5.5E+01 0.45 [0.21 , 0.45] 3.8E-04 0.17 [0.07 , 0.17] 1.9E-03 Cd4 0.79 [0.53 , 1.04] 5.6E+01 0.54 [0.17 , 0.54] 5.1E-03 0.16 [0.06 , 0.16] 3.6E-03 Il1b 0.78 [-0.01 , 1.56] 5.7E+01 0.55 [-0.22 , 0.55] 5.0E-01 0.25 [-0.07 , 0.25] 4.1E-01 Tnfrsf13c 0.78 [0.06 , 1.50] 5.8E+01 0.67 [-0.05 , 0.67] 1.6E-01 n.d [n.d , n.d] n.d Cd83 0.77 [0.55 , 0.99] 5.9E+01 0.48 [0.27 , 0.48] 3.9E-05 0.15 [0.07 , 0.15] 4.9E-04 Ccr2 0.77 [0.48 , 1.06] 6.0E+01 0.43 [0.18 , 0.43] 1.1E-03 0.20 [0.06 , 0.20] 9.9E-03 Tlr7 0.77 [0.56 , 0.98] 6.1E+01 0.55 [0.19 , 0.55] 3.5E-03 0.12 [0.05 , 0.12] 2.6E-03 H2-DMa 0.77 [0.47 , 1.06] 6.2E+01 0.61 [0.52 , 0.61] 3.0E-10 0.15 [0.11 , 0.15] 3.9E-07 Nlrp3 0.77 [0.27 , 1.26] 6.3E+01 0.65 [0.17 , 0.65] 1.1E-02 0.23 [0.03 , 0.23] 5.2E-02 Fcer1g 0.76 [0.49 , 1.02] 6.4E+01 0.50 [0.26 , 0.50] 1.4E-04 0.21 [0.12 , 0.21] 4.5E-05 Lag3 0.75 [0.40 , 1.10] 6.5E+01 0.32 [0.03 , 0.32] 6.4E-02 0.11 [-0.01 , 0.11] 2.0E-01 H2-D1 0.74 [0.51 , 0.97] 6.6E+01 0.51 [0.32 , 0.51] 5.9E-06 0.08 [0.04 , 0.08] 2.4E-04 Pou2af1 0.73 [0.25 , 1.21] 6.7E+01 0.65 [0.24 , 0.65] 2.6E-03 0.15 [0.01 , 0.15] 9.4E-02 Csf3r 0.71 [-0.05 , 1.46] 6.8E+01 0.71 [-0.02 , 0.71] 1.3E-01 0.30 [0.01 , 0.30] 1.1E-01 H2-Eb1 0.70 [0.50 , 0.91] 6.9E+01 0.43 [0.30 , 0.43] 9.4E-07 0.16 [0.11 , 0.16] 2.9E-06 Csf1r 0.70 [0.52 , 0.88] 7.0E+01 0.43 [0.26 , 0.43] 1.0E-05 0.09 [0.04 , 0.09] 1.1E-03 339 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Emr1 0.70 [0.51 , 0.89] 7.1E+01 0.39 [0.19 , 0.39] 3.1E-04 0.13 [0.08 , 0.13] 1.0E-05 Cd200r1 0.70 [0.49 , 0.90] 7.2E+01 0.61 [0.40 , 0.61] 3.4E-06 0.17 [0.10 , 0.17] 6.9E-05 Il21r 0.69 [0.42 , 0.97] 7.3E+01 0.41 [0.04 , 0.41] 5.1E-02 0.12 [0.01 , 0.12] 7.5E-02 Cd44 0.69 [0.46 , 0.93] 7.4E+01 0.54 [0.28 , 0.54] 1.5E-04 0.13 [0.04 , 0.13] 1.0E-02 Cd5 0.69 [0.43 , 0.95] 7.5E+01 0.54 [0.26 , 0.54] 2.6E-04 0.17 [0.08 , 0.17] 2.7E-04 Stat4 0.68 [0.44 , 0.92] 7.6E+01 0.57 [0.29 , 0.57] 1.7E-04 0.14 [0.04 , 0.14] 1.0E-02 Itgae 0.68 [0.51 , 0.86] 7.7E+01 0.56 [0.38 , 0.56] 1.1E-06 0.22 [0.13 , 0.22] 4.6E-05 H2-Ab1 0.68 [0.40 , 0.95] 7.8E+01 0.57 [0.38 , 0.57] 1.4E-06 0.15 [0.07 , 0.15] 3.3E-04 Cd80 0.67 [0.40 , 0.94] 7.9E+01 0.57 [0.32 , 0.57] 4.7E-05 0.19 [0.09 , 0.19] 5.2E-04 H2-K1 0.67 [0.40 , 0.93] 8.0E+01 0.42 [0.14 , 0.42] 4.7E-03 0.07 [0.01 , 0.07] 4.3E-02 Havcr2 0.66 [0.45 , 0.88] 8.1E+01 0.49 [0.23 , 0.49] 3.6E-04 0.15 [0.08 , 0.15] 2.5E-04 Il7r 0.66 [0.45 , 0.87] 8.2E+01 0.52 [0.28 , 0.52] 9.9E-05 0.15 [0.06 , 0.15] 1.6E-03 Tnfrsf11b 0.66 [0.07 , 1.24] 8.3E+01 0.87 [0.28 , 0.87] 4.8E-03 0.19 [-0.07 , 0.19] 4.9E-01 Cybb 0.65 [0.38 , 0.93] 8.4E+01 0.26 [-0.01 , 0.26] 1.4E-01 0.12 [0.04 , 0.12] 3.9E-03 Il1rl2 0.65 [0.44 , 0.86] 8.5E+01 0.37 [0.17 , 0.37] 6.0E-04 0.16 [0.08 , 0.16] 2.0E-04 Cxcl9 0.65 [-0.17 , 1.47] 8.6E+01 0.71 [-0.24 , 0.71] 4.3E-01 0.19 [-0.13 , 0.19] 8.2E-01 Osm 0.65 [0.33 , 0.97] 8.7E+01 0.57 [0.23 , 0.57] 1.6E-03 0.21 [0.10 , 0.21] 3.5E-04 340 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Irf4 0.65 [0.24 , 1.06] 8.8E+01 0.50 [0.14 , 0.50] 1.0E-02 0.22 [0.08 , 0.22] 3.3E-03 H2-Aa 0.64 [0.45 , 0.84] 8.9E+01 0.45 [0.17 , 0.45] 2.1E-03 0.17 [0.10 , 0.17] 4.3E-05 Ticam2 0.64 [0.44 , 0.83] 9.0E+01 0.50 [0.27 , 0.50] 7.2E-05 0.17 [0.07 , 0.17] 1.2E-03 Tnfrsf11a 0.63 [0.46 , 0.81] 9.1E+01 0.41 [0.24 , 0.41] 3.9E-05 0.17 [0.11 , 0.17] 1.1E-05 Ccr7 0.63 [0.41 , 0.85] 9.2E+01 0.43 [0.15 , 0.43] 4.0E-03 0.17 [0.07 , 0.17] 1.7E-03 Cd207 0.63 [0.17 , 1.09] 9.3E+01 0.75 [0.30 , 0.75] 1.5E-03 0.33 [0.10 , 0.33] 7.4E-03 Arg1 0.63 [0.02 , 1.25] 9.4E+01 0.73 [-0.37 , 0.73] 6.1E-01 0.16 [-0.12 , 0.16] 8.6E-01 Gzmk 0.63 [-0.11 , 1.37] 9.5E+01 0.61 [-0.17 , 0.61] 3.5E-01 n.d [n.d , n.d] n.d Cd84 0.63 [0.39 , 0.87] 9.6E+01 0.39 [0.18 , 0.39] 5.8E-04 0.15 [0.08 , 0.15] 1.4E-04 Spp1 0.63 [0.28 , 0.98] 9.7E+01 0.77 [0.32 , 0.77] 1.2E-03 0.25 [0.11 , 0.25] 1.4E-03 Ly86 0.62 [0.37 , 0.88] 9.8E+01 0.29 [-0.01 , 0.29] 1.4E-01 0.16 [0.06 , 0.16] 2.8E-03 Ccrl2 0.62 [0.43 , 0.81] 9.9E+01 0.44 [0.33 , 0.44] 1.4E-07 0.06 [0.01 , 0.06] 3.7E-02 Psmb9 0.62 [0.42 , 0.83] 1.0E+02 0.32 [0.11 , 0.32] 3.2E-03 0.07 [0.00 , 0.07] 1.7E-01 Pla2g1b 0.62 [0.41 , 0.84] 1.0E+02 0.52 [0.33 , 0.52] 6.1E-06 0.15 [0.08 , 0.15] 2.7E-04 Lgals3 0.62 [0.35 , 0.89] 1.0E+02 0.53 [0.32 , 0.53] 1.7E-05 0.18 [0.10 , 0.18] 6.6E-05 H2-DMb2 0.62 [0.31 , 0.93] 1.0E+02 0.41 [0.17 , 0.41] 1.1E-03 0.18 [0.09 , 0.18] 2.3E-04 Cfp 0.61 [0.39 , 0.84] 1.0E+02 0.42 [0.13 , 0.42] 6.2E-03 0.19 [0.11 , 0.19] 4.6E-05 341 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ddx60 0.61 [0.26 , 0.96] 1.1E+02 0.42 [0.07 , 0.42] 3.2E-02 0.06 [-0.05 , 0.06] 9.2E-01 Gpr183 0.61 [0.38 , 0.83] 1.1E+02 0.43 [0.16 , 0.43] 2.2E-03 0.13 [0.03 , 0.13] 2.1E-02 Btk 0.60 [0.41 , 0.79] 1.1E+02 0.37 [0.18 , 0.37] 2.9E-04 0.12 [0.05 , 0.12] 2.5E-03 Pik3cg 0.60 [0.39 , 0.80] 1.1E+02 0.37 [0.17 , 0.37] 5.0E-04 0.12 [0.05 , 0.12] 1.3E-03 H2-T23 0.59 [0.38 , 0.81] 1.1E+02 0.36 [0.11 , 0.36] 6.7E-03 0.04 [-0.02 , 0.04] 5.4E-01 Irf5 0.59 [0.46 , 0.73] 1.1E+02 0.32 [0.17 , 0.32] 9.9E-05 0.17 [0.10 , 0.17] 2.5E-05 Cd86 0.59 [0.33 , 0.85] 1.1E+02 0.47 [0.18 , 0.47] 2.3E-03 0.17 [0.07 , 0.17] 1.6E-03 Itgb2 0.59 [0.40 , 0.77] 1.1E+02 0.47 [0.25 , 0.47] 1.1E-04 0.14 [0.07 , 0.14] 1.1E-04 Ikzf2 0.58 [0.43 , 0.74] 1.1E+02 0.43 [0.17 , 0.43] 1.4E-03 0.12 [0.06 , 0.12] 1.1E-04 Abca1 0.58 [0.39 , 0.78] 1.1E+02 0.40 [0.19 , 0.40] 3.9E-04 0.11 [0.05 , 0.11] 7.1E-04 Batf 0.58 [0.29 , 0.88] 1.2E+02 0.38 [0.11 , 0.38] 7.2E-03 0.13 [0.03 , 0.13] 1.7E-02 Ulbp1 0.58 [0.18 , 0.98] 1.2E+02 0.49 [0.15 , 0.49] 6.7E-03 0.17 [0.04 , 0.17] 1.8E-02 Slamf6 0.58 [0.21 , 0.96] 1.2E+02 0.43 [0.05 , 0.43] 4.8E-02 0.14 [0.02 , 0.14] 6.0E-02 Il18rap 0.57 [0.28 , 0.86] 1.2E+02 0.35 [0.06 , 0.35] 3.4E-02 0.07 [-0.05 , 0.07] 7.6E-01 Tlr8 0.57 [0.35 , 0.78] 1.2E+02 0.37 [0.13 , 0.37] 4.2E-03 0.07 [0.01 , 0.07] 6.5E-02 Psmb8 0.56 [0.36 , 0.77] 1.2E+02 0.33 [0.14 , 0.33] 9.0E-04 0.08 [0.02 , 0.08] 1.8E-02 Psen2 0.56 [0.37 , 0.76] 1.2E+02 0.45 [0.34 , 0.45] 1.0E-07 0.07 [0.02 , 0.07] 3.7E-03 342 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value H2-M3 0.56 [0.39 , 0.73] 1.2E+02 0.29 [0.11 , 0.29] 1.6E-03 0.08 [0.03 , 0.08] 1.6E-03 Cxcr4 0.55 [0.26 , 0.84] 1.2E+02 0.38 [0.13 , 0.38] 3.8E-03 0.19 [0.08 , 0.19] 9.9E-04 Mefv 0.55 [-0.06 , 1.17] 1.2E+02 0.64 [0.15 , 0.64] 1.4E-02 0.16 [-0.06 , 0.16] 4.8E-01 Tlr1 0.55 [0.20 , 0.89] 1.3E+02 0.41 [0.08 , 0.41] 2.6E-02 0.11 [-0.03 , 0.11] 3.6E-01 Hcst 0.54 [0.14 , 0.95] 1.3E+02 0.52 [0.12 , 0.52] 1.7E-02 0.12 [-0.03 , 0.12] 3.9E-01 Fpr2 0.54 [0.24 , 0.85] 1.3E+02 0.24 [-0.09 , 0.24] 4.5E-01 0.13 [0.01 , 0.13] 9.3E-02 Itgam 0.54 [0.29 , 0.78] 1.3E+02 0.44 [0.12 , 0.44] 8.9E-03 0.15 [0.06 , 0.15] 2.4E-03 Xaf1 0.53 [0.26 , 0.81] 1.3E+02 0.24 [-0.07 , 0.24] 3.6E-01 0.02 [-0.05 , 0.02] 5.8E-01 Il6 0.53 [-0.30 , 1.36] 1.3E+02 0.49 [-0.35 , 0.49] 8.1E-01 0.20 [-0.17 , 0.20] 9.1E-01 Cd37 0.53 [0.33 , 0.74] 1.3E+02 0.30 [0.09 , 0.30] 6.4E-03 0.09 [0.01 , 0.09] 7.8E-02 Bst2 0.53 [0.25 , 0.80] 1.3E+02 0.33 [-0.02 , 0.33] 1.6E-01 0.06 [-0.01 , 0.06] 3.8E-01 Ccl19 0.52 [0.17 , 0.87] 1.3E+02 0.43 [0.09 , 0.43] 2.1E-02 0.09 [-0.06 , 0.09] 8.4E-01 Txnip 0.52 [0.35 , 0.70] 1.3E+02 0.34 [0.16 , 0.34] 3.7E-04 0.09 [0.01 , 0.09] 6.4E-02 Herc6 0.52 [0.31 , 0.73] 1.4E+02 0.36 [0.00 , 0.36] 1.1E-01 0.06 [-0.01 , 0.06] 3.6E-01 Cmpk2 0.51 [0.22 , 0.81] 1.4E+02 0.22 [-0.11 , 0.22] 6.1E-01 0.02 [-0.08 , 0.02] 3.7E-01 Il1rl1 0.51 [0.26 , 0.76] 1.4E+02 0.49 [0.22 , 0.49] 5.4E-04 0.24 [0.17 , 0.24] 8.0E-07 Xcr1 0.51 [0.28 , 0.73] 1.4E+02 0.46 [0.19 , 0.46] 1.3E-03 0.19 [0.09 , 0.19] 6.4E-04 343 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value C1s1 0.51 [0.36 , 0.66] 1.4E+02 0.36 [0.22 , 0.36] 1.4E-05 0.12 [0.07 , 0.12] 2.8E-05 Ly9 0.50 [0.25 , 0.76] 1.4E+02 0.40 [0.09 , 0.40] 1.8E-02 0.07 [-0.01 , 0.07] 3.3E-01 Cd96 0.50 [0.04 , 0.97] 1.4E+02 0.53 [0.06 , 0.53] 4.9E-02 0.20 [0.03 , 0.20] 5.0E-02 Lbp 0.50 [0.31 , 0.69] 1.4E+02 0.43 [0.26 , 0.43] 2.1E-05 0.11 [0.03 , 0.11] 8.2E-03 H2-Ea-ps 0.50 [0.30 , 0.70] 1.4E+02 0.32 [0.15 , 0.32] 5.8E-04 0.17 [0.10 , 0.17] 3.2E-05 Runx3 0.50 [-0.01 , 1.00] 1.4E+02 0.43 [-0.09 , 0.43] 2.9E-01 0.10 [-0.06 , 0.10] 7.3E-01 Sh2b2 0.49 [0.00 , 0.99] 1.5E+02 0.43 [-0.01 , 0.43] 1.2E-01 0.17 [-0.01 , 0.17] 1.7E-01 Tnfsf13 0.49 [0.31 , 0.66] 1.5E+02 0.42 [0.22 , 0.42] 1.1E-04 0.13 [0.05 , 0.13] 2.1E-03 Lcp1 0.49 [0.28 , 0.69] 1.5E+02 0.26 [0.04 , 0.26] 3.2E-02 0.12 [0.04 , 0.12] 4.5E-03 H2-Q2 0.49 [0.28 , 0.69] 1.5E+02 0.35 [-0.21 , 0.35] 6.9E-01 0.08 [-0.01 , 0.08] 2.1E-01 Foxj1 0.48 [0.20 , 0.77] 1.5E+02 0.35 [0.09 , 0.35] 1.1E-02 0.14 [0.03 , 0.14] 3.1E-02 Selplg 0.48 [0.32 , 0.64] 1.5E+02 0.31 [0.14 , 0.31] 6.4E-04 0.13 [0.06 , 0.13] 3.3E-04 Ltb 0.48 [0.21 , 0.75] 1.5E+02 0.33 [0.04 , 0.33] 4.5E-02 0.17 [0.03 , 0.17] 3.4E-02 Inpp5d 0.48 [0.28 , 0.69] 1.5E+02 0.33 [0.13 , 0.33] 1.9E-03 0.10 [0.02 , 0.10] 3.3E-02 Nfatc2 0.48 [0.25 , 0.71] 1.5E+02 0.26 [0.02 , 0.26] 6.5E-02 0.10 [-0.01 , 0.10] 1.8E-01 Rsad2 0.47 [-0.17 , 1.11] 1.5E+02 0.37 [-0.08 , 0.37] 2.8E-01 -0.03 [-0.21 , -0.03] 4.7E-02 Pou2f2 0.47 [0.19 , 0.75] 1.6E+02 0.18 [-0.14 , 0.18] 8.7E-01 0.06 [-0.10 , 0.06] 7.4E-01 344 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tyk2 0.47 [0.29 , 0.64] 1.6E+02 0.28 [0.14 , 0.28] 3.2E-04 0.03 [-0.04 , 0.03] 8.0E-01 Pdcd1lg2 0.46 [0.06 , 0.87] 1.6E+02 0.58 [0.16 , 0.58] 1.0E-02 0.18 [0.02 , 0.18] 5.3E-02 Relb 0.46 [0.26 , 0.66] 1.6E+02 0.26 [0.06 , 0.26] 1.9E-02 0.07 [-0.02 , 0.07] 3.8E-01 Tnfrsf13b 0.46 [0.20 , 0.71] 1.6E+02 0.25 [-0.03 , 0.25] 2.0E-01 0.11 [0.01 , 0.11] 9.1E-02 Sh2d1a 0.46 [0.19 , 0.73] 1.6E+02 0.34 [0.07 , 0.34] 2.2E-02 0.09 [-0.04 , 0.09] 5.8E-01 Tnfrsf1b 0.46 [0.26 , 0.66] 1.6E+02 0.32 [0.13 , 0.32] 1.4E-03 0.10 [0.03 , 0.10] 8.1E-03 Tnfsf15 0.45 [0.21 , 0.70] 1.6E+02 0.40 [0.07 , 0.40] 2.8E-02 0.15 [-0.01 , 0.15] 2.2E-01 Flt3 0.45 [0.21 , 0.69] 1.6E+02 0.33 [0.05 , 0.33] 4.0E-02 0.19 [0.10 , 0.19] 3.4E-04 C4b 0.45 [0.26 , 0.64] 1.6E+02 0.31 [0.13 , 0.31] 1.2E-03 0.08 [0.00 , 0.08] 1.3E-01 Socs3 0.45 [0.02 , 0.87] 1.7E+02 0.58 [0.32 , 0.58] 5.9E-05 0.01 [-0.14 , 0.01] 2.0E-01 Ifih1 0.44 [0.29 , 0.60] 1.7E+02 0.28 [0.05 , 0.28] 2.3E-02 0.00 [-0.05 , 0.00] 2.0E-01 Spn 0.44 [0.21 , 0.67] 1.7E+02 0.25 [0.08 , 0.25] 6.1E-03 0.06 [-0.03 , 0.06] 6.1E-01 Tnfsf14 0.44 [0.11 , 0.77] 1.7E+02 0.24 [-0.08 , 0.24] 4.3E-01 0.15 [0.02 , 0.15] 5.3E-02 Cd247 0.44 [0.20 , 0.68] 1.7E+02 0.35 [0.10 , 0.35] 7.6E-03 0.09 [-0.01 , 0.09] 2.5E-01 Stat2 0.44 [0.19 , 0.69] 1.7E+02 0.22 [-0.02 , 0.22] 1.7E-01 -0.02 [-0.08 , -0.02] 2.5E-02 Nfatc4 0.44 [0.16 , 0.72] 1.7E+02 0.42 [0.21 , 0.42] 1.8E-04 0.02 [-0.07 , 0.02] 4.8E-01 Cxcr5 0.44 [-0.03 , 0.91] 1.7E+02 0.36 [-0.04 , 0.36] 2.0E-01 0.08 [-0.07 , 0.08] 8.8E-01 345 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd3d 0.44 [0.27 , 0.60] 1.7E+02 0.30 [0.14 , 0.30] 4.6E-04 0.11 [0.04 , 0.11] 5.2E-03 Il1a 0.44 [0.13 , 0.74] 1.7E+02 0.32 [0.04 , 0.32] 4.1E-02 0.12 [0.04 , 0.12] 8.2E-03 H2-Ob 0.43 [0.01 , 0.85] 1.8E+02 0.42 [0.09 , 0.42] 1.7E-02 0.06 [-0.05 , 0.06] 8.7E-01 Cd69 0.43 [0.17 , 0.68] 1.8E+02 0.30 [0.08 , 0.30] 9.4E-03 0.14 [0.03 , 0.14] 1.9E-02 Ido1 0.43 [-0.28 , 1.14] 1.8E+02 0.67 [0.09 , 0.67] 4.1E-02 n.d [n.d , n.d] n.d Il2rg 0.42 [0.20 , 0.65] 1.8E+02 0.34 [0.13 , 0.34] 2.2E-03 0.05 [-0.01 , 0.05] 2.5E-01 Cd3e 0.42 [0.15 , 0.70] 1.8E+02 0.38 [0.08 , 0.38] 2.0E-02 0.08 [-0.01 , 0.08] 2.6E-01 Pik3cd 0.42 [0.21 , 0.63] 1.8E+02 0.18 [-0.03 , 0.18] 2.7E-01 0.12 [0.01 , 0.12] 6.0E-02 Abcg1 0.42 [0.18 , 0.66] 1.8E+02 0.34 [0.14 , 0.34] 1.2E-03 0.13 [0.05 , 0.13] 3.6E-03 Syk 0.41 [0.23 , 0.58] 1.8E+02 0.22 [0.05 , 0.22] 1.4E-02 0.08 [0.03 , 0.08] 3.6E-03 Cxcr3 0.41 [0.16 , 0.66] 1.8E+02 0.30 [-0.08 , 0.30] 3.6E-01 0.12 [0.03 , 0.12] 1.5E-02 Tapbp 0.40 [0.20 , 0.61] 1.8E+02 0.35 [0.26 , 0.35] 2.7E-07 0.05 [0.02 , 0.05] 2.0E-03 Col3a1 0.40 [0.14 , 0.67] 1.9E+02 0.48 [0.06 , 0.48] 4.7E-02 0.12 [0.05 , 0.12] 1.2E-03 Tlr2 0.40 [0.06 , 0.74] 1.9E+02 0.17 [-0.02 , 0.17] 2.1E-01 0.14 [0.06 , 0.14] 1.6E-03 Cd274 0.40 [0.14 , 0.66] 1.9E+02 0.30 [0.01 , 0.30] 7.8E-02 0.07 [-0.03 , 0.07] 5.2E-01 Ccr9 0.40 [0.11 , 0.69] 1.9E+02 0.38 [0.02 , 0.38] 8.2E-02 0.03 [-0.10 , 0.03] 3.7E-01 Sh2d1b1 0.40 [0.23 , 0.57] 1.9E+02 0.20 [-0.09 , 0.20] 5.7E-01 0.10 [0.02 , 0.10] 4.3E-02 346 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Traf3 0.40 [0.27 , 0.52] 1.9E+02 0.33 [-0.04 , 0.33] 2.0E-01 0.06 [0.01 , 0.06] 5.2E-02 Prkcd 0.39 [0.28 , 0.51] 1.9E+02 0.26 [0.15 , 0.26] 2.1E-05 0.06 [0.02 , 0.06] 8.2E-03 Lyz2 0.39 [0.18 , 0.61] 1.9E+02 0.38 [0.15 , 0.38] 1.5E-03 0.16 [0.08 , 0.16] 1.1E-04 Ikbke 0.39 [0.22 , 0.56] 1.9E+02 0.33 [0.12 , 0.33] 2.8E-03 0.09 [0.01 , 0.09] 4.6E-02 Blk 0.39 [0.01 , 0.77] 1.9E+02 0.36 [-0.05 , 0.36] 2.3E-01 0.11 [-0.07 , 0.11] 7.6E-01 Ly96 0.38 [0.20 , 0.57] 2.0E+02 0.26 [0.07 , 0.26] 1.3E-02 0.08 [0.02 , 0.08] 1.9E-02 Cd276 0.38 [0.24 , 0.52] 2.0E+02 0.33 [0.18 , 0.33] 7.1E-05 0.09 [0.02 , 0.09] 2.1E-02 Cd19 0.38 [-0.09 , 0.85] 2.0E+02 0.35 [-0.02 , 0.35] 1.5E-01 0.08 [-0.08 , 0.08] 9.6E-01 Cd79b 0.38 [0.03 , 0.73] 2.0E+02 0.22 [-0.13 , 0.22] 6.7E-01 0.11 [-0.03 , 0.11] 3.9E-01 Muc1 0.37 [0.24 , 0.50] 2.0E+02 0.30 [0.19 , 0.30] 4.0E-06 0.09 [0.05 , 0.09] 8.6E-05 Cd99 0.37 [0.15 , 0.59] 2.0E+02 0.43 [0.18 , 0.43] 1.3E-03 0.10 [0.01 , 0.10] 5.7E-02 Cd28 0.37 [-0.20 , 0.94] 2.0E+02 0.43 [-0.04 , 0.43] 1.7E-01 0.16 [-0.04 , 0.16] 3.9E-01 Cxcr6 0.37 [0.10 , 0.64] 2.0E+02 0.30 [0.05 , 0.30] 3.2E-02 0.12 [0.04 , 0.12] 9.5E-03 Cd244 0.37 [-0.13 , 0.86] 2.0E+02 0.34 [-0.28 , 0.34] 9.0E-01 0.07 [-0.13 , 0.07] 6.9E-01 Maf 0.36 [0.14 , 0.58] 2.0E+02 0.25 [-0.01 , 0.25] 1.4E-01 0.07 [-0.03 , 0.07] 5.0E-01 Trp53 0.36 [0.10 , 0.62] 2.1E+02 0.38 [0.17 , 0.38] 6.2E-04 0.07 [-0.01 , 0.07] 2.2E-01 Fap 0.36 [0.10 , 0.62] 2.1E+02 0.36 [0.03 , 0.36] 5.5E-02 0.09 [-0.05 , 0.09] 6.6E-01 347 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Nfkbia 0.36 [0.14 , 0.57] 2.1E+02 0.28 [0.05 , 0.28] 2.4E-02 0.07 [-0.01 , 0.07] 3.1E-01 Pparg 0.35 [0.11 , 0.60] 2.1E+02 0.33 [0.13 , 0.33] 1.5E-03 0.08 [-0.01 , 0.08] 2.7E-01 Cd53 0.35 [0.09 , 0.61] 2.1E+02 0.39 [0.12 , 0.39] 6.3E-03 0.11 [0.01 , 0.11] 7.4E-02 Ifitm2 0.35 [0.21 , 0.50] 2.1E+02 0.34 [0.24 , 0.34] 5.6E-07 0.06 [0.02 , 0.06] 9.8E-03 Cd200 0.35 [0.23 , 0.47] 2.1E+02 0.33 [0.19 , 0.33] 4.7E-05 0.06 [0.00 , 0.06] 1.0E-01 C6 0.35 [0.12 , 0.58] 2.1E+02 0.41 [0.17 , 0.41] 1.2E-03 0.13 [0.05 , 0.13] 2.0E-03 Cfi 0.35 [-0.14 , 0.84] 2.1E+02 0.41 [0.05 , 0.41] 4.9E-02 0.19 [0.05 , 0.19] 1.2E-02 Rrad 0.35 [-0.22 , 0.91] 2.1E+02 0.42 [-0.18 , 0.42] 5.2E-01 0.17 [-0.10 , 0.17] 7.3E-01 Ifit2 0.35 [0.04 , 0.65] 2.2E+02 0.17 [-0.22 , 0.17] 8.2E-01 0.04 [-0.07 , 0.04] 6.3E-01 Ambp 0.35 [-0.64 , 1.33] 2.2E+02 0.45 [-0.43 , 0.45] 9.8E-01 n.d [n.d , n.d] n.d Tdo2 0.34 [-0.22 , 0.90] 2.2E+02 0.34 [0.02 , 0.34] 7.8E-02 0.18 [-0.02 , 0.18] 2.3E-01 Il3ra 0.34 [0.08 , 0.60] 2.2E+02 0.42 [0.22 , 0.42] 1.3E-04 0.12 [0.04 , 0.12] 4.8E-03 Fcer1a 0.34 [-0.12 , 0.80] 2.2E+02 0.39 [0.02 , 0.39] 7.7E-02 0.19 [0.06 , 0.19] 8.2E-03 St6gal1 0.33 [0.22 , 0.45] 2.2E+02 0.22 [0.09 , 0.22] 1.1E-03 0.06 [0.02 , 0.06] 3.2E-03 Cd63 0.33 [0.16 , 0.51] 2.2E+02 0.37 [0.20 , 0.37] 8.1E-05 0.11 [0.04 , 0.11] 2.1E-03 Tap2 0.33 [0.12 , 0.54] 2.2E+02 0.14 [-0.05 , 0.14] 4.1E-01 0.04 [-0.04 , 0.04] 9.3E-01 Serping1 0.33 [0.17 , 0.49] 2.2E+02 0.30 [0.20 , 0.30] 2.3E-06 0.07 [0.03 , 0.07] 7.4E-04 348 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il17ra 0.33 [0.19 , 0.47] 2.2E+02 0.22 [0.04 , 0.22] 2.7E-02 0.04 [-0.03 , 0.04] 7.9E-01 Btla 0.33 [0.04 , 0.62] 2.3E+02 0.24 [-0.05 , 0.24] 3.0E-01 0.07 [-0.05 , 0.07] 7.7E-01 Casp3 0.32 [0.20 , 0.44] 2.3E+02 0.26 [0.09 , 0.26] 3.3E-03 0.11 [0.04 , 0.11] 2.8E-03 Ms4a1 0.32 [-0.27 , 0.90] 2.3E+02 0.21 [-0.26 , 0.21] 8.7E-01 0.10 [-0.12 , 0.10] 8.8E-01 Tlr6 0.32 [0.16 , 0.47] 2.3E+02 0.22 [0.01 , 0.22] 7.6E-02 0.10 [0.03 , 0.10] 8.4E-03 Il16 0.32 [0.18 , 0.46] 2.3E+02 0.23 [0.09 , 0.23] 1.3E-03 0.07 [0.00 , 0.07] 8.7E-02 Sigirr 0.32 [0.16 , 0.47] 2.3E+02 0.34 [0.22 , 0.34] 4.1E-06 0.05 [0.00 , 0.05] 2.2E-01 Amica1 0.31 [0.08 , 0.55] 2.3E+02 0.25 [0.04 , 0.25] 3.1E-02 0.16 [0.06 , 0.16] 2.9E-03 Mapk3 0.31 [0.11 , 0.51] 2.3E+02 0.32 [0.27 , 0.32] 3.0E-10 0.05 [0.02 , 0.05] 2.4E-03 Cd180 0.31 [0.04 , 0.57] 2.3E+02 0.21 [-0.12 , 0.21] 6.6E-01 0.06 [-0.06 , 0.06] 9.3E-01 Abl1 0.30 [0.14 , 0.47] 2.3E+02 0.27 [0.17 , 0.27] 1.5E-05 0.04 [0.01 , 0.04] 3.2E-02 Il5ra 0.29 [-0.16 , 0.74] 2.4E+02 0.43 [0.08 , 0.43] 2.4E-02 0.12 [-0.01 , 0.12] 1.7E-01 Vhl 0.29 [0.13 , 0.46] 2.4E+02 0.27 [0.14 , 0.27] 1.4E-04 0.07 [0.02 , 0.07] 3.8E-03 Axl 0.29 [0.19 , 0.39] 2.4E+02 0.16 [0.05 , 0.16] 6.3E-03 0.03 [0.00 , 0.03] 6.7E-02 Klra2 0.29 [0.05 , 0.53] 2.4E+02 0.10 [-0.15 , 0.10] 7.4E-01 0.02 [-0.09 , 0.02] 3.8E-01 Nlrc5 0.29 [-0.06 , 0.64] 2.4E+02 0.13 [-0.21 , 0.13] 7.1E-01 -0.03 [-0.13 , -0.03] 2.3E-02 Pdgfc 0.29 [0.08 , 0.49] 2.4E+02 0.17 [-0.31 , 0.17] 6.6E-01 0.03 [-0.03 , 0.03] 9.2E-01 349 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd3g 0.28 [0.08 , 0.49] 2.4E+02 0.21 [-0.05 , 0.21] 3.2E-01 0.10 [0.00 , 0.10] 9.8E-02 Tlr5 0.28 [0.02 , 0.55] 2.4E+02 0.43 [0.22 , 0.43] 1.3E-04 0.03 [-0.06 , 0.03] 6.7E-01 Cd48 0.28 [0.11 , 0.46] 2.4E+02 0.25 [0.06 , 0.25] 1.4E-02 0.10 [0.02 , 0.10] 2.9E-02 Itgb3 0.28 [0.09 , 0.48] 2.4E+02 0.26 [0.07 , 0.26] 1.0E-02 0.05 [-0.02 , 0.05] 6.0E-01 Lamp1 0.28 [0.19 , 0.38] 2.5E+02 0.26 [0.16 , 0.26] 6.1E-06 0.04 [0.02 , 0.04] 1.3E-03 Itga4 0.28 [0.08 , 0.49] 2.5E+02 0.19 [-0.01 , 0.19] 1.4E-01 0.03 [-0.05 , 0.03] 6.9E-01 Il34 0.28 [0.06 , 0.50] 2.5E+02 0.37 [0.21 , 0.37] 3.9E-05 0.08 [0.04 , 0.08] 4.9E-04 Csf1 0.28 [0.09 , 0.48] 2.5E+02 0.18 [-0.05 , 0.18] 3.4E-01 0.08 [-0.04 , 0.08] 6.6E-01 Ifnar2 0.28 [0.19 , 0.36] 2.5E+02 0.19 [0.10 , 0.19] 1.1E-04 0.06 [0.03 , 0.06] 4.1E-04 Tnfaip3 0.28 [0.05 , 0.50] 2.5E+02 0.13 [-0.08 , 0.13] 7.2E-01 0.08 [0.00 , 0.08] 9.9E-02 Casp1 0.28 [0.13 , 0.43] 2.5E+02 0.10 [-0.03 , 0.10] 4.2E-01 0.09 [0.03 , 0.09] 9.1E-03 Nfkb2 0.27 [0.10 , 0.45] 2.5E+02 0.20 [0.00 , 0.20] 1.0E-01 0.04 [-0.05 , 0.04] 7.9E-01 Jak3 0.27 [0.09 , 0.45] 2.5E+02 0.25 [0.09 , 0.25] 3.4E-03 0.01 [-0.06 , 0.01] 3.3E-01 Ncam1 0.27 [-0.10 , 0.64] 2.5E+02 0.43 [0.09 , 0.43] 2.0E-02 0.08 [-0.07 , 0.08] 9.2E-01 Tgfbr1 0.27 [0.11 , 0.43] 2.6E+02 0.15 [0.03 , 0.15] 1.7E-02 0.05 [0.01 , 0.05] 1.2E-02 Ccl22 0.27 [-0.13 , 0.66] 2.6E+02 0.49 [0.04 , 0.49] 5.8E-02 0.23 [0.05 , 0.23] 2.7E-02 Cxcl16 0.26 [0.10 , 0.43] 2.6E+02 0.16 [0.01 , 0.16] 6.6E-02 0.06 [0.01 , 0.06] 6.0E-02 350 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Fas 0.26 [-0.03 , 0.56] 2.6E+02 0.42 [0.18 , 0.42] 9.7E-04 0.07 [-0.06 , 0.07] 9.4E-01 Irak1 0.26 [-0.04 , 0.55] 2.6E+02 0.37 [0.27 , 0.37] 2.2E-07 -0.01 [-0.04 , -0.01] 5.7E-02 Ccr3 0.26 [-1.18 , 1.70] 2.6E+02 0.83 [-0.69 , 0.83] 8.9E-01 n.d [n.d , n.d] n.d Lyn 0.26 [0.16 , 0.35] 2.6E+02 0.11 [0.01 , 0.11] 7.9E-02 0.04 [0.00 , 0.04] 2.2E-01 Irf8 0.26 [0.09 , 0.42] 2.6E+02 0.29 [0.06 , 0.29] 2.1E-02 0.08 [0.01 , 0.08] 8.7E-02 Cd47 0.26 [0.15 , 0.36] 2.6E+02 0.25 [-0.08 , 0.25] 4.1E-01 0.03 [0.01 , 0.03] 3.2E-02 Sbno2 0.26 [0.01 , 0.50] 2.6E+02 0.26 [0.02 , 0.26] 6.3E-02 0.05 [-0.06 , 0.05] 9.1E-01 Xcl1 0.25 [-0.15 , 0.66] 2.7E+02 0.20 [-0.19 , 0.20] 9.6E-01 0.04 [-0.12 , 0.04] 4.6E-01 Vcam1 0.25 [-0.04 , 0.54] 2.7E+02 0.30 [0.04 , 0.30] 4.8E-02 0.07 [-0.04 , 0.07] 7.9E-01 Ciita 0.25 [-0.06 , 0.55] 2.7E+02 0.20 [-0.08 , 0.20] 4.9E-01 0.09 [-0.04 , 0.09] 5.6E-01 Rorc 0.24 [0.10 , 0.39] 2.7E+02 0.18 [0.03 , 0.18] 2.6E-02 0.04 [-0.01 , 0.04] 3.0E-01 Hck 0.24 [-0.04 , 0.53] 2.7E+02 0.17 [-0.11 , 0.17] 7.4E-01 0.07 [-0.07 , 0.07] 9.9E-01 Casp8 0.24 [0.15 , 0.34] 2.7E+02 0.12 [0.04 , 0.12] 6.7E-03 0.07 [0.03 , 0.07] 1.5E-03 Ccl24 0.23 [-0.17 , 0.63] 2.7E+02 0.51 [0.03 , 0.51] 6.9E-02 0.06 [-0.08 , 0.06] 9.1E-01 Tfeb 0.23 [0.10 , 0.36] 2.7E+02 0.27 [0.14 , 0.27] 1.8E-04 0.09 [0.04 , 0.09] 4.9E-04 Nfatc1 0.23 [0.02 , 0.43] 2.7E+02 0.27 [0.15 , 0.27] 5.1E-05 -0.01 [-0.06 , -0.01] 3.2E-02 Pdgfrb 0.23 [-0.01 , 0.46] 2.7E+02 0.28 [0.14 , 0.28] 3.5E-04 0.04 [-0.01 , 0.04] 3.9E-01 351 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Rel 0.22 [0.10 , 0.35] 2.8E+02 0.17 [0.07 , 0.17] 9.5E-04 0.05 [0.00 , 0.05] 1.9E-01 Il15ra 0.22 [-0.14 , 0.58] 2.8E+02 0.21 [-0.17 , 0.21] 8.7E-01 0.14 [0.02 , 0.14] 4.0E-02 Icosl 0.22 [0.08 , 0.36] 2.8E+02 0.15 [-0.02 , 0.15] 2.5E-01 0.06 [-0.01 , 0.06] 3.7E-01 Tab1 0.21 [0.02 , 0.41] 2.8E+02 0.28 [0.13 , 0.28] 6.2E-04 0.06 [-0.01 , 0.06] 3.5E-01 Cd2 0.21 [0.00 , 0.42] 2.8E+02 0.11 [-0.19 , 0.11] 6.7E-01 0.01 [-0.09 , 0.01] 2.5E-01 Ctsl 0.21 [0.06 , 0.36] 2.8E+02 0.11 [0.00 , 0.11] 1.2E-01 0.04 [0.01 , 0.04] 5.3E-02 Ptprc 0.21 [0.04 , 0.38] 2.8E+02 0.15 [-0.04 , 0.15] 3.7E-01 0.05 [-0.03 , 0.05] 6.7E-01 Bst1 0.21 [-0.01 , 0.44] 2.8E+02 0.16 [-0.02 , 0.16] 2.0E-01 0.08 [0.01 , 0.08] 8.7E-02 Vim 0.21 [0.07 , 0.34] 2.8E+02 0.24 [0.14 , 0.24] 4.7E-05 0.07 [0.03 , 0.07] 4.9E-04 Snai1 0.21 [-0.23 , 0.64] 2.8E+02 0.40 [0.11 , 0.40] 1.1E-02 0.02 [-0.11 , 0.02] 2.8E-01 Cd38 0.21 [-0.04 , 0.45] 2.9E+02 0.22 [0.02 , 0.22] 5.2E-02 0.02 [-0.05 , 0.02] 5.6E-01 C2 0.21 [0.00 , 0.41] 2.9E+02 0.16 [-0.17 , 0.16] 9.7E-01 0.07 [-0.01 , 0.07] 2.1E-01 Ripk2 0.20 [0.10 , 0.31] 2.9E+02 0.16 [0.01 , 0.16] 6.3E-02 0.03 [-0.03 , 0.03] 9.7E-01 Pml 0.20 [0.01 , 0.39] 2.9E+02 0.10 [-0.07 , 0.10] 7.7E-01 0.00 [-0.05 , 0.00] 1.9E-01 Zap70 0.20 [-0.05 , 0.45] 2.9E+02 0.26 [0.06 , 0.26] 1.8E-02 0.05 [-0.05 , 0.05] 9.1E-01 Cd1d1 0.20 [0.04 , 0.36] 2.9E+02 0.17 [0.01 , 0.17] 7.2E-02 0.05 [-0.01 , 0.05] 2.2E-01 Atg12 0.20 [0.10 , 0.30] 2.9E+02 0.18 [0.05 , 0.18] 9.3E-03 0.03 [0.00 , 0.03] 9.4E-02 352 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Flt3l 0.20 [0.05 , 0.35] 2.9E+02 0.16 [-0.09 , 0.16] 6.6E-01 0.01 [-0.06 , 0.01] 2.4E-01 Smad2 0.20 [0.00 , 0.39] 2.9E+02 0.25 [0.09 , 0.25] 3.9E-03 0.04 [-0.02 , 0.04] 6.9E-01 Stat1 0.20 [-0.05 , 0.44] 2.9E+02 0.04 [-0.25 , 0.04] 2.6E-01 -0.01 [-0.08 , -0.01] 7.6E-02 Il1r1 0.20 [0.07 , 0.33] 3.0E+02 0.13 [0.00 , 0.13] 1.1E-01 0.05 [-0.01 , 0.05] 2.5E-01 H2-DMb1 0.20 [-0.09 , 0.48] 3.0E+02 0.05 [-0.18 , 0.05] 3.8E-01 0.16 [0.09 , 0.16] 3.5E-05 Ddx58 0.20 [0.04 , 0.35] 3.0E+02 0.06 [-0.10 , 0.06] 6.8E-01 0.01 [-0.04 , 0.01] 5.3E-01 Nfkb1 0.20 [0.12 , 0.28] 3.0E+02 0.16 [0.08 , 0.16] 2.2E-04 0.04 [0.00 , 0.04] 2.2E-01 Irak2 0.20 [0.08 , 0.31] 3.0E+02 0.10 [-0.01 , 0.10] 2.0E-01 0.05 [0.01 , 0.05] 2.4E-02 Il2rb 0.20 [-0.04 , 0.43] 3.0E+02 0.24 [-0.03 , 0.24] 2.2E-01 0.00 [-0.09 , 0.00] 9.8E-02 Cr2 0.20 [-0.68 , 1.07] 3.0E+02 0.30 [-0.62 , 0.30] 5.9E-01 0.15 [-0.15 , 0.15] 1.0E+00 Lilra5 0.19 [-0.04 , 0.43] 3.0E+02 0.09 [-0.19 , 0.09] 5.5E-01 0.04 [-0.05 , 0.04] 9.6E-01 Mrc1 0.19 [-0.05 , 0.43] 3.0E+02 0.19 [0.01 , 0.19] 7.3E-02 0.07 [0.01 , 0.07] 6.0E-02 Cmklr1 0.19 [-0.04 , 0.42] 3.0E+02 0.23 [0.00 , 0.23] 1.2E-01 0.05 [-0.06 , 0.05] 8.8E-01 Traf2 0.18 [0.09 , 0.27] 3.1E+02 0.17 [0.07 , 0.17] 6.1E-04 0.04 [-0.02 , 0.04] 6.9E-01 Tfe3 0.18 [0.08 , 0.28] 3.1E+02 0.15 [0.01 , 0.15] 5.5E-02 0.02 [-0.02 , 0.02] 9.3E-01 Mef2c 0.18 [-0.02 , 0.38] 3.1E+02 0.16 [-0.03 , 0.16] 2.9E-01 0.05 [-0.04 , 0.05] 9.6E-01 Fcgr4 0.18 [-0.06 , 0.42] 3.1E+02 0.13 [-0.16 , 0.13] 8.9E-01 0.04 [-0.06 , 0.04] 7.6E-01 353 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Adora2a 0.18 [-0.15 , 0.50] 3.1E+02 0.25 [-0.08 , 0.25] 4.3E-01 0.08 [-0.09 , 0.08] 9.1E-01 Ifnar1 0.17 [0.10 , 0.24] 3.1E+02 0.11 [0.04 , 0.11] 4.3E-03 0.03 [-0.01 , 0.03] 4.0E-01 Ctsw 0.17 [-0.07 , 0.41] 3.1E+02 0.31 [0.07 , 0.31] 1.4E-02 0.04 [-0.02 , 0.04] 7.5E-01 Mertk 0.17 [-0.02 , 0.35] 3.1E+02 0.19 [0.02 , 0.19] 5.4E-02 0.04 [-0.06 , 0.04] 8.2E-01 Pvrl2 0.16 [0.03 , 0.30] 3.1E+02 0.16 [0.03 , 0.16] 3.3E-02 0.07 [0.02 , 0.07] 6.3E-03 Jak2 0.16 [0.07 , 0.25] 3.1E+02 0.14 [0.01 , 0.14] 5.5E-02 0.02 [-0.01 , 0.02] 7.9E-01 Abcb1a 0.16 [-0.13 , 0.45] 3.2E+02 -0.02 [-0.18 , -0.02] 4.4E-02 0.01 [-0.07 , 0.01] 3.0E-01 C5ar1 0.16 [-0.11 , 0.44] 3.2E+02 0.23 [-0.04 , 0.23] 2.7E-01 0.06 [-0.05 , 0.06] 8.5E-01 Slamf1 0.16 [-0.12 , 0.44] 3.2E+02 0.22 [-0.14 , 0.22] 7.2E-01 0.05 [-0.06 , 0.05] 8.7E-01 C7 0.16 [0.05 , 0.27] 3.2E+02 0.23 [0.09 , 0.23] 1.5E-03 0.08 [0.02 , 0.08] 1.0E-02 Raet1c 0.16 [-0.04 , 0.36] 3.2E+02 0.15 [-0.04 , 0.15] 3.2E-01 0.10 [0.03 , 0.10] 5.3E-03 Myd88 0.16 [0.01 , 0.31] 3.2E+02 0.20 [-0.32 , 0.20] 7.2E-01 0.01 [-0.05 , 0.01] 2.4E-01 Irak3 0.16 [-0.04 , 0.36] 3.2E+02 0.03 [-0.11 , 0.03] 3.9E-01 0.03 [-0.02 , 0.03] 9.2E-01 Hras 0.16 [-0.22 , 0.53] 3.2E+02 0.10 [-0.25 , 0.10] 5.0E-01 0.14 [-0.01 , 0.14] 2.0E-01 Il1rap 0.16 [-0.03 , 0.34] 3.2E+02 0.10 [-0.02 , 0.10] 3.0E-01 0.04 [-0.01 , 0.04] 3.9E-01 Cebpb 0.15 [0.01 , 0.29] 3.2E+02 0.18 [0.04 , 0.18] 1.5E-02 0.06 [0.00 , 0.06] 9.8E-02 Card11 0.15 [-0.01 , 0.31] 3.3E+02 0.10 [-0.05 , 0.10] 6.6E-01 0.01 [-0.10 , 0.01] 2.3E-01 354 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Nfatc3 0.15 [-0.04 , 0.34] 3.3E+02 -0.01 [-0.10 , -0.01] 7.4E-02 0.00 [-0.03 , 0.00] 1.4E-01 Lck 0.15 [-0.05 , 0.34] 3.3E+02 0.11 [-0.16 , 0.11] 8.0E-01 0.07 [-0.02 , 0.07] 3.8E-01 Ada 0.14 [-0.22 , 0.51] 3.3E+02 0.19 [-0.16 , 0.19] 8.7E-01 0.06 [-0.08 , 0.06] 8.2E-01 Apoe 0.14 [-0.05 , 0.34] 3.3E+02 0.00 [-0.12 , 0.00] 1.1E-01 0.02 [-0.01 , 0.02] 7.9E-01 Psmb7 0.14 [0.01 , 0.27] 3.3E+02 0.20 [0.09 , 0.20] 7.4E-04 0.04 [0.00 , 0.04] 2.2E-01 Ctsh 0.14 [-0.25 , 0.53] 3.3E+02 -0.02 [-0.25 , -0.02] 5.5E-02 0.12 [0.07 , 0.12] 1.3E-05 Tgfbr2 0.14 [0.00 , 0.28] 3.3E+02 0.07 [-0.05 , 0.07] 8.4E-01 0.04 [-0.02 , 0.04] 5.6E-01 Il4ra 0.14 [-0.05 , 0.33] 3.3E+02 0.19 [0.01 , 0.19] 6.3E-02 0.02 [-0.05 , 0.02] 5.6E-01 Socs1 0.14 [-0.16 , 0.44] 3.3E+02 0.02 [-0.30 , 0.02] 1.6E-01 0.07 [-0.07 , 0.07] 9.9E-01 Atm 0.14 [0.03 , 0.24] 3.4E+02 0.12 [-0.02 , 0.12] 2.4E-01 0.02 [-0.04 , 0.02] 5.3E-01 Ikzf1 0.13 [-0.10 , 0.36] 3.4E+02 0.19 [0.03 , 0.19] 4.3E-02 0.02 [-0.06 , 0.02] 4.3E-01 Map2k1 0.13 [0.03 , 0.23] 3.4E+02 0.18 [0.08 , 0.18] 5.3E-04 0.03 [0.01 , 0.03] 1.9E-02 Icam1 0.13 [-0.04 , 0.30] 3.4E+02 0.15 [-0.03 , 0.15] 3.0E-01 0.01 [-0.06 , 0.01] 3.4E-01 Tnfsf10 0.13 [-0.13 , 0.39] 3.4E+02 0.12 [-0.18 , 0.12] 7.3E-01 0.03 [-0.13 , 0.03] 3.6E-01 Ewsr1 0.12 [0.02 , 0.23] 3.4E+02 0.04 [-0.03 , 0.04] 8.4E-01 0.03 [0.00 , 0.03] 8.2E-02 Map4k2 0.12 [-0.01 , 0.26] 3.4E+02 0.06 [-0.09 , 0.06] 7.0E-01 0.02 [-0.03 , 0.02] 8.4E-01 Cyfip2 0.12 [-0.12 , 0.36] 3.4E+02 0.19 [-0.01 , 0.19] 1.5E-01 0.00 [-0.07 , 0.00] 1.5E-01 355 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tgfb1 0.12 [-0.01 , 0.24] 3.4E+02 0.04 [-0.08 , 0.04] 6.4E-01 0.03 [-0.01 , 0.03] 4.3E-01 Il6ra 0.12 [-0.06 , 0.29] 3.4E+02 0.16 [-0.02 , 0.16] 2.3E-01 0.03 [-0.06 , 0.03] 6.9E-01 Bcl10 0.11 [0.03 , 0.20] 3.5E+02 0.12 [0.02 , 0.12] 3.5E-02 0.03 [0.00 , 0.03] 5.3E-02 Cklf 0.11 [-0.16 , 0.39] 3.5E+02 0.17 [-0.11 , 0.17] 7.4E-01 0.09 [-0.03 , 0.09] 5.2E-01 Mr1 0.11 [-0.11 , 0.33] 3.5E+02 0.08 [-0.10 , 0.08] 8.7E-01 0.06 [-0.01 , 0.06] 2.1E-01 Ptgs2 0.11 [-0.43 , 0.65] 3.5E+02 -0.13 [-0.44 , -0.13] 6.3E-03 0.02 [-0.09 , 0.02] 3.1E-01 Mst1r 0.11 [-0.29 , 0.51] 3.5E+02 0.21 [-0.13 , 0.21] 7.2E-01 0.06 [-0.07 , 0.06] 9.2E-01 Il15 0.10 [-0.16 , 0.37] 3.5E+02 0.09 [-0.19 , 0.09] 5.9E-01 0.06 [-0.03 , 0.06] 6.3E-01 Cyld 0.10 [-0.02 , 0.22] 3.5E+02 0.16 [-0.01 , 0.16] 1.4E-01 0.06 [0.01 , 0.06] 2.1E-02 Cd1d2 0.10 [-0.28 , 0.48] 3.5E+02 0.19 [-0.10 , 0.19] 6.1E-01 0.05 [-0.08 , 0.05] 7.6E-01 Cd97 0.10 [-0.05 , 0.24] 3.5E+02 0.11 [-0.03 , 0.11] 3.7E-01 0.00 [-0.07 , 0.00] 8.7E-02 Alcam 0.09 [-0.01 , 0.20] 3.5E+02 0.16 [0.05 , 0.16] 5.2E-03 0.06 [0.02 , 0.06] 1.2E-02 Ltbr 0.09 [-0.03 , 0.21] 3.6E+02 0.06 [-0.04 , 0.06] 7.2E-01 0.03 [-0.02 , 0.03] 7.5E-01 Itgal 0.09 [-0.12 , 0.29] 3.6E+02 0.03 [-0.17 , 0.03] 2.4E-01 0.01 [-0.07 , 0.01] 3.3E-01 Tlr4 0.09 [-0.09 , 0.27] 3.6E+02 0.03 [-0.05 , 0.03] 5.9E-01 0.03 [-0.02 , 0.03] 9.2E-01 Irf3 0.09 [-0.02 , 0.19] 3.6E+02 0.13 [0.04 , 0.13] 6.2E-03 0.00 [-0.04 , 0.00] 1.4E-01 App 0.09 [0.00 , 0.17] 3.6E+02 0.11 [0.03 , 0.11] 6.2E-03 0.00 [-0.02 , 0.00] 2.0E-01 356 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tbx21 0.09 [-0.44 , 0.61] 3.6E+02 0.17 [-0.33 , 0.17] 6.3E-01 0.04 [-0.12 , 0.04] 4.5E-01 Col4a1 0.08 [-0.12 , 0.29] 3.6E+02 0.13 [-0.06 , 0.13] 5.9E-01 0.04 [-0.06 , 0.04] 7.6E-01 Tbk1 0.08 [-0.06 , 0.22] 3.6E+02 0.01 [-0.07 , 0.01] 1.9E-01 0.00 [-0.02 , 0.00] 3.4E-01 Cd40lg 0.08 [-0.31 , 0.47] 3.6E+02 0.13 [-0.30 , 0.13] 5.2E-01 0.09 [-0.07 , 0.09] 8.6E-01 Tnfrsf10b 0.08 [-0.24 , 0.39] 3.6E+02 0.15 [-0.13 , 0.15] 9.3E-01 0.02 [-0.11 , 0.02] 2.6E-01 Litaf 0.08 [-0.06 , 0.21] 3.7E+02 0.06 [-0.10 , 0.06] 7.3E-01 0.00 [-0.06 , 0.00] 8.2E-02 Nod1 0.07 [-0.06 , 0.21] 3.7E+02 0.17 [0.03 , 0.17] 3.1E-02 0.00 [-0.06 , 0.00] 1.5E-01 Tcf7 0.07 [-0.09 , 0.24] 3.7E+02 0.17 [-0.12 , 0.17] 7.6E-01 0.01 [-0.07 , 0.01] 2.3E-01 Gbp2b 0.07 [-0.33 , 0.47] 3.7E+02 0.11 [-0.16 , 0.11] 7.9E-01 -0.01 [-0.16 , -0.01] 8.7E-02 Klra17 0.07 [-0.17 , 0.31] 3.7E+02 0.05 [-0.18 , 0.05] 3.9E-01 0.05 [-0.07 , 0.05] 8.5E-01 Dock9 0.07 [-0.04 , 0.18] 3.7E+02 0.16 [0.04 , 0.16] 1.4E-02 0.02 [-0.03 , 0.02] 8.6E-01 Tank 0.07 [-0.08 , 0.22] 3.7E+02 0.07 [-0.06 , 0.07] 9.3E-01 0.03 [-0.02 , 0.03] 6.3E-01 Irgm2 0.07 [-0.35 , 0.48] 3.7E+02 -0.03 [-0.45 , -0.03] 6.9E-02 -0.02 [-0.17 , -0.02] 4.6E-02 Il17b 0.07 [-0.34 , 0.47] 3.7E+02 0.36 [-0.02 , 0.36] 1.4E-01 0.12 [-0.06 , 0.12] 6.9E-01 Il7 0.07 [-0.15 , 0.28] 3.7E+02 0.28 [-0.04 , 0.28] 2.2E-01 0.03 [-0.07 , 0.03] 5.3E-01 Irak4 0.06 [-0.04 , 0.17] 3.8E+02 0.08 [-0.04 , 0.08] 5.4E-01 0.02 [-0.04 , 0.02] 7.5E-01 Tnfrsf1a 0.06 [-0.08 , 0.20] 3.8E+02 0.08 [-0.07 , 0.08] 9.3E-01 0.01 [-0.06 , 0.01] 2.2E-01 357 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tlr3 0.06 [-0.14 , 0.26] 3.8E+02 -0.04 [-0.29 , -0.04] 4.0E-02 0.03 [-0.02 , 0.03] 6.9E-01 Nt5e 0.06 [-0.21 , 0.33] 3.8E+02 0.05 [-0.20 , 0.05] 3.1E-01 0.02 [-0.06 , 0.02] 3.9E-01 Map3k1 0.06 [-0.14 , 0.26] 3.8E+02 0.05 [-0.14 , 0.05] 4.4E-01 0.02 [-0.06 , 0.02] 5.2E-01 Map2k4 0.06 [-0.03 , 0.15] 3.8E+02 0.07 [-0.06 , 0.07] 8.4E-01 0.03 [-0.01 , 0.03] 3.7E-01 Pin1 0.06 [-0.11 , 0.23] 3.8E+02 0.11 [-0.05 , 0.11] 5.7E-01 0.01 [-0.05 , 0.01] 3.7E-01 Kit 0.05 [-0.20 , 0.30] 3.8E+02 0.17 [-0.07 , 0.17] 5.3E-01 0.08 [-0.02 , 0.08] 3.4E-01 Fn1 0.05 [-0.18 , 0.28] 3.8E+02 0.12 [-0.12 , 0.12] 9.8E-01 0.05 [-0.06 , 0.05] 8.9E-01 Psma2 0.05 [0.01 , 0.10] 3.8E+02 0.08 [0.03 , 0.08] 2.0E-03 0.03 [0.01 , 0.03] 1.4E-02 Cd163 0.05 [-0.36 , 0.46] 3.9E+02 0.33 [-0.06 , 0.33] 2.5E-01 0.09 [-0.07 , 0.09] 8.6E-01 Prdm1 0.05 [-0.11 , 0.21] 3.9E+02 0.06 [-0.15 , 0.06] 5.0E-01 0.02 [-0.07 , 0.02] 5.1E-01 Cd164 0.05 [-0.02 , 0.11] 3.9E+02 0.07 [-0.01 , 0.07] 2.6E-01 0.02 [0.00 , 0.02] 2.2E-01 Ltk 0.04 [-0.58 , 0.67] 3.9E+02 0.23 [-0.29 , 0.23] 8.7E-01 0.13 [-0.06 , 0.13] 6.3E-01 Lamp3 0.04 [-0.14 , 0.23] 3.9E+02 0.18 [-0.01 , 0.18] 1.7E-01 0.08 [0.01 , 0.08] 4.3E-02 Yy1 0.04 [-0.04 , 0.12] 3.9E+02 0.03 [-0.05 , 0.03] 7.9E-01 0.01 [-0.01 , 0.01] 9.3E-01 Egr3 0.04 [-0.34 , 0.42] 3.9E+02 0.32 [-0.03 , 0.32] 1.9E-01 0.02 [-0.12 , 0.02] 2.7E-01 Itgb4 0.04 [-0.22 , 0.29] 3.9E+02 0.12 [-0.16 , 0.12] 8.4E-01 0.00 [-0.11 , 0.00] 1.0E-01 Stat6 0.04 [-0.04 , 0.11] 3.9E+02 0.03 [-0.04 , 0.03] 9.3E-01 0.02 [-0.02 , 0.02] 9.0E-01 358 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Thy1 0.03 [-0.13 , 0.20] 3.9E+02 0.07 [-0.15 , 0.07] 5.8E-01 0.05 [-0.03 , 0.05] 7.5E-01 Mapk8 0.03 [-0.10 , 0.16] 4.0E+02 0.04 [-0.08 , 0.04] 5.9E-01 0.01 [-0.04 , 0.01] 2.7E-01 Ccl5 0.03 [-0.15 , 0.21] 4.0E+02 0.02 [-0.17 , 0.02] 2.1E-01 -0.02 [-0.10 , -0.02] 3.4E-02 Lrrn3 0.03 [-0.20 , 0.26] 4.0E+02 0.17 [-0.10 , 0.17] 6.7E-01 0.06 [-0.08 , 0.06] 7.9E-01 Tnfrsf14 0.03 [-0.11 , 0.16] 4.0E+02 0.08 [-0.10 , 0.08] 8.9E-01 -0.02 [-0.07 , -0.02] 1.2E-02 Lrp1 0.03 [-0.17 , 0.22] 4.0E+02 0.07 [-0.11 , 0.07] 6.9E-01 0.00 [-0.10 , 0.00] 1.4E-01 Gtf3c1 0.02 [-0.08 , 0.13] 4.0E+02 -0.03 [-0.10 , -0.03] 8.9E-03 0.00 [-0.04 , 0.00] 2.1E-01 Ilf3 0.02 [-0.11 , 0.15] 4.0E+02 0.10 [-0.05 , 0.10] 6.3E-01 0.01 [-0.05 , 0.01] 2.7E-01 Mapk14 0.02 [-0.07 , 0.11] 4.0E+02 0.03 [-0.05 , 0.03] 7.5E-01 0.03 [-0.01 , 0.03] 3.8E-01 Isg20 0.02 [-0.30 , 0.34] 4.0E+02 0.18 [-0.05 , 0.18] 3.5E-01 0.04 [-0.05 , 0.04] 9.1E-01 Tnfsf12 0.02 [-0.16 , 0.20] 4.0E+02 0.15 [0.00 , 0.15] 9.0E-02 0.05 [0.01 , 0.05] 3.6E-02 Anp32b 0.02 [-0.06 , 0.09] 4.1E+02 0.04 [-0.04 , 0.04] 8.9E-01 0.03 [0.00 , 0.03] 1.9E-01 Cd22 0.02 [-0.57 , 0.60] 4.1E+02 0.11 [-0.85 , 0.11] 2.1E-01 0.01 [-0.23 , 0.01] 1.6E-01 Bid 0.02 [-0.26 , 0.29] 4.1E+02 -0.04 [-0.27 , -0.04] 3.4E-02 0.03 [-0.04 , 0.03] 9.4E-01 Ceacam1 0.02 [-0.16 , 0.19] 4.1E+02 0.10 [-0.13 , 0.10] 8.7E-01 0.05 [-0.01 , 0.05] 3.7E-01 Psen1 0.02 [-0.08 , 0.11] 4.1E+02 0.04 [-0.06 , 0.04] 7.2E-01 0.02 [-0.02 , 0.02] 9.6E-01 Erbb2 0.02 [-0.09 , 0.12] 4.1E+02 0.10 [-0.02 , 0.10] 3.0E-01 0.00 [-0.05 , 0.00] 1.4E-01 359 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Prkce 0.01 [-0.26 , 0.29] 4.1E+02 0.14 [-0.09 , 0.14] 7.2E-01 0.02 [-0.07 , 0.02] 3.9E-01 Tmem173 0.01 [-0.28 , 0.31] 4.1E+02 -0.05 [-0.37 , -0.05] 3.7E-02 0.06 [-0.06 , 0.06] 9.4E-01 Lamp2 0.01 [-0.12 , 0.14] 4.1E+02 0.01 [-0.06 , 0.01] 2.0E-01 0.02 [0.00 , 0.02] 9.2E-02 Cxcl12 0.01 [-0.15 , 0.17] 4.1E+02 0.03 [-0.14 , 0.03] 2.9E-01 0.02 [-0.05 , 0.02] 6.0E-01 Colec12 0.01 [-0.19 , 0.21] 4.2E+02 0.23 [0.04 , 0.23] 2.5E-02 0.06 [0.00 , 0.06] 2.0E-01 Tnfsf13b 0.01 [-0.25 , 0.27] 4.2E+02 0.22 [-0.05 , 0.22] 2.9E-01 0.11 [0.00 , 0.11] 1.2E-01 Ifi27 0.01 [-0.19 , 0.21] 4.2E+02 -0.05 [-0.21 , -0.05] 1.4E-02 0.06 [0.00 , 0.06] 9.6E-02 Zfp13 0.01 [-0.19 , 0.21] 4.2E+02 0.13 [-0.15 , 0.13] 9.0E-01 0.07 [-0.01 , 0.07] 2.5E-01 Il13ra1 0.01 [-0.16 , 0.18] 4.2E+02 0.02 [-0.09 , 0.02] 3.1E-01 0.01 [-0.03 , 0.01] 5.3E-01 Cd8b1 0.01 [-0.51 , 0.53] 4.2E+02 0.30 [-0.16 , 0.30] 6.5E-01 0.08 [-0.10 , 0.08] 9.2E-01 Il18r1 0.01 [-0.22 , 0.23] 4.2E+02 -0.04 [-0.15 , -0.04] 1.2E-02 -0.03 [-0.08 , -0.03] 2.8E-03 Hif1a 0.00 [-0.11 , 0.12] 4.2E+02 0.02 [-0.07 , 0.02] 4.2E-01 0.02 [-0.01 , 0.02] 7.5E-01 Clu 0.00 [-0.11 , 0.12] 4.2E+02 0.11 [-0.05 , 0.11] 5.2E-01 0.04 [-0.01 , 0.04] 5.2E-01 Map2k2 0.00 [-0.05 , 0.05] 4.2E+02 0.06 [-0.02 , 0.06] 5.0E-01 0.02 [0.00 , 0.02] 4.2E-02 Mif 0.00 [-0.10 , 0.10] 4.3E+02 0.05 [-0.07 , 0.05] 8.1E-01 0.03 [-0.02 , 0.03] 7.6E-01 Irf2 0.00 [-0.10 , 0.10] 4.3E+02 0.00 [-0.07 , 0.00] 1.4E-01 -0.03 [-0.06 , -0.03] 4.6E-05 Traf6 0.00 [-0.08 , 0.07] 4.3E+02 0.08 [0.02 , 0.08] 2.2E-02 0.00 [-0.04 , 0.00] 7.1E-02 360 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd9 0.00 [-0.13 , 0.13] 4.3E+02 0.06 [-0.05 , 0.06] 9.0E-01 0.03 [0.01 , 0.03] 2.6E-02 Bax 0.00 [-0.15 , 0.15] 4.3E+02 0.07 [-0.17 , 0.07] 5.1E-01 0.09 [0.05 , 0.09] 1.0E-04 Tgfb2 0.00 [-0.33 , 0.33] 4.3E+02 0.06 [-0.32 , 0.06] 2.8E-01 0.02 [-0.13 , 0.02] 2.6E-01 Bcl6 0.00 [-0.12 , 0.11] 4.3E+02 0.05 [-0.06 , 0.05] 9.7E-01 -0.04 [-0.09 , -0.04] 7.3E-04 Tirap -0.01 [-0.14 , 0.13] 4.3E+02 -0.02 [-0.29 , -0.02] 6.0E-02 0.01 [-0.04 , 0.01] 4.6E-01 Igf2r -0.01 [-0.10 , 0.09] 4.3E+02 0.12 [0.00 , 0.12] 1.1E-01 0.00 [-0.05 , 0.00] 2.1E-01 Reps1 -0.01 [-0.07 , 0.05] 4.3E+02 -0.02 [-0.08 , -0.02] 1.9E-02 0.02 [-0.02 , 0.02] 8.4E-01 Ythdf2 -0.01 [-0.11 , 0.08] 4.4E+02 0.02 [-0.07 , 0.02] 3.3E-01 0.00 [-0.03 , 0.00] 4.3E-02 Ifi35 -0.01 [-0.10 , 0.08] 4.4E+02 0.01 [-0.11 , 0.01] 1.6E-01 0.00 [-0.02 , 0.00] 2.6E-01 Klrb1c -0.02 [-0.35 , 0.32] 4.4E+02 0.18 [-0.10 , 0.18] 6.5E-01 -0.03 [-0.13 , -0.03] 2.4E-02 Cd55 -0.02 [-0.15 , 0.12] 4.4E+02 0.02 [-0.14 , 0.02] 2.0E-01 0.03 [-0.02 , 0.03] 6.2E-01 Myc -0.02 [-0.22 , 0.18] 4.4E+02 0.10 [-0.16 , 0.10] 7.2E-01 0.01 [-0.07 , 0.01] 2.5E-01 Ccl25 -0.02 [-0.42 , 0.37] 4.4E+02 0.36 [0.00 , 0.36] 1.1E-01 0.10 [-0.04 , 0.10] 5.4E-01 Mapkapk2 -0.02 [-0.12 , 0.07] 4.4E+02 0.01 [-0.07 , 0.01] 2.8E-01 0.02 [-0.01 , 0.02] 4.5E-01 Map3k7 -0.03 [-0.21 , 0.15] 4.4E+02 0.00 [-0.40 , 0.00] 1.2E-01 0.03 [0.00 , 0.03] 1.5E-01 Map3k5 -0.03 [-0.12 , 0.07] 4.4E+02 -0.01 [-0.09 , -0.01] 6.5E-02 -0.02 [-0.06 , -0.02] 3.9E-03 Atf1 -0.03 [-0.26 , 0.20] 4.4E+02 -0.01 [-0.23 , -0.01] 7.7E-02 0.05 [0.00 , 0.05] 1.0E-01 361 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mavs -0.03 [-0.14 , 0.08] 4.5E+02 0.03 [-0.06 , 0.03] 5.3E-01 0.05 [0.00 , 0.05] 7.6E-02 Atg10 -0.03 [-0.15 , 0.08] 4.5E+02 0.07 [-0.07 , 0.07] 9.8E-01 0.05 [0.00 , 0.05] 1.8E-01 Atg7 -0.03 [-0.10 , 0.04] 4.5E+02 0.06 [-0.01 , 0.06] 3.2E-01 0.02 [0.00 , 0.02] 2.7E-01 Ikbkg -0.03 [-0.18 , 0.11] 4.5E+02 0.01 [-0.13 , 0.01] 2.0E-01 0.03 [-0.02 , 0.03] 5.8E-01 Cd3eap -0.03 [-0.25 , 0.18] 4.5E+02 0.05 [-0.19 , 0.05] 3.5E-01 0.08 [0.00 , 0.08] 1.4E-01 Bcl2 -0.03 [-0.24 , 0.17] 4.5E+02 0.10 [-0.04 , 0.10] 4.5E-01 -0.03 [-0.08 , -0.03] 4.1E-03 Pax5 -0.03 [-0.56 , 0.50] 4.5E+02 0.13 [-0.55 , 0.13] 3.1E-01 0.09 [-0.14 , 0.09] 7.6E-01 Chuk -0.03 [-0.15 , 0.08] 4.5E+02 -0.04 [-0.10 , -0.04] 1.2E-03 -0.01 [-0.03 , -0.01] 2.4E-02 F13a1 -0.04 [-0.39 , 0.30] 4.5E+02 0.27 [-0.15 , 0.27] 6.6E-01 0.06 [-0.07 , 0.06] 8.9E-01 Tollip -0.05 [-0.18 , 0.09] 4.5E+02 -0.06 [-0.12 , -0.06] 3.3E-04 0.01 [-0.01 , 0.01] 7.7E-01 Hspb2 -0.05 [-0.27 , 0.18] 4.6E+02 0.13 [-0.12 , 0.13] 9.4E-01 0.05 [-0.05 , 0.05] 9.9E-01 Nod2 -0.05 [-0.40 , 0.31] 4.6E+02 0.01 [-0.31 , 0.01] 1.3E-01 0.02 [-0.12 , 0.02] 2.5E-01 Cd8a -0.05 [-0.35 , 0.26] 4.6E+02 0.06 [-0.35 , 0.06] 2.5E-01 0.05 [-0.09 , 0.05] 6.9E-01 Il18 -0.05 [-0.26 , 0.16] 4.6E+02 0.04 [-0.21 , 0.04] 2.7E-01 0.06 [-0.04 , 0.06] 8.5E-01 Ep300 -0.05 [-0.14 , 0.04] 4.6E+02 -0.03 [-0.11 , -0.03] 5.7E-03 -0.01 [-0.04 , -0.01] 1.9E-02 Smpd3 -0.05 [-0.38 , 0.28] 4.6E+02 -0.16 [-0.50 , -0.16] 5.9E-03 -0.01 [-0.11 , -0.01] 8.8E-02 Smn1 -0.06 [-0.22 , 0.10] 4.6E+02 -0.01 [-0.15 , -0.01] 5.5E-02 0.03 [-0.02 , 0.03] 7.5E-01 362 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ikbkb -0.06 [-0.17 , 0.05] 4.6E+02 -0.03 [-0.16 , -0.03] 2.5E-02 0.01 [-0.04 , 0.01] 2.8E-01 Ubc -0.06 [-0.31 , 0.19] 4.6E+02 0.18 [-0.03 , 0.18] 2.4E-01 0.03 [-0.06 , 0.03] 7.1E-01 Stat5b -0.06 [-0.21 , 0.09] 4.6E+02 -0.06 [-0.17 , -0.06] 3.2E-03 0.00 [-0.03 , 0.00] 2.1E-01 Sell -0.06 [-0.39 , 0.28] 4.7E+02 0.04 [-0.36 , 0.04] 1.9E-01 0.05 [-0.10 , 0.05] 6.3E-01 Fos -0.06 [-0.23 , 0.11] 4.7E+02 0.14 [-0.10 , 0.14] 7.5E-01 0.00 [-0.08 , 0.00] 1.2E-01 Klrk1 -0.06 [-0.23 , 0.11] 4.7E+02 -0.03 [-0.26 , -0.03] 5.5E-02 -0.04 [-0.14 , -0.04] 9.1E-03 Runx1 -0.06 [-0.17 , 0.04] 4.7E+02 -0.01 [-0.13 , -0.01] 5.4E-02 -0.04 [-0.06 , -0.04] 4.5E-05 Klra7 -0.06 [-0.60 , 0.47] 4.7E+02 0.10 [-0.51 , 0.10] 2.8E-01 -0.06 [-0.52 , -0.06] 6.3E-02 Ifngr1 -0.07 [-0.23 , 0.10] 4.7E+02 -0.10 [-0.26 , -0.10] 2.2E-03 0.01 [-0.03 , 0.01] 3.9E-01 Irf1 -0.07 [-0.19 , 0.06] 4.7E+02 0.01 [-0.13 , 0.01] 1.4E-01 -0.03 [-0.09 , -0.03] 9.0E-03 Elk1 -0.07 [-0.28 , 0.14] 4.7E+02 0.06 [-0.18 , 0.06] 3.9E-01 0.00 [-0.10 , 0.00] 9.9E-02 Epcam -0.07 [-0.24 , 0.10] 4.7E+02 -0.05 [-0.17 , -0.05] 1.1E-02 0.00 [-0.03 , 0.00] 1.9E-01 Ecsit -0.07 [-0.32 , 0.17] 4.7E+02 0.07 [-0.17 , 0.07] 5.3E-01 0.06 [-0.04 , 0.06] 8.8E-01 Vwf -0.07 [-0.32 , 0.17] 4.8E+02 0.01 [-0.20 , 0.01] 1.3E-01 -0.02 [-0.12 , -0.02] 4.1E-02 Fadd -0.07 [-0.24 , 0.09] 4.8E+02 0.06 [-0.07 , 0.06] 9.6E-01 0.03 [-0.03 , 0.03] 9.3E-01 Il12a -0.08 [-0.36 , 0.20] 4.8E+02 0.04 [-0.35 , 0.04] 2.1E-01 0.03 [-0.11 , 0.03] 4.0E-01 Itk -0.08 [-0.40 , 0.23] 4.8E+02 0.14 [-0.14 , 0.14] 1.0E+00 0.06 [-0.04 , 0.06] 8.1E-01 363 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cdh1 -0.09 [-0.17 , -0.01] 4.8E+02 0.05 [-0.05 , 0.05] 9.3E-01 0.00 [-0.02 , 0.00] 2.4E-01 Rora -0.09 [-0.26 , 0.08] 4.8E+02 0.01 [-0.15 , 0.01] 1.5E-01 0.01 [-0.07 , 0.01] 2.2E-01 Syt17 -0.09 [-0.49 , 0.31] 4.8E+02 0.25 [-0.08 , 0.25] 4.1E-01 0.06 [-0.06 , 0.06] 9.9E-01 Cd40 -0.09 [-0.28 , 0.09] 4.8E+02 -0.09 [-0.25 , -0.09] 3.8E-03 0.01 [-0.07 , 0.01] 2.4E-01 Fyn -0.10 [-0.27 , 0.08] 4.8E+02 0.02 [-0.15 , 0.02] 2.2E-01 0.00 [-0.06 , 0.00] 1.2E-01 Cfh -0.10 [-0.24 , 0.04] 4.8E+02 -0.01 [-0.12 , -0.01] 5.5E-02 0.03 [-0.01 , 0.03] 6.7E-01 Crebbp -0.10 [-0.19 , -0.01] 4.9E+02 -0.07 [-0.14 , -0.07] 4.3E-04 -0.05 [-0.09 , -0.05] 2.1E-05 Ppbp -0.10 [-0.63 , 0.43] 4.9E+02 0.04 [-0.49 , 0.04] 1.7E-01 0.05 [-0.15 , 0.05] 4.9E-01 Jak1 -0.10 [-0.19 , -0.02] 4.9E+02 -0.03 [-0.13 , -0.03] 1.5E-02 0.00 [-0.03 , 0.00] 1.0E-01 Col1a1 -0.11 [-0.33 , 0.11] 4.9E+02 -0.07 [-0.29 , -0.07] 1.5E-02 0.03 [-0.05 , 0.03] 7.5E-01 Il12rb2 -0.11 [-0.49 , 0.27] 4.9E+02 0.12 [-0.25 , 0.12] 5.9E-01 0.10 [-0.04 , 0.10] 5.8E-01 Smad3 -0.11 [-0.24 , 0.01] 4.9E+02 0.06 [-0.08 , 0.06] 8.1E-01 -0.02 [-0.08 , -0.02] 1.6E-02 Ccl21a -0.12 [-0.46 , 0.23] 4.9E+02 0.22 [-0.13 , 0.22] 6.8E-01 0.08 [-0.04 , 0.08] 6.8E-01 Psmd7 -0.12 [-0.18 , -0.07] 4.9E+02 -0.05 [-0.10 , -0.05] 3.8E-04 -0.01 [-0.02 , -0.01] 5.1E-03 Mapk1 -0.12 [-0.23 , -0.01] 4.9E+02 -0.06 [-0.15 , -0.06] 1.9E-03 0.01 [-0.02 , 0.01] 7.9E-01 Il22ra2 -0.12 [-0.51 , 0.26] 4.9E+02 0.02 [-0.33 , 0.02] 1.6E-01 0.08 [-0.05 , 0.08] 7.9E-01 Cmah -0.13 [-0.37 , 0.11] 5.0E+02 0.00 [-0.27 , 0.00] 1.2E-01 0.02 [-0.10 , 0.02] 3.5E-01 364 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Rela -0.13 [-0.24 , -0.02] 5.0E+02 -0.02 [-0.17 , -0.02] 3.8E-02 -0.02 [-0.06 , -0.02] 6.6E-03 Cd7 -0.13 [-0.35 , 0.10] 5.0E+02 0.02 [-0.26 , 0.02] 1.6E-01 0.00 [-0.09 , 0.00] 1.5E-01 Itga2 -0.13 [-0.27 , 0.01] 5.0E+02 -0.05 [-0.17 , -0.05] 9.8E-03 -0.02 [-0.10 , -0.02] 1.6E-02 Thbd -0.13 [-0.32 , 0.06] 5.0E+02 0.06 [-0.15 , 0.06] 5.0E-01 0.00 [-0.09 , 0.00] 9.3E-02 Cdh5 -0.13 [-0.23 , -0.03] 5.0E+02 -0.03 [-0.15 , -0.03] 1.5E-02 -0.03 [-0.07 , -0.03] 6.3E-04 Itga6 -0.13 [-0.31 , 0.05] 5.0E+02 0.08 [-0.10 , 0.08] 8.8E-01 0.00 [-0.07 , 0.00] 1.8E-01 Atg16l1 -0.14 [-0.30 , 0.02] 5.0E+02 -0.08 [-0.19 , -0.08] 9.8E-04 0.00 [-0.03 , 0.00] 2.2E-01 Il22ra1 -0.14 [-0.59 , 0.31] 5.0E+02 0.26 [-0.15 , 0.26] 6.9E-01 0.10 [-0.06 , 0.10] 6.9E-01 Nup107 -0.14 [-0.24 , -0.04] 5.0E+02 -0.01 [-0.11 , -0.01] 5.9E-02 0.01 [-0.05 , 0.01] 2.2E-01 Lyve1 -0.14 [-0.55 , 0.27] 5.1E+02 0.28 [-0.11 , 0.28] 4.8E-01 0.07 [-0.10 , 0.07] 8.2E-01 Egfr -0.14 [-0.29 , 0.01] 5.1E+02 0.02 [-0.14 , 0.02] 2.6E-01 -0.02 [-0.09 , -0.02] 3.3E-02 Prg2 -0.14 [-0.47 , 0.18] 5.1E+02 0.05 [-0.13 , 0.05] 4.8E-01 -0.03 [-0.12 , -0.03] 1.6E-02 Gata3 -0.14 [-0.45 , 0.17] 5.1E+02 -0.07 [-0.37 , -0.07] 2.8E-02 0.08 [-0.04 , 0.08] 5.9E-01 Atg5 -0.15 [-0.22 , -0.07] 5.1E+02 -0.02 [-0.12 , -0.02] 3.3E-02 0.01 [-0.04 , 0.01] 2.8E-01 Tmed1 -0.15 [-0.29 , 0.00] 5.1E+02 0.05 [-0.13 , 0.05] 4.5E-01 0.02 [-0.05 , 0.02] 5.8E-01 Cspg4 -0.15 [-0.43 , 0.13] 5.1E+02 0.03 [-0.23 , 0.03] 2.3E-01 0.00 [-0.12 , 0.00] 1.3E-01 Cd36 -0.15 [-0.35 , 0.04] 5.1E+02 -0.04 [-0.18 , -0.04] 1.9E-02 0.00 [-0.05 , 0.00] 2.1E-01 365 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Pnma1 -0.16 [-0.52 , 0.21] 5.1E+02 0.18 [-0.04 , 0.18] 3.1E-01 0.03 [-0.08 , 0.03] 4.5E-01 Fez1 -0.16 [-0.60 , 0.28] 5.1E+02 -0.08 [-0.59 , -0.08] 4.2E-02 0.09 [-0.08 , 0.09] 9.3E-01 Itch -0.16 [-0.29 , -0.03] 5.2E+02 -0.05 [-0.16 , -0.05] 5.1E-03 -0.03 [-0.06 , -0.03] 4.2E-04 Ccl27a -0.16 [-0.32 , 0.00] 5.2E+02 0.02 [-0.15 , 0.02] 2.3E-01 0.01 [-0.05 , 0.01] 2.3E-01 Gpi1 -0.17 [-0.32 , -0.01] 5.2E+02 -0.13 [-0.20 , -0.13] 4.7E-06 0.02 [-0.01 , 0.02] 4.7E-01 Cd81 -0.17 [-0.28 , -0.05] 5.2E+02 0.00 [-0.11 , 0.00] 1.1E-01 -0.01 [-0.05 , -0.01] 3.7E-02 Lif -0.17 [-0.59 , 0.26] 5.2E+02 -0.04 [-0.35 , -0.04] 4.9E-02 0.00 [-0.18 , 0.00] 1.6E-01 Ccnd3 -0.17 [-0.37 , 0.03] 5.2E+02 0.03 [-0.14 , 0.03] 3.1E-01 0.00 [-0.06 , 0.00] 8.2E-02 Mme -0.17 [-0.29 , -0.06] 5.2E+02 0.01 [-0.10 , 0.01] 2.0E-01 -0.02 [-0.07 , -0.02] 5.0E-03 Txk -0.17 [-0.52 , 0.17] 5.2E+02 0.10 [-0.22 , 0.10] 5.3E-01 0.02 [-0.10 , 0.02] 3.7E-01 Egr2 -0.18 [-0.60 , 0.25] 5.2E+02 0.08 [-0.47 , 0.08] 2.5E-01 0.12 [-0.08 , 0.12] 7.9E-01 Nrp1 -0.18 [-0.35 , -0.01] 5.2E+02 -0.08 [-0.21 , -0.08] 2.2E-03 -0.02 [-0.09 , -0.02] 1.8E-02 Pla2g6 -0.18 [-0.32 , -0.04] 5.3E+02 -0.05 [-0.17 , -0.05] 7.3E-03 -0.03 [-0.08 , -0.03] 1.4E-03 Anxa1 -0.18 [-0.42 , 0.06] 5.3E+02 -0.02 [-0.19 , -0.02] 5.0E-02 0.02 [-0.04 , 0.02] 5.8E-01 Tie1 -0.19 [-0.31 , -0.07] 5.3E+02 -0.13 [-0.24 , -0.13] 7.8E-05 -0.03 [-0.07 , -0.03] 1.4E-03 Nos2 -0.19 [-0.54 , 0.17] 5.3E+02 0.07 [-0.33 , 0.07] 2.8E-01 0.11 [-0.05 , 0.11] 6.4E-01 Stat3 -0.19 [-0.43 , 0.04] 5.3E+02 -0.16 [-0.28 , -0.16] 4.7E-05 0.00 [-0.05 , 0.00] 1.9E-01 366 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccl17 -0.20 [-0.57 , 0.18] 5.3E+02 0.36 [-0.01 , 0.36] 1.2E-01 0.23 [0.08 , 0.23] 3.9E-03 Itga2b -0.20 [-0.47 , 0.07] 5.3E+02 0.04 [-0.26 , 0.04] 2.3E-01 0.08 [-0.03 , 0.08] 5.3E-01 Hmgb1 -0.20 [-0.35 , -0.06] 5.3E+02 -0.01 [-0.17 , -0.01] 8.6E-02 0.01 [-0.04 , 0.01] 3.7E-01 Cma1 -0.20 [-0.64 , 0.23] 5.3E+02 -0.04 [-0.35 , -0.04] 4.4E-02 -0.05 [-0.18 , -0.05] 1.0E-02 Pvr -0.21 [-0.37 , -0.04] 5.3E+02 -0.06 [-0.24 , -0.06] 1.5E-02 -0.04 [-0.12 , -0.04] 5.3E-03 Bmi1 -0.21 [-0.37 , -0.04] 5.4E+02 -0.03 [-0.45 , -0.03] 6.5E-02 0.00 [-0.04 , 0.00] 1.8E-01 Akt3 -0.21 [-0.35 , -0.07] 5.4E+02 -0.15 [-0.28 , -0.15] 5.9E-05 -0.01 [-0.05 , -0.01] 2.1E-02 Klrd1 -0.21 [-0.37 , -0.05] 5.4E+02 -0.11 [-0.28 , -0.11] 1.9E-03 -0.04 [-0.12 , -0.04] 8.2E-03 Rps6 -0.21 [-0.31 , -0.11] 5.4E+02 -0.01 [-0.20 , -0.01] 6.9E-02 0.00 [-0.04 , 0.00] 2.3E-01 Gbp5 -0.21 [-0.68 , 0.27] 5.4E+02 -0.24 [-0.63 , -0.24] 2.1E-03 -0.07 [-0.24 , -0.07] 1.1E-02 Plaur -0.21 [-0.56 , 0.13] 5.4E+02 0.15 [-0.20 , 0.15] 8.2E-01 0.01 [-0.13 , 0.01] 2.0E-01 C1qbp -0.21 [-0.36 , -0.07] 5.4E+02 -0.11 [-0.22 , -0.11] 2.5E-04 -0.02 [-0.06 , -0.02] 5.8E-03 Mfge8 -0.21 [-0.36 , -0.06] 5.4E+02 -0.04 [-0.25 , -0.04] 3.7E-02 0.02 [-0.04 , 0.02] 7.9E-01 Igf1r -0.21 [-0.32 , -0.11] 5.4E+02 -0.05 [-0.16 , -0.05] 6.4E-03 -0.04 [-0.08 , -0.04] 5.9E-05 Pecam1 -0.22 [-0.33 , -0.10] 5.4E+02 -0.11 [-0.22 , -0.11] 2.5E-04 -0.04 [-0.08 , -0.04] 3.0E-04 Entpd1 -0.22 [-0.55 , 0.11] 5.5E+02 0.09 [-0.30 , 0.09] 4.0E-01 0.04 [-0.11 , 0.04] 5.0E-01 Icam4 -0.22 [-0.57 , 0.13] 5.5E+02 0.16 [-0.14 , 0.16] 9.4E-01 0.03 [-0.08 , 0.03] 4.9E-01 367 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cdkn1a -0.22 [-0.67 , 0.22] 5.5E+02 0.03 [-0.48 , 0.03] 1.6E-01 0.07 [-0.20 , 0.07] 5.0E-01 Itga1 -0.22 [-0.36 , -0.09] 5.5E+02 -0.17 [-0.26 , -0.17] 2.8E-06 -0.04 [-0.08 , -0.04] 4.6E-04 Plau -0.22 [-0.57 , 0.12] 5.5E+02 -0.09 [-0.42 , -0.09] 1.7E-02 0.04 [-0.09 , 0.04] 6.2E-01 Angpt1 -0.24 [-0.48 , 0.01] 5.5E+02 -0.12 [-0.32 , -0.12] 2.0E-03 0.00 [-0.08 , 0.00] 1.1E-01 Dusp4 -0.24 [-0.46 , -0.02] 5.5E+02 -0.08 [-0.27 , -0.08] 6.3E-03 -0.05 [-0.16 , -0.05] 8.4E-03 Tgfb3 -0.24 [-0.48 , 0.00] 5.5E+02 -0.02 [-0.25 , -0.02] 6.4E-02 0.05 [-0.06 , 0.05] 8.3E-01 Ccl11 -0.24 [-0.54 , 0.06] 5.5E+02 0.06 [-0.30 , 0.06] 2.8E-01 0.07 [-0.09 , 0.07] 9.2E-01 Il11ra1 -0.25 [-0.56 , 0.06] 5.5E+02 0.16 [-0.14 , 0.16] 9.4E-01 0.01 [-0.12 , 0.01] 2.0E-01 Cx3cl1 -0.25 [-0.53 , 0.03] 5.6E+02 -0.03 [-0.31 , -0.03] 6.1E-02 0.02 [-0.08 , 0.02] 4.1E-01 Dpp4 -0.26 [-0.42 , -0.09] 5.6E+02 -0.10 [-0.24 , -0.10] 9.8E-04 -0.01 [-0.08 , -0.01] 4.3E-02 Gzmb -0.26 [-0.59 , 0.08] 5.6E+02 0.03 [-0.26 , 0.03] 1.9E-01 0.01 [-0.10 , 0.01] 2.5E-01 Dll4 -0.26 [-0.52 , 0.00] 5.6E+02 0.02 [-0.26 , 0.02] 1.6E-01 -0.03 [-0.14 , -0.03] 3.2E-02 Ets1 -0.27 [-0.45 , -0.08] 5.6E+02 -0.24 [-0.35 , -0.24] 9.4E-07 0.00 [-0.05 , 0.00] 1.3E-01 Tek -0.27 [-0.48 , -0.05] 5.6E+02 -0.21 [-0.34 , -0.21] 1.3E-05 -0.04 [-0.09 , -0.04] 2.3E-03 Cxcl15 -0.27 [-0.47 , -0.07] 5.6E+02 0.08 [-0.17 , 0.08] 5.5E-01 0.06 [-0.02 , 0.06] 5.2E-01 Atf2 -0.28 [-0.53 , -0.03] 5.6E+02 -0.24 [-0.31 , -0.24] 1.7E-08 0.01 [-0.02 , 0.01] 4.4E-01 Notch1 -0.28 [-0.42 , -0.13] 5.6E+02 -0.08 [-0.20 , -0.08] 2.5E-03 -0.07 [-0.12 , -0.07] 5.9E-05 368 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mapk11 -0.29 [-0.46 , -0.12] 5.6E+02 0.01 [-0.18 , 0.01] 1.4E-01 0.01 [-0.11 , 0.01] 2.1E-01 Eng -0.29 [-0.46 , -0.12] 5.7E+02 -0.09 [-0.28 , -0.09] 4.3E-03 -0.03 [-0.09 , -0.03] 9.8E-03 Smad4 -0.30 [-0.48 , -0.13] 5.7E+02 -0.21 [-0.30 , -0.21] 7.8E-07 -0.04 [-0.08 , -0.04] 7.5E-05 Ticam1 -0.31 [-0.50 , -0.12] 5.7E+02 -0.22 [-0.39 , -0.22] 4.7E-05 -0.01 [-0.06 , -0.01] 2.1E-02 Hsd11b1 -0.31 [-0.50 , -0.11] 5.7E+02 -0.22 [-0.35 , -0.22] 1.0E-05 -0.02 [-0.06 , -0.02] 1.2E-02 F2rl1 -0.31 [-0.55 , -0.08] 5.7E+02 -0.12 [-0.29 , -0.12] 1.2E-03 -0.02 [-0.09 , -0.02] 2.0E-02 Jam3 -0.32 [-0.48 , -0.15] 5.7E+02 -0.07 [-0.21 , -0.07] 6.3E-03 0.00 [-0.06 , 0.00] 9.0E-02 Itgb1 -0.32 [-0.42 , -0.22] 5.7E+02 -0.17 [-0.24 , -0.17] 1.1E-06 -0.04 [-0.07 , -0.04] 2.2E-05 Hc -0.33 [-0.58 , -0.08] 5.7E+02 -0.03 [-0.32 , -0.03] 5.5E-02 0.06 [-0.03 , 0.06] 7.0E-01 Eomes -0.33 [-0.65 , 0.00] 5.7E+02 -0.10 [-0.48 , -0.10] 2.1E-02 -0.04 [-0.18 , -0.04] 2.4E-02 Icam2 -0.33 [-0.44 , -0.22] 5.7E+02 -0.22 [-0.33 , -0.22] 2.7E-06 -0.03 [-0.07 , -0.03] 7.5E-04 Itga5 -0.33 [-0.46 , -0.20] 5.8E+02 -0.11 [-0.22 , -0.11] 1.8E-04 -0.05 [-0.11 , -0.05] 1.4E-03 Klrc1 -0.33 [-0.60 , -0.06] 5.8E+02 -0.10 [-0.48 , -0.10] 2.1E-02 -0.10 [-0.21 , -0.10] 4.3E-04 Il6st -0.33 [-0.62 , -0.05] 5.8E+02 -0.29 [-0.40 , -0.29] 3.2E-07 0.01 [-0.05 , 0.01] 2.2E-01 Gzma -0.34 [-0.64 , -0.03] 5.8E+02 -0.09 [-0.36 , -0.09] 1.5E-02 -0.10 [-0.24 , -0.10] 1.5E-03 Mill2 -0.34 [-0.69 , 0.01] 5.8E+02 0.05 [-0.28 , 0.05] 2.6E-01 0.00 [-0.13 , 0.00] 1.1E-01 Cd34 -0.36 [-0.46 , -0.25] 5.8E+02 -0.19 [-0.29 , -0.19] 2.7E-06 -0.01 [-0.07 , -0.01] 2.2E-02 369 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Kdr -0.37 [-0.55 , -0.19] 5.8E+02 -0.22 [-0.43 , -0.22] 1.5E-04 -0.02 [-0.13 , -0.02] 4.3E-02 Masp1 -0.37 [-0.64 , -0.11] 5.8E+02 -0.18 [-0.46 , -0.18] 2.1E-03 0.01 [-0.08 , 0.01] 2.4E-01 Prf1 -0.38 [-0.72 , -0.05] 5.8E+02 -0.11 [-0.45 , -0.11] 1.3E-02 -0.10 [-0.23 , -0.10] 1.2E-03 Klrc2 -0.39 [-0.65 , -0.13] 5.8E+02 -0.20 [-0.48 , -0.20] 1.2E-03 -0.11 [-0.20 , -0.11] 1.1E-04 Msln -0.39 [-0.71 , -0.07] 5.9E+02 -0.19 [-0.44 , -0.19] 8.3E-04 0.06 [-0.07 , 0.06] 9.0E-01 Xbp1 -0.41 [-0.53 , -0.29] 5.9E+02 -0.23 [-0.31 , -0.23] 3.2E-07 -0.03 [-0.08 , -0.03] 2.1E-03 Creb5 -0.42 [-0.84 , 0.00] 5.9E+02 -0.06 [-0.50 , -0.06] 4.6E-02 -0.02 [-0.20 , -0.02] 7.2E-02 Tal1 -0.43 [-0.68 , -0.19] 5.9E+02 -0.24 [-0.45 , -0.24] 9.2E-05 -0.03 [-0.10 , -0.03] 5.7E-03 Angpt2 -0.44 [-0.61 , -0.27] 5.9E+02 -0.09 [-0.31 , -0.09] 6.7E-03 0.01 [-0.07 , 0.01] 3.2E-01 Ncr1 -0.44 [-0.69 , -0.20] 5.9E+02 -0.17 [-0.40 , -0.17] 8.6E-04 -0.09 [-0.21 , -0.09] 1.5E-03 Masp2 -0.45 [-0.89 , 0.00] 5.9E+02 0.01 [-0.44 , 0.01] 1.3E-01 0.10 [-0.06 , 0.10] 7.6E-01 Camp -0.46 [-1.13 , 0.21] 5.9E+02 0.04 [-0.61 , 0.04] 1.6E-01 0.07 [-0.17 , 0.07] 5.4E-01 Vegfa -0.47 [-0.59 , -0.36] 5.9E+02 -0.24 [-0.46 , -0.24] 1.1E-04 -0.07 [-0.11 , -0.07] 3.5E-05 Timd4 -0.49 [-0.81 , -0.17] 5.9E+02 -0.13 [-0.46 , -0.13] 9.4E-03 -0.14 [-0.27 , -0.14] 2.0E-04 Jun -0.54 [-0.82 , -0.26] 6.0E+02 -0.35 [-0.51 , -0.35] 9.2E-07 -0.04 [-0.08 , -0.04] 9.3E-04 Tfrc -0.57 [-0.75 , -0.39] 6.0E+02 -0.17 [-0.35 , -0.17] 3.8E-04 -0.02 [-0.10 , -0.02] 3.9E-02 Hamp -0.57 [-1.09 , -0.06] 6.0E+02 -0.33 [-1.17 , -0.33] 8.2E-03 0.20 [-0.17 , 0.20] 9.1E-01 370 Table 31 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 1 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl14 -0.61 [-1.16 , -0.05] 6.0E+02 -0.38 [-0.78 , -0.38] 3.1E-04 -0.01 [-0.16 , -0.01] 9.4E-02 Dusp6 -0.61 [-0.95 , -0.28] 6.0E+02 -0.40 [-0.59 , -0.40] 1.6E-06 -0.03 [-0.09 , -0.03] 7.9E-03 Mcam -0.62 [-0.79 , -0.45] 6.0E+02 -0.34 [-0.52 , -0.34] 2.6E-06 -0.06 [-0.13 , -0.06] 8.7E-04 Vegfc -0.62 [-1.04 , -0.20] 6.0E+02 -0.48 [-0.67 , -0.48] 2.7E-07 -0.01 [-0.06 , -0.01] 4.3E-02 Egr1 -0.63 [-0.93 , -0.34] 6.0E+02 -0.09 [-0.54 , -0.09] 2.9E-02 -0.01 [-0.20 , -0.01] 1.0E-01 Klra4 -0.67 [-1.16 , -0.19] 6.0E+02 -0.27 [-0.46 , -0.27] 2.8E-05 -0.07 [-0.16 , -0.07] 1.3E-03 Bcl2l1 -0.75 [-1.30 , -0.21] 6.0E+02 -0.49 [-0.82 , -0.49] 1.5E-05 0.02 [-0.14 , 0.02] 2.6E-01 Sele -0.76 [-1.43 , -0.09] 6.1E+02 -0.29 [-0.92 , -0.29] 6.1E-03 -0.11 [-0.37 , -0.11] 8.1E-03 Tnfrsf12a -0.82 [-1.08 , -0.57] 6.1E+02 -0.43 [-0.71 , -0.43] 1.2E-05 -0.09 [-0.19 , -0.09] 2.6E-04 Thbs1 -0.88 [-1.31 , -0.46] 6.1E+02 -0.29 [-0.72 , -0.29] 1.5E-03 -0.13 [-0.35 , -0.13] 2.8E-03 Creb1 -0.94 [-1.42 , -0.46] 6.1E+02 -0.75 [-0.88 , -0.75] 2.5E-10 -0.06 [-0.10 , -0.06] 1.1E-05 Cfd -1.17 [-2.97 , 0.64] 6.1E+02 0.43 [-1.56 , 0.43] 3.6E-01 0.25 [-0.52 , 0.25] 6.3E-01 Defb1 -1.38 [-2.26 , -0.51] 6.1E+02 -0.10 [-1.06 , -0.10] 5.7E-02 n.d [n.d , n.d] n.d 371 Table 32. Complete mRNA transcript profile in lung at 5 wk post final exposure in cSiO 2-exposed NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log2(fold change) relative to VEH + CON mice. Expression data are included for all mRNA transcripts that were above background threshold. Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl5 4.76 [4.23 , 5.30] 8.1E-08 2.06 [1.66 , 2.47] 3.0E-06 0.87 [0.77 , 0.97] 2.7E-08 Cxcl3 4.03 [3.50 , 4.56] 1.3E-07 1.63 [1.35 , 1.90] 8.3E-07 0.62 [0.52 , 0.72] 7.7E-07 Lcn2 3.49 [3.04 , 3.95] 1.3E-07 1.70 [1.53 , 1.88] 2.5E-08 0.67 [0.60 , 0.74] 2.4E-08 Ccl3 3.40 [2.99 , 3.80] 9.0E-08 1.62 [1.47 , 1.77] 1.3E-08 0.69 [0.64 , 0.75] 1.6E-09 Ccl8 2.99 [2.21 , 3.77] 3.0E-05 1.27 [0.89 , 1.65] 1.2E-04 0.33 [0.19 , 0.48] 1.7E-03 Ccl2 2.92 [2.22 , 3.62] 1.5E-05 1.04 [0.77 , 1.31] 3.0E-05 0.41 [0.32 , 0.50] 6.6E-06 Cxcl2 2.89 [2.46 , 3.32] 4.6E-07 1.46 [1.29 , 1.63] 6.0E-08 0.53 [0.45 , 0.60] 2.6E-07 Il1rn 2.88 [2.28 , 3.48] 4.9E-06 1.24 [0.96 , 1.53] 1.2E-05 0.48 [0.38 , 0.59] 7.1E-06 Cxcl10 2.85 [1.80 , 3.91] 5.0E-04 0.63 [0.23 , 1.04] 2.2E-02 0.20 [0.05 , 0.34] 3.6E-02 Cxcl1 2.71 [2.21 , 3.20] 1.9E-06 0.97 [0.62 , 1.31] 4.9E-04 0.44 [0.36 , 0.52] 9.5E-07 Cxcl9 2.70 [1.59 , 3.82] 1.1E-03 0.71 [0.24 , 1.17] 2.4E-02 0.17 [-0.01 , 0.34] 1.5E-01 Fcer2a 2.65 [2.15 , 3.15] 2.4E-06 1.19 [1.00 , 1.38] 5.6E-07 0.44 [0.37 , 0.52] 9.1E-07 Clec5a 2.49 [2.09 , 2.90] 9.3E-07 1.11 [0.93 , 1.30] 7.8E-07 0.47 [0.40 , 0.55] 3.4E-07 372 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Msr1 2.36 [1.85 , 2.86] 6.1E-06 0.98 [0.77 , 1.19] 6.9E-06 0.40 [0.33 , 0.47] 1.1E-06 Ccl4 2.34 [1.69 , 2.99] 5.4E-05 n.d [n.d , n.d] n.d 0.40 [0.32 , 0.48] 3.2E-06 Pdcd1 2.33 [1.86 , 2.80] 4.6E-06 1.11 [0.85 , 1.37] 1.3E-05 0.42 [0.32 , 0.52] 1.4E-05 Mx1 2.25 [1.60 , 2.91] 7.7E-05 0.56 [0.40 , 0.72] 6.5E-05 0.16 [0.11 , 0.22] 3.1E-04 Marco 2.24 [1.76 , 2.71] 5.7E-06 1.24 [1.06 , 1.42] 3.5E-07 0.56 [0.48 , 0.64] 2.6E-07 Ccl7 2.24 [1.45 , 3.02] 3.4E-04 0.57 [0.28 , 0.87] 6.2E-03 0.22 [0.12 , 0.31] 2.9E-03 C3ar1 2.23 [1.74 , 2.73] 7.8E-06 0.85 [0.72 , 0.98] 4.0E-07 0.35 [0.32 , 0.38] 7.6E-09 Spink5 2.20 [1.85 , 2.55] 8.4E-07 1.07 [0.89 , 1.25] 8.3E-07 0.40 [0.33 , 0.47] 9.4E-07 Tnfrsf9 2.16 [1.65 , 2.66] 1.2E-05 1.01 [0.79 , 1.24] 9.3E-06 0.35 [0.25 , 0.45] 7.5E-05 Irf7 2.16 [1.33 , 3.00] 7.1E-04 0.40 [0.19 , 0.60] 6.7E-03 0.05 [-0.01 , 0.11] 1.9E-01 Cfb 2.15 [1.80 , 2.50] 9.6E-07 0.80 [0.68 , 0.93] 5.0E-07 0.28 [0.22 , 0.33] 1.9E-06 Clec4n 2.11 [1.78 , 2.45] 8.4E-07 0.90 [0.75 , 1.06] 1.1E-06 0.36 [0.29 , 0.42] 1.3E-06 Ctla4 2.10 [1.74 , 2.47] 1.3E-06 0.88 [0.72 , 1.05] 2.2E-06 0.37 [0.30 , 0.44] 1.9E-06 Ifi44 2.06 [1.38 , 2.74] 2.3E-04 0.28 [0.09 , 0.47] 3.0E-02 -0.03 [-0.08 , 0.03] 4.6E-01 Oas2 2.04 [1.46 , 2.62] 6.3E-05 0.25 [0.08 , 0.43] 3.3E-02 0.00 [-0.07 , 0.06] 9.0E-01 Oasl1 2.02 [1.20 , 2.83] 1.0E-03 0.47 [0.18 , 0.76] 1.7E-02 0.03 [-0.06 , 0.12] 6.6E-01 Chil3 2.01 [1.21 , 2.81] 8.7E-04 1.49 [1.11 , 1.87] 2.8E-05 0.79 [0.62 , 0.97] 6.6E-06 373 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il12b 2.00 [1.48 , 2.52] 3.1E-05 0.94 [0.72 , 1.17] 1.7E-05 0.35 [0.26 , 0.43] 1.9E-05 Ccl9 1.98 [1.68 , 2.28] 6.5E-07 0.91 [0.75 , 1.07] 1.1E-06 0.32 [0.27 , 0.38] 1.0E-06 Tnfsf11 1.98 [1.55 , 2.40] 6.7E-06 0.79 [0.64 , 0.93] 1.9E-06 0.29 [0.24 , 0.34] 9.4E-07 Ccl12 1.97 [1.28 , 2.65] 3.2E-04 0.39 [0.10 , 0.69] 4.3E-02 0.18 [0.08 , 0.28] 8.3E-03 Mmp9 1.96 [1.71 , 2.22] 1.3E-07 0.98 [0.86 , 1.10] 6.4E-08 0.42 [0.38 , 0.46] 7.6E-09 Ambp 1.95 [0.58 , 3.32] 2.6E-02 0.64 [-0.04 , 1.31] 1.5E-01 0.29 [0.03 , 0.55] 8.6E-02 Trem2 1.93 [1.61 , 2.25] 1.0E-06 0.98 [0.87 , 1.10] 6.0E-08 0.46 [0.41 , 0.51] 2.0E-08 Csf2 1.92 [1.57 , 2.26] 1.7E-06 0.81 [0.61 , 1.01] 2.0E-05 0.38 [0.32 , 0.44] 8.4E-07 Zbp1 1.85 [1.21 , 2.49] 3.1E-04 0.25 [0.08 , 0.42] 2.9E-02 0.00 [-0.05 , 0.05] 9.6E-01 Spp1 1.83 [1.40 , 2.27] 1.3E-05 0.86 [0.63 , 1.08] 4.2E-05 0.37 [0.29 , 0.45] 7.0E-06 Clec7a 1.83 [1.23 , 2.43] 2.1E-04 0.72 [0.41 , 1.02] 2.0E-03 0.29 [0.17 , 0.42] 1.4E-03 Isg15 1.78 [1.06 , 2.50] 9.5E-04 0.31 [0.14 , 0.48] 8.0E-03 0.04 [-0.01 , 0.08] 1.9E-01 Foxp3 1.77 [1.34 , 2.20] 1.6E-05 0.80 [0.61 , 0.98] 1.2E-05 0.33 [0.25 , 0.41] 2.0E-05 Ccl6 1.77 [1.32 , 2.23] 2.7E-05 0.97 [0.73 , 1.21] 2.3E-05 0.41 [0.32 , 0.50] 7.7E-06 Ccl20 1.73 [0.69 , 2.77] 1.2E-02 0.39 [-0.13 , 0.91] 2.5E-01 0.01 [-0.19 , 0.21] 9.5E-01 Tigit 1.71 [1.25 , 2.16] 3.6E-05 0.60 [0.40 , 0.80] 2.7E-04 0.19 [0.12 , 0.25] 2.5E-04 Oas3 1.71 [0.97 , 2.45] 1.6E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d 374 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value C1qa 1.69 [1.37 , 2.01] 2.4E-06 0.71 [0.62 , 0.80] 1.8E-07 0.23 [0.19 , 0.26] 4.9E-07 S100a8 1.65 [1.29 , 2.01] 7.1E-06 0.68 [0.50 , 0.86] 4.3E-05 0.30 [0.23 , 0.38] 1.5E-05 Ccr6 1.64 [1.06 , 2.23] 3.5E-04 0.43 [0.27 , 0.59] 7.8E-04 0.15 [0.10 , 0.19] 1.3E-04 Il1a 1.61 [1.33 , 1.90] 1.7E-06 0.59 [0.48 , 0.69] 1.1E-06 0.26 [0.21 , 0.30] 6.8E-07 Osm 1.60 [1.18 , 2.02] 3.4E-05 0.69 [0.54 , 0.84] 6.9E-06 0.23 [0.17 , 0.29] 4.5E-05 Fcgr1 1.59 [1.07 , 2.11] 2.1E-04 0.32 [0.18 , 0.46] 2.1E-03 0.08 [0.03 , 0.13] 1.6E-02 Ifit1 1.59 [1.00 , 2.18] 5.1E-04 0.23 [0.09 , 0.37] 1.5E-02 0.02 [-0.03 , 0.07] 5.6E-01 Birc5 1.58 [1.10 , 2.05] 9.9E-05 0.67 [0.50 , 0.84] 2.7E-05 0.26 [0.20 , 0.33] 2.4E-05 Ctss 1.57 [1.14 , 2.00] 4.2E-05 0.58 [0.40 , 0.76] 1.7E-04 0.24 [0.17 , 0.32] 1.1E-04 C1qb 1.56 [1.24 , 1.88] 4.7E-06 0.59 [0.50 , 0.68] 4.0E-07 0.18 [0.15 , 0.21] 1.4E-06 Ccr1 1.55 [1.23 , 1.87] 4.8E-06 0.77 [0.60 , 0.94] 8.9E-06 0.30 [0.23 , 0.37] 8.5E-06 Usp18 1.52 [0.97 , 2.06] 4.3E-04 0.30 [0.17 , 0.44] 2.8E-03 0.05 [0.00 , 0.09] 8.5E-02 Blnk 1.51 [1.00 , 2.02] 2.5E-04 0.45 [0.32 , 0.58] 1.1E-04 0.19 [0.15 , 0.22] 3.7E-06 Ccl22 1.50 [0.98 , 2.03] 3.2E-04 0.92 [0.70 , 1.14] 2.0E-05 0.34 [0.25 , 0.43] 2.4E-05 Cd14 1.49 [1.20 , 1.78] 3.2E-06 0.76 [0.61 , 0.91] 3.3E-06 0.27 [0.22 , 0.31] 7.7E-07 Fcgr2b 1.47 [1.19 , 1.75] 2.4E-06 0.67 [0.56 , 0.79] 1.1E-06 0.25 [0.20 , 0.29] 1.0E-06 Mx2 1.45 [0.76 , 2.14] 2.9E-03 0.33 [0.14 , 0.52] 1.3E-02 0.03 [-0.04 , 0.10] 5.0E-01 375 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cfi 1.43 [1.06 , 1.80] 2.8E-05 0.49 [0.29 , 0.70] 1.5E-03 0.17 [0.09 , 0.25] 2.4E-03 Fcgr3 1.40 [0.95 , 1.86] 1.9E-04 0.62 [0.39 , 0.85] 7.4E-04 0.24 [0.15 , 0.33] 7.1E-04 Cd83 1.39 [1.13 , 1.65] 2.4E-06 0.48 [0.38 , 0.58] 5.5E-06 0.21 [0.18 , 0.23] 3.0E-08 Slc11a1 1.37 [1.09 , 1.66] 4.8E-06 0.67 [0.53 , 0.80] 5.0E-06 0.28 [0.22 , 0.34] 4.5E-06 Tnfrsf4 1.36 [0.97 , 1.74] 6.3E-05 0.61 [0.42 , 0.81] 1.8E-04 0.24 [0.18 , 0.30] 2.6E-05 Ccr5 1.36 [0.85 , 1.87] 5.3E-04 0.41 [0.24 , 0.57] 1.1E-03 0.15 [0.08 , 0.21] 3.1E-03 Arg2 1.35 [1.01 , 1.68] 2.3E-05 0.88 [0.66 , 1.10] 2.6E-05 0.33 [0.26 , 0.40] 6.5E-06 Il23r 1.35 [0.94 , 1.76] 1.1E-04 0.68 [0.48 , 0.87] 7.3E-05 0.22 [0.13 , 0.31] 1.4E-03 Tlr2 1.34 [1.11 , 1.58] 1.7E-06 0.50 [0.38 , 0.63] 1.7E-05 0.23 [0.19 , 0.27] 1.5E-06 Cxcr1 1.34 [1.01 , 1.67] 1.9E-05 0.73 [0.52 , 0.93] 6.8E-05 0.34 [0.29 , 0.39] 3.4E-07 Icos 1.34 [0.99 , 1.70] 3.6E-05 0.37 [0.25 , 0.50] 3.2E-04 0.15 [0.10 , 0.19] 2.5E-04 H2-Q2 1.34 [0.86 , 1.83] 4.3E-04 0.27 [0.00 , 0.54] 1.3E-01 0.06 [-0.03 , 0.15] 3.0E-01 Emr1 1.33 [0.90 , 1.77] 2.1E-04 0.50 [0.28 , 0.71] 1.9E-03 0.19 [0.11 , 0.27] 2.2E-03 Il1b 1.31 [1.04 , 1.58] 4.8E-06 0.42 [0.26 , 0.58] 9.0E-04 0.20 [0.15 , 0.26] 3.7E-05 Ly86 1.30 [0.97 , 1.63] 2.8E-05 0.35 [0.20 , 0.49] 1.5E-03 0.13 [0.08 , 0.18] 5.4E-04 H2-DMb2 1.29 [0.84 , 1.74] 3.4E-04 0.39 [0.24 , 0.53] 7.4E-04 0.14 [0.09 , 0.18] 5.3E-04 Il1r2 1.27 [0.86 , 1.69] 2.0E-04 0.91 [0.64 , 1.18] 1.1E-04 0.28 [0.20 , 0.37] 8.7E-05 376 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Lta 1.27 [0.83 , 1.71] 3.2E-04 0.41 [0.25 , 0.57] 8.6E-04 0.18 [0.13 , 0.22] 4.5E-05 Cx3cr1 1.25 [0.84 , 1.66] 2.1E-04 0.31 [0.07 , 0.55] 4.9E-02 0.13 [0.05 , 0.21] 1.4E-02 Slamf7 1.25 [0.77 , 1.74] 7.0E-04 0.34 [0.23 , 0.45] 2.5E-04 0.14 [0.10 , 0.18] 2.6E-05 Cd68 1.24 [1.04 , 1.44] 8.7E-07 0.60 [0.52 , 0.69] 3.5E-07 0.27 [0.24 , 0.30] 5.8E-08 Pou2af1 1.23 [0.63 , 1.82] 3.5E-03 0.23 [0.04 , 0.43] 6.6E-02 0.09 [0.02 , 0.17] 6.0E-02 Cd86 1.21 [0.93 , 1.49] 1.1E-05 0.56 [0.44 , 0.68] 5.9E-06 0.18 [0.14 , 0.23] 1.3E-05 Cd80 1.21 [0.85 , 1.57] 8.8E-05 0.57 [0.43 , 0.70] 1.6E-05 0.20 [0.16 , 0.25] 4.0E-06 Rsad2 1.21 [0.55 , 1.86] 6.6E-03 0.16 [-0.01 , 0.34] 1.5E-01 0.07 [0.02 , 0.12] 3.3E-02 Ncf4 1.20 [0.98 , 1.42] 1.7E-06 0.40 [0.30 , 0.50] 2.0E-05 0.17 [0.14 , 0.20] 1.7E-06 Ifitm1 1.19 [0.94 , 1.44] 4.9E-06 0.71 [0.59 , 0.83] 8.3E-07 0.24 [0.20 , 0.28] 9.1E-07 Clec4a2 1.19 [0.78 , 1.60] 3.0E-04 0.35 [0.15 , 0.55] 1.3E-02 0.13 [0.05 , 0.21] 1.7E-02 Cd53 1.17 [1.00 , 1.34] 4.0E-07 0.37 [0.30 , 0.44] 2.1E-06 0.15 [0.12 , 0.18] 3.4E-06 Fcer1g 1.17 [0.95 , 1.40] 2.5E-06 0.45 [0.36 , 0.53] 3.0E-06 0.18 [0.14 , 0.21] 4.9E-06 Cybb 1.17 [0.89 , 1.45] 1.6E-05 0.31 [0.17 , 0.45] 3.2E-03 0.12 [0.06 , 0.17] 3.2E-03 Cxcr2 1.17 [0.87 , 1.48] 3.2E-05 0.59 [0.42 , 0.76] 8.4E-05 0.26 [0.18 , 0.33] 7.2E-05 Cd180 1.17 [0.72 , 1.62] 6.5E-04 0.29 [0.11 , 0.46] 1.6E-02 0.09 [0.05 , 0.13] 1.8E-03 Cfp 1.16 [0.81 , 1.52] 1.1E-04 0.48 [0.31 , 0.64] 3.9E-04 0.15 [0.09 , 0.20] 8.1E-04 377 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Fcgr4 1.16 [0.67 , 1.66] 1.4E-03 0.27 [0.05 , 0.50] 6.5E-02 0.09 [0.00 , 0.18] 1.0E-01 Cd200r1 1.15 [0.91 , 1.39] 5.7E-06 0.43 [0.34 , 0.52] 4.5E-06 0.22 [0.18 , 0.25] 3.4E-07 Ifit3 1.15 [0.63 , 1.67] 2.1E-03 0.01 [-0.16 , 0.18] 9.2E-01 -0.02 [-0.04 , 0.01] 2.7E-01 Pik3cg 1.14 [0.88 , 1.40] 1.1E-05 0.38 [0.31 , 0.44] 8.3E-07 0.13 [0.10 , 0.17] 9.2E-06 Ly9 1.14 [0.75 , 1.54] 3.0E-04 0.26 [0.12 , 0.39] 6.5E-03 0.13 [0.08 , 0.17] 4.5E-04 Tnfrsf13c 1.14 [0.39 , 1.89] 1.9E-02 0.15 [-0.12 , 0.42] 3.9E-01 0.05 [-0.06 , 0.16] 4.7E-01 Il2ra 1.12 [0.74 , 1.50] 2.6E-04 0.55 [0.41 , 0.70] 3.3E-05 0.23 [0.17 , 0.30] 4.5E-05 Ulbp1 1.09 [0.85 , 1.32] 7.0E-06 0.38 [0.25 , 0.50] 3.4E-04 0.13 [0.09 , 0.17] 1.6E-04 Ebi3 1.08 [0.86 , 1.30] 4.6E-06 0.28 [0.18 , 0.39] 5.8E-04 0.12 [0.09 , 0.16] 6.7E-05 Tnfrsf11a 1.08 [0.78 , 1.38] 4.9E-05 0.51 [0.39 , 0.62] 1.0E-05 0.21 [0.16 , 0.25] 4.5E-06 Ddx60 1.07 [0.59 , 1.56] 2.1E-03 0.19 [0.08 , 0.31] 1.4E-02 0.05 [0.01 , 0.08] 6.3E-02 H2-Q10 1.07 [0.52 , 1.61] 4.7E-03 0.42 [0.16 , 0.68] 1.7E-02 0.18 [0.07 , 0.28] 1.4E-02 Cd74 1.06 [0.84 , 1.28] 4.9E-06 0.54 [0.46 , 0.63] 4.0E-07 0.19 [0.16 , 0.22] 8.5E-07 Ccr8 1.06 [0.79 , 1.34] 3.0E-05 0.47 [0.30 , 0.64] 4.8E-04 0.15 [0.09 , 0.20] 3.9E-04 Ms4a1 1.06 [0.58 , 1.55] 2.1E-03 0.09 [-0.14 , 0.31] 5.5E-01 0.03 [-0.03 , 0.08] 4.9E-01 Csf2rb 1.05 [0.70 , 1.41] 2.3E-04 0.61 [0.43 , 0.79] 1.1E-04 0.26 [0.19 , 0.33] 3.4E-05 Itgam 1.04 [0.75 , 1.32] 5.4E-05 0.42 [0.28 , 0.56] 3.2E-04 0.17 [0.12 , 0.22] 4.0E-05 378 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Slamf6 1.04 [0.61 , 1.47] 1.2E-03 0.19 [0.08 , 0.31] 1.5E-02 0.08 [0.03 , 0.13] 1.2E-02 Lgals3 1.03 [0.80 , 1.26] 7.9E-06 0.44 [0.33 , 0.54] 2.0E-05 0.20 [0.16 , 0.25] 7.3E-06 Ltb 1.02 [0.67 , 1.38] 3.3E-04 0.15 [-0.02 , 0.33] 1.7E-01 0.07 [0.03 , 0.11] 1.2E-02 Xcl1 1.02 [0.56 , 1.49] 2.2E-03 0.14 [-0.11 , 0.38] 4.0E-01 0.05 [-0.04 , 0.14] 4.0E-01 Csf3r 1.01 [0.66 , 1.36] 3.2E-04 0.49 [0.29 , 0.69] 1.3E-03 0.25 [0.18 , 0.33] 8.1E-05 Irf4 1.01 [0.41 , 1.61] 1.1E-02 0.39 [0.20 , 0.57] 3.7E-03 0.16 [0.09 , 0.22] 1.5E-03 Lamp3 1.00 [0.78 , 1.22] 7.1E-06 0.41 [0.30 , 0.52] 4.0E-05 0.17 [0.12 , 0.21] 2.7E-05 Sh2b2 1.00 [0.60 , 1.39] 8.3E-04 0.31 [0.16 , 0.45] 4.5E-03 0.10 [0.03 , 0.17] 3.1E-02 Cd84 0.99 [0.82 , 1.15] 1.1E-06 0.32 [0.25 , 0.38] 2.7E-06 0.15 [0.12 , 0.18] 1.9E-06 C1ra 0.98 [0.79 , 1.17] 3.2E-06 0.44 [0.37 , 0.51] 5.5E-07 0.15 [0.12 , 0.18] 1.5E-06 Tnfrsf13b 0.97 [0.65 , 1.30] 2.5E-04 0.25 [0.10 , 0.40] 1.5E-02 0.09 [0.04 , 0.14] 6.5E-03 Lyz2 0.97 [0.80 , 1.13] 1.3E-06 0.47 [0.40 , 0.54] 4.0E-07 0.20 [0.17 , 0.23] 1.7E-07 Cd3g 0.96 [0.63 , 1.29] 3.1E-04 0.21 [0.10 , 0.33] 9.4E-03 0.06 [0.02 , 0.10] 3.0E-02 Trem1 0.96 [0.53 , 1.39] 2.0E-03 0.57 [0.35 , 0.79] 9.4E-04 0.20 [0.11 , 0.29] 2.0E-03 Tlr1 0.95 [0.57 , 1.33] 9.1E-04 0.15 [-0.01 , 0.32] 1.6E-01 0.07 [0.03 , 0.12] 1.6E-02 Bst2 0.95 [0.54 , 1.37] 1.6E-03 0.19 [0.09 , 0.28] 5.0E-03 0.01 [-0.01 , 0.04] 3.7E-01 Casp1 0.95 [0.70 , 1.20] 3.5E-05 0.17 [0.06 , 0.29] 2.2E-02 0.08 [0.04 , 0.12] 7.0E-03 379 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Klrg1 0.94 [0.64 , 1.24] 1.7E-04 0.48 [0.34 , 0.62] 8.2E-05 0.22 [0.16 , 0.27] 2.8E-05 Havcr2 0.94 [0.75 , 1.13] 4.5E-06 0.39 [0.28 , 0.50] 6.4E-05 0.16 [0.12 , 0.20] 2.0E-05 Mefv 0.94 [0.37 , 1.51] 1.2E-02 0.16 [-0.12 , 0.43] 3.8E-01 0.07 [-0.03 , 0.18] 2.9E-01 Tlr9 0.93 [0.48 , 1.39] 3.3E-03 0.28 [0.13 , 0.44] 8.9E-03 0.09 [0.03 , 0.16] 2.2E-02 Herc6 0.93 [0.56 , 1.31] 9.8E-04 0.26 [0.18 , 0.33] 1.4E-04 0.10 [0.07 , 0.13] 1.2E-04 Tnf 0.93 [0.55 , 1.31] 1.0E-03 0.39 [0.19 , 0.59] 6.7E-03 0.21 [0.15 , 0.27] 8.2E-05 H2-Aa 0.93 [0.73 , 1.12] 5.7E-06 0.41 [0.33 , 0.49] 2.7E-06 0.13 [0.10 , 0.16] 6.6E-06 Cd5 0.92 [0.64 , 1.20] 1.1E-04 0.26 [0.17 , 0.35] 5.1E-04 0.09 [0.05 , 0.13] 1.8E-03 Lag3 0.91 [0.45 , 1.38] 4.7E-03 0.18 [0.07 , 0.29] 1.7E-02 -0.01 [-0.09 , 0.07] 9.0E-01 Il21r 0.91 [0.33 , 1.50] 1.6E-02 0.21 [0.03 , 0.40] 7.5E-02 0.06 [-0.01 , 0.13] 1.5E-01 Cd207 0.91 [0.41 , 1.41] 7.3E-03 0.79 [0.63 , 0.95] 3.8E-06 0.28 [0.23 , 0.33] 1.1E-06 Glycam1 0.90 [0.33 , 1.48] 1.6E-02 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Ccr2 0.90 [0.71 , 1.10] 7.0E-06 0.11 [-0.08 , 0.29] 3.7E-01 0.08 [0.03 , 0.13] 1.8E-02 Raet1c 0.90 [0.69 , 1.11] 1.2E-05 0.39 [0.29 , 0.49] 3.3E-05 0.14 [0.11 , 0.18] 6.8E-06 Gzmk 0.90 [0.38 , 1.41] 9.3E-03 0.19 [-0.01 , 0.39] 1.4E-01 0.03 [-0.03 , 0.10] 4.2E-01 C6 0.89 [0.71 , 1.07] 4.6E-06 0.47 [0.38 , 0.56] 1.7E-06 0.21 [0.18 , 0.24] 2.1E-07 H2-DMb1 0.89 [0.70 , 1.07] 4.8E-06 0.41 [0.32 , 0.50] 8.9E-06 0.14 [0.11 , 0.18] 1.4E-05 380 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Irf5 0.88 [0.66 , 1.10] 2.6E-05 0.30 [0.20 , 0.41] 4.8E-04 0.14 [0.10 , 0.18] 3.4E-05 Ccl17 0.88 [0.40 , 1.36] 6.6E-03 0.79 [0.63 , 0.96] 5.5E-06 0.33 [0.26 , 0.40] 4.5E-06 C3 0.87 [0.68 , 1.07] 8.4E-06 0.45 [0.37 , 0.54] 1.5E-06 0.18 [0.15 , 0.22] 1.4E-06 Cmpk2 0.87 [0.48 , 1.27] 2.2E-03 0.04 [-0.12 , 0.19] 7.2E-01 -0.02 [-0.05 , 0.01] 3.7E-01 H2-Ab1 0.87 [0.68 , 1.06] 7.8E-06 0.44 [0.37 , 0.50] 4.0E-07 0.15 [0.12 , 0.17] 9.1E-07 Tnfaip3 0.87 [0.66 , 1.08] 1.5E-05 0.26 [0.19 , 0.33] 3.5E-05 0.11 [0.08 , 0.14] 7.1E-05 H2-Ea-ps 0.86 [0.68 , 1.05] 6.1E-06 0.34 [0.27 , 0.41] 3.0E-06 0.11 [0.08 , 0.13] 6.6E-06 Tnfrsf18 0.86 [0.39 , 1.33] 6.6E-03 0.35 [0.20 , 0.50] 1.9E-03 0.11 [0.06 , 0.16] 2.4E-03 Nlrc5 0.86 [0.41 , 1.31] 5.4E-03 0.11 [-0.04 , 0.25] 2.5E-01 0.02 [-0.03 , 0.08] 5.0E-01 Ccl19 0.85 [0.41 , 1.29] 4.8E-03 0.25 [0.08 , 0.42] 3.0E-02 0.07 [0.02 , 0.12] 2.6E-02 Cxcr6 0.85 [0.62 , 1.09] 4.8E-05 0.29 [0.19 , 0.39] 5.0E-04 0.12 [0.08 , 0.17] 6.7E-04 Cd6 0.85 [0.43 , 1.27] 3.7E-03 0.25 [0.10 , 0.39] 1.5E-02 0.08 [0.02 , 0.15] 5.4E-02 Flt3 0.85 [0.63 , 1.06] 2.4E-05 0.38 [0.27 , 0.49] 8.4E-05 0.16 [0.11 , 0.21] 9.8E-05 Cd48 0.84 [0.62 , 1.06] 3.0E-05 0.16 [0.03 , 0.28] 5.4E-02 0.07 [0.04 , 0.10] 2.9E-03 Ikzf2 0.84 [0.53 , 1.15] 5.2E-04 0.32 [0.21 , 0.42] 2.4E-04 0.13 [0.10 , 0.16] 7.1E-06 Cd274 0.83 [0.54 , 1.13] 3.5E-04 0.25 [0.17 , 0.33] 2.8E-04 0.14 [0.11 , 0.17] 7.3E-06 Nlrp3 0.83 [0.48 , 1.18] 1.2E-03 0.39 [0.22 , 0.56] 2.3E-03 0.17 [0.10 , 0.25] 2.1E-03 381 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Sh2d1a 0.83 [0.48 , 1.18] 1.4E-03 0.15 [0.05 , 0.24] 2.3E-02 0.01 [-0.01 , 0.04] 4.2E-01 Cxcl15 0.83 [0.64 , 1.01] 8.8E-06 0.34 [0.25 , 0.43] 4.0E-05 0.12 [0.08 , 0.15] 8.2E-05 Ticam2 0.82 [0.54 , 1.10] 2.6E-04 0.15 [-0.02 , 0.31] 1.6E-01 0.08 [0.03 , 0.13] 1.5E-02 Cxcl13 0.81 [-0.08 , 1.71] 1.3E-01 0.24 [-0.26 , 0.73] 4.6E-01 -0.03 [-0.22 , 0.16] 8.0E-01 Cxcr3 0.81 [0.23 , 1.40] 2.9E-02 0.14 [0.02 , 0.27] 8.3E-02 0.04 [-0.01 , 0.08] 1.7E-01 H2-K1 0.81 [0.46 , 1.15] 1.6E-03 0.37 [0.24 , 0.50] 4.6E-04 0.13 [0.09 , 0.17] 2.2E-04 Cd33 0.80 [0.53 , 1.08] 3.0E-04 0.48 [0.35 , 0.62] 6.5E-05 0.22 [0.16 , 0.27] 1.7E-05 Psmb9 0.80 [0.46 , 1.14] 1.5E-03 0.23 [0.13 , 0.34] 3.1E-03 0.09 [0.05 , 0.13] 2.6E-03 Cxcl16 0.80 [0.65 , 0.94] 1.7E-06 0.26 [0.20 , 0.31] 5.9E-06 0.08 [0.06 , 0.10] 1.3E-05 Xaf1 0.79 [0.39 , 1.20] 4.8E-03 0.10 [0.01 , 0.20] 8.7E-02 0.00 [-0.04 , 0.03] 8.9E-01 Btk 0.78 [0.56 , 1.00] 6.6E-05 0.18 [0.10 , 0.26] 2.2E-03 0.10 [0.08 , 0.12] 1.9E-06 Batf 0.77 [0.51 , 1.04] 3.0E-04 0.12 [-0.02 , 0.27] 1.8E-01 0.06 [0.01 , 0.11] 4.2E-02 Muc1 0.77 [0.64 , 0.90] 1.2E-06 0.36 [0.31 , 0.41] 1.8E-07 0.15 [0.13 , 0.17] 1.7E-07 Fap 0.77 [0.49 , 1.05] 4.9E-04 0.29 [0.15 , 0.43] 4.9E-03 0.05 [0.00 , 0.10] 1.0E-01 Selplg 0.77 [0.64 , 0.89] 9.6E-07 0.28 [0.23 , 0.34] 2.6E-06 0.11 [0.09 , 0.13] 2.0E-06 Col3a1 0.77 [0.54 , 1.00] 1.0E-04 0.19 [0.08 , 0.30] 1.1E-02 0.03 [-0.01 , 0.07] 3.0E-01 Itgae 0.77 [0.48 , 1.05] 4.9E-04 0.54 [0.41 , 0.66] 1.0E-05 0.21 [0.16 , 0.26] 7.1E-06 382 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Bid 0.76 [0.53 , 0.99] 1.1E-04 0.15 [0.08 , 0.22] 3.6E-03 0.06 [0.03 , 0.09] 6.6E-03 Cd19 0.76 [0.24 , 1.28] 2.3E-02 0.08 [-0.11 , 0.27] 5.0E-01 0.06 [0.00 , 0.11] 8.8E-02 Ccl21a 0.76 [0.45 , 1.07] 1.1E-03 0.23 [0.11 , 0.35] 6.4E-03 0.05 [0.01 , 0.09] 5.1E-02 C1s1 0.76 [0.66 , 0.85] 1.3E-07 0.30 [0.25 , 0.36] 1.9E-06 0.12 [0.10 , 0.14] 8.3E-07 Psmb8 0.76 [0.46 , 1.05] 8.4E-04 0.19 [0.11 , 0.26] 1.7E-03 0.06 [0.03 , 0.09] 9.0E-03 Tnfrsf1b 0.75 [0.45 , 1.05] 1.0E-03 0.20 [0.05 , 0.34] 4.2E-02 0.09 [0.03 , 0.15] 1.9E-02 Cd4 0.75 [0.32 , 1.18] 9.3E-03 0.30 [0.12 , 0.48] 1.4E-02 0.11 [0.04 , 0.18] 2.5E-02 Ddx58 0.74 [0.50 , 0.98] 2.0E-04 0.07 [0.01 , 0.13] 6.1E-02 0.02 [0.00 , 0.04] 8.2E-02 Cd59b 0.74 [0.43 , 1.05] 1.3E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Plau 0.73 [0.43 , 1.04] 1.1E-03 0.16 [0.05 , 0.27] 2.5E-02 0.10 [0.06 , 0.13] 3.3E-04 Cd79b 0.73 [0.36 , 1.11] 4.4E-03 0.10 [-0.05 , 0.25] 3.0E-01 0.03 [-0.01 , 0.07] 1.9E-01 Csf1r 0.73 [0.51 , 0.94] 8.8E-05 0.33 [0.23 , 0.43] 1.7E-04 0.13 [0.08 , 0.17] 3.0E-04 Itgax 0.72 [0.42 , 1.03] 1.2E-03 0.47 [0.30 , 0.63] 4.0E-04 0.20 [0.13 , 0.26] 2.0E-04 Xcr1 0.72 [0.38 , 1.05] 2.8E-03 0.49 [0.34 , 0.64] 1.6E-04 0.15 [0.09 , 0.21] 9.7E-04 Tap1 0.72 [0.26 , 1.17] 1.5E-02 0.30 [0.16 , 0.45] 4.3E-03 0.11 [0.05 , 0.17] 7.1E-03 Tnfsf14 0.71 [0.43 , 0.99] 8.4E-04 0.21 [0.06 , 0.35] 3.0E-02 0.09 [0.04 , 0.14] 1.4E-02 Syk 0.71 [0.53 , 0.89] 2.4E-05 0.22 [0.17 , 0.28] 2.8E-05 0.11 [0.08 , 0.13] 4.7E-06 383 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd96 0.70 [0.49 , 0.91] 1.1E-04 0.15 [0.02 , 0.27] 8.4E-02 0.03 [-0.02 , 0.07] 3.8E-01 Tnfsf15 0.70 [0.47 , 0.93] 2.2E-04 0.15 [-0.04 , 0.34] 2.2E-01 0.10 [0.06 , 0.15] 2.5E-03 Gpr183 0.69 [0.33 , 1.06] 5.5E-03 0.32 [0.18 , 0.46] 2.0E-03 0.13 [0.08 , 0.18] 6.8E-04 Tlr6 0.69 [0.48 , 0.90] 1.3E-04 0.28 [0.19 , 0.37] 1.7E-04 0.13 [0.09 , 0.17] 7.9E-05 Pdcd1lg2 0.69 [0.40 , 0.97] 1.2E-03 0.25 [0.11 , 0.40] 1.1E-02 0.14 [0.10 , 0.18] 9.6E-05 Stat1 0.68 [0.26 , 1.11] 1.4E-02 0.14 [0.00 , 0.28] 1.2E-01 0.05 [-0.01 , 0.10] 1.8E-01 Ly96 0.68 [0.53 , 0.84] 1.1E-05 0.18 [0.08 , 0.27] 6.8E-03 0.06 [0.03 , 0.09] 2.2E-03 Siglec1 0.67 [0.12 , 1.22] 5.2E-02 0.35 [0.10 , 0.60] 3.6E-02 0.11 [0.02 , 0.19] 5.0E-02 Cxcr5 0.66 [0.17 , 1.15] 3.4E-02 0.08 [-0.13 , 0.28] 5.7E-01 0.02 [-0.06 , 0.10] 6.9E-01 Klra2 0.66 [0.43 , 0.89] 3.2E-04 -0.09 [-0.23 , 0.05] 3.0E-01 -0.05 [-0.10 , 0.00] 1.5E-01 Itgal 0.66 [0.44 , 0.87] 2.3E-04 0.16 [0.04 , 0.29] 5.1E-02 0.04 [0.00 , 0.09] 1.2E-01 H2-T23 0.66 [0.32 , 0.99] 4.5E-03 0.21 [0.12 , 0.30] 2.4E-03 0.08 [0.04 , 0.11] 2.0E-03 Vcam1 0.65 [0.37 , 0.94] 1.5E-03 0.22 [0.10 , 0.33] 6.9E-03 0.05 [0.01 , 0.10] 7.6E-02 H2-Ob 0.65 [0.17 , 1.14] 3.3E-02 0.14 [-0.06 , 0.33] 2.9E-01 0.07 [0.00 , 0.14] 1.2E-01 Abca1 0.65 [0.47 , 0.82] 3.9E-05 0.26 [0.17 , 0.36] 4.5E-04 0.11 [0.07 , 0.15] 3.4E-04 Lcp1 0.64 [0.50 , 0.79] 7.0E-06 0.22 [0.17 , 0.28] 2.1E-05 0.09 [0.07 , 0.11] 6.6E-06 Tlr7 0.64 [0.42 , 0.86] 2.8E-04 0.25 [0.16 , 0.34] 4.5E-04 0.11 [0.08 , 0.14] 4.5E-05 384 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ada 0.64 [0.25 , 1.03] 1.2E-02 0.23 [0.06 , 0.40] 4.4E-02 0.08 [0.05 , 0.11] 5.4E-04 Il15 0.63 [0.44 , 0.81] 8.8E-05 0.11 [-0.06 , 0.28] 3.2E-01 0.09 [0.06 , 0.12] 3.4E-04 H2-D1 0.63 [0.35 , 0.91] 1.9E-03 0.26 [0.16 , 0.36] 8.4E-04 0.10 [0.06 , 0.13] 7.1E-04 Itgb2 0.63 [0.40 , 0.85] 4.1E-04 0.38 [0.24 , 0.52] 7.4E-04 0.16 [0.12 , 0.21] 3.0E-05 Ikbke 0.63 [0.33 , 0.92] 2.9E-03 0.24 [0.12 , 0.36] 5.8E-03 0.10 [0.05 , 0.16] 8.9E-03 Ptprc 0.62 [0.38 , 0.86] 7.7E-04 0.09 [-0.01 , 0.18] 1.5E-01 0.04 [0.01 , 0.07] 3.6E-02 Il7 0.61 [0.43 , 0.79] 9.7E-05 0.24 [0.09 , 0.39] 1.7E-02 0.10 [0.06 , 0.14] 7.3E-04 Cr2 0.61 [0.13 , 1.09] 4.3E-02 0.03 [-0.20 , 0.25] 8.5E-01 0.01 [-0.06 , 0.09] 7.9E-01 Fut7 0.61 [0.24 , 0.97] 1.2E-02 0.14 [-0.06 , 0.33] 2.9E-01 0.06 [-0.01 , 0.13] 1.6E-01 Cxcr4 0.60 [0.46 , 0.75] 1.3E-05 0.22 [0.11 , 0.32] 5.7E-03 0.13 [0.09 , 0.17] 1.9E-04 Pik3cd 0.60 [0.35 , 0.86] 1.4E-03 0.16 [0.03 , 0.29] 6.5E-02 0.09 [0.04 , 0.14] 1.2E-02 Amica1 0.60 [0.47 , 0.73] 6.7E-06 0.32 [0.27 , 0.37] 5.2E-07 0.10 [0.08 , 0.13] 1.4E-05 Ifih1 0.60 [0.38 , 0.81] 4.4E-04 0.02 [-0.03 , 0.06] 5.2E-01 0.00 [-0.01 , 0.02] 7.8E-01 Arg1 0.60 [-0.05 , 1.24] 1.3E-01 0.25 [-0.11 , 0.60] 2.9E-01 0.17 [0.02 , 0.31] 7.9E-02 Pou2f2 0.59 [0.13 , 1.04] 4.1E-02 -0.03 [-0.23 , 0.16] 8.0E-01 -0.01 [-0.08 , 0.06] 8.6E-01 Lck 0.58 [0.25 , 0.91] 8.4E-03 0.04 [-0.05 , 0.14] 4.5E-01 -0.01 [-0.03 , 0.02] 7.9E-01 Fcer1a 0.57 [0.26 , 0.89] 7.0E-03 0.13 [-0.01 , 0.26] 1.5E-01 -0.01 [-0.05 , 0.03] 7.3E-01 385 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Gbp2b 0.57 [0.22 , 0.92] 1.3E-02 -0.03 [-0.18 , 0.11] 7.2E-01 -0.02 [-0.07 , 0.03] 5.4E-01 H2-DMa 0.57 [0.37 , 0.77] 3.2E-04 0.37 [0.29 , 0.44] 3.8E-06 0.12 [0.09 , 0.16] 3.4E-05 Klra17 0.57 [0.27 , 0.87] 5.6E-03 -0.08 [-0.26 , 0.10] 4.9E-01 -0.02 [-0.08 , 0.04] 5.9E-01 Cmah 0.57 [0.29 , 0.85] 3.7E-03 -0.01 [-0.10 , 0.07] 8.3E-01 -0.02 [-0.05 , 0.00] 2.0E-01 Abcg1 0.57 [0.38 , 0.75] 1.8E-04 0.24 [0.17 , 0.32] 1.4E-04 0.14 [0.10 , 0.17] 8.2E-06 Blk 0.57 [0.12 , 1.02] 4.5E-02 0.12 [-0.05 , 0.30] 2.8E-01 0.06 [-0.01 , 0.13] 2.0E-01 Card9 0.56 [0.14 , 0.98] 3.6E-02 0.25 [0.10 , 0.41] 1.5E-02 0.13 [0.05 , 0.21] 1.4E-02 Socs1 0.56 [0.24 , 0.88] 8.7E-03 0.01 [-0.14 , 0.17] 8.9E-01 0.02 [-0.01 , 0.05] 3.1E-01 Angpt2 0.55 [0.26 , 0.85] 6.3E-03 0.23 [0.09 , 0.36] 1.3E-02 0.10 [0.03 , 0.17] 2.7E-02 Tnfsf13b 0.55 [0.37 , 0.73] 1.9E-04 0.32 [0.23 , 0.40] 3.1E-05 0.06 [0.03 , 0.09] 1.1E-02 Tdo2 0.55 [-0.15 , 1.25] 1.9E-01 0.18 [-0.15 , 0.52] 4.0E-01 0.11 [-0.04 , 0.26] 2.4E-01 Apoe 0.55 [0.45 , 0.65] 2.5E-06 0.17 [0.10 , 0.24] 1.3E-03 0.09 [0.06 , 0.11] 1.1E-04 Egr3 0.55 [-0.03 , 1.13] 1.2E-01 0.10 [-0.18 , 0.37] 5.9E-01 0.01 [-0.10 , 0.13] 8.9E-01 Icosl 0.55 [0.40 , 0.70] 4.6E-05 0.18 [0.13 , 0.24] 6.5E-05 0.08 [0.06 , 0.10] 5.9E-05 Slc7a11 0.54 [0.23 , 0.85] 8.7E-03 0.27 [0.12 , 0.42] 1.1E-02 0.11 [0.06 , 0.16] 2.1E-03 Cd3e 0.53 [0.18 , 0.89] 2.0E-02 0.21 [0.12 , 0.30] 1.7E-03 0.05 [0.02 , 0.08] 2.2E-02 Pdgfc 0.52 [0.20 , 0.85] 1.4E-02 0.24 [0.12 , 0.37] 7.3E-03 0.06 [0.00 , 0.12] 9.8E-02 386 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il5ra 0.52 [0.09 , 0.96] 5.3E-02 0.18 [-0.01 , 0.36] 1.4E-01 0.08 [0.01 , 0.16] 9.4E-02 H2-M3 0.52 [0.21 , 0.84] 1.2E-02 0.20 [0.09 , 0.30] 7.2E-03 0.07 [0.03 , 0.11] 8.2E-03 Stat2 0.52 [0.20 , 0.83] 1.2E-02 -0.04 [-0.15 , 0.07] 5.8E-01 -0.03 [-0.06 , 0.00] 1.6E-01 Bst1 0.51 [0.30 , 0.73] 1.3E-03 0.20 [0.07 , 0.33] 2.5E-02 0.09 [0.05 , 0.13] 3.2E-03 Zap70 0.51 [0.23 , 0.80] 7.9E-03 0.23 [0.14 , 0.32] 6.9E-04 0.08 [0.04 , 0.13] 6.9E-03 Il12rb2 0.51 [0.20 , 0.83] 1.4E-02 0.24 [0.10 , 0.38] 1.3E-02 0.07 [0.01 , 0.12] 6.4E-02 Fpr2 0.51 [0.13 , 0.89] 3.3E-02 0.03 [-0.17 , 0.22] 8.4E-01 0.03 [-0.04 , 0.11] 4.6E-01 Il34 0.51 [0.30 , 0.71] 9.1E-04 0.26 [0.16 , 0.36] 8.4E-04 0.09 [0.04 , 0.14] 5.0E-03 Hc 0.50 [0.31 , 0.70] 7.4E-04 0.22 [0.15 , 0.29] 1.9E-04 0.07 [0.04 , 0.10] 2.9E-03 Irf8 0.50 [0.27 , 0.73] 2.2E-03 0.24 [0.11 , 0.36] 6.6E-03 0.10 [0.06 , 0.14] 6.0E-04 Cyfip2 0.50 [0.24 , 0.76] 4.9E-03 0.11 [0.04 , 0.18] 2.0E-02 0.03 [0.00 , 0.05] 1.5E-01 Ifnar2 0.50 [0.38 , 0.62] 1.5E-05 0.17 [0.11 , 0.24] 8.8E-04 0.07 [0.05 , 0.10] 2.7E-04 Clu 0.50 [0.29 , 0.71] 1.3E-03 0.15 [0.08 , 0.21] 2.0E-03 0.02 [0.00 , 0.04] 2.2E-01 Relb 0.50 [0.24 , 0.75] 4.8E-03 0.17 [0.07 , 0.27] 1.4E-02 0.11 [0.06 , 0.15] 7.4E-04 Il10ra 0.50 [0.18 , 0.81] 1.6E-02 0.19 [0.04 , 0.34] 4.8E-02 0.09 [0.03 , 0.15] 2.2E-02 Egr2 0.48 [0.12 , 0.85] 3.5E-02 0.13 [-0.06 , 0.32] 3.0E-01 0.07 [0.00 , 0.13] 1.2E-01 Isg20 0.48 [0.31 , 0.65] 3.4E-04 0.14 [0.09 , 0.19] 3.3E-04 0.02 [0.00 , 0.04] 1.5E-01 387 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value F13a1 0.48 [0.24 , 0.72] 3.8E-03 0.12 [0.00 , 0.25] 1.3E-01 0.05 [0.00 , 0.10] 1.5E-01 Tlr8 0.48 [0.24 , 0.73] 4.2E-03 0.10 [-0.02 , 0.21] 2.0E-01 0.04 [-0.02 , 0.09] 2.7E-01 Csf1 0.48 [0.34 , 0.62] 1.0E-04 0.09 [-0.07 , 0.25] 4.0E-01 0.07 [0.03 , 0.10] 4.3E-03 Slamf1 0.48 [0.09 , 0.86] 4.8E-02 0.07 [-0.07 , 0.21] 4.2E-01 0.04 [-0.02 , 0.11] 2.7E-01 Nod2 0.47 [0.11 , 0.84] 3.9E-02 0.20 [0.04 , 0.36] 5.3E-02 0.07 [0.00 , 0.13] 1.0E-01 Rel 0.47 [0.34 , 0.60] 4.6E-05 0.20 [0.14 , 0.26] 1.3E-04 0.07 [0.05 , 0.09] 2.9E-05 Lilra5 0.47 [0.10 , 0.84] 4.5E-02 0.11 [-0.06 , 0.28] 3.0E-01 0.05 [-0.02 , 0.11] 2.9E-01 Ikzf1 0.46 [0.13 , 0.80] 2.9E-02 0.02 [-0.13 , 0.17] 8.3E-01 -0.01 [-0.06 , 0.04] 8.4E-01 Ifi27 0.46 [0.28 , 0.63] 6.3E-04 0.20 [0.12 , 0.28] 1.3E-03 0.06 [0.02 , 0.09] 9.9E-03 Ccl11 0.46 [0.10 , 0.81] 4.0E-02 0.31 [0.15 , 0.47] 6.5E-03 0.09 [0.02 , 0.15] 5.4E-02 Ms4a2 0.46 [0.05 , 0.87] 6.9E-02 0.19 [-0.01 , 0.39] 1.3E-01 n.d [n.d , n.d] n.d C5ar1 0.45 [0.17 , 0.74] 1.6E-02 0.13 [-0.02 , 0.28] 1.9E-01 0.03 [-0.03 , 0.08] 4.7E-01 Cd37 0.45 [0.13 , 0.77] 2.6E-02 0.15 [0.03 , 0.26] 5.6E-02 0.07 [0.03 , 0.11] 6.2E-03 Cd244 0.45 [0.20 , 0.70] 6.9E-03 0.05 [-0.15 , 0.24] 7.2E-01 0.01 [-0.04 , 0.07] 7.1E-01 Ccr9 0.45 [0.09 , 0.81] 4.8E-02 0.05 [-0.13 , 0.23] 7.0E-01 0.01 [-0.04 , 0.06] 7.5E-01 Il1rl1 0.44 [0.18 , 0.71] 1.2E-02 0.38 [0.29 , 0.47] 1.3E-05 0.18 [0.15 , 0.22] 1.4E-06 Cd3d 0.44 [0.15 , 0.72] 1.8E-02 0.11 [-0.01 , 0.22] 1.6E-01 0.01 [-0.04 , 0.05] 8.4E-01 388 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Itga4 0.44 [0.21 , 0.66] 4.5E-03 -0.01 [-0.09 , 0.07] 8.7E-01 0.01 [-0.02 , 0.03] 7.5E-01 Irgm2 0.43 [0.14 , 0.73] 2.3E-02 -0.12 [-0.28 , 0.04] 2.6E-01 -0.02 [-0.07 , 0.03] 5.1E-01 Tnfsf13 0.43 [0.15 , 0.71] 1.8E-02 0.16 [0.03 , 0.30] 6.2E-02 0.11 [0.06 , 0.16] 1.9E-03 Ciita 0.42 [0.13 , 0.71] 2.3E-02 0.06 [-0.09 , 0.21] 5.5E-01 0.06 [0.00 , 0.11] 9.4E-02 Lbp 0.42 [0.23 , 0.62] 2.4E-03 0.28 [0.18 , 0.39] 6.7E-04 0.08 [0.04 , 0.12] 6.5E-03 Ifit2 0.42 [0.04 , 0.81] 7.4E-02 -0.14 [-0.24 , -0.04] 3.2E-02 -0.06 [-0.09 , -0.04] 6.1E-04 Nfkb2 0.42 [0.11 , 0.73] 3.0E-02 0.10 [-0.04 , 0.24] 2.8E-01 0.06 [0.00 , 0.11] 8.8E-02 Casp3 0.42 [0.27 , 0.57] 4.9E-04 0.13 [0.08 , 0.19] 9.4E-04 0.06 [0.05 , 0.08] 1.3E-05 Mef2c 0.41 [0.23 , 0.59] 1.7E-03 -0.02 [-0.11 , 0.07] 7.2E-01 0.01 [-0.01 , 0.04] 4.7E-01 Irak3 0.40 [0.28 , 0.51] 8.8E-05 0.08 [0.02 , 0.15] 4.6E-02 0.05 [0.03 , 0.08] 3.0E-03 Cd44 0.40 [0.13 , 0.66] 2.0E-02 0.33 [0.20 , 0.45] 1.1E-03 0.11 [0.04 , 0.18] 1.8E-02 Il1rl2 0.39 [0.25 , 0.54] 5.7E-04 0.32 [0.24 , 0.39] 1.3E-05 0.12 [0.09 , 0.15] 8.7E-06 Ccrl2 0.39 [0.22 , 0.56] 1.6E-03 0.18 [0.11 , 0.25] 1.3E-03 0.10 [0.07 , 0.14] 3.2E-04 Tfrc 0.39 [0.21 , 0.57] 2.1E-03 0.14 [0.02 , 0.26] 8.4E-02 0.08 [0.05 , 0.10] 3.2E-04 Atg10 0.39 [0.26 , 0.52] 2.1E-04 0.07 [0.00 , 0.15] 1.3E-01 0.03 [0.01 , 0.06] 4.9E-02 Pml 0.39 [0.09 , 0.68] 3.7E-02 -0.01 [-0.07 , 0.05] 8.3E-01 0.00 [-0.02 , 0.02] 9.6E-01 Gbp5 0.38 [0.01 , 0.75] 8.8E-02 -0.20 [-0.34 , -0.05] 4.2E-02 -0.06 [-0.11 , -0.01] 8.0E-02 389 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Pla2g1b 0.38 [0.17 , 0.59] 7.9E-03 0.40 [0.30 , 0.50] 3.4E-05 0.17 [0.13 , 0.20] 7.7E-06 C4b 0.36 [0.16 , 0.57] 8.8E-03 0.15 [0.06 , 0.24] 2.0E-02 0.06 [0.03 , 0.10] 6.9E-03 Il2rg 0.36 [0.15 , 0.58] 1.1E-02 0.12 [0.04 , 0.20] 2.4E-02 0.03 [0.00 , 0.06] 1.2E-01 Lif 0.36 [-0.11 , 0.83] 2.1E-01 0.08 [-0.13 , 0.30] 5.6E-01 -0.04 [-0.11 , 0.03] 3.8E-01 Pax5 0.36 [-0.16 , 0.88] 2.5E-01 -0.09 [-0.35 , 0.16] 5.8E-01 -0.02 [-0.09 , 0.05] 7.2E-01 Psmb10 0.36 [0.02 , 0.69] 8.2E-02 0.08 [-0.05 , 0.21] 3.3E-01 0.03 [-0.02 , 0.09] 2.9E-01 Ctsh 0.36 [0.16 , 0.55] 6.8E-03 0.21 [0.11 , 0.31] 3.3E-03 0.10 [0.06 , 0.14] 1.4E-03 Lyn 0.35 [0.19 , 0.52] 2.5E-03 0.08 [-0.01 , 0.16] 1.9E-01 0.04 [0.01 , 0.07] 5.6E-02 Mr1 0.34 [0.21 , 0.48] 8.4E-04 0.14 [0.07 , 0.20] 2.7E-03 0.06 [0.04 , 0.09] 4.7E-04 Il13ra1 0.34 [0.30 , 0.38] 8.1E-08 0.10 [0.06 , 0.14] 1.4E-03 0.03 [0.02 , 0.05] 6.5E-04 Tgfbr1 0.34 [0.24 , 0.44] 6.5E-05 0.11 [0.08 , 0.14] 1.0E-04 0.05 [0.04 , 0.06] 6.3E-05 Ceacam1 0.34 [0.24 , 0.44] 1.2E-04 0.17 [0.12 , 0.21] 4.7E-05 0.06 [0.04 , 0.07] 6.0E-05 Flt3l 0.34 [0.12 , 0.56] 1.7E-02 0.15 [0.04 , 0.26] 3.3E-02 0.12 [0.08 , 0.17] 4.1E-04 Cd276 0.33 [0.03 , 0.63] 6.9E-02 0.30 [0.14 , 0.46] 7.5E-03 0.11 [0.06 , 0.16] 2.6E-03 Tnfsf10 0.33 [-0.02 , 0.68] 1.2E-01 0.02 [-0.26 , 0.30] 9.1E-01 0.05 [-0.03 , 0.14] 3.5E-01 H2-Eb1 0.33 [-0.17 , 0.83] 2.8E-01 0.41 [0.16 , 0.66] 1.7E-02 0.15 [0.05 , 0.24] 1.7E-02 Casp8 0.32 [0.20 , 0.44] 5.4E-04 0.13 [0.08 , 0.18] 8.7E-04 0.06 [0.04 , 0.08] 2.5E-04 390 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Masp2 0.32 [-0.05 , 0.69] 1.5E-01 0.16 [-0.06 , 0.37] 2.6E-01 0.07 [-0.02 , 0.16] 2.0E-01 Tlr4 0.32 [0.23 , 0.41] 3.8E-05 0.03 [-0.02 , 0.08] 3.2E-01 0.04 [0.03 , 0.05] 3.2E-04 Ctsw 0.32 [0.12 , 0.51] 1.3E-02 -0.01 [-0.08 , 0.06] 8.9E-01 0.03 [0.00 , 0.06] 7.6E-02 Tab1 0.31 [0.08 , 0.54] 3.4E-02 0.23 [0.12 , 0.34] 4.5E-03 0.09 [0.04 , 0.13] 7.1E-03 Tank 0.31 [0.18 , 0.44] 1.4E-03 0.10 [0.05 , 0.16] 7.2E-03 0.05 [0.03 , 0.07] 3.9E-04 Tmem173 0.31 [0.04 , 0.58] 6.5E-02 -0.07 [-0.17 , 0.03] 2.8E-01 -0.03 [-0.06 , 0.01] 2.0E-01 Tapbp 0.31 [0.10 , 0.51] 1.9E-02 0.10 [0.02 , 0.18] 6.2E-02 0.01 [-0.02 , 0.05] 5.5E-01 Gzmb 0.30 [-0.04 , 0.65] 1.5E-01 0.12 [-0.04 , 0.27] 2.7E-01 -0.05 [-0.11 , 0.01] 2.2E-01 Cd1d1 0.30 [0.16 , 0.45] 3.3E-03 0.11 [0.03 , 0.18] 3.4E-02 0.09 [0.06 , 0.11] 1.4E-04 Maf 0.30 [0.05 , 0.55] 5.3E-02 0.06 [-0.05 , 0.18] 4.1E-01 0.06 [0.02 , 0.10] 3.7E-02 Jak2 0.30 [0.22 , 0.37] 3.2E-05 0.08 [0.04 , 0.12] 7.5E-03 0.04 [0.02 , 0.05] 6.7E-04 Ctsl 0.29 [0.14 , 0.45] 5.2E-03 0.06 [-0.02 , 0.14] 2.6E-01 0.04 [0.01 , 0.08] 3.9E-02 Cd38 0.29 [0.14 , 0.44] 4.3E-03 0.08 [0.00 , 0.15] 1.2E-01 0.03 [0.00 , 0.05] 7.1E-02 Spn 0.28 [0.00 , 0.56] 1.0E-01 0.03 [-0.09 , 0.15] 7.2E-01 -0.01 [-0.06 , 0.04] 7.4E-01 Alcam 0.27 [0.21 , 0.33] 1.2E-05 0.14 [0.10 , 0.17] 1.2E-05 0.05 [0.04 , 0.06] 1.9E-06 Bax 0.27 [0.15 , 0.38] 1.6E-03 0.04 [-0.02 , 0.11] 3.4E-01 0.02 [-0.01 , 0.04] 3.0E-01 Hck 0.27 [0.08 , 0.45] 2.4E-02 -0.03 [-0.17 , 0.11] 7.7E-01 0.01 [-0.02 , 0.05] 5.1E-01 391 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cklf 0.27 [0.05 , 0.49] 5.2E-02 0.01 [-0.09 , 0.12] 8.6E-01 0.02 [-0.03 , 0.06] 5.6E-01 Tgfb1 0.26 [0.12 , 0.41] 6.8E-03 0.12 [0.05 , 0.19] 1.4E-02 0.06 [0.04 , 0.09] 1.5E-03 Mrc1 0.26 [0.00 , 0.52] 9.5E-02 0.19 [0.08 , 0.30] 1.2E-02 0.07 [0.03 , 0.11] 1.0E-02 Hif1a 0.26 [0.17 , 0.35] 3.4E-04 0.04 [-0.01 , 0.09] 2.1E-01 0.01 [0.00 , 0.02] 2.8E-01 St6gal1 0.26 [0.05 , 0.47] 4.8E-02 0.14 [0.05 , 0.22] 1.6E-02 0.07 [0.04 , 0.10] 1.9E-03 Cd69 0.26 [0.02 , 0.49] 7.6E-02 -0.05 [-0.15 , 0.05] 4.2E-01 0.03 [-0.01 , 0.07] 1.9E-01 Cd247 0.26 [-0.03 , 0.54] 1.4E-01 0.11 [0.00 , 0.22] 1.2E-01 0.03 [-0.01 , 0.07] 2.1E-01 Il18 0.26 [-0.10 , 0.61] 2.4E-01 -0.04 [-0.21 , 0.13] 7.2E-01 -0.01 [-0.07 , 0.06] 8.5E-01 Btla 0.25 [-0.16 , 0.66] 3.0E-01 0.16 [0.01 , 0.32] 1.1E-01 0.07 [0.03 , 0.12] 1.6E-02 Thy1 0.25 [0.08 , 0.42] 2.0E-02 0.03 [-0.04 , 0.09] 4.9E-01 -0.01 [-0.04 , 0.01] 3.3E-01 Stat4 0.25 [0.02 , 0.47] 7.0E-02 0.16 [0.07 , 0.26] 1.0E-02 0.06 [0.03 , 0.08] 3.4E-03 Irak2 0.24 [0.12 , 0.37] 4.5E-03 0.21 [0.14 , 0.29] 4.5E-04 0.07 [0.05 , 0.09] 2.2E-04 Ccl25 0.24 [0.06 , 0.42] 3.3E-02 0.21 [0.09 , 0.33] 1.2E-02 0.05 [0.00 , 0.10] 9.8E-02 Traf3 0.24 [0.04 , 0.44] 5.0E-02 0.07 [-0.03 , 0.17] 2.7E-01 0.06 [0.03 , 0.10] 1.2E-02 Cd63 0.24 [0.13 , 0.36] 3.1E-03 0.14 [0.09 , 0.19] 7.4E-04 0.06 [0.04 , 0.09] 7.5E-04 Atg7 0.24 [0.15 , 0.32] 4.1E-04 0.12 [0.07 , 0.16] 1.3E-03 0.04 [0.03 , 0.06] 1.5E-03 Cd55 0.23 [0.12 , 0.34] 2.8E-03 0.09 [0.05 , 0.14] 3.1E-03 0.03 [0.01 , 0.04] 6.1E-03 392 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Psmb7 0.23 [0.13 , 0.33] 1.9E-03 0.06 [0.00 , 0.12] 1.3E-01 0.00 [-0.02 , 0.03] 7.9E-01 Cd22 0.23 [-0.27 , 0.72] 4.5E-01 -0.06 [-0.34 , 0.21] 7.3E-01 -0.02 [-0.12 , 0.07] 7.5E-01 Traf2 0.23 [0.14 , 0.31] 5.5E-04 -0.02 [-0.09 , 0.05] 7.2E-01 0.03 [0.01 , 0.04] 3.0E-02 Prdm1 0.22 [-0.12 , 0.56] 2.8E-01 0.19 [0.05 , 0.32] 3.7E-02 0.06 [0.01 , 0.11] 7.6E-02 Cxcl12 0.22 [-0.04 , 0.47] 1.6E-01 0.05 [-0.05 , 0.15] 4.1E-01 -0.02 [-0.07 , 0.03] 4.4E-01 C2 0.21 [0.04 , 0.39] 5.1E-02 -0.01 [-0.10 , 0.08] 9.0E-01 -0.01 [-0.04 , 0.02] 6.8E-01 Ifi35 0.21 [0.08 , 0.35] 1.6E-02 0.03 [0.01 , 0.05] 4.8E-02 -0.02 [-0.03 , -0.01] 6.3E-03 Il17ra 0.21 [0.04 , 0.38] 4.5E-02 0.08 [-0.01 , 0.17] 1.7E-01 0.05 [0.02 , 0.08] 2.3E-02 Irak4 0.21 [0.09 , 0.33] 8.5E-03 0.01 [-0.06 , 0.08] 8.4E-01 0.01 [-0.01 , 0.04] 4.0E-01 Lamp1 0.21 [0.13 , 0.29] 7.6E-04 0.15 [0.12 , 0.18] 1.2E-06 0.07 [0.06 , 0.08] 2.6E-07 Inpp5d 0.21 [-0.01 , 0.42] 1.1E-01 0.27 [0.14 , 0.39] 3.3E-03 0.11 [0.08 , 0.14] 3.0E-05 Sh2d1b1 0.21 [-0.08 , 0.50] 2.4E-01 0.11 [-0.01 , 0.22] 1.5E-01 0.04 [-0.01 , 0.08] 1.8E-01 Nfatc3 0.20 [-0.03 , 0.43] 1.5E-01 -0.01 [-0.06 , 0.04] 8.3E-01 0.02 [0.01 , 0.03] 3.2E-02 Psma2 0.20 [0.10 , 0.29] 3.0E-03 0.04 [-0.02 , 0.09] 3.3E-01 0.00 [-0.03 , 0.02] 9.0E-01 Psen2 0.20 [-0.07 , 0.46] 2.2E-01 0.19 [0.05 , 0.33] 3.9E-02 0.08 [0.03 , 0.13] 1.3E-02 Serping1 0.19 [0.06 , 0.33] 2.8E-02 0.14 [0.08 , 0.20] 2.6E-03 0.05 [0.03 , 0.08] 2.5E-03 Cd40lg 0.19 [-0.16 , 0.55] 3.7E-01 -0.02 [-0.19 , 0.15] 8.6E-01 -0.05 [-0.12 , 0.03] 3.6E-01 393 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il12a 0.19 [-0.05 , 0.44] 1.9E-01 0.04 [-0.11 , 0.19] 6.8E-01 0.00 [-0.04 , 0.05] 9.4E-01 Map2k1 0.19 [0.11 , 0.27] 1.1E-03 0.05 [0.02 , 0.09] 2.4E-02 0.02 [0.01 , 0.03] 3.6E-02 Icam1 0.19 [0.03 , 0.35] 5.7E-02 0.00 [-0.07 , 0.08] 9.2E-01 0.00 [-0.02 , 0.03] 8.1E-01 Col1a1 0.18 [-0.12 , 0.48] 3.3E-01 0.07 [-0.07 , 0.22] 4.5E-01 0.01 [-0.05 , 0.07] 8.0E-01 Ccl5 0.18 [0.01 , 0.35] 8.7E-02 -0.06 [-0.16 , 0.03] 2.9E-01 -0.04 [-0.08 , 0.00] 1.0E-01 Prkcd 0.17 [0.03 , 0.31] 5.5E-02 0.18 [0.11 , 0.25] 8.5E-04 0.10 [0.07 , 0.12] 3.1E-05 Hmgb1 0.16 [0.03 , 0.29] 4.4E-02 -0.01 [-0.06 , 0.04] 7.6E-01 -0.01 [-0.03 , 0.01] 4.2E-01 Nfatc2 0.16 [-0.19 , 0.52] 4.5E-01 0.06 [-0.09 , 0.22] 5.3E-01 0.03 [-0.04 , 0.11] 4.7E-01 Colec12 0.16 [-0.03 , 0.35] 1.6E-01 0.08 [0.02 , 0.15] 5.5E-02 0.01 [-0.02 , 0.04] 5.4E-01 Il7r 0.16 [-0.12 , 0.44] 3.5E-01 0.21 [0.06 , 0.36] 3.7E-02 0.08 [0.04 , 0.13] 8.2E-03 Il22ra1 0.16 [-0.13 , 0.44] 3.6E-01 0.14 [-0.02 , 0.31] 1.8E-01 0.06 [0.02 , 0.10] 3.8E-02 Camp 0.16 [-0.21 , 0.52] 4.9E-01 0.10 [-0.12 , 0.31] 5.0E-01 0.14 [0.05 , 0.22] 2.2E-02 Adora2a 0.15 [-0.12 , 0.42] 3.5E-01 -0.02 [-0.16 , 0.12] 8.1E-01 0.01 [-0.04 , 0.06] 7.9E-01 Ifnar1 0.15 [0.01 , 0.29] 7.2E-02 0.05 [-0.02 , 0.12] 2.6E-01 0.04 [0.01 , 0.07] 2.1E-02 Myc 0.15 [-0.09 , 0.39] 3.0E-01 -0.07 [-0.17 , 0.04] 3.3E-01 0.02 [-0.01 , 0.06] 3.4E-01 Sell 0.14 [0.01 , 0.28] 8.6E-02 -0.08 [-0.19 , 0.04] 3.0E-01 -0.01 [-0.04 , 0.01] 4.0E-01 Epcam 0.14 [-0.01 , 0.29] 1.2E-01 0.09 [0.01 , 0.17] 7.9E-02 0.02 [-0.01 , 0.05] 3.3E-01 394 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd27 0.14 [-0.16 , 0.43] 4.5E-01 -0.06 [-0.23 , 0.12] 6.2E-01 -0.01 [-0.08 , 0.06] 7.5E-01 Pla2g6 0.13 [0.03 , 0.24] 4.2E-02 0.14 [0.07 , 0.21] 5.8E-03 0.04 [0.01 , 0.06] 1.2E-02 Tbx21 0.13 [-0.27 , 0.54] 5.9E-01 -0.05 [-0.26 , 0.16] 7.2E-01 -0.02 [-0.10 , 0.05] 6.6E-01 Ccr7 0.13 [-0.06 , 0.32] 2.4E-01 0.16 [0.07 , 0.25] 9.0E-03 0.10 [0.07 , 0.14] 4.4E-04 Lamp2 0.13 [0.04 , 0.22] 2.3E-02 0.01 [-0.04 , 0.06] 6.9E-01 0.01 [-0.02 , 0.03] 6.8E-01 Fez1 0.13 [-0.16 , 0.41] 4.6E-01 0.07 [-0.09 , 0.24] 4.9E-01 0.03 [-0.03 , 0.08] 4.4E-01 Runx1 0.12 [0.03 , 0.22] 3.4E-02 0.02 [-0.02 , 0.06] 5.4E-01 0.01 [-0.01 , 0.02] 5.1E-01 Ifitm2 0.12 [0.01 , 0.23] 6.4E-02 0.11 [0.06 , 0.17] 6.0E-03 0.04 [0.02 , 0.06] 8.5E-03 Atm 0.12 [-0.01 , 0.25] 1.2E-01 -0.06 [-0.15 , 0.02] 2.7E-01 0.00 [-0.03 , 0.02] 8.2E-01 Serpinb2 0.12 [-0.75 , 0.98] 8.3E-01 -0.08 [-0.53 , 0.38] 8.0E-01 0.01 [-0.18 , 0.19] 9.6E-01 Pvrl2 0.12 [-0.06 , 0.30] 2.8E-01 0.15 [0.06 , 0.23] 1.7E-02 0.08 [0.04 , 0.11] 2.1E-03 Nfkb1 0.12 [-0.04 , 0.27] 2.3E-01 0.09 [0.01 , 0.18] 9.0E-02 0.06 [0.03 , 0.09] 5.9E-03 Jak3 0.11 [-0.31 , 0.54] 6.6E-01 0.10 [-0.10 , 0.30] 4.3E-01 0.06 [-0.02 , 0.14] 2.6E-01 Cd40 0.11 [-0.01 , 0.22] 1.2E-01 -0.05 [-0.14 , 0.05] 4.5E-01 0.03 [0.00 , 0.05] 1.2E-01 Mapk1 0.11 [0.04 , 0.18] 2.0E-02 0.01 [-0.03 , 0.05] 7.8E-01 0.00 [-0.01 , 0.02] 9.5E-01 Tnfsf18 0.10 [-0.35 , 0.55] 7.2E-01 -0.20 [-0.39 , -0.01] 1.0E-01 -0.13 [-0.21 , -0.04] 2.2E-02 Il3ra 0.10 [-0.14 , 0.33] 5.0E-01 0.16 [0.04 , 0.27] 3.9E-02 0.06 [0.02 , 0.11] 3.9E-02 395 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Psen1 0.10 [0.02 , 0.17] 3.5E-02 0.02 [-0.02 , 0.06] 4.9E-01 0.02 [0.01 , 0.03] 1.7E-02 Card11 0.10 [-0.14 , 0.33] 5.1E-01 -0.09 [-0.23 , 0.04] 2.9E-01 -0.02 [-0.06 , 0.02] 4.0E-01 Irf1 0.09 [-0.04 , 0.22] 2.4E-01 -0.05 [-0.17 , 0.06] 4.7E-01 -0.01 [-0.05 , 0.02] 5.7E-01 Tfeb 0.09 [-0.12 , 0.30] 4.8E-01 0.11 [0.01 , 0.21] 9.2E-02 0.08 [0.04 , 0.12] 3.1E-03 Lrrn3 0.09 [-0.17 , 0.34] 5.8E-01 -0.06 [-0.26 , 0.14] 6.6E-01 0.03 [-0.02 , 0.09] 3.1E-01 Xbp1 0.08 [-0.07 , 0.24] 3.7E-01 -0.14 [-0.20 , -0.07] 5.1E-03 -0.05 [-0.07 , -0.04] 1.9E-04 Zfp13 0.08 [-0.15 , 0.31] 5.6E-01 0.06 [-0.07 , 0.18] 4.7E-01 0.06 [0.03 , 0.09] 7.5E-03 Gata3 0.08 [-0.12 , 0.27] 5.2E-01 0.03 [-0.17 , 0.23] 8.3E-01 0.02 [-0.03 , 0.07] 4.5E-01 Cd8a 0.07 [-0.23 , 0.37] 7.0E-01 0.01 [-0.12 , 0.14] 9.2E-01 -0.05 [-0.11 , 0.01] 1.9E-01 Cdh1 0.07 [-0.06 , 0.20] 3.6E-01 0.09 [0.05 , 0.13] 2.1E-03 0.02 [0.01 , 0.04] 3.2E-02 Cd8b1 0.07 [-0.23 , 0.37] 7.1E-01 -0.02 [-0.16 , 0.11] 8.0E-01 -0.07 [-0.17 , 0.02] 2.4E-01 Hras 0.07 [-0.34 , 0.47] 7.9E-01 -0.09 [-0.25 , 0.06] 3.6E-01 0.02 [-0.06 , 0.09] 7.8E-01 Map3k5 0.07 [-0.04 , 0.17] 3.1E-01 0.02 [-0.03 , 0.07] 6.3E-01 0.02 [0.00 , 0.04] 1.4E-01 Tlr3 0.06 [-0.10 , 0.23] 5.3E-01 0.07 [-0.01 , 0.15] 2.0E-01 0.06 [0.03 , 0.09] 9.4E-03 Atg5 0.06 [-0.16 , 0.28] 6.5E-01 0.10 [-0.02 , 0.23] 2.2E-01 0.06 [0.02 , 0.10] 3.7E-02 Il1r1 0.06 [-0.02 , 0.14] 2.2E-01 -0.02 [-0.06 , 0.02] 4.9E-01 0.00 [-0.01 , 0.02] 7.5E-01 Il1rap 0.06 [-0.07 , 0.19] 4.6E-01 0.07 [0.03 , 0.12] 2.0E-02 0.04 [0.02 , 0.05] 1.8E-03 396 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd1d2 0.06 [-0.19 , 0.31] 7.1E-01 -0.10 [-0.21 , 0.02] 2.0E-01 0.01 [-0.03 , 0.06] 6.5E-01 C7 0.06 [-0.14 , 0.25] 6.3E-01 -0.01 [-0.08 , 0.07] 9.0E-01 0.01 [-0.01 , 0.04] 3.9E-01 Ripk2 0.06 [-0.06 , 0.17] 4.3E-01 -0.02 [-0.10 , 0.06] 7.2E-01 0.02 [0.00 , 0.04] 2.2E-01 Kit 0.05 [-0.27 , 0.38] 7.9E-01 0.08 [-0.15 , 0.30] 6.1E-01 0.08 [0.01 , 0.15] 7.6E-02 Il16 0.05 [-0.11 , 0.21] 5.8E-01 0.04 [-0.04 , 0.13] 4.7E-01 0.03 [0.00 , 0.05] 1.2E-01 Cd9 0.05 [-0.21 , 0.31] 7.5E-01 0.07 [-0.05 , 0.20] 3.6E-01 0.04 [-0.01 , 0.09] 1.6E-01 Itch 0.05 [-0.06 , 0.15] 4.5E-01 -0.02 [-0.08 , 0.04] 6.4E-01 -0.01 [-0.03 , 0.01] 5.3E-01 Cd34 0.05 [-0.08 , 0.17] 5.4E-01 0.00 [-0.05 , 0.06] 9.7E-01 -0.03 [-0.05 , -0.01] 7.8E-03 Tnfrsf11b 0.05 [-0.38 , 0.47] 8.7E-01 n.d [n.d , n.d] n.d 0.04 [-0.03 , 0.10] 3.6E-01 Rorc 0.04 [-0.10 , 0.19] 6.1E-01 -0.04 [-0.14 , 0.06] 5.5E-01 -0.02 [-0.04 , 0.01] 3.6E-01 Stat6 0.04 [-0.03 , 0.11] 3.0E-01 0.01 [-0.02 , 0.05] 5.0E-01 0.02 [0.01 , 0.03] 2.7E-03 Creb1 0.04 [-0.11 , 0.19] 6.5E-01 -0.12 [-0.22 , -0.02] 6.2E-02 -0.03 [-0.05 , -0.01] 6.0E-02 Map2k4 0.04 [-0.02 , 0.10] 2.9E-01 0.04 [0.00 , 0.08] 1.6E-01 0.01 [0.00 , 0.03] 6.5E-02 Entpd1 0.04 [-0.08 , 0.15] 5.7E-01 0.14 [0.02 , 0.27] 7.9E-02 0.00 [-0.02 , 0.02] 7.3E-01 Ncam1 0.03 [-0.30 , 0.36] 8.9E-01 0.05 [-0.11 , 0.21] 6.3E-01 0.06 [-0.02 , 0.13] 2.1E-01 Ilf3 0.03 [-0.10 , 0.16] 7.2E-01 0.01 [-0.04 , 0.07] 7.7E-01 0.00 [-0.03 , 0.02] 9.1E-01 Vim 0.03 [-0.17 , 0.22] 8.2E-01 0.16 [0.05 , 0.27] 3.3E-02 0.07 [0.04 , 0.11] 4.0E-03 397 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value C1qbp 0.03 [-0.04 , 0.09] 5.2E-01 -0.03 [-0.06 , 0.00] 1.7E-01 -0.02 [-0.04 , -0.01] 5.2E-03 Cx3cl1 0.02 [-0.24 , 0.29] 9.0E-01 0.06 [-0.09 , 0.21] 5.4E-01 0.02 [-0.03 , 0.08] 4.6E-01 Il6 0.02 [-0.68 , 0.72] 9.6E-01 -0.39 [-0.68 , -0.10] 4.4E-02 -0.15 [-0.28 , -0.03] 5.6E-02 Fadd 0.02 [-0.18 , 0.22] 8.8E-01 -0.02 [-0.12 , 0.07] 7.2E-01 0.02 [-0.01 , 0.06] 2.9E-01 Anp32b 0.02 [-0.09 , 0.13] 7.9E-01 -0.01 [-0.05 , 0.03] 6.5E-01 -0.01 [-0.03 , 0.00] 1.7E-01 Il6st 0.02 [-0.13 , 0.17] 8.5E-01 -0.04 [-0.11 , 0.03] 3.8E-01 0.00 [-0.03 , 0.03] 9.2E-01 Ikbkb 0.02 [-0.05 , 0.08] 6.6E-01 -0.02 [-0.04 , 0.01] 3.5E-01 -0.01 [-0.02 , 0.00] 1.9E-01 Hcst 0.02 [-0.49 , 0.53] 9.5E-01 -0.09 [-0.37 , 0.19] 6.3E-01 0.04 [-0.04 , 0.12] 4.4E-01 Timd4 0.02 [-0.42 , 0.46] 9.5E-01 -0.13 [-0.38 , 0.12] 4.3E-01 -0.12 [-0.22 , -0.02] 5.5E-02 Nfatc1 0.02 [-0.21 , 0.25] 9.1E-01 0.10 [-0.01 , 0.21] 1.6E-01 0.03 [-0.01 , 0.07] 2.1E-01 Cd3eap 0.02 [-0.32 , 0.35] 9.4E-01 -0.07 [-0.23 , 0.09] 4.8E-01 0.05 [-0.01 , 0.11] 1.8E-01 Elk1 0.02 [-0.24 , 0.27] 9.2E-01 -0.04 [-0.18 , 0.10] 7.1E-01 -0.01 [-0.07 , 0.05] 8.6E-01 Bmi1 0.01 [-0.08 , 0.11] 8.4E-01 -0.07 [-0.13 , -0.02] 3.7E-02 -0.03 [-0.05 , -0.02] 4.9E-03 Map2k2 0.01 [-0.07 , 0.09] 8.4E-01 0.00 [-0.03 , 0.04] 8.5E-01 0.00 [-0.02 , 0.01] 5.7E-01 Litaf 0.01 [-0.15 , 0.17] 9.2E-01 0.05 [-0.04 , 0.14] 4.2E-01 0.03 [0.00 , 0.06] 1.6E-01 Cfh 0.01 [-0.11 , 0.13] 9.2E-01 -0.01 [-0.06 , 0.03] 6.1E-01 -0.01 [-0.03 , 0.01] 3.5E-01 Tnfrsf14 0.01 [-0.32 , 0.34] 9.7E-01 0.09 [-0.07 , 0.25] 3.8E-01 0.04 [-0.02 , 0.10] 3.3E-01 398 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ewsr1 0.01 [-0.05 , 0.06] 8.9E-01 -0.05 [-0.08 , -0.02] 8.9E-03 0.00 [-0.01 , 0.01] 4.9E-01 Chuk 0.01 [-0.06 , 0.07] 9.0E-01 -0.04 [-0.08 , 0.00] 1.3E-01 -0.01 [-0.02 , 0.00] 2.8E-01 Vhl 0.00 [-0.19 , 0.20] 9.7E-01 0.05 [-0.03 , 0.14] 3.5E-01 0.03 [0.00 , 0.07] 9.3E-02 Tap2 0.00 [-0.29 , 0.28] 9.9E-01 0.00 [-0.11 , 0.11] 9.4E-01 0.03 [-0.02 , 0.07] 3.3E-01 Yy1 0.00 [-0.08 , 0.07] 9.4E-01 0.00 [-0.05 , 0.04] 9.0E-01 0.01 [-0.01 , 0.02] 3.6E-01 Axl 0.00 [-0.12 , 0.11] 9.5E-01 0.02 [-0.02 , 0.06] 4.9E-01 0.01 [-0.01 , 0.03] 2.6E-01 Pparg 0.00 [-0.26 , 0.25] 9.7E-01 -0.03 [-0.15 , 0.08] 6.7E-01 0.05 [0.00 , 0.09] 1.1E-01 Atg16l1 -0.01 [-0.14 , 0.13] 9.4E-01 -0.07 [-0.14 , 0.00] 1.2E-01 -0.01 [-0.04 , 0.01] 4.0E-01 Irf2 -0.01 [-0.10 , 0.08] 9.0E-01 -0.09 [-0.16 , -0.02] 6.3E-02 -0.01 [-0.03 , 0.01] 3.0E-01 Anxa1 -0.01 [-0.18 , 0.16] 9.2E-01 -0.07 [-0.14 , 0.00] 1.1E-01 -0.03 [-0.05 , 0.00] 1.6E-01 Irf3 -0.02 [-0.12 , 0.09] 8.1E-01 -0.02 [-0.08 , 0.03] 5.0E-01 0.01 [-0.01 , 0.02] 6.3E-01 Stat5b -0.02 [-0.12 , 0.09] 8.1E-01 -0.04 [-0.11 , 0.02] 3.1E-01 0.00 [-0.02 , 0.02] 7.8E-01 Ythdf2 -0.02 [-0.08 , 0.05] 6.7E-01 -0.04 [-0.08 , -0.01] 4.3E-02 0.00 [-0.02 , 0.01] 5.0E-01 Smn1 -0.02 [-0.09 , 0.06] 7.1E-01 -0.06 [-0.11 , -0.01] 6.3E-02 -0.02 [-0.04 , -0.01] 1.3E-02 Mavs -0.02 [-0.21 , 0.17] 8.8E-01 0.08 [-0.01 , 0.17] 1.7E-01 0.05 [0.02 , 0.08] 2.1E-02 Bcl10 -0.02 [-0.15 , 0.10] 7.6E-01 -0.01 [-0.06 , 0.04] 7.6E-01 0.00 [-0.03 , 0.02] 8.8E-01 Atg12 -0.02 [-0.16 , 0.12] 7.9E-01 0.02 [-0.05 , 0.08] 7.2E-01 0.01 [-0.01 , 0.04] 3.6E-01 399 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Itgb3 -0.03 [-0.33 , 0.28] 9.0E-01 0.15 [0.00 , 0.30] 1.1E-01 0.04 [-0.01 , 0.10] 2.3E-01 Cd47 -0.03 [-0.10 , 0.04] 5.1E-01 0.03 [-0.01 , 0.07] 2.4E-01 0.02 [0.00 , 0.03] 8.2E-02 Cd164 -0.03 [-0.12 , 0.06] 6.0E-01 -0.02 [-0.03 , 0.00] 1.6E-01 0.00 [-0.01 , 0.00] 3.9E-01 Itk -0.03 [-0.24 , 0.17] 8.1E-01 -0.02 [-0.15 , 0.10] 8.0E-01 -0.05 [-0.11 , 0.01] 2.2E-01 Mapk14 -0.03 [-0.12 , 0.05] 5.4E-01 -0.05 [-0.11 , 0.02] 2.8E-01 -0.01 [-0.03 , 0.02] 6.7E-01 Myd88 -0.04 [-0.15 , 0.08] 6.0E-01 0.00 [-0.05 , 0.06] 9.3E-01 0.01 [-0.01 , 0.03] 5.2E-01 Cd36 -0.04 [-0.23 , 0.15] 7.4E-01 -0.03 [-0.09 , 0.03] 4.2E-01 -0.01 [-0.03 , 0.01] 3.8E-01 Smad2 -0.04 [-0.14 , 0.05] 4.7E-01 0.05 [0.00 , 0.09] 1.5E-01 0.03 [0.01 , 0.05] 5.7E-02 Tfe3 -0.04 [-0.22 , 0.13] 6.8E-01 0.04 [-0.04 , 0.11] 4.8E-01 0.04 [0.01 , 0.07] 3.5E-02 Jam3 -0.04 [-0.18 , 0.09] 5.8E-01 -0.03 [-0.09 , 0.03] 4.0E-01 -0.05 [-0.07 , -0.04] 7.4E-05 Atf1 -0.05 [-0.18 , 0.08] 5.6E-01 -0.05 [-0.12 , 0.01] 2.3E-01 -0.02 [-0.04 , 0.00] 2.1E-01 Rps6 -0.05 [-0.18 , 0.08] 5.5E-01 -0.04 [-0.11 , 0.03] 3.9E-01 -0.02 [-0.05 , 0.00] 1.8E-01 Nup107 -0.05 [-0.19 , 0.09] 5.4E-01 -0.04 [-0.09 , 0.01] 2.3E-01 -0.02 [-0.04 , 0.00] 8.8E-02 Psmd7 -0.05 [-0.15 , 0.04] 3.5E-01 -0.12 [-0.17 , -0.07] 1.9E-03 -0.04 [-0.06 , -0.03] 9.7E-04 Nrp1 -0.06 [-0.18 , 0.06] 4.1E-01 -0.11 [-0.21 , -0.01] 8.5E-02 -0.02 [-0.04 , 0.00] 1.8E-01 Sigirr -0.06 [-0.17 , 0.05] 3.8E-01 0.01 [-0.03 , 0.06] 6.8E-01 0.02 [0.01 , 0.04] 2.6E-02 Tgfbr2 -0.06 [-0.16 , 0.04] 3.2E-01 -0.01 [-0.04 , 0.03] 8.3E-01 0.02 [0.00 , 0.03] 1.2E-01 400 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd2 -0.06 [-0.31 , 0.18] 6.6E-01 -0.07 [-0.17 , 0.04] 3.6E-01 -0.02 [-0.05 , 0.02] 5.0E-01 Jak1 -0.07 [-0.15 , 0.02] 1.8E-01 -0.04 [-0.07 , -0.01] 3.1E-02 -0.02 [-0.04 , -0.01] 1.5E-02 Nos2 -0.07 [-0.48 , 0.34] 7.9E-01 -0.09 [-0.26 , 0.08] 4.1E-01 0.00 [-0.06 , 0.07] 9.2E-01 Itga2 -0.07 [-0.26 , 0.12] 5.5E-01 -0.05 [-0.15 , 0.06] 5.0E-01 0.00 [-0.05 , 0.04] 9.0E-01 Map3k1 -0.07 [-0.23 , 0.09] 4.7E-01 -0.08 [-0.17 , 0.01] 1.6E-01 -0.01 [-0.04 , 0.01] 4.6E-01 Atf2 -0.08 [-0.21 , 0.04] 2.9E-01 -0.06 [-0.12 , -0.01] 8.4E-02 -0.01 [-0.03 , 0.01] 3.8E-01 Igf2r -0.08 [-0.24 , 0.08] 4.0E-01 0.04 [-0.05 , 0.12] 5.1E-01 0.02 [-0.02 , 0.05] 4.0E-01 Nfkbia -0.08 [-0.35 , 0.18] 6.1E-01 0.04 [-0.10 , 0.18] 6.5E-01 0.06 [0.01 , 0.10] 6.4E-02 Il18rap -0.09 [-0.25 , 0.07] 3.6E-01 0.14 [0.02 , 0.26] 8.5E-02 0.05 [0.01 , 0.10] 6.4E-02 Mill2 -0.09 [-0.28 , 0.10] 4.5E-01 -0.04 [-0.10 , 0.03] 3.8E-01 -0.03 [-0.06 , 0.00] 1.5E-01 Mfge8 -0.09 [-0.23 , 0.05] 3.1E-01 -0.04 [-0.11 , 0.02] 3.2E-01 0.00 [-0.03 , 0.02] 7.9E-01 Eomes -0.10 [-0.34 , 0.15] 5.1E-01 -0.33 [-0.47 , -0.18] 2.3E-03 -0.12 [-0.17 , -0.07] 9.7E-04 Gzma -0.10 [-0.36 , 0.16] 5.3E-01 -0.22 [-0.34 , -0.09] 1.5E-02 -0.10 [-0.15 , -0.06] 1.5E-03 Tbk1 -0.10 [-0.19 , -0.02] 4.8E-02 -0.08 [-0.14 , -0.02] 3.2E-02 -0.03 [-0.04 , -0.01] 2.6E-02 Mapk11 -0.10 [-0.32 , 0.11] 4.3E-01 0.00 [-0.09 , 0.10] 9.6E-01 0.01 [-0.03 , 0.05] 7.3E-01 Tmed1 -0.11 [-0.23 , 0.02] 1.6E-01 -0.07 [-0.12 , -0.01] 6.2E-02 -0.03 [-0.06 , -0.01] 3.1E-02 Plaur -0.11 [-0.33 , 0.12] 4.4E-01 0.03 [-0.10 , 0.16] 7.2E-01 0.02 [-0.02 , 0.06] 3.9E-01 401 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Masp1 -0.11 [-0.35 , 0.14] 4.9E-01 -0.09 [-0.21 , 0.04] 2.8E-01 -0.02 [-0.07 , 0.04] 6.7E-01 Cma1 -0.11 [-0.55 , 0.34] 7.0E-01 0.01 [-0.16 , 0.19] 9.0E-01 -0.04 [-0.12 , 0.04] 4.2E-01 Il2rb -0.11 [-0.52 , 0.30] 6.7E-01 -0.07 [-0.23 , 0.08] 4.6E-01 -0.03 [-0.08 , 0.02] 3.4E-01 Dpp4 -0.11 [-0.28 , 0.06] 2.9E-01 -0.07 [-0.14 , 0.01] 1.5E-01 -0.01 [-0.04 , 0.02] 5.6E-01 Abcb1a -0.11 [-0.30 , 0.07] 3.0E-01 -0.11 [-0.21 , -0.02] 7.5E-02 0.02 [-0.01 , 0.05] 2.6E-01 Mapk8 -0.12 [-0.22 , -0.01] 6.6E-02 -0.12 [-0.17 , -0.07] 1.0E-03 -0.03 [-0.05 , -0.02] 4.8E-03 Il11ra1 -0.12 [-0.47 , 0.23] 5.7E-01 0.02 [-0.11 , 0.15] 8.1E-01 0.03 [-0.02 , 0.08] 3.5E-01 Ep300 -0.12 [-0.23 , -0.01] 7.3E-02 -0.09 [-0.15 , -0.04] 1.2E-02 -0.02 [-0.04 , 0.00] 9.5E-02 Fn1 -0.12 [-0.35 , 0.11] 3.8E-01 -0.01 [-0.11 , 0.10] 9.2E-01 0.00 [-0.05 , 0.04] 8.7E-01 Ltbr -0.12 [-0.26 , 0.01] 1.4E-01 -0.04 [-0.11 , 0.02] 3.1E-01 -0.01 [-0.03 , 0.01] 3.0E-01 Fyn -0.12 [-0.19 , -0.06] 4.6E-03 -0.01 [-0.06 , 0.05] 8.9E-01 -0.02 [-0.03 , -0.01] 2.2E-03 Traf6 -0.13 [-0.22 , -0.03] 3.6E-02 0.00 [-0.07 , 0.07] 9.3E-01 -0.01 [-0.03 , 0.01] 3.9E-01 Tyk2 -0.13 [-0.52 , 0.26] 5.8E-01 0.04 [-0.14 , 0.22] 7.2E-01 0.05 [-0.03 , 0.13] 3.0E-01 Ecsit -0.13 [-0.32 , 0.05] 2.4E-01 -0.04 [-0.12 , 0.04] 4.2E-01 0.00 [-0.04 , 0.04] 9.4E-01 Socs3 -0.13 [-0.78 , 0.51] 7.4E-01 -0.13 [-0.43 , 0.18] 5.2E-01 -0.05 [-0.17 , 0.07] 5.5E-01 Crebbp -0.14 [-0.25 , -0.03] 4.8E-02 -0.08 [-0.13 , -0.03] 2.2E-02 -0.01 [-0.03 , 0.00] 2.3E-01 Ikbkg -0.14 [-0.28 , 0.01] 1.2E-01 -0.03 [-0.09 , 0.03] 4.1E-01 -0.01 [-0.03 , 0.02] 7.6E-01 402 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il18r1 -0.14 [-0.25 , -0.03] 4.0E-02 -0.18 [-0.25 , -0.11] 9.4E-04 -0.06 [-0.07 , -0.04] 5.8E-05 Icam4 -0.14 [-0.61 , 0.32] 6.1E-01 -0.01 [-0.20 , 0.18] 9.4E-01 0.03 [-0.04 , 0.11] 5.2E-01 Cyld -0.15 [-0.34 , 0.04] 1.9E-01 -0.11 [-0.20 , -0.03] 4.8E-02 -0.03 [-0.07 , 0.01] 2.0E-01 Cd160 -0.15 [-0.57 , 0.26] 5.5E-01 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Tnfsf12 -0.15 [-0.34 , 0.03] 1.6E-01 0.07 [-0.02 , 0.16] 2.1E-01 0.05 [0.02 , 0.09] 1.6E-02 Gpi1 -0.16 [-0.25 , -0.07] 8.8E-03 0.00 [-0.05 , 0.06] 9.4E-01 0.01 [-0.02 , 0.03] 6.8E-01 Pin1 -0.16 [-0.29 , -0.02] 5.5E-02 -0.07 [-0.15 , 0.01] 1.5E-01 -0.01 [-0.04 , 0.02] 7.2E-01 Tollip -0.16 [-0.25 , -0.07] 9.7E-03 -0.01 [-0.04 , 0.03] 7.5E-01 0.01 [-0.01 , 0.02] 4.0E-01 Il15ra -0.16 [-0.39 , 0.07] 2.4E-01 -0.01 [-0.16 , 0.14] 9.1E-01 0.01 [-0.05 , 0.07] 8.0E-01 Mme -0.17 [-0.36 , 0.02] 1.5E-01 -0.08 [-0.14 , -0.02] 3.8E-02 -0.01 [-0.03 , 0.01] 5.5E-01 Tlr5 -0.17 [-0.54 , 0.20] 4.5E-01 0.10 [-0.09 , 0.30] 4.2E-01 0.04 [-0.03 , 0.11] 3.6E-01 Syt17 -0.18 [-0.40 , 0.04] 1.7E-01 -0.15 [-0.27 , -0.02] 7.3E-02 0.02 [-0.02 , 0.06] 5.0E-01 Ccl27a -0.18 [-0.42 , 0.06] 2.2E-01 0.05 [-0.11 , 0.21] 6.6E-01 0.05 [0.01 , 0.10] 6.5E-02 Tcf7 -0.18 [-0.36 , 0.00] 9.8E-02 -0.05 [-0.14 , 0.04] 4.1E-01 -0.02 [-0.05 , 0.01] 2.2E-01 Ncr1 -0.18 [-0.40 , 0.04] 1.7E-01 -0.28 [-0.42 , -0.14] 6.4E-03 -0.10 [-0.14 , -0.05] 2.8E-03 Gtf3c1 -0.18 [-0.33 , -0.04] 4.7E-02 0.02 [-0.05 , 0.08] 7.0E-01 0.04 [0.01 , 0.07] 3.6E-02 Klrc1 -0.18 [-0.44 , 0.07] 2.3E-01 -0.34 [-0.48 , -0.20] 1.3E-03 -0.12 [-0.18 , -0.07] 1.8E-03 403 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Stat3 -0.19 [-0.28 , -0.09] 4.4E-03 -0.06 [-0.12 , 0.00] 1.5E-01 -0.03 [-0.04 , -0.01] 2.4E-02 Il22ra2 -0.19 [-0.51 , 0.13] 3.2E-01 -0.19 [-0.38 , -0.01] 1.0E-01 -0.01 [-0.06 , 0.05] 8.5E-01 Tnfrsf10b -0.19 [-0.53 , 0.14] 3.4E-01 -0.05 [-0.25 , 0.15] 7.0E-01 -0.01 [-0.06 , 0.05] 8.9E-01 Cd200 -0.20 [-0.30 , -0.10] 4.3E-03 -0.13 [-0.19 , -0.08] 1.4E-03 -0.02 [-0.04 , -0.01] 4.0E-02 Foxj1 -0.20 [-0.62 , 0.22] 4.4E-01 0.13 [-0.06 , 0.31] 2.8E-01 0.04 [-0.03 , 0.12] 3.5E-01 Map3k7 -0.20 [-0.32 , -0.08] 1.1E-02 0.07 [0.02 , 0.12] 5.1E-02 0.04 [0.02 , 0.06] 2.0E-03 Irak1 -0.20 [-0.32 , -0.09] 9.3E-03 -0.04 [-0.09 , 0.02] 3.0E-01 -0.01 [-0.03 , 0.01] 4.3E-01 Ccnd3 -0.21 [-0.41 , 0.00] 9.5E-02 -0.02 [-0.11 , 0.08] 8.0E-01 -0.03 [-0.07 , 0.00] 1.5E-01 Reps1 -0.21 [-0.36 , -0.06] 2.6E-02 -0.02 [-0.09 , 0.04] 6.4E-01 0.00 [-0.02 , 0.02] 9.9E-01 Ets1 -0.21 [-0.35 , -0.08] 1.4E-02 -0.09 [-0.19 , 0.01] 1.5E-01 -0.01 [-0.03 , 0.01] 4.7E-01 Bcl6 -0.22 [-0.33 , -0.10] 4.9E-03 -0.10 [-0.16 , -0.05] 8.0E-03 0.01 [0.00 , 0.03] 2.0E-01 Smad4 -0.22 [-0.32 , -0.12] 2.5E-03 -0.05 [-0.08 , -0.01] 6.9E-02 -0.03 [-0.04 , -0.01] 3.1E-03 Klra7 -0.22 [-0.76 , 0.32] 5.0E-01 -0.20 [-0.51 , 0.10] 3.0E-01 -0.15 [-0.24 , -0.07] 7.8E-03 Smad3 -0.22 [-0.45 , 0.00] 1.0E-01 -0.04 [-0.14 , 0.05] 5.1E-01 0.00 [-0.03 , 0.04] 8.5E-01 Mif -0.22 [-0.50 , 0.05] 1.8E-01 0.05 [-0.08 , 0.18] 5.7E-01 0.01 [-0.04 , 0.06] 7.8E-01 Klrk1 -0.23 [-0.42 , -0.04] 5.6E-02 -0.25 [-0.35 , -0.15] 9.4E-04 -0.09 [-0.13 , -0.05] 2.1E-03 Ptgs2 -0.23 [-0.50 , 0.04] 1.6E-01 -0.27 [-0.49 , -0.05] 6.2E-02 -0.07 [-0.13 , -0.01] 7.4E-02 404 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Trp53 -0.23 [-0.58 , 0.12] 2.7E-01 -0.02 [-0.19 , 0.16] 8.8E-01 0.00 [-0.07 , 0.06] 9.2E-01 Mapkapk2 -0.24 [-0.41 , -0.07] 2.5E-02 0.06 [-0.05 , 0.17] 3.8E-01 0.04 [0.01 , 0.07] 5.8E-02 Ccl24 -0.24 [-0.69 , 0.20] 3.7E-01 0.13 [-0.08 , 0.33] 3.6E-01 -0.01 [-0.10 , 0.07] 8.1E-01 Ticam1 -0.24 [-0.47 , -0.01] 8.3E-02 -0.05 [-0.15 , 0.04] 3.7E-01 0.00 [-0.05 , 0.04] 8.9E-01 Klrd1 -0.25 [-0.52 , 0.03] 1.5E-01 -0.27 [-0.44 , -0.11] 1.4E-02 -0.11 [-0.17 , -0.06] 4.7E-03 Mapk3 -0.25 [-0.45 , -0.04] 5.3E-02 0.03 [-0.06 , 0.11] 6.5E-01 0.02 [-0.01 , 0.06] 3.0E-01 Creb5 -0.25 [-0.47 , -0.03] 6.5E-02 0.03 [-0.23 , 0.30] 8.5E-01 -0.04 [-0.09 , 0.00] 1.2E-01 Rrad -0.25 [-0.47 , -0.03] 6.6E-02 -0.22 [-0.42 , -0.01] 1.0E-01 -0.05 [-0.08 , -0.02] 1.3E-02 Txnip -0.25 [-0.41 , -0.10] 1.3E-02 0.03 [-0.06 , 0.11] 6.5E-01 0.03 [0.00 , 0.05] 9.3E-02 Rela -0.26 [-0.40 , -0.12] 7.4E-03 -0.06 [-0.12 , 0.00] 1.1E-01 -0.03 [-0.05 , 0.00] 1.4E-01 Lrp1 -0.26 [-0.45 , -0.07] 3.0E-02 -0.05 [-0.11 , 0.01] 2.4E-01 0.01 [-0.02 , 0.04] 7.6E-01 Nt5e -0.26 [-0.41 , -0.11] 8.8E-03 -0.11 [-0.18 , -0.05] 1.2E-02 -0.04 [-0.06 , -0.01] 3.4E-02 Thbd -0.26 [-0.52 , 0.00] 1.0E-01 -0.05 [-0.12 , 0.01] 2.1E-01 -0.04 [-0.07 , -0.02] 9.9E-03 Klra4 -0.26 [-0.55 , 0.03] 1.4E-01 -0.33 [-0.47 , -0.20] 1.2E-03 -0.14 [-0.20 , -0.08] 2.4E-03 Prg2 -0.26 [-0.51 , -0.01] 8.8E-02 -0.24 [-0.35 , -0.13] 3.0E-03 -0.07 [-0.12 , -0.02] 4.0E-02 Sbno2 -0.26 [-0.64 , 0.11] 2.5E-01 -0.01 [-0.19 , 0.17] 9.2E-01 0.04 [-0.03 , 0.11] 3.3E-01 Dock9 -0.27 [-0.44 , -0.09] 1.8E-02 0.04 [0.00 , 0.08] 1.3E-01 0.03 [0.01 , 0.05] 2.4E-02 405 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Txk -0.27 [-0.50 , -0.04] 6.0E-02 -0.14 [-0.28 , 0.00] 1.1E-01 -0.09 [-0.13 , -0.04] 4.5E-03 Ubc -0.27 [-0.41 , -0.12] 6.3E-03 0.09 [-0.03 , 0.20] 2.5E-01 0.02 [-0.01 , 0.04] 3.7E-01 Dusp4 -0.28 [-0.82 , 0.26] 3.9E-01 -0.20 [-0.38 , -0.03] 7.9E-02 -0.11 [-0.16 , -0.06] 4.0E-03 Cd7 -0.29 [-0.55 , -0.04] 6.4E-02 -0.11 [-0.22 , 0.00] 1.1E-01 -0.04 [-0.09 , 0.00] 1.6E-01 Erbb2 -0.30 [-0.55 , -0.04] 5.9E-02 0.01 [-0.09 , 0.11] 8.8E-01 0.01 [-0.03 , 0.05] 6.2E-01 Itga6 -0.30 [-0.41 , -0.19] 5.0E-04 -0.05 [-0.14 , 0.05] 4.5E-01 -0.02 [-0.04 , 0.00] 1.8E-01 Map4k2 -0.30 [-0.46 , -0.13] 7.7E-03 0.01 [-0.07 , 0.08] 8.7E-01 0.03 [0.00 , 0.06] 1.6E-01 Cd81 -0.31 [-0.45 , -0.17] 1.8E-03 -0.06 [-0.10 , -0.01] 6.0E-02 -0.02 [-0.04 , 0.00] 9.7E-02 App -0.31 [-0.47 , -0.15] 4.5E-03 0.03 [-0.03 , 0.10] 4.5E-01 0.03 [0.00 , 0.05] 9.6E-02 Itgb1 -0.32 [-0.47 , -0.16] 3.1E-03 -0.18 [-0.24 , -0.12] 3.8E-04 -0.08 [-0.10 , -0.05] 9.9E-05 Cebpb -0.32 [-0.55 , -0.08] 3.1E-02 -0.06 [-0.16 , 0.04] 3.9E-01 0.02 [-0.02 , 0.05] 4.1E-01 Prkce -0.32 [-0.58 , -0.06] 5.1E-02 0.05 [-0.03 , 0.12] 3.3E-01 0.01 [-0.02 , 0.05] 5.5E-01 Mst1r -0.32 [-0.64 , 0.00] 1.0E-01 -0.04 [-0.17 , 0.09] 6.5E-01 -0.03 [-0.08 , 0.02] 3.0E-01 Pecam1 -0.32 [-0.50 , -0.14] 7.2E-03 -0.14 [-0.20 , -0.09] 9.8E-04 -0.06 [-0.08 , -0.04] 2.7E-04 Smpd3 -0.33 [-0.70 , 0.05] 1.5E-01 -0.26 [-0.47 , -0.06] 4.6E-02 -0.12 [-0.17 , -0.07] 2.2E-03 Pnma1 -0.34 [-0.73 , 0.06] 1.6E-01 -0.03 [-0.24 , 0.17] 8.0E-01 0.03 [-0.05 , 0.10] 5.9E-01 Bcl2 -0.34 [-0.65 , -0.03] 7.0E-02 0.06 [-0.09 , 0.20] 5.6E-01 0.00 [-0.06 , 0.06] 9.8E-01 406 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Itga1 -0.34 [-0.57 , -0.11] 2.2E-02 -0.16 [-0.23 , -0.10] 1.1E-03 -0.05 [-0.07 , -0.03] 2.5E-03 Akt3 -0.35 [-0.51 , -0.19] 2.5E-03 -0.25 [-0.31 , -0.18] 6.9E-05 -0.06 [-0.09 , -0.04] 2.2E-04 Dll4 -0.36 [-0.69 , -0.02] 8.3E-02 0.03 [-0.11 , 0.16] 7.7E-01 0.01 [-0.06 , 0.08] 8.0E-01 Tie1 -0.36 [-0.57 , -0.15] 1.1E-02 -0.13 [-0.21 , -0.05] 1.6E-02 -0.03 [-0.06 , -0.01] 4.9E-02 Tek -0.36 [-0.59 , -0.12] 1.9E-02 -0.19 [-0.29 , -0.09] 9.2E-03 -0.04 [-0.07 , -0.02] 1.9E-02 Ifngr1 -0.37 [-0.53 , -0.20] 1.9E-03 -0.17 [-0.25 , -0.08] 6.4E-03 -0.06 [-0.09 , -0.02] 1.9E-02 Prf1 -0.37 [-0.65 , -0.09] 3.8E-02 -0.22 [-0.37 , -0.07] 3.1E-02 -0.11 [-0.17 , -0.04] 1.2E-02 Cd99 -0.37 [-0.80 , 0.06] 1.5E-01 0.12 [-0.08 , 0.31] 3.7E-01 0.06 [-0.01 , 0.14] 1.9E-01 Tirap -0.38 [-0.54 , -0.21] 1.8E-03 -0.16 [-0.27 , -0.06] 2.5E-02 -0.05 [-0.08 , -0.02] 9.8E-03 Cd97 -0.39 [-0.60 , -0.17] 7.3E-03 -0.07 [-0.16 , 0.02] 2.5E-01 0.00 [-0.03 , 0.02] 7.8E-01 Abl1 -0.39 [-0.64 , -0.14] 1.7E-02 0.10 [0.00 , 0.20] 1.4E-01 0.03 [-0.01 , 0.07] 1.9E-01 Rora -0.39 [-0.58 , -0.20] 3.5E-03 -0.12 [-0.23 , -0.02] 6.8E-02 -0.06 [-0.10 , -0.03] 6.6E-03 Nfatc4 -0.40 [-0.62 , -0.18] 6.7E-03 -0.04 [-0.14 , 0.05] 4.9E-01 0.02 [-0.02 , 0.06] 4.0E-01 Tal1 -0.40 [-0.63 , -0.17] 8.7E-03 -0.20 [-0.29 , -0.11] 3.0E-03 -0.03 [-0.06 , 0.00] 7.4E-02 Klrb1c -0.40 [-0.74 , -0.06] 5.8E-02 -0.29 [-0.45 , -0.14] 7.5E-03 -0.09 [-0.16 , -0.02] 4.1E-02 Cfd -0.41 [-1.92 , 1.11] 6.6E-01 0.26 [-0.42 , 0.93] 5.6E-01 -0.04 [-0.36 , 0.27] 8.4E-01 Il6ra -0.41 [-0.61 , -0.21] 3.7E-03 -0.01 [-0.08 , 0.05] 7.5E-01 0.01 [-0.01 , 0.04] 3.6E-01 407 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Vegfa -0.41 [-0.61 , -0.21] 3.1E-03 -0.16 [-0.22 , -0.10] 4.3E-04 -0.07 [-0.09 , -0.05] 1.6E-04 Msln -0.41 [-0.74 , -0.08] 4.5E-02 -0.20 [-0.34 , -0.06] 3.0E-02 -0.08 [-0.14 , -0.03] 1.9E-02 Pdgfrb -0.42 [-0.70 , -0.15] 1.7E-02 0.05 [-0.06 , 0.17] 4.6E-01 0.07 [0.02 , 0.12] 3.0E-02 Cmklr1 -0.43 [-0.62 , -0.24] 2.0E-03 -0.15 [-0.24 , -0.07] 7.1E-03 -0.02 [-0.05 , 0.00] 9.1E-02 Itga5 -0.46 [-0.60 , -0.32] 1.1E-04 -0.09 [-0.18 , -0.01] 9.1E-02 -0.02 [-0.05 , 0.00] 1.4E-01 Tnfrsf1a -0.46 [-0.67 , -0.25] 2.2E-03 -0.10 [-0.19 , -0.01] 9.0E-02 -0.01 [-0.04 , 0.03] 7.2E-01 Kdr -0.47 [-0.73 , -0.22] 5.9E-03 -0.25 [-0.38 , -0.12] 6.9E-03 -0.06 [-0.10 , -0.02] 3.6E-02 Nod1 -0.48 [-0.78 , -0.19] 1.2E-02 -0.08 [-0.22 , 0.06] 3.9E-01 0.01 [-0.05 , 0.06] 8.0E-01 Sele -0.49 [-1.00 , 0.03] 1.2E-01 -0.56 [-0.85 , -0.28] 5.9E-03 -0.25 [-0.34 , -0.17] 2.7E-04 Hamp -0.49 [-1.39 , 0.41] 3.7E-01 0.08 [-0.35 , 0.51] 7.7E-01 0.00 [-0.20 , 0.20] 9.9E-01 Vwf -0.51 [-0.76 , -0.26] 3.7E-03 -0.15 [-0.21 , -0.09] 7.8E-04 -0.03 [-0.05 , -0.02] 6.5E-03 Lyve1 -0.52 [-0.80 , -0.25] 5.5E-03 -0.05 [-0.28 , 0.19] 7.7E-01 -0.12 [-0.16 , -0.08] 3.2E-04 Fos -0.53 [-0.99 , -0.07] 6.3E-02 -0.22 [-0.41 , -0.02] 9.2E-02 -0.08 [-0.16 , -0.01] 6.9E-02 Cxcl14 -0.54 [-0.76 , -0.32] 1.1E-03 -0.24 [-0.33 , -0.15] 8.1E-04 -0.11 [-0.14 , -0.07] 4.7E-04 Vegfc -0.54 [-0.83 , -0.24] 7.0E-03 -0.18 [-0.29 , -0.07] 1.7E-02 -0.08 [-0.12 , -0.04] 7.0E-03 Cdh5 -0.54 [-0.78 , -0.30] 2.1E-03 -0.13 [-0.19 , -0.06] 4.9E-03 -0.05 [-0.07 , -0.02] 7.7E-03 F2rl1 -0.55 [-0.70 , -0.39] 5.4E-05 -0.22 [-0.30 , -0.14] 4.5E-04 -0.08 [-0.11 , -0.05] 4.9E-04 408 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mcam -0.56 [-0.75 , -0.37] 2.4E-04 -0.18 [-0.30 , -0.07] 1.8E-02 -0.10 [-0.12 , -0.07] 2.4E-05 Snai1 -0.56 [-0.85 , -0.27] 5.5E-03 0.00 [-0.14 , 0.13] 9.5E-01 0.01 [-0.06 , 0.07] 8.9E-01 Cd163 -0.56 [-0.83 , -0.30] 2.5E-03 0.07 [-0.08 , 0.23] 4.8E-01 0.01 [-0.05 , 0.06] 8.7E-01 Notch1 -0.58 [-0.82 , -0.34] 1.2E-03 -0.08 [-0.19 , 0.02] 2.0E-01 -0.02 [-0.06 , 0.02] 5.3E-01 Klrc2 -0.59 [-0.82 , -0.36] 8.3E-04 -0.44 [-0.51 , -0.37] 8.0E-07 -0.15 [-0.19 , -0.11] 2.8E-05 Mertk -0.59 [-0.76 , -0.43] 6.3E-05 -0.08 [-0.18 , 0.02] 2.0E-01 -0.02 [-0.04 , 0.00] 8.5E-02 Jun -0.60 [-0.79 , -0.40] 2.3E-04 -0.25 [-0.31 , -0.19] 1.7E-05 -0.07 [-0.09 , -0.05] 1.2E-04 Angpt1 -0.60 [-0.91 , -0.28] 6.1E-03 -0.27 [-0.42 , -0.13] 7.1E-03 -0.05 [-0.09 , -0.01] 7.4E-02 Icam2 -0.60 [-0.82 , -0.38] 5.3E-04 -0.26 [-0.33 , -0.19] 2.8E-05 -0.11 [-0.13 , -0.08] 9.7E-06 Dusp6 -0.60 [-0.78 , -0.42] 1.1E-04 -0.32 [-0.46 , -0.18] 1.9E-03 -0.07 [-0.12 , -0.02] 4.0E-02 Il4ra -0.60 [-0.97 , -0.24] 1.2E-02 -0.01 [-0.22 , 0.19] 9.1E-01 -0.01 [-0.08 , 0.06] 8.0E-01 Itga2b -0.61 [-0.90 , -0.32] 3.0E-03 0.08 [-0.09 , 0.26] 4.6E-01 0.03 [-0.03 , 0.10] 4.0E-01 Egfr -0.61 [-0.91 , -0.31] 3.7E-03 -0.04 [-0.19 , 0.10] 6.4E-01 -0.05 [-0.09 , 0.00] 1.0E-01 Tgfb3 -0.61 [-0.79 , -0.43] 9.2E-05 -0.24 [-0.32 , -0.17] 1.3E-04 -0.09 [-0.13 , -0.05] 3.1E-03 Col4a1 -0.62 [-0.98 , -0.25] 1.1E-02 -0.13 [-0.29 , 0.03] 2.1E-01 -0.03 [-0.09 , 0.04] 5.3E-01 Pvr -0.62 [-0.80 , -0.44] 8.8E-05 -0.11 [-0.23 , 0.01] 1.5E-01 -0.04 [-0.07 , -0.01] 2.1E-02 Igf1r -0.62 [-0.87 , -0.37] 8.7E-04 -0.10 [-0.19 , 0.00] 1.1E-01 -0.05 [-0.08 , -0.02] 2.1E-02 409 Table 32 (cont’d) Gene expression of cSiO2-treated NZBWF1 mice relative to VEH + CON mice in lung at 5 wk post exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Hspb2 -0.62 [-0.93 , -0.31] 3.7E-03 -0.06 [-0.21 , 0.08] 4.8E-01 0.00 [-0.05 , 0.05] 9.9E-01 Fas -0.63 [-0.79 , -0.47] 2.8E-05 -0.15 [-0.30 , 0.01] 1.4E-01 -0.06 [-0.09 , -0.03] 8.5E-03 Ppbp -0.65 [-1.07 , -0.23] 1.8E-02 -0.08 [-0.25 , 0.10] 5.0E-01 -0.04 [-0.11 , 0.04] 4.3E-01 Itgb4 -0.66 [-1.08 , -0.25] 1.5E-02 -0.16 [-0.36 , 0.04] 2.3E-01 -0.09 [-0.17 , -0.01] 6.9E-02 Hsd11b1 -0.69 [-0.93 , -0.45] 3.2E-04 -0.27 [-0.33 , -0.21] 9.3E-06 -0.07 [-0.09 , -0.05] 1.3E-04 Eng -0.70 [-1.10 , -0.31] 7.9E-03 -0.04 [-0.21 , 0.13] 7.3E-01 -0.03 [-0.10 , 0.04] 4.8E-01 Cspg4 -0.77 [-1.16 , -0.39] 3.7E-03 -0.24 [-0.40 , -0.08] 2.5E-02 -0.04 [-0.11 , 0.02] 2.9E-01 Egr1 -0.83 [-1.17 , -0.49] 9.8E-04 -0.37 [-0.51 , -0.23] 6.9E-04 -0.16 [-0.22 , -0.11] 2.5E-04 Tgfb2 -0.93 [-1.18 , -0.68] 3.9E-05 -0.43 [-0.53 , -0.34] 6.0E-06 -0.14 [-0.19 , -0.09] 3.2E-04 Bcl2l1 -0.96 [-1.20 , -0.72] 2.4E-05 -0.26 [-0.48 , -0.04] 6.9E-02 -0.11 [-0.15 , -0.06] 2.3E-03 Tnfrsf12a -1.11 [-1.36 , -0.87] 6.9E-06 -0.56 [-0.74 , -0.37] 2.6E-04 -0.25 [-0.31 , -0.19] 2.0E-05 Cdkn1a -1.49 [-1.82 , -1.16] 7.9E-06 -0.27 [-0.67 , 0.13] 2.9E-01 -0.14 [-0.21 , -0.06] 8.6E-03 Thbs1 -1.54 [-1.90 , -1.18] 1.2E-05 -0.79 [-0.94 , -0.64] 2.3E-06 -0.30 [-0.37 , -0.23] 1.0E-05 410 Table 33. Complete mRNA transcript profile in lung at 9 wk post final exposure in cSiO 2-exposed NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log2(fold change) relative to VEH + CON mice. Expression data are included for all mRNA transcripts that were above background threshold. Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl5 4.87 [4.30 , 5.45] 6.4E-09 2.49 [2.28 , 2.70] 2.8E-10 0.93 [0.84 , 1.03] 3.6E-09 Ccl2 4.07 [3.46 , 4.67] 5.3E-08 2.07 [1.77 , 2.37] 3.8E-08 0.66 [0.53 , 0.78] 8.9E-07 Lcn2 3.82 [3.43 , 4.22] 3.9E-09 2.00 [1.87 , 2.13] 2.5E-11 0.74 [0.69 , 0.79] 9.6E-11 Cxcl3 3.68 [2.93 , 4.42] 1.0E-06 1.87 [1.51 , 2.23] 7.1E-07 0.66 [0.52 , 0.80] 2.9E-06 Cxcl10 3.43 [2.97 , 3.89] 2.1E-08 1.27 [0.89 , 1.65] 5.2E-05 0.23 [0.07 , 0.38] 2.2E-02 Cxcl1 3.40 [3.00 , 3.80] 6.4E-09 1.67 [1.50 , 1.84] 1.9E-09 0.63 [0.55 , 0.71] 1.7E-08 Ccl7 3.37 [2.77 , 3.97] 3.1E-07 1.67 [1.35 , 1.98] 6.3E-07 0.48 [0.35 , 0.61] 3.3E-05 Cxcl9 3.24 [2.52 , 3.95] 2.2E-06 1.10 [0.68 , 1.52] 3.9E-04 0.21 [0.02 , 0.41] 8.2E-02 Ccl3 3.15 [2.77 , 3.54] 8.9E-09 1.63 [1.46 , 1.81] 3.1E-09 0.58 [0.49 , 0.67] 1.9E-07 Ccl8 3.11 [2.39 , 3.83] 3.2E-06 1.39 [0.99 , 1.79] 3.5E-05 0.39 [0.21 , 0.57] 2.2E-03 Il1rn 2.97 [2.63 , 3.31] 6.4E-09 1.50 [1.34 , 1.65] 2.4E-09 0.53 [0.46 , 0.59] 1.7E-08 Oasl1 2.87 [2.36 , 3.38] 3.1E-07 1.10 [0.69 , 1.50] 3.3E-04 0.12 [-0.03 , 0.28] 2.0E-01 C3ar1 2.83 [2.48 , 3.19] 1.2E-08 1.39 [1.18 , 1.60] 5.5E-08 0.41 [0.32 , 0.50] 4.2E-06 411 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Msr1 2.72 [2.31 , 3.13] 6.3E-08 1.40 [1.22 , 1.59] 1.4E-08 0.46 [0.38 , 0.54] 3.1E-07 Irf7 2.63 [2.04 , 3.22] 2.5E-06 1.07 [0.59 , 1.55] 1.5E-03 0.11 [-0.06 , 0.27] 2.9E-01 Pdcd1 2.61 [2.15 , 3.08] 3.2E-07 1.20 [0.95 , 1.44] 1.3E-06 0.37 [0.25 , 0.48] 9.2E-05 Ccl12 2.55 [2.16 , 2.94] 6.7E-08 0.97 [0.71 , 1.24] 2.1E-05 0.32 [0.22 , 0.41] 8.8E-05 Isg15 2.54 [2.10 , 2.98] 2.5E-07 0.99 [0.59 , 1.38] 6.0E-04 0.12 [-0.01 , 0.26] 1.3E-01 Ccl4 2.54 [2.08 , 3.01] 3.6E-07 0.98 [0.65 , 1.30] 1.2E-04 0.26 [0.12 , 0.40] 6.0E-03 Trem2 2.47 [2.26 , 2.68] 9.7E-10 1.34 [1.22 , 1.47] 7.0E-10 0.47 [0.42 , 0.52] 3.6E-09 Ifi44 2.46 [1.99 , 2.93] 6.1E-07 0.93 [0.51 , 1.34] 1.5E-03 0.08 [-0.07 , 0.23] 4.0E-01 Fcer2a 2.46 [1.88 , 3.03] 3.9E-06 1.21 [0.98 , 1.43] 5.8E-07 0.43 [0.35 , 0.52] 1.6E-06 Clec5a 2.42 [2.15 , 2.68] 5.2E-09 1.28 [1.16 , 1.39] 6.4E-10 0.47 [0.42 , 0.53] 1.2E-08 Cfb 2.42 [2.05 , 2.80] 7.9E-08 1.12 [0.89 , 1.36] 1.7E-06 0.35 [0.28 , 0.43] 2.4E-06 Oas2 2.41 [1.89 , 2.93] 1.8E-06 0.96 [0.63 , 1.29] 1.7E-04 0.11 [-0.02 , 0.23] 1.6E-01 Zbp1 2.37 [1.99 , 2.75] 1.2E-07 0.89 [0.53 , 1.24] 6.0E-04 0.14 [0.02 , 0.27] 7.1E-02 Cxcl2 2.37 [1.97 , 2.78] 2.2E-07 1.12 [0.89 , 1.34] 1.0E-06 0.40 [0.32 , 0.47] 5.0E-07 Mx1 2.24 [1.80 , 2.67] 6.8E-07 0.82 [0.46 , 1.18] 1.2E-03 0.14 [0.02 , 0.26] 5.6E-02 Cxcl13 2.24 [1.57 , 2.92] 3.6E-05 0.52 [0.07 , 0.98] 5.9E-02 0.14 [-0.07 , 0.34] 2.9E-01 Tnfrsf9 2.20 [1.81 , 2.58] 2.7E-07 1.15 [1.00 , 1.30] 1.3E-08 0.36 [0.31 , 0.40] 3.7E-08 412 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Spp1 2.19 [1.94 , 2.45] 6.4E-09 1.19 [1.05 , 1.33] 4.6E-09 0.41 [0.36 , 0.47] 3.7E-08 Spink5 2.16 [1.68 , 2.64] 2.2E-06 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Ccr5 2.13 [1.82 , 2.44] 4.5E-08 0.83 [0.64 , 1.01] 3.5E-06 0.22 [0.15 , 0.30] 2.4E-04 Oas3 2.12 [1.46 , 2.79] 5.1E-05 0.83 [0.44 , 1.22] 1.9E-03 0.08 [-0.07 , 0.23] 4.0E-01 Il12b 2.10 [1.65 , 2.56] 1.8E-06 0.99 [0.77 , 1.21] 3.3E-06 0.37 [0.28 , 0.46] 9.9E-06 C1qa 2.07 [1.79 , 2.34] 1.9E-08 0.85 [0.71 , 0.98] 1.2E-07 0.25 [0.19 , 0.32] 1.9E-05 Clec4n 2.05 [1.85 , 2.24] 3.3E-09 1.05 [0.96 , 1.13] 2.5E-10 0.37 [0.33 , 0.42] 1.2E-08 Ifit1 2.01 [1.65 , 2.37] 3.2E-07 0.73 [0.39 , 1.07] 1.8E-03 0.08 [-0.03 , 0.19] 2.4E-01 Fcgr1 2.01 [1.65 , 2.37] 3.2E-07 0.76 [0.46 , 1.05] 5.0E-04 0.15 [0.06 , 0.24] 1.2E-02 C1qb 1.99 [1.69 , 2.29] 6.2E-08 0.78 [0.64 , 0.92] 3.1E-07 0.24 [0.17 , 0.31] 6.6E-05 Mx2 1.92 [1.56 , 2.28] 4.6E-07 0.76 [0.45 , 1.06] 6.4E-04 -0.02 [-0.17 , 0.14] 8.7E-01 Marco 1.85 [1.38 , 2.32] 8.2E-06 1.14 [0.88 , 1.40] 4.5E-06 0.57 [0.49 , 0.65] 6.7E-08 Fcgr2b 1.84 [1.57 , 2.11] 5.2E-08 0.98 [0.88 , 1.09] 3.1E-09 0.32 [0.28 , 0.37] 1.0E-07 Tigit 1.83 [1.32 , 2.34] 1.7E-05 0.84 [0.55 , 1.12] 1.8E-04 0.19 [0.07 , 0.31] 1.6E-02 Ccl9 1.77 [1.56 , 1.98] 6.4E-09 0.88 [0.77 , 0.99] 7.9E-09 0.30 [0.25 , 0.35] 2.7E-07 Csf2 1.77 [1.48 , 2.07] 1.5E-07 0.94 [0.81 , 1.06] 1.4E-08 0.36 [0.32 , 0.40] 9.5E-09 Il21 1.72 [1.03 , 2.42] 4.8E-04 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d 413 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il1a 1.71 [1.49 , 1.93] 1.5E-08 0.89 [0.78 , 1.00] 1.1E-08 0.31 [0.26 , 0.35] 7.0E-08 Tnfsf11 1.70 [1.40 , 2.01] 3.3E-07 0.87 [0.76 , 0.97] 8.7E-09 0.26 [0.20 , 0.32] 4.1E-06 Foxp3 1.70 [1.35 , 2.06] 1.4E-06 0.98 [0.78 , 1.19] 1.6E-06 0.27 [0.20 , 0.34] 2.3E-05 Slamf7 1.70 [1.31 , 2.09] 3.1E-06 0.74 [0.49 , 0.99] 1.6E-04 0.17 [0.07 , 0.28] 1.3E-02 Ctss 1.67 [1.46 , 1.89] 1.4E-08 0.80 [0.65 , 0.96] 1.0E-06 0.24 [0.17 , 0.30] 3.1E-05 Cd14 1.67 [1.38 , 1.97] 3.0E-07 0.84 [0.74 , 0.95] 7.9E-09 0.28 [0.23 , 0.34] 5.2E-07 Siglec1 1.67 [1.27 , 2.07] 4.5E-06 0.83 [0.63 , 1.03] 6.8E-06 0.19 [0.14 , 0.23] 9.7E-06 Aicda 1.66 [0.81 , 2.52] 3.1E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Cd68 1.65 [1.48 , 1.82] 3.9E-09 0.91 [0.82 , 1.00] 1.3E-09 0.31 [0.27 , 0.35] 1.3E-08 Slc11a1 1.62 [1.41 , 1.83] 1.5E-08 0.74 [0.60 , 0.88] 6.2E-07 0.25 [0.19 , 0.31] 1.2E-05 Ccl6 1.62 [1.39 , 1.84] 3.4E-08 0.94 [0.81 , 1.07] 2.6E-08 0.35 [0.32 , 0.39] 3.6E-09 Cxcr1 1.62 [1.18 , 2.06] 1.5E-05 1.00 [0.79 , 1.21] 1.8E-06 0.39 [0.30 , 0.47] 3.9E-06 Fcgr4 1.61 [1.36 , 1.86] 7.6E-08 0.37 [0.28 , 0.46] 1.0E-05 0.15 [0.07 , 0.22] 3.7E-03 Gzmk 1.61 [1.10 , 2.12] 6.3E-05 0.42 [0.10 , 0.74] 3.5E-02 0.09 [-0.06 , 0.24] 3.2E-01 Birc5 1.61 [1.08 , 2.14] 7.9E-05 0.64 [0.37 , 0.92] 9.9E-04 0.19 [0.07 , 0.30] 1.4E-02 Usp18 1.59 [1.19 , 1.99] 6.5E-06 0.61 [0.30 , 0.92] 3.5E-03 0.04 [-0.07 , 0.14] 5.9E-01 Ifit3 1.58 [1.27 , 1.89] 7.2E-07 0.62 [0.33 , 0.90] 2.0E-03 0.07 [-0.01 , 0.16] 1.4E-01 414 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tlr2 1.51 [1.30 , 1.72] 3.2E-08 0.73 [0.60 , 0.86] 3.2E-07 0.25 [0.20 , 0.29] 3.7E-07 Arg2 1.51 [1.23 , 1.80] 5.0E-07 0.82 [0.72 , 0.92] 8.0E-09 0.35 [0.29 , 0.41] 2.3E-07 Pou2af1 1.50 [1.13 , 1.88] 5.9E-06 0.48 [0.27 , 0.70] 1.4E-03 0.13 [0.03 , 0.23] 4.4E-02 Ccl20 1.50 [0.49 , 2.52] 1.7E-02 0.39 [-0.09 , 0.87] 1.7E-01 0.08 [-0.12 , 0.28] 5.4E-01 Blnk 1.49 [1.13 , 1.85] 4.7E-06 0.59 [0.39 , 0.80] 1.6E-04 0.15 [0.07 , 0.24] 9.5E-03 Plau 1.48 [1.27 , 1.69] 3.7E-08 0.79 [0.68 , 0.91] 4.5E-08 0.25 [0.20 , 0.30] 1.3E-06 Ccr1 1.44 [1.15 , 1.74] 1.2E-06 0.83 [0.71 , 0.95] 2.9E-08 0.28 [0.23 , 0.32] 4.5E-07 Il6 1.43 [0.78 , 2.08] 1.2E-03 0.72 [0.39 , 1.05] 1.6E-03 0.13 [0.00 , 0.26] 1.2E-01 Ccr6 1.42 [0.88 , 1.97] 3.3E-04 0.48 [0.22 , 0.74] 4.7E-03 0.11 [0.00 , 0.22] 9.7E-02 Irf4 1.40 [0.97 , 1.82] 4.2E-05 0.57 [0.32 , 0.83] 1.3E-03 0.12 [0.01 , 0.22] 8.6E-02 Tnfrsf17 1.40 [0.89 , 1.92] 2.2E-04 0.67 [0.42 , 0.93] 3.9E-04 n.d [n.d , n.d] n.d Ctla4 1.39 [0.96 , 1.81] 4.2E-05 0.80 [0.59 , 1.01] 1.4E-05 0.21 [0.13 , 0.28] 2.8E-04 Cd200r1 1.38 [1.22 , 1.54] 6.4E-09 0.76 [0.68 , 0.84] 2.4E-09 0.28 [0.24 , 0.31] 1.7E-08 Cx3cr1 1.38 [1.14 , 1.62] 2.7E-07 0.38 [0.27 , 0.50] 6.1E-05 0.17 [0.09 , 0.24] 1.5E-03 Cfi 1.37 [1.08 , 1.66] 1.5E-06 0.68 [0.51 , 0.85] 9.0E-06 0.25 [0.19 , 0.31] 9.9E-06 Rsad2 1.37 [0.87 , 1.87] 2.1E-04 0.47 [0.13 , 0.82] 2.8E-02 0.03 [-0.09 , 0.14] 7.1E-01 Tnf 1.36 [1.00 , 1.72] 1.1E-05 0.65 [0.47 , 0.83] 3.0E-05 0.23 [0.15 , 0.31] 2.8E-04 415 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfrsf4 1.36 [0.99 , 1.72] 1.5E-05 0.65 [0.48 , 0.82] 1.3E-05 0.20 [0.13 , 0.27] 2.1E-04 Lag3 1.36 [0.93 , 1.80] 6.3E-05 0.50 [0.27 , 0.73] 1.7E-03 0.07 [-0.03 , 0.18] 2.7E-01 Lgals3 1.35 [1.15 , 1.55] 5.3E-08 0.83 [0.73 , 0.93] 4.6E-09 0.29 [0.25 , 0.33] 3.7E-08 Fcer1g 1.33 [1.18 , 1.48] 6.4E-09 0.50 [0.40 , 0.60] 1.1E-06 0.19 [0.14 , 0.24] 1.2E-05 Clec7a 1.33 [1.10 , 1.56] 2.2E-07 0.59 [0.48 , 0.70] 4.5E-07 0.21 [0.18 , 0.25] 4.4E-07 Osm 1.33 [0.93 , 1.73] 3.9E-05 0.67 [0.47 , 0.86] 5.4E-05 0.17 [0.10 , 0.24] 8.8E-04 Csf2rb 1.31 [1.13 , 1.50] 3.4E-08 0.68 [0.57 , 0.80] 2.3E-07 0.25 [0.22 , 0.29] 1.6E-08 Itgam 1.30 [1.05 , 1.54] 5.0E-07 0.55 [0.41 , 0.70] 1.3E-05 0.16 [0.10 , 0.21] 4.9E-04 Il2ra 1.30 [0.97 , 1.63] 7.0E-06 0.70 [0.54 , 0.87] 4.7E-06 0.20 [0.15 , 0.26] 3.0E-05 Il21r 1.29 [0.92 , 1.66] 2.6E-05 0.44 [0.21 , 0.68] 4.8E-03 0.09 [0.00 , 0.19] 1.2E-01 Bst2 1.26 [0.98 , 1.54] 2.5E-06 0.53 [0.30 , 0.76] 1.2E-03 0.07 [0.01 , 0.14] 7.6E-02 Slc7a11 1.25 [0.92 , 1.58] 1.1E-05 0.86 [0.70 , 1.03] 6.7E-07 0.35 [0.28 , 0.41] 1.2E-06 Cxcr3 1.25 [0.84 , 1.66] 7.6E-05 0.48 [0.20 , 0.75] 8.4E-03 0.05 [-0.06 , 0.16] 4.6E-01 Emr1 1.22 [1.03 , 1.40] 6.7E-08 0.49 [0.38 , 0.59] 2.5E-06 0.15 [0.10 , 0.20] 1.2E-04 Cd274 1.21 [1.02 , 1.40] 1.0E-07 0.59 [0.47 , 0.72] 2.1E-06 0.20 [0.15 , 0.24] 3.9E-06 Tnfrsf11a 1.21 [1.00 , 1.42] 2.6E-07 0.65 [0.57 , 0.73] 7.9E-09 0.21 [0.16 , 0.25] 2.9E-06 Cd180 1.21 [0.99 , 1.43] 4.0E-07 0.43 [0.27 , 0.58] 3.0E-04 0.11 [0.04 , 0.18] 1.4E-02 416 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd83 1.21 [0.97 , 1.46] 9.5E-07 0.56 [0.48 , 0.64] 2.7E-08 0.18 [0.13 , 0.22] 9.5E-06 Il1r2 1.21 [0.77 , 1.66] 2.3E-04 0.66 [0.47 , 0.85] 4.2E-05 0.25 [0.15 , 0.36] 1.0E-03 Ly9 1.20 [0.94 , 1.46] 1.8E-06 0.63 [0.49 , 0.76] 2.0E-06 0.15 [0.08 , 0.21] 1.7E-03 Tap1 1.20 [0.94 , 1.46] 1.9E-06 0.51 [0.33 , 0.70] 3.0E-04 0.10 [0.04 , 0.16] 1.7E-02 Icos 1.20 [0.88 , 1.51] 1.0E-05 0.54 [0.34 , 0.74] 3.0E-04 0.12 [0.05 , 0.18] 8.5E-03 Fcgr3 1.19 [1.02 , 1.37] 5.3E-08 0.60 [0.48 , 0.72] 1.1E-06 0.22 [0.18 , 0.25] 1.1E-07 Tnfrsf13c 1.19 [0.71 , 1.67] 4.5E-04 0.36 [0.10 , 0.61] 2.5E-02 0.05 [-0.10 , 0.20] 6.0E-01 Ada 1.18 [0.93 , 1.44] 1.5E-06 0.39 [0.23 , 0.54] 5.6E-04 0.08 [0.00 , 0.16] 1.1E-01 Il1b 1.18 [0.89 , 1.47] 5.3E-06 0.38 [0.27 , 0.50] 4.6E-05 0.15 [0.10 , 0.21] 5.4E-04 H2-K1 1.17 [0.92 , 1.41] 1.5E-06 0.49 [0.31 , 0.67] 3.4E-04 0.09 [0.04 , 0.15] 1.2E-02 Clec4a2 1.16 [0.98 , 1.35] 9.6E-08 0.35 [0.27 , 0.42] 4.5E-06 0.13 [0.08 , 0.18] 3.4E-04 Ncf4 1.16 [0.94 , 1.38] 5.3E-07 0.47 [0.36 , 0.58] 6.0E-06 0.16 [0.11 , 0.21] 8.3E-05 Cd86 1.15 [0.96 , 1.35] 2.5E-07 0.53 [0.42 , 0.63] 1.1E-06 0.17 [0.13 , 0.20] 6.0E-06 Lta 1.15 [0.86 , 1.45] 7.5E-06 0.51 [0.36 , 0.65] 4.1E-05 0.13 [0.05 , 0.20] 1.1E-02 Slamf6 1.15 [0.81 , 1.50] 3.1E-05 0.48 [0.28 , 0.69] 7.8E-04 0.09 [0.01 , 0.17] 8.8E-02 Nlrc5 1.14 [0.85 , 1.44] 8.2E-06 0.37 [0.16 , 0.58] 6.7E-03 0.02 [-0.07 , 0.11] 7.6E-01 Ulbp1 1.13 [0.85 , 1.40] 5.9E-06 0.46 [0.37 , 0.56] 1.4E-06 0.18 [0.14 , 0.22] 2.1E-06 417 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ddx60 1.13 [0.80 , 1.47] 3.3E-05 0.47 [0.23 , 0.70] 3.3E-03 0.03 [-0.04 , 0.10] 4.5E-01 Sh2b2 1.11 [0.93 , 1.30] 2.1E-07 0.52 [0.43 , 0.61] 3.4E-07 0.14 [0.07 , 0.20] 2.6E-03 Itgax 1.10 [0.92 , 1.27] 1.1E-07 0.44 [0.29 , 0.59] 2.0E-04 0.18 [0.14 , 0.23] 1.6E-05 Ccr8 1.09 [0.85 , 1.34] 2.6E-06 0.59 [0.44 , 0.75] 1.3E-05 0.15 [0.07 , 0.23] 5.4E-03 Herc6 1.09 [0.83 , 1.36] 4.8E-06 0.45 [0.27 , 0.63] 7.0E-04 0.07 [0.01 , 0.14] 8.2E-02 Trem1 1.08 [0.68 , 1.48] 2.4E-04 0.28 [0.10 , 0.46] 1.3E-02 0.18 [0.13 , 0.23] 8.7E-05 Chil3 1.07 [0.31 , 1.84] 2.2E-02 0.79 [0.45 , 1.13] 1.2E-03 0.48 [0.34 , 0.62] 5.7E-05 Cybb 1.06 [0.90 , 1.22] 5.3E-08 0.41 [0.29 , 0.52] 4.1E-05 0.16 [0.10 , 0.22] 3.1E-04 C3 1.06 [0.87 , 1.25] 2.9E-07 0.56 [0.46 , 0.65] 2.5E-07 0.20 [0.16 , 0.24] 1.3E-06 Itgb2 1.05 [0.93 , 1.16] 5.2E-09 0.44 [0.37 , 0.52] 2.6E-07 0.16 [0.13 , 0.19] 2.0E-06 Tlr9 1.05 [0.81 , 1.30] 3.6E-06 0.34 [0.13 , 0.56] 1.2E-02 0.06 [-0.01 , 0.13] 1.5E-01 Psmb9 1.04 [0.82 , 1.26] 1.3E-06 0.40 [0.24 , 0.56] 6.8E-04 0.06 [0.00 , 0.13] 1.1E-01 Cfp 1.04 [0.74 , 1.34] 2.7E-05 0.40 [0.22 , 0.58] 1.4E-03 0.09 [0.03 , 0.16] 2.5E-02 Psmb8 1.03 [0.84 , 1.23] 5.0E-07 0.39 [0.24 , 0.54] 3.9E-04 0.08 [0.02 , 0.13] 2.6E-02 Ly86 1.03 [0.83 , 1.23] 8.1E-07 0.32 [0.16 , 0.48] 2.7E-03 0.09 [0.04 , 0.14] 8.7E-03 Stat1 1.03 [0.80 , 1.26] 2.4E-06 0.33 [0.13 , 0.54] 1.1E-02 0.02 [-0.05 , 0.09] 7.1E-01 Ikbke 1.02 [0.80 , 1.25] 2.0E-06 0.36 [0.21 , 0.52] 8.2E-04 0.13 [0.07 , 0.19] 2.7E-03 418 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Pik3cg 1.01 [0.79 , 1.24] 2.6E-06 0.37 [0.26 , 0.48] 4.1E-05 0.14 [0.08 , 0.20] 1.1E-03 H2-Q2 1.01 [0.70 , 1.31] 4.2E-05 0.49 [0.34 , 0.65] 6.9E-05 0.08 [0.03 , 0.13] 1.3E-02 Cd74 1.00 [0.82 , 1.18] 3.1E-07 0.45 [0.37 , 0.53] 2.6E-07 0.14 [0.11 , 0.17] 6.8E-06 Cd80 1.00 [0.70 , 1.30] 3.5E-05 0.52 [0.40 , 0.64] 4.7E-06 0.16 [0.11 , 0.21] 1.2E-04 Ebi3 0.99 [0.75 , 1.22] 4.4E-06 0.34 [0.22 , 0.46] 1.9E-04 0.14 [0.08 , 0.20] 1.7E-03 Batf 0.99 [0.77 , 1.20] 1.7E-06 0.43 [0.32 , 0.53] 8.3E-06 0.14 [0.10 , 0.18] 4.8E-05 Xaf1 0.98 [0.72 , 1.24] 1.2E-05 0.39 [0.17 , 0.61] 6.5E-03 0.02 [-0.04 , 0.09] 5.5E-01 Cmpk2 0.98 [0.77 , 1.18] 1.6E-06 0.33 [0.12 , 0.53] 1.3E-02 0.01 [-0.04 , 0.07] 7.4E-01 Ccl19 0.98 [0.66 , 1.29] 7.2E-05 0.29 [0.15 , 0.44] 3.1E-03 0.05 [-0.03 , 0.12] 3.0E-01 Ccl22 0.98 [0.56 , 1.40] 8.3E-04 0.57 [0.37 , 0.77] 1.9E-04 0.24 [0.15 , 0.33] 4.5E-04 Lcp1 0.97 [0.86 , 1.08] 5.5E-09 0.42 [0.37 , 0.47] 8.1E-09 0.18 [0.15 , 0.20] 2.1E-07 Egr2 0.97 [0.52 , 1.41] 1.3E-03 0.43 [0.19 , 0.68] 6.7E-03 0.19 [0.10 , 0.28] 2.5E-03 Tlr1 0.96 [0.66 , 1.27] 5.6E-05 0.34 [0.17 , 0.52] 3.6E-03 0.08 [0.00 , 0.15] 9.9E-02 Lyz2 0.95 [0.83 , 1.07] 1.5E-08 0.54 [0.47 , 0.61] 1.2E-08 0.20 [0.18 , 0.22] 1.2E-08 H2-DMb2 0.95 [0.61 , 1.29] 1.9E-04 0.34 [0.15 , 0.54] 6.7E-03 0.08 [0.02 , 0.15] 3.9E-02 Cdk1 0.94 [0.58 , 1.31] 3.5E-04 0.37 [0.23 , 0.51] 4.5E-04 0.17 [0.11 , 0.23] 4.5E-04 H2-T23 0.94 [0.70 , 1.17] 7.1E-06 0.41 [0.23 , 0.58] 1.1E-03 0.07 [0.02 , 0.12] 3.4E-02 419 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfrsf18 0.93 [0.55 , 1.31] 6.0E-04 0.47 [0.31 , 0.63] 1.5E-04 0.11 [0.06 , 0.16] 3.6E-03 Ifitm1 0.92 [0.73 , 1.11] 1.2E-06 0.50 [0.40 , 0.60] 1.0E-06 0.19 [0.15 , 0.22] 8.9E-07 Cd53 0.92 [0.72 , 1.12] 1.7E-06 0.50 [0.43 , 0.58] 8.7E-08 0.17 [0.13 , 0.21] 6.8E-06 Cd4 0.91 [0.64 , 1.18] 3.2E-05 0.43 [0.29 , 0.57] 1.0E-04 0.07 [0.01 , 0.13] 7.2E-02 H2-D1 0.90 [0.73 , 1.08] 8.4E-07 0.41 [0.29 , 0.53] 5.0E-05 0.09 [0.05 , 0.12] 9.2E-04 Lif 0.90 [0.69 , 1.11] 3.5E-06 0.40 [0.21 , 0.59] 2.0E-03 0.18 [0.13 , 0.22] 1.4E-05 C1ra 0.90 [0.72 , 1.08] 9.2E-07 0.44 [0.35 , 0.52] 1.0E-06 0.12 [0.09 , 0.16] 2.4E-05 Cd84 0.89 [0.71 , 1.07] 1.0E-06 0.50 [0.42 , 0.58] 1.1E-07 0.16 [0.12 , 0.19] 5.9E-06 Tlr7 0.88 [0.75 , 1.00] 4.5E-08 0.32 [0.17 , 0.46] 1.7E-03 0.11 [0.07 , 0.15] 3.5E-04 Tmem173 0.88 [0.69 , 1.06] 1.7E-06 0.39 [0.30 , 0.48] 4.7E-06 0.14 [0.09 , 0.18] 9.5E-05 Fpr2 0.87 [0.54 , 1.20] 2.9E-04 0.09 [-0.08 , 0.27] 3.6E-01 0.13 [0.02 , 0.24] 7.1E-02 Socs1 0.87 [0.65 , 1.09] 7.4E-06 0.39 [0.24 , 0.54] 4.9E-04 0.09 [0.04 , 0.15] 1.2E-02 Btk 0.85 [0.67 , 1.03] 1.7E-06 0.33 [0.23 , 0.43] 5.0E-05 0.13 [0.09 , 0.17] 2.1E-04 Pou2f2 0.84 [0.53 , 1.15] 2.4E-04 0.09 [-0.06 , 0.24] 3.0E-01 0.04 [-0.05 , 0.12] 4.9E-01 Casp1 0.84 [0.65 , 1.02] 2.4E-06 0.31 [0.20 , 0.42] 2.1E-04 0.10 [0.06 , 0.15] 2.0E-03 H2-Eb1 0.84 [0.64 , 1.03] 4.3E-06 0.31 [0.21 , 0.41] 1.0E-04 0.09 [0.06 , 0.13] 5.7E-04 C6 0.82 [0.67 , 0.97] 4.1E-07 0.57 [0.50 , 0.64] 1.3E-08 0.22 [0.18 , 0.25] 2.2E-07 420 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Bst1 0.82 [0.65 , 0.98] 8.9E-07 0.48 [0.39 , 0.56] 3.9E-07 0.14 [0.11 , 0.18] 9.9E-06 Pdcd1lg2 0.82 [0.54 , 1.10] 1.2E-04 0.37 [0.27 , 0.47] 2.0E-05 0.15 [0.10 , 0.21] 1.9E-04 Tnfrsf8 0.82 [0.50 , 1.14] 4.0E-04 n.d [n.d , n.d] n.d 0.13 [0.06 , 0.20] 7.2E-03 Cxcr6 0.82 [0.57 , 1.06] 4.1E-05 0.37 [0.23 , 0.52] 4.4E-04 0.14 [0.08 , 0.19] 6.3E-04 H2-Ea-ps 0.81 [0.63 , 0.99] 2.1E-06 0.36 [0.29 , 0.44] 1.1E-06 0.12 [0.09 , 0.15] 1.7E-05 Stat2 0.81 [0.55 , 1.07] 7.0E-05 0.30 [0.08 , 0.52] 2.8E-02 0.02 [-0.04 , 0.07] 6.5E-01 Havcr2 0.81 [0.64 , 0.98] 1.6E-06 0.35 [0.26 , 0.45] 1.8E-05 0.15 [0.11 , 0.18] 2.4E-05 Ltb 0.81 [0.59 , 1.02] 1.1E-05 0.23 [0.08 , 0.39] 1.8E-02 0.07 [0.01 , 0.12] 5.2E-02 Tnfaip3 0.80 [0.56 , 1.05] 3.9E-05 0.39 [0.27 , 0.51] 7.1E-05 0.12 [0.07 , 0.16] 9.3E-04 Ticam2 0.80 [0.52 , 1.08] 1.4E-04 0.30 [0.20 , 0.40] 1.9E-04 0.13 [0.08 , 0.17] 4.1E-04 Cxcl16 0.80 [0.64 , 0.96] 7.8E-07 0.32 [0.23 , 0.41] 4.1E-05 0.11 [0.08 , 0.14] 3.5E-05 H2-Aa 0.80 [0.62 , 0.97] 2.3E-06 0.32 [0.25 , 0.40] 7.0E-06 0.11 [0.08 , 0.14] 2.0E-05 Gbp2b 0.79 [0.43 , 1.16] 1.3E-03 0.14 [-0.14 , 0.41] 4.1E-01 0.02 [-0.04 , 0.09] 5.7E-01 Cd5 0.79 [0.56 , 1.03] 3.4E-05 0.35 [0.23 , 0.47] 1.6E-04 0.05 [-0.02 , 0.11] 2.3E-01 Raet1c 0.79 [0.55 , 1.02] 3.8E-05 0.40 [0.31 , 0.49] 4.0E-06 0.18 [0.15 , 0.22] 3.1E-06 Cd48 0.78 [0.63 , 0.94] 7.7E-07 0.20 [0.07 , 0.33] 1.4E-02 0.07 [0.02 , 0.12] 2.2E-02 Relb 0.78 [0.62 , 0.94] 1.4E-06 0.42 [0.34 , 0.51] 9.9E-07 0.13 [0.11 , 0.16] 4.5E-07 421 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value H2-M3 0.77 [0.60 , 0.93] 1.8E-06 0.32 [0.19 , 0.44] 7.0E-04 0.06 [0.02 , 0.11] 2.5E-02 Irf5 0.77 [0.61 , 0.93] 1.3E-06 0.29 [0.19 , 0.38] 1.4E-04 0.10 [0.07 , 0.14] 1.8E-04 Ikzf2 0.77 [0.57 , 0.96] 7.7E-06 0.42 [0.31 , 0.53] 1.3E-05 0.12 [0.07 , 0.16] 4.2E-04 H2-Ab1 0.75 [0.54 , 0.95] 1.5E-05 0.32 [0.22 , 0.41] 4.6E-05 0.10 [0.07 , 0.13] 2.0E-04 Lamp3 0.74 [0.58 , 0.91] 2.4E-06 0.42 [0.36 , 0.48] 4.1E-08 0.16 [0.13 , 0.18] 3.2E-07 Ddx58 0.73 [0.57 , 0.90] 2.8E-06 0.30 [0.16 , 0.43] 1.6E-03 0.05 [0.02 , 0.09] 2.1E-02 Pdgfc 0.72 [0.47 , 0.97] 1.2E-04 0.44 [0.35 , 0.53] 1.5E-06 0.07 [0.03 , 0.12] 8.3E-03 Mefv 0.72 [0.31 , 1.13] 6.5E-03 0.23 [0.00 , 0.45] 9.7E-02 0.12 [0.02 , 0.22] 5.9E-02 Spn 0.71 [0.42 , 0.99] 4.7E-04 0.02 [-0.10 , 0.14] 7.8E-01 0.03 [-0.04 , 0.10] 5.6E-01 Il10ra 0.71 [0.53 , 0.89] 7.5E-06 0.20 [0.09 , 0.30] 3.8E-03 0.05 [0.01 , 0.10] 5.9E-02 Cd6 0.70 [0.35 , 1.05] 2.4E-03 0.30 [0.14 , 0.46] 5.3E-03 0.05 [-0.03 , 0.13] 3.5E-01 Socs3 0.70 [0.47 , 0.93] 8.3E-05 0.41 [0.28 , 0.55] 1.3E-04 0.12 [0.06 , 0.17] 2.1E-03 Ms4a1 0.70 [0.27 , 1.13] 1.0E-02 0.13 [-0.12 , 0.39] 3.9E-01 0.01 [-0.07 , 0.10] 8.3E-01 Cd33 0.68 [0.51 , 0.85] 7.1E-06 0.43 [0.37 , 0.50] 7.4E-08 0.17 [0.15 , 0.19] 6.1E-08 Tnfrsf1b 0.68 [0.41 , 0.95] 4.5E-04 0.28 [0.16 , 0.40] 1.0E-03 0.09 [0.04 , 0.14] 1.2E-02 Bid 0.68 [0.47 , 0.89] 5.0E-05 0.29 [0.19 , 0.40] 3.0E-04 0.08 [0.03 , 0.12] 1.0E-02 Ly96 0.68 [0.56 , 0.79] 2.1E-07 0.30 [0.23 , 0.37] 4.5E-06 0.10 [0.07 , 0.14] 8.8E-05 422 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Nfkb2 0.67 [0.54 , 0.81] 9.2E-07 0.35 [0.28 , 0.42] 8.2E-07 0.11 [0.08 , 0.14] 2.2E-05 Muc1 0.67 [0.53 , 0.81] 1.5E-06 0.43 [0.38 , 0.49] 1.4E-08 0.14 [0.12 , 0.16] 2.9E-07 Fap 0.67 [0.41 , 0.92] 2.9E-04 0.30 [0.15 , 0.44] 2.4E-03 0.08 [0.02 , 0.14] 2.8E-02 Gbp5 0.67 [0.43 , 0.90] 1.7E-04 0.25 [0.09 , 0.41] 1.3E-02 0.02 [-0.04 , 0.07] 6.3E-01 Ifit2 0.66 [0.48 , 0.83] 1.3E-05 0.15 [-0.06 , 0.35] 2.2E-01 -0.01 [-0.06 , 0.04] 7.0E-01 Xcr1 0.65 [0.45 , 0.86] 6.4E-05 0.28 [0.17 , 0.39] 6.0E-04 0.15 [0.11 , 0.18] 6.4E-06 Nod2 0.65 [0.40 , 0.90] 3.5E-04 0.16 [0.05 , 0.27] 2.2E-02 0.05 [0.00 , 0.10] 9.9E-02 Mmp9 0.65 [0.31 , 0.99] 3.4E-03 0.42 [0.27 , 0.57] 2.4E-04 0.19 [0.13 , 0.25] 1.1E-04 Cd63 0.64 [0.55 , 0.73] 3.2E-08 0.40 [0.36 , 0.45] 4.6E-09 0.14 [0.12 , 0.16] 1.2E-07 Xcl1 0.64 [0.35 , 0.94] 1.4E-03 0.30 [0.13 , 0.48] 7.5E-03 0.05 [-0.04 , 0.14] 3.4E-01 Csf1r 0.64 [0.43 , 0.84] 7.1E-05 0.17 [0.09 , 0.25] 1.3E-03 0.10 [0.05 , 0.14] 2.7E-03 Tnfrsf13b 0.63 [0.41 , 0.86] 1.7E-04 0.32 [0.17 , 0.47] 2.0E-03 0.08 [0.02 , 0.13] 3.5E-02 Ifih1 0.63 [0.47 , 0.79] 7.1E-06 0.23 [0.08 , 0.38] 1.6E-02 0.03 [0.00 , 0.06] 7.5E-02 Gpr183 0.63 [0.42 , 0.84] 1.0E-04 0.27 [0.18 , 0.35] 1.1E-04 0.08 [0.04 , 0.11] 1.4E-03 Hck 0.63 [0.41 , 0.84] 1.1E-04 0.15 [0.05 , 0.24] 1.5E-02 0.09 [0.03 , 0.14] 1.3E-02 Cd276 0.62 [0.46 , 0.78] 8.3E-06 0.40 [0.33 , 0.48] 4.9E-07 0.11 [0.08 , 0.14] 1.7E-05 Itgae 0.62 [0.45 , 0.79] 1.6E-05 0.39 [0.30 , 0.48] 4.7E-06 0.16 [0.13 , 0.19] 4.6E-07 423 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Nlrp3 0.61 [0.46 , 0.77] 7.1E-06 0.31 [0.20 , 0.43] 3.0E-04 0.13 [0.09 , 0.17] 1.4E-04 Syk 0.61 [0.46 , 0.76] 5.8E-06 0.25 [0.17 , 0.33] 9.7E-05 0.08 [0.05 , 0.12] 1.2E-03 Cd3g 0.61 [0.35 , 0.87] 7.6E-04 0.19 [0.04 , 0.34] 3.8E-02 0.03 [-0.03 , 0.09] 4.9E-01 Ptprc 0.61 [0.43 , 0.79] 2.9E-05 0.10 [0.01 , 0.20] 7.6E-02 0.03 [-0.02 , 0.09] 2.7E-01 Il23r 0.60 [0.30 , 0.90] 2.4E-03 0.29 [0.14 , 0.44] 3.1E-03 0.15 [0.08 , 0.22] 2.3E-03 Ciita 0.60 [0.38 , 0.81] 1.9E-04 0.33 [0.20 , 0.46] 4.9E-04 0.09 [0.04 , 0.15] 7.6E-03 Selplg 0.59 [0.48 , 0.70] 5.0E-07 0.18 [0.11 , 0.25] 4.1E-04 0.06 [0.03 , 0.09] 1.5E-03 Cr2 0.59 [-0.03 , 1.20] 1.1E-01 0.06 [-0.25 , 0.37] 7.4E-01 0.00 [-0.11 , 0.12] 9.4E-01 C1s1 0.58 [0.44 , 0.73] 6.5E-06 0.29 [0.21 , 0.36] 1.8E-05 0.10 [0.07 , 0.14] 1.3E-04 Ctsh 0.58 [0.46 , 0.70] 1.2E-06 0.38 [0.32 , 0.43] 5.5E-08 0.15 [0.13 , 0.17] 7.8E-08 H2-DMb1 0.56 [0.38 , 0.75] 7.8E-05 0.24 [0.16 , 0.32] 1.2E-04 0.10 [0.08 , 0.13] 2.4E-06 H2-DMa 0.55 [0.36 , 0.75] 1.3E-04 0.21 [0.13 , 0.29] 5.2E-04 0.08 [0.06 , 0.10] 4.1E-05 Irgm2 0.55 [0.32 , 0.79] 8.3E-04 0.20 [0.02 , 0.39] 6.5E-02 -0.02 [-0.08 , 0.05] 7.1E-01 Sh2d1a 0.55 [0.24 , 0.86] 6.3E-03 0.17 [0.01 , 0.33] 8.3E-02 -0.03 [-0.09 , 0.04] 5.7E-01 Jak3 0.55 [0.36 , 0.73] 1.1E-04 0.25 [0.15 , 0.34] 4.3E-04 0.05 [0.02 , 0.08] 1.1E-02 Tlr6 0.55 [0.38 , 0.72] 5.2E-05 0.20 [0.14 , 0.27] 1.2E-04 0.08 [0.04 , 0.12] 5.2E-03 Cxcl15 0.54 [0.41 , 0.68] 4.8E-06 0.31 [0.23 , 0.39] 1.3E-05 0.13 [0.10 , 0.16] 9.6E-06 424 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Lbp 0.52 [0.41 , 0.64] 2.1E-06 0.30 [0.25 , 0.35] 2.3E-07 0.10 [0.08 , 0.13] 3.5E-06 C4b 0.52 [0.30 , 0.75] 8.6E-04 0.28 [0.16 , 0.40] 1.0E-03 0.07 [0.03 , 0.11] 1.4E-02 Litaf 0.52 [0.41 , 0.63] 1.1E-06 0.35 [0.30 , 0.41] 4.5E-08 0.13 [0.11 , 0.15] 2.2E-07 Tlr8 0.52 [0.40 , 0.64] 3.8E-06 0.12 [0.02 , 0.23] 4.9E-02 0.06 [0.03 , 0.10] 1.2E-02 Pla2g1b 0.51 [0.34 , 0.68] 9.1E-05 0.33 [0.25 , 0.41] 6.0E-06 0.13 [0.10 , 0.17] 5.7E-06 Pml 0.51 [0.33 , 0.69] 1.6E-04 0.17 [0.03 , 0.31] 5.0E-02 0.01 [-0.03 , 0.05] 7.6E-01 Cd19 0.51 [0.13 , 0.89] 2.9E-02 0.14 [-0.05 , 0.32] 2.1E-01 0.02 [-0.05 , 0.08] 7.0E-01 Abca1 0.51 [0.36 , 0.65] 2.8E-05 0.30 [0.23 , 0.37] 5.9E-06 0.11 [0.08 , 0.14] 2.3E-05 Nfatc2 0.50 [0.26 , 0.73] 1.8E-03 0.20 [0.10 , 0.31] 3.6E-03 0.05 [0.00 , 0.10] 8.1E-02 Card9 0.49 [0.21 , 0.78] 6.9E-03 0.17 [-0.01 , 0.36] 1.2E-01 0.11 [0.05 , 0.17] 5.3E-03 Cd37 0.49 [0.27 , 0.71] 1.2E-03 0.17 [0.06 , 0.29] 1.9E-02 0.04 [0.00 , 0.08] 1.2E-01 Abcg1 0.49 [0.33 , 0.64] 5.4E-05 0.30 [0.18 , 0.42] 5.4E-04 0.12 [0.09 , 0.15] 9.9E-06 Clu 0.48 [0.21 , 0.76] 6.4E-03 0.25 [0.12 , 0.37] 2.9E-03 0.04 [0.01 , 0.08] 5.9E-02 Tank 0.48 [0.38 , 0.57] 9.2E-07 0.21 [0.15 , 0.26] 2.5E-05 0.07 [0.05 , 0.09] 1.9E-05 Klra2 0.47 [0.28 , 0.67] 6.0E-04 -0.07 [-0.17 , 0.04] 3.1E-01 -0.01 [-0.06 , 0.05] 8.3E-01 Casp3 0.47 [0.30 , 0.63] 1.6E-04 0.19 [0.11 , 0.28] 1.2E-03 0.06 [0.03 , 0.08] 2.0E-03 H2-Ob 0.47 [0.12 , 0.81] 2.7E-02 0.17 [0.04 , 0.30] 3.6E-02 0.03 [-0.03 , 0.09] 3.8E-01 425 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tgfbr1 0.47 [0.35 , 0.58] 5.9E-06 0.16 [0.12 , 0.20] 1.2E-05 0.07 [0.05 , 0.09] 1.6E-04 Sbno2 0.46 [0.31 , 0.61] 7.1E-05 0.29 [0.19 , 0.39] 1.9E-04 0.09 [0.06 , 0.11] 5.5E-05 Vcam1 0.46 [0.32 , 0.60] 4.2E-05 0.15 [0.10 , 0.21] 3.1E-04 0.01 [-0.02 , 0.03] 7.4E-01 Cd28 0.45 [0.19 , 0.71] 6.4E-03 0.05 [-0.06 , 0.17] 4.4E-01 -0.02 [-0.09 , 0.06] 7.2E-01 Rel 0.45 [0.30 , 0.60] 1.1E-04 0.18 [0.12 , 0.25] 2.2E-04 0.06 [0.04 , 0.09] 4.9E-04 Pycard 0.45 [0.23 , 0.66] 2.1E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Hc 0.45 [0.27 , 0.62] 3.7E-04 0.32 [0.22 , 0.43] 1.1E-04 0.10 [0.07 , 0.14] 2.7E-04 Lck 0.45 [0.22 , 0.67] 2.5E-03 0.13 [-0.01 , 0.27] 1.1E-01 -0.01 [-0.07 , 0.04] 7.2E-01 Ifi27 0.44 [0.34 , 0.55] 2.8E-06 0.21 [0.15 , 0.26] 2.9E-05 0.04 [0.01 , 0.07] 3.4E-02 Cd79b 0.44 [0.14 , 0.74] 1.9E-02 0.14 [-0.05 , 0.33] 2.0E-01 0.04 [-0.03 , 0.10] 3.4E-01 Irak3 0.44 [0.24 , 0.63] 1.1E-03 0.16 [0.06 , 0.26] 1.4E-02 0.09 [0.05 , 0.13] 1.2E-03 Serpinb2 0.44 [-0.48 , 1.35] 4.1E-01 0.04 [-0.46 , 0.55] 8.8E-01 -0.06 [-0.23 , 0.12] 6.2E-01 Klrg1 0.43 [0.19 , 0.67] 5.2E-03 0.35 [0.24 , 0.46] 1.1E-04 0.11 [0.06 , 0.15] 7.5E-04 Il1rl2 0.43 [0.28 , 0.58] 1.7E-04 0.30 [0.24 , 0.35] 6.9E-07 0.12 [0.09 , 0.15] 9.0E-06 Fut7 0.43 [0.16 , 0.70] 1.2E-02 0.04 [-0.11 , 0.20] 6.2E-01 -0.01 [-0.07 , 0.04] 7.0E-01 Itgal 0.42 [0.15 , 0.70] 1.2E-02 -0.09 [-0.17 , 0.00] 8.2E-02 0.02 [-0.05 , 0.08] 7.1E-01 Isg20 0.41 [0.28 , 0.54] 6.4E-05 0.13 [0.00 , 0.25] 9.8E-02 0.03 [0.00 , 0.06] 1.6E-01 426 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Apoe 0.41 [0.27 , 0.54] 6.8E-05 0.24 [0.18 , 0.29] 9.3E-06 0.10 [0.07 , 0.13] 2.0E-05 Ltk 0.40 [0.04 , 0.77] 6.2E-02 0.17 [0.04 , 0.30] 3.5E-02 0.01 [-0.06 , 0.09] 7.5E-01 C5ar1 0.40 [0.24 , 0.57] 5.9E-04 0.06 [-0.02 , 0.13] 1.8E-01 0.06 [0.02 , 0.10] 1.3E-02 Irak2 0.40 [0.29 , 0.51] 1.2E-05 0.24 [0.19 , 0.28] 4.9E-07 0.09 [0.07 , 0.10] 2.9E-06 Csf1 0.40 [0.25 , 0.55] 2.5E-04 0.29 [0.24 , 0.34] 5.2E-07 0.07 [0.04 , 0.11] 1.9E-03 Traf3 0.40 [0.30 , 0.50] 7.7E-06 0.22 [0.17 , 0.26] 1.1E-06 0.07 [0.05 , 0.09] 9.5E-06 Flt3 0.40 [0.19 , 0.60] 3.1E-03 0.26 [0.18 , 0.34] 1.1E-04 0.09 [0.05 , 0.12] 1.4E-03 Ctsl 0.39 [0.29 , 0.49] 7.5E-06 0.29 [0.25 , 0.34] 5.5E-08 0.10 [0.08 , 0.11] 4.4E-07 Myd88 0.39 [0.25 , 0.53] 1.9E-04 0.17 [0.13 , 0.22] 2.8E-05 0.05 [0.03 , 0.08] 4.2E-03 Casp8 0.39 [0.30 , 0.48] 2.7E-06 0.20 [0.17 , 0.22] 1.4E-08 0.06 [0.05 , 0.07] 4.4E-07 Col3a1 0.39 [0.19 , 0.58] 2.5E-03 0.15 [0.05 , 0.24] 1.6E-02 0.02 [-0.02 , 0.05] 4.5E-01 Adora2a 0.38 [0.21 , 0.55] 1.1E-03 0.21 [0.12 , 0.29] 6.8E-04 0.11 [0.05 , 0.16] 6.0E-03 Il2rg 0.38 [0.26 , 0.51] 7.5E-05 0.14 [0.06 , 0.21] 7.6E-03 0.03 [0.00 , 0.06] 1.3E-01 Hif1a 0.37 [0.28 , 0.46] 5.4E-06 0.21 [0.18 , 0.25] 8.8E-08 0.07 [0.06 , 0.09] 8.8E-07 Il34 0.37 [0.24 , 0.50] 1.4E-04 0.25 [0.19 , 0.30] 3.5E-06 0.07 [0.04 , 0.09] 3.2E-04 Cd44 0.37 [0.17 , 0.57] 4.3E-03 0.19 [0.12 , 0.27] 4.1E-04 0.06 [0.03 , 0.10] 7.0E-03 Cd244 0.37 [0.07 , 0.67] 4.0E-02 -0.08 [-0.22 , 0.06] 3.3E-01 0.04 [-0.03 , 0.11] 3.7E-01 427 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Runx3 0.37 [0.13 , 0.60] 1.2E-02 0.16 [0.00 , 0.31] 8.8E-02 -0.01 [-0.07 , 0.05] 7.8E-01 Tnfsf15 0.36 [0.17 , 0.55] 3.5E-03 0.20 [0.10 , 0.31] 3.6E-03 0.10 [0.06 , 0.14] 5.6E-04 Ifnar2 0.36 [0.29 , 0.43] 7.8E-07 0.19 [0.14 , 0.23] 6.8E-06 0.08 [0.06 , 0.09] 2.4E-07 Ikzf1 0.36 [0.21 , 0.50] 5.1E-04 -0.01 [-0.09 , 0.08] 8.6E-01 0.01 [-0.04 , 0.05] 8.1E-01 Tap2 0.35 [0.19 , 0.51] 1.4E-03 0.13 [0.06 , 0.20] 3.8E-03 0.00 [-0.02 , 0.02] 8.3E-01 Cyfip2 0.35 [0.15 , 0.55] 7.0E-03 0.04 [-0.07 , 0.15] 5.2E-01 0.01 [-0.04 , 0.06] 7.3E-01 Irf8 0.35 [0.25 , 0.45] 1.8E-05 0.12 [0.05 , 0.19] 8.7E-03 0.05 [0.03 , 0.07] 2.0E-03 Itga4 0.35 [0.06 , 0.63] 4.3E-02 -0.01 [-0.12 , 0.09] 8.1E-01 0.02 [-0.04 , 0.08] 6.0E-01 Sh2d1b1 0.34 [0.10 , 0.58] 2.0E-02 -0.14 [-0.23 , -0.05] 1.5E-02 -0.01 [-0.06 , 0.05] 8.7E-01 Il7 0.34 [0.09 , 0.59] 2.4E-02 0.25 [0.14 , 0.36] 1.5E-03 0.09 [0.03 , 0.14] 1.3E-02 Tlr4 0.34 [0.25 , 0.42] 7.1E-06 0.19 [0.15 , 0.23] 1.8E-06 0.08 [0.06 , 0.10] 3.9E-05 Tapbp 0.34 [0.21 , 0.46] 3.1E-04 0.14 [0.05 , 0.23] 1.3E-02 0.02 [0.00 , 0.04] 8.0E-02 Cxcl12 0.33 [0.18 , 0.48] 1.3E-03 0.18 [0.10 , 0.27] 2.1E-03 0.08 [0.04 , 0.12] 2.9E-03 Glycam1 0.33 [-0.38 , 1.03] 4.2E-01 0.11 [-0.25 , 0.47] 6.0E-01 -0.01 [-0.16 , 0.14] 9.2E-01 Cxcr5 0.32 [-0.02 , 0.66] 1.0E-01 0.08 [-0.11 , 0.27] 4.8E-01 0.00 [-0.07 , 0.07] 9.5E-01 Psmb10 0.32 [0.23 , 0.41] 2.0E-05 0.05 [-0.05 , 0.16] 4.0E-01 0.00 [-0.03 , 0.03] 9.9E-01 Lyn 0.32 [0.22 , 0.41] 4.6E-05 0.07 [0.02 , 0.12] 1.8E-02 0.04 [0.02 , 0.06] 8.8E-03 428 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cmah 0.32 [0.06 , 0.57] 4.1E-02 0.07 [-0.08 , 0.23] 4.2E-01 -0.01 [-0.07 , 0.05] 7.5E-01 Mrc1 0.31 [0.17 , 0.45] 1.1E-03 0.21 [0.10 , 0.32] 3.6E-03 0.09 [0.06 , 0.12] 3.4E-04 Zap70 0.31 [0.05 , 0.58] 4.6E-02 0.19 [0.05 , 0.32] 2.6E-02 0.02 [-0.03 , 0.06] 6.3E-01 Arg1 0.31 [0.01 , 0.62] 8.2E-02 0.17 [0.03 , 0.32] 4.7E-02 0.13 [0.04 , 0.22] 3.1E-02 Pik3cd 0.31 [0.13 , 0.50] 8.1E-03 0.09 [-0.04 , 0.22] 2.4E-01 0.04 [0.00 , 0.07] 7.9E-02 Bax 0.31 [0.19 , 0.42] 2.5E-04 0.16 [0.08 , 0.24] 3.5E-03 0.06 [0.05 , 0.08] 7.9E-06 Myc 0.30 [0.11 , 0.50] 1.2E-02 0.17 [0.08 , 0.25] 3.4E-03 0.03 [0.00 , 0.07] 1.3E-01 Cd3d 0.30 [0.05 , 0.56] 4.9E-02 0.14 [-0.01 , 0.28] 1.1E-01 -0.01 [-0.07 , 0.06] 8.5E-01 Cd27 0.30 [0.04 , 0.56] 5.4E-02 0.11 [-0.05 , 0.26] 2.4E-01 -0.05 [-0.12 , 0.02] 2.2E-01 Gfi1 0.29 [-0.08 , 0.67] 1.7E-01 0.11 [-0.04 , 0.26] 2.1E-01 -0.01 [-0.07 , 0.05] 8.3E-01 Traf2 0.29 [0.19 , 0.39] 9.0E-05 0.17 [0.12 , 0.22] 6.8E-05 0.05 [0.03 , 0.07] 1.8E-04 Tnfsf14 0.29 [0.12 , 0.46] 6.4E-03 0.18 [0.09 , 0.27] 3.1E-03 0.04 [0.00 , 0.08] 1.1E-01 Cd22 0.29 [-0.04 , 0.62] 1.3E-01 -0.23 [-0.36 , -0.09] 8.5E-03 -0.08 [-0.18 , 0.01] 1.5E-01 Pax5 0.29 [-0.09 , 0.67] 1.9E-01 0.06 [-0.14 , 0.26] 6.0E-01 -0.01 [-0.09 , 0.06] 7.7E-01 Tnfsf13 0.29 [0.16 , 0.42] 1.2E-03 0.18 [0.10 , 0.26] 1.3E-03 0.08 [0.05 , 0.12] 2.0E-03 Xbp1 0.29 [0.19 , 0.38] 8.7E-05 0.06 [-0.03 , 0.14] 2.5E-01 0.01 [-0.02 , 0.04] 6.0E-01 Prkcd 0.28 [0.21 , 0.35] 1.1E-05 0.12 [0.08 , 0.16] 6.1E-05 0.06 [0.05 , 0.08] 2.0E-05 429 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mif 0.28 [0.20 , 0.35] 1.4E-05 0.15 [0.07 , 0.24] 5.3E-03 0.04 [0.02 , 0.05] 7.8E-04 Inpp5d 0.27 [0.14 , 0.40] 2.1E-03 0.05 [-0.01 , 0.12] 1.8E-01 0.03 [0.00 , 0.06] 7.9E-02 C2 0.27 [0.08 , 0.46] 2.0E-02 0.08 [-0.05 , 0.22] 3.1E-01 0.04 [0.00 , 0.07] 8.8E-02 Ifitm2 0.27 [0.17 , 0.37] 3.2E-04 0.13 [0.09 , 0.18] 1.1E-04 0.06 [0.04 , 0.07] 1.2E-05 Tgfb1 0.26 [0.18 , 0.34] 4.7E-05 0.16 [0.12 , 0.20] 7.3E-06 0.06 [0.04 , 0.08] 8.1E-05 Amica1 0.25 [0.08 , 0.42] 1.6E-02 0.07 [-0.03 , 0.18] 2.4E-01 0.04 [0.00 , 0.07] 1.2E-01 Ccl11 0.25 [0.05 , 0.45] 3.6E-02 0.29 [0.19 , 0.39] 2.0E-04 0.06 [0.02 , 0.10] 1.8E-02 Cd247 0.25 [0.00 , 0.50] 8.9E-02 0.07 [-0.05 , 0.19] 3.5E-01 -0.03 [-0.08 , 0.02] 3.6E-01 Nos2 0.24 [0.00 , 0.48] 8.6E-02 0.11 [-0.06 , 0.29] 2.7E-01 0.13 [0.07 , 0.18] 8.7E-04 Il7r 0.24 [-0.09 , 0.57] 2.1E-01 0.27 [0.14 , 0.41] 2.6E-03 0.05 [-0.01 , 0.10] 1.6E-01 Ccl17 0.24 [-0.16 , 0.63] 3.0E-01 0.31 [0.02 , 0.59] 7.5E-02 0.18 [0.08 , 0.28] 8.3E-03 Pvrl2 0.24 [0.13 , 0.34] 8.5E-04 0.22 [0.18 , 0.26] 3.0E-07 0.06 [0.04 , 0.08] 4.1E-04 Irf1 0.23 [0.17 , 0.29] 7.9E-06 0.07 [0.01 , 0.12] 5.2E-02 0.02 [0.00 , 0.03] 8.0E-02 Il3ra 0.23 [0.10 , 0.36] 5.1E-03 0.14 [0.08 , 0.20] 1.5E-03 0.04 [0.01 , 0.07] 4.2E-02 Axl 0.23 [0.14 , 0.32] 4.9E-04 0.09 [0.03 , 0.15] 2.1E-02 0.04 [0.02 , 0.05] 2.5E-03 Icosl 0.23 [0.06 , 0.39] 2.3E-02 0.12 [0.05 , 0.19] 9.1E-03 0.06 [0.03 , 0.08] 1.0E-03 Tnfrsf10b 0.22 [-0.04 , 0.49] 1.5E-01 0.16 [0.03 , 0.28] 4.3E-02 0.03 [-0.02 , 0.07] 3.1E-01 430 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd207 0.22 [-0.17 , 0.61] 3.3E-01 0.13 [-0.06 , 0.32] 2.6E-01 0.12 [0.07 , 0.18] 2.5E-03 Ifnar1 0.21 [0.13 , 0.30] 6.2E-04 0.13 [0.09 , 0.17] 6.4E-05 0.03 [0.02 , 0.05] 4.7E-04 Vhl 0.21 [0.08 , 0.35] 1.1E-02 0.15 [0.08 , 0.22] 1.6E-03 0.05 [0.02 , 0.08] 1.2E-02 Ripk2 0.21 [0.11 , 0.32] 2.6E-03 0.07 [0.03 , 0.12] 1.5E-02 0.05 [0.03 , 0.06] 1.0E-03 Jak2 0.21 [0.12 , 0.31] 1.1E-03 0.14 [0.08 , 0.19] 6.0E-04 0.04 [0.03 , 0.06] 1.3E-04 Stat4 0.21 [-0.04 , 0.46] 1.4E-01 0.05 [-0.06 , 0.17] 4.3E-01 0.01 [-0.03 , 0.06] 6.3E-01 Cxcr4 0.21 [-0.11 , 0.53] 2.5E-01 0.13 [-0.03 , 0.29] 1.7E-01 -0.04 [-0.10 , 0.02] 2.5E-01 Ifi35 0.21 [0.11 , 0.30] 1.2E-03 0.04 [-0.05 , 0.13] 4.3E-01 -0.01 [-0.04 , 0.01] 4.0E-01 Btla 0.21 [-0.12 , 0.53] 2.7E-01 0.06 [-0.09 , 0.22] 5.0E-01 0.01 [-0.06 , 0.08] 8.1E-01 Creb5 0.21 [0.02 , 0.39] 5.9E-02 0.09 [0.00 , 0.17] 8.2E-02 0.07 [0.03 , 0.11] 8.3E-03 Gzmb 0.21 [-0.12 , 0.53] 2.7E-01 -0.05 [-0.21 , 0.11] 5.7E-01 0.01 [-0.05 , 0.08] 7.7E-01 Blk 0.21 [-0.10 , 0.51] 2.4E-01 0.05 [-0.09 , 0.20] 5.3E-01 0.01 [-0.04 , 0.06] 7.3E-01 Il1rl1 0.20 [0.06 , 0.35] 2.0E-02 0.26 [0.16 , 0.35] 4.3E-04 0.09 [0.05 , 0.14] 4.4E-03 Prdm1 0.20 [0.03 , 0.37] 4.8E-02 0.04 [-0.05 , 0.14] 4.5E-01 -0.02 [-0.07 , 0.03] 4.9E-01 Tfe3 0.20 [0.11 , 0.28] 8.1E-04 0.11 [0.04 , 0.19] 2.0E-02 0.05 [0.03 , 0.07] 7.9E-04 S100a8 0.20 [-0.30 , 0.69] 4.9E-01 0.15 [-0.07 , 0.38] 2.6E-01 0.08 [-0.02 , 0.18] 1.8E-01 Tnfsf13b 0.20 [0.01 , 0.38] 7.4E-02 -0.06 [-0.14 , 0.03] 2.6E-01 -0.04 [-0.08 , -0.01] 6.0E-02 431 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Hcst 0.19 [-0.28 , 0.67] 4.7E-01 0.11 [-0.13 , 0.35] 4.3E-01 0.01 [-0.08 , 0.10] 8.7E-01 Serping1 0.19 [0.12 , 0.26] 3.8E-04 0.14 [0.08 , 0.21] 1.0E-03 0.04 [0.03 , 0.05] 9.5E-05 Lilra5 0.19 [0.05 , 0.33] 2.3E-02 -0.02 [-0.14 , 0.10] 8.0E-01 0.05 [0.01 , 0.08] 4.5E-02 Cd96 0.19 [-0.15 , 0.52] 3.3E-01 0.13 [-0.02 , 0.29] 1.4E-01 -0.01 [-0.07 , 0.05] 7.7E-01 Il17ra 0.19 [0.03 , 0.34] 4.5E-02 -0.02 [-0.08 , 0.03] 4.2E-01 0.02 [-0.01 , 0.04] 2.6E-01 Cd3e 0.18 [-0.05 , 0.42] 1.8E-01 0.09 [-0.04 , 0.22] 2.3E-01 -0.03 [-0.09 , 0.03] 3.6E-01 Icam1 0.18 [0.08 , 0.29] 6.8E-03 0.12 [0.07 , 0.17] 1.2E-03 0.03 [0.01 , 0.05] 3.6E-02 Ctsw 0.18 [0.03 , 0.32] 4.6E-02 0.09 [0.01 , 0.17] 6.0E-02 0.00 [-0.04 , 0.03] 9.8E-01 Il2rb 0.17 [-0.04 , 0.39] 1.7E-01 0.05 [-0.11 , 0.20] 6.2E-01 -0.06 [-0.12 , 0.00] 8.9E-02 St6gal1 0.17 [0.03 , 0.31] 3.7E-02 0.07 [-0.02 , 0.17] 2.0E-01 0.02 [-0.01 , 0.05] 2.8E-01 Lamp1 0.17 [0.11 , 0.23] 1.1E-04 0.12 [0.10 , 0.14] 1.1E-06 0.05 [0.04 , 0.06] 5.0E-07 Csf3r 0.17 [-0.08 , 0.42] 2.4E-01 0.05 [-0.07 , 0.16] 5.1E-01 0.07 [0.03 , 0.10] 5.1E-03 Cklf 0.17 [0.01 , 0.32] 6.4E-02 0.10 [0.02 , 0.18] 5.1E-02 0.02 [0.00 , 0.05] 1.2E-01 Vim 0.16 [0.06 , 0.26] 8.5E-03 0.14 [0.07 , 0.20] 2.5E-03 0.06 [0.04 , 0.08] 5.4E-04 Il5ra 0.16 [-0.18 , 0.49] 4.1E-01 0.19 [0.04 , 0.35] 4.0E-02 0.05 [-0.03 , 0.13] 2.8E-01 Mef2c 0.15 [-0.01 , 0.32] 1.2E-01 0.07 [-0.01 , 0.15] 1.3E-01 0.02 [-0.01 , 0.05] 3.6E-01 Cd8a 0.15 [-0.14 , 0.45] 3.7E-01 -0.14 [-0.26 , -0.02] 5.2E-02 -0.08 [-0.16 , 0.00] 1.2E-01 432 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il13ra1 0.15 [0.06 , 0.24] 1.0E-02 0.07 [0.03 , 0.11] 7.5E-03 0.04 [0.02 , 0.06] 3.1E-03 Psma2 0.15 [0.09 , 0.20] 2.8E-04 0.06 [0.02 , 0.10] 9.9E-03 0.03 [0.02 , 0.04] 2.3E-03 Map2k1 0.15 [0.09 , 0.20] 3.5E-04 0.04 [0.00 , 0.07] 5.9E-02 0.03 [0.01 , 0.04] 7.0E-03 Ccl21a 0.14 [0.00 , 0.28] 9.1E-02 0.04 [-0.04 , 0.13] 3.7E-01 0.00 [-0.03 , 0.02] 8.5E-01 Mavs 0.13 [0.06 , 0.21] 5.4E-03 0.08 [0.04 , 0.12] 1.9E-03 0.04 [0.02 , 0.05] 3.3E-03 Anp32b 0.13 [0.09 , 0.17] 6.4E-05 0.06 [0.04 , 0.09] 6.6E-04 0.03 [0.02 , 0.04] 2.9E-06 Lamp2 0.13 [0.08 , 0.19] 6.7E-04 0.12 [0.09 , 0.15] 2.9E-05 0.05 [0.04 , 0.06] 1.8E-05 Il1r1 0.13 [0.03 , 0.22] 2.9E-02 0.15 [0.10 , 0.20] 8.0E-05 0.06 [0.04 , 0.07] 8.1E-05 Atg7 0.12 [0.05 , 0.19] 6.4E-03 0.10 [0.06 , 0.15] 1.2E-03 0.04 [0.02 , 0.05] 1.9E-03 Bcl10 0.12 [0.03 , 0.21] 2.7E-02 0.08 [0.04 , 0.11] 1.9E-03 0.03 [0.01 , 0.04] 1.3E-02 Cd40lg 0.11 [-0.10 , 0.33] 3.6E-01 0.01 [-0.14 , 0.16] 8.9E-01 -0.04 [-0.08 , 0.00] 1.2E-01 Psmb7 0.11 [0.01 , 0.21] 5.7E-02 0.04 [-0.01 , 0.10] 1.9E-01 0.03 [0.02 , 0.04] 8.7E-04 Tyk2 0.11 [-0.03 , 0.25] 1.6E-01 0.03 [-0.07 , 0.13] 6.2E-01 0.00 [-0.02 , 0.03] 8.1E-01 Irak4 0.11 [0.03 , 0.19] 2.6E-02 0.01 [-0.03 , 0.06] 6.4E-01 0.02 [0.00 , 0.04] 9.9E-02 Alcam 0.11 [0.03 , 0.18] 2.6E-02 0.11 [0.06 , 0.16] 1.2E-03 0.04 [0.03 , 0.06] 5.7E-05 Pparg 0.11 [-0.09 , 0.30] 3.5E-01 -0.02 [-0.11 , 0.07] 7.1E-01 0.02 [-0.03 , 0.06] 5.4E-01 Map3k1 0.10 [0.01 , 0.20] 7.3E-02 0.10 [0.05 , 0.16] 5.4E-03 0.03 [0.00 , 0.05] 8.0E-02 433 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tdo2 0.10 [-0.29 , 0.50] 6.5E-01 -0.11 [-0.30 , 0.09] 3.6E-01 0.15 [0.04 , 0.26] 3.6E-02 Rrad 0.10 [-0.14 , 0.34] 4.5E-01 0.21 [0.08 , 0.33] 9.7E-03 0.12 [0.05 , 0.18] 8.5E-03 Stat3 0.10 [0.03 , 0.17] 2.0E-02 0.08 [0.05 , 0.10] 5.2E-05 0.03 [0.03 , 0.04] 4.7E-06 Mapkapk2 0.10 [0.04 , 0.16] 6.8E-03 0.06 [0.03 , 0.09] 5.4E-03 0.05 [0.04 , 0.06] 2.0E-05 H2-Q10 0.10 [-0.30 , 0.50] 6.7E-01 0.03 [-0.17 , 0.23] 7.9E-01 0.11 [0.02 , 0.19] 5.2E-02 Klra17 0.10 [-0.14 , 0.34] 4.7E-01 -0.12 [-0.25 , 0.02] 1.4E-01 -0.04 [-0.09 , 0.01] 1.7E-01 Psen2 0.10 [-0.02 , 0.22] 1.6E-01 0.11 [0.04 , 0.18] 1.3E-02 0.03 [0.01 , 0.05] 8.8E-03 Tfeb 0.10 [-0.02 , 0.22] 1.7E-01 0.05 [-0.02 , 0.11] 2.4E-01 0.03 [0.00 , 0.05] 5.8E-02 Ccr2 0.10 [-0.10 , 0.29] 3.9E-01 0.09 [0.00 , 0.18] 9.7E-02 0.06 [0.02 , 0.11] 2.1E-02 Plaur 0.09 [-0.07 , 0.25] 3.3E-01 0.08 [0.02 , 0.15] 4.3E-02 0.03 [0.01 , 0.05] 2.1E-02 Cxcr2 0.09 [-0.20 , 0.37] 5.9E-01 0.04 [-0.14 , 0.21] 7.1E-01 0.06 [0.01 , 0.12] 7.3E-02 Ticam1 0.09 [-0.05 , 0.22] 2.8E-01 -0.02 [-0.09 , 0.05] 6.8E-01 0.01 [-0.01 , 0.04] 4.4E-01 Nfkb1 0.08 [0.00 , 0.16] 7.6E-02 0.04 [0.01 , 0.07] 4.3E-02 0.02 [0.01 , 0.03] 2.7E-03 Cd3eap 0.08 [-0.25 , 0.41] 6.8E-01 0.05 [-0.06 , 0.16] 4.4E-01 0.02 [-0.04 , 0.08] 5.7E-01 Trp53 0.08 [-0.03 , 0.18] 1.9E-01 0.05 [-0.03 , 0.13] 2.5E-01 0.02 [-0.01 , 0.04] 3.2E-01 Card11 0.07 [-0.08 , 0.22] 4.0E-01 0.00 [-0.08 , 0.08] 9.2E-01 -0.03 [-0.06 , 0.01] 2.9E-01 Atg5 0.07 [-0.03 , 0.17] 2.1E-01 0.01 [-0.05 , 0.06] 8.5E-01 0.01 [-0.01 , 0.04] 3.1E-01 434 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfrsf14 0.07 [-0.07 , 0.21] 4.0E-01 -0.05 [-0.11 , 0.02] 2.4E-01 -0.04 [-0.07 , -0.01] 3.9E-02 Il16 0.07 [-0.06 , 0.20] 3.6E-01 -0.04 [-0.12 , 0.05] 4.5E-01 -0.01 [-0.05 , 0.03] 7.1E-01 Rorc 0.07 [-0.12 , 0.25] 5.3E-01 -0.03 [-0.09 , 0.02] 3.4E-01 -0.01 [-0.04 , 0.01] 4.5E-01 Cd1d1 0.07 [-0.05 , 0.18] 3.2E-01 0.06 [0.00 , 0.13] 1.2E-01 0.05 [0.02 , 0.08] 1.2E-02 Igf2r 0.06 [-0.02 , 0.15] 2.1E-01 0.12 [0.06 , 0.17] 1.5E-03 0.02 [0.00 , 0.04] 1.3E-01 Ccr9 0.06 [-0.26 , 0.37] 7.6E-01 0.04 [-0.20 , 0.28] 7.8E-01 -0.03 [-0.09 , 0.04] 5.1E-01 Il1rap 0.06 [-0.02 , 0.13] 2.0E-01 0.07 [0.02 , 0.12] 2.5E-02 0.05 [0.03 , 0.06] 1.7E-05 Ceacam1 0.05 [-0.13 , 0.24] 6.1E-01 -0.03 [-0.10 , 0.04] 5.0E-01 0.02 [-0.01 , 0.05] 2.5E-01 Ccrl2 0.05 [-0.05 , 0.15] 3.8E-01 0.03 [-0.03 , 0.09] 4.5E-01 0.03 [0.01 , 0.05] 3.6E-02 Atf1 0.05 [-0.04 , 0.14] 3.4E-01 0.07 [0.02 , 0.13] 3.4E-02 0.04 [0.01 , 0.06] 1.3E-02 Mapk11 0.04 [-0.12 , 0.20] 6.4E-01 0.04 [-0.07 , 0.16] 5.0E-01 0.02 [-0.02 , 0.05] 4.8E-01 Fadd 0.04 [-0.12 , 0.20] 6.8E-01 0.06 [-0.03 , 0.14] 2.5E-01 0.04 [0.01 , 0.07] 3.9E-02 Masp2 0.03 [-0.31 , 0.37] 8.8E-01 -0.02 [-0.12 , 0.08] 7.7E-01 0.02 [-0.03 , 0.08] 5.4E-01 Slamf1 0.03 [-0.30 , 0.36] 8.9E-01 -0.11 [-0.29 , 0.07] 2.9E-01 -0.03 [-0.09 , 0.03] 4.5E-01 Cd9 0.03 [-0.06 , 0.12] 6.0E-01 0.04 [-0.03 , 0.12] 3.5E-01 0.05 [0.03 , 0.07] 3.3E-04 Nfatc1 0.03 [-0.09 , 0.14] 7.2E-01 -0.01 [-0.09 , 0.08] 8.9E-01 0.00 [-0.02 , 0.01] 6.9E-01 Egr3 0.02 [-0.36 , 0.40] 9.2E-01 0.13 [-0.03 , 0.30] 1.8E-01 -0.03 [-0.10 , 0.03] 4.2E-01 435 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Fn1 0.02 [-0.13 , 0.17] 8.0E-01 0.09 [0.02 , 0.17] 4.2E-02 0.00 [-0.03 , 0.03] 8.8E-01 Il18 0.02 [-0.15 , 0.18] 8.8E-01 -0.12 [-0.24 , 0.00] 9.7E-02 0.02 [-0.01 , 0.05] 3.7E-01 Tfrc 0.02 [-0.14 , 0.18] 8.8E-01 0.09 [-0.01 , 0.18] 1.3E-01 0.05 [0.02 , 0.08] 2.0E-02 Psen1 0.01 [-0.05 , 0.08] 7.0E-01 -0.02 [-0.05 , 0.02] 4.1E-01 0.01 [-0.01 , 0.02] 5.4E-01 Il12rb2 0.01 [-0.28 , 0.31] 9.5E-01 -0.07 [-0.17 , 0.04] 3.0E-01 0.00 [-0.05 , 0.04] 9.0E-01 Smn1 0.01 [-0.05 , 0.07] 7.2E-01 0.05 [0.02 , 0.08] 1.5E-02 0.02 [0.01 , 0.03] 1.4E-02 Atm 0.01 [-0.10 , 0.12] 8.6E-01 -0.05 [-0.11 , 0.01] 1.7E-01 -0.01 [-0.03 , 0.02] 6.1E-01 Il15ra 0.01 [-0.18 , 0.20] 9.2E-01 0.03 [-0.07 , 0.13] 5.6E-01 0.00 [-0.04 , 0.04] 9.5E-01 Tlr3 0.01 [-0.08 , 0.10] 8.6E-01 0.02 [-0.03 , 0.07] 4.5E-01 -0.02 [-0.05 , 0.01] 2.2E-01 Smad2 0.00 [-0.11 , 0.12] 9.5E-01 -0.02 [-0.11 , 0.07] 7.1E-01 0.01 [-0.01 , 0.04] 3.5E-01 Atg10 0.00 [-0.12 , 0.12] 9.6E-01 0.01 [-0.03 , 0.06] 6.0E-01 0.00 [-0.01 , 0.02] 7.5E-01 Maf 0.00 [-0.19 , 0.20] 9.9E-01 0.07 [-0.02 , 0.16] 1.8E-01 0.01 [-0.02 , 0.04] 5.1E-01 Il4ra 0.00 [-0.10 , 0.10] 1.0E+00 0.03 [-0.06 , 0.12] 5.6E-01 0.01 [-0.01 , 0.04] 3.4E-01 Masp1 0.00 [-0.16 , 0.16] 9.9E-01 -0.05 [-0.17 , 0.06] 4.4E-01 -0.03 [-0.08 , 0.02] 3.2E-01 Map2k2 0.00 [-0.05 , 0.05] 9.6E-01 0.04 [0.01 , 0.06] 1.3E-02 0.01 [0.00 , 0.02] 3.7E-02 Itga5 0.00 [-0.12 , 0.12] 9.9E-01 0.03 [-0.04 , 0.11] 4.2E-01 0.01 [-0.01 , 0.03] 5.3E-01 Runx1 0.00 [-0.05 , 0.05] 9.1E-01 0.03 [0.00 , 0.05] 7.4E-02 0.02 [0.01 , 0.03] 3.0E-03 436 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd40 -0.01 [-0.09 , 0.08] 9.2E-01 -0.02 [-0.07 , 0.03] 5.6E-01 0.02 [-0.01 , 0.04] 2.1E-01 Cx3cl1 -0.01 [-0.24 , 0.23] 9.6E-01 -0.06 [-0.17 , 0.04] 3.0E-01 -0.03 [-0.08 , 0.01] 2.5E-01 Atg12 -0.01 [-0.09 , 0.07] 8.1E-01 0.06 [0.01 , 0.12] 5.8E-02 0.02 [0.01 , 0.03] 7.8E-04 Fcer1a -0.02 [-0.32 , 0.29] 9.3E-01 -0.21 [-0.37 , -0.04] 3.9E-02 -0.08 [-0.13 , -0.02] 3.3E-02 Cd69 -0.02 [-0.31 , 0.27] 9.1E-01 0.00 [-0.13 , 0.13] 9.5E-01 -0.07 [-0.13 , -0.02] 3.4E-02 Map2k4 -0.02 [-0.08 , 0.04] 5.1E-01 0.01 [-0.03 , 0.06] 6.3E-01 0.02 [0.01 , 0.03] 5.1E-03 C1qbp -0.02 [-0.08 , 0.03] 4.6E-01 -0.02 [-0.05 , 0.01] 2.2E-01 0.00 [0.00 , 0.01] 3.9E-01 Ccl5 -0.03 [-0.23 , 0.17] 8.1E-01 -0.08 [-0.22 , 0.06] 3.1E-01 -0.05 [-0.10 , 0.00] 1.4E-01 Cebpb -0.03 [-0.18 , 0.11] 7.0E-01 -0.01 [-0.07 , 0.06] 8.5E-01 0.00 [-0.03 , 0.02] 7.5E-01 Mapk1 -0.03 [-0.09 , 0.03] 3.3E-01 0.00 [-0.02 , 0.03] 9.5E-01 0.01 [0.00 , 0.02] 1.9E-01 Col1a1 -0.04 [-0.20 , 0.13] 7.0E-01 0.02 [-0.07 , 0.10] 7.5E-01 -0.03 [-0.05 , 0.00] 9.5E-02 Stat6 -0.04 [-0.09 , 0.01] 2.1E-01 0.01 [-0.02 , 0.04] 6.0E-01 0.02 [0.01 , 0.04] 1.1E-02 Il15 -0.04 [-0.21 , 0.14] 7.0E-01 -0.05 [-0.14 , 0.05] 4.0E-01 -0.04 [-0.08 , 0.00] 9.9E-02 Ltbr -0.04 [-0.11 , 0.03] 3.3E-01 0.03 [-0.02 , 0.09] 3.3E-01 0.03 [0.01 , 0.04] 4.7E-03 Chuk -0.04 [-0.10 , 0.02] 2.1E-01 0.01 [-0.02 , 0.03] 7.5E-01 0.02 [0.01 , 0.03] 1.4E-02 Tbk1 -0.04 [-0.13 , 0.04] 4.1E-01 0.01 [-0.03 , 0.04] 7.8E-01 0.02 [0.01 , 0.03] 1.1E-02 Epcam -0.04 [-0.13 , 0.05] 4.1E-01 0.07 [0.04 , 0.11] 2.9E-03 0.03 [0.01 , 0.04] 1.9E-02 437 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Gpi1 -0.04 [-0.09 , 0.01] 1.3E-01 0.01 [-0.01 , 0.03] 5.6E-01 0.02 [0.01 , 0.04] 5.9E-03 Eomes -0.05 [-0.30 , 0.21] 7.6E-01 -0.14 [-0.30 , 0.03] 1.8E-01 -0.10 [-0.18 , -0.03] 3.6E-02 Ewsr1 -0.05 [-0.10 , 0.01] 1.4E-01 0.03 [0.01 , 0.06] 4.4E-02 0.02 [0.01 , 0.03] 3.6E-03 Tnfsf10 -0.05 [-0.20 , 0.10] 5.8E-01 -0.09 [-0.19 , 0.01] 1.2E-01 -0.04 [-0.08 , -0.01] 3.4E-02 Pla2g6 -0.05 [-0.17 , 0.08] 5.0E-01 -0.02 [-0.08 , 0.04] 5.9E-01 0.01 [-0.02 , 0.04] 5.3E-01 Ilf3 -0.05 [-0.16 , 0.05] 3.9E-01 -0.06 [-0.10 , -0.02] 2.5E-02 0.00 [-0.02 , 0.01] 7.7E-01 Thy1 -0.06 [-0.25 , 0.12] 5.6E-01 -0.08 [-0.17 , 0.01] 1.6E-01 -0.05 [-0.09 , 0.00] 7.5E-02 Map3k7 -0.06 [-0.12 , 0.00] 7.7E-02 -0.02 [-0.05 , 0.02] 4.4E-01 0.01 [0.00 , 0.02] 3.6E-01 Tgfbr2 -0.07 [-0.14 , 0.00] 1.1E-01 0.03 [-0.02 , 0.07] 3.1E-01 0.02 [0.01 , 0.04] 3.6E-02 Ikbkb -0.07 [-0.15 , 0.01] 1.4E-01 -0.06 [-0.09 , -0.02] 1.1E-02 -0.01 [-0.02 , 0.01] 5.1E-01 Il18rap -0.07 [-0.29 , 0.15] 5.7E-01 -0.01 [-0.07 , 0.06] 8.1E-01 -0.01 [-0.04 , 0.02] 5.2E-01 Ythdf2 -0.08 [-0.15 , -0.01] 6.2E-02 0.00 [-0.03 , 0.03] 8.6E-01 0.01 [0.00 , 0.02] 5.3E-02 Cd1d2 -0.08 [-0.30 , 0.14] 5.4E-01 -0.05 [-0.18 , 0.07] 4.5E-01 0.01 [-0.03 , 0.05] 7.0E-01 Mr1 -0.08 [-0.17 , 0.01] 1.3E-01 0.04 [0.00 , 0.09] 8.6E-02 0.04 [0.02 , 0.06] 5.0E-03 Ubc -0.08 [-0.17 , 0.00] 9.7E-02 0.02 [-0.02 , 0.06] 4.2E-01 0.00 [-0.02 , 0.01] 7.4E-01 Sigirr -0.08 [-0.17 , 0.00] 7.2E-02 -0.03 [-0.07 , 0.02] 2.8E-01 0.00 [-0.01 , 0.02] 8.3E-01 Ccl25 -0.09 [-0.28 , 0.11] 4.4E-01 0.03 [-0.05 , 0.11] 4.8E-01 0.04 [0.01 , 0.08] 5.4E-02 438 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd34 -0.09 [-0.19 , 0.01] 1.1E-01 -0.06 [-0.12 , -0.01] 6.2E-02 0.01 [-0.02 , 0.04] 4.8E-01 Tnfrsf11b -0.09 [-0.34 , 0.16] 5.2E-01 0.13 [0.01 , 0.25] 7.2E-02 -0.02 [-0.07 , 0.04] 6.5E-01 Rela -0.09 [-0.21 , 0.03] 1.8E-01 0.00 [-0.05 , 0.06] 9.2E-01 0.00 [-0.02 , 0.02] 8.5E-01 Cyld -0.09 [-0.28 , 0.09] 3.9E-01 -0.02 [-0.10 , 0.06] 6.4E-01 0.00 [-0.03 , 0.04] 9.5E-01 Pin1 -0.09 [-0.25 , 0.06] 3.0E-01 0.02 [-0.05 , 0.09] 6.0E-01 0.01 [-0.02 , 0.03] 5.7E-01 Bcl2 -0.09 [-0.26 , 0.07] 3.2E-01 -0.09 [-0.17 , 0.00] 9.1E-02 -0.03 [-0.06 , 0.00] 1.2E-01 Elk1 -0.10 [-0.33 , 0.14] 4.8E-01 0.01 [-0.07 , 0.10] 7.8E-01 0.03 [0.00 , 0.06] 1.5E-01 Tab1 -0.10 [-0.18 , -0.01] 4.9E-02 0.00 [-0.04 , 0.05] 8.7E-01 0.01 [-0.01 , 0.02] 5.3E-01 Nup107 -0.10 [-0.18 , -0.01] 5.5E-02 -0.04 [-0.08 , 0.00] 1.1E-01 0.00 [-0.01 , 0.01] 8.0E-01 Ambp -0.11 [-1.01 , 0.80] 8.5E-01 -0.22 [-0.61 , 0.16] 3.2E-01 0.15 [-0.06 , 0.36] 2.5E-01 Fyn -0.11 [-0.17 , -0.04] 1.1E-02 -0.08 [-0.12 , -0.05] 7.3E-04 -0.02 [-0.04 , -0.01] 2.9E-02 Nfatc3 -0.11 [-0.18 , -0.04] 1.1E-02 -0.08 [-0.14 , -0.03] 1.7E-02 0.00 [-0.02 , 0.01] 4.8E-01 Itch -0.12 [-0.17 , -0.06] 3.1E-03 -0.07 [-0.10 , -0.04] 1.6E-03 0.00 [-0.02 , 0.01] 6.2E-01 Icam4 -0.12 [-0.40 , 0.17] 4.7E-01 -0.09 [-0.24 , 0.06] 2.9E-01 -0.01 [-0.06 , 0.04] 7.2E-01 Tirap -0.12 [-0.23 , -0.01] 5.8E-02 -0.01 [-0.06 , 0.03] 6.1E-01 0.01 [-0.02 , 0.03] 6.6E-01 Cd38 -0.12 [-0.20 , -0.04] 2.0E-02 -0.14 [-0.19 , -0.09] 3.0E-04 -0.04 [-0.06 , -0.02] 1.1E-03 Anxa1 -0.12 [-0.22 , -0.02] 4.8E-02 -0.04 [-0.12 , 0.03] 3.6E-01 0.01 [-0.01 , 0.03] 4.2E-01 439 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Nfkbia -0.12 [-0.30 , 0.06] 2.5E-01 -0.03 [-0.12 , 0.05] 5.2E-01 -0.02 [-0.05 , 0.01] 3.1E-01 Psmd7 -0.12 [-0.16 , -0.08] 7.5E-05 -0.02 [-0.04 , 0.00] 1.6E-01 0.00 [-0.01 , 0.01] 7.6E-01 Cd47 -0.13 [-0.18 , -0.07] 1.2E-03 -0.09 [-0.16 , -0.01] 6.4E-02 -0.01 [-0.02 , 0.00] 8.9E-02 Erbb2 -0.13 [-0.24 , -0.02] 4.8E-02 0.08 [0.00 , 0.17] 7.9E-02 0.01 [-0.01 , 0.04] 3.0E-01 Yy1 -0.14 [-0.20 , -0.07] 1.9E-03 -0.01 [-0.05 , 0.03] 5.6E-01 0.01 [-0.01 , 0.02] 3.6E-01 Cxcl14 -0.14 [-0.32 , 0.04] 1.8E-01 -0.05 [-0.16 , 0.06] 4.3E-01 0.00 [-0.04 , 0.04] 9.4E-01 Itgb3 -0.14 [-0.34 , 0.06] 2.1E-01 -0.02 [-0.12 , 0.07] 6.9E-01 0.00 [-0.03 , 0.03] 8.1E-01 Traf6 -0.14 [-0.24 , -0.05] 1.4E-02 -0.04 [-0.08 , 0.00] 6.4E-02 -0.01 [-0.03 , 0.00] 2.1E-01 Jak1 -0.15 [-0.18 , -0.11] 5.9E-06 -0.08 [-0.11 , -0.05] 1.9E-04 -0.02 [-0.03 , -0.01] 1.6E-02 Colec12 -0.15 [-0.23 , -0.07] 5.0E-03 -0.10 [-0.16 , -0.05] 4.9E-03 -0.03 [-0.05 , -0.01] 1.3E-02 Col4a1 -0.15 [-0.33 , 0.03] 1.6E-01 0.05 [-0.07 , 0.17] 4.5E-01 0.04 [0.00 , 0.08] 9.8E-02 Reps1 -0.15 [-0.21 , -0.09] 5.1E-04 -0.07 [-0.10 , -0.04] 1.4E-03 -0.01 [-0.02 , 0.01] 4.1E-01 Tbx21 -0.15 [-0.36 , 0.06] 2.2E-01 -0.23 [-0.43 , -0.03] 5.7E-02 -0.05 [-0.11 , 0.01] 1.5E-01 Cd200 -0.15 [-0.23 , -0.08] 2.4E-03 -0.05 [-0.08 , -0.02] 7.0E-03 -0.01 [-0.02 , 0.00] 3.1E-01 Ifngr1 -0.15 [-0.25 , -0.06] 1.2E-02 -0.08 [-0.12 , -0.04] 2.4E-03 0.02 [0.00 , 0.05] 1.6E-01 Cd55 -0.16 [-0.26 , -0.06] 8.5E-03 -0.05 [-0.11 , 0.02] 2.4E-01 0.01 [-0.01 , 0.03] 4.0E-01 Irf2 -0.16 [-0.22 , -0.11] 1.7E-04 -0.08 [-0.11 , -0.05] 3.1E-04 -0.01 [-0.02 , 0.00] 2.4E-01 440 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Entpd1 -0.17 [-0.28 , -0.06] 1.3E-02 -0.10 [-0.17 , -0.03] 1.7E-02 -0.03 [-0.06 , 0.00] 9.8E-02 Cd8b1 -0.18 [-0.48 , 0.13] 3.2E-01 -0.19 [-0.33 , -0.06] 2.1E-02 -0.10 [-0.17 , -0.03] 2.0E-02 Timd4 -0.18 [-0.55 , 0.20] 4.1E-01 -0.20 [-0.45 , 0.05] 1.9E-01 -0.11 [-0.17 , -0.04] 1.3E-02 Flt3l -0.18 [-0.32 , -0.03] 4.1E-02 -0.07 [-0.14 , 0.01] 1.4E-01 -0.02 [-0.05 , 0.01] 3.6E-01 Mapk8 -0.18 [-0.28 , -0.08] 5.0E-03 -0.05 [-0.08 , -0.02] 1.8E-02 -0.01 [-0.03 , 0.00] 2.4E-01 Cdh1 -0.18 [-0.27 , -0.09] 3.1E-03 -0.03 [-0.07 , 0.01] 2.4E-01 0.00 [-0.01 , 0.02] 7.9E-01 Tollip -0.18 [-0.28 , -0.09] 3.8E-03 -0.05 [-0.09 , 0.00] 7.2E-02 0.01 [-0.01 , 0.02] 4.9E-01 Rps6 -0.19 [-0.31 , -0.06] 1.4E-02 -0.11 [-0.16 , -0.05] 4.5E-03 -0.02 [-0.03 , 0.00] 5.1E-02 Mapk3 -0.19 [-0.33 , -0.05] 2.7E-02 -0.06 [-0.16 , 0.04] 3.2E-01 -0.01 [-0.03 , 0.02] 6.8E-01 Smad3 -0.19 [-0.27 , -0.11] 8.5E-04 0.00 [-0.05 , 0.05] 9.4E-01 -0.01 [-0.03 , 0.01] 6.3E-01 Cd2 -0.19 [-0.40 , 0.02] 1.3E-01 -0.03 [-0.11 , 0.04] 4.5E-01 -0.05 [-0.09 , 0.00] 8.0E-02 Atg16l1 -0.19 [-0.24 , -0.14] 1.7E-05 -0.06 [-0.10 , -0.02] 1.9E-02 -0.01 [-0.02 , 0.00] 3.2E-01 Ikbkg -0.19 [-0.30 , -0.09] 4.1E-03 -0.07 [-0.13 , -0.02] 3.1E-02 -0.01 [-0.03 , 0.02] 6.6E-01 Il22ra1 -0.20 [-0.57 , 0.18] 3.6E-01 -0.06 [-0.19 , 0.08] 4.6E-01 -0.04 [-0.09 , 0.02] 2.6E-01 Bmi1 -0.20 [-0.26 , -0.14] 4.2E-05 -0.07 [-0.12 , -0.02] 2.5E-02 0.00 [-0.02 , 0.01] 8.5E-01 Mapk14 -0.20 [-0.29 , -0.12] 7.1E-04 -0.12 [-0.16 , -0.08] 2.1E-04 -0.02 [-0.04 , -0.01] 2.7E-02 Cd164 -0.21 [-0.27 , -0.15] 2.0E-05 -0.06 [-0.09 , -0.02] 1.2E-02 -0.01 [-0.02 , 0.01] 4.9E-01 441 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd99 -0.21 [-0.37 , -0.05] 2.8E-02 -0.05 [-0.17 , 0.07] 4.8E-01 0.00 [-0.03 , 0.03] 9.5E-01 Irak1 -0.21 [-0.27 , -0.16] 7.1E-06 -0.09 [-0.13 , -0.05] 2.0E-03 -0.03 [-0.04 , -0.01] 1.7E-03 Ecsit -0.21 [-0.39 , -0.04] 4.0E-02 -0.11 [-0.20 , -0.01] 6.9E-02 -0.05 [-0.09 , -0.01] 3.8E-02 Ep300 -0.21 [-0.29 , -0.14] 1.2E-04 -0.07 [-0.11 , -0.03] 3.8E-03 -0.02 [-0.03 , 0.00] 1.1E-01 Il12a -0.22 [-0.38 , -0.05] 3.1E-02 -0.06 [-0.17 , 0.05] 3.7E-01 -0.02 [-0.05 , 0.01] 3.2E-01 Il18r1 -0.22 [-0.32 , -0.12] 8.7E-04 -0.10 [-0.15 , -0.06] 1.4E-03 -0.02 [-0.04 , 0.00] 1.1E-01 Tnfsf12 -0.22 [-0.34 , -0.10] 4.1E-03 -0.04 [-0.12 , 0.03] 3.2E-01 -0.02 [-0.04 , 0.01] 2.2E-01 Atf2 -0.23 [-0.29 , -0.17] 6.5E-06 -0.07 [-0.11 , -0.04] 1.2E-03 -0.01 [-0.02 , 0.01] 5.4E-01 Sell -0.23 [-0.44 , -0.02] 6.9E-02 -0.12 [-0.23 , -0.02] 5.9E-02 -0.04 [-0.08 , 0.01] 1.8E-01 Map3k5 -0.23 [-0.32 , -0.14] 4.5E-04 -0.15 [-0.18 , -0.11] 4.6E-06 -0.03 [-0.05 , -0.02] 2.0E-03 Tnfrsf1a -0.23 [-0.33 , -0.13] 7.8E-04 -0.06 [-0.13 , 0.01] 1.6E-01 0.00 [-0.02 , 0.02] 9.6E-01 Irf3 -0.23 [-0.30 , -0.16] 4.7E-05 -0.12 [-0.15 , -0.08] 5.7E-05 -0.02 [-0.03 , -0.01] 5.8E-03 Tmed1 -0.23 [-0.33 , -0.14] 6.7E-04 -0.13 [-0.17 , -0.09] 3.6E-05 -0.03 [-0.05 , -0.02] 6.8E-03 Hmgb1 -0.24 [-0.31 , -0.16] 3.7E-05 -0.13 [-0.17 , -0.10] 3.6E-05 -0.03 [-0.04 , -0.02] 2.3E-04 Stat5b -0.24 [-0.31 , -0.16] 4.0E-05 -0.13 [-0.17 , -0.09] 8.0E-05 -0.03 [-0.04 , -0.01] 1.1E-02 Gata3 -0.24 [-0.41 , -0.07] 1.9E-02 -0.06 [-0.14 , 0.02] 1.9E-01 -0.03 [-0.06 , 0.01] 2.8E-01 Zfp13 -0.24 [-0.45 , -0.04] 4.5E-02 -0.14 [-0.23 , -0.05] 1.4E-02 0.00 [-0.04 , 0.05] 9.2E-01 442 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cfh -0.25 [-0.34 , -0.15] 3.7E-04 -0.09 [-0.15 , -0.04] 9.9E-03 -0.01 [-0.03 , 0.02] 7.1E-01 Prg2 -0.25 [-0.52 , 0.02] 1.1E-01 -0.19 [-0.33 , -0.05] 2.8E-02 -0.07 [-0.13 , -0.02] 4.6E-02 App -0.25 [-0.35 , -0.15] 3.5E-04 -0.07 [-0.12 , -0.02] 3.2E-02 -0.02 [-0.04 , 0.01] 3.1E-01 Pvr -0.25 [-0.36 , -0.14] 1.2E-03 -0.04 [-0.13 , 0.05] 4.7E-01 -0.01 [-0.03 , 0.01] 3.1E-01 Tgfb3 -0.25 [-0.45 , -0.06] 3.4E-02 0.00 [-0.10 , 0.11] 9.5E-01 0.01 [-0.04 , 0.06] 7.5E-01 Ccnd3 -0.26 [-0.41 , -0.11] 6.8E-03 -0.21 [-0.26 , -0.16] 1.0E-05 -0.05 [-0.07 , -0.03] 4.9E-04 Ptgs2 -0.26 [-0.50 , -0.02] 6.6E-02 -0.12 [-0.21 , -0.04] 2.2E-02 -0.01 [-0.05 , 0.02] 6.1E-01 Itga6 -0.26 [-0.35 , -0.17] 1.2E-04 -0.11 [-0.16 , -0.06] 1.8E-03 -0.02 [-0.04 , 0.01] 2.5E-01 Msln -0.26 [-0.57 , 0.04] 1.4E-01 -0.23 [-0.37 , -0.09] 1.2E-02 -0.04 [-0.10 , 0.02] 3.1E-01 Lrp1 -0.27 [-0.39 , -0.14] 1.5E-03 -0.06 [-0.12 , 0.01] 1.4E-01 0.00 [-0.03 , 0.03] 9.4E-01 C7 -0.27 [-0.39 , -0.16] 5.9E-04 -0.08 [-0.15 , -0.01] 7.6E-02 0.00 [-0.03 , 0.02] 7.7E-01 Gtf3c1 -0.28 [-0.35 , -0.20] 1.5E-05 -0.08 [-0.12 , -0.04] 3.5E-03 -0.02 [-0.04 , 0.00] 9.7E-02 Fos -0.28 [-0.66 , 0.11] 2.1E-01 -0.05 [-0.27 , 0.16] 6.8E-01 -0.13 [-0.20 , -0.05] 1.2E-02 Abl1 -0.28 [-0.40 , -0.16] 8.5E-04 -0.07 [-0.14 , -0.01] 7.5E-02 -0.03 [-0.05 , 0.00] 5.8E-02 Smpd3 -0.28 [-0.55 , -0.01] 7.6E-02 -0.20 [-0.31 , -0.09] 5.8E-03 -0.08 [-0.13 , -0.02] 3.5E-02 Il6st -0.29 [-0.36 , -0.21] 1.3E-05 -0.08 [-0.13 , -0.02] 2.6E-02 -0.02 [-0.04 , 0.00] 1.9E-01 Crebbp -0.29 [-0.37 , -0.21] 1.7E-05 -0.10 [-0.14 , -0.06] 3.4E-04 -0.02 [-0.04 , -0.01] 2.3E-02 443 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Creb1 -0.29 [-0.38 , -0.21] 2.4E-05 -0.17 [-0.21 , -0.13] 5.2E-06 -0.04 [-0.06 , -0.02] 1.1E-02 Dpp4 -0.31 [-0.40 , -0.23] 1.5E-05 -0.12 [-0.19 , -0.05] 8.0E-03 -0.03 [-0.05 , -0.01] 1.6E-02 F13a1 -0.32 [-0.64 , 0.01] 9.5E-02 0.16 [0.06 , 0.25] 9.0E-03 0.02 [-0.03 , 0.08] 4.9E-01 Nrp1 -0.32 [-0.38 , -0.26] 5.2E-07 -0.15 [-0.20 , -0.10] 1.1E-04 -0.03 [-0.06 , -0.01] 1.3E-02 Cd81 -0.32 [-0.38 , -0.27] 2.5E-07 -0.16 [-0.20 , -0.11] 6.8E-05 -0.04 [-0.06 , -0.02] 3.1E-03 Itgb1 -0.33 [-0.43 , -0.23] 4.9E-05 -0.14 [-0.19 , -0.10] 5.3E-05 -0.02 [-0.04 , 0.00] 7.7E-02 Cd36 -0.34 [-0.44 , -0.23] 5.5E-05 -0.10 [-0.18 , -0.03] 2.5E-02 -0.01 [-0.03 , 0.01] 5.1E-01 Fez1 -0.34 [-0.62 , -0.06] 4.5E-02 -0.28 [-0.40 , -0.17] 7.1E-04 -0.04 [-0.10 , 0.02] 3.0E-01 Smad4 -0.34 [-0.44 , -0.25] 1.9E-05 -0.12 [-0.16 , -0.07] 3.9E-04 -0.02 [-0.04 , 0.00] 6.8E-02 Nfatc4 -0.34 [-0.46 , -0.22] 1.4E-04 -0.04 [-0.16 , 0.07] 4.9E-01 -0.03 [-0.06 , 0.00] 7.6E-02 Ccr7 -0.35 [-0.58 , -0.11] 1.7E-02 -0.05 [-0.14 , 0.03] 2.9E-01 -0.05 [-0.09 , -0.01] 4.7E-02 Il22ra2 -0.35 [-0.64 , -0.06] 4.5E-02 -0.15 [-0.28 , -0.01] 7.3E-02 -0.08 [-0.14 , -0.02] 2.8E-02 Ncam1 -0.35 [-0.60 , -0.10] 2.1E-02 -0.03 [-0.16 , 0.09] 6.2E-01 -0.04 [-0.09 , 0.01] 2.4E-01 Nt5e -0.36 [-0.46 , -0.25] 2.6E-05 -0.14 [-0.25 , -0.04] 2.6E-02 -0.02 [-0.04 , -0.01] 4.2E-02 Ets1 -0.36 [-0.46 , -0.26] 1.8E-05 -0.14 [-0.21 , -0.08] 1.7E-03 -0.03 [-0.05 , 0.00] 1.1E-01 Txnip -0.37 [-0.50 , -0.24] 1.7E-04 -0.12 [-0.17 , -0.06] 1.6E-03 -0.09 [-0.12 , -0.06] 8.7E-05 Mst1r -0.37 [-0.66 , -0.08] 3.4E-02 -0.31 [-0.48 , -0.13] 8.0E-03 -0.11 [-0.17 , -0.06] 3.3E-03 444 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd160 -0.37 [-0.88 , 0.15] 2.1E-01 -0.17 [-0.35 , 0.02] 1.4E-01 -0.06 [-0.14 , 0.02] 1.9E-01 Itga2 -0.37 [-0.46 , -0.29] 3.6E-06 -0.18 [-0.26 , -0.09] 2.6E-03 -0.06 [-0.10 , -0.03] 6.0E-03 Abcb1a -0.38 [-0.47 , -0.28] 8.6E-06 -0.11 [-0.20 , -0.02] 4.2E-02 0.03 [0.01 , 0.06] 3.4E-02 Thbs1 -0.38 [-0.68 , -0.07] 3.9E-02 -0.06 [-0.29 , 0.17] 6.4E-01 -0.04 [-0.11 , 0.04] 4.3E-01 Bcl6 -0.38 [-0.55 , -0.21] 1.3E-03 -0.22 [-0.28 , -0.17] 9.6E-06 -0.04 [-0.08 , -0.01] 5.7E-02 Dusp6 -0.38 [-0.56 , -0.21] 1.1E-03 -0.22 [-0.33 , -0.10] 4.2E-03 -0.04 [-0.07 , 0.00] 1.1E-01 Cspg4 -0.39 [-0.63 , -0.14] 1.1E-02 -0.14 [-0.28 , 0.01] 1.2E-01 -0.01 [-0.05 , 0.04] 8.6E-01 Ccl27a -0.40 [-0.56 , -0.23] 7.0E-04 -0.22 [-0.39 , -0.05] 3.7E-02 -0.04 [-0.08 , 0.00] 8.8E-02 Pnma1 -0.40 [-0.69 , -0.12] 2.2E-02 -0.03 [-0.19 , 0.12] 7.0E-01 0.01 [-0.04 , 0.07] 6.8E-01 Fasl -0.40 [-0.75 , -0.06] 4.7E-02 -0.26 [-0.42 , -0.11] 7.5E-03 -0.11 [-0.17 , -0.06] 4.8E-03 Ptgdr2 -0.41 [-0.80 , -0.01] 8.4E-02 -0.04 [-0.23 , 0.16] 7.6E-01 -0.05 [-0.12 , 0.02] 2.5E-01 Nod1 -0.41 [-0.56 , -0.26] 2.5E-04 -0.16 [-0.23 , -0.10] 5.7E-04 -0.04 [-0.06 , -0.02] 2.5E-03 Map4k2 -0.42 [-0.54 , -0.30] 1.7E-05 -0.24 [-0.31 , -0.17] 6.5E-05 -0.07 [-0.10 , -0.03] 4.2E-03 Hras -0.42 [-0.82 , -0.02] 7.6E-02 -0.17 [-0.35 , 0.01] 1.2E-01 -0.06 [-0.12 , 0.00] 1.1E-01 Egr1 -0.42 [-0.97 , 0.13] 1.9E-01 -0.23 [-0.53 , 0.06] 1.9E-01 -0.15 [-0.26 , -0.03] 4.5E-02 Cd7 -0.42 [-0.61 , -0.23] 1.3E-03 -0.27 [-0.38 , -0.16] 5.2E-04 -0.09 [-0.14 , -0.04] 1.0E-02 Itk -0.42 [-0.72 , -0.12] 2.2E-02 -0.18 [-0.34 , -0.02] 6.5E-02 -0.08 [-0.12 , -0.03] 1.2E-02 445 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il17b -0.42 [-0.74 , -0.11] 2.8E-02 -0.27 [-0.43 , -0.11] 8.6E-03 n.d [n.d , n.d] n.d Foxj1 -0.43 [-0.65 , -0.20] 3.9E-03 -0.08 [-0.17 , 0.02] 1.7E-01 -0.02 [-0.06 , 0.02] 4.5E-01 Dock9 -0.43 [-0.56 , -0.29] 6.5E-05 -0.17 [-0.24 , -0.09] 1.5E-03 -0.04 [-0.06 , -0.01] 5.3E-02 Tcf7 -0.43 [-0.55 , -0.31] 2.1E-05 -0.24 [-0.32 , -0.16] 1.2E-04 -0.09 [-0.12 , -0.06] 8.4E-05 Gzma -0.44 [-0.73 , -0.16] 1.3E-02 -0.37 [-0.54 , -0.19] 1.9E-03 -0.12 [-0.19 , -0.05] 1.1E-02 Ms4a2 -0.45 [-0.87 , -0.04] 6.4E-02 -0.53 [-0.78 , -0.27] 2.6E-03 -0.18 [-0.27 , -0.09] 4.2E-03 Pecam1 -0.46 [-0.59 , -0.32] 3.3E-05 -0.25 [-0.33 , -0.17] 9.0E-05 -0.05 [-0.07 , -0.02] 4.7E-03 Akt3 -0.47 [-0.61 , -0.34] 1.7E-05 -0.21 [-0.30 , -0.13] 6.3E-04 -0.02 [-0.05 , 0.01] 1.8E-01 Mfge8 -0.49 [-0.60 , -0.37] 5.1E-06 -0.25 [-0.31 , -0.20] 3.4E-06 -0.07 [-0.09 , -0.05] 1.2E-05 Prf1 -0.49 [-0.79 , -0.18] 1.1E-02 -0.36 [-0.56 , -0.16] 5.7E-03 -0.12 [-0.19 , -0.05] 1.1E-02 Il6ra -0.49 [-0.71 , -0.28] 8.3E-04 -0.21 [-0.28 , -0.14] 1.6E-04 -0.05 [-0.08 , -0.02] 2.2E-02 Kit -0.49 [-0.60 , -0.39] 2.2E-06 -0.29 [-0.40 , -0.18] 3.6E-04 -0.10 [-0.13 , -0.07] 1.7E-04 Vwf -0.50 [-0.72 , -0.28] 8.8E-04 -0.21 [-0.29 , -0.13] 3.4E-04 -0.04 [-0.08 , 0.00] 8.1E-02 Cd97 -0.50 [-0.65 , -0.35] 3.3E-05 -0.26 [-0.34 , -0.19] 2.5E-05 -0.06 [-0.09 , -0.02] 9.8E-03 Dll4 -0.50 [-0.65 , -0.34] 4.9E-05 -0.28 [-0.42 , -0.14] 2.9E-03 -0.10 [-0.14 , -0.06] 6.3E-04 Jam3 -0.50 [-0.65 , -0.36] 2.2E-05 -0.23 [-0.34 , -0.12] 1.8E-03 -0.07 [-0.11 , -0.04] 8.8E-04 Rora -0.51 [-0.62 , -0.39] 3.0E-06 -0.20 [-0.24 , -0.17] 7.5E-07 -0.04 [-0.06 , -0.01] 1.4E-02 446 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Snai1 -0.51 [-0.96 , -0.06] 5.5E-02 -0.13 [-0.32 , 0.06] 2.4E-01 -0.01 [-0.08 , 0.06] 8.1E-01 Egfr -0.52 [-0.67 , -0.36] 4.4E-05 -0.29 [-0.36 , -0.21] 1.9E-05 -0.07 [-0.09 , -0.04] 7.6E-04 Tie1 -0.52 [-0.66 , -0.37] 2.3E-05 -0.22 [-0.30 , -0.13] 6.0E-04 -0.04 [-0.07 , -0.01] 5.8E-02 F2rl1 -0.52 [-0.74 , -0.30] 6.2E-04 -0.24 [-0.32 , -0.15] 2.2E-04 -0.03 [-0.06 , 0.00] 9.7E-02 Klra4 -0.52 [-0.77 , -0.27] 1.8E-03 -0.27 [-0.41 , -0.13] 4.4E-03 -0.11 [-0.16 , -0.06] 2.4E-03 Icam2 -0.52 [-0.70 , -0.35] 9.5E-05 -0.29 [-0.36 , -0.22] 1.1E-05 -0.05 [-0.08 , -0.02] 1.4E-02 Mertk -0.53 [-0.77 , -0.29] 1.2E-03 -0.23 [-0.33 , -0.13] 1.3E-03 -0.06 [-0.09 , -0.03] 3.4E-03 Dusp4 -0.54 [-0.89 , -0.20] 1.2E-02 -0.16 [-0.36 , 0.04] 1.9E-01 -0.11 [-0.21 , -0.02] 5.3E-02 Tgfb2 -0.54 [-0.71 , -0.37] 6.4E-05 -0.13 [-0.24 , -0.02] 5.6E-02 -0.03 [-0.09 , 0.02] 3.4E-01 Cma1 -0.55 [-0.94 , -0.16] 2.2E-02 -0.19 [-0.41 , 0.02] 1.3E-01 -0.13 [-0.23 , -0.03] 4.0E-02 Ncr1 -0.55 [-0.84 , -0.27] 3.3E-03 -0.36 [-0.52 , -0.20] 1.4E-03 -0.10 [-0.17 , -0.03] 2.0E-02 Mcam -0.55 [-0.68 , -0.43] 2.6E-06 -0.27 [-0.36 , -0.18] 1.3E-04 -0.05 [-0.09 , -0.02] 2.8E-02 Vegfc -0.56 [-0.83 , -0.28] 2.3E-03 -0.29 [-0.39 , -0.20] 1.1E-04 -0.03 [-0.08 , 0.02] 3.0E-01 Klrk1 -0.57 [-0.76 , -0.38] 9.2E-05 -0.28 [-0.42 , -0.15] 2.1E-03 -0.14 [-0.17 , -0.10] 1.3E-05 Tlr5 -0.58 [-0.82 , -0.35] 5.0E-04 -0.20 [-0.34 , -0.06] 2.5E-02 -0.09 [-0.15 , -0.03] 2.3E-02 Notch1 -0.58 [-0.72 , -0.45] 2.9E-06 -0.27 [-0.35 , -0.18] 9.0E-05 -0.08 [-0.11 , -0.05] 2.1E-04 Mme -0.59 [-0.74 , -0.43] 1.1E-05 -0.23 [-0.32 , -0.14] 5.3E-04 -0.05 [-0.08 , -0.02] 1.9E-02 447 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Txk -0.59 [-0.75 , -0.44] 7.5E-06 -0.31 [-0.41 , -0.22] 6.5E-05 -0.10 [-0.13 , -0.06] 3.1E-04 Mill2 -0.60 [-0.73 , -0.47] 1.9E-06 -0.30 [-0.37 , -0.24] 1.8E-06 -0.07 [-0.11 , -0.04] 2.0E-03 Hspb2 -0.60 [-0.81 , -0.39] 1.4E-04 -0.25 [-0.33 , -0.17] 9.6E-05 -0.06 [-0.09 , -0.02] 1.2E-02 Cd163 -0.61 [-0.98 , -0.24] 9.5E-03 -0.01 [-0.19 , 0.18] 9.4E-01 -0.06 [-0.14 , 0.02] 1.9E-01 Klrc1 -0.61 [-0.84 , -0.39] 1.9E-04 -0.41 [-0.57 , -0.24] 6.4E-04 -0.12 [-0.17 , -0.08] 3.6E-04 Angpt2 -0.62 [-0.74 , -0.50] 7.1E-07 -0.30 [-0.40 , -0.21] 6.9E-05 -0.06 [-0.09 , -0.03] 2.9E-03 Itgb4 -0.63 [-0.77 , -0.49] 2.7E-06 -0.23 [-0.32 , -0.14] 4.1E-04 -0.09 [-0.12 , -0.06] 1.3E-04 Igf1r -0.63 [-0.80 , -0.46] 1.4E-05 -0.26 [-0.35 , -0.17] 2.7E-04 -0.07 [-0.10 , -0.04] 7.7E-04 Cmklr1 -0.63 [-0.76 , -0.50] 1.0E-06 -0.24 [-0.32 , -0.17] 8.9E-05 -0.04 [-0.08 , -0.01] 4.2E-02 Prkce -0.63 [-0.79 , -0.47] 7.4E-06 -0.38 [-0.47 , -0.29] 6.3E-06 -0.09 [-0.12 , -0.06] 3.3E-04 Cdh5 -0.66 [-0.84 , -0.47] 2.0E-05 -0.29 [-0.39 , -0.19] 1.9E-04 -0.05 [-0.08 , -0.02] 1.9E-02 Itga1 -0.66 [-0.82 , -0.50] 4.8E-06 -0.31 [-0.40 , -0.23] 2.8E-05 -0.06 [-0.09 , -0.03] 5.0E-03 Fas -0.66 [-0.84 , -0.49] 8.9E-06 -0.32 [-0.38 , -0.26] 6.6E-07 -0.08 [-0.11 , -0.05] 6.5E-04 Lrrn3 -0.67 [-0.82 , -0.51] 3.6E-06 -0.33 [-0.42 , -0.25] 1.3E-05 -0.11 [-0.14 , -0.08] 3.5E-05 Pdgfrb -0.68 [-0.83 , -0.53] 2.6E-06 -0.25 [-0.35 , -0.16] 4.0E-04 -0.06 [-0.10 , -0.02] 1.6E-02 Kdr -0.68 [-0.81 , -0.56] 3.5E-07 -0.33 [-0.41 , -0.24] 1.3E-05 -0.09 [-0.13 , -0.06] 5.9E-04 Tek -0.69 [-0.85 , -0.52] 4.3E-06 -0.32 [-0.42 , -0.22] 6.9E-05 -0.07 [-0.11 , -0.04] 2.0E-03 448 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Klrd1 -0.69 [-0.86 , -0.52] 5.1E-06 -0.36 [-0.47 , -0.26] 5.3E-05 -0.12 [-0.17 , -0.06] 2.0E-03 Tal1 -0.69 [-0.91 , -0.47] 5.2E-05 -0.29 [-0.42 , -0.16] 1.8E-03 -0.08 [-0.11 , -0.05] 4.5E-04 Vegfa -0.70 [-0.83 , -0.57] 5.0E-07 -0.36 [-0.46 , -0.25] 5.4E-05 -0.09 [-0.12 , -0.05] 8.2E-04 Ccl24 -0.72 [-1.16 , -0.28] 1.0E-02 -0.21 [-0.39 , -0.04] 4.6E-02 -0.11 [-0.20 , -0.01] 7.3E-02 Hsd11b1 -0.73 [-0.92 , -0.54] 9.7E-06 -0.35 [-0.42 , -0.27] 4.0E-06 -0.06 [-0.10 , -0.02] 1.3E-02 Thbd -0.74 [-0.91 , -0.58] 2.7E-06 -0.45 [-0.55 , -0.36] 1.5E-06 -0.11 [-0.14 , -0.07] 1.7E-04 Jun -0.74 [-0.88 , -0.60] 5.0E-07 -0.37 [-0.46 , -0.28] 4.7E-06 -0.08 [-0.11 , -0.06] 1.2E-04 Tnfrsf12a -0.75 [-0.98 , -0.51] 5.6E-05 -0.31 [-0.44 , -0.18] 8.7E-04 -0.07 [-0.12 , -0.03] 1.3E-02 Eng -0.75 [-0.98 , -0.53] 3.7E-05 -0.45 [-0.55 , -0.34] 7.5E-06 -0.15 [-0.18 , -0.11] 1.4E-05 Bcl2l1 -0.77 [-1.01 , -0.53] 5.7E-05 -0.36 [-0.45 , -0.27] 1.1E-05 -0.10 [-0.14 , -0.06] 1.1E-03 Syt17 -0.78 [-1.09 , -0.48] 3.4E-04 -0.29 [-0.41 , -0.16] 1.4E-03 -0.08 [-0.14 , -0.03] 1.3E-02 Cdkn1a -0.81 [-1.17 , -0.46] 8.8E-04 -0.44 [-0.59 , -0.30] 1.3E-04 -0.13 [-0.19 , -0.06] 4.8E-03 Il11ra1 -0.82 [-1.08 , -0.55] 7.0E-05 -0.42 [-0.53 , -0.31] 1.5E-05 -0.15 [-0.18 , -0.11] 1.2E-05 Lyve1 -0.86 [-1.10 , -0.62] 1.9E-05 -0.56 [-0.71 , -0.41] 2.0E-05 -0.22 [-0.27 , -0.16] 1.7E-05 Klrb1c -0.88 [-1.13 , -0.62] 3.0E-05 -0.46 [-0.60 , -0.33] 3.7E-05 -0.16 [-0.24 , -0.09] 2.4E-03 Angpt1 -0.90 [-1.11 , -0.69] 3.3E-06 -0.38 [-0.47 , -0.29] 4.5E-06 -0.08 [-0.12 , -0.04] 6.2E-03 Klra7 -0.90 [-1.47 , -0.33] 1.2E-02 -0.45 [-0.67 , -0.23] 3.0E-03 -0.28 [-0.51 , -0.04] 6.0E-02 449 Table 33 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 9 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Klrc2 -0.92 [-1.16 , -0.68] 8.8E-06 -0.53 [-0.66 , -0.40] 6.7E-06 -0.15 [-0.21 , -0.09] 1.1E-03 Itga2b -1.04 [-1.39 , -0.68] 1.1E-04 -0.33 [-0.52 , -0.14] 8.0E-03 -0.03 [-0.10 , 0.03] 4.2E-01 Camp -1.20 [-1.93 , -0.48] 8.5E-03 -0.41 [-0.70 , -0.12] 2.5E-02 -0.05 [-0.20 , 0.10] 6.0E-01 Ccr3 -1.25 [-1.96 , -0.55] 5.8E-03 -0.64 [-0.99 , -0.29] 5.4E-03 -0.18 [-0.32 , -0.04] 4.4E-02 Ppbp -1.40 [-2.01 , -0.79] 8.8E-04 -0.64 [-0.89 , -0.39] 5.2E-04 -0.15 [-0.24 , -0.06] 1.4E-02 Hamp -1.51 [-1.98 , -1.04] 4.8E-05 -0.65 [-0.90 , -0.39] 6.0E-04 -0.28 [-0.41 , -0.15] 2.1E-03 Cfd -2.53 [-3.67 , -1.39] 1.2E-03 -0.74 [-1.40 , -0.09] 6.3E-02 -0.54 [-0.81 , -0.27] 4.2E-03 450 Table 34. Complete mRNA transcript profile in lung at 13 wk post final exposure in cSiO 2-exposed NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log2(fold change) relative to VEH + CON mice. Expression data are included for all mRNA transcripts that were above background threshold. Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl5 4.99 [3.95 , 6.03] 1.4E-05 2.94 [2.60 , 3.27] 5.9E-08 0.97 [0.81 , 1.14] 1.6E-06 Cxcl3 4.46 [3.43 , 5.49] 2.6E-05 2.22 [1.78 , 2.67] 6.1E-06 0.83 [0.67 , 0.99] 4.9E-06 Lcn2 3.99 [3.55 , 4.43] 1.1E-07 2.38 [2.15 , 2.60] 9.5E-09 0.93 [0.84 , 1.01] 1.4E-08 Ccl2 3.87 [3.38 , 4.35] 3.0E-07 2.33 [1.97 , 2.69] 8.1E-07 0.91 [0.79 , 1.03] 2.1E-07 Ccl3 3.46 [2.87 , 4.05] 3.9E-06 2.11 [1.84 , 2.38] 1.8E-07 0.82 [0.70 , 0.95] 6.6E-07 Ccl8 3.41 [2.19 , 4.62] 7.6E-04 1.57 [0.96 , 2.18] 9.9E-04 0.64 [0.40 , 0.87] 7.1E-04 Cxcl13 3.24 [2.11 , 4.37] 6.8E-04 1.20 [0.58 , 1.82] 6.2E-03 0.49 [0.26 , 0.72] 3.6E-03 C3ar1 3.17 [2.47 , 3.88] 2.1E-05 1.57 [1.33 , 1.81] 8.1E-07 0.53 [0.42 , 0.64] 6.6E-06 Ccl7 3.15 [2.58 , 3.73] 4.8E-06 1.87 [1.52 , 2.22] 3.1E-06 0.70 [0.57 , 0.83] 2.8E-06 Cxcl9 3.15 [1.84 , 4.45] 2.1E-03 1.17 [0.62 , 1.72] 3.6E-03 0.40 [0.19 , 0.60] 6.9E-03 Cxcl1 2.89 [2.30 , 3.48] 1.1E-05 1.61 [1.38 , 1.84] 4.9E-07 0.57 [0.47 , 0.67] 1.8E-06 Cxcl10 2.86 [1.88 , 3.84] 6.2E-04 1.39 [0.79 , 2.00] 2.2E-03 0.48 [0.23 , 0.72] 6.6E-03 Ccl12 2.85 [2.19 , 3.52] 2.8E-05 1.46 [1.13 , 1.79] 1.7E-05 0.52 [0.39 , 0.65] 3.0E-05 451 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccl4 2.81 [1.82 , 3.79] 7.0E-04 1.58 [1.10 , 2.06] 1.7E-04 0.52 [0.32 , 0.72] 9.4E-04 Pdcd1 2.81 [1.70 , 3.93] 1.5E-03 1.34 [1.00 , 1.68] 4.1E-05 0.46 [0.31 , 0.61] 2.6E-04 C1qb 2.70 [1.96 , 3.45] 1.2E-04 1.06 [0.74 , 1.39] 1.7E-04 0.37 [0.25 , 0.48] 2.0E-04 Trem2 2.56 [2.11 , 3.02] 4.3E-06 1.34 [1.15 , 1.53] 4.6E-07 0.53 [0.45 , 0.61] 6.6E-07 Tigit 2.53 [1.57 , 3.48] 1.1E-03 1.01 [0.68 , 1.35] 3.3E-04 0.37 [0.23 , 0.50] 6.6E-04 Gzmk 2.49 [1.45 , 3.53] 2.1E-03 0.94 [0.45 , 1.43] 6.4E-03 0.30 [0.12 , 0.47] 1.4E-02 Il1rn 2.48 [2.10 , 2.86] 2.2E-06 1.51 [1.24 , 1.78] 2.6E-06 0.59 [0.49 , 0.69] 1.7E-06 Cfb 2.48 [1.99 , 2.96] 8.6E-06 1.32 [1.08 , 1.56] 2.5E-06 0.49 [0.37 , 0.61] 2.9E-05 Ccr5 2.47 [1.60 , 3.35] 7.2E-04 0.97 [0.61 , 1.32] 7.1E-04 0.28 [0.17 , 0.40] 1.5E-03 Spp1 2.46 [1.83 , 3.10] 6.6E-05 1.54 [1.26 , 1.82] 2.8E-06 0.60 [0.51 , 0.69] 5.1E-07 Cxcl2 2.45 [1.53 , 3.37] 1.1E-03 1.54 [1.11 , 1.97] 8.8E-05 0.53 [0.35 , 0.72] 4.6E-04 Fcgr4 2.44 [1.74 , 3.15] 1.8E-04 0.79 [0.33 , 1.25] 1.3E-02 0.22 [0.09 , 0.35] 1.5E-02 C1qa 2.44 [1.63 , 3.25] 5.0E-04 0.98 [0.66 , 1.29] 2.8E-04 0.35 [0.23 , 0.46] 3.4E-04 Oasl1 2.39 [1.43 , 3.35] 1.7E-03 1.14 [0.67 , 1.61] 1.6E-03 0.30 [0.02 , 0.58] 9.4E-02 Tnfrsf17 2.39 [1.38 , 3.41] 2.3E-03 0.98 [0.46 , 1.50] 7.4E-03 0.34 [0.20 , 0.47] 1.5E-03 Chil3 2.33 [0.32 , 4.35] 6.9E-02 1.96 [0.91 , 3.01] 7.7E-03 0.46 [0.22 , 0.69] 7.2E-03 Msr1 2.26 [1.83 , 2.69] 7.3E-06 1.29 [1.07 , 1.52] 2.2E-06 0.50 [0.41 , 0.59] 1.7E-06 452 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Aicda 2.23 [0.42 , 4.03] 5.5E-02 0.57 [-0.25 , 1.39] 2.7E-01 0.30 [0.01 , 0.59] 1.1E-01 Prg2 2.17 [1.26 , 3.08] 2.3E-03 0.93 [0.37 , 1.48] 1.4E-02 0.25 [0.12 , 0.38] 7.9E-03 Tnfsf11 2.12 [1.50 , 2.74] 1.8E-04 1.05 [0.82 , 1.28] 1.3E-05 0.38 [0.28 , 0.48] 5.2E-05 Isg15 2.09 [1.29 , 2.89] 1.3E-03 0.95 [0.49 , 1.41] 4.3E-03 0.26 [0.01 , 0.50] 9.9E-02 Cfi 2.05 [1.22 , 2.89] 1.8E-03 1.13 [0.93 , 1.34] 2.6E-06 0.36 [0.30 , 0.42] 1.6E-06 Slamf7 2.05 [1.20 , 2.90] 2.1E-03 0.84 [0.56 , 1.12] 3.3E-04 0.30 [0.19 , 0.41] 6.5E-04 Oas3 2.01 [1.15 , 2.87] 2.5E-03 0.85 [0.45 , 1.24] 3.3E-03 0.22 [0.02 , 0.43] 9.3E-02 Oas2 2.00 [1.22 , 2.78] 1.4E-03 0.83 [0.46 , 1.19] 2.4E-03 0.18 [0.00 , 0.35] 1.1E-01 Irf7 2.00 [1.04 , 2.96] 4.9E-03 0.92 [0.48 , 1.36] 4.0E-03 0.25 [-0.01 , 0.50] 1.3E-01 Pou2af1 1.97 [0.97 , 2.98] 6.8E-03 0.57 [0.16 , 0.99] 3.4E-02 0.20 [0.07 , 0.34] 2.7E-02 Ccr6 1.96 [0.96 , 2.95] 6.8E-03 0.50 [0.13 , 0.86] 3.9E-02 0.17 [0.05 , 0.28] 3.1E-02 Birc5 1.95 [1.31 , 2.59] 4.7E-04 0.69 [0.43 , 0.95] 8.0E-04 0.28 [0.19 , 0.36] 1.8E-04 Clec4n 1.91 [1.50 , 2.33] 1.8E-05 1.21 [1.09 , 1.32] 9.5E-09 0.47 [0.40 , 0.54] 4.5E-07 Lag3 1.88 [0.94 , 2.83] 6.2E-03 0.53 [0.18 , 0.89] 2.5E-02 0.19 [0.09 , 0.30] 8.3E-03 Il10 1.88 [0.93 , 2.82] 6.2E-03 0.83 [0.44 , 1.21] 3.4E-03 0.33 [0.20 , 0.46] 1.1E-03 Zbp1 1.79 [1.14 , 2.44] 8.0E-04 0.76 [0.41 , 1.11] 3.2E-03 0.22 [0.06 , 0.37] 3.5E-02 Clec5a 1.73 [1.36 , 2.10] 1.7E-05 1.17 [0.98 , 1.36] 9.5E-07 0.48 [0.42 , 0.55] 2.1E-07 453 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Blnk 1.73 [0.94 , 2.53] 3.8E-03 0.57 [0.27 , 0.88] 7.9E-03 0.21 [0.12 , 0.30] 2.5E-03 Fcgr1 1.71 [0.99 , 2.43] 2.3E-03 0.67 [0.37 , 0.97] 2.5E-03 0.16 [0.02 , 0.30] 7.0E-02 Ifi44 1.68 [0.88 , 2.47] 4.4E-03 0.79 [0.37 , 1.22] 7.9E-03 0.20 [-0.02 , 0.42] 1.6E-01 Foxp3 1.64 [1.00 , 2.27] 1.3E-03 0.87 [0.58 , 1.16] 3.6E-04 0.31 [0.19 , 0.43] 1.1E-03 Ctss 1.63 [1.04 , 2.22] 8.2E-04 0.81 [0.62 , 1.00] 2.1E-05 0.27 [0.20 , 0.35] 7.9E-05 Pdcd1lg2 1.63 [1.01 , 2.25] 1.1E-03 0.65 [0.39 , 0.91] 1.3E-03 0.23 [0.14 , 0.33] 1.4E-03 Ifit1 1.60 [0.90 , 2.29] 2.7E-03 0.80 [0.37 , 1.23] 7.7E-03 0.21 [0.01 , 0.40] 9.5E-02 Irf4 1.60 [0.85 , 2.35] 4.3E-03 0.43 [0.08 , 0.78] 5.9E-02 0.16 [0.06 , 0.26] 2.0E-02 Clec4a2 1.58 [1.06 , 2.10] 4.5E-04 0.57 [0.24 , 0.90] 1.3E-02 0.13 [0.03 , 0.24] 5.4E-02 Fcgr2b 1.57 [1.31 , 1.83] 3.7E-06 1.06 [0.88 , 1.24] 1.6E-06 0.40 [0.32 , 0.48] 4.7E-06 Il6 1.57 [1.07 , 2.06] 3.3E-04 0.85 [0.55 , 1.15] 6.0E-04 0.27 [0.16 , 0.38] 1.2E-03 Ccr1 1.54 [1.26 , 1.81] 4.5E-06 0.95 [0.72 , 1.17] 2.5E-05 0.35 [0.29 , 0.41] 1.6E-06 Ccl9 1.54 [1.18 , 1.89] 2.6E-05 0.87 [0.68 , 1.07] 1.5E-05 0.32 [0.23 , 0.42] 1.3E-04 Arg2 1.53 [1.17 , 1.89] 2.9E-05 1.08 [0.82 , 1.34] 2.6E-05 0.45 [0.38 , 0.52] 8.2E-07 Il21r 1.53 [0.66 , 2.41] 1.2E-02 0.39 [0.02 , 0.76] 1.0E-01 0.15 [0.03 , 0.28] 5.3E-02 Cd14 1.50 [1.22 , 1.77] 4.8E-06 0.92 [0.73 , 1.12] 9.7E-06 0.33 [0.25 , 0.41] 2.5E-05 Slc11a1 1.50 [0.95 , 2.06] 9.6E-04 0.78 [0.54 , 1.01] 1.7E-04 0.29 [0.20 , 0.38] 2.0E-04 454 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd68 1.46 [1.21 , 1.72] 4.3E-06 0.85 [0.70 , 0.99] 1.6E-06 0.32 [0.27 , 0.36] 6.4E-07 Tnfrsf4 1.46 [0.97 , 1.95] 5.1E-04 0.65 [0.44 , 0.87] 3.0E-04 0.25 [0.19 , 0.32] 4.5E-05 Tnf 1.45 [0.92 , 1.99] 8.9E-04 0.65 [0.31 , 0.99] 6.6E-03 0.32 [0.21 , 0.42] 2.7E-04 Emr1 1.44 [1.09 , 1.79] 3.7E-05 0.60 [0.38 , 0.81] 6.1E-04 0.18 [0.09 , 0.25] 3.0E-03 Cxcr3 1.44 [0.75 , 2.13] 4.6E-03 0.37 [-0.01 , 0.74] 1.2E-01 0.15 [0.05 , 0.25] 2.5E-02 Il1b 1.41 [0.95 , 1.87] 4.4E-04 0.55 [0.35 , 0.75] 6.4E-04 0.10 [-0.01 , 0.21] 1.5E-01 Pik3cg 1.40 [0.96 , 1.83] 3.1E-04 0.54 [0.32 , 0.76] 1.6E-03 0.19 [0.12 , 0.26] 6.9E-04 Itgam 1.39 [0.87 , 1.92] 1.1E-03 0.66 [0.42 , 0.90] 7.1E-04 0.21 [0.12 , 0.30] 1.5E-03 Ccr8 1.37 [0.63 , 2.10] 9.0E-03 0.83 [0.53 , 1.13] 6.0E-04 0.26 [0.13 , 0.39] 6.4E-03 Il1r2 1.34 [0.76 , 1.93] 2.7E-03 0.77 [0.44 , 1.11] 2.2E-03 0.28 [0.15 , 0.40] 2.5E-03 Ada 1.34 [0.73 , 1.96] 3.7E-03 0.39 [0.09 , 0.68] 4.4E-02 0.15 [0.06 , 0.24] 1.7E-02 Slamf6 1.32 [0.53 , 2.12] 1.6E-02 0.38 [0.11 , 0.65] 3.4E-02 0.16 [0.07 , 0.26] 1.4E-02 Ccl6 1.31 [0.95 , 1.68] 1.4E-04 0.97 [0.82 , 1.12] 8.8E-07 0.40 [0.34 , 0.46] 6.4E-07 Tlr2 1.30 [1.02 , 1.57] 1.7E-05 0.74 [0.56 , 0.91] 2.5E-05 0.26 [0.19 , 0.34] 7.5E-05 Fcer2a 1.30 [0.45 , 2.15] 2.4E-02 1.00 [0.61 , 1.40] 1.2E-03 0.36 [0.21 , 0.51] 1.9E-03 Fcgr3 1.29 [1.07 , 1.51] 3.9E-06 0.71 [0.57 , 0.84] 4.2E-06 0.23 [0.17 , 0.29] 6.6E-05 Cd200r1 1.29 [0.99 , 1.58] 2.6E-05 0.79 [0.68 , 0.90] 4.9E-07 0.29 [0.24 , 0.33] 6.6E-07 455 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Fpr2 1.28 [1.02 , 1.53] 9.5E-06 0.45 [0.13 , 0.77] 3.3E-02 0.14 [-0.01 , 0.29] 1.5E-01 Cx3cr1 1.28 [0.84 , 1.72] 6.5E-04 0.38 [0.07 , 0.69] 6.1E-02 0.10 [-0.03 , 0.23] 2.4E-01 Itgax 1.27 [0.99 , 1.56] 2.2E-05 0.56 [0.33 , 0.79] 1.6E-03 0.16 [0.11 , 0.22] 4.8E-04 Rsad2 1.27 [0.46 , 2.09] 2.1E-02 0.73 [0.28 , 1.18] 1.6E-02 0.19 [-0.06 , 0.43] 2.4E-01 Il1a 1.26 [0.86 , 1.67] 4.1E-04 0.80 [0.62 , 0.98] 1.3E-05 0.30 [0.24 , 0.37] 1.2E-05 Marco 1.26 [0.63 , 1.90] 6.1E-03 1.02 [0.77 , 1.27] 3.4E-05 0.46 [0.34 , 0.58] 6.6E-05 Sh2d1b1 1.25 [0.76 , 1.75] 1.6E-03 0.25 [-0.15 , 0.64] 3.2E-01 0.01 [-0.08 , 0.09] 9.4E-01 Il2ra 1.25 [0.75 , 1.75] 1.7E-03 0.77 [0.53 , 1.01] 1.8E-04 0.29 [0.20 , 0.38] 2.3E-04 Ulbp1 1.25 [0.72 , 1.77] 2.3E-03 0.61 [0.39 , 0.82] 5.7E-04 0.22 [0.14 , 0.30] 5.1E-04 Ctla4 1.25 [0.51 , 1.99] 1.5E-02 0.51 [0.22 , 0.80] 1.2E-02 0.23 [0.14 , 0.32] 1.1E-03 Cd83 1.24 [0.86 , 1.62] 2.8E-04 0.53 [0.35 , 0.71] 4.1E-04 0.16 [0.09 , 0.23] 2.2E-03 Usp18 1.24 [0.65 , 1.82] 4.4E-03 0.61 [0.32 , 0.89] 3.8E-03 0.17 [0.03 , 0.31] 7.0E-02 Cfp 1.23 [0.65 , 1.81] 4.3E-03 0.46 [0.23 , 0.68] 4.6E-03 0.17 [0.08 , 0.26] 6.0E-03 Lgals3 1.21 [0.98 , 1.44] 7.3E-06 0.85 [0.67 , 1.04] 1.3E-05 0.34 [0.30 , 0.38] 1.8E-07 Csf2rb 1.21 [0.94 , 1.48] 2.2E-05 0.71 [0.60 , 0.82] 8.1E-07 0.27 [0.22 , 0.31] 1.6E-06 Fcer1g 1.21 [0.88 , 1.54] 1.1E-04 0.51 [0.31 , 0.70] 1.0E-03 0.16 [0.09 , 0.23] 2.0E-03 Sh2b2 1.21 [0.73 , 1.69] 1.5E-03 0.58 [0.34 , 0.83] 2.0E-03 0.20 [0.15 , 0.25] 3.9E-05 456 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Plau 1.20 [0.83 , 1.57] 2.6E-04 0.79 [0.62 , 0.95] 9.7E-06 0.29 [0.23 , 0.34] 4.2E-06 Il12b 1.19 [0.42 , 1.96] 2.3E-02 0.70 [0.40 , 1.01] 2.3E-03 0.26 [0.14 , 0.37] 2.5E-03 C3 1.18 [0.84 , 1.52] 1.8E-04 0.74 [0.60 , 0.88] 4.1E-06 0.27 [0.22 , 0.32] 2.6E-06 Mx1 1.18 [0.45 , 1.90] 1.8E-02 0.61 [0.25 , 0.97] 1.4E-02 0.10 [-0.13 , 0.33] 5.0E-01 Siglec1 1.17 [0.64 , 1.69] 3.2E-03 0.59 [0.31 , 0.87] 4.0E-03 0.18 [0.09 , 0.27] 6.6E-03 Tlr1 1.17 [0.33 , 2.00] 3.3E-02 0.27 [-0.10 , 0.63] 2.5E-01 0.08 [-0.02 , 0.19] 2.3E-01 Tnfrsf13c 1.16 [0.29 , 2.02] 4.1E-02 0.07 [-0.34 , 0.49] 7.8E-01 0.04 [-0.10 , 0.18] 7.0E-01 Cd274 1.14 [0.83 , 1.45] 1.2E-04 0.65 [0.49 , 0.81] 3.2E-05 0.22 [0.15 , 0.30] 4.6E-04 Cd86 1.14 [0.81 , 1.48] 1.9E-04 0.52 [0.39 , 0.65] 3.9E-05 0.19 [0.13 , 0.25] 1.5E-04 Tmem173 1.14 [0.74 , 1.53] 6.4E-04 0.50 [0.30 , 0.69] 1.0E-03 0.16 [0.09 , 0.22] 1.6E-03 Csf2 1.12 [0.63 , 1.61] 2.8E-03 0.95 [0.71 , 1.18] 3.6E-05 0.38 [0.29 , 0.46] 1.3E-05 Ly86 1.10 [0.48 , 1.72] 1.1E-02 0.35 [0.09 , 0.61] 4.0E-02 0.08 [-0.01 , 0.16] 1.6E-01 Serpinb2 1.10 [-0.66 , 2.87] 3.1E-01 0.09 [-0.53 , 0.71] 8.3E-01 0.08 [-0.13 , 0.28] 5.6E-01 Tlr9 1.09 [0.37 , 1.80] 2.4E-02 0.29 [-0.02 , 0.61] 1.4E-01 0.08 [-0.04 , 0.19] 3.2E-01 Tnfrsf11a 1.08 [0.84 , 1.33] 2.6E-05 0.65 [0.52 , 0.78] 5.9E-06 0.23 [0.18 , 0.28] 1.3E-05 Mx2 1.08 [0.07 , 2.09] 9.0E-02 0.52 [0.09 , 0.96] 6.3E-02 0.12 [-0.07 , 0.31] 3.4E-01 Clec7a 1.07 [0.75 , 1.39] 2.1E-04 0.69 [0.51 , 0.87] 6.2E-05 0.22 [0.16 , 0.28] 3.9E-05 457 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd180 1.06 [0.47 , 1.64] 1.1E-02 0.33 [0.04 , 0.62] 7.2E-02 0.10 [0.00 , 0.20] 1.2E-01 Cd80 1.05 [0.79 , 1.31] 4.2E-05 0.59 [0.47 , 0.70] 4.1E-06 0.20 [0.15 , 0.24] 2.0E-05 C6 1.05 [0.70 , 1.40] 5.0E-04 0.73 [0.51 , 0.95] 1.5E-04 0.27 [0.21 , 0.33] 1.2E-05 Ifit3 1.05 [0.37 , 1.73] 2.3E-02 0.61 [0.26 , 0.97] 1.2E-02 0.14 [-0.06 , 0.34] 2.7E-01 Cybb 1.03 [0.80 , 1.26] 2.5E-05 0.39 [0.16 , 0.61] 1.2E-02 0.15 [0.07 , 0.22] 7.1E-03 H2-DMb2 1.03 [0.37 , 1.69] 2.2E-02 0.25 [-0.08 , 0.57] 2.3E-01 0.11 [0.02 , 0.20] 5.9E-02 Ccl11 1.02 [0.30 , 1.74] 3.2E-02 0.82 [0.57 , 1.07] 1.8E-04 0.22 [0.14 , 0.29] 3.7E-04 Arg1 1.02 [-0.44 , 2.48] 2.6E-01 1.03 [0.20 , 1.86] 5.6E-02 0.20 [0.06 , 0.34] 2.9E-02 Pdgfc 1.01 [0.63 , 1.39] 1.1E-03 0.43 [0.25 , 0.60] 1.6E-03 0.16 [0.10 , 0.22] 9.8E-04 Ly9 1.01 [0.44 , 1.59] 1.2E-02 0.41 [0.20 , 0.61] 5.3E-03 0.16 [0.09 , 0.22] 1.2E-03 Cxcl16 1.00 [0.71 , 1.30] 1.9E-04 0.47 [0.36 , 0.57] 1.7E-05 0.16 [0.12 , 0.20] 5.5E-05 Casp1 0.99 [0.65 , 1.32] 6.1E-04 0.38 [0.21 , 0.56] 2.8E-03 0.11 [0.05 , 0.16] 7.5E-03 H2-Eb1 0.99 [0.54 , 1.43] 3.5E-03 0.26 [0.11 , 0.41] 1.3E-02 0.07 [0.01 , 0.13] 5.6E-02 Socs3 0.98 [0.55 , 1.42] 3.0E-03 0.51 [0.23 , 0.78] 7.9E-03 0.21 [0.12 , 0.29] 1.4E-03 Ncf4 0.98 [0.70 , 1.25] 1.4E-04 0.45 [0.24 , 0.66] 3.4E-03 0.16 [0.10 , 0.23] 1.1E-03 Osm 0.97 [0.44 , 1.50] 1.0E-02 0.70 [0.38 , 1.02] 2.8E-03 0.25 [0.14 , 0.36] 2.5E-03 Pou2f2 0.96 [0.51 , 1.41] 4.4E-03 0.17 [-0.21 , 0.55] 4.8E-01 0.05 [-0.07 , 0.16] 5.3E-01 458 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccl19 0.96 [0.41 , 1.50] 1.2E-02 0.29 [0.02 , 0.56] 9.8E-02 0.10 [-0.01 , 0.21] 1.7E-01 Btk 0.95 [0.63 , 1.28] 6.1E-04 0.30 [0.11 , 0.49] 1.9E-02 0.11 [0.06 , 0.16] 2.6E-03 Cd48 0.95 [0.54 , 1.35] 2.4E-03 0.35 [0.14 , 0.55] 1.3E-02 0.09 [0.03 , 0.15] 3.7E-02 Ikbke 0.94 [0.57 , 1.31] 1.5E-03 0.42 [0.24 , 0.61] 2.1E-03 0.18 [0.11 , 0.25] 8.0E-04 Tap1 0.93 [0.52 , 1.34] 3.0E-03 0.36 [0.20 , 0.52] 2.6E-03 0.12 [0.05 , 0.18] 1.0E-02 Icos 0.93 [0.38 , 1.47] 1.5E-02 0.37 [0.18 , 0.56] 6.6E-03 0.14 [0.08 , 0.20] 2.1E-03 Cd74 0.92 [0.51 , 1.34] 3.4E-03 0.41 [0.31 , 0.50] 1.9E-05 0.14 [0.09 , 0.19] 3.1E-04 Klra2 0.91 [0.42 , 1.40] 9.5E-03 0.09 [-0.19 , 0.36] 6.3E-01 -0.06 [-0.14 , 0.03] 2.8E-01 H2-K1 0.90 [0.59 , 1.22] 7.0E-04 0.40 [0.27 , 0.54] 3.1E-04 0.13 [0.07 , 0.18] 3.2E-03 Tnfrsf8 0.90 [0.14 , 1.66] 6.5E-02 0.26 [-0.14 , 0.67] 3.0E-01 0.13 [-0.03 , 0.30] 2.1E-01 Bst2 0.90 [0.49 , 1.31] 3.8E-03 0.42 [0.21 , 0.63] 5.3E-03 0.12 [0.01 , 0.23] 8.8E-02 Ccl22 0.89 [0.32 , 1.47] 2.1E-02 0.74 [0.53 , 0.95] 1.0E-04 0.30 [0.21 , 0.39] 1.5E-04 Ebi3 0.88 [0.65 , 1.11] 9.0E-05 0.34 [0.10 , 0.58] 3.2E-02 0.10 [0.04 , 0.16] 1.8E-02 Psmb9 0.88 [0.48 , 1.28] 3.6E-03 0.28 [0.14 , 0.41] 4.4E-03 0.07 [0.02 , 0.13] 5.0E-02 Bid 0.87 [0.56 , 1.19] 8.0E-04 0.37 [0.22 , 0.51] 1.2E-03 0.11 [0.06 , 0.17] 4.7E-03 Cd84 0.87 [0.62 , 1.13] 1.8E-04 0.55 [0.42 , 0.67] 1.7E-05 0.19 [0.15 , 0.24] 1.3E-05 Ms4a1 0.87 [0.11 , 1.64] 7.3E-02 0.02 [-0.39 , 0.42] 9.6E-01 0.04 [-0.09 , 0.16] 6.8E-01 459 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Psmb8 0.86 [0.44 , 1.29] 5.4E-03 0.32 [0.15 , 0.49] 8.2E-03 0.10 [0.04 , 0.16] 1.3E-02 H2-Q2 0.86 [0.21 , 1.50] 4.2E-02 0.49 [0.29 , 0.69] 1.3E-03 0.18 [0.09 , 0.27] 5.7E-03 H2-Ea-ps 0.85 [0.46 , 1.24] 3.8E-03 0.36 [0.26 , 0.46] 7.2E-05 0.12 [0.08 , 0.16] 7.6E-04 Tnfrsf13b 0.85 [0.36 , 1.34] 1.3E-02 0.22 [-0.02 , 0.47] 1.5E-01 0.12 [0.04 , 0.19] 2.0E-02 Lcp1 0.85 [0.67 , 1.03] 1.7E-05 0.44 [0.32 , 0.56] 9.1E-05 0.15 [0.12 , 0.18] 1.0E-05 Nlrc5 0.84 [0.33 , 1.35] 1.7E-02 0.28 [0.06 , 0.49] 4.8E-02 0.08 [0.00 , 0.16] 1.2E-01 Itgb2 0.83 [0.63 , 1.04] 4.1E-05 0.40 [0.24 , 0.55] 1.2E-03 0.14 [0.09 , 0.19] 5.3E-04 Lta 0.83 [0.26 , 1.40] 2.9E-02 0.28 [0.01 , 0.55] 1.0E-01 0.09 [-0.01 , 0.19] 1.6E-01 Cxcr1 0.82 [0.17 , 1.48] 5.1E-02 0.61 [0.23 , 0.98] 1.7E-02 0.30 [0.21 , 0.38] 1.2E-04 Syk 0.82 [0.50 , 1.13] 1.3E-03 0.28 [0.08 , 0.48] 3.4E-02 0.10 [0.05 , 0.15] 3.8E-03 Batf 0.82 [0.46 , 1.17] 2.5E-03 0.41 [0.27 , 0.55] 3.9E-04 0.12 [0.07 , 0.16] 8.2E-04 Herc6 0.81 [0.47 , 1.16] 2.4E-03 0.43 [0.22 , 0.65] 5.3E-03 0.12 [-0.01 , 0.25] 1.5E-01 Slc7a11 0.80 [0.26 , 1.35] 2.8E-02 0.83 [0.63 , 1.03] 2.6E-05 0.36 [0.29 , 0.43] 4.4E-06 Il17rb 0.79 [0.20 , 1.39] 4.2E-02 n.d [n.d , n.d] n.d 0.15 [0.05 , 0.25] 2.8E-02 H2-M3 0.78 [0.47 , 1.10] 1.6E-03 0.22 [0.12 , 0.32] 3.6E-03 0.06 [0.02 , 0.10] 1.7E-02 Cd53 0.78 [0.50 , 1.05] 7.6E-04 0.46 [0.32 , 0.61] 1.8E-04 0.15 [0.10 , 0.20] 3.8E-04 Trem1 0.77 [0.14 , 1.41] 5.6E-02 0.46 [0.15 , 0.76] 2.4E-02 0.14 [0.03 , 0.25] 5.3E-02 460 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value H2-Aa 0.77 [0.40 , 1.14] 4.6E-03 0.33 [0.23 , 0.42] 1.7E-04 0.11 [0.06 , 0.15] 9.4E-04 H2-Ab1 0.76 [0.39 , 1.13] 5.1E-03 0.33 [0.25 , 0.42] 6.4E-05 0.12 [0.07 , 0.16] 5.4E-04 Xcr1 0.75 [0.18 , 1.32] 4.4E-02 0.26 [0.08 , 0.43] 2.7E-02 0.08 [0.02 , 0.14] 4.9E-02 Stat1 0.75 [0.35 , 1.14] 8.9E-03 0.25 [0.03 , 0.48] 7.4E-02 0.03 [-0.04 , 0.11] 5.1E-01 Ctsh 0.74 [0.58 , 0.91] 1.8E-05 0.44 [0.34 , 0.53] 1.6E-05 0.17 [0.14 , 0.19] 8.5E-07 Nfkb2 0.74 [0.52 , 0.96] 2.4E-04 0.37 [0.26 , 0.49] 1.7E-04 0.12 [0.10 , 0.15] 6.5E-06 Cd19 0.74 [-0.16 , 1.64] 1.9E-01 -0.09 [-0.56 , 0.38] 7.8E-01 n.d [n.d , n.d] n.d Tlr7 0.74 [0.51 , 0.96] 3.0E-04 0.31 [0.10 , 0.51] 2.5E-02 0.09 [0.02 , 0.17] 6.2E-02 Gbp5 0.73 [0.41 , 1.06] 2.8E-03 0.36 [0.19 , 0.52] 3.5E-03 0.09 [0.02 , 0.16] 5.6E-02 Ticam2 0.73 [0.47 , 1.00] 8.4E-04 0.29 [0.09 , 0.49] 2.7E-02 0.09 [0.03 , 0.15] 2.0E-02 Slamf1 0.72 [0.14 , 1.30] 5.4E-02 0.05 [-0.18 , 0.29] 7.2E-01 -0.02 [-0.09 , 0.05] 7.2E-01 Hck 0.71 [0.41 , 1.01] 2.2E-03 0.26 [0.03 , 0.49] 7.3E-02 0.07 [-0.02 , 0.17] 2.4E-01 Tlr8 0.71 [0.44 , 0.98] 1.1E-03 0.18 [-0.03 , 0.40] 1.8E-01 0.02 [-0.05 , 0.08] 7.3E-01 Lyz2 0.70 [0.54 , 0.87] 3.3E-05 0.52 [0.41 , 0.63] 1.1E-05 0.20 [0.16 , 0.24] 7.3E-06 Ptprc 0.70 [0.33 , 1.05] 7.5E-03 0.16 [-0.07 , 0.40] 2.7E-01 0.04 [-0.03 , 0.11] 3.5E-01 Ccl20 0.69 [-0.81 , 2.19] 4.6E-01 0.20 [-0.54 , 0.95] 6.8E-01 0.01 [-0.27 , 0.29] 9.6E-01 Cd276 0.69 [0.49 , 0.88] 1.4E-04 0.43 [0.31 , 0.56] 1.3E-04 0.16 [0.10 , 0.22] 6.0E-04 461 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfaip3 0.69 [0.45 , 0.93] 7.0E-04 0.40 [0.27 , 0.53] 2.5E-04 0.12 [0.08 , 0.16] 3.1E-04 Il23r 0.69 [0.13 , 1.24] 5.5E-02 0.46 [0.18 , 0.75] 1.7E-02 0.22 [0.12 , 0.32] 3.2E-03 H2-D1 0.68 [0.46 , 0.91] 4.7E-04 0.31 [0.21 , 0.42] 3.1E-04 0.10 [0.06 , 0.15] 2.0E-03 Havcr2 0.68 [0.51 , 0.85] 4.9E-05 0.43 [0.31 , 0.55] 1.0E-04 0.14 [0.08 , 0.20] 2.6E-03 Spn 0.68 [0.30 , 1.06] 1.1E-02 0.14 [-0.17 , 0.45] 4.8E-01 0.04 [-0.04 , 0.13] 4.5E-01 Xbp1 0.68 [0.32 , 1.03] 7.7E-03 0.18 [0.04 , 0.32] 4.8E-02 0.05 [0.00 , 0.09] 1.3E-01 Relb 0.67 [0.43 , 0.91] 7.6E-04 0.38 [0.27 , 0.49] 1.2E-04 0.15 [0.10 , 0.19] 1.0E-04 Tank 0.67 [0.47 , 0.87] 1.9E-04 0.35 [0.25 , 0.45] 8.2E-05 0.10 [0.08 , 0.13] 1.7E-05 Ifitm1 0.67 [0.37 , 0.97] 3.0E-03 0.50 [0.34 , 0.65] 2.1E-04 0.17 [0.10 , 0.24] 1.5E-03 Ltb 0.66 [0.16 , 1.17] 4.4E-02 0.10 [-0.17 , 0.37] 5.6E-01 0.01 [-0.08 , 0.11] 8.1E-01 Ly96 0.66 [0.43 , 0.89] 6.4E-04 0.28 [0.16 , 0.40] 2.0E-03 0.10 [0.06 , 0.13] 4.6E-04 H2-T23 0.66 [0.34 , 0.98] 5.1E-03 0.27 [0.15 , 0.39] 2.4E-03 0.07 [0.01 , 0.14] 9.4E-02 H2-Ob 0.66 [-0.06 , 1.38] 1.4E-01 0.03 [-0.31 , 0.37] 8.9E-01 0.03 [-0.06 , 0.12] 6.4E-01 Ikzf2 0.65 [0.19 , 1.11] 3.2E-02 0.33 [0.14 , 0.51] 1.2E-02 0.11 [0.05 , 0.16] 9.3E-03 H2-DMb1 0.64 [0.30 , 0.99] 9.3E-03 0.33 [0.23 , 0.44] 2.4E-04 0.09 [0.05 , 0.13] 1.7E-03 Cd79b 0.64 [-0.11 , 1.39] 1.7E-01 -0.05 [-0.41 , 0.31] 8.3E-01 0.03 [-0.08 , 0.13] 7.3E-01 Xaf1 0.64 [0.21 , 1.07] 2.5E-02 0.30 [0.08 , 0.52] 3.9E-02 0.05 [-0.08 , 0.17] 5.7E-01 462 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Litaf 0.64 [0.44 , 0.85] 3.7E-04 0.43 [0.31 , 0.56] 1.4E-04 0.15 [0.11 , 0.19] 5.5E-05 Casp3 0.64 [0.33 , 0.95] 5.1E-03 0.25 [0.12 , 0.37] 5.3E-03 0.08 [0.03 , 0.12] 2.0E-02 Klra17 0.63 [0.02 , 1.24] 9.8E-02 -0.04 [-0.31 , 0.24] 8.5E-01 -0.07 [-0.16 , 0.01] 1.9E-01 Tgfbr1 0.63 [0.36 , 0.90] 2.5E-03 0.23 [0.12 , 0.35] 5.3E-03 0.08 [0.05 , 0.11] 1.1E-03 Tlr6 0.63 [0.41 , 0.85] 7.0E-04 0.26 [0.14 , 0.37] 2.3E-03 0.10 [0.04 , 0.16] 1.8E-02 Cd28 0.62 [-0.10 , 1.35] 1.7E-01 0.10 [-0.18 , 0.38] 5.9E-01 0.10 [-0.03 , 0.22] 2.3E-01 Cd244 0.60 [0.26 , 0.94] 1.2E-02 0.08 [-0.13 , 0.29] 5.6E-01 0.05 [-0.05 , 0.15] 4.5E-01 Bst1 0.60 [0.34 , 0.86] 2.7E-03 0.50 [0.34 , 0.66] 2.6E-04 0.17 [0.13 , 0.22] 5.5E-05 Ddx60 0.59 [0.14 , 1.05] 4.4E-02 0.29 [0.06 , 0.52] 5.3E-02 0.07 [-0.05 , 0.19] 4.0E-01 Gfi1 0.59 [0.03 , 1.14] 9.4E-02 0.17 [-0.19 , 0.54] 4.5E-01 0.09 [-0.02 , 0.20] 2.1E-01 Cdk1 0.58 [-0.19 , 1.34] 2.2E-01 0.39 [0.20 , 0.59] 5.0E-03 0.15 [0.06 , 0.24] 1.4E-02 Cd63 0.58 [0.38 , 0.78] 6.4E-04 0.44 [0.31 , 0.57] 1.2E-04 0.19 [0.14 , 0.23] 2.0E-05 Irak3 0.58 [0.34 , 0.81] 2.1E-03 0.26 [0.11 , 0.41] 1.1E-02 0.09 [0.03 , 0.15] 3.1E-02 Cd4 0.58 [0.20 , 0.96] 2.4E-02 0.23 [0.09 , 0.36] 1.5E-02 0.07 [0.01 , 0.13] 7.4E-02 Runx3 0.57 [0.06 , 1.08] 7.6E-02 0.15 [-0.10 , 0.39] 3.5E-01 0.05 [-0.04 , 0.14] 3.6E-01 Itgae 0.57 [0.25 , 0.90] 1.2E-02 0.46 [0.32 , 0.60] 1.7E-04 0.18 [0.12 , 0.24] 4.1E-04 Jak3 0.57 [0.33 , 0.82] 2.5E-03 0.20 [0.09 , 0.31] 8.6E-03 0.08 [0.04 , 0.11] 1.8E-03 463 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfrsf1b 0.57 [0.19 , 0.95] 2.6E-02 0.31 [0.08 , 0.54] 3.7E-02 0.08 [0.00 , 0.15] 9.8E-02 C4b 0.57 [0.18 , 0.96] 2.7E-02 0.35 [0.17 , 0.53] 6.7E-03 0.13 [0.07 , 0.19] 3.8E-03 Socs1 0.57 [0.18 , 0.95] 2.7E-02 0.27 [0.07 , 0.46] 3.8E-02 0.03 [-0.06 , 0.12] 6.0E-01 Ciita 0.57 [0.12 , 1.01] 4.8E-02 0.19 [-0.01 , 0.38] 1.4E-01 0.05 [-0.01 , 0.11] 1.7E-01 Il10ra 0.56 [0.14 , 0.98] 4.2E-02 0.16 [-0.04 , 0.37] 2.2E-01 -0.01 [-0.08 , 0.05] 7.8E-01 Cyfip2 0.54 [0.17 , 0.92] 3.0E-02 0.12 [-0.08 , 0.32] 3.6E-01 0.06 [0.01 , 0.11] 6.6E-02 Cmah 0.54 [0.01 , 1.07] 1.1E-01 -0.01 [-0.28 , 0.26] 9.7E-01 0.01 [-0.05 , 0.07] 8.2E-01 Selplg 0.54 [0.28 , 0.80] 4.8E-03 0.18 [0.02 , 0.34] 8.6E-02 0.02 [-0.03 , 0.07] 5.5E-01 Flt3 0.54 [-0.10 , 1.17] 1.8E-01 0.18 [-0.05 , 0.40] 2.2E-01 0.12 [0.02 , 0.22] 5.8E-02 H2-DMa 0.53 [0.24 , 0.83] 1.1E-02 0.22 [0.11 , 0.33] 5.5E-03 0.06 [0.03 , 0.09] 2.9E-03 Pml 0.53 [0.15 , 0.92] 3.6E-02 0.10 [-0.09 , 0.28] 4.1E-01 0.01 [-0.05 , 0.08] 7.7E-01 Ccl17 0.53 [-0.38 , 1.44] 3.5E-01 0.71 [0.36 , 1.06] 4.8E-03 0.32 [0.23 , 0.42] 1.2E-04 Lamp3 0.52 [0.36 , 0.68] 3.0E-04 0.48 [0.34 , 0.62] 1.5E-04 0.17 [0.14 , 0.20] 6.6E-07 Irf5 0.52 [0.28 , 0.76] 4.3E-03 0.23 [0.07 , 0.38] 2.7E-02 0.06 [0.01 , 0.12] 7.2E-02 Apoe 0.51 [0.32 , 0.70] 9.6E-04 0.25 [0.10 , 0.39] 1.4E-02 0.08 [0.05 , 0.11] 5.1E-04 Tdo2 0.50 [-0.45 , 1.44] 4.0E-01 0.51 [0.01 , 1.01] 1.1E-01 0.23 [0.10 , 0.36] 1.2E-02 Tnfrsf18 0.49 [-0.38 , 1.36] 3.6E-01 0.38 [0.22 , 0.55] 2.4E-03 0.16 [0.08 , 0.23] 3.7E-03 464 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Myc 0.49 [0.22 , 0.76] 1.0E-02 0.16 [0.05 , 0.27] 2.8E-02 0.08 [0.03 , 0.12] 1.4E-02 Sbno2 0.49 [0.28 , 0.70] 2.5E-03 0.30 [0.18 , 0.42] 1.6E-03 0.08 [0.04 , 0.12] 3.6E-03 Nod2 0.49 [0.03 , 0.95] 9.1E-02 0.23 [-0.01 , 0.47] 1.4E-01 0.04 [-0.04 , 0.12] 5.0E-01 Cxcr5 0.49 [-0.13 , 1.11] 2.1E-01 -0.17 [-0.51 , 0.17] 4.3E-01 -0.01 [-0.12 , 0.10] 9.1E-01 Cd33 0.49 [0.10 , 0.87] 5.1E-02 0.44 [0.26 , 0.62] 1.5E-03 0.17 [0.12 , 0.21] 7.6E-05 Cd37 0.48 [-0.06 , 1.03] 1.6E-01 0.01 [-0.21 , 0.24] 9.3E-01 0.03 [-0.04 , 0.10] 5.3E-01 Csf1r 0.48 [0.26 , 0.71] 3.9E-03 0.19 [0.01 , 0.37] 1.1E-01 0.05 [-0.03 , 0.13] 3.4E-01 Col3a1 0.48 [0.04 , 0.92] 8.6E-02 0.25 [0.08 , 0.41] 2.7E-02 0.10 [0.04 , 0.17] 2.3E-02 Lyn 0.48 [0.29 , 0.67] 1.5E-03 0.13 [0.03 , 0.24] 5.1E-02 0.02 [-0.02 , 0.06] 4.3E-01 Cxcr6 0.48 [-0.11 , 1.06] 1.9E-01 0.44 [0.22 , 0.66] 5.3E-03 0.12 [0.03 , 0.22] 4.4E-02 Ccr3 0.48 [-0.46 , 1.42] 4.1E-01 0.27 [-0.15 , 0.68] 3.2E-01 -0.01 [-0.21 , 0.19] 9.8E-01 Cmpk2 0.47 [0.11 , 0.84] 4.4E-02 0.26 [0.01 , 0.51] 1.0E-01 0.03 [-0.10 , 0.17] 7.2E-01 Tnfsf14 0.47 [-0.03 , 0.98] 1.3E-01 0.35 [0.16 , 0.54] 7.9E-03 0.08 [0.01 , 0.16] 8.1E-02 Il5ra 0.47 [-0.15 , 1.09] 2.2E-01 0.26 [-0.04 , 0.57] 1.8E-01 0.14 [-0.02 , 0.31] 1.8E-01 Card9 0.47 [-0.01 , 0.94] 1.2E-01 0.16 [0.00 , 0.32] 1.3E-01 0.06 [-0.03 , 0.15] 3.0E-01 C1ra 0.47 [0.21 , 0.72] 9.7E-03 0.39 [0.25 , 0.53] 5.5E-04 0.16 [0.11 , 0.20] 1.2E-04 Itga4 0.46 [0.15 , 0.77] 2.6E-02 0.09 [-0.13 , 0.30] 5.3E-01 0.04 [-0.04 , 0.12] 4.6E-01 465 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Fap 0.46 [0.03 , 0.90] 9.0E-02 0.13 [-0.09 , 0.36] 3.6E-01 0.10 [0.02 , 0.17] 4.7E-02 Vcam1 0.46 [0.18 , 0.75] 1.8E-02 0.13 [0.01 , 0.24] 1.0E-01 0.02 [-0.02 , 0.06] 5.3E-01 Egr2 0.46 [-0.21 , 1.12] 2.7E-01 0.39 [0.16 , 0.63] 1.4E-02 0.13 [0.01 , 0.24] 8.2E-02 Abca1 0.45 [0.20 , 0.70] 1.0E-02 0.24 [0.10 , 0.38] 1.3E-02 0.11 [0.07 , 0.14] 8.0E-04 Camp 0.44 [-0.73 , 1.62] 5.5E-01 0.54 [-0.13 , 1.22] 2.1E-01 0.19 [0.00 , 0.38] 1.1E-01 Raet1c 0.44 [0.11 , 0.77] 4.1E-02 0.49 [0.27 , 0.70] 2.3E-03 0.19 [0.13 , 0.25] 1.5E-04 Cd3g 0.44 [-0.13 , 1.01] 2.1E-01 0.13 [-0.08 , 0.34] 3.2E-01 0.02 [-0.05 , 0.09] 6.9E-01 Tbx21 0.44 [-0.16 , 1.04] 2.4E-01 -0.04 [-0.37 , 0.29] 8.7E-01 0.00 [-0.12 , 0.13] 9.7E-01 Cxcl15 0.44 [0.27 , 0.61] 1.3E-03 0.41 [0.25 , 0.56] 8.1E-04 0.14 [0.11 , 0.17] 7.7E-06 Nlrp3 0.43 [-0.05 , 0.91] 1.5E-01 0.30 [0.12 , 0.49] 1.5E-02 0.06 [-0.05 , 0.16] 4.0E-01 Cr2 0.42 [-0.47 , 1.31] 4.5E-01 -0.11 [-0.54 , 0.33] 7.1E-01 -0.07 [-0.20 , 0.07] 4.5E-01 Cd207 0.41 [-0.31 , 1.12] 3.6E-01 0.30 [0.06 , 0.54] 5.1E-02 0.10 [0.00 , 0.21] 1.2E-01 Adora2a 0.41 [0.05 , 0.76] 7.3E-02 0.12 [-0.07 , 0.31] 3.3E-01 0.10 [0.02 , 0.18] 5.1E-02 Nos2 0.41 [-0.07 , 0.88] 1.7E-01 0.43 [0.19 , 0.67] 9.1E-03 0.14 [0.08 , 0.21] 2.2E-03 Stat2 0.40 [0.08 , 0.73] 5.2E-02 0.20 [-0.03 , 0.43] 1.7E-01 0.02 [-0.06 , 0.11] 6.7E-01 Ctsl 0.40 [0.19 , 0.62] 8.8E-03 0.34 [0.17 , 0.51] 4.9E-03 0.12 [0.08 , 0.17] 5.2E-04 Cd22 0.40 [0.04 , 0.76] 7.7E-02 -0.05 [-0.38 , 0.28] 8.2E-01 -0.03 [-0.13 , 0.07] 6.4E-01 466 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ddx58 0.40 [0.18 , 0.62] 9.6E-03 0.22 [0.07 , 0.37] 2.4E-02 0.05 [-0.02 , 0.11] 2.9E-01 C1s1 0.39 [0.21 , 0.58] 4.8E-03 0.34 [0.24 , 0.44] 1.7E-04 0.15 [0.12 , 0.18] 1.3E-05 Il2rg 0.39 [0.05 , 0.73] 6.9E-02 0.13 [0.00 , 0.25] 1.1E-01 0.03 [-0.01 , 0.07] 2.2E-01 Abcg1 0.38 [0.17 , 0.60] 1.1E-02 0.22 [0.11 , 0.34] 6.7E-03 0.07 [0.03 , 0.11] 1.1E-02 Muc1 0.38 [0.18 , 0.58] 7.5E-03 0.40 [0.28 , 0.53] 1.7E-04 0.15 [0.12 , 0.18] 1.3E-05 Cd5 0.38 [-0.21 , 0.97] 3.0E-01 0.07 [-0.19 , 0.34] 6.8E-01 0.04 [-0.03 , 0.12] 3.8E-01 Pax5 0.38 [-0.51 , 1.26] 4.9E-01 -0.33 [-0.76 , 0.10] 2.3E-01 -0.06 [-0.18 , 0.06] 4.4E-01 Btla 0.38 [-0.25 , 1.00] 3.3E-01 -0.02 [-0.31 , 0.28] 9.4E-01 0.02 [-0.04 , 0.08] 6.1E-01 Clu 0.37 [0.09 , 0.65] 4.1E-02 0.25 [0.10 , 0.39] 1.3E-02 0.08 [0.05 , 0.12] 2.0E-03 Pparg 0.37 [0.16 , 0.57] 1.1E-02 0.14 [-0.06 , 0.35] 2.8E-01 0.02 [-0.03 , 0.07] 5.8E-01 Hif1a 0.37 [0.22 , 0.51] 1.8E-03 0.28 [0.20 , 0.35] 7.3E-05 0.10 [0.08 , 0.12] 2.3E-05 S100a8 0.37 [-0.79 , 1.51] 6.1E-01 0.39 [-0.10 , 0.87] 2.1E-01 0.11 [-0.03 , 0.24] 2.1E-01 Myd88 0.36 [0.14 , 0.59] 1.8E-02 0.26 [0.16 , 0.35] 7.6E-04 0.07 [0.04 , 0.10] 1.6E-03 Isg20 0.36 [0.11 , 0.61] 2.9E-02 0.16 [0.02 , 0.29] 6.6E-02 0.03 [-0.05 , 0.10] 5.9E-01 Ifih1 0.36 [0.12 , 0.60] 2.5E-02 0.21 [0.05 , 0.36] 3.9E-02 0.05 [-0.03 , 0.13] 3.6E-01 Tlr4 0.35 [0.26 , 0.44] 6.1E-05 0.29 [0.20 , 0.39] 3.1E-04 0.09 [0.06 , 0.12] 4.0E-04 Zap70 0.35 [-0.05 , 0.75] 1.6E-01 0.11 [-0.02 , 0.24] 1.7E-01 0.04 [0.00 , 0.07] 1.5E-01 467 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Inpp5d 0.35 [0.13 , 0.56] 1.9E-02 0.12 [-0.01 , 0.25] 1.4E-01 0.04 [0.00 , 0.08] 9.6E-02 St6gal1 0.35 [-0.11 , 0.80] 2.2E-01 0.01 [-0.15 , 0.16] 9.6E-01 0.03 [-0.01 , 0.07] 3.0E-01 Ifit2 0.35 [-0.16 , 0.85] 2.7E-01 0.23 [-0.06 , 0.51] 2.1E-01 0.02 [-0.14 , 0.19] 8.3E-01 Xcl1 0.33 [-0.43 , 1.10] 4.8E-01 0.24 [-0.05 , 0.52] 1.9E-01 0.06 [-0.08 , 0.21] 5.0E-01 Axl 0.33 [0.15 , 0.51] 1.1E-02 0.07 [-0.07 , 0.21] 4.2E-01 0.01 [-0.02 , 0.04] 7.5E-01 Rel 0.33 [0.08 , 0.58] 4.4E-02 0.14 [0.05 , 0.22] 2.2E-02 0.03 [-0.02 , 0.07] 3.4E-01 Nfatc2 0.33 [-0.07 , 0.73] 1.9E-01 0.06 [-0.20 , 0.32] 7.2E-01 0.02 [-0.05 , 0.08] 7.2E-01 Cd6 0.33 [-0.34 , 0.99] 4.3E-01 0.14 [-0.12 , 0.41] 4.0E-01 0.06 [-0.02 , 0.13] 2.3E-01 Mif 0.32 [0.05 , 0.59] 6.3E-02 0.14 [0.09 , 0.20] 9.7E-04 0.04 [0.02 , 0.06] 7.5E-03 Il1rl1 0.32 [-0.02 , 0.66] 1.3E-01 0.31 [0.11 , 0.52] 2.3E-02 0.12 [0.08 , 0.17] 5.7E-04 Cxcr4 0.32 [-0.16 , 0.80] 2.8E-01 0.06 [-0.09 , 0.20] 5.4E-01 -0.01 [-0.06 , 0.04] 7.8E-01 Creb5 0.32 [-0.08 , 0.72] 2.0E-01 0.15 [-0.01 , 0.32] 1.4E-01 0.08 [0.02 , 0.14] 5.0E-02 Stat4 0.32 [-0.04 , 0.68] 1.6E-01 0.08 [-0.03 , 0.20] 2.5E-01 0.04 [-0.01 , 0.09] 2.5E-01 Irgm2 0.32 [-0.12 , 0.74] 2.4E-01 0.20 [-0.03 , 0.44] 1.6E-01 0.02 [-0.07 , 0.11] 7.3E-01 Hcst 0.31 [-0.71 , 1.34] 6.2E-01 0.10 [-0.16 , 0.36] 5.4E-01 0.07 [-0.02 , 0.16] 2.5E-01 Maf 0.31 [-0.14 , 0.76] 2.7E-01 0.11 [-0.06 , 0.29] 2.9E-01 0.03 [-0.04 , 0.09] 5.3E-01 Itgal 0.30 [-0.02 , 0.62] 1.4E-01 0.03 [-0.20 , 0.27] 8.3E-01 -0.01 [-0.11 , 0.09] 8.9E-01 468 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Csf3r 0.30 [-0.29 , 0.89] 4.1E-01 0.16 [-0.08 , 0.41] 3.0E-01 0.01 [-0.09 , 0.10] 9.2E-01 Ticam1 0.30 [-0.04 , 0.64] 1.6E-01 0.09 [-0.06 , 0.25] 3.4E-01 0.03 [-0.03 , 0.08] 4.5E-01 Hc 0.29 [0.03 , 0.56] 7.7E-02 0.30 [0.16 , 0.44] 3.4E-03 0.10 [0.04 , 0.16] 1.6E-02 Prdm1 0.29 [0.00 , 0.59] 1.1E-01 -0.05 [-0.27 , 0.17] 7.2E-01 -0.03 [-0.09 , 0.04] 5.5E-01 Bax 0.29 [0.15 , 0.43] 5.1E-03 0.16 [0.09 , 0.24] 3.0E-03 0.06 [0.04 , 0.08] 8.4E-04 Ifi27 0.29 [0.06 , 0.53] 5.5E-02 0.17 [0.05 , 0.28] 2.9E-02 0.04 [-0.01 , 0.08] 1.7E-01 Lbp 0.29 [0.02 , 0.56] 9.2E-02 0.32 [0.20 , 0.44] 9.7E-04 0.12 [0.09 , 0.16] 6.6E-05 Mrc1 0.28 [-0.02 , 0.57] 1.4E-01 0.26 [0.13 , 0.40] 6.0E-03 0.10 [0.06 , 0.14] 1.1E-03 Lck 0.27 [-0.26 , 0.81] 4.1E-01 -0.05 [-0.27 , 0.16] 7.2E-01 -0.03 [-0.09 , 0.04] 5.5E-01 Sh2d1a 0.27 [-0.34 , 0.88] 4.7E-01 -0.01 [-0.26 , 0.25] 9.7E-01 0.00 [-0.09 , 0.09] 9.7E-01 Fut7 0.26 [-0.23 , 0.74] 4.0E-01 -0.03 [-0.28 , 0.22] 8.6E-01 n.d [n.d , n.d] n.d Gpr183 0.24 [-0.11 , 0.60] 2.7E-01 0.12 [0.00 , 0.24] 1.2E-01 0.05 [0.00 , 0.10] 1.4E-01 Lilra5 0.24 [0.04 , 0.44] 6.0E-02 0.03 [-0.12 , 0.17] 7.8E-01 -0.03 [-0.09 , 0.03] 4.9E-01 Ifitm2 0.24 [0.05 , 0.43] 5.3E-02 0.24 [0.14 , 0.34] 1.7E-03 0.10 [0.06 , 0.13] 4.7E-04 Mef2c 0.24 [-0.08 , 0.55] 2.3E-01 -0.02 [-0.16 , 0.12] 7.9E-01 -0.02 [-0.07 , 0.03] 5.7E-01 Ikzf1 0.22 [-0.10 , 0.55] 2.7E-01 -0.04 [-0.20 , 0.13] 7.4E-01 -0.01 [-0.04 , 0.02] 6.4E-01 Blk 0.21 [-0.63 , 1.05] 6.8E-01 -0.09 [-0.48 , 0.30] 7.3E-01 0.01 [-0.14 , 0.15] 9.5E-01 469 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Card11 0.21 [-0.26 , 0.69] 4.7E-01 -0.12 [-0.39 , 0.15] 4.8E-01 -0.07 [-0.14 , 0.00] 1.4E-01 Fcer1a 0.21 [-0.45 , 0.87] 6.1E-01 0.02 [-0.26 , 0.31] 9.2E-01 0.00 [-0.08 , 0.09] 9.8E-01 Irf8 0.20 [-0.06 , 0.47] 2.1E-01 0.02 [-0.09 , 0.14] 7.8E-01 0.00 [-0.06 , 0.06] 9.8E-01 C5ar1 0.20 [-0.15 , 0.56] 3.5E-01 0.12 [-0.03 , 0.27] 2.1E-01 0.03 [-0.06 , 0.11] 6.6E-01 Traf3 0.20 [-0.05 , 0.45] 2.0E-01 0.16 [0.07 , 0.25] 1.2E-02 0.05 [0.02 , 0.08] 1.5E-02 Irak2 0.20 [0.08 , 0.32] 1.6E-02 0.16 [0.07 , 0.26] 1.2E-02 0.07 [0.05 , 0.10] 1.6E-04 Amica1 0.20 [-0.14 , 0.54] 3.5E-01 0.14 [-0.02 , 0.30] 1.7E-01 0.02 [-0.04 , 0.08] 6.3E-01 Tgfb1 0.20 [0.04 , 0.35] 5.3E-02 0.13 [0.03 , 0.23] 3.8E-02 0.04 [0.00 , 0.07] 9.5E-02 Ccl5 0.20 [-0.28 , 0.68] 5.1E-01 0.01 [-0.23 , 0.24] 9.8E-01 -0.04 [-0.11 , 0.04] 4.5E-01 Pik3cd 0.20 [-0.14 , 0.53] 3.4E-01 0.02 [-0.15 , 0.19] 8.5E-01 -0.01 [-0.07 , 0.04] 6.8E-01 Casp8 0.19 [0.09 , 0.29] 8.8E-03 0.07 [0.01 , 0.14] 7.0E-02 0.01 [-0.01 , 0.03] 3.5E-01 Prkcd 0.19 [0.00 , 0.37] 1.1E-01 0.13 [0.05 , 0.22] 2.1E-02 0.05 [0.02 , 0.08] 1.1E-02 Traf2 0.18 [0.03 , 0.33] 6.6E-02 0.12 [0.06 , 0.19] 7.9E-03 0.04 [0.01 , 0.07] 2.7E-02 Ltk 0.18 [-0.32 , 0.67] 5.7E-01 0.06 [-0.15 , 0.27] 6.8E-01 -0.02 [-0.10 , 0.06] 6.9E-01 Tap2 0.17 [-0.21 , 0.55] 4.6E-01 0.00 [-0.14 , 0.13] 9.8E-01 0.01 [-0.04 , 0.06] 8.0E-01 Col1a1 0.17 [-0.24 , 0.58] 5.0E-01 0.09 [-0.06 , 0.24] 3.2E-01 0.05 [0.00 , 0.10] 1.3E-01 Map2k1 0.17 [0.08 , 0.25] 5.3E-03 0.12 [0.05 , 0.18] 7.9E-03 0.02 [0.00 , 0.04] 5.5E-02 470 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cklf 0.16 [-0.13 , 0.46] 3.7E-01 0.09 [-0.05 , 0.24] 3.1E-01 0.03 [-0.01 , 0.08] 2.8E-01 Map3k1 0.16 [-0.08 , 0.40] 2.8E-01 0.02 [-0.09 , 0.12] 7.9E-01 -0.01 [-0.04 , 0.03] 8.1E-01 Ifnar2 0.16 [0.00 , 0.32] 1.1E-01 0.17 [0.08 , 0.25] 5.3E-03 0.05 [0.02 , 0.07] 2.4E-02 Tnfsf15 0.16 [-0.13 , 0.45] 3.8E-01 0.22 [0.11 , 0.34] 7.4E-03 0.05 [0.01 , 0.09] 5.3E-02 Gbp2b 0.16 [-0.25 , 0.56] 5.3E-01 0.12 [-0.08 , 0.32] 3.4E-01 0.03 [-0.06 , 0.13] 5.7E-01 Il34 0.16 [-0.12 , 0.44] 3.7E-01 0.25 [0.09 , 0.41] 1.9E-02 0.10 [0.05 , 0.14] 4.7E-03 Il2rb 0.16 [-0.32 , 0.63] 6.1E-01 -0.04 [-0.26 , 0.18] 7.8E-01 -0.02 [-0.08 , 0.03] 5.8E-01 Irak4 0.15 [0.01 , 0.30] 9.3E-02 0.09 [-0.01 , 0.18] 1.6E-01 0.01 [-0.02 , 0.05] 5.1E-01 Mmp9 0.15 [-0.70 , 1.00] 7.7E-01 0.57 [0.13 , 1.02] 4.6E-02 0.21 [0.09 , 0.33] 9.9E-03 Psmb10 0.15 [-0.11 , 0.41] 3.6E-01 0.04 [-0.07 , 0.15] 5.7E-01 -0.01 [-0.06 , 0.03] 6.8E-01 Tnfrsf10b 0.14 [-0.19 , 0.47] 4.9E-01 0.05 [-0.10 , 0.21] 6.0E-01 -0.01 [-0.06 , 0.05] 8.5E-01 Nfatc1 0.14 [-0.06 , 0.33] 2.5E-01 0.01 [-0.08 , 0.10] 8.7E-01 0.00 [-0.03 , 0.04] 9.2E-01 Klrg1 0.14 [-0.28 , 0.56] 6.1E-01 0.15 [-0.12 , 0.42] 3.7E-01 0.10 [0.04 , 0.17] 1.7E-02 Il1rl2 0.13 [-0.11 , 0.37] 3.8E-01 0.32 [0.15 , 0.48] 7.4E-03 0.14 [0.10 , 0.17] 1.8E-05 Eomes 0.13 [-0.53 , 0.79] 7.5E-01 -0.13 [-0.41 , 0.15] 4.7E-01 -0.08 [-0.17 , 0.02] 2.2E-01 Ms4a2 0.13 [-0.76 , 1.01] 8.1E-01 -0.12 [-0.50 , 0.26] 6.2E-01 0.01 [-0.12 , 0.13] 9.4E-01 Psma2 0.12 [0.01 , 0.23] 7.3E-02 0.12 [0.05 , 0.19] 8.8E-03 0.04 [0.02 , 0.06] 4.8E-03 471 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Rrad 0.12 [-0.26 , 0.50] 6.1E-01 n.d [n.d , n.d] n.d -0.03 [-0.12 , 0.06] 6.6E-01 Ripk2 0.11 [-0.05 , 0.28] 2.6E-01 0.05 [-0.06 , 0.15] 4.7E-01 0.02 [-0.02 , 0.06] 5.0E-01 Timd4 0.11 [-0.66 , 0.88] 8.1E-01 -0.04 [-0.39 , 0.31] 8.9E-01 0.05 [-0.07 , 0.17] 5.4E-01 Anp32b 0.11 [-0.03 , 0.25] 2.0E-01 0.07 [-0.01 , 0.14] 1.5E-01 0.02 [0.00 , 0.04] 2.1E-01 C1qbp 0.11 [-0.04 , 0.26] 2.5E-01 0.07 [0.01 , 0.13] 9.1E-02 0.02 [-0.01 , 0.04] 2.4E-01 Cxcl12 0.11 [-0.21 , 0.42] 5.9E-01 0.27 [0.16 , 0.38] 1.7E-03 0.08 [0.04 , 0.12] 3.2E-03 Mefv 0.11 [-0.38 , 0.59] 7.2E-01 0.17 [-0.19 , 0.53] 4.7E-01 0.01 [-0.13 , 0.14] 9.6E-01 Bcl10 0.11 [-0.05 , 0.27] 2.9E-01 0.04 [-0.02 , 0.10] 2.8E-01 0.00 [-0.02 , 0.03] 7.8E-01 Col4a1 0.10 [-0.24 , 0.44] 6.3E-01 0.09 [-0.09 , 0.27] 4.5E-01 0.05 [0.02 , 0.07] 1.1E-02 Tfe3 0.10 [-0.19 , 0.39] 5.9E-01 0.08 [-0.04 , 0.21] 3.0E-01 0.01 [-0.02 , 0.04] 5.3E-01 Il7 0.10 [-0.34 , 0.54] 7.2E-01 0.18 [-0.12 , 0.47] 3.4E-01 0.10 [-0.01 , 0.21] 1.7E-01 Il16 0.10 [-0.20 , 0.39] 6.0E-01 -0.15 [-0.29 , -0.01] 9.8E-02 -0.08 [-0.13 , -0.03] 1.7E-02 Cd3d 0.09 [-0.44 , 0.63] 7.7E-01 -0.05 [-0.25 , 0.16] 7.2E-01 -0.03 [-0.12 , 0.05] 5.4E-01 Pvrl2 0.09 [-0.06 , 0.24] 3.4E-01 0.16 [0.08 , 0.24] 4.0E-03 0.05 [0.02 , 0.08] 9.1E-03 Jak2 0.09 [-0.03 , 0.21] 2.1E-01 0.15 [0.08 , 0.21] 2.8E-03 0.06 [0.03 , 0.08] 2.5E-03 Tapbp 0.09 [-0.10 , 0.28] 4.5E-01 0.06 [-0.05 , 0.17] 4.0E-01 0.01 [-0.03 , 0.05] 6.0E-01 Il3ra 0.09 [-0.26 , 0.43] 6.8E-01 0.06 [-0.07 , 0.19] 4.7E-01 0.02 [-0.02 , 0.07] 3.6E-01 472 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Lamp2 0.08 [-0.05 , 0.22] 3.1E-01 0.16 [0.08 , 0.24] 5.7E-03 0.06 [0.04 , 0.08] 3.8E-04 Cd44 0.08 [-0.25 , 0.42] 6.8E-01 0.34 [0.22 , 0.47] 6.4E-04 0.08 [0.00 , 0.16] 1.4E-01 Egr3 0.08 [-0.55 , 0.71] 8.2E-01 0.00 [-0.28 , 0.27] 9.8E-01 -0.05 [-0.15 , 0.05] 4.3E-01 Vhl 0.08 [-0.22 , 0.37] 6.7E-01 0.16 [0.03 , 0.28] 6.3E-02 0.07 [0.03 , 0.11] 1.2E-02 Cd3e 0.08 [-0.58 , 0.73] 8.4E-01 -0.11 [-0.34 , 0.13] 4.7E-01 -0.04 [-0.11 , 0.03] 3.5E-01 Pla2g1b 0.06 [-0.16 , 0.29] 6.6E-01 0.23 [0.09 , 0.37] 1.6E-02 0.07 [0.04 , 0.10] 2.9E-03 Tnfsf13b 0.06 [-0.18 , 0.30] 6.9E-01 -0.05 [-0.18 , 0.07] 4.9E-01 -0.06 [-0.11 , -0.01] 8.3E-02 Cd69 0.05 [-0.69 , 0.80] 9.0E-01 -0.27 [-0.61 , 0.07] 2.1E-01 -0.10 [-0.19 , -0.01] 9.9E-02 Cyld 0.05 [-0.21 , 0.31] 7.4E-01 0.00 [-0.15 , 0.16] 9.7E-01 0.00 [-0.05 , 0.04] 9.8E-01 Icosl 0.05 [-0.18 , 0.28] 7.2E-01 0.03 [-0.09 , 0.14] 7.4E-01 0.03 [-0.02 , 0.08] 4.1E-01 Cxcr2 0.05 [-0.93 , 1.04] 9.3E-01 0.19 [-0.20 , 0.58] 4.5E-01 0.04 [-0.06 , 0.14] 5.5E-01 Psmb7 0.05 [-0.23 , 0.32] 7.7E-01 0.12 [-0.01 , 0.24] 1.5E-01 0.06 [0.01 , 0.10] 4.7E-02 Cdkn1a 0.05 [-0.47 , 0.57] 8.8E-01 0.02 [-0.23 , 0.27] 9.3E-01 0.02 [-0.09 , 0.14] 7.9E-01 Il17ra 0.04 [-0.17 , 0.25] 7.4E-01 -0.02 [-0.15 , 0.11] 8.2E-01 -0.02 [-0.07 , 0.02] 4.5E-01 Nfkbia 0.04 [-0.32 , 0.41] 8.4E-01 0.01 [-0.16 , 0.18] 9.2E-01 -0.01 [-0.07 , 0.05] 8.0E-01 Stat3 0.04 [-0.09 , 0.17] 6.3E-01 0.08 [0.03 , 0.13] 2.7E-02 0.02 [0.01 , 0.04] 9.3E-03 Il13ra1 0.04 [-0.23 , 0.31] 8.1E-01 0.18 [0.05 , 0.31] 3.3E-02 0.05 [0.02 , 0.09] 3.7E-02 473 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Lif 0.04 [-0.26 , 0.33] 8.2E-01 0.13 [-0.08 , 0.33] 3.2E-01 0.03 [-0.05 , 0.12] 5.4E-01 Mavs 0.04 [-0.17 , 0.24] 7.6E-01 0.05 [-0.04 , 0.15] 3.4E-01 0.01 [-0.02 , 0.05] 5.3E-01 Psen2 0.04 [-0.11 , 0.19] 6.9E-01 0.00 [-0.11 , 0.10] 9.7E-01 0.02 [-0.01 , 0.05] 3.4E-01 Tnfrsf14 0.02 [-0.17 , 0.21] 8.6E-01 -0.06 [-0.16 , 0.03] 2.9E-01 -0.04 [-0.07 , -0.01] 6.9E-02 Cebpb 0.02 [-0.30 , 0.33] 9.3E-01 0.10 [-0.05 , 0.24] 2.9E-01 0.03 [-0.04 , 0.09] 5.4E-01 Psen1 0.01 [-0.09 , 0.12] 8.1E-01 -0.01 [-0.06 , 0.03] 6.3E-01 -0.01 [-0.03 , 0.00] 2.0E-01 Tfeb 0.00 [-0.21 , 0.21] 9.9E-01 -0.02 [-0.11 , 0.07] 7.8E-01 -0.01 [-0.05 , 0.02] 5.3E-01 Tirap 0.00 [-0.19 , 0.18] 9.7E-01 0.00 [-0.11 , 0.10] 9.7E-01 -0.02 [-0.06 , 0.02] 3.6E-01 Atg7 -0.01 [-0.20 , 0.17] 9.0E-01 0.12 [0.05 , 0.19] 1.2E-02 0.02 [-0.01 , 0.04] 2.7E-01 Vim -0.02 [-0.22 , 0.19] 8.8E-01 0.05 [-0.04 , 0.15] 4.0E-01 0.03 [0.01 , 0.06] 5.6E-02 Lamp1 -0.02 [-0.17 , 0.13] 8.1E-01 0.08 [0.01 , 0.16] 9.7E-02 0.03 [0.01 , 0.05] 4.6E-02 Il1r1 -0.02 [-0.26 , 0.21] 8.6E-01 0.11 [-0.02 , 0.23] 1.8E-01 0.04 [0.01 , 0.08] 7.0E-02 Nfkb1 -0.02 [-0.16 , 0.11] 7.5E-01 0.03 [-0.03 , 0.08] 4.7E-01 0.00 [-0.02 , 0.02] 9.4E-01 Mapk1 -0.03 [-0.15 , 0.09] 7.3E-01 0.04 [-0.01 , 0.08] 2.2E-01 0.01 [0.00 , 0.02] 2.2E-01 Cd40lg -0.03 [-0.62 , 0.57] 9.3E-01 -0.10 [-0.41 , 0.21] 6.2E-01 -0.06 [-0.14 , 0.02] 2.4E-01 Tfrc -0.03 [-0.28 , 0.22] 8.4E-01 0.22 [0.07 , 0.38] 2.8E-02 0.09 [0.06 , 0.13] 6.2E-04 Ifi35 -0.03 [-0.23 , 0.16] 7.8E-01 0.05 [-0.05 , 0.15] 4.3E-01 0.00 [-0.06 , 0.06] 9.6E-01 474 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd247 -0.03 [-0.54 , 0.47] 9.0E-01 -0.06 [-0.26 , 0.15] 6.8E-01 -0.01 [-0.08 , 0.06] 7.8E-01 Nfatc3 -0.04 [-0.31 , 0.23] 8.0E-01 -0.09 [-0.22 , 0.03] 2.5E-01 -0.02 [-0.05 , 0.01] 3.6E-01 Entpd1 -0.04 [-0.21 , 0.13] 6.8E-01 -0.02 [-0.12 , 0.08] 7.4E-01 0.01 [-0.02 , 0.04] 6.9E-01 Mapkapk2 -0.04 [-0.22 , 0.13] 6.9E-01 0.06 [-0.02 , 0.14] 2.5E-01 0.03 [0.00 , 0.05] 1.4E-01 Igf2r -0.05 [-0.20 , 0.11] 6.3E-01 0.10 [0.01 , 0.20] 9.2E-02 0.04 [0.01 , 0.07] 7.1E-02 Cd40 -0.05 [-0.21 , 0.11] 6.2E-01 -0.01 [-0.08 , 0.05] 7.5E-01 -0.02 [-0.09 , 0.05] 6.0E-01 Trp53 -0.05 [-0.30 , 0.20] 7.5E-01 -0.08 [-0.20 , 0.03] 2.6E-01 -0.04 [-0.07 , 0.00] 8.3E-02 Smn1 -0.05 [-0.19 , 0.09] 5.7E-01 0.01 [-0.03 , 0.06] 6.0E-01 0.00 [-0.02 , 0.02] 9.9E-01 Mapk11 -0.05 [-0.36 , 0.25] 7.7E-01 0.03 [-0.10 , 0.16] 7.1E-01 0.01 [-0.04 , 0.07] 7.1E-01 Epcam -0.05 [-0.28 , 0.17] 6.9E-01 0.12 [-0.01 , 0.24] 1.3E-01 0.04 [0.00 , 0.08] 8.2E-02 Atf1 -0.05 [-0.36 , 0.26] 7.7E-01 0.08 [-0.05 , 0.22] 3.2E-01 0.01 [-0.05 , 0.06] 8.4E-01 Chuk -0.06 [-0.16 , 0.05] 3.7E-01 0.04 [-0.02 , 0.09] 3.4E-01 0.02 [0.00 , 0.03] 7.1E-02 Fn1 -0.06 [-0.35 , 0.23] 7.5E-01 0.06 [-0.06 , 0.18] 4.5E-01 0.02 [-0.02 , 0.06] 5.2E-01 Cd160 -0.06 [-0.68 , 0.57] 8.8E-01 -0.04 [-0.36 , 0.29] 8.7E-01 -0.05 [-0.13 , 0.02] 2.5E-01 Map2k2 -0.06 [-0.25 , 0.13] 6.2E-01 0.04 [-0.02 , 0.10] 2.7E-01 0.01 [-0.02 , 0.03] 7.6E-01 Itga5 -0.06 [-0.34 , 0.21] 7.2E-01 0.05 [-0.12 , 0.22] 6.5E-01 0.03 [-0.02 , 0.07] 3.2E-01 C2 -0.06 [-0.43 , 0.31] 7.7E-01 0.08 [-0.06 , 0.22] 3.7E-01 0.03 [-0.02 , 0.09] 3.1E-01 475 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Pla2g6 -0.07 [-0.29 , 0.16] 6.4E-01 0.05 [-0.06 , 0.17] 4.8E-01 0.02 [-0.01 , 0.05] 2.3E-01 Plaur -0.07 [-0.42 , 0.29] 7.5E-01 0.19 [0.04 , 0.35] 5.2E-02 0.08 [0.03 , 0.12] 1.9E-02 Ythdf2 -0.07 [-0.16 , 0.02] 2.0E-01 -0.01 [-0.05 , 0.02] 5.5E-01 -0.01 [-0.02 , 0.01] 5.4E-01 Atg5 -0.08 [-0.27 , 0.11] 4.8E-01 n.d [n.d , n.d] n.d 0.01 [-0.03 , 0.04] 7.1E-01 Il18 -0.08 [-0.41 , 0.24] 6.8E-01 -0.08 [-0.26 , 0.10] 5.0E-01 -0.07 [-0.15 , 0.00] 1.3E-01 Irf1 -0.08 [-0.25 , 0.08] 4.2E-01 -0.01 [-0.12 , 0.10] 8.7E-01 -0.03 [-0.07 , 0.01] 2.8E-01 Gpi1 -0.09 [-0.20 , 0.01] 1.6E-01 0.00 [-0.03 , 0.04] 8.9E-01 0.01 [-0.01 , 0.02] 5.0E-01 Cd9 -0.10 [-0.44 , 0.25] 6.6E-01 0.09 [0.00 , 0.17] 1.2E-01 0.04 [0.02 , 0.07] 1.7E-02 Cd38 -0.10 [-0.29 , 0.09] 4.1E-01 -0.12 [-0.25 , 0.01] 1.6E-01 -0.02 [-0.05 , 0.02] 4.5E-01 Csf1 -0.11 [-0.45 , 0.24] 6.2E-01 0.19 [0.10 , 0.28] 4.5E-03 0.04 [0.01 , 0.08] 6.0E-02 Tlr3 -0.11 [-0.37 , 0.15] 5.0E-01 0.03 [-0.11 , 0.17] 7.3E-01 0.00 [-0.04 , 0.04] 9.9E-01 Cd3eap -0.11 [-0.47 , 0.25] 6.2E-01 0.12 [-0.09 , 0.33] 3.6E-01 0.02 [-0.05 , 0.09] 6.2E-01 Serping1 -0.12 [-0.37 , 0.14] 4.7E-01 0.03 [-0.07 , 0.14] 6.3E-01 0.01 [-0.02 , 0.04] 5.3E-01 Tab1 -0.12 [-0.35 , 0.12] 4.2E-01 0.04 [-0.06 , 0.14] 5.3E-01 0.03 [-0.02 , 0.07] 3.1E-01 Fyn -0.12 [-0.22 , -0.02] 6.3E-02 -0.13 [-0.20 , -0.05] 1.4E-02 -0.05 [-0.07 , -0.03] 5.2E-04 Ambp -0.12 [-2.09 , 1.86] 9.2E-01 0.10 [-0.72 , 0.92] 8.6E-01 0.16 [-0.07 , 0.38] 2.8E-01 Atm -0.12 [-0.28 , 0.03] 2.1E-01 -0.09 [-0.17 , 0.00] 1.2E-01 -0.03 [-0.06 , 0.00] 1.2E-01 476 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Anxa1 -0.12 [-0.36 , 0.12] 4.2E-01 0.08 [-0.04 , 0.21] 3.0E-01 0.02 [0.00 , 0.04] 1.0E-01 Bcl6 -0.12 [-0.42 , 0.18] 5.1E-01 -0.03 [-0.11 , 0.05] 5.4E-01 -0.02 [-0.07 , 0.03] 5.2E-01 Ccl21a -0.12 [-0.58 , 0.33] 6.6E-01 -0.09 [-0.26 , 0.08] 3.8E-01 -0.07 [-0.11 , -0.02] 2.7E-02 Ifnar1 -0.13 [-0.21 , -0.04] 2.2E-02 0.02 [-0.02 , 0.05] 4.9E-01 0.00 [-0.01 , 0.02] 8.1E-01 Ep300 -0.13 [-0.24 , -0.01] 9.3E-02 -0.05 [-0.12 , 0.02] 2.5E-01 -0.02 [-0.04 , 0.00] 5.8E-02 Runx1 -0.13 [-0.34 , 0.09] 3.4E-01 0.05 [-0.02 , 0.12] 2.4E-01 0.02 [0.00 , 0.04] 1.6E-01 Cd1d1 -0.13 [-0.33 , 0.07] 3.0E-01 -0.08 [-0.19 , 0.03] 2.5E-01 -0.01 [-0.03 , 0.02] 7.0E-01 Tbk1 -0.13 [-0.33 , 0.08] 3.2E-01 -0.03 [-0.11 , 0.05] 5.3E-01 0.00 [-0.03 , 0.03] 8.3E-01 Tyk2 -0.13 [-0.56 , 0.29] 6.2E-01 -0.10 [-0.25 , 0.05] 2.7E-01 -0.05 [-0.11 , 0.01] 2.4E-01 Ewsr1 -0.14 [-0.29 , 0.02] 1.5E-01 -0.04 [-0.08 , 0.00] 1.3E-01 0.00 [-0.02 , 0.02] 7.4E-01 Gzmb -0.14 [-0.67 , 0.40] 6.8E-01 -0.06 [-0.35 , 0.23] 7.4E-01 -0.02 [-0.09 , 0.06] 7.4E-01 Il1rap -0.14 [-0.49 , 0.22] 5.3E-01 0.10 [0.00 , 0.19] 1.2E-01 0.06 [0.03 , 0.09] 4.7E-03 Alcam -0.14 [-0.33 , 0.05] 2.4E-01 0.08 [-0.04 , 0.20] 3.1E-01 0.03 [0.00 , 0.07] 9.5E-02 Nup107 -0.14 [-0.31 , 0.03] 1.7E-01 -0.09 [-0.16 , -0.02] 5.6E-02 -0.03 [-0.06 , 0.00] 9.5E-02 Irf2 -0.14 [-0.30 , 0.01] 1.4E-01 -0.04 [-0.10 , 0.02] 2.8E-01 -0.02 [-0.04 , 0.00] 1.8E-01 Cd47 -0.15 [-0.27 , -0.02] 6.4E-02 -0.08 [-0.13 , -0.03] 2.0E-02 -0.04 [-0.07 , -0.01] 6.0E-02 Tnfsf13 -0.15 [-0.51 , 0.22] 5.2E-01 0.11 [-0.06 , 0.29] 3.1E-01 0.05 [0.00 , 0.09] 1.2E-01 477 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Thbs1 -0.15 [-0.70 , 0.41] 6.8E-01 -0.01 [-0.25 , 0.23] 9.7E-01 -0.01 [-0.08 , 0.06] 8.7E-01 Fadd -0.15 [-0.43 , 0.14] 4.1E-01 0.01 [-0.13 , 0.15] 9.2E-01 -0.01 [-0.05 , 0.02] 5.7E-01 Il18rap -0.15 [-0.57 , 0.27] 5.6E-01 0.10 [-0.09 , 0.30] 4.1E-01 0.05 [0.00 , 0.10] 1.3E-01 Ccl25 -0.16 [-0.62 , 0.30] 5.8E-01 0.01 [-0.18 , 0.20] 9.4E-01 0.02 [-0.05 , 0.08] 7.1E-01 Ubc -0.16 [-0.33 , 0.01] 1.3E-01 -0.03 [-0.11 , 0.06] 6.3E-01 -0.01 [-0.03 , 0.02] 7.1E-01 Atg12 -0.16 [-0.30 , -0.03] 6.1E-02 0.00 [-0.10 , 0.09] 9.7E-01 -0.01 [-0.02 , 0.01] 4.4E-01 Icam1 -0.17 [-0.44 , 0.09] 3.0E-01 0.03 [-0.08 , 0.13] 6.9E-01 0.01 [-0.01 , 0.03] 4.0E-01 Psmd7 -0.17 [-0.31 , -0.04] 5.1E-02 0.01 [-0.04 , 0.07] 7.6E-01 0.00 [-0.01 , 0.02] 9.9E-01 Cd200 -0.18 [-0.37 , 0.02] 1.4E-01 0.00 [-0.08 , 0.09] 9.7E-01 0.01 [-0.02 , 0.03] 7.0E-01 Il15 -0.18 [-0.55 , 0.19] 4.3E-01 -0.01 [-0.15 , 0.13] 9.6E-01 -0.06 [-0.11 , -0.01] 5.3E-02 Il12rb2 -0.18 [-0.53 , 0.17] 4.0E-01 0.01 [-0.16 , 0.19] 9.2E-01 0.00 [-0.07 , 0.07] 9.8E-01 Ikbkg -0.18 [-0.49 , 0.12] 3.3E-01 -0.04 [-0.15 , 0.08] 6.3E-01 -0.01 [-0.05 , 0.03] 7.8E-01 Itgb3 -0.19 [-0.50 , 0.13] 3.4E-01 -0.03 [-0.14 , 0.07] 6.4E-01 -0.01 [-0.05 , 0.04] 8.1E-01 Il4ra -0.19 [-0.49 , 0.11] 3.0E-01 -0.09 [-0.19 , 0.01] 1.6E-01 -0.03 [-0.06 , 0.01] 2.5E-01 Cd27 -0.19 [-0.94 , 0.55] 6.8E-01 -0.23 [-0.62 , 0.15] 3.4E-01 -0.06 [-0.17 , 0.05] 4.1E-01 Yy1 -0.20 [-0.32 , -0.07] 2.0E-02 -0.04 [-0.10 , 0.01] 2.1E-01 -0.02 [-0.03 , 0.00] 9.2E-02 Tgfbr2 -0.20 [-0.33 , -0.06] 2.6E-02 -0.02 [-0.07 , 0.03] 5.5E-01 -0.01 [-0.02 , 0.01] 5.6E-01 478 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tgfb3 -0.20 [-0.52 , 0.13] 3.2E-01 -0.09 [-0.21 , 0.03] 2.4E-01 -0.01 [-0.06 , 0.03] 6.6E-01 Atg10 -0.20 [-0.54 , 0.14] 3.4E-01 -0.01 [-0.13 , 0.12] 9.2E-01 -0.01 [-0.05 , 0.03] 7.3E-01 Stat6 -0.20 [-0.39 , -0.02] 7.7E-02 -0.04 [-0.08 , 0.01] 2.1E-01 -0.02 [-0.04 , 0.00] 1.6E-01 Smpd3 -0.21 [-0.77 , 0.36] 5.6E-01 n.d [n.d , n.d] n.d 0.01 [-0.06 , 0.09] 7.8E-01 Ceacam1 -0.21 [-0.67 , 0.25] 4.6E-01 0.00 [-0.13 , 0.13] 9.8E-01 -0.01 [-0.08 , 0.05] 7.8E-01 Map2k4 -0.21 [-0.43 , 0.00] 1.1E-01 -0.03 [-0.11 , 0.05] 5.5E-01 0.00 [-0.03 , 0.02] 8.3E-01 Atg16l1 -0.22 [-0.36 , -0.07] 3.0E-02 -0.06 [-0.12 , 0.00] 1.2E-01 -0.02 [-0.04 , 0.00] 6.1E-02 Map3k7 -0.23 [-0.68 , 0.23] 4.2E-01 -0.01 [-0.07 , 0.06] 9.1E-01 0.00 [-0.03 , 0.03] 9.8E-01 Ccr2 -0.23 [-0.69 , 0.24] 4.3E-01 -0.06 [-0.24 , 0.12] 5.9E-01 -0.07 [-0.16 , 0.01] 1.9E-01 Itgb1 -0.23 [-0.47 , 0.02] 1.4E-01 -0.07 [-0.12 , -0.03] 1.3E-02 -0.03 [-0.04 , -0.01] 1.1E-02 Tnfrsf11b -0.23 [-1.05 , 0.60] 6.6E-01 -0.23 [-0.55 , 0.09] 2.6E-01 -0.04 [-0.16 , 0.08] 6.0E-01 Ifngr1 -0.23 [-0.50 , 0.04] 1.7E-01 -0.03 [-0.09 , 0.03] 3.7E-01 -0.01 [-0.04 , 0.01] 4.7E-01 Ikbkb -0.24 [-0.38 , -0.11] 1.2E-02 -0.13 [-0.19 , -0.07] 3.6E-03 -0.05 [-0.07 , -0.02] 6.1E-03 Ctsw -0.24 [-0.58 , 0.09] 2.4E-01 -0.02 [-0.19 , 0.14] 8.4E-01 n.d [n.d , n.d] n.d Cxcl14 -0.25 [-0.41 , -0.08] 2.5E-02 -0.07 [-0.14 , 0.01] 1.6E-01 0.00 [-0.03 , 0.03] 1.0E+00 Cd55 -0.25 [-0.49 , -0.01] 9.4E-02 -0.04 [-0.13 , 0.05] 5.2E-01 n.d [n.d , n.d] n.d Ecsit -0.25 [-0.47 , -0.03] 7.5E-02 -0.18 [-0.28 , -0.08] 8.9E-03 -0.07 [-0.11 , -0.03] 1.0E-02 479 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cspg4 -0.25 [-0.72 , 0.21] 3.8E-01 -0.07 [-0.21 , 0.07] 4.6E-01 -0.01 [-0.06 , 0.05] 8.9E-01 Ltbr -0.25 [-0.51 , 0.01] 1.2E-01 -0.01 [-0.11 , 0.08] 8.5E-01 0.00 [-0.03 , 0.02] 9.2E-01 Il15ra -0.26 [-0.68 , 0.16] 3.2E-01 -0.09 [-0.30 , 0.13] 5.3E-01 0.05 [-0.01 , 0.12] 2.1E-01 H2-Q10 -0.26 [-1.37 , 0.84] 7.0E-01 0.10 [-0.37 , 0.57] 7.4E-01 0.09 [0.00 , 0.17] 1.0E-01 Mapk8 -0.27 [-0.47 , -0.06] 4.5E-02 -0.08 [-0.16 , -0.01] 7.7E-02 -0.04 [-0.07 , -0.01] 6.3E-02 Il7r -0.27 [-0.71 , 0.18] 3.3E-01 0.17 [-0.03 , 0.38] 1.9E-01 0.09 [0.03 , 0.15] 3.2E-02 Bcl2 -0.27 [-0.45 , -0.10] 2.2E-02 -0.24 [-0.34 , -0.14] 1.6E-03 -0.09 [-0.13 , -0.04] 4.5E-03 Ilf3 -0.28 [-0.50 , -0.05] 5.4E-02 -0.09 [-0.18 , 0.01] 1.6E-01 -0.03 [-0.07 , 0.01] 2.3E-01 Itch -0.28 [-0.43 , -0.13] 9.1E-03 -0.07 [-0.11 , -0.03] 9.1E-03 -0.02 [-0.04 , 0.00] 8.6E-02 Smad2 -0.28 [-0.65 , 0.09] 2.2E-01 -0.04 [-0.19 , 0.12] 7.2E-01 0.00 [-0.05 , 0.06] 9.3E-01 Traf6 -0.29 [-0.41 , -0.16] 3.4E-03 -0.11 [-0.20 , -0.02] 5.0E-02 -0.02 [-0.04 , 0.00] 8.8E-02 Irak1 -0.29 [-0.49 , -0.08] 3.3E-02 -0.12 [-0.21 , -0.04] 3.3E-02 -0.03 [-0.06 , 0.00] 1.6E-01 Thy1 -0.29 [-0.58 , 0.00] 1.1E-01 -0.18 [-0.33 , -0.03] 6.7E-02 -0.08 [-0.15 , -0.01] 7.3E-02 Icam4 -0.30 [-0.76 , 0.17] 3.0E-01 -0.18 [-0.45 , 0.09] 2.9E-01 -0.09 [-0.15 , -0.02] 3.7E-02 Jak1 -0.30 [-0.44 , -0.17] 2.7E-03 -0.10 [-0.12 , -0.07] 3.2E-05 -0.04 [-0.05 , -0.02] 7.7E-04 Map3k5 -0.31 [-0.53 , -0.08] 3.6E-02 -0.14 [-0.19 , -0.09] 7.2E-04 -0.07 [-0.09 , -0.04] 9.6E-04 Irf3 -0.31 [-0.52 , -0.10] 2.7E-02 -0.12 [-0.18 , -0.06] 5.3E-03 -0.04 [-0.07 , -0.02] 1.3E-02 480 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tmed1 -0.32 [-0.53 , -0.11] 2.5E-02 -0.14 [-0.26 , -0.03] 5.6E-02 -0.05 [-0.08 , -0.01] 4.0E-02 Rela -0.32 [-0.59 , -0.06] 5.5E-02 -0.11 [-0.18 , -0.04] 1.7E-02 -0.04 [-0.07 , -0.02] 1.1E-02 Atf2 -0.33 [-0.58 , -0.08] 4.4E-02 -0.10 [-0.18 , -0.02] 6.2E-02 -0.05 [-0.08 , -0.01] 5.7E-02 Smad3 -0.33 [-0.51 , -0.15] 1.0E-02 -0.12 [-0.20 , -0.03] 3.1E-02 -0.04 [-0.07 , -0.01] 2.5E-02 Pvr -0.33 [-0.58 , -0.08] 4.3E-02 -0.04 [-0.17 , 0.08] 6.1E-01 -0.03 [-0.06 , 0.00] 1.6E-01 Hmgb1 -0.34 [-0.50 , -0.18] 4.1E-03 -0.09 [-0.15 , -0.03] 2.3E-02 -0.04 [-0.06 , -0.03] 3.8E-04 Cd164 -0.34 [-0.54 , -0.14] 1.5E-02 -0.09 [-0.17 , -0.02] 5.6E-02 -0.03 [-0.06 , -0.01] 4.2E-02 Mapk14 -0.35 [-0.55 , -0.15] 1.2E-02 -0.14 [-0.17 , -0.11] 3.2E-05 -0.06 [-0.09 , -0.03] 3.6E-03 Cd34 -0.36 [-0.61 , -0.10] 3.4E-02 -0.06 [-0.19 , 0.06] 4.0E-01 -0.01 [-0.03 , 0.01] 3.2E-01 Sell -0.36 [-0.75 , 0.03] 1.4E-01 -0.29 [-0.43 , -0.14] 5.3E-03 -0.11 [-0.19 , -0.03] 4.1E-02 Cd81 -0.36 [-0.55 , -0.17] 8.5E-03 -0.15 [-0.22 , -0.08] 2.4E-03 -0.07 [-0.09 , -0.05] 8.9E-05 Reps1 -0.37 [-0.50 , -0.24] 7.0E-04 -0.13 [-0.17 , -0.08] 1.1E-03 -0.04 [-0.05 , -0.02] 8.1E-04 Il18r1 -0.37 [-0.68 , -0.05] 6.7E-02 -0.10 [-0.19 , -0.02] 6.4E-02 -0.05 [-0.08 , -0.02] 1.8E-02 Mr1 -0.37 [-0.59 , -0.14] 1.7E-02 -0.04 [-0.16 , 0.09] 6.5E-01 0.02 [-0.03 , 0.06] 5.7E-01 Cd2 -0.37 [-0.88 , 0.14] 2.5E-01 -0.45 [-0.76 , -0.14] 2.8E-02 -0.11 [-0.20 , -0.02] 7.0E-02 Tnfsf12 -0.37 [-0.64 , -0.11] 3.5E-02 -0.09 [-0.15 , -0.02] 3.8E-02 -0.04 [-0.07 , -0.02] 1.2E-02 Stat5b -0.38 [-0.56 , -0.19] 5.4E-03 -0.18 [-0.24 , -0.12] 4.0E-04 -0.06 [-0.09 , -0.03] 3.8E-03 481 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Elk1 -0.38 [-0.62 , -0.14] 2.1E-02 -0.13 [-0.36 , 0.10] 3.8E-01 -0.08 [-0.15 , -0.02] 5.9E-02 Itga2b -0.38 [-1.27 , 0.52] 4.9E-01 -0.16 [-0.50 , 0.18] 4.7E-01 -0.03 [-0.11 , 0.06] 6.6E-01 Bmi1 -0.39 [-0.69 , -0.08] 5.3E-02 -0.11 [-0.22 , -0.01] 9.1E-02 -0.05 [-0.08 , -0.02] 9.8E-03 Klrc1 -0.39 [-0.89 , 0.11] 2.1E-01 -0.15 [-0.32 , 0.03] 1.9E-01 -0.03 [-0.11 , 0.04] 5.0E-01 Creb1 -0.40 [-0.61 , -0.18] 1.0E-02 -0.16 [-0.25 , -0.07] 1.3E-02 -0.06 [-0.08 , -0.04] 7.1E-04 Pin1 -0.40 [-0.67 , -0.12] 3.0E-02 -0.17 [-0.30 , -0.04] 4.2E-02 -0.03 [-0.07 , 0.01] 2.9E-01 Crebbp -0.40 [-0.59 , -0.21] 4.4E-03 -0.14 [-0.19 , -0.09] 4.9E-04 -0.04 [-0.06 , -0.02] 4.8E-03 Itga6 -0.40 [-0.61 , -0.20] 6.8E-03 -0.13 [-0.22 , -0.03] 3.6E-02 -0.04 [-0.07 , -0.02] 1.7E-02 Fos -0.41 [-0.94 , 0.12] 2.1E-01 -0.38 [-0.66 , -0.09] 4.1E-02 -0.17 [-0.28 , -0.07] 1.3E-02 Mapk3 -0.41 [-0.74 , -0.08] 5.3E-02 -0.13 [-0.22 , -0.04] 2.7E-02 -0.05 [-0.08 , -0.02] 6.6E-03 Sigirr -0.41 [-0.65 , -0.18] 1.1E-02 -0.20 [-0.32 , -0.07] 1.7E-02 -0.06 [-0.09 , -0.04] 1.2E-03 Fas -0.42 [-0.77 , -0.07] 6.0E-02 -0.13 [-0.26 , 0.00] 1.2E-01 -0.01 [-0.08 , 0.07] 9.2E-01 Erbb2 -0.42 [-0.78 , -0.07] 6.4E-02 -0.06 [-0.18 , 0.06] 4.6E-01 0.00 [-0.03 , 0.02] 8.5E-01 Tollip -0.42 [-0.75 , -0.10] 4.4E-02 -0.13 [-0.21 , -0.06] 9.4E-03 -0.03 [-0.05 , -0.01] 3.4E-02 Mertk -0.42 [-0.76 , -0.09] 4.7E-02 -0.24 [-0.40 , -0.07] 2.9E-02 -0.05 [-0.12 , 0.02] 2.4E-01 Rps6 -0.43 [-0.69 , -0.16] 1.9E-02 -0.17 [-0.28 , -0.06] 2.4E-02 -0.06 [-0.10 , -0.02] 3.3E-02 Cfh -0.43 [-0.85 , -0.01] 1.1E-01 -0.08 [-0.19 , 0.03] 2.7E-01 -0.02 [-0.06 , 0.01] 2.9E-01 482 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccnd3 -0.44 [-0.69 , -0.19] 1.2E-02 -0.20 [-0.27 , -0.13] 4.6E-04 -0.09 [-0.12 , -0.07] 6.6E-05 Cd99 -0.45 [-0.75 , -0.14] 2.9E-02 -0.15 [-0.27 , -0.03] 6.2E-02 -0.06 [-0.09 , -0.03] 2.2E-03 Il6ra -0.45 [-0.80 , -0.09] 5.0E-02 -0.16 [-0.29 , -0.03] 6.4E-02 -0.06 [-0.11 , -0.02] 3.7E-02 Ppbp -0.45 [-1.64 , 0.74] 5.5E-01 -0.21 [-0.61 , 0.19] 4.1E-01 -0.07 [-0.20 , 0.05] 3.4E-01 Tnfrsf1a -0.45 [-0.74 , -0.17] 2.0E-02 -0.12 [-0.20 , -0.05] 1.7E-02 -0.04 [-0.07 , -0.02] 1.5E-02 Cd1d2 -0.46 [-1.14 , 0.22] 2.8E-01 -0.06 [-0.28 , 0.16] 6.8E-01 -0.06 [-0.13 , 0.01] 1.7E-01 Ptgs2 -0.46 [-1.00 , 0.08] 1.7E-01 -0.16 [-0.45 , 0.13] 3.8E-01 -0.08 [-0.15 , -0.01] 9.3E-02 Rorc -0.46 [-0.87 , -0.06] 7.4E-02 -0.14 [-0.31 , 0.03] 1.9E-01 -0.05 [-0.09 , 0.00] 9.3E-02 Cdh1 -0.47 [-0.85 , -0.10] 5.2E-02 -0.05 [-0.16 , 0.05] 4.4E-01 -0.03 [-0.05 , 0.00] 1.4E-01 App -0.48 [-0.75 , -0.20] 1.3E-02 -0.17 [-0.26 , -0.08] 8.9E-03 -0.05 [-0.07 , -0.04] 2.4E-04 Nod1 -0.48 [-0.79 , -0.17] 2.3E-02 -0.18 [-0.30 , -0.06] 2.3E-02 -0.08 [-0.12 , -0.04] 8.2E-03 Smad4 -0.48 [-0.77 , -0.18] 1.9E-02 -0.16 [-0.25 , -0.07] 8.4E-03 -0.05 [-0.08 , -0.02] 1.4E-02 Foxj1 -0.49 [-1.00 , 0.01] 1.2E-01 -0.07 [-0.29 , 0.15] 6.3E-01 -0.03 [-0.11 , 0.05] 5.7E-01 Cd96 -0.50 [-1.17 , 0.18] 2.4E-01 -0.15 [-0.48 , 0.17] 4.7E-01 -0.05 [-0.16 , 0.05] 4.5E-01 Fasl -0.50 [-1.22 , 0.23] 2.7E-01 -0.39 [-0.73 , -0.04] 8.2E-02 -0.14 [-0.27 , -0.01] 9.5E-02 Mfge8 -0.50 [-0.83 , -0.16] 2.6E-02 -0.25 [-0.35 , -0.16] 8.7E-04 -0.09 [-0.13 , -0.05] 2.6E-03 Lrp1 -0.50 [-0.90 , -0.09] 5.6E-02 -0.07 [-0.16 , 0.01] 1.9E-01 -0.01 [-0.04 , 0.02] 6.3E-01 483 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Colec12 -0.51 [-0.84 , -0.17] 2.5E-02 -0.15 [-0.23 , -0.08] 5.3E-03 -0.06 [-0.09 , -0.03] 2.2E-03 Bcl2l1 -0.51 [-0.90 , -0.12] 4.6E-02 -0.15 [-0.33 , 0.02] 1.7E-01 -0.02 [-0.12 , 0.07] 7.2E-01 Glycam1 -0.51 [-2.34 , 1.31] 6.6E-01 -0.35 [-1.06 , 0.36] 4.4E-01 -0.21 [-0.47 , 0.05] 2.1E-01 Nt5e -0.52 [-0.92 , -0.11] 4.8E-02 -0.11 [-0.27 , 0.05] 2.7E-01 -0.02 [-0.06 , 0.02] 5.3E-01 Ccl27a -0.52 [-0.82 , -0.22] 1.3E-02 -0.32 [-0.60 , -0.05] 6.3E-02 -0.12 [-0.20 , -0.05] 1.4E-02 Abl1 -0.52 [-0.78 , -0.27] 4.9E-03 -0.18 [-0.29 , -0.06] 2.2E-02 -0.05 [-0.08 , -0.01] 4.1E-02 Prf1 -0.53 [-0.94 , -0.12] 4.5E-02 -0.36 [-0.59 , -0.13] 2.0E-02 -0.10 [-0.18 , -0.03] 4.4E-02 Ccr9 -0.53 [-1.04 , -0.02] 9.8E-02 -0.18 [-0.38 , 0.02] 1.6E-01 -0.04 [-0.18 , 0.11] 7.0E-01 Nfatc4 -0.54 [-1.01 , -0.06] 7.4E-02 -0.29 [-0.49 , -0.09] 2.7E-02 -0.05 [-0.10 , 0.00] 9.3E-02 Dusp6 -0.54 [-1.00 , -0.08] 6.5E-02 -0.16 [-0.33 , 0.02] 1.6E-01 -0.09 [-0.15 , -0.02] 3.7E-02 Egr1 -0.54 [-1.04 , -0.04] 8.6E-02 -0.29 [-0.53 , -0.05] 6.3E-02 -0.13 [-0.23 , -0.02] 6.0E-02 C7 -0.54 [-1.08 , 0.00] 1.1E-01 -0.06 [-0.21 , 0.08] 5.1E-01 -0.03 [-0.08 , 0.01] 2.7E-01 Il22ra1 -0.54 [-1.11 , 0.02] 1.2E-01 -0.08 [-0.35 , 0.20] 6.6E-01 0.02 [-0.07 , 0.11] 7.5E-01 Ccl24 -0.56 [-1.42 , 0.30] 3.0E-01 0.12 [-0.27 , 0.50] 6.5E-01 -0.04 [-0.15 , 0.07] 6.2E-01 Pecam1 -0.56 [-0.98 , -0.14] 4.1E-02 -0.22 [-0.32 , -0.12] 3.4E-03 -0.08 [-0.11 , -0.04] 3.0E-03 F2rl1 -0.57 [-0.95 , -0.19] 2.5E-02 -0.33 [-0.45 , -0.21] 6.4E-04 -0.12 [-0.18 , -0.06] 3.5E-03 Gtf3c1 -0.58 [-0.82 , -0.33] 2.4E-03 -0.24 [-0.34 , -0.13] 2.3E-03 -0.09 [-0.11 , -0.06] 2.5E-04 484 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mcam -0.58 [-1.05 , -0.10] 5.7E-02 -0.27 [-0.37 , -0.17] 8.7E-04 -0.08 [-0.12 , -0.04] 9.4E-03 Akt3 -0.58 [-0.89 , -0.27] 8.6E-03 -0.27 [-0.37 , -0.17] 6.0E-04 -0.08 [-0.12 , -0.05] 1.7E-03 Rora -0.58 [-0.96 , -0.21] 2.2E-02 -0.20 [-0.34 , -0.06] 2.7E-02 -0.04 [-0.08 , -0.01] 5.0E-02 Il17b -0.59 [-1.39 , 0.22] 2.4E-01 -0.22 [-0.63 , 0.20] 4.2E-01 -0.06 [-0.19 , 0.06] 4.5E-01 Mst1r -0.59 [-1.13 , -0.05] 8.6E-02 -0.33 [-0.66 , -0.01] 1.1E-01 -0.07 [-0.16 , 0.02] 2.4E-01 Cd163 -0.59 [-1.36 , 0.17] 2.1E-01 -0.12 [-0.49 , 0.24] 6.0E-01 0.00 [-0.10 , 0.10] 9.7E-01 Cx3cl1 -0.60 [-1.01 , -0.19] 2.9E-02 -0.33 [-0.47 , -0.20] 1.6E-03 -0.12 [-0.19 , -0.06] 7.2E-03 Ncam1 -0.60 [-1.06 , -0.14] 4.4E-02 -0.22 [-0.52 , 0.08] 2.4E-01 -0.04 [-0.13 , 0.04] 4.5E-01 Cma1 -0.62 [-1.53 , 0.29] 2.7E-01 -0.34 [-0.69 , 0.01] 1.3E-01 -0.09 [-0.19 , 0.02] 1.8E-01 Abcb1a -0.63 [-0.98 , -0.27] 1.2E-02 -0.13 [-0.25 , 0.00] 1.1E-01 -0.05 [-0.09 , 0.00] 1.0E-01 Il6st -0.63 [-0.98 , -0.27] 1.2E-02 -0.21 [-0.31 , -0.12] 2.5E-03 -0.08 [-0.11 , -0.06] 3.0E-04 Ets1 -0.63 [-0.83 , -0.43] 3.4E-04 -0.32 [-0.39 , -0.25] 1.3E-05 -0.11 [-0.15 , -0.07] 8.9E-04 Nrp1 -0.64 [-0.96 , -0.33] 5.1E-03 -0.21 [-0.30 , -0.12] 2.1E-03 -0.08 [-0.11 , -0.06] 1.2E-04 Tgfb2 -0.65 [-1.08 , -0.22] 2.5E-02 -0.17 [-0.33 , -0.01] 9.9E-02 -0.05 [-0.10 , 0.00] 9.5E-02 Msln -0.65 [-1.23 , -0.06] 8.0E-02 -0.25 [-0.38 , -0.12] 7.5E-03 -0.07 [-0.13 , -0.01] 8.1E-02 Gzma -0.65 [-1.23 , -0.07] 7.7E-02 -0.40 [-0.58 , -0.23] 2.4E-03 -0.17 [-0.25 , -0.10] 2.5E-03 Cd7 -0.65 [-0.94 , -0.37] 2.8E-03 -0.40 [-0.58 , -0.22] 2.5E-03 -0.14 [-0.20 , -0.08] 1.9E-03 485 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Dpp4 -0.66 [-0.94 , -0.38] 2.4E-03 -0.25 [-0.36 , -0.14] 2.4E-03 -0.10 [-0.12 , -0.08] 1.2E-05 F13a1 -0.66 [-1.39 , 0.07] 1.5E-01 -0.02 [-0.28 , 0.24] 9.0E-01 0.01 [-0.06 , 0.09] 7.8E-01 Klrk1 -0.66 [-0.95 , -0.37] 2.7E-03 -0.33 [-0.42 , -0.23] 1.7E-04 -0.09 [-0.16 , -0.02] 4.5E-02 Tnfsf10 -0.67 [-1.12 , -0.21] 2.7E-02 -0.32 [-0.40 , -0.23] 7.2E-05 -0.14 [-0.20 , -0.08] 1.7E-03 Ccrl2 -0.67 [-1.17 , -0.17] 4.0E-02 -0.23 [-0.43 , -0.02] 8.2E-02 -0.09 [-0.18 , 0.01] 1.4E-01 Il22ra2 -0.68 [-1.44 , 0.08] 1.5E-01 -0.25 [-0.65 , 0.15] 3.2E-01 -0.03 [-0.13 , 0.07] 6.9E-01 Vwf -0.69 [-1.29 , -0.08] 7.4E-02 -0.22 [-0.38 , -0.07] 3.2E-02 -0.07 [-0.12 , -0.02] 2.5E-02 Dock9 -0.69 [-1.14 , -0.25] 2.2E-02 -0.19 [-0.35 , -0.03] 6.6E-02 -0.06 [-0.10 , -0.02] 3.7E-02 Cd36 -0.69 [-1.12 , -0.26] 1.9E-02 -0.19 [-0.32 , -0.05] 3.8E-02 -0.05 [-0.07 , -0.02] 1.0E-02 Ptgdr2 -0.69 [-1.70 , 0.31] 2.7E-01 -0.26 [-0.46 , -0.06] 4.9E-02 -0.11 [-0.21 , -0.01] 7.6E-02 Itgb4 -0.70 [-1.33 , -0.07] 8.0E-02 -0.22 [-0.39 , -0.06] 3.8E-02 -0.05 [-0.14 , 0.03] 3.0E-01 Txnip -0.71 [-1.01 , -0.40] 2.5E-03 -0.30 [-0.45 , -0.16] 4.7E-03 -0.12 [-0.16 , -0.07] 1.1E-03 Klra4 -0.71 [-1.25 , -0.16] 4.7E-02 -0.44 [-0.62 , -0.26] 1.5E-03 -0.12 [-0.19 , -0.05] 1.2E-02 Egfr -0.72 [-1.21 , -0.22] 3.0E-02 -0.32 [-0.51 , -0.13] 1.4E-02 -0.11 [-0.17 , -0.05] 1.0E-02 Map4k2 -0.72 [-1.12 , -0.33] 9.6E-03 -0.34 [-0.47 , -0.22] 7.6E-04 -0.11 [-0.16 , -0.06] 4.3E-03 Cd8a -0.73 [-1.51 , 0.05] 1.4E-01 -0.58 [-0.94 , -0.23] 1.5E-02 -0.19 [-0.29 , -0.08] 9.5E-03 Gata3 -0.74 [-1.06 , -0.42] 2.5E-03 -0.40 [-0.58 , -0.22] 2.8E-03 -0.14 [-0.21 , -0.07] 6.7E-03 486 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfrsf12a -0.74 [-1.26 , -0.22] 3.2E-02 -0.31 [-0.51 , -0.10] 2.4E-02 -0.09 [-0.15 , -0.03] 2.0E-02 Vegfc -0.74 [-1.25 , -0.23] 3.0E-02 -0.40 [-0.51 , -0.29] 7.2E-05 -0.09 [-0.14 , -0.04] 7.8E-03 Itga2 -0.74 [-1.20 , -0.29] 1.8E-02 -0.30 [-0.40 , -0.20] 2.5E-04 -0.12 [-0.16 , -0.08] 4.5E-04 Zfp13 -0.75 [-1.19 , -0.30] 1.6E-02 -0.21 [-0.46 , 0.04] 1.9E-01 -0.08 [-0.15 , 0.00] 9.9E-02 Flt3l -0.76 [-1.07 , -0.45] 1.8E-03 -0.35 [-0.52 , -0.17] 5.3E-03 -0.12 [-0.17 , -0.07] 1.1E-03 Jam3 -0.77 [-1.06 , -0.47] 1.4E-03 -0.29 [-0.46 , -0.12] 1.5E-02 -0.12 [-0.16 , -0.08] 5.1E-04 Kit -0.77 [-1.27 , -0.27] 2.3E-02 -0.33 [-0.47 , -0.19] 2.1E-03 -0.12 [-0.15 , -0.08] 1.5E-04 Snai1 -0.77 [-1.45 , -0.09] 7.4E-02 -0.28 [-0.56 , 0.00] 1.2E-01 -0.11 [-0.23 , 0.01] 1.5E-01 Cd97 -0.77 [-1.31 , -0.24] 2.9E-02 -0.37 [-0.48 , -0.27] 9.5E-05 -0.15 [-0.19 , -0.10] 2.4E-04 Tlr5 -0.80 [-1.48 , -0.11] 6.7E-02 -0.27 [-0.48 , -0.06] 4.9E-02 -0.10 [-0.22 , 0.02] 2.0E-01 Notch1 -0.80 [-1.23 , -0.37] 9.1E-03 -0.31 [-0.46 , -0.16] 4.5E-03 -0.12 [-0.16 , -0.07] 8.2E-04 Pdgfrb -0.80 [-1.18 , -0.43] 4.1E-03 -0.26 [-0.40 , -0.12] 8.1E-03 -0.09 [-0.14 , -0.04] 7.9E-03 Mme -0.82 [-1.26 , -0.38] 9.0E-03 -0.35 [-0.49 , -0.22] 8.9E-04 -0.13 [-0.18 , -0.09] 4.6E-04 Masp1 -0.82 [-1.26 , -0.38] 9.0E-03 -0.08 [-0.36 , 0.21] 6.9E-01 -0.11 [-0.19 , -0.03] 3.7E-02 Hras -0.85 [-1.78 , 0.08] 1.4E-01 -0.23 [-0.47 , 0.01] 1.3E-01 -0.11 [-0.22 , -0.01] 9.2E-02 Tcf7 -0.85 [-1.22 , -0.49] 2.5E-03 -0.54 [-0.73 , -0.34] 7.3E-04 -0.18 [-0.27 , -0.10] 2.9E-03 Hsd11b1 -0.87 [-1.45 , -0.30] 2.4E-02 -0.42 [-0.56 , -0.27] 4.3E-04 -0.15 [-0.21 , -0.09] 7.7E-04 487 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Icam2 -0.88 [-1.46 , -0.30] 2.5E-02 -0.41 [-0.51 , -0.30] 5.0E-05 -0.16 [-0.21 , -0.12] 1.2E-04 Jun -0.88 [-1.40 , -0.36] 1.4E-02 -0.36 [-0.45 , -0.26] 6.3E-05 -0.15 [-0.20 , -0.11] 1.1E-04 Cmklr1 -0.88 [-1.38 , -0.39] 1.1E-02 -0.29 [-0.40 , -0.18] 1.1E-03 -0.10 [-0.16 , -0.04] 1.0E-02 Igf1r -0.89 [-1.45 , -0.33] 1.9E-02 -0.37 [-0.50 , -0.24] 5.4E-04 -0.13 [-0.17 , -0.09] 1.6E-04 Cdh5 -0.91 [-1.55 , -0.27] 3.2E-02 -0.35 [-0.51 , -0.19] 2.6E-03 -0.13 [-0.17 , -0.08] 5.4E-04 Pnma1 -0.91 [-1.72 , -0.09] 7.8E-02 -0.13 [-0.36 , 0.11] 4.0E-01 -0.06 [-0.16 , 0.05] 3.9E-01 Itga1 -0.91 [-1.49 , -0.33] 2.1E-02 -0.38 [-0.50 , -0.25] 3.4E-04 -0.15 [-0.20 , -0.10] 3.8E-04 Il12a -0.91 [-1.37 , -0.45] 6.1E-03 -0.35 [-0.48 , -0.22] 9.3E-04 -0.10 [-0.18 , -0.02] 5.4E-02 Itk -0.92 [-1.45 , -0.38] 1.4E-02 -0.58 [-0.79 , -0.36] 8.1E-04 -0.17 [-0.25 , -0.09] 2.8E-03 Prkce -0.94 [-1.34 , -0.55] 2.3E-03 -0.48 [-0.59 , -0.37] 1.9E-05 -0.15 [-0.19 , -0.12] 5.6E-06 Ncr1 -0.95 [-1.60 , -0.29] 3.0E-02 -0.43 [-0.57 , -0.29] 2.8E-04 -0.15 [-0.21 , -0.09] 1.4E-03 Tal1 -0.97 [-1.63 , -0.31] 2.7E-02 -0.41 [-0.59 , -0.22] 2.8E-03 -0.16 [-0.21 , -0.11] 2.3E-04 Tie1 -0.98 [-1.52 , -0.43] 1.1E-02 -0.42 [-0.55 , -0.29] 1.7E-04 -0.17 [-0.20 , -0.13] 1.4E-05 Mill2 -0.98 [-1.59 , -0.37] 1.9E-02 -0.32 [-0.50 , -0.15] 8.6E-03 -0.15 [-0.19 , -0.10] 1.5E-04 Thbd -0.99 [-1.68 , -0.30] 3.1E-02 -0.42 [-0.57 , -0.26] 7.2E-04 -0.17 [-0.22 , -0.12] 1.4E-04 Hspb2 -0.99 [-1.48 , -0.51] 5.2E-03 -0.38 [-0.54 , -0.22] 1.6E-03 -0.10 [-0.16 , -0.04] 1.1E-02 Dusp4 -1.04 [-3.76 , 1.69] 5.4E-01 -0.72 [-2.33 , 0.90] 4.9E-01 -0.42 [-1.05 , 0.21] 3.0E-01 488 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Vegfa -1.05 [-1.59 , -0.52] 6.8E-03 -0.43 [-0.55 , -0.31] 7.2E-05 -0.15 [-0.19 , -0.12] 3.4E-05 Fez1 -1.06 [-1.59 , -0.53] 5.8E-03 -0.44 [-0.72 , -0.16] 1.9E-02 -0.16 [-0.24 , -0.08] 6.3E-03 Klrc2 -1.06 [-1.67 , -0.46] 1.2E-02 -0.60 [-0.81 , -0.38] 6.1E-04 -0.16 [-0.24 , -0.07] 9.0E-03 Klrb1c -1.08 [-1.67 , -0.49] 1.0E-02 -0.53 [-0.75 , -0.30] 1.9E-03 -0.17 [-0.25 , -0.10] 2.1E-03 Eng -1.08 [-1.69 , -0.46] 1.2E-02 -0.62 [-0.86 , -0.39] 8.5E-04 -0.25 [-0.30 , -0.19] 1.1E-05 Txk -1.09 [-1.47 , -0.71] 7.0E-04 -0.59 [-0.75 , -0.43] 7.2E-05 -0.17 [-0.26 , -0.09] 3.6E-03 Klrd1 -1.12 [-1.50 , -0.73] 6.4E-04 -0.55 [-0.66 , -0.45] 4.2E-06 -0.19 [-0.25 , -0.12] 3.2E-04 Lrrn3 -1.15 [-1.43 , -0.87] 3.7E-05 -0.44 [-0.58 , -0.30] 2.1E-04 -0.19 [-0.26 , -0.11] 1.2E-03 Syt17 -1.16 [-1.72 , -0.60] 5.1E-03 -0.47 [-0.64 , -0.30] 7.2E-04 -0.21 [-0.32 , -0.10] 7.8E-03 Tek -1.16 [-1.86 , -0.47] 1.5E-02 -0.48 [-0.64 , -0.32] 3.4E-04 -0.19 [-0.24 , -0.15] 2.9E-05 Ccr7 -1.18 [-1.76 , -0.59] 5.7E-03 -0.57 [-0.85 , -0.29] 4.5E-03 -0.18 [-0.30 , -0.07] 1.7E-02 Lyve1 -1.23 [-1.76 , -0.69] 2.8E-03 -0.57 [-0.78 , -0.36] 7.1E-04 -0.13 [-0.22 , -0.04] 2.6E-02 Il11ra1 -1.23 [-2.00 , -0.46] 1.9E-02 -0.54 [-0.78 , -0.31] 2.2E-03 -0.20 [-0.26 , -0.14] 1.3E-04 Angpt1 -1.25 [-1.93 , -0.57] 1.0E-02 -0.49 [-0.70 , -0.29] 1.7E-03 -0.16 [-0.24 , -0.09] 3.4E-03 Kdr -1.26 [-1.86 , -0.67] 4.4E-03 -0.57 [-0.69 , -0.44] 1.4E-05 -0.23 [-0.30 , -0.16] 1.3E-04 Dll4 -1.33 [-1.78 , -0.88] 5.8E-04 -0.64 [-0.84 , -0.43] 2.5E-04 -0.23 [-0.29 , -0.17] 6.8E-05 Cd8b1 -1.41 [-2.16 , -0.66] 8.6E-03 -0.82 [-1.19 , -0.44] 3.4E-03 -0.29 [-0.42 , -0.16] 2.9E-03 489 Table 34 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Klra7 -1.59 [-3.20 , 0.01] 1.1E-01 -0.39 [-0.75 , -0.03] 9.3E-02 -0.12 [-0.21 , -0.04] 3.3E-02 Angpt2 -1.60 [-2.18 , -1.02] 8.0E-04 -0.59 [-0.70 , -0.49] 2.8E-06 -0.22 [-0.27 , -0.17] 1.9E-05 Hamp -2.28 [-3.99 , -0.56] 4.2E-02 -0.88 [-1.82 , 0.06] 1.4E-01 -0.16 [-0.34 , 0.03] 1.8E-01 Cfd -3.00 [-5.20 , -0.80] 3.8E-02 -1.03 [-2.17 , 0.12] 1.6E-01 -0.19 [-0.47 , 0.09] 2.9E-01 490 Table 35. Complete mRNA transcript profile in spleen at 13 wk post final exposure in cSiO 2-exposed NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log 2(fold change) relative to VEH + CON mice. Expression data are included for all mRNA transcripts that were above background threshold. Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl14 3.87 [2.47 , 5.27] 6.6E-04 1.71 [1.10 , 2.32] 2.5E-03 0.41 [0.18 , 0.64] 7.4E-02 Ccl8 3.32 [2.72 , 3.91] 2.2E-05 1.36 [1.03 , 1.70] 3.4E-04 0.41 [0.22 , 0.59] 3.4E-02 Fcgr4 2.93 [2.11 , 3.76] 1.2E-04 1.19 [0.77 , 1.61] 2.4E-03 0.26 [0.11 , 0.41] 7.8E-02 Ccl12 2.24 [1.66 , 2.83] 8.3E-05 0.95 [0.64 , 1.25] 1.7E-03 0.24 [0.11 , 0.38] 7.4E-02 Klra2 2.06 [1.54 , 2.57] 6.2E-05 0.80 [0.48 , 1.12] 4.7E-03 0.12 [0.01 , 0.22] 2.1E-01 C3ar1 1.98 [1.30 , 2.65] 4.4E-04 0.84 [0.50 , 1.18] 5.0E-03 0.21 [0.09 , 0.33] 7.4E-02 Il1b 1.92 [1.37 , 2.48] 1.5E-04 0.62 [0.33 , 0.92] 1.1E-02 0.06 [-0.07 , 0.19] 6.8E-01 Ccl4 1.66 [1.12 , 2.20] 3.1E-04 0.63 [0.32 , 0.94] 1.3E-02 0.14 [0.04 , 0.24] 1.2E-01 Gzmk 1.63 [1.10 , 2.16] 2.9E-04 0.65 [0.37 , 0.94] 7.1E-03 0.16 [0.01 , 0.31] 2.5E-01 Il12b 1.50 [0.99 , 2.01] 4.2E-04 0.54 [0.26 , 0.81] 1.4E-02 0.16 [0.07 , 0.26] 7.8E-02 Il21 1.49 [1.02 , 1.96] 2.4E-04 0.48 [0.21 , 0.75] 2.3E-02 0.10 [-0.04 , 0.25] 4.5E-01 Il1rn 1.45 [1.14 , 1.76] 3.0E-05 0.51 [0.33 , 0.69] 2.4E-03 0.08 [-0.01 , 0.17] 3.1E-01 Ccl2 1.45 [1.02 , 1.88] 1.7E-04 0.42 [0.15 , 0.68] 3.6E-02 0.09 [-0.02 , 0.20] 3.9E-01 491 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Itgax 1.45 [0.87 , 2.04] 1.3E-03 0.54 [0.22 , 0.86] 2.6E-02 0.10 [0.00 , 0.20] 2.5E-01 Fpr2 1.44 [0.89 , 2.00] 1.0E-03 0.62 [0.37 , 0.87] 4.7E-03 0.15 [0.05 , 0.25] 9.9E-02 Ccl3 1.42 [1.12 , 1.72] 3.0E-05 0.61 [0.32 , 0.90] 1.1E-02 0.09 [-0.01 , 0.19] 3.1E-01 Lag3 1.40 [1.10 , 1.69] 3.0E-05 0.47 [0.33 , 0.61] 1.4E-03 0.15 [0.08 , 0.22] 4.5E-02 Msr1 1.37 [0.84 , 1.90] 1.0E-03 0.48 [0.26 , 0.70] 9.3E-03 0.11 [0.04 , 0.19] 9.6E-02 Tnfrsf8 1.35 [1.06 , 1.63] 3.0E-05 0.47 [0.33 , 0.61] 1.4E-03 0.17 [0.12 , 0.22] 4.3E-03 Oas2 1.34 [1.02 , 1.67] 6.2E-05 0.44 [0.27 , 0.62] 4.7E-03 0.09 [0.01 , 0.16] 2.1E-01 Socs3 1.30 [0.92 , 1.68] 1.7E-04 0.46 [0.26 , 0.66] 7.5E-03 0.13 [0.05 , 0.21] 7.8E-02 Cx3cr1 1.28 [0.90 , 1.66] 1.7E-04 0.50 [0.29 , 0.72] 7.1E-03 0.10 [0.02 , 0.17] 1.7E-01 Il10 1.27 [0.84 , 1.70] 4.3E-04 0.43 [0.23 , 0.63] 9.9E-03 0.09 [0.02 , 0.16] 1.6E-01 Ccr5 1.18 [0.80 , 1.57] 2.9E-04 0.41 [0.19 , 0.63] 1.9E-02 0.08 [0.02 , 0.14] 1.6E-01 Ifi44 1.16 [0.93 , 1.39] 3.0E-05 0.40 [0.27 , 0.52] 1.5E-03 0.09 [0.03 , 0.16] 1.1E-01 Cfb 1.14 [0.87 , 1.41] 5.4E-05 0.37 [0.22 , 0.51] 4.7E-03 0.07 [0.01 , 0.14] 2.2E-01 Fos 1.14 [0.75 , 1.54] 4.8E-04 0.22 [-0.03 , 0.46] 2.1E-01 -0.01 [-0.10 , 0.07] 9.0E-01 Tigit 1.05 [0.76 , 1.34] 1.1E-04 0.35 [0.20 , 0.51] 7.7E-03 0.07 [0.00 , 0.14] 2.5E-01 Dusp4 1.04 [0.74 , 1.35] 1.7E-04 0.33 [0.16 , 0.50] 1.4E-02 0.07 [0.01 , 0.13] 2.3E-01 Batf 1.02 [0.68 , 1.36] 3.7E-04 0.37 [0.19 , 0.56] 1.3E-02 0.07 [0.00 , 0.14] 2.5E-01 492 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccr6 1.02 [0.66 , 1.37] 5.1E-04 0.34 [0.18 , 0.50] 1.1E-02 0.05 [-0.01 , 0.12] 3.9E-01 Xbp1 1.00 [0.72 , 1.27] 1.1E-04 0.34 [0.26 , 0.43] 3.4E-04 0.11 [0.05 , 0.16] 5.8E-02 Zbp1 0.97 [0.75 , 1.19] 3.7E-05 0.33 [0.21 , 0.45] 2.6E-03 0.06 [0.00 , 0.12] 2.3E-01 Pdcd1 0.97 [0.68 , 1.25] 1.7E-04 0.30 [0.13 , 0.48] 2.5E-02 0.04 [-0.03 , 0.11] 5.5E-01 Cxcl1 0.96 [0.34 , 1.58] 2.2E-02 0.21 [-0.06 , 0.49] 2.8E-01 0.07 [-0.05 , 0.19] 5.5E-01 Itgam 0.96 [0.58 , 1.34] 1.3E-03 0.46 [0.25 , 0.68] 9.1E-03 0.09 [0.03 , 0.15] 9.9E-02 Cxcl9 0.95 [0.67 , 1.24] 1.8E-04 0.26 [0.07 , 0.44] 6.4E-02 0.00 [-0.07 , 0.07] 9.9E-01 Cxcr3 0.95 [0.70 , 1.20] 9.3E-05 0.30 [0.15 , 0.45] 1.3E-02 0.08 [0.01 , 0.14] 1.9E-01 Cd70 0.93 [0.51 , 1.35] 2.7E-03 0.24 [0.03 , 0.46] 1.1E-01 0.01 [-0.06 , 0.08] 8.9E-01 Tnfrsf4 0.92 [0.65 , 1.19] 1.7E-04 0.38 [0.25 , 0.50] 2.1E-03 0.10 [0.04 , 0.15] 7.8E-02 Fcgr1 0.91 [0.65 , 1.17] 1.4E-04 0.32 [0.19 , 0.44] 4.7E-03 0.09 [0.04 , 0.14] 7.8E-02 Tlr8 0.87 [0.59 , 1.16] 3.1E-04 0.30 [0.16 , 0.44] 1.1E-02 0.08 [0.02 , 0.13] 1.2E-01 Clec7a 0.87 [0.63 , 1.11] 1.1E-04 0.32 [0.21 , 0.42] 2.1E-03 0.06 [0.02 , 0.10] 9.4E-02 Irf7 0.86 [0.65 , 1.08] 6.2E-05 0.34 [0.22 , 0.47] 2.6E-03 0.09 [0.04 , 0.14] 7.8E-02 Cxcl16 0.84 [0.59 , 1.09] 1.7E-04 0.27 [0.10 , 0.44] 3.5E-02 0.07 [0.03 , 0.12] 7.8E-02 Cd200r1 0.84 [0.46 , 1.21] 2.7E-03 0.33 [0.14 , 0.52] 2.6E-02 0.07 [0.01 , 0.12] 1.9E-01 C5ar1 0.82 [0.49 , 1.15] 1.3E-03 0.39 [0.24 , 0.55] 4.7E-03 0.12 [0.04 , 0.19] 9.7E-02 493 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Pycard 0.81 [0.47 , 1.15] 1.8E-03 0.18 [0.03 , 0.34] 1.1E-01 0.03 [-0.05 , 0.11] 7.5E-01 Socs1 0.80 [0.59 , 1.00] 7.8E-05 0.25 [0.11 , 0.38] 2.1E-02 0.02 [-0.03 , 0.07] 6.8E-01 Csf3r 0.80 [0.53 , 1.07] 4.2E-04 0.35 [0.23 , 0.47] 2.2E-03 0.06 [0.02 , 0.11] 1.6E-01 Tnfsf8 0.80 [0.44 , 1.15] 2.8E-03 0.25 [0.05 , 0.45] 9.7E-02 0.03 [-0.05 , 0.10] 7.6E-01 Arg2 0.79 [0.45 , 1.13] 1.9E-03 0.24 [0.07 , 0.41] 6.1E-02 0.11 [0.05 , 0.17] 5.8E-02 Mme 0.78 [0.53 , 1.03] 2.4E-04 0.31 [0.14 , 0.49] 2.3E-02 0.10 [0.03 , 0.16] 9.9E-02 Fcer1g 0.77 [0.49 , 1.05] 6.6E-04 0.30 [0.15 , 0.45] 1.5E-02 0.06 [0.00 , 0.11] 2.3E-01 Klra17 0.76 [0.41 , 1.10] 3.1E-03 0.33 [0.11 , 0.55] 4.6E-02 0.07 [0.01 , 0.13] 2.3E-01 C7 0.74 [0.20 , 1.27] 3.9E-02 0.35 [0.13 , 0.56] 3.2E-02 0.11 [0.03 , 0.19] 1.2E-01 Il6 0.73 [0.23 , 1.23] 2.9E-02 0.34 [0.08 , 0.60] 7.5E-02 n.d [n.d , n.d] n.d Slamf7 0.73 [0.54 , 0.92] 8.3E-05 0.22 [0.14 , 0.31] 2.8E-03 0.08 [0.04 , 0.12] 5.4E-02 Jun 0.72 [0.53 , 0.91] 8.3E-05 0.17 [0.04 , 0.29] 7.4E-02 0.02 [-0.02 , 0.07] 6.0E-01 Tnfsf13b 0.72 [0.41 , 1.03] 1.8E-03 0.32 [0.11 , 0.52] 4.0E-02 0.06 [-0.01 , 0.12] 3.3E-01 Tbx21 0.71 [0.42 , 1.01] 1.5E-03 0.15 [-0.02 , 0.32] 2.2E-01 -0.02 [-0.10 , 0.06] 8.5E-01 Aicda 0.71 [0.41 , 1.01] 1.8E-03 0.21 [-0.02 , 0.44] 1.9E-01 0.06 [-0.06 , 0.18] 6.6E-01 Tap1 0.70 [0.51 , 0.89] 9.3E-05 0.24 [0.14 , 0.35] 7.4E-03 0.07 [0.03 , 0.11] 7.4E-02 C3 0.70 [0.54 , 0.85] 3.4E-05 0.23 [0.13 , 0.33] 7.1E-03 0.07 [0.03 , 0.10] 5.4E-02 494 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Sh2d1b1 0.70 [0.30 , 1.10] 1.2E-02 0.27 [0.07 , 0.47] 7.4E-02 0.03 [-0.04 , 0.11] 6.6E-01 Pou2f2 0.69 [0.45 , 0.94] 5.6E-04 0.26 [0.12 , 0.41] 2.1E-02 0.05 [0.00 , 0.10] 2.7E-01 Pdgfc 0.69 [0.41 , 0.97] 1.5E-03 0.25 [0.09 , 0.41] 4.0E-02 0.09 [0.02 , 0.16] 1.6E-01 Ctla4 0.68 [0.42 , 0.95] 1.1E-03 0.16 [0.03 , 0.30] 1.0E-01 0.05 [0.01 , 0.10] 1.7E-01 Itgae 0.68 [0.37 , 0.98] 2.8E-03 0.17 [-0.01 , 0.35] 1.8E-01 0.02 [-0.03 , 0.08] 7.1E-01 St6gal1 0.67 [0.46 , 0.88] 2.1E-04 0.15 [0.06 , 0.24] 2.6E-02 0.05 [0.01 , 0.09] 1.7E-01 Stat1 0.66 [0.50 , 0.83] 6.2E-05 0.18 [0.08 , 0.29] 2.5E-02 0.01 [-0.03 , 0.05] 8.5E-01 Lif 0.66 [0.19 , 1.13] 3.4E-02 0.18 [-0.05 , 0.41] 2.6E-01 0.09 [-0.02 , 0.19] 3.9E-01 Nlrc5 0.65 [0.43 , 0.87] 4.0E-04 0.18 [0.06 , 0.30] 4.6E-02 0.04 [-0.01 , 0.08] 3.7E-01 Il21r 0.65 [0.34 , 0.97] 4.2E-03 0.17 [-0.02 , 0.35] 2.0E-01 0.02 [-0.05 , 0.09] 8.2E-01 Psmb9 0.64 [0.45 , 0.84] 1.8E-04 0.14 [0.03 , 0.25] 7.9E-02 0.03 [-0.01 , 0.07] 5.1E-01 Ada 0.64 [0.42 , 0.86] 4.5E-04 0.21 [0.14 , 0.29] 2.4E-03 0.08 [0.05 , 0.12] 2.2E-02 Plau 0.63 [0.37 , 0.89] 1.6E-03 0.18 [0.06 , 0.30] 3.9E-02 0.03 [-0.01 , 0.07] 3.8E-01 Cd276 0.63 [0.37 , 0.89] 1.7E-03 0.28 [0.15 , 0.41] 1.1E-02 0.04 [-0.02 , 0.11] 5.2E-01 Foxp3 0.62 [0.39 , 0.86] 8.1E-04 0.27 [0.12 , 0.42] 2.1E-02 0.04 [-0.03 , 0.10] 5.5E-01 Ctss 0.62 [0.41 , 0.83] 4.3E-04 0.22 [0.11 , 0.33] 1.3E-02 0.05 [0.01 , 0.09] 1.6E-01 Mx1 0.62 [0.29 , 0.96] 7.9E-03 0.27 [0.08 , 0.45] 4.8E-02 0.09 [0.04 , 0.15] 7.4E-02 495 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Slamf6 0.62 [0.38 , 0.87] 1.2E-03 0.17 [0.05 , 0.30] 6.1E-02 0.04 [-0.01 , 0.09] 3.2E-01 Pik3cg 0.62 [0.37 , 0.87] 1.3E-03 0.24 [0.17 , 0.30] 1.0E-03 0.10 [0.07 , 0.12] 4.9E-03 Cd274 0.62 [0.46 , 0.78] 8.3E-05 0.18 [0.08 , 0.28] 2.3E-02 0.03 [-0.01 , 0.07] 4.9E-01 Ccl6 0.61 [0.30 , 0.92] 5.4E-03 0.27 [0.12 , 0.43] 2.3E-02 0.10 [0.03 , 0.16] 1.2E-01 Cebpb 0.60 [0.44 , 0.77] 1.1E-04 0.21 [0.13 , 0.28] 2.6E-03 0.04 [0.01 , 0.08] 1.6E-01 Il1r2 0.60 [0.19 , 1.01] 2.9E-02 0.37 [0.16 , 0.59] 2.5E-02 0.11 [0.04 , 0.18] 9.9E-02 Ikzf2 0.60 [0.42 , 0.78] 1.7E-04 0.20 [0.08 , 0.32] 3.0E-02 0.03 [-0.02 , 0.07] 5.4E-01 Gzmb 0.59 [0.38 , 0.81] 6.4E-04 0.03 [-0.17 , 0.22] 8.9E-01 -0.02 [-0.13 , 0.10] 9.3E-01 Hck 0.59 [0.41 , 0.78] 2.4E-04 0.22 [0.10 , 0.34] 2.1E-02 0.04 [0.00 , 0.07] 3.1E-01 Pdcd1lg2 0.59 [0.28 , 0.90] 6.5E-03 0.24 [0.12 , 0.37] 1.6E-02 0.05 [-0.01 , 0.11] 3.9E-01 Pou2af1 0.59 [0.40 , 0.78] 2.4E-04 0.22 [0.15 , 0.28] 1.4E-03 0.08 [0.04 , 0.11] 3.4E-02 Egr2 0.59 [0.21 , 0.97] 2.2E-02 0.19 [-0.03 , 0.41] 2.3E-01 0.07 [-0.01 , 0.15] 3.6E-01 Alcam 0.59 [0.41 , 0.77] 2.0E-04 0.20 [0.13 , 0.26] 1.7E-03 0.06 [0.03 , 0.09] 5.4E-02 H2-K1 0.58 [0.44 , 0.73] 6.2E-05 0.18 [0.10 , 0.26] 7.1E-03 0.04 [0.01 , 0.07] 1.9E-01 Gbp5 0.58 [0.41 , 0.75] 1.7E-04 0.18 [0.07 , 0.29] 2.7E-02 0.01 [-0.03 , 0.05] 8.8E-01 Hif1a 0.58 [0.39 , 0.77] 3.0E-04 0.18 [0.09 , 0.27] 1.3E-02 0.05 [0.02 , 0.07] 8.3E-02 Ctsh 0.58 [0.33 , 0.83] 1.9E-03 0.21 [0.09 , 0.33] 2.4E-02 0.05 [0.01 , 0.09] 1.9E-01 496 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Creb5 0.58 [0.07 , 1.09] 8.0E-02 0.17 [-0.10 , 0.45] 3.8E-01 0.04 [-0.02 , 0.10] 5.3E-01 Oasl1 0.58 [0.26 , 0.89] 9.0E-03 0.20 [0.02 , 0.37] 1.1E-01 0.02 [-0.04 , 0.09] 7.8E-01 Fut7 0.57 [0.24 , 0.91] 1.3E-02 0.33 [0.11 , 0.55] 4.5E-02 0.07 [0.00 , 0.13] 2.6E-01 Bcl6 0.57 [0.30 , 0.84] 3.8E-03 0.18 [0.02 , 0.34] 1.3E-01 0.01 [-0.04 , 0.07] 8.3E-01 Myd88 0.56 [0.38 , 0.74] 2.4E-04 0.20 [0.12 , 0.29] 6.8E-03 0.04 [0.01 , 0.08] 1.9E-01 Il2ra 0.56 [0.31 , 0.81] 2.5E-03 0.26 [0.14 , 0.38] 9.7E-03 0.06 [0.02 , 0.11] 1.5E-01 Itgb2 0.56 [0.39 , 0.72] 1.7E-04 0.20 [0.11 , 0.28] 6.8E-03 0.04 [0.02 , 0.07] 8.9E-02 Pparg 0.56 [0.19 , 0.92] 2.4E-02 0.21 [0.02 , 0.39] 1.2E-01 0.01 [-0.05 , 0.07] 8.8E-01 Icos 0.55 [0.36 , 0.75] 5.0E-04 0.12 [0.01 , 0.22] 1.4E-01 0.03 [0.00 , 0.07] 3.1E-01 Irf1 0.55 [0.35 , 0.76] 8.3E-04 0.14 [0.02 , 0.26] 1.0E-01 0.02 [-0.03 , 0.07] 6.5E-01 Bst2 0.55 [0.43 , 0.67] 3.2E-05 0.16 [0.08 , 0.24] 1.3E-02 0.04 [0.01 , 0.07] 1.1E-01 Psmb8 0.55 [0.38 , 0.71] 1.8E-04 0.15 [0.05 , 0.24] 3.8E-02 0.02 [-0.02 , 0.06] 5.5E-01 Ulbp1 0.54 [0.30 , 0.79] 2.8E-03 0.26 [0.13 , 0.39] 1.5E-02 0.06 [0.03 , 0.09] 5.4E-02 Ccl9 0.54 [0.18 , 0.91] 2.7E-02 0.35 [0.15 , 0.55] 2.3E-02 0.10 [0.02 , 0.18] 1.9E-01 Ifit3 0.54 [0.33 , 0.75] 9.7E-04 0.19 [0.06 , 0.32] 4.5E-02 0.02 [-0.04 , 0.07] 8.4E-01 Tgfbr1 0.54 [0.30 , 0.77] 2.4E-03 0.17 [0.04 , 0.30] 7.5E-02 0.03 [-0.02 , 0.07] 5.3E-01 Rrad 0.53 [0.29 , 0.77] 3.0E-03 0.13 [0.00 , 0.27] 1.6E-01 0.02 [-0.04 , 0.07] 8.1E-01 497 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ifih1 0.53 [0.31 , 0.74] 1.5E-03 0.21 [0.10 , 0.32] 1.8E-02 0.06 [0.03 , 0.10] 7.8E-02 Tnfsf11 0.53 [0.16 , 0.90] 3.3E-02 0.20 [0.01 , 0.39] 1.4E-01 0.01 [-0.07 , 0.09] 9.3E-01 Csf2rb 0.52 [0.39 , 0.65] 6.2E-05 0.26 [0.17 , 0.34] 1.8E-03 0.08 [0.05 , 0.11] 1.6E-02 Ccl22 0.52 [0.18 , 0.86] 2.3E-02 0.18 [0.01 , 0.34] 1.3E-01 0.03 [-0.04 , 0.11] 6.7E-01 Fap 0.52 [0.18 , 0.85] 2.2E-02 0.15 [0.00 , 0.30] 1.7E-01 0.08 [0.03 , 0.14] 1.1E-01 Jak3 0.52 [0.31 , 0.73] 1.5E-03 0.15 [0.04 , 0.25] 6.8E-02 0.01 [-0.04 , 0.07] 8.2E-01 Col1a1 0.52 [0.23 , 0.81] 1.0E-02 0.16 [0.03 , 0.29] 1.0E-01 -0.01 [-0.06 , 0.04] 9.3E-01 Irf4 0.51 [0.33 , 0.70] 6.6E-04 0.19 [0.12 , 0.25] 2.4E-03 0.07 [0.03 , 0.11] 7.8E-02 Raet1c 0.51 [0.27 , 0.75] 3.9E-03 0.17 [0.06 , 0.27] 3.8E-02 0.05 [0.01 , 0.10] 2.1E-01 H2-D1 0.51 [0.39 , 0.62] 3.7E-05 0.14 [0.06 , 0.22] 2.6E-02 0.03 [0.00 , 0.06] 2.4E-01 Ticam2 0.51 [0.37 , 0.65] 1.1E-04 0.20 [0.13 , 0.28] 2.6E-03 0.04 [0.01 , 0.07] 1.8E-01 Clec4a2 0.50 [0.06 , 0.94] 8.0E-02 0.26 [0.07 , 0.45] 6.8E-02 0.07 [-0.01 , 0.14] 3.1E-01 Stat3 0.50 [0.36 , 0.64] 1.1E-04 0.16 [0.07 , 0.24] 2.0E-02 0.03 [0.00 , 0.06] 2.3E-01 Ifitm1 0.50 [0.25 , 0.74] 5.1E-03 0.19 [0.06 , 0.32] 5.0E-02 0.06 [0.01 , 0.10] 1.6E-01 Il13ra2 0.50 [0.18 , 0.81] 2.0E-02 0.16 [-0.02 , 0.34] 2.1E-01 0.05 [0.00 , 0.10] 2.5E-01 C1qb 0.49 [0.33 , 0.66] 4.0E-04 0.17 [0.06 , 0.28] 4.0E-02 0.06 [0.02 , 0.09] 7.8E-02 C1qa 0.49 [0.31 , 0.67] 7.1E-04 0.14 [0.03 , 0.25] 8.7E-02 0.04 [0.01 , 0.07] 1.9E-01 498 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mx2 0.49 [0.16 , 0.82] 2.7E-02 0.18 [0.01 , 0.35] 1.4E-01 0.05 [-0.03 , 0.12] 5.2E-01 Cybb 0.49 [0.35 , 0.63] 1.2E-04 0.23 [0.18 , 0.29] 3.4E-04 0.06 [0.04 , 0.09] 1.3E-02 Ccr8 0.48 [0.07 , 0.90] 7.4E-02 0.15 [-0.04 , 0.35] 2.7E-01 0.05 [-0.02 , 0.12] 5.0E-01 Tnfsf10 0.48 [0.35 , 0.61] 9.3E-05 0.16 [0.09 , 0.24] 7.7E-03 0.03 [0.00 , 0.06] 1.9E-01 Abcg1 0.48 [0.30 , 0.66] 8.4E-04 0.18 [0.07 , 0.28] 2.7E-02 0.05 [0.01 , 0.08] 1.9E-01 Col3a1 0.48 [0.13 , 0.83] 4.1E-02 0.06 [-0.15 , 0.26] 7.3E-01 -0.05 [-0.11 , 0.01] 3.9E-01 Sbno2 0.48 [0.30 , 0.65] 8.1E-04 0.17 [0.07 , 0.26] 2.6E-02 0.02 [-0.02 , 0.05] 6.3E-01 Tmem173 0.47 [0.25 , 0.69] 3.1E-03 0.17 [0.03 , 0.30] 9.1E-02 0.02 [-0.03 , 0.07] 6.7E-01 Cd83 0.46 [0.27 , 0.66] 1.6E-03 0.11 [0.02 , 0.20] 9.8E-02 0.00 [-0.03 , 0.02] 9.7E-01 Cd53 0.46 [0.24 , 0.69] 3.9E-03 0.13 [0.00 , 0.25] 1.6E-01 0.02 [-0.02 , 0.06] 7.1E-01 Lamp3 0.46 [0.01 , 0.91] 1.1E-01 0.08 [-0.13 , 0.30] 6.1E-01 0.05 [-0.02 , 0.12] 5.0E-01 Itgal 0.45 [0.31 , 0.59] 2.2E-04 0.14 [0.07 , 0.21] 1.5E-02 0.01 [-0.02 , 0.04] 6.8E-01 Tnf 0.44 [0.21 , 0.68] 7.2E-03 0.15 [0.02 , 0.29] 1.2E-01 0.00 [-0.05 , 0.06] 9.9E-01 Ifit1 0.44 [0.16 , 0.73] 2.1E-02 0.21 [0.03 , 0.40] 1.1E-01 0.04 [-0.02 , 0.10] 4.4E-01 Vim 0.44 [0.30 , 0.57] 1.8E-04 0.17 [0.09 , 0.24] 9.7E-03 0.02 [-0.01 , 0.05] 4.0E-01 H2-M3 0.43 [0.28 , 0.59] 5.8E-04 0.08 [0.00 , 0.16] 1.7E-01 0.01 [-0.02 , 0.05] 6.8E-01 Tirap 0.43 [0.26 , 0.61] 1.4E-03 0.14 [0.07 , 0.21] 1.3E-02 0.07 [0.03 , 0.11] 8.9E-02 499 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ddx60 0.43 [0.26 , 0.60] 1.1E-03 0.11 [0.01 , 0.21] 1.4E-01 0.03 [-0.01 , 0.06] 4.4E-01 H2-Q2 0.43 [0.22 , 0.65] 5.1E-03 0.02 [-0.10 , 0.14] 8.4E-01 0.01 [-0.04 , 0.05] 9.3E-01 Tlr9 0.43 [0.24 , 0.62] 2.8E-03 0.11 [0.00 , 0.23] 1.6E-01 0.02 [-0.02 , 0.06] 6.7E-01 Cfi 0.42 [0.19 , 0.65] 8.4E-03 0.26 [0.08 , 0.44] 4.9E-02 0.06 [-0.01 , 0.13] 3.1E-01 Tlr7 0.42 [0.26 , 0.59] 1.1E-03 0.14 [0.06 , 0.21] 2.3E-02 0.04 [0.01 , 0.08] 1.9E-01 Tnfrsf17 0.42 [0.15 , 0.70] 2.4E-02 0.25 [0.13 , 0.37] 1.3E-02 0.07 [-0.05 , 0.18] 5.5E-01 Prdm1 0.42 [0.20 , 0.64] 6.0E-03 0.19 [0.09 , 0.30] 2.0E-02 0.08 [0.03 , 0.14] 8.8E-02 Tgfb3 0.42 [0.18 , 0.66] 1.1E-02 0.07 [-0.10 , 0.25] 6.0E-01 0.05 [0.01 , 0.09] 2.1E-01 Trem2 0.42 [-0.17 , 1.01] 2.6E-01 0.20 [-0.02 , 0.41] 2.0E-01 0.11 [0.04 , 0.19] 1.1E-01 Epcam 0.41 [0.14 , 0.69] 2.7E-02 0.18 [0.02 , 0.35] 1.3E-01 0.03 [-0.03 , 0.08] 6.7E-01 Spn 0.41 [0.27 , 0.56] 5.8E-04 0.11 [0.01 , 0.22] 1.3E-01 0.01 [-0.02 , 0.05] 7.8E-01 Il4ra 0.41 [0.16 , 0.66] 1.6E-02 0.10 [-0.03 , 0.22] 3.0E-01 0.02 [-0.03 , 0.06] 7.9E-01 H2-T23 0.41 [0.31 , 0.51] 6.2E-05 0.12 [0.07 , 0.17] 7.4E-03 0.04 [0.02 , 0.06] 5.4E-02 Cd48 0.41 [0.20 , 0.61] 5.7E-03 0.09 [-0.02 , 0.20] 2.6E-01 0.00 [-0.04 , 0.05] 9.4E-01 Nt5e 0.40 [0.21 , 0.60] 4.1E-03 0.10 [0.01 , 0.19] 1.1E-01 0.04 [0.00 , 0.08] 2.3E-01 Tmed1 0.40 [0.29 , 0.51] 9.3E-05 0.12 [0.06 , 0.19] 1.6E-02 0.03 [0.00 , 0.06] 2.8E-01 Tnfsf15 0.40 [0.04 , 0.77] 8.8E-02 0.11 [-0.07 , 0.28] 4.2E-01 0.01 [-0.07 , 0.08] 9.5E-01 500 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ptprc 0.40 [0.21 , 0.59] 4.4E-03 0.08 [-0.03 , 0.19] 3.0E-01 n.d [n.d , n.d] n.d Syk 0.40 [0.25 , 0.55] 8.6E-04 0.14 [0.07 , 0.22] 1.7E-02 0.04 [0.01 , 0.07] 1.2E-01 Egr1 0.40 [0.18 , 0.61] 8.1E-03 0.07 [-0.06 , 0.20] 4.6E-01 0.02 [-0.04 , 0.07] 8.1E-01 Il10ra 0.40 [0.17 , 0.62] 1.0E-02 0.11 [0.01 , 0.22] 1.4E-01 -0.01 [-0.05 , 0.02] 7.2E-01 Cxcl11 0.39 [-0.18 , 0.95] 2.8E-01 0.09 [-0.20 , 0.38] 7.1E-01 -0.01 [-0.12 , 0.11] 9.7E-01 Psmb10 0.39 [0.25 , 0.53] 6.6E-04 0.06 [-0.01 , 0.14] 2.5E-01 -0.01 [-0.04 , 0.03] 8.9E-01 Il3ra 0.39 [0.20 , 0.57] 3.8E-03 0.14 [0.02 , 0.26] 1.0E-01 0.04 [-0.01 , 0.08] 4.0E-01 Xaf1 0.38 [0.24 , 0.53] 9.9E-04 0.14 [0.06 , 0.22] 2.7E-02 0.02 [-0.01 , 0.05] 5.1E-01 Siglec1 0.38 [0.22 , 0.53] 1.5E-03 0.12 [0.02 , 0.23] 1.1E-01 0.01 [-0.03 , 0.04] 8.9E-01 Lcp1 0.37 [0.23 , 0.51] 9.7E-04 0.11 [0.02 , 0.19] 9.7E-02 0.01 [-0.02 , 0.04] 8.7E-01 Casp1 0.37 [0.14 , 0.60] 1.8E-02 0.11 [-0.01 , 0.22] 1.9E-01 0.02 [-0.03 , 0.06] 7.6E-01 Ceacam1 0.37 [0.16 , 0.59] 1.2E-02 0.18 [0.07 , 0.28] 2.5E-02 0.05 [0.02 , 0.08] 1.1E-01 Tlr2 0.37 [0.17 , 0.56] 7.7E-03 0.12 [0.01 , 0.23] 1.4E-01 0.03 [-0.02 , 0.08] 5.9E-01 Cd80 0.37 [0.23 , 0.50] 8.0E-04 0.06 [-0.01 , 0.13] 2.1E-01 0.04 [0.00 , 0.07] 2.4E-01 Lgals3 0.36 [0.18 , 0.55] 4.9E-03 0.16 [0.08 , 0.23] 1.3E-02 0.04 [0.01 , 0.07] 1.6E-01 Cmah 0.36 [0.18 , 0.54] 5.3E-03 0.09 [0.00 , 0.17] 1.5E-01 0.04 [0.00 , 0.08] 3.1E-01 Tnfrsf1b 0.36 [0.22 , 0.51] 1.3E-03 0.09 [0.03 , 0.15] 5.1E-02 0.00 [-0.03 , 0.03] 9.4E-01 501 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfsf18 0.36 [-0.01 , 0.73] 1.3E-01 0.14 [0.00 , 0.28] 1.7E-01 0.12 [0.06 , 0.19] 5.8E-02 Cdkn1a 0.36 [0.20 , 0.52] 2.5E-03 0.12 [0.03 , 0.21] 7.7E-02 0.02 [-0.01 , 0.05] 5.5E-01 Ly96 0.36 [0.21 , 0.50] 1.4E-03 0.10 [0.03 , 0.17] 4.8E-02 0.03 [0.00 , 0.05] 2.1E-01 Il12rb1 0.35 [0.00 , 0.71] 1.2E-01 0.20 [0.05 , 0.35] 6.8E-02 0.05 [-0.01 , 0.11] 3.6E-01 Stat2 0.35 [0.19 , 0.51] 3.3E-03 0.12 [0.03 , 0.22] 8.3E-02 0.00 [-0.04 , 0.04] 9.9E-01 Selplg 0.35 [0.21 , 0.48] 1.1E-03 0.09 [0.01 , 0.17] 1.1E-01 0.02 [-0.01 , 0.05] 5.9E-01 Nrp1 0.35 [0.24 , 0.45] 2.1E-04 0.10 [0.04 , 0.17] 3.9E-02 0.03 [0.00 , 0.05] 3.2E-01 Ifit2 0.34 [0.14 , 0.55] 1.6E-02 0.15 [0.02 , 0.28] 1.0E-01 0.04 [0.00 , 0.08] 3.2E-01 Pml 0.34 [0.22 , 0.46] 4.9E-04 0.13 [0.05 , 0.20] 2.5E-02 0.03 [0.00 , 0.05] 3.0E-01 Lta 0.34 [0.08 , 0.61] 4.8E-02 0.11 [-0.06 , 0.27] 3.7E-01 -0.01 [-0.07 , 0.04] 8.7E-01 Cd14 0.34 [0.03 , 0.66] 9.4E-02 0.03 [-0.09 , 0.16] 7.6E-01 0.06 [-0.02 , 0.13] 4.0E-01 Irak3 0.34 [0.13 , 0.54] 1.7E-02 0.12 [0.02 , 0.21] 9.8E-02 0.04 [0.00 , 0.08] 2.3E-01 Igf2r 0.33 [0.18 , 0.49] 3.1E-03 0.06 [-0.01 , 0.14] 2.3E-01 0.01 [-0.03 , 0.04] 8.8E-01 Il15ra 0.33 [0.10 , 0.57] 3.3E-02 0.15 [0.02 , 0.29] 1.1E-01 0.08 [0.04 , 0.13] 4.7E-02 Lyn 0.33 [0.20 , 0.46] 1.5E-03 0.11 [0.05 , 0.17] 2.5E-02 0.02 [0.00 , 0.04] 3.1E-01 Mif 0.33 [0.21 , 0.44] 4.7E-04 0.09 [0.04 , 0.14] 1.8E-02 0.01 [-0.02 , 0.03] 8.5E-01 Cd63 0.32 [0.15 , 0.50] 8.2E-03 0.11 [0.02 , 0.20] 1.0E-01 0.02 [-0.01 , 0.05] 4.4E-01 502 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Fcgr3 0.32 [0.14 , 0.51] 1.2E-02 0.14 [0.06 , 0.23] 3.0E-02 0.07 [0.03 , 0.11] 5.4E-02 Mefv 0.32 [0.13 , 0.51] 1.5E-02 0.13 [0.05 , 0.21] 3.5E-02 0.07 [0.03 , 0.10] 6.5E-02 Cd69 0.32 [0.08 , 0.56] 4.5E-02 0.03 [-0.09 , 0.16] 7.4E-01 -0.02 [-0.07 , 0.03] 7.0E-01 Ddx58 0.32 [0.20 , 0.44] 1.0E-03 0.09 [0.03 , 0.16] 6.3E-02 0.00 [-0.03 , 0.04] 9.1E-01 Colec12 0.32 [0.17 , 0.47] 2.9E-03 0.11 [0.01 , 0.20] 1.3E-01 0.04 [0.01 , 0.06] 1.5E-01 Cxcl10 0.32 [0.07 , 0.56] 4.8E-02 0.03 [-0.10 , 0.16] 8.2E-01 -0.05 [-0.11 , 0.01] 3.9E-01 Bax 0.32 [0.19 , 0.44] 1.0E-03 0.11 [0.06 , 0.17] 1.3E-02 0.05 [0.03 , 0.07] 1.3E-02 Fcgr2b 0.31 [0.11 , 0.52] 2.3E-02 0.07 [-0.01 , 0.16] 2.3E-01 0.00 [-0.03 , 0.04] 9.8E-01 Ifi35 0.31 [0.23 , 0.40] 1.1E-04 0.10 [0.05 , 0.15] 1.8E-02 0.02 [0.00 , 0.04] 3.8E-01 Ciita 0.31 [0.04 , 0.58] 7.4E-02 0.06 [-0.08 , 0.19] 5.9E-01 0.01 [-0.05 , 0.07] 8.9E-01 Tlr1 0.31 [0.03 , 0.58] 8.2E-02 0.07 [-0.07 , 0.22] 4.9E-01 0.01 [-0.05 , 0.06] 9.3E-01 C1ra 0.31 [0.12 , 0.50] 1.8E-02 0.08 [-0.03 , 0.19] 3.1E-01 0.01 [-0.03 , 0.05] 8.2E-01 Litaf 0.31 [0.16 , 0.45] 4.1E-03 0.16 [0.08 , 0.23] 9.3E-03 0.04 [0.01 , 0.07] 1.1E-01 Nlrp3 0.31 [0.15 , 0.46] 5.4E-03 0.11 [0.04 , 0.19] 3.9E-02 0.01 [-0.04 , 0.05] 9.3E-01 Ifitm2 0.30 [0.18 , 0.42] 1.3E-03 0.13 [0.06 , 0.19] 1.7E-02 0.04 [0.03 , 0.06] 1.6E-02 Tnfrsf12a 0.30 [0.09 , 0.52] 3.6E-02 0.14 [0.02 , 0.27] 1.1E-01 0.00 [-0.05 , 0.05] 9.9E-01 Cxcl5 0.30 [-0.11 , 0.71] 2.5E-01 0.08 [-0.10 , 0.26] 5.4E-01 0.06 [-0.02 , 0.13] 4.5E-01 503 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd22 0.30 [-0.02 , 0.62] 1.4E-01 0.09 [-0.06 , 0.25] 4.0E-01 0.02 [-0.05 , 0.08] 8.3E-01 Tnfrsf11a 0.30 [0.18 , 0.42] 1.3E-03 0.10 [0.01 , 0.19] 1.2E-01 0.03 [0.00 , 0.06] 3.3E-01 Serping1 0.30 [0.18 , 0.42] 1.5E-03 0.08 [-0.01 , 0.16] 2.1E-01 0.02 [-0.01 , 0.04] 5.0E-01 Mef2c 0.30 [0.16 , 0.43] 2.7E-03 0.08 [0.02 , 0.13] 5.2E-02 0.02 [-0.01 , 0.04] 5.1E-01 Icam1 0.29 [0.14 , 0.44] 5.5E-03 0.06 [-0.03 , 0.15] 3.5E-01 -0.01 [-0.05 , 0.03] 8.9E-01 Slc7a11 0.29 [0.14 , 0.44] 7.7E-03 0.11 [-0.03 , 0.25] 2.7E-01 0.02 [-0.02 , 0.06] 6.0E-01 Card9 0.29 [-0.05 , 0.63] 1.9E-01 0.15 [-0.10 , 0.41] 4.2E-01 0.03 [-0.04 , 0.10] 6.9E-01 Cd38 0.28 [0.12 , 0.45] 1.1E-02 0.07 [0.00 , 0.14] 1.9E-01 0.02 [-0.01 , 0.05] 5.9E-01 Irak4 0.28 [0.16 , 0.40] 2.4E-03 0.10 [0.04 , 0.15] 2.5E-02 0.02 [-0.01 , 0.04] 5.2E-01 Blnk 0.28 [0.15 , 0.41] 3.8E-03 0.06 [-0.01 , 0.13] 2.1E-01 0.02 [-0.01 , 0.05] 6.0E-01 Map3k5 0.28 [0.13 , 0.43] 9.5E-03 0.14 [0.07 , 0.21] 1.5E-02 0.04 [0.00 , 0.08] 2.4E-01 Map2k1 0.27 [0.15 , 0.40] 2.9E-03 0.08 [0.03 , 0.13] 4.3E-02 0.01 [-0.01 , 0.03] 7.5E-01 Sh2d1a 0.27 [0.01 , 0.54] 1.1E-01 n.d [n.d , n.d] n.d -0.02 [-0.08 , 0.03] 7.1E-01 Gbp2b 0.27 [-0.09 , 0.63] 2.3E-01 -0.07 [-0.27 , 0.14] 6.8E-01 -0.03 [-0.10 , 0.05] 7.8E-01 C2 0.27 [0.17 , 0.37] 6.4E-04 0.06 [0.01 , 0.12] 1.1E-01 0.03 [0.02 , 0.05] 2.2E-02 Cd68 0.27 [0.15 , 0.39] 2.5E-03 0.07 [-0.01 , 0.16] 2.3E-01 0.01 [-0.02 , 0.04] 6.8E-01 Nfkb2 0.27 [0.08 , 0.45] 3.3E-02 0.04 [-0.06 , 0.14] 6.5E-01 -0.02 [-0.06 , 0.02] 5.3E-01 504 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ikbke 0.26 [0.00 , 0.53] 1.2E-01 0.03 [-0.11 , 0.16] 8.0E-01 0.01 [-0.04 , 0.06] 8.9E-01 Egr3 0.26 [-0.01 , 0.54] 1.3E-01 0.13 [-0.02 , 0.28] 2.1E-01 -0.03 [-0.09 , 0.03] 6.6E-01 Ltbr 0.26 [0.13 , 0.39] 5.5E-03 0.10 [0.04 , 0.16] 3.5E-02 0.03 [-0.01 , 0.07] 5.0E-01 C1s1 0.26 [0.12 , 0.40] 9.0E-03 0.07 [-0.03 , 0.18] 3.4E-01 0.03 [0.00 , 0.05] 3.1E-01 Cfh 0.26 [0.04 , 0.48] 7.2E-02 0.12 [-0.02 , 0.26] 2.2E-01 0.05 [0.01 , 0.09] 1.8E-01 Ccl1 0.26 [-0.13 , 0.65] 2.9E-01 0.12 [-0.15 , 0.40] 5.7E-01 -0.02 [-0.10 , 0.06] 8.4E-01 Tapbp 0.25 [0.14 , 0.36] 2.4E-03 0.07 [0.00 , 0.13] 1.4E-01 0.01 [-0.01 , 0.03] 7.7E-01 Itgb1 0.25 [0.06 , 0.44] 4.4E-02 0.09 [0.01 , 0.17] 1.1E-01 0.04 [0.01 , 0.07] 1.6E-01 Ebi3 0.25 [0.06 , 0.44] 4.6E-02 0.18 [0.08 , 0.27] 2.2E-02 0.04 [-0.02 , 0.10] 5.1E-01 Prkcd 0.25 [0.16 , 0.35] 8.9E-04 0.11 [0.07 , 0.15] 4.7E-03 0.03 [0.01 , 0.06] 9.9E-02 Pecam1 0.25 [0.12 , 0.38] 6.4E-03 0.06 [0.00 , 0.12] 1.7E-01 0.03 [0.01 , 0.05] 1.0E-01 Slc11a1 0.25 [0.11 , 0.39] 9.3E-03 0.03 [-0.05 , 0.12] 6.1E-01 0.02 [-0.01 , 0.05] 4.0E-01 Tnfrsf10b 0.25 [0.02 , 0.47] 8.6E-02 0.11 [0.00 , 0.23] 1.6E-01 0.02 [-0.05 , 0.09] 7.9E-01 Fyn 0.25 [0.13 , 0.37] 3.9E-03 0.05 [-0.03 , 0.12] 4.0E-01 0.02 [-0.01 , 0.04] 5.4E-01 Zap70 0.25 [-0.04 , 0.54] 1.8E-01 0.02 [-0.14 , 0.17] 9.1E-01 -0.01 [-0.07 , 0.05] 9.4E-01 Psen1 0.25 [0.17 , 0.32] 2.3E-04 0.07 [0.02 , 0.12] 7.4E-02 0.02 [0.01 , 0.04] 1.6E-01 App 0.25 [0.15 , 0.34] 9.0E-04 0.05 [-0.02 , 0.12] 3.0E-01 0.01 [-0.01 , 0.03] 6.2E-01 505 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Entpd1 0.25 [0.13 , 0.36] 3.2E-03 0.05 [-0.04 , 0.14] 4.2E-01 0.04 [0.01 , 0.08] 1.9E-01 Tfeb 0.25 [0.09 , 0.40] 2.0E-02 0.06 [-0.03 , 0.15] 3.4E-01 -0.01 [-0.04 , 0.02] 7.5E-01 Dusp6 0.24 [0.11 , 0.37] 8.4E-03 0.03 [-0.04 , 0.10] 6.1E-01 -0.01 [-0.04 , 0.01] 5.8E-01 H2-Eb1 0.24 [0.04 , 0.44] 6.2E-02 0.05 [-0.05 , 0.14] 5.1E-01 0.00 [-0.04 , 0.04] 9.7E-01 Lamp2 0.24 [0.04 , 0.45] 7.2E-02 0.09 [0.00 , 0.19] 1.6E-01 0.04 [0.01 , 0.08] 2.0E-01 Casp8 0.24 [0.14 , 0.34] 1.6E-03 0.04 [-0.01 , 0.09] 2.7E-01 0.01 [-0.01 , 0.03] 6.3E-01 Lamp1 0.24 [0.14 , 0.33] 1.6E-03 0.07 [0.02 , 0.11] 4.0E-02 0.02 [0.00 , 0.03] 2.1E-01 Cd200 0.24 [0.06 , 0.41] 3.8E-02 0.08 [-0.01 , 0.17] 2.3E-01 0.02 [-0.01 , 0.05] 5.5E-01 Mapkapk2 0.23 [0.11 , 0.36] 8.2E-03 0.10 [0.04 , 0.15] 2.1E-02 0.03 [0.00 , 0.05] 2.3E-01 Tnfrsf18 0.23 [0.01 , 0.45] 1.0E-01 0.06 [-0.04 , 0.17] 4.2E-01 0.00 [-0.05 , 0.05] 9.9E-01 Sell 0.23 [-0.05 , 0.51] 1.9E-01 0.02 [-0.13 , 0.17] 8.8E-01 0.01 [-0.04 , 0.06] 8.9E-01 Irf2 0.23 [0.11 , 0.35] 8.6E-03 0.08 [0.01 , 0.14] 1.1E-01 0.01 [-0.01 , 0.03] 5.8E-01 Runx1 0.22 [0.10 , 0.35] 1.1E-02 0.09 [0.02 , 0.15] 8.8E-02 0.02 [0.00 , 0.04] 3.2E-01 Nod2 0.22 [0.02 , 0.42] 8.6E-02 0.01 [-0.07 , 0.09] 9.1E-01 -0.05 [-0.09 , -0.01] 2.1E-01 Amica1 0.22 [0.02 , 0.42] 8.8E-02 0.04 [-0.05 , 0.13] 5.6E-01 0.01 [-0.02 , 0.04] 7.9E-01 Ctsl 0.22 [0.04 , 0.40] 5.8E-02 0.09 [-0.01 , 0.18] 1.9E-01 0.05 [0.02 , 0.08] 1.1E-01 Il12a 0.22 [0.00 , 0.43] 1.2E-01 0.13 [0.02 , 0.23] 9.4E-02 0.05 [0.01 , 0.09] 1.9E-01 506 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Herc6 0.22 [-0.05 , 0.48] 2.1E-01 0.10 [-0.02 , 0.23] 2.5E-01 0.05 [-0.01 , 0.10] 3.4E-01 Cd40 0.21 [-0.07 , 0.50] 2.4E-01 -0.01 [-0.15 , 0.12] 9.2E-01 -0.04 [-0.10 , 0.01] 4.0E-01 H2-DMb1 0.21 [0.02 , 0.40] 8.2E-02 0.05 [-0.06 , 0.15] 5.4E-01 n.d [n.d , n.d] n.d Cd180 0.21 [-0.03 , 0.45] 1.8E-01 0.06 [-0.05 , 0.18] 4.7E-01 n.d [n.d , n.d] n.d Cxcr2 0.21 [-0.07 , 0.49] 2.4E-01 0.17 [0.01 , 0.33] 1.3E-01 0.03 [-0.01 , 0.08] 4.7E-01 Sigirr 0.21 [-0.01 , 0.42] 1.3E-01 0.07 [-0.04 , 0.17] 3.7E-01 0.04 [0.00 , 0.09] 3.1E-01 Ticam1 0.21 [0.07 , 0.34] 2.8E-02 0.06 [0.00 , 0.13] 1.8E-01 0.02 [-0.01 , 0.04] 6.1E-01 Gpi1 0.20 [0.13 , 0.28] 8.3E-04 0.10 [0.05 , 0.14] 7.7E-03 0.02 [0.00 , 0.04] 3.9E-01 Chuk 0.20 [0.07 , 0.33] 2.1E-02 0.05 [0.00 , 0.11] 1.6E-01 0.02 [0.00 , 0.04] 2.5E-01 H2-Ab1 0.20 [-0.01 , 0.41] 1.4E-01 0.04 [-0.06 , 0.13] 6.3E-01 -0.01 [-0.05 , 0.03] 8.7E-01 Xcr1 0.20 [-0.09 , 0.49] 2.8E-01 0.02 [-0.18 , 0.21] 9.2E-01 -0.01 [-0.07 , 0.05] 9.0E-01 Tap2 0.20 [-0.06 , 0.46] 2.3E-01 0.03 [-0.12 , 0.17] 8.3E-01 -0.02 [-0.07 , 0.04] 7.8E-01 Cd97 0.20 [0.05 , 0.35] 4.7E-02 0.04 [-0.05 , 0.13] 5.5E-01 0.00 [-0.04 , 0.03] 9.3E-01 Sh2b2 0.20 [-0.03 , 0.42] 1.8E-01 0.12 [0.02 , 0.23] 9.8E-02 0.01 [-0.02 , 0.04] 8.3E-01 H2-DMa 0.19 [0.03 , 0.36] 7.3E-02 0.05 [-0.03 , 0.12] 3.8E-01 0.00 [-0.03 , 0.04] 9.7E-01 Csf2 0.19 [-0.12 , 0.51] 3.3E-01 0.03 [-0.11 , 0.17] 8.3E-01 n.d [n.d , n.d] n.d Ccl5 0.19 [0.02 , 0.36] 8.7E-02 0.06 [-0.03 , 0.15] 3.9E-01 0.00 [-0.03 , 0.04] 9.4E-01 507 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cyfip2 0.19 [0.07 , 0.31] 2.3E-02 0.06 [0.00 , 0.12] 1.5E-01 0.02 [-0.01 , 0.05] 5.2E-01 Cd44 0.19 [0.01 , 0.37] 1.1E-01 0.07 [-0.02 , 0.16] 3.0E-01 0.04 [0.01 , 0.07] 1.2E-01 Cd19 0.19 [-0.06 , 0.43] 2.3E-01 0.04 [-0.08 , 0.15] 6.7E-01 -0.01 [-0.06 , 0.04] 9.1E-01 Ncf4 0.19 [0.06 , 0.31] 2.4E-02 0.09 [0.05 , 0.13] 6.9E-03 0.01 [-0.02 , 0.03] 8.3E-01 Ccl25 0.18 [-0.12 , 0.49] 3.3E-01 0.13 [0.00 , 0.25] 1.6E-01 0.07 [0.02 , 0.12] 1.2E-01 Tnfaip3 0.18 [0.01 , 0.35] 9.2E-02 0.05 [-0.03 , 0.14] 3.9E-01 -0.02 [-0.05 , 0.01] 4.3E-01 Relb 0.18 [0.00 , 0.36] 1.2E-01 0.05 [-0.04 , 0.14] 4.4E-01 0.00 [-0.04 , 0.05] 9.4E-01 Slamf1 0.18 [-0.06 , 0.42] 2.3E-01 0.09 [-0.01 , 0.19] 1.9E-01 0.04 [-0.01 , 0.08] 3.6E-01 Psen2 0.18 [0.04 , 0.31] 4.5E-02 0.08 [-0.01 , 0.17] 2.1E-01 0.02 [-0.01 , 0.05] 5.5E-01 Clec4n 0.18 [-0.08 , 0.43] 2.7E-01 0.04 [-0.09 , 0.18] 7.0E-01 0.04 [-0.01 , 0.10] 4.0E-01 Cd79a 0.18 [-0.08 , 0.43] 2.7E-01 -0.03 [-0.14 , 0.09] 8.0E-01 -0.02 [-0.07 , 0.04] 8.0E-01 Irf8 0.18 [0.05 , 0.30] 3.4E-02 0.03 [-0.04 , 0.10] 5.9E-01 0.01 [-0.01 , 0.03] 7.2E-01 Col4a1 0.17 [0.03 , 0.32] 7.1E-02 0.02 [-0.06 , 0.09] 8.0E-01 -0.02 [-0.05 , 0.01] 5.1E-01 Lyz2 0.17 [-0.06 , 0.40] 2.4E-01 0.09 [-0.02 , 0.20] 2.3E-01 -0.01 [-0.05 , 0.04] 9.0E-01 Il2rg 0.17 [-0.07 , 0.40] 2.6E-01 0.02 [-0.09 , 0.14] 8.1E-01 -0.01 [-0.05 , 0.04] 8.9E-01 Traf6 0.17 [0.09 , 0.24] 3.0E-03 0.01 [-0.02 , 0.05] 6.3E-01 0.00 [-0.02 , 0.02] 9.7E-01 Bcl10 0.17 [0.04 , 0.29] 4.2E-02 0.05 [0.00 , 0.11] 1.7E-01 0.01 [-0.02 , 0.03] 8.7E-01 508 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tank 0.16 [0.00 , 0.32] 1.1E-01 0.04 [-0.04 , 0.11] 5.3E-01 -0.01 [-0.03 , 0.02] 8.5E-01 Tlr4 0.16 [-0.08 , 0.40] 2.9E-01 0.01 [-0.11 , 0.13] 9.3E-01 -0.01 [-0.05 , 0.04] 9.3E-01 Nfkb1 0.16 [0.04 , 0.28] 4.0E-02 0.07 [0.00 , 0.13] 1.5E-01 0.00 [-0.03 , 0.03] 9.4E-01 Eomes 0.16 [-0.11 , 0.43] 3.5E-01 -0.14 [-0.26 , -0.02] 1.1E-01 -0.03 [-0.08 , 0.02] 5.4E-01 Tgfbr2 0.16 [-0.01 , 0.33] 1.5E-01 0.01 [-0.08 , 0.11] 8.7E-01 -0.01 [-0.04 , 0.03] 8.9E-01 H2-Ea-ps 0.16 [-0.09 , 0.41] 3.2E-01 0.04 [-0.08 , 0.15] 7.0E-01 0.00 [-0.05 , 0.05] 9.8E-01 H2-Aa 0.16 [-0.10 , 0.41] 3.3E-01 0.03 [-0.09 , 0.15] 7.4E-01 0.00 [-0.05 , 0.05] 9.7E-01 Cd74 0.16 [-0.04 , 0.35] 2.2E-01 0.03 [-0.06 , 0.11] 6.8E-01 -0.01 [-0.05 , 0.03] 8.9E-01 Tlr6 0.16 [-0.06 , 0.38] 2.7E-01 0.05 [-0.06 , 0.16] 5.3E-01 0.00 [-0.05 , 0.04] 9.7E-01 Btla 0.16 [-0.08 , 0.39] 3.0E-01 0.01 [-0.11 , 0.13] 9.2E-01 0.03 [-0.02 , 0.07] 5.2E-01 Cd2 0.15 [-0.15 , 0.45] 4.1E-01 -0.07 [-0.23 , 0.09] 5.8E-01 0.00 [-0.07 , 0.07] 9.9E-01 H2-Q10 0.15 [-0.26 , 0.57] 5.7E-01 0.21 [0.00 , 0.42] 1.6E-01 0.05 [-0.03 , 0.13] 5.2E-01 Cd4 0.15 [-0.14 , 0.45] 4.1E-01 -0.02 [-0.18 , 0.13] 8.7E-01 0.00 [-0.07 , 0.06] 9.7E-01 H2-DMb2 0.15 [-0.08 , 0.39] 3.1E-01 0.02 [-0.09 , 0.13] 8.1E-01 -0.02 [-0.06 , 0.03] 7.6E-01 Nod1 0.15 [0.01 , 0.29] 9.7E-02 0.03 [-0.05 , 0.10] 6.7E-01 -0.01 [-0.05 , 0.03] 8.8E-01 Traf2 0.15 [0.02 , 0.27] 7.8E-02 0.02 [-0.06 , 0.09] 7.9E-01 0.00 [-0.03 , 0.03] 9.5E-01 Apoe 0.15 [0.00 , 0.29] 1.3E-01 0.08 [0.03 , 0.13] 3.6E-02 0.05 [0.03 , 0.08] 3.4E-02 509 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Hsd11b1 0.15 [-0.07 , 0.36] 2.8E-01 -0.03 [-0.13 , 0.08] 7.4E-01 n.d [n.d , n.d] n.d Psma2 0.14 [-0.05 , 0.34] 2.5E-01 0.03 [-0.06 , 0.11] 7.1E-01 0.01 [-0.03 , 0.04] 8.7E-01 Tnfrsf1a 0.14 [0.04 , 0.24] 3.8E-02 0.02 [-0.03 , 0.07] 5.9E-01 0.00 [-0.01 , 0.02] 8.5E-01 Ly86 0.14 [-0.08 , 0.36] 3.2E-01 0.02 [-0.09 , 0.13] 8.0E-01 -0.01 [-0.05 , 0.04] 9.1E-01 Il17ra 0.14 [0.00 , 0.28] 1.3E-01 0.05 [-0.03 , 0.12] 3.7E-01 0.01 [-0.02 , 0.04] 8.8E-01 Il2rb 0.14 [-0.02 , 0.29] 1.6E-01 -0.03 [-0.11 , 0.05] 6.1E-01 -0.02 [-0.05 , 0.01] 4.9E-01 Nfkbia 0.14 [-0.02 , 0.29] 1.6E-01 0.02 [-0.06 , 0.11] 7.3E-01 -0.02 [-0.06 , 0.02] 5.5E-01 Plaur 0.14 [-0.02 , 0.29] 1.7E-01 0.00 [-0.06 , 0.05] 9.4E-01 -0.02 [-0.05 , 0.00] 3.6E-01 Btk 0.14 [0.04 , 0.23] 3.3E-02 0.05 [0.01 , 0.10] 9.4E-02 0.01 [-0.01 , 0.03] 5.0E-01 C4b 0.14 [-0.04 , 0.31] 2.3E-01 -0.01 [-0.11 , 0.10] 9.2E-01 0.00 [-0.04 , 0.04] 9.7E-01 Smn1 0.13 [0.05 , 0.22] 2.2E-02 0.03 [0.00 , 0.06] 1.4E-01 0.00 [-0.01 , 0.02] 9.0E-01 Map3k1 0.13 [0.03 , 0.23] 4.6E-02 0.05 [0.00 , 0.09] 1.9E-01 0.03 [0.01 , 0.06] 9.9E-02 Irf5 0.13 [-0.04 , 0.30] 2.2E-01 0.04 [-0.04 , 0.11] 5.1E-01 0.00 [-0.04 , 0.04] 9.7E-01 Ripk2 0.13 [-0.05 , 0.31] 2.5E-01 0.03 [-0.04 , 0.10] 6.1E-01 0.00 [-0.04 , 0.03] 9.4E-01 H2-Ob 0.13 [-0.10 , 0.36] 3.6E-01 0.02 [-0.08 , 0.12] 8.0E-01 n.d [n.d , n.d] n.d Fcer1a 0.13 [-0.26 , 0.52] 6.0E-01 -0.04 [-0.22 , 0.15] 8.2E-01 -0.01 [-0.08 , 0.07] 9.4E-01 Ly9 0.13 [-0.04 , 0.29] 2.3E-01 0.01 [-0.07 , 0.09] 9.1E-01 0.00 [-0.03 , 0.04] 9.3E-01 510 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Elk1 0.13 [-0.14 , 0.39] 4.4E-01 0.08 [-0.06 , 0.22] 4.2E-01 0.01 [-0.05 , 0.06] 9.4E-01 Il13ra1 0.12 [-0.08 , 0.33] 3.4E-01 0.09 [-0.01 , 0.19] 2.1E-01 0.02 [-0.02 , 0.05] 6.4E-01 Usp18 0.12 [-0.09 , 0.34] 3.6E-01 0.08 [-0.04 , 0.21] 3.6E-01 -0.01 [-0.07 , 0.04] 8.7E-01 Tnfrsf13b 0.12 [-0.03 , 0.28] 2.2E-01 0.03 [-0.06 , 0.11] 6.9E-01 0.00 [-0.04 , 0.05] 9.5E-01 Cd3g 0.12 [-0.21 , 0.45] 5.6E-01 -0.10 [-0.27 , 0.07] 4.1E-01 -0.04 [-0.11 , 0.03] 5.3E-01 Icam2 0.12 [-0.10 , 0.34] 3.8E-01 -0.04 [-0.15 , 0.07] 6.7E-01 -0.02 [-0.07 , 0.03] 6.9E-01 Il34 0.12 [0.00 , 0.24] 1.3E-01 -0.01 [-0.09 , 0.07] 8.8E-01 -0.01 [-0.04 , 0.01] 6.4E-01 Cd5 0.12 [-0.23 , 0.46] 6.0E-01 -0.06 [-0.23 , 0.12] 6.9E-01 -0.01 [-0.09 , 0.06] 9.2E-01 Pvr 0.12 [-0.06 , 0.30] 3.0E-01 0.02 [-0.07 , 0.10] 8.5E-01 -0.01 [-0.05 , 0.02] 7.7E-01 Cd244 0.11 [-0.10 , 0.32] 4.0E-01 0.02 [-0.07 , 0.11] 8.1E-01 0.01 [-0.03 , 0.05] 8.5E-01 Bmi1 0.11 [-0.02 , 0.24] 2.0E-01 0.03 [-0.02 , 0.09] 4.3E-01 0.01 [-0.02 , 0.04] 7.0E-01 Cd99 0.10 [-0.06 , 0.27] 3.1E-01 0.04 [-0.04 , 0.13] 4.5E-01 0.03 [-0.01 , 0.06] 4.0E-01 Osm 0.10 [-0.15 , 0.35] 5.1E-01 0.06 [-0.07 , 0.19] 5.7E-01 0.01 [-0.04 , 0.05] 8.9E-01 Atm 0.10 [-0.08 , 0.28] 3.6E-01 -0.03 [-0.11 , 0.05] 6.0E-01 -0.01 [-0.05 , 0.02] 7.7E-01 Pla2g6 0.10 [-0.08 , 0.29] 3.6E-01 0.02 [-0.07 , 0.10] 8.3E-01 0.04 [-0.01 , 0.09] 3.6E-01 Pik3cd 0.10 [-0.07 , 0.27] 3.3E-01 0.01 [-0.08 , 0.09] 9.4E-01 -0.01 [-0.05 , 0.03] 8.5E-01 Itga4 0.10 [0.01 , 0.19] 9.5E-02 0.04 [-0.01 , 0.09] 3.1E-01 0.04 [0.03 , 0.06] 1.5E-02 511 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tbk1 0.10 [0.03 , 0.17] 4.0E-02 0.03 [-0.01 , 0.07] 2.3E-01 0.02 [0.00 , 0.03] 2.4E-01 Cd86 0.09 [-0.05 , 0.24] 3.1E-01 -0.05 [-0.13 , 0.02] 3.4E-01 0.01 [-0.01 , 0.04] 6.8E-01 Ms4a2 0.09 [-0.34 , 0.52] 7.6E-01 0.06 [-0.16 , 0.29] 7.3E-01 -0.02 [-0.09 , 0.06] 8.9E-01 Clu 0.09 [-0.01 , 0.19] 1.4E-01 0.09 [0.02 , 0.17] 9.7E-02 0.03 [0.00 , 0.06] 3.4E-01 Traf3 0.09 [-0.08 , 0.26] 4.0E-01 0.00 [-0.09 , 0.09] 9.6E-01 -0.02 [-0.06 , 0.02] 6.3E-01 Itga5 0.09 [-0.11 , 0.29] 4.8E-01 0.00 [-0.13 , 0.13] 9.8E-01 -0.01 [-0.06 , 0.04] 8.9E-01 Mfge8 0.09 [-0.08 , 0.26] 4.1E-01 0.09 [-0.03 , 0.20] 3.0E-01 0.00 [-0.04 , 0.04] 9.9E-01 Flt3 0.09 [-0.11 , 0.29] 4.9E-01 0.06 [-0.06 , 0.17] 5.1E-01 0.06 [0.02 , 0.10] 9.9E-02 Stat6 0.09 [-0.04 , 0.22] 2.9E-01 0.00 [-0.07 , 0.06] 9.6E-01 -0.01 [-0.04 , 0.02] 7.6E-01 Rela 0.09 [-0.07 , 0.24] 3.6E-01 0.01 [-0.07 , 0.09] 8.8E-01 0.01 [-0.03 , 0.05] 8.1E-01 Psmb7 0.08 [-0.10 , 0.27] 4.8E-01 0.02 [-0.07 , 0.10] 8.0E-01 0.01 [-0.03 , 0.05] 8.2E-01 Rel 0.08 [-0.12 , 0.28] 5.1E-01 0.01 [-0.08 , 0.09] 9.2E-01 -0.02 [-0.06 , 0.02] 5.5E-01 Reps1 0.08 [0.01 , 0.16] 8.5E-02 0.01 [-0.03 , 0.06] 6.8E-01 0.01 [-0.02 , 0.03] 8.5E-01 Adora2a 0.08 [-0.12 , 0.28] 5.2E-01 0.02 [-0.04 , 0.08] 6.9E-01 0.04 [0.00 , 0.07] 2.3E-01 Inpp5d 0.08 [-0.05 , 0.21] 3.2E-01 0.00 [-0.07 , 0.06] 9.7E-01 0.01 [-0.03 , 0.04] 8.9E-01 Irak1 0.08 [-0.01 , 0.17] 1.7E-01 0.04 [-0.01 , 0.09] 2.2E-01 0.02 [-0.01 , 0.04] 5.3E-01 Ifnar1 0.08 [-0.02 , 0.18] 2.1E-01 0.01 [-0.03 , 0.06] 7.2E-01 0.01 [-0.01 , 0.03] 7.5E-01 512 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd6 0.08 [-0.25 , 0.40] 7.3E-01 -0.03 [-0.18 , 0.12] 7.9E-01 -0.01 [-0.08 , 0.05] 8.8E-01 Psmd7 0.08 [-0.05 , 0.20] 3.2E-01 0.02 [-0.03 , 0.06] 6.7E-01 0.00 [-0.02 , 0.02] 9.7E-01 Vhl 0.07 [-0.07 , 0.21] 4.4E-01 0.02 [-0.05 , 0.10] 6.9E-01 0.03 [0.00 , 0.07] 2.2E-01 Klrb1 0.07 [-0.29 , 0.43] 7.8E-01 -0.02 [-0.16 , 0.12] 8.4E-01 0.01 [-0.05 , 0.07] 8.9E-01 Atg5 0.07 [-0.12 , 0.25] 5.7E-01 -0.01 [-0.09 , 0.07] 9.2E-01 -0.03 [-0.06 , 0.01] 4.1E-01 Tnfrsf13c 0.07 [-0.17 , 0.30] 6.6E-01 n.d [n.d , n.d] n.d -0.02 [-0.06 , 0.03] 7.3E-01 Cd81 0.07 [-0.02 , 0.15] 2.1E-01 -0.01 [-0.06 , 0.03] 6.9E-01 0.01 [-0.01 , 0.02] 8.2E-01 Stat5b 0.06 [-0.06 , 0.18] 3.9E-01 0.02 [-0.04 , 0.08] 6.7E-01 0.00 [-0.03 , 0.03] 9.7E-01 Cd28 0.06 [-0.24 , 0.36] 7.6E-01 -0.07 [-0.25 , 0.10] 5.9E-01 -0.01 [-0.06 , 0.05] 9.3E-01 Lilra5 0.06 [-0.16 , 0.28] 6.8E-01 0.05 [-0.05 , 0.14] 5.1E-01 0.04 [0.01 , 0.08] 2.1E-01 Gtf3c1 0.06 [-0.07 , 0.19] 4.8E-01 n.d [n.d , n.d] n.d -0.02 [-0.04 , 0.01] 5.2E-01 Cd164 0.06 [-0.04 , 0.16] 3.5E-01 -0.01 [-0.06 , 0.04] 7.3E-01 0.00 [-0.02 , 0.02] 9.5E-01 Ilf3 0.06 [-0.05 , 0.16] 3.8E-01 0.00 [-0.06 , 0.06] 9.8E-01 0.00 [-0.02 , 0.03] 9.7E-01 C8g 0.05 [-0.28 , 0.39] 8.2E-01 0.12 [-0.05 , 0.28] 3.2E-01 0.01 [-0.05 , 0.08] 8.9E-01 Abca1 0.05 [-0.12 , 0.23] 6.5E-01 0.04 [-0.05 , 0.12] 5.9E-01 0.05 [0.00 , 0.09] 2.5E-01 Card11 0.05 [-0.20 , 0.30] 7.7E-01 -0.03 [-0.16 , 0.10] 7.8E-01 -0.03 [-0.09 , 0.02] 5.6E-01 Ecsit 0.05 [-0.13 , 0.23] 6.8E-01 0.04 [-0.05 , 0.14] 5.8E-01 0.00 [-0.04 , 0.03] 9.9E-01 513 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il1rap 0.05 [-0.14 , 0.23] 7.1E-01 0.03 [-0.07 , 0.12] 7.3E-01 0.00 [-0.03 , 0.04] 9.5E-01 C1qbp 0.04 [-0.02 , 0.11] 3.0E-01 0.04 [-0.01 , 0.08] 2.2E-01 -0.01 [-0.02 , 0.01] 6.0E-01 Csf1r 0.04 [-0.08 , 0.17] 6.2E-01 -0.05 [-0.14 , 0.04] 4.5E-01 0.02 [-0.01 , 0.04] 4.9E-01 Cd33 0.04 [-0.14 , 0.22] 7.4E-01 0.02 [-0.09 , 0.12] 8.6E-01 0.00 [-0.03 , 0.04] 9.7E-01 Nup107 0.04 [-0.10 , 0.18] 6.8E-01 0.00 [-0.06 , 0.07] 9.4E-01 0.00 [-0.02 , 0.03] 9.3E-01 Ltb 0.03 [-0.25 , 0.32] 8.6E-01 -0.04 [-0.18 , 0.09] 6.9E-01 -0.05 [-0.11 , 0.01] 3.5E-01 Prkce 0.03 [-0.17 , 0.23] 8.1E-01 0.02 [-0.06 , 0.11] 7.3E-01 0.01 [-0.02 , 0.05] 7.6E-01 Il18 0.03 [-0.17 , 0.23] 8.2E-01 -0.03 [-0.15 , 0.08] 7.2E-01 0.04 [0.00 , 0.07] 2.8E-01 Icosl 0.03 [-0.24 , 0.30] 8.7E-01 -0.04 [-0.17 , 0.09] 6.9E-01 -0.03 [-0.08 , 0.03] 6.4E-01 Gfi1 0.03 [-0.22 , 0.27] 8.7E-01 -0.02 [-0.12 , 0.08] 7.8E-01 0.00 [-0.03 , 0.04] 9.7E-01 Ikbkb 0.03 [-0.16 , 0.21] 8.3E-01 -0.04 [-0.13 , 0.06] 6.0E-01 -0.02 [-0.06 , 0.02] 6.3E-01 Cd84 0.03 [-0.15 , 0.20] 8.2E-01 -0.03 [-0.12 , 0.06] 6.7E-01 0.01 [-0.02 , 0.05] 7.9E-01 Irgm2 0.03 [-0.16 , 0.22] 8.3E-01 0.07 [-0.03 , 0.16] 3.4E-01 0.02 [-0.01 , 0.06] 5.2E-01 Aire 0.03 [-0.30 , 0.35] 9.0E-01 0.05 [-0.12 , 0.22] 6.9E-01 0.04 [-0.01 , 0.09] 4.4E-01 Ifi27 0.02 [-0.10 , 0.15] 7.9E-01 0.03 [-0.02 , 0.08] 4.6E-01 -0.01 [-0.03 , 0.02] 8.5E-01 Irf3 0.02 [-0.11 , 0.15] 8.2E-01 -0.02 [-0.09 , 0.04] 6.3E-01 -0.01 [-0.03 , 0.02] 8.6E-01 Tfe3 0.02 [-0.08 , 0.12] 7.6E-01 0.04 [0.00 , 0.09] 1.4E-01 0.00 [-0.02 , 0.03] 9.4E-01 514 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Atg7 0.02 [-0.07 , 0.11] 7.6E-01 0.02 [-0.04 , 0.08] 6.7E-01 0.00 [-0.02 , 0.02] 9.8E-01 Ubc 0.02 [-0.10 , 0.13] 8.2E-01 0.01 [-0.04 , 0.06] 8.3E-01 0.02 [-0.01 , 0.05] 5.3E-01 Mr1 0.01 [-0.16 , 0.19] 9.0E-01 0.00 [-0.09 , 0.10] 9.6E-01 0.03 [-0.01 , 0.07] 5.1E-01 Casp3 0.01 [-0.18 , 0.20] 9.3E-01 0.00 [-0.08 , 0.08] 9.6E-01 -0.01 [-0.05 , 0.03] 8.4E-01 Rora 0.01 [-0.19 , 0.21] 9.4E-01 -0.03 [-0.14 , 0.08] 7.0E-01 0.01 [-0.02 , 0.05] 8.3E-01 Mapk11 0.01 [-0.29 , 0.30] 9.6E-01 -0.01 [-0.21 , 0.18] 9.3E-01 0.02 [-0.06 , 0.09] 8.7E-01 Cd37 0.01 [-0.22 , 0.23] 9.6E-01 -0.02 [-0.11 , 0.07] 8.4E-01 -0.01 [-0.06 , 0.04] 8.9E-01 Cd247 0.00 [-0.37 , 0.38] 9.9E-01 -0.10 [-0.30 , 0.09] 4.7E-01 -0.02 [-0.09 , 0.06] 8.5E-01 Ccl27a 0.00 [-0.25 , 0.26] 9.8E-01 -0.01 [-0.15 , 0.13] 9.2E-01 -0.02 [-0.08 , 0.05] 8.5E-01 Ythdf2 0.00 [-0.07 , 0.07] 9.4E-01 -0.02 [-0.05 , 0.02] 5.3E-01 -0.01 [-0.02 , 0.01] 5.9E-01 Smad4 0.00 [-0.11 , 0.11] 9.9E-01 -0.02 [-0.08 , 0.04] 6.9E-01 0.00 [-0.03 , 0.03] 9.9E-01 Ifngr1 0.00 [-0.12 , 0.12] 9.6E-01 0.01 [-0.04 , 0.07] 7.3E-01 0.02 [-0.01 , 0.04] 5.0E-01 Itch -0.01 [-0.11 , 0.10] 9.3E-01 -0.01 [-0.05 , 0.04] 8.8E-01 0.01 [-0.01 , 0.03] 6.3E-01 Bid -0.01 [-0.13 , 0.11] 9.3E-01 0.03 [-0.02 , 0.08] 4.9E-01 0.00 [-0.02 , 0.02] 9.4E-01 Pvrl2 -0.01 [-0.17 , 0.16] 9.4E-01 -0.06 [-0.13 , 0.01] 2.2E-01 -0.01 [-0.04 , 0.02] 8.4E-01 Map3k7 -0.01 [-0.12 , 0.10] 9.0E-01 0.02 [-0.04 , 0.07] 7.0E-01 0.01 [0.00 , 0.03] 4.4E-01 Map2k2 -0.01 [-0.11 , 0.09] 8.8E-01 -0.01 [-0.05 , 0.04] 9.0E-01 0.00 [-0.02 , 0.02] 9.6E-01 515 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Abcb1a -0.01 [-0.18 , 0.16] 9.3E-01 -0.05 [-0.14 , 0.04] 4.6E-01 -0.03 [-0.06 , 0.00] 3.0E-01 Map2k4 -0.01 [-0.10 , 0.07] 8.3E-01 -0.01 [-0.05 , 0.02] 5.9E-01 0.01 [0.00 , 0.02] 5.1E-01 Havcr2 -0.01 [-0.21 , 0.19] 9.3E-01 -0.07 [-0.17 , 0.03] 3.5E-01 -0.03 [-0.07 , 0.01] 4.2E-01 Tyk2 -0.01 [-0.11 , 0.09] 8.5E-01 -0.02 [-0.07 , 0.02] 5.0E-01 -0.02 [-0.05 , 0.01] 5.4E-01 Itga1 -0.01 [-0.16 , 0.13] 9.0E-01 -0.09 [-0.17 , -0.01] 1.3E-01 0.00 [-0.03 , 0.03] 9.5E-01 Masp2 -0.01 [-0.48 , 0.45] 9.6E-01 0.01 [-0.21 , 0.23] 9.6E-01 -0.01 [-0.10 , 0.08] 9.4E-01 Lck -0.02 [-0.31 , 0.28] 9.3E-01 -0.10 [-0.24 , 0.04] 3.4E-01 -0.03 [-0.09 , 0.03] 6.0E-01 Stat4 -0.02 [-0.32 , 0.27] 9.0E-01 -0.11 [-0.23 , 0.01] 2.1E-01 -0.01 [-0.06 , 0.04] 8.9E-01 Cdh1 -0.02 [-0.19 , 0.14] 8.3E-01 -0.16 [-0.30 , -0.02] 1.3E-01 -0.02 [-0.06 , 0.02] 5.5E-01 Cdh5 -0.02 [-0.18 , 0.13] 8.2E-01 -0.01 [-0.09 , 0.08] 9.1E-01 0.01 [-0.03 , 0.04] 8.9E-01 Il6ra -0.02 [-0.13 , 0.08] 7.4E-01 0.00 [-0.06 , 0.05] 9.3E-01 0.02 [-0.01 , 0.05] 4.0E-01 Mapk8 -0.03 [-0.20 , 0.15] 8.2E-01 -0.05 [-0.13 , 0.04] 4.5E-01 0.00 [-0.03 , 0.03] 9.5E-01 Tnfsf12 -0.03 [-0.23 , 0.18] 8.4E-01 0.00 [-0.09 , 0.08] 9.6E-01 0.01 [-0.03 , 0.04] 9.2E-01 Gpr183 -0.03 [-0.31 , 0.26] 8.8E-01 -0.12 [-0.25 , 0.01] 1.9E-01 -0.06 [-0.11 , 0.00] 2.6E-01 Nfatc1 -0.03 [-0.22 , 0.16] 8.2E-01 0.00 [-0.09 , 0.10] 9.5E-01 -0.01 [-0.07 , 0.04] 8.5E-01 Fas -0.03 [-0.39 , 0.32] 9.0E-01 0.09 [-0.16 , 0.34] 6.7E-01 0.01 [-0.06 , 0.07] 9.5E-01 Pdgfrb -0.03 [-0.28 , 0.22] 8.6E-01 -0.07 [-0.24 , 0.11] 6.4E-01 0.01 [-0.06 , 0.07] 9.3E-01 516 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mapk1 -0.03 [-0.14 , 0.08] 6.6E-01 -0.01 [-0.06 , 0.04] 8.3E-01 0.00 [-0.02 , 0.02] 9.4E-01 Isg15 -0.03 [-0.49 , 0.42] 9.1E-01 0.13 [-0.08 , 0.33] 4.2E-01 0.05 [-0.05 , 0.14] 6.4E-01 Il22ra2 -0.04 [-0.55 , 0.48] 9.2E-01 -0.03 [-0.24 , 0.19] 8.9E-01 0.04 [-0.06 , 0.13] 7.4E-01 Ms4a1 -0.04 [-0.33 , 0.26] 8.5E-01 -0.06 [-0.18 , 0.06] 5.1E-01 -0.04 [-0.09 , 0.02] 4.6E-01 Cxcl13 -0.04 [-0.27 , 0.20] 8.2E-01 -0.08 [-0.20 , 0.05] 4.2E-01 -0.05 [-0.10 , 0.00] 2.7E-01 Runx3 -0.04 [-0.30 , 0.23] 8.3E-01 -0.07 [-0.18 , 0.05] 4.2E-01 -0.04 [-0.09 , 0.00] 3.1E-01 Fadd -0.04 [-0.15 , 0.08] 6.0E-01 -0.03 [-0.09 , 0.03] 5.3E-01 -0.02 [-0.04 , 0.01] 4.9E-01 Jak1 -0.04 [-0.15 , 0.06] 5.2E-01 -0.06 [-0.11 , -0.01] 1.0E-01 0.00 [-0.03 , 0.02] 9.4E-01 Yy1 -0.04 [-0.11 , 0.03] 3.3E-01 0.00 [-0.03 , 0.02] 9.2E-01 0.01 [0.00 , 0.02] 2.6E-01 Cd79b -0.04 [-0.29 , 0.20] 7.9E-01 -0.04 [-0.15 , 0.06] 6.0E-01 -0.02 [-0.07 , 0.03] 7.8E-01 Il23r -0.04 [-0.50 , 0.41] 8.8E-01 -0.12 [-0.35 , 0.12] 5.1E-01 0.05 [-0.03 , 0.13] 5.5E-01 Pin1 -0.05 [-0.24 , 0.15] 7.3E-01 -0.01 [-0.12 , 0.10] 9.2E-01 -0.01 [-0.05 , 0.04] 9.4E-01 Ifnar2 -0.05 [-0.12 , 0.02] 3.0E-01 n.d [n.d , n.d] n.d 0.02 [0.00 , 0.03] 2.0E-01 Tgfb1 -0.05 [-0.20 , 0.11] 6.5E-01 0.01 [-0.06 , 0.07] 9.2E-01 0.00 [-0.03 , 0.03] 9.9E-01 Nos2 -0.05 [-0.46 , 0.36] 8.6E-01 -0.14 [-0.34 , 0.07] 3.7E-01 -0.04 [-0.12 , 0.04] 5.7E-01 Bst1 -0.05 [-0.29 , 0.19] 7.6E-01 0.04 [-0.09 , 0.18] 6.9E-01 -0.01 [-0.05 , 0.03] 8.4E-01 Crebbp -0.05 [-0.14 , 0.04] 3.7E-01 -0.01 [-0.04 , 0.01] 5.7E-01 0.01 [-0.01 , 0.03] 7.9E-01 517 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Smad2 -0.06 [-0.18 , 0.07] 4.7E-01 -0.06 [-0.12 , 0.00] 1.7E-01 -0.01 [-0.04 , 0.02] 7.4E-01 Cd3e -0.06 [-0.42 , 0.30] 8.2E-01 -0.14 [-0.32 , 0.04] 2.7E-01 -0.05 [-0.12 , 0.02] 4.7E-01 Akt3 -0.06 [-0.22 , 0.10] 5.7E-01 -0.01 [-0.09 , 0.07] 9.1E-01 0.01 [-0.01 , 0.04] 6.7E-01 Cyld -0.06 [-0.26 , 0.14] 6.6E-01 -0.16 [-0.26 , -0.06] 3.8E-02 -0.08 [-0.13 , -0.03] 1.1E-01 Klra7 -0.06 [-0.36 , 0.25] 7.8E-01 -0.13 [-0.32 , 0.07] 3.6E-01 -0.01 [-0.04 , 0.03] 8.6E-01 Il15 -0.06 [-0.28 , 0.16] 6.9E-01 -0.04 [-0.16 , 0.08] 6.7E-01 -0.03 [-0.08 , 0.02] 5.2E-01 Cmklr1 -0.06 [-0.22 , 0.10] 5.6E-01 -0.03 [-0.11 , 0.05] 5.9E-01 -0.01 [-0.04 , 0.02] 8.0E-01 Gata3 -0.06 [-0.36 , 0.24] 7.6E-01 -0.06 [-0.20 , 0.08] 5.6E-01 -0.02 [-0.08 , 0.03] 7.1E-01 Nfatc3 -0.06 [-0.20 , 0.07] 4.7E-01 -0.03 [-0.09 , 0.04] 5.8E-01 0.00 [-0.03 , 0.03] 9.5E-01 Tlr3 -0.06 [-0.33 , 0.21] 7.4E-01 -0.12 [-0.28 , 0.05] 3.4E-01 -0.04 [-0.09 , 0.02] 4.9E-01 Mertk -0.06 [-0.20 , 0.07] 4.4E-01 -0.10 [-0.22 , 0.03] 2.9E-01 0.02 [-0.01 , 0.04] 5.3E-01 Eng -0.06 [-0.15 , 0.02] 2.4E-01 -0.04 [-0.10 , 0.02] 3.2E-01 -0.01 [-0.03 , 0.02] 8.5E-01 Itga6 -0.06 [-0.25 , 0.12] 5.9E-01 -0.01 [-0.12 , 0.09] 8.9E-01 0.01 [-0.04 , 0.06] 8.8E-01 Smad3 -0.07 [-0.20 , 0.07] 4.2E-01 -0.04 [-0.11 , 0.04] 5.1E-01 -0.01 [-0.04 , 0.03] 8.5E-01 Il16 -0.07 [-0.25 , 0.12] 5.8E-01 -0.06 [-0.16 , 0.03] 3.4E-01 -0.02 [-0.06 , 0.02] 5.9E-01 Oas3 -0.07 [-0.49 , 0.36] 8.2E-01 0.14 [-0.02 , 0.30] 2.2E-01 0.06 [-0.02 , 0.14] 4.0E-01 Lrp1 -0.07 [-0.20 , 0.07] 4.2E-01 -0.06 [-0.17 , 0.04] 4.0E-01 0.02 [-0.01 , 0.04] 5.0E-01 518 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Atg10 -0.07 [-0.26 , 0.12] 5.8E-01 -0.01 [-0.11 , 0.09] 9.1E-01 0.00 [-0.04 , 0.04] 9.5E-01 Ccr4 -0.07 [-0.40 , 0.26] 7.6E-01 -0.14 [-0.28 , 0.00] 1.7E-01 -0.08 [-0.15 , 0.00] 3.0E-01 Ikbkg -0.07 [-0.19 , 0.05] 3.6E-01 -0.04 [-0.10 , 0.02] 3.4E-01 n.d [n.d , n.d] n.d Hmgb1 -0.07 [-0.29 , 0.14] 6.0E-01 -0.03 [-0.12 , 0.07] 7.2E-01 0.00 [-0.04 , 0.04] 9.9E-01 Atg12 -0.07 [-0.23 , 0.08] 4.3E-01 -0.04 [-0.10 , 0.02] 3.7E-01 0.00 [-0.02 , 0.03] 9.1E-01 Creb1 -0.08 [-0.25 , 0.09] 4.7E-01 -0.06 [-0.13 , 0.01] 2.3E-01 -0.03 [-0.06 , 0.00] 3.0E-01 Irak2 -0.08 [-0.26 , 0.10] 4.8E-01 -0.05 [-0.13 , 0.04] 4.6E-01 -0.03 [-0.07 , 0.02] 5.3E-01 Notch1 -0.08 [-0.31 , 0.15] 5.8E-01 -0.10 [-0.20 , 0.01] 1.9E-01 -0.03 [-0.07 , 0.02] 5.3E-01 Ep300 -0.09 [-0.18 , 0.01] 1.6E-01 -0.05 [-0.08 , -0.02] 3.7E-02 -0.01 [-0.03 , 0.01] 5.5E-01 Cdk1 -0.09 [-0.54 , 0.36] 7.7E-01 0.04 [-0.23 , 0.31] 8.6E-01 0.07 [0.00 , 0.15] 2.8E-01 Map4k2 -0.09 [-0.31 , 0.13] 5.1E-01 -0.03 [-0.13 , 0.08] 7.6E-01 0.01 [-0.05 , 0.06] 9.4E-01 Ewsr1 -0.09 [-0.16 , -0.03] 3.9E-02 -0.01 [-0.05 , 0.02] 7.1E-01 0.01 [-0.01 , 0.03] 5.3E-01 Xcl1 -0.09 [-0.46 , 0.27] 7.0E-01 -0.15 [-0.31 , 0.01] 2.0E-01 -0.06 [-0.12 , -0.01] 2.3E-01 Klra4 -0.09 [-0.43 , 0.25] 6.8E-01 -0.14 [-0.26 , -0.01] 1.4E-01 -0.01 [-0.06 , 0.04] 8.7E-01 Tollip -0.10 [-0.18 , -0.01] 9.1E-02 -0.02 [-0.07 , 0.04] 7.1E-01 0.02 [0.00 , 0.04] 2.9E-01 Bcl2 -0.10 [-0.22 , 0.02] 2.1E-01 -0.02 [-0.09 , 0.06] 8.0E-01 n.d [n.d , n.d] n.d Ccl11 -0.10 [-0.45 , 0.25] 6.7E-01 -0.01 [-0.14 , 0.13] 9.6E-01 0.04 [-0.01 , 0.09] 3.9E-01 519 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Nfatc2 -0.11 [-0.35 , 0.14] 4.8E-01 -0.03 [-0.15 , 0.08] 6.9E-01 -0.04 [-0.09 , 0.01] 4.7E-01 Dpp4 -0.11 [-0.35 , 0.14] 4.8E-01 -0.03 [-0.15 , 0.09] 7.8E-01 -0.01 [-0.06 , 0.04] 8.6E-01 Il7 -0.11 [-0.46 , 0.24] 6.3E-01 -0.01 [-0.17 , 0.15] 9.4E-01 0.03 [-0.03 , 0.10] 6.0E-01 Mapk14 -0.11 [-0.19 , -0.03] 4.2E-02 -0.01 [-0.06 , 0.04] 8.9E-01 0.00 [-0.02 , 0.03] 9.3E-01 F2rl1 -0.11 [-0.59 , 0.37] 7.3E-01 -0.12 [-0.42 , 0.18] 6.1E-01 -0.02 [-0.13 , 0.08] 8.8E-01 Atg16l1 -0.11 [-0.18 , -0.05] 1.0E-02 -0.05 [-0.09 , -0.01] 6.1E-02 -0.01 [-0.03 , 0.01] 6.0E-01 Atf2 -0.12 [-0.23 , 0.00] 1.1E-01 -0.02 [-0.07 , 0.03] 6.1E-01 0.01 [-0.01 , 0.03] 7.4E-01 Dock9 -0.12 [-0.32 , 0.08] 3.4E-01 -0.10 [-0.21 , 0.02] 2.3E-01 -0.02 [-0.07 , 0.02] 5.7E-01 Zfp13 -0.12 [-0.49 , 0.25] 6.2E-01 0.03 [-0.20 , 0.25] 8.9E-01 -0.02 [-0.08 , 0.05] 8.3E-01 Tnfsf13 -0.12 [-0.46 , 0.22] 5.8E-01 -0.06 [-0.22 , 0.11] 6.7E-01 -0.02 [-0.07 , 0.02] 6.1E-01 Jak2 -0.12 [-0.23 , -0.01] 9.7E-02 -0.01 [-0.07 , 0.06] 9.2E-01 0.03 [0.00 , 0.05] 2.3E-01 Spp1 -0.12 [-0.45 , 0.21] 5.7E-01 -0.26 [-0.44 , -0.09] 4.5E-02 -0.14 [-0.20 , -0.07] 5.4E-02 Klrk1 -0.12 [-0.37 , 0.13] 4.3E-01 -0.12 [-0.24 , 0.00] 1.8E-01 -0.02 [-0.06 , 0.02] 7.1E-01 Ets1 -0.12 [-0.37 , 0.12] 4.2E-01 -0.08 [-0.19 , 0.03] 3.2E-01 -0.01 [-0.06 , 0.04] 8.9E-01 Cfp -0.13 [-0.31 , 0.06] 2.7E-01 -0.06 [-0.16 , 0.04] 4.2E-01 0.01 [-0.02 , 0.05] 7.6E-01 Vegfc -0.13 [-0.32 , 0.07] 3.0E-01 -0.03 [-0.12 , 0.06] 6.5E-01 -0.02 [-0.05 , 0.01] 4.4E-01 Tie1 -0.13 [-0.30 , 0.04] 2.2E-01 -0.08 [-0.16 , 0.01] 2.0E-01 -0.02 [-0.06 , 0.03] 7.1E-01 520 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd3eap -0.13 [-0.41 , 0.14] 4.3E-01 -0.12 [-0.27 , 0.04] 2.8E-01 -0.06 [-0.10 , -0.02] 9.9E-02 Pax5 -0.14 [-0.39 , 0.11] 3.8E-01 -0.09 [-0.20 , 0.01] 2.2E-01 -0.06 [-0.11 , -0.01] 1.9E-01 Cd47 -0.14 [-0.35 , 0.07] 2.9E-01 0.00 [-0.09 , 0.10] 9.5E-01 0.05 [0.01 , 0.08] 1.6E-01 Cklf -0.15 [-0.30 , 0.01] 1.4E-01 -0.09 [-0.16 , -0.02] 9.4E-02 -0.04 [-0.07 , -0.01] 1.6E-01 Ccr2 -0.15 [-0.44 , 0.15] 4.2E-01 0.08 [-0.10 , 0.25] 5.7E-01 0.00 [-0.06 , 0.05] 9.7E-01 Atf1 -0.15 [-0.31 , 0.01] 1.5E-01 -0.04 [-0.12 , 0.04] 5.0E-01 0.03 [0.00 , 0.06] 3.1E-01 Kdr -0.15 [-0.31 , 0.00] 1.2E-01 -0.02 [-0.12 , 0.08] 7.9E-01 -0.01 [-0.05 , 0.04] 9.3E-01 Jam3 -0.15 [-0.33 , 0.02] 1.7E-01 -0.07 [-0.18 , 0.03] 3.4E-01 -0.03 [-0.07 , 0.00] 3.0E-01 Tlr5 -0.16 [-0.40 , 0.08] 3.0E-01 -0.08 [-0.22 , 0.06] 4.4E-01 -0.01 [-0.06 , 0.04] 9.4E-01 Ikzf1 -0.17 [-0.28 , -0.05] 3.0E-02 -0.12 [-0.17 , -0.07] 4.7E-03 -0.06 [-0.08 , -0.03] 3.4E-02 Egfr -0.17 [-0.41 , 0.07] 2.7E-01 -0.07 [-0.19 , 0.06] 4.7E-01 n.d [n.d , n.d] n.d Fcer2a -0.17 [-0.66 , 0.32] 5.9E-01 -0.09 [-0.29 , 0.12] 5.9E-01 -0.05 [-0.13 , 0.04] 5.8E-01 Emr1 -0.17 [-0.39 , 0.05] 2.2E-01 -0.08 [-0.18 , 0.02] 2.8E-01 -0.01 [-0.04 , 0.03] 9.1E-01 Timd4 -0.17 [-0.39 , 0.04] 2.0E-01 -0.07 [-0.19 , 0.04] 3.6E-01 -0.02 [-0.05 , 0.02] 6.4E-01 Cd55 -0.17 [-0.47 , 0.12] 3.4E-01 -0.13 [-0.27 , 0.02] 2.1E-01 -0.01 [-0.07 , 0.05] 9.4E-01 C6 -0.18 [-0.51 , 0.16] 4.0E-01 -0.09 [-0.28 , 0.11] 5.6E-01 0.06 [0.00 , 0.11] 2.7E-01 Igf1r -0.18 [-0.33 , -0.03] 6.5E-02 0.01 [-0.07 , 0.09] 8.7E-01 0.02 [-0.01 , 0.04] 5.4E-01 521 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il1r1 -0.18 [-0.27 , -0.09] 4.1E-03 -0.08 [-0.13 , -0.03] 3.5E-02 0.00 [-0.02 , 0.03] 9.1E-01 Mapk3 -0.18 [-0.32 , -0.04] 4.9E-02 0.00 [-0.08 , 0.07] 9.5E-01 0.01 [-0.02 , 0.03] 8.6E-01 Lbp -0.18 [-0.35 , -0.01] 9.1E-02 -0.03 [-0.12 , 0.07] 7.3E-01 0.03 [-0.01 , 0.07] 4.7E-01 Cxcr4 -0.18 [-0.42 , 0.05] 2.2E-01 -0.05 [-0.14 , 0.04] 5.0E-01 -0.03 [-0.07 , 0.00] 3.2E-01 Trp53 -0.19 [-0.35 , -0.03] 7.3E-02 -0.07 [-0.14 , 0.00] 1.4E-01 -0.04 [-0.08 , -0.01] 1.6E-01 Ccr1 -0.19 [-0.39 , 0.01] 1.5E-01 0.02 [-0.14 , 0.18] 8.8E-01 -0.02 [-0.07 , 0.04] 8.5E-01 Thbd -0.19 [-0.36 , -0.02] 7.9E-02 -0.09 [-0.16 , -0.02] 8.3E-02 -0.05 [-0.08 , -0.01] 1.1E-01 Cd34 -0.19 [-0.41 , 0.03] 1.7E-01 -0.05 [-0.19 , 0.08] 6.0E-01 0.00 [-0.05 , 0.04] 9.5E-01 Vcam1 -0.20 [-0.41 , 0.01] 1.3E-01 -0.11 [-0.27 , 0.04] 2.9E-01 0.03 [-0.02 , 0.07] 5.5E-01 Abl1 -0.20 [-0.31 , -0.09] 8.2E-03 -0.08 [-0.14 , -0.01] 1.0E-01 -0.02 [-0.06 , 0.01] 4.4E-01 Dll4 -0.20 [-0.51 , 0.10] 2.9E-01 -0.12 [-0.28 , 0.03] 2.6E-01 -0.02 [-0.08 , 0.04] 7.4E-01 Tab1 -0.21 [-0.35 , -0.06] 3.0E-02 -0.04 [-0.10 , 0.02] 3.9E-01 0.03 [0.00 , 0.06] 2.4E-01 Cd160 -0.21 [-0.52 , 0.10] 2.8E-01 -0.16 [-0.33 , 0.00] 1.7E-01 -0.04 [-0.09 , 0.00] 3.1E-01 Cd3d -0.22 [-0.59 , 0.16] 3.6E-01 -0.20 [-0.39 , -0.01] 1.4E-01 -0.06 [-0.14 , 0.02] 3.9E-01 Il11ra1 -0.22 [-0.59 , 0.15] 3.5E-01 -0.12 [-0.26 , 0.03] 2.5E-01 -0.01 [-0.06 , 0.05] 9.4E-01 Mcam -0.22 [-0.45 , 0.02] 1.5E-01 -0.13 [-0.26 , 0.00] 1.5E-01 -0.03 [-0.07 , 0.01] 4.3E-01 Hspb2 -0.22 [-0.50 , 0.06] 2.3E-01 -0.15 [-0.31 , 0.00] 1.7E-01 -0.01 [-0.06 , 0.04] 8.9E-01 522 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccr7 -0.22 [-0.57 , 0.14] 3.3E-01 -0.19 [-0.37 , -0.01] 1.5E-01 -0.03 [-0.11 , 0.04] 7.1E-01 Cd40lg -0.22 [-0.62 , 0.18] 3.8E-01 -0.17 [-0.38 , 0.04] 2.4E-01 -0.06 [-0.15 , 0.02] 4.4E-01 Rps6 -0.22 [-0.38 , -0.06] 3.9E-02 -0.14 [-0.22 , -0.07] 1.4E-02 -0.05 [-0.08 , -0.03] 4.5E-02 Il6st -0.22 [-0.39 , -0.06] 4.4E-02 -0.08 [-0.17 , 0.00] 1.9E-01 -0.01 [-0.06 , 0.04] 9.0E-01 Tnfsf4 -0.22 [-0.68 , 0.24] 4.4E-01 -0.04 [-0.25 , 0.17] 8.2E-01 -0.06 [-0.17 , 0.05] 5.5E-01 Maf -0.22 [-0.40 , -0.04] 5.8E-02 -0.11 [-0.17 , -0.05] 2.1E-02 -0.02 [-0.05 , 0.02] 6.1E-01 Tnfsf14 -0.23 [-0.50 , 0.04] 1.9E-01 -0.06 [-0.20 , 0.08] 5.7E-01 0.00 [-0.05 , 0.05] 9.7E-01 Cd9 -0.23 [-0.50 , 0.04] 1.9E-01 -0.03 [-0.18 , 0.13] 8.3E-01 0.02 [-0.04 , 0.08] 8.4E-01 Itga2b -0.24 [-0.63 , 0.16] 3.4E-01 0.02 [-0.21 , 0.24] 9.3E-01 0.05 [-0.03 , 0.13] 5.5E-01 Fn1 -0.24 [-0.48 , 0.01] 1.3E-01 -0.10 [-0.23 , 0.03] 2.7E-01 -0.03 [-0.08 , 0.02] 5.1E-01 Lrrn3 -0.24 [-0.56 , 0.08] 2.4E-01 -0.07 [-0.25 , 0.10] 5.9E-01 0.02 [-0.04 , 0.08] 7.6E-01 Tek -0.24 [-0.41 , -0.08] 2.9E-02 -0.09 [-0.19 , 0.01] 2.1E-01 -0.03 [-0.07 , 0.01] 4.9E-01 Itga2 -0.24 [-0.45 , -0.04] 6.6E-02 -0.05 [-0.19 , 0.09] 6.4E-01 0.00 [-0.05 , 0.06] 9.9E-01 Axl -0.25 [-0.38 , -0.11] 9.3E-03 -0.14 [-0.24 , -0.03] 7.4E-02 -0.01 [-0.04 , 0.02] 7.4E-01 Muc1 -0.25 [-0.65 , 0.16] 3.3E-01 n.d [n.d , n.d] n.d -0.06 [-0.15 , 0.03] 5.0E-01 Myc -0.26 [-0.46 , -0.06] 4.8E-02 -0.11 [-0.20 , -0.01] 1.1E-01 -0.07 [-0.11 , -0.03] 7.8E-02 Tcf7 -0.26 [-0.71 , 0.19] 3.6E-01 -0.25 [-0.45 , -0.04] 9.7E-02 -0.04 [-0.13 , 0.06] 7.4E-01 523 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd27 -0.27 [-0.68 , 0.15] 3.1E-01 -0.18 [-0.37 , 0.01] 1.8E-01 -0.05 [-0.12 , 0.03] 5.3E-01 Cxcr5 -0.27 [-0.56 , 0.01] 1.3E-01 -0.14 [-0.25 , -0.02] 1.1E-01 -0.06 [-0.12 , 0.00] 2.3E-01 Klrc2 -0.28 [-0.58 , 0.03] 1.6E-01 -0.22 [-0.38 , -0.07] 5.4E-02 0.00 [-0.05 , 0.04] 9.7E-01 Trem1 -0.28 [-0.77 , 0.21] 3.6E-01 0.12 [-0.02 , 0.27] 2.3E-01 -0.04 [-0.13 , 0.04] 6.4E-01 Blk -0.29 [-0.54 , -0.03] 8.0E-02 -0.06 [-0.19 , 0.06] 4.9E-01 -0.02 [-0.07 , 0.04] 8.5E-01 Itgb3 -0.29 [-0.66 , 0.08] 2.3E-01 -0.02 [-0.23 , 0.20] 9.2E-01 0.03 [-0.05 , 0.10] 7.6E-01 Il1a -0.29 [-0.58 , 0.00] 1.3E-01 -0.09 [-0.27 , 0.08] 4.8E-01 0.00 [-0.06 , 0.06] 9.9E-01 Serpinb2 -0.29 [-0.72 , 0.13] 2.8E-01 -0.05 [-0.29 , 0.20] 8.3E-01 0.02 [-0.07 , 0.11] 8.7E-01 Prf1 -0.30 [-0.64 , 0.05] 1.8E-01 -0.21 [-0.37 , -0.05] 8.3E-02 -0.04 [-0.10 , 0.02] 4.9E-01 Thbs1 -0.30 [-0.69 , 0.10] 2.4E-01 -0.02 [-0.25 , 0.21] 9.1E-01 0.01 [-0.08 , 0.09] 9.7E-01 Anp32b -0.32 [-0.56 , -0.08] 4.4E-02 -0.04 [-0.15 , 0.07] 6.1E-01 0.01 [-0.03 , 0.05] 8.6E-01 Mrc1 -0.32 [-0.56 , -0.08] 4.4E-02 -0.12 [-0.29 , 0.04] 3.0E-01 0.05 [0.00 , 0.09] 2.4E-01 Flt3l -0.32 [-0.57 , -0.07] 5.0E-02 -0.12 [-0.25 , 0.01] 1.9E-01 -0.02 [-0.09 , 0.04] 7.8E-01 Il18rap -0.32 [-0.61 , -0.03] 8.7E-02 -0.10 [-0.23 , 0.03] 3.0E-01 0.01 [-0.04 , 0.05] 9.3E-01 Vwf -0.32 [-0.70 , 0.05] 1.8E-01 -0.03 [-0.25 , 0.19] 8.8E-01 0.03 [-0.05 , 0.10] 7.9E-01 Ltk -0.33 [-0.66 , 0.01] 1.3E-01 -0.10 [-0.25 , 0.05] 3.4E-01 -0.06 [-0.11 , 0.00] 2.7E-01 Klrg1 -0.33 [-0.62 , -0.04] 8.2E-02 -0.02 [-0.21 , 0.17] 9.0E-01 0.00 [-0.05 , 0.05] 9.6E-01 524 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value F13a1 -0.33 [-0.92 , 0.26] 3.7E-01 0.10 [-0.26 , 0.45] 7.3E-01 0.06 [-0.06 , 0.17] 6.2E-01 Ccrl2 -0.33 [-0.65 , -0.02] 1.0E-01 -0.07 [-0.16 , 0.02] 2.7E-01 0.01 [-0.02 , 0.05] 7.5E-01 Ppbp -0.33 [-0.77 , 0.10] 2.3E-01 -0.03 [-0.28 , 0.21] 8.8E-01 0.03 [-0.06 , 0.12] 8.1E-01 Angpt1 -0.34 [-0.78 , 0.10] 2.3E-01 -0.01 [-0.24 , 0.23] 9.7E-01 0.03 [-0.06 , 0.13] 7.6E-01 Csf1 -0.34 [-0.61 , -0.07] 5.6E-02 -0.19 [-0.32 , -0.06] 5.0E-02 -0.06 [-0.11 , 0.00] 2.3E-01 Clec5a -0.34 [-0.81 , 0.13] 2.5E-01 0.04 [-0.25 , 0.33] 8.8E-01 0.00 [-0.09 , 0.10] 9.9E-01 Il18r1 -0.35 [-0.57 , -0.12] 2.2E-02 -0.21 [-0.32 , -0.09] 2.2E-02 -0.04 [-0.09 , 0.02] 5.3E-01 Kit -0.35 [-0.53 , -0.18] 4.2E-03 -0.08 [-0.22 , 0.05] 4.2E-01 -0.03 [-0.08 , 0.02] 5.9E-01 Cd7 -0.35 [-0.76 , 0.05] 1.7E-01 -0.21 [-0.38 , -0.04] 9.7E-02 -0.05 [-0.11 , 0.00] 2.7E-01 Txnip -0.37 [-0.48 , -0.26] 2.0E-04 -0.19 [-0.25 , -0.13] 1.6E-03 -0.05 [-0.09 , -0.02] 1.1E-01 Cmpk2 -0.37 [-0.73 , -0.01] 1.1E-01 n.d [n.d , n.d] n.d 0.03 [-0.04 , 0.10] 6.7E-01 Ccl19 -0.37 [-0.64 , -0.10] 3.8E-02 -0.15 [-0.27 , -0.03] 8.8E-02 -0.05 [-0.10 , -0.01] 1.9E-01 Klrd1 -0.37 [-0.70 , -0.04] 8.3E-02 -0.23 [-0.37 , -0.09] 3.0E-02 -0.03 [-0.09 , 0.03] 5.8E-01 Rorc -0.37 [-0.69 , -0.06] 7.0E-02 -0.11 [-0.19 , -0.02] 9.7E-02 0.04 [-0.01 , 0.09] 4.0E-01 Angpt2 -0.37 [-0.63 , -0.12] 2.8E-02 -0.21 [-0.37 , -0.04] 9.1E-02 -0.05 [-0.10 , -0.01] 1.9E-01 Nfatc4 -0.37 [-0.68 , -0.07] 5.9E-02 -0.16 [-0.34 , 0.02] 2.1E-01 0.00 [-0.07 , 0.08] 9.8E-01 Hcst -0.39 [-0.68 , -0.10] 4.3E-02 -0.19 [-0.33 , -0.05] 7.1E-02 -0.05 [-0.12 , 0.01] 3.8E-01 525 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Vegfa -0.40 [-0.65 , -0.15] 1.8E-02 -0.17 [-0.30 , -0.04] 6.8E-02 -0.05 [-0.12 , 0.01] 3.9E-01 Mavs -0.40 [-0.54 , -0.26] 5.6E-04 -0.17 [-0.27 , -0.08] 1.8E-02 -0.04 [-0.07 , -0.01] 2.1E-01 Smpd3 -0.41 [-0.72 , -0.09] 4.8E-02 -0.10 [-0.22 , 0.02] 2.2E-01 -0.05 [-0.12 , 0.01] 3.8E-01 Erbb2 -0.41 [-0.59 , -0.23] 2.3E-03 -0.16 [-0.29 , -0.02] 1.1E-01 -0.01 [-0.04 , 0.03] 8.9E-01 Hras -0.42 [-0.81 , -0.02] 1.0E-01 -0.10 [-0.32 , 0.12] 5.7E-01 -0.07 [-0.14 , 0.00] 3.0E-01 Tpsab1 -0.42 [-0.73 , -0.10] 4.7E-02 -0.11 [-0.30 , 0.08] 4.4E-01 -0.01 [-0.09 , 0.06] 8.9E-01 Mmp9 -0.43 [-0.94 , 0.09] 1.9E-01 0.06 [-0.24 , 0.37] 8.0E-01 0.02 [-0.08 , 0.13] 8.8E-01 Itgb4 -0.43 [-0.78 , -0.09] 5.5E-02 -0.10 [-0.25 , 0.05] 3.5E-01 -0.07 [-0.13 , -0.02] 1.7E-01 Il12rb2 -0.44 [-0.65 , -0.22] 5.3E-03 -0.24 [-0.36 , -0.13] 1.2E-02 -0.06 [-0.11 , -0.02] 1.2E-01 Il5ra -0.44 [-0.83 , -0.05] 8.0E-02 0.07 [-0.15 , 0.29] 6.7E-01 0.10 [0.01 , 0.20] 2.3E-01 Cd96 -0.45 [-0.83 , -0.07] 6.6E-02 -0.26 [-0.43 , -0.09] 4.0E-02 -0.06 [-0.12 , 0.01] 3.1E-01 Ctsw -0.47 [-0.77 , -0.16] 2.3E-02 -0.26 [-0.39 , -0.13] 1.3E-02 -0.07 [-0.13 , -0.01] 1.9E-01 Gzma -0.47 [-0.82 , -0.11] 4.6E-02 -0.33 [-0.52 , -0.15] 2.0E-02 -0.12 [-0.19 , -0.06] 6.5E-02 Lyve1 -0.47 [-0.93 , -0.02] 1.1E-01 0.01 [-0.18 , 0.20] 9.4E-01 0.01 [-0.08 , 0.11] 9.3E-01 Tgfb2 -0.48 [-0.77 , -0.19] 1.5E-02 -0.09 [-0.29 , 0.11] 5.5E-01 0.01 [-0.07 , 0.08] 9.5E-01 Mill2 -0.50 [-0.71 , -0.28] 2.1E-03 -0.18 [-0.32 , -0.05] 7.0E-02 -0.03 [-0.06 , -0.01] 1.6E-01 Il4 -0.51 [-0.94 , -0.08] 6.7E-02 -0.19 [-0.44 , 0.06] 3.0E-01 -0.02 [-0.10 , 0.06] 8.4E-01 526 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Igll1 -0.52 [-1.30 , 0.26] 2.9E-01 0.46 [-0.06 , 0.99] 2.1E-01 0.18 [0.02 , 0.33] 2.1E-01 Il17rb -0.53 [-0.92 , -0.13] 4.4E-02 0.02 [-0.15 , 0.18] 9.2E-01 0.04 [-0.02 , 0.09] 5.2E-01 Ncr1 -0.53 [-0.84 , -0.22] 1.3E-02 -0.28 [-0.39 , -0.17] 4.7E-03 -0.05 [-0.10 , 0.00] 2.8E-01 Cd207 -0.53 [-0.95 , -0.11] 5.2E-02 -0.28 [-0.51 , -0.05] 9.7E-02 -0.06 [-0.15 , 0.04] 5.3E-01 Marco -0.55 [-0.89 , -0.20] 2.0E-02 -0.34 [-0.54 , -0.14] 2.6E-02 -0.04 [-0.11 , 0.03] 5.3E-01 Cd1d2 -0.55 [-0.88 , -0.21] 1.7E-02 -0.22 [-0.39 , -0.05] 7.8E-02 -0.03 [-0.10 , 0.03] 6.3E-01 Itk -0.56 [-1.06 , -0.05] 8.8E-02 -0.37 [-0.59 , -0.15] 2.7E-02 -0.07 [-0.18 , 0.04] 5.0E-01 Anxa1 -0.56 [-1.22 , 0.10] 1.9E-01 0.03 [-0.38 , 0.44] 9.2E-01 0.04 [-0.09 , 0.17] 8.0E-01 Ccnd3 -0.56 [-0.83 , -0.29] 4.2E-03 -0.12 [-0.26 , 0.02] 2.3E-01 -0.01 [-0.07 , 0.04] 8.7E-01 Thy1 -0.57 [-1.01 , -0.12] 5.3E-02 -0.35 [-0.54 , -0.16] 1.8E-02 -0.08 [-0.17 , 0.01] 3.1E-01 Il1rl2 -0.57 [-0.72 , -0.42] 8.3E-05 -0.11 [-0.22 , 0.01] 1.9E-01 0.00 [-0.04 , 0.04] 9.9E-01 Ccl21a -0.58 [-0.90 , -0.26] 9.1E-03 -0.10 [-0.30 , 0.10] 5.2E-01 0.01 [-0.03 , 0.05] 8.5E-01 Fasl -0.58 [-0.89 , -0.27] 8.0E-03 -0.27 [-0.42 , -0.12] 2.3E-02 -0.07 [-0.12 , -0.01] 1.9E-01 Ccr3 -0.59 [-0.98 , -0.21] 2.2E-02 -0.21 [-0.41 , -0.02] 1.3E-01 -0.01 [-0.06 , 0.04] 9.0E-01 Cd36 -0.60 [-0.88 , -0.31] 3.8E-03 -0.14 [-0.27 , 0.00] 1.8E-01 0.02 [-0.03 , 0.07] 7.2E-01 Cd1d1 -0.60 [-0.93 , -0.28] 7.7E-03 -0.24 [-0.41 , -0.08] 4.6E-02 -0.07 [-0.12 , -0.02] 1.6E-01 Birc5 -0.61 [-1.10 , -0.11] 6.0E-02 -0.05 [-0.26 , 0.16] 7.6E-01 0.05 [-0.04 , 0.14] 6.1E-01 527 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfrsf14 -0.61 [-0.92 , -0.30] 5.5E-03 -0.15 [-0.28 , -0.01] 1.5E-01 0.00 [-0.06 , 0.06] 9.9E-01 Txk -0.61 [-1.05 , -0.18] 3.4E-02 -0.33 [-0.53 , -0.12] 3.5E-02 -0.07 [-0.15 , 0.01] 3.4E-01 Klra6 -0.64 [-1.20 , -0.08] 8.0E-02 -0.25 [-0.50 , 0.00] 1.6E-01 -0.07 [-0.16 , 0.02] 4.0E-01 Klrc1 -0.64 [-0.91 , -0.38] 1.5E-03 -0.33 [-0.49 , -0.18] 1.1E-02 -0.06 [-0.11 , -0.01] 1.6E-01 Ncam1 -0.65 [-1.06 , -0.23] 2.1E-02 -0.17 [-0.42 , 0.08] 3.4E-01 -0.01 [-0.07 , 0.05] 9.0E-01 Ccr9 -0.65 [-1.02 , -0.28] 1.2E-02 -0.28 [-0.46 , -0.10] 3.8E-02 -0.05 [-0.12 , 0.03] 5.2E-01 Cxcl12 -0.68 [-0.94 , -0.41] 1.1E-03 -0.31 [-0.44 , -0.17] 7.7E-03 -0.08 [-0.13 , -0.03] 9.7E-02 Cd163 -0.69 [-1.01 , -0.37] 3.1E-03 -0.19 [-0.40 , 0.02] 2.0E-01 0.07 [0.01 , 0.13] 2.2E-01 Il7r -0.72 [-1.09 , -0.36] 5.1E-03 -0.29 [-0.47 , -0.10] 3.8E-02 0.01 [-0.08 , 0.09] 9.5E-01 Cxcr6 -0.73 [-1.08 , -0.37] 4.6E-03 -0.33 [-0.51 , -0.15] 1.8E-02 -0.11 [-0.19 , -0.04] 1.1E-01 Cr2 -0.73 [-1.14 , -0.31] 1.2E-02 -0.28 [-0.47 , -0.09] 4.8E-02 -0.12 [-0.19 , -0.05] 7.8E-02 Cd8a -0.74 [-1.31 , -0.16] 5.1E-02 -0.52 [-0.79 , -0.26] 1.4E-02 -0.10 [-0.22 , 0.01] 3.3E-01 Mst1r -0.75 [-1.18 , -0.33] 1.0E-02 -0.27 [-0.54 , 0.00] 1.6E-01 -0.13 [-0.25 , 0.00] 2.8E-01 Prg2 -0.78 [-1.75 , 0.19] 2.1E-01 0.08 [-0.54 , 0.71] 8.8E-01 0.03 [-0.15 , 0.21] 8.9E-01 Cma1 -0.78 [-1.22 , -0.34] 1.1E-02 -0.39 [-0.56 , -0.23] 5.5E-03 -0.10 [-0.17 , -0.03] 1.1E-01 Pnma1 -0.82 [-1.25 , -0.38] 7.6E-03 -0.34 [-0.53 , -0.14] 2.6E-02 -0.05 [-0.13 , 0.03] 5.5E-01 Isg20 -0.82 [-1.35 , -0.29] 2.2E-02 -0.12 [-0.36 , 0.12] 5.1E-01 0.05 [-0.04 , 0.14] 5.6E-01 528 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Klrb1c -0.89 [-1.20 , -0.58] 5.4E-04 -0.40 [-0.54 , -0.25] 2.6E-03 -0.10 [-0.16 , -0.04] 9.1E-02 Msln -0.90 [-1.79 , -0.01] 1.2E-01 -0.15 [-0.44 , 0.13] 4.8E-01 0.00 [-0.07 , 0.06] 9.7E-01 Cd209e -0.94 [-1.56 , -0.32] 2.4E-02 -0.34 [-0.56 , -0.12] 3.8E-02 -0.12 [-0.21 , -0.02] 1.9E-01 Bcl2l1 -0.96 [-1.39 , -0.52] 2.9E-03 -0.07 [-0.29 , 0.14] 6.7E-01 0.07 [0.00 , 0.15] 3.0E-01 Il22ra1 -1.00 [-1.47 , -0.52] 3.9E-03 -0.30 [-0.50 , -0.10] 4.6E-02 -0.01 [-0.09 , 0.07] 9.3E-01 S100a8 -1.03 [-1.93 , -0.13] 8.0E-02 -0.03 [-0.59 , 0.52] 9.4E-01 0.06 [-0.11 , 0.23] 7.6E-01 Mpo -1.07 [-1.90 , -0.24] 5.0E-02 -0.07 [-0.60 , 0.47] 8.9E-01 0.04 [-0.11 , 0.20] 8.4E-01 Lcn2 -1.12 [-2.00 , -0.24] 5.1E-02 -0.04 [-0.59 , 0.50] 9.2E-01 0.06 [-0.11 , 0.22] 7.6E-01 Cd8b1 -1.14 [-1.85 , -0.44] 1.8E-02 -0.67 [-1.00 , -0.33] 1.3E-02 -0.15 [-0.30 , 0.01] 3.0E-01 Elane -1.17 [-2.05 , -0.30] 4.3E-02 -0.05 [-0.59 , 0.50] 9.2E-01 0.04 [-0.12 , 0.20] 8.5E-01 Cd59b -1.18 [-1.65 , -0.71] 1.3E-03 -0.25 [-0.43 , -0.07] 6.2E-02 -0.02 [-0.11 , 0.06] 8.3E-01 Camp -1.19 [-2.10 , -0.27] 4.9E-02 -0.08 [-0.66 , 0.50] 8.8E-01 0.06 [-0.11 , 0.23] 7.9E-01 Ltf -1.20 [-1.81 , -0.59] 5.6E-03 -0.14 [-0.60 , 0.31] 6.9E-01 -0.04 [-0.15 , 0.07] 7.8E-01 Ctsg -1.22 [-2.10 , -0.35] 3.6E-02 -0.07 [-0.63 , 0.49] 8.9E-01 0.04 [-0.13 , 0.21] 8.5E-01 Il1rl1 -1.33 [-1.97 , -0.68] 4.2E-03 -0.32 [-0.57 , -0.07] 8.1E-02 -0.08 [-0.19 , 0.03] 4.4E-01 Tal1 -1.40 [-2.04 , -0.75] 3.1E-03 -0.22 [-0.49 , 0.05] 2.5E-01 0.02 [-0.08 , 0.12] 8.9E-01 Ccl24 -1.52 [-2.13 , -0.91] 1.3E-03 -0.51 [-0.83 , -0.18] 3.8E-02 -0.10 [-0.23 , 0.02] 3.6E-01 529 Table 35 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in spleen at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tfrc -1.57 [-2.33 , -0.81] 4.3E-03 -0.26 [-0.54 , 0.02] 2.0E-01 0.01 [-0.10 , 0.12] 9.4E-01 Rsad2 -1.69 [-2.57 , -0.81] 6.7E-03 -0.05 [-0.33 , 0.22] 8.2E-01 0.10 [-0.01 , 0.20] 3.1E-01 Icam4 -2.14 [-3.12 , -1.16] 3.0E-03 -0.30 [-0.63 , 0.03] 2.0E-01 0.02 [-0.10 , 0.14] 9.1E-01 Cxcl15 -2.66 [-3.78 , -1.54] 1.7E-03 -1.29 [-1.76 , -0.81] 2.8E-03 -0.61 [-0.80 , -0.41] 6.1E-03 530 Table 36. Complete mRNA transcript profile in kidney at 13 wk post final exposure in cSiO 2-exposed NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log2(fold change) relative to VEH + CON mice. Expression data are included for all mRNA transcripts that were above background threshold. Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Fpr2 3.76 [2.83 , 4.68] 5.1E-04 1.36 [0.89 , 1.84] 1.5E-02 0.40 [0.17 , 0.64] 7.6E-02 Itgam 3.22 [2.13 , 4.31] 9.4E-04 0.97 [0.37 , 1.57] 4.5E-02 0.20 [0.01 , 0.39] 2.7E-01 Fcgr4 3.13 [2.16 , 4.09] 9.4E-04 0.94 [0.44 , 1.44] 3.3E-02 0.22 [0.06 , 0.38] 1.5E-01 Lcn2 3.04 [1.07 , 5.02] 1.9E-02 0.79 [-0.09 , 1.67] 2.1E-01 0.00 [-0.08 , 0.09] 9.5E-01 Ccl8 3.01 [1.03 , 4.99] 2.0E-02 0.08 [-0.87 , 1.03] 9.1E-01 -0.16 [-0.56 , 0.23] 6.6E-01 Ccl12 2.97 [2.05 , 3.88] 9.4E-04 0.87 [0.43 , 1.31] 3.3E-02 0.10 [-0.07 , 0.28] 5.2E-01 Clec4a2 2.95 [1.99 , 3.91] 9.4E-04 0.92 [0.40 , 1.43] 3.5E-02 0.17 [0.04 , 0.31] 1.8E-01 Fcgr1 2.78 [1.91 , 3.64] 9.4E-04 0.81 [0.40 , 1.21] 3.3E-02 0.12 [0.03 , 0.20] 1.4E-01 C3ar1 2.78 [1.89 , 3.66] 9.4E-04 0.89 [0.49 , 1.30] 3.0E-02 0.19 [0.11 , 0.28] 2.5E-02 Ccr5 2.77 [1.47 , 4.08] 4.2E-03 0.94 [0.35 , 1.53] 4.7E-02 0.15 [-0.07 , 0.38] 4.6E-01 Tlr8 2.63 [1.73 , 3.54] 9.4E-04 0.80 [0.39 , 1.22] 3.3E-02 0.15 [0.04 , 0.25] 1.4E-01 Ccl2 2.60 [1.74 , 3.45] 9.4E-04 0.88 [0.48 , 1.28] 3.0E-02 0.15 [0.00 , 0.31] 2.8E-01 Il1b 2.59 [1.93 , 3.26] 5.1E-04 0.62 [0.31 , 0.93] 3.3E-02 0.19 [0.07 , 0.32] 1.2E-01 531 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Sele 2.57 [1.94 , 3.20] 5.1E-04 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Klra2 2.57 [1.75 , 3.38] 9.4E-04 0.69 [0.29 , 1.08] 3.6E-02 0.14 [0.04 , 0.23] 1.3E-01 Ccl9 2.53 [1.66 , 3.41] 9.4E-04 0.80 [0.34 , 1.26] 3.6E-02 0.19 [0.06 , 0.32] 1.4E-01 Ccl5 2.49 [1.36 , 3.62] 3.5E-03 0.53 [0.01 , 1.04] 1.6E-01 0.04 [-0.09 , 0.16] 7.7E-01 Irf7 2.47 [1.58 , 3.36] 1.1E-03 0.89 [0.47 , 1.31] 3.0E-02 0.17 [0.03 , 0.30] 1.9E-01 Ccl3 2.45 [1.60 , 3.29] 9.4E-04 0.82 [0.39 , 1.24] 3.3E-02 0.27 [0.13 , 0.40] 4.9E-02 Ctss 2.44 [1.55 , 3.33] 1.1E-03 0.64 [0.24 , 1.05] 4.7E-02 0.08 [-0.01 , 0.17] 3.5E-01 Hck 2.43 [1.78 , 3.07] 5.1E-04 0.70 [0.33 , 1.06] 3.3E-02 0.20 [0.04 , 0.36] 1.9E-01 C1qb 2.41 [1.57 , 3.25] 9.4E-04 0.68 [0.29 , 1.08] 3.6E-02 0.10 [-0.01 , 0.20] 3.2E-01 C1qa 2.33 [1.53 , 3.14] 9.4E-04 0.64 [0.26 , 1.02] 4.1E-02 0.08 [-0.01 , 0.18] 3.3E-01 Oas2 2.31 [1.60 , 3.03] 9.4E-04 0.69 [0.41 , 0.97] 2.1E-02 0.09 [0.02 , 0.16] 1.8E-01 Emr1 2.27 [1.38 , 3.16] 1.9E-03 0.64 [0.22 , 1.06] 5.4E-02 0.08 [-0.03 , 0.19] 4.1E-01 Zbp1 2.25 [1.53 , 2.97] 9.4E-04 0.49 [0.16 , 0.83] 6.1E-02 0.04 [-0.09 , 0.18] 7.5E-01 Cx3cr1 2.24 [1.48 , 3.01] 9.4E-04 0.60 [0.23 , 0.96] 4.5E-02 0.08 [0.00 , 0.16] 2.8E-01 Tnfsf13b 2.22 [1.46 , 2.99] 9.4E-04 0.62 [0.23 , 1.01] 4.7E-02 0.14 [0.00 , 0.28] 2.9E-01 Ncf4 2.21 [1.54 , 2.87] 9.4E-04 0.58 [0.23 , 0.92] 4.2E-02 0.10 [-0.04 , 0.24] 4.4E-01 Col1a1 2.21 [0.93 , 3.50] 1.2E-02 0.42 [-0.29 , 1.13] 4.0E-01 0.01 [-0.09 , 0.12] 9.1E-01 532 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Pou2f2 2.19 [1.44 , 2.94] 9.4E-04 0.59 [0.20 , 0.97] 5.4E-02 0.13 [0.00 , 0.26] 2.8E-01 Fcer1g 2.18 [1.42 , 2.94] 9.8E-04 0.67 [0.35 , 1.00] 3.1E-02 0.12 [0.02 , 0.21] 1.9E-01 Cxcl10 2.18 [1.40 , 2.96] 1.1E-03 0.74 [0.36 , 1.12] 3.3E-02 0.18 [0.01 , 0.34] 2.4E-01 Isg15 2.18 [1.40 , 2.96] 1.1E-03 0.68 [0.36 , 1.00] 3.0E-02 0.12 [-0.02 , 0.25] 3.7E-01 Cxcl9 2.18 [1.26 , 3.11] 2.6E-03 0.37 [-0.09 , 0.83] 2.6E-01 0.04 [-0.14 , 0.22] 8.1E-01 Lif 2.18 [0.88 , 3.48] 1.3E-02 0.58 [0.09 , 1.07] 1.1E-01 0.10 [0.02 , 0.18] 1.9E-01 Oasl1 2.17 [1.17 , 3.17] 3.8E-03 0.81 [0.35 , 1.27] 3.5E-02 0.15 [-0.01 , 0.31] 3.0E-01 Ifit1 2.16 [1.38 , 2.93] 1.1E-03 0.78 [0.39 , 1.17] 3.3E-02 0.14 [-0.02 , 0.30] 3.5E-01 Fcgr3 2.15 [1.38 , 2.92] 1.1E-03 0.58 [0.13 , 1.02] 8.6E-02 0.07 [-0.02 , 0.16] 3.7E-01 Mx2 2.12 [1.36 , 2.87] 1.0E-03 0.79 [0.49 , 1.10] 1.7E-02 0.12 [-0.05 , 0.29] 4.4E-01 Slc11a1 2.08 [1.34 , 2.82] 1.0E-03 0.58 [0.26 , 0.90] 3.4E-02 0.10 [-0.03 , 0.24] 4.0E-01 Mrc1 2.05 [1.12 , 2.97] 3.4E-03 0.54 [0.12 , 0.95] 8.5E-02 0.07 [0.02 , 0.12] 1.3E-01 Socs3 2.05 [1.01 , 3.10] 6.1E-03 0.50 [0.08 , 0.92] 1.1E-01 0.03 [-0.04 , 0.10] 6.5E-01 C5ar1 2.01 [1.38 , 2.64] 9.4E-04 0.67 [0.37 , 0.97] 3.0E-02 0.15 [0.04 , 0.26] 1.7E-01 Cybb 2.01 [1.37 , 2.64] 9.4E-04 0.56 [0.27 , 0.84] 3.3E-02 0.14 [0.04 , 0.24] 1.4E-01 Ccl28 2.01 [0.68 , 3.35] 2.0E-02 0.38 [-0.16 , 0.92] 3.2E-01 0.13 [0.01 , 0.24] 2.2E-01 Itgal 1.99 [1.46 , 2.51] 5.1E-04 0.45 [0.15 , 0.74] 5.6E-02 0.11 [-0.03 , 0.26] 4.0E-01 533 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Lyz2 1.99 [1.16 , 2.82] 2.4E-03 0.53 [0.13 , 0.93] 8.1E-02 0.05 [-0.09 , 0.19] 7.2E-01 Il21r 1.97 [1.13 , 2.82] 2.7E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Tlr9 1.95 [1.32 , 2.57] 9.4E-04 0.51 [0.27 , 0.75] 3.0E-02 0.05 [-0.05 , 0.14] 6.2E-01 C7 1.95 [0.97 , 2.93] 5.8E-03 0.48 [0.05 , 0.90] 1.3E-01 0.06 [-0.01 , 0.13] 3.8E-01 Ccr2 1.94 [1.05 , 2.83] 3.7E-03 0.52 [0.10 , 0.93] 9.9E-02 0.05 [-0.01 , 0.12] 3.8E-01 Cd68 1.93 [1.28 , 2.58] 9.4E-04 0.46 [0.12 , 0.80] 7.9E-02 0.06 [-0.01 , 0.13] 3.6E-01 Cd48 1.93 [1.21 , 2.65] 1.3E-03 0.46 [0.13 , 0.79] 7.2E-02 0.02 [-0.09 , 0.13] 8.8E-01 Ly86 1.92 [1.18 , 2.65] 1.6E-03 0.44 [0.06 , 0.83] 1.2E-01 0.02 [-0.07 , 0.11] 8.2E-01 Cd84 1.91 [1.10 , 2.73] 2.6E-03 0.34 [-0.06 , 0.74] 2.3E-01 0.04 [-0.06 , 0.14] 6.6E-01 Msr1 1.89 [0.99 , 2.78] 4.3E-03 0.55 [0.09 , 1.00] 1.1E-01 0.06 [-0.04 , 0.15] 5.1E-01 Col3a1 1.87 [0.76 , 2.97] 1.2E-02 0.36 [-0.27 , 0.98] 4.2E-01 -0.03 [-0.11 , 0.04] 6.6E-01 Ccl20 1.87 [0.34 , 3.41] 4.9E-02 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Cxcl1 1.86 [0.57 , 3.15] 2.5E-02 0.53 [-0.05 , 1.11] 2.0E-01 0.05 [-0.08 , 0.17] 6.8E-01 Cxcl13 1.84 [0.62 , 3.06] 2.1E-02 0.18 [-0.28 , 0.64] 5.9E-01 -0.01 [-0.19 , 0.16] 9.3E-01 Ticam2 1.83 [1.22 , 2.44] 9.4E-04 0.50 [0.21 , 0.80] 3.8E-02 0.09 [-0.04 , 0.21] 4.5E-01 Itgb2 1.83 [1.20 , 2.46] 9.4E-04 0.41 [0.11 , 0.72] 7.9E-02 0.08 [-0.05 , 0.20] 5.0E-01 Vcam1 1.83 [0.88 , 2.79] 6.8E-03 0.53 [0.07 , 0.98] 1.2E-01 0.05 [-0.01 , 0.12] 3.7E-01 534 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Spn 1.82 [1.18 , 2.46] 1.0E-03 0.47 [0.18 , 0.77] 4.7E-02 0.10 [-0.04 , 0.24] 4.2E-01 Ptprc 1.80 [1.12 , 2.48] 1.5E-03 0.39 [0.09 , 0.69] 8.3E-02 0.05 [-0.05 , 0.15] 6.3E-01 Runx1 1.80 [0.93 , 2.67] 4.6E-03 0.38 [-0.12 , 0.88] 3.0E-01 0.00 [-0.08 , 0.08] 9.9E-01 Il10ra 1.79 [1.08 , 2.50] 2.0E-03 0.47 [0.22 , 0.73] 3.3E-02 0.06 [-0.03 , 0.15] 4.9E-01 Cd14 1.79 [0.87 , 2.71] 6.5E-03 0.45 [-0.02 , 0.92] 1.9E-01 0.05 [0.01 , 0.08] 1.9E-01 Csf3r 1.77 [1.00 , 2.53] 2.8E-03 0.47 [0.11 , 0.82] 8.4E-02 0.07 [-0.04 , 0.18] 4.8E-01 Nlrc5 1.77 [1.00 , 2.55] 2.9E-03 0.32 [0.00 , 0.64] 1.7E-01 0.01 [-0.11 , 0.14] 9.1E-01 Selplg 1.74 [1.03 , 2.45] 2.2E-03 0.28 [-0.02 , 0.58] 1.9E-01 0.01 [-0.08 , 0.11] 8.9E-01 Syk 1.73 [1.04 , 2.43] 2.0E-03 0.32 [-0.05 , 0.70] 2.2E-01 0.03 [-0.07 , 0.13] 7.7E-01 Cd4 1.73 [0.79 , 2.66] 8.3E-03 0.19 [-0.24 , 0.61] 5.6E-01 -0.15 [-0.28 , -0.02] 2.0E-01 H2-DMb2 1.71 [0.26 , 3.16] 5.5E-02 0.32 [-0.24 , 0.88] 4.2E-01 -0.06 [-0.20 , 0.08] 6.4E-01 Klra17 1.70 [0.77 , 2.64] 9.0E-03 0.37 [-0.05 , 0.80] 2.2E-01 -0.01 [-0.08 , 0.07] 9.2E-01 Ccl4 1.69 [0.90 , 2.49] 4.2E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Tlr7 1.66 [0.95 , 2.37] 2.7E-03 0.39 [0.09 , 0.69] 8.4E-02 0.05 [-0.04 , 0.14] 5.2E-01 Ifit3 1.66 [0.89 , 2.43] 3.8E-03 0.65 [0.31 , 0.99] 3.3E-02 0.11 [-0.05 , 0.26] 4.8E-01 Ly9 1.65 [0.94 , 2.35] 2.7E-03 0.20 [-0.16 , 0.55] 4.3E-01 -0.04 [-0.16 , 0.08] 7.2E-01 Ddx60 1.64 [0.97 , 2.30] 2.2E-03 0.50 [0.31 , 0.68] 1.5E-02 0.08 [0.00 , 0.17] 2.9E-01 535 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Havcr2 1.63 [1.19 , 2.08] 5.5E-04 0.45 [0.28 , 0.62] 1.5E-02 n.d [n.d , n.d] n.d Sh2d1b1 1.61 [1.01 , 2.20] 1.3E-03 0.45 [0.17 , 0.73] 4.7E-02 0.03 [-0.06 , 0.13] 7.3E-01 Tap1 1.61 [1.00 , 2.23] 1.6E-03 0.41 [0.17 , 0.64] 3.8E-02 0.07 [0.00 , 0.13] 2.8E-01 Cd74 1.61 [0.86 , 2.37] 4.1E-03 0.31 [0.01 , 0.61] 1.6E-01 0.00 [-0.06 , 0.07] 9.5E-01 C4b 1.60 [0.80 , 2.39] 5.3E-03 0.48 [0.24 , 0.72] 3.3E-02 0.08 [0.01 , 0.14] 2.1E-01 Il2rg 1.59 [1.07 , 2.11] 9.4E-04 0.18 [-0.04 , 0.41] 2.6E-01 0.06 [-0.05 , 0.16] 5.6E-01 Psmb8 1.59 [0.89 , 2.28] 2.9E-03 0.36 [0.06 , 0.65] 1.1E-01 0.01 [-0.07 , 0.09] 9.1E-01 Btk 1.58 [0.92 , 2.24] 2.4E-03 0.35 [0.02 , 0.69] 1.5E-01 0.01 [-0.10 , 0.12] 9.2E-01 Itga4 1.56 [1.03 , 2.09] 9.4E-04 0.32 [0.04 , 0.60] 1.2E-01 0.09 [-0.02 , 0.20] 3.6E-01 Lilra5 1.56 [1.02 , 2.11] 9.8E-04 0.45 [0.21 , 0.69] 3.3E-02 0.08 [0.02 , 0.14] 1.8E-01 Cd53 1.55 [0.87 , 2.23] 2.9E-03 0.33 [0.00 , 0.65] 1.7E-01 0.03 [-0.09 , 0.14] 8.1E-01 Csf2rb 1.54 [0.88 , 2.19] 2.6E-03 0.34 [-0.03 , 0.71] 2.0E-01 0.03 [-0.09 , 0.15] 8.2E-01 Cd180 1.54 [0.85 , 2.24] 3.4E-03 0.32 [0.05 , 0.60] 1.1E-01 -0.01 [-0.11 , 0.09] 9.3E-01 H2-Eb1 1.54 [0.82 , 2.26] 4.1E-03 0.25 [0.00 , 0.50] 1.7E-01 -0.01 [-0.08 , 0.06] 8.8E-01 Tmem173 1.54 [0.74 , 2.35] 7.0E-03 0.33 [-0.01 , 0.68] 1.9E-01 0.07 [0.01 , 0.14] 2.0E-01 H2-Aa 1.53 [0.79 , 2.27] 4.5E-03 0.22 [-0.04 , 0.49] 2.3E-01 0.00 [-0.07 , 0.06] 9.5E-01 Cd5 1.53 [0.63 , 2.43] 1.2E-02 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d 536 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Bst2 1.52 [0.96 , 2.09] 1.3E-03 0.49 [0.23 , 0.75] 3.3E-02 0.07 [0.00 , 0.15] 2.9E-01 Itgax 1.52 [0.91 , 2.13] 2.0E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Usp18 1.51 [0.87 , 2.14] 2.5E-03 0.45 [0.21 , 0.69] 3.3E-02 0.05 [-0.06 , 0.16] 6.2E-01 H2-Ab1 1.50 [0.80 , 2.21] 4.2E-03 0.28 [-0.02 , 0.58] 1.9E-01 0.01 [-0.04 , 0.06] 8.6E-01 Lcp1 1.49 [0.95 , 2.04] 1.1E-03 0.30 [0.05 , 0.55] 1.0E-01 0.07 [-0.02 , 0.15] 3.8E-01 Xaf1 1.49 [0.95 , 2.03] 1.1E-03 0.42 [0.22 , 0.61] 3.0E-02 0.06 [-0.02 , 0.13] 4.0E-01 Pik3cd 1.49 [0.88 , 2.10] 2.2E-03 0.39 [0.11 , 0.67] 7.2E-02 0.06 [-0.02 , 0.14] 4.1E-01 H2-Ea-ps 1.49 [0.78 , 2.19] 4.3E-03 0.24 [-0.02 , 0.50] 2.0E-01 0.00 [-0.06 , 0.05] 9.9E-01 H2-K1 1.48 [0.80 , 2.17] 3.8E-03 0.41 [0.12 , 0.70] 6.6E-02 0.03 [-0.03 , 0.08] 6.0E-01 Slamf7 1.48 [0.63 , 2.32] 1.1E-02 0.08 [-0.26 , 0.42] 7.6E-01 -0.02 [-0.13 , 0.09] 8.8E-01 Psmb9 1.47 [0.87 , 2.07] 2.2E-03 0.33 [0.10 , 0.56] 6.5E-02 0.03 [-0.05 , 0.11] 7.4E-01 Ccl6 1.47 [0.72 , 2.21] 6.0E-03 0.46 [0.13 , 0.79] 7.2E-02 0.10 [0.04 , 0.16] 7.6E-02 Gbp5 1.46 [0.86 , 2.05] 2.2E-03 0.37 [0.13 , 0.61] 5.3E-02 0.04 [-0.05 , 0.12] 6.6E-01 Cd200r1 1.46 [0.54 , 2.39] 1.7E-02 0.32 [-0.11 , 0.75] 3.1E-01 0.01 [-0.09 , 0.11] 9.1E-01 Tnfrsf1b 1.45 [0.99 , 1.91] 9.4E-04 0.27 [0.04 , 0.50] 1.1E-01 0.06 [-0.03 , 0.16] 4.5E-01 Ebi3 1.45 [0.87 , 2.04] 2.0E-03 0.41 [0.13 , 0.70] 6.3E-02 0.07 [-0.04 , 0.17] 4.9E-01 Cd3g 1.45 [0.45 , 2.45] 2.4E-02 -0.08 [-0.48 , 0.32] 8.0E-01 -0.12 [-0.25 , 0.02] 3.5E-01 537 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcr6 1.44 [0.63 , 2.24] 9.7E-03 0.13 [-0.11 , 0.38] 4.4E-01 -0.05 [-0.14 , 0.03] 4.9E-01 Clec5a 1.42 [0.78 , 2.06] 3.4E-03 0.35 [0.05 , 0.65] 1.1E-01 0.04 [-0.03 , 0.11] 6.0E-01 Itgae 1.42 [0.75 , 2.10] 4.3E-03 0.19 [-0.09 , 0.47] 3.3E-01 -0.03 [-0.09 , 0.03] 6.6E-01 Irf4 1.42 [0.68 , 2.17] 7.0E-03 0.25 [-0.10 , 0.60] 3.1E-01 n.d [n.d , n.d] n.d Cd86 1.41 [0.81 , 2.01] 2.6E-03 0.27 [0.09 , 0.45] 5.9E-02 0.04 [-0.03 , 0.11] 5.4E-01 H2-DMa 1.40 [0.73 , 2.06] 4.3E-03 0.30 [-0.02 , 0.62] 1.9E-01 0.01 [-0.04 , 0.06] 8.8E-01 Il2rb 1.38 [0.63 , 2.12] 8.2E-03 0.14 [-0.08 , 0.36] 3.8E-01 -0.01 [-0.10 , 0.08] 9.1E-01 Ltb 1.38 [0.32 , 2.45] 3.8E-02 0.02 [-0.43 , 0.47] 9.5E-01 -0.13 [-0.30 , 0.05] 4.2E-01 Spp1 1.38 [0.30 , 2.47] 4.0E-02 0.31 [-0.20 , 0.83] 3.9E-01 -0.01 [-0.03 , 0.01] 6.7E-01 Cfp 1.37 [0.76 , 1.98] 3.1E-03 0.22 [-0.18 , 0.62] 4.4E-01 -0.01 [-0.10 , 0.09] 9.5E-01 Lag3 1.37 [0.70 , 2.04] 5.1E-03 0.31 [0.09 , 0.52] 6.9E-02 0.02 [-0.07 , 0.12] 8.1E-01 Pik3cg 1.36 [0.61 , 2.10] 9.0E-03 0.31 [0.01 , 0.61] 1.5E-01 0.04 [-0.08 , 0.15] 7.3E-01 Cxcr3 1.36 [0.61 , 2.11] 9.0E-03 0.13 [-0.15 , 0.40] 5.2E-01 -0.06 [-0.17 , 0.04] 5.0E-01 Clec7a 1.35 [0.66 , 2.04] 6.1E-03 0.37 [0.12 , 0.63] 6.5E-02 0.07 [0.03 , 0.12] 7.7E-02 C3 1.35 [0.37 , 2.33] 3.0E-02 0.52 [0.12 , 0.93] 8.7E-02 0.09 [0.01 , 0.17] 2.0E-01 Cfd 1.35 [-0.86 , 3.56] 2.9E-01 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Irak3 1.34 [0.92 , 1.77] 9.4E-04 0.36 [0.16 , 0.57] 3.6E-02 0.10 [0.03 , 0.16] 1.3E-01 538 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cdkn1a 1.34 [0.57 , 2.12] 1.1E-02 0.39 [0.09 , 0.69] 8.7E-02 0.11 [0.00 , 0.22] 2.9E-01 Casp1 1.33 [0.78 , 1.88] 2.4E-03 0.18 [-0.08 , 0.44] 3.3E-01 0.03 [-0.04 , 0.10] 6.6E-01 Ikzf1 1.33 [0.69 , 1.97] 4.5E-03 0.19 [-0.09 , 0.47] 3.4E-01 -0.02 [-0.11 , 0.06] 7.9E-01 Socs1 1.33 [0.66 , 2.00] 5.8E-03 0.28 [0.01 , 0.55] 1.5E-01 -0.01 [-0.13 , 0.11] 9.5E-01 Cxcl11 1.33 [0.65 , 2.01] 6.4E-03 0.41 [0.14 , 0.69] 5.8E-02 0.05 [-0.02 , 0.13] 4.3E-01 Cd33 1.33 [0.56 , 2.11] 1.2E-02 0.34 [0.05 , 0.63] 1.1E-01 0.07 [0.01 , 0.13] 2.1E-01 Cd83 1.31 [0.88 , 1.75] 9.4E-04 0.26 [0.04 , 0.47] 1.1E-01 0.03 [-0.03 , 0.10] 6.1E-01 Gzma 1.31 [0.79 , 1.84] 2.0E-03 0.29 [0.08 , 0.49] 7.1E-02 0.02 [-0.06 , 0.11] 8.1E-01 Gpr183 1.31 [0.59 , 2.04] 9.2E-03 0.32 [-0.02 , 0.66] 2.0E-01 0.02 [-0.04 , 0.09] 7.4E-01 Icam1 1.30 [0.78 , 1.82] 2.0E-03 0.42 [0.20 , 0.64] 3.3E-02 0.09 [0.04 , 0.15] 9.6E-02 Batf 1.29 [0.79 , 1.79] 1.7E-03 0.24 [-0.02 , 0.50] 2.1E-01 -0.03 [-0.10 , 0.04] 6.6E-01 H2-Ob 1.29 [0.56 , 2.01] 9.7E-03 0.16 [-0.19 , 0.50] 5.5E-01 -0.02 [-0.10 , 0.06] 8.1E-01 Card11 1.29 [0.48 , 2.09] 1.6E-02 0.02 [-0.31 , 0.36] 9.3E-01 -0.03 [-0.16 , 0.10] 8.1E-01 Fn1 1.29 [0.36 , 2.22] 2.9E-02 0.38 [-0.14 , 0.89] 3.1E-01 0.00 [-0.06 , 0.06] 9.7E-01 Tnfrsf12a 1.29 [0.32 , 2.25] 3.4E-02 0.27 [-0.14 , 0.69] 3.5E-01 0.03 [-0.01 , 0.07] 4.2E-01 Amica1 1.28 [0.67 , 1.89] 4.3E-03 0.17 [-0.09 , 0.44] 3.5E-01 -0.01 [-0.06 , 0.03] 7.5E-01 Ctsw 1.28 [0.48 , 2.09] 1.6E-02 -0.07 [-0.37 , 0.23] 7.5E-01 -0.13 [-0.23 , -0.03] 1.7E-01 539 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd276 1.28 [0.46 , 2.11] 1.8E-02 0.42 [0.05 , 0.80] 1.3E-01 0.06 [-0.01 , 0.13] 3.8E-01 Sh2b2 1.27 [0.85 , 1.69] 9.4E-04 n.d n.d n.d -0.02 [-0.10 , 0.06] 8.1E-01 Vim 1.27 [0.67 , 1.87] 4.3E-03 0.30 [-0.04 , 0.64] 2.2E-01 0.02 [-0.03 , 0.08] 6.6E-01 Irf1 1.26 [0.75 , 1.77] 2.0E-03 0.31 [0.11 , 0.52] 5.4E-02 0.05 [-0.01 , 0.11] 3.4E-01 Tnfrsf14 1.25 [0.75 , 1.75] 2.0E-03 0.25 [0.07 , 0.43] 7.4E-02 0.02 [-0.03 , 0.08] 6.0E-01 Il1rl2 1.25 [0.64 , 1.85] 4.7E-03 0.48 [0.18 , 0.78] 4.7E-02 0.10 [0.03 , 0.16] 1.3E-01 H2-DMb1 1.25 [0.59 , 1.92] 7.4E-03 0.21 [-0.07 , 0.50] 3.1E-01 0.01 [-0.04 , 0.07] 8.1E-01 Zap70 1.25 [0.32 , 2.18] 3.3E-02 n.d [n.d , n.d] n.d -0.02 [-0.12 , 0.07] 8.1E-01 Csf1 1.24 [0.81 , 1.67] 9.4E-04 0.34 [0.10 , 0.58] 6.7E-02 0.05 [-0.01 , 0.11] 3.6E-01 Herc6 1.24 [0.60 , 1.88] 6.5E-03 0.38 [0.18 , 0.59] 3.3E-02 0.07 [-0.03 , 0.17] 4.5E-01 Ciita 1.24 [0.51 , 1.98] 1.2E-02 0.18 [-0.15 , 0.50] 4.4E-01 -0.02 [-0.11 , 0.07] 8.1E-01 Irgm2 1.21 [0.81 , 1.61] 9.4E-04 0.39 [0.21 , 0.57] 3.0E-02 0.05 [-0.04 , 0.14] 5.3E-01 Inpp5d 1.21 [0.67 , 1.74] 3.1E-03 0.28 [0.04 , 0.51] 1.1E-01 0.04 [-0.01 , 0.09] 3.9E-01 Xcr1 1.21 [0.60 , 1.83] 6.0E-03 0.21 [-0.03 , 0.46] 2.3E-01 -0.07 [-0.15 , 0.00] 2.9E-01 Stat1 1.20 [0.74 , 1.66] 1.6E-03 0.31 [0.14 , 0.49] 3.5E-02 0.03 [-0.04 , 0.11] 6.6E-01 Cd8a 1.20 [0.56 , 1.84] 7.4E-03 0.08 [-0.16 , 0.32] 6.5E-01 -0.03 [-0.13 , 0.07] 7.5E-01 Tnfrsf13b 1.20 [0.53 , 1.87] 9.7E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d 540 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Icos 1.20 [0.31 , 2.09] 3.2E-02 -0.02 [-0.32 , 0.28] 9.3E-01 -0.07 [-0.19 , 0.04] 5.0E-01 Cd3d 1.20 [0.30 , 2.09] 3.4E-02 0.04 [-0.34 , 0.42] 9.0E-01 -0.10 [-0.25 , 0.06] 4.9E-01 H2-M3 1.19 [0.65 , 1.73] 3.5E-03 0.30 [0.13 , 0.47] 3.6E-02 0.01 [-0.03 , 0.06] 8.1E-01 Klrk1 1.19 [0.36 , 2.02] 2.5E-02 0.11 [-0.16 , 0.38] 5.7E-01 -0.01 [-0.09 , 0.07] 9.0E-01 Xcl1 1.19 [0.26 , 2.12] 4.0E-02 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Ifit2 1.17 [0.48 , 1.86] 1.2E-02 0.39 [0.18 , 0.61] 3.3E-02 0.07 [-0.04 , 0.18] 4.9E-01 H2-D1 1.16 [0.66 , 1.66] 2.7E-03 0.31 [0.11 , 0.52] 5.5E-02 0.02 [-0.01 , 0.06] 5.2E-01 H2-Q2 1.16 [0.61 , 1.71] 4.3E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Cd69 1.16 [0.42 , 1.91] 1.8E-02 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Creb5 1.15 [0.31 , 1.99] 3.0E-02 0.34 [-0.06 , 0.73] 2.3E-01 -0.01 [-0.09 , 0.07] 9.0E-01 Ccr8 1.14 [0.56 , 1.73] 6.4E-03 0.29 [-0.01 , 0.59] 1.9E-01 -0.02 [-0.09 , 0.06] 8.1E-01 Flt3 1.13 [0.50 , 1.77] 9.8E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Trem2 1.12 [0.43 , 1.81] 1.5E-02 0.09 [-0.21 , 0.39] 6.9E-01 -0.06 [-0.17 , 0.04] 5.0E-01 Cd3e 1.12 [0.40 , 1.85] 1.9E-02 0.01 [-0.30 , 0.31] 9.8E-01 -0.07 [-0.18 , 0.04] 4.8E-01 Thy1 1.12 [0.38 , 1.86] 2.0E-02 0.05 [-0.38 , 0.48] 8.9E-01 -0.12 [-0.21 , -0.04] 1.4E-01 Anxa1 1.11 [0.37 , 1.85] 2.1E-02 0.25 [-0.11 , 0.60] 3.2E-01 -0.02 [-0.07 , 0.02] 5.8E-01 Tlr4 1.10 [0.62 , 1.58] 3.0E-03 0.34 [0.15 , 0.53] 3.5E-02 0.04 [0.00 , 0.08] 3.3E-01 541 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Entpd1 1.09 [0.63 , 1.54] 2.5E-03 0.28 [0.17 , 0.39] 1.7E-02 0.12 [0.07 , 0.16] 2.3E-02 Relb 1.09 [0.54 , 1.64] 6.0E-03 0.29 [0.01 , 0.56] 1.5E-01 0.04 [0.00 , 0.09] 3.3E-01 Ncr1 1.08 [0.43 , 1.74] 1.4E-02 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Ccl17 1.08 [0.26 , 1.90] 3.6E-02 0.28 [-0.12 , 0.68] 3.2E-01 0.04 [-0.05 , 0.13] 6.6E-01 Psmb10 1.07 [0.60 , 1.55] 3.1E-03 0.28 [0.06 , 0.49] 8.7E-02 -0.02 [-0.08 , 0.03] 6.8E-01 Serping1 1.07 [0.52 , 1.62] 6.6E-03 0.26 [0.03 , 0.49] 1.3E-01 0.00 [-0.04 , 0.04] 9.8E-01 Lck 1.07 [0.19 , 1.96] 4.9E-02 -0.06 [-0.40 , 0.28] 8.1E-01 -0.09 [-0.20 , 0.02] 3.8E-01 Stat2 1.06 [0.64 , 1.48] 2.0E-03 0.28 [0.13 , 0.43] 3.3E-02 0.04 [-0.02 , 0.09] 4.5E-01 Itgb3 1.06 [0.39 , 1.74] 1.8E-02 0.51 [0.24 , 0.79] 3.3E-02 0.08 [-0.01 , 0.17] 3.3E-01 Btla 1.06 [0.36 , 1.77] 2.0E-02 0.08 [-0.17 , 0.32] 6.7E-01 -0.06 [-0.16 , 0.04] 5.3E-01 Cd247 1.06 [0.08 , 2.04] 7.6E-02 -0.13 [-0.55 , 0.29] 6.7E-01 -0.14 [-0.31 , 0.03] 3.7E-01 Clu 1.05 [0.14 , 1.96] 5.9E-02 0.20 [-0.26 , 0.66] 5.6E-01 -0.02 [-0.06 , 0.03] 6.6E-01 Tnfaip3 1.04 [0.62 , 1.46] 2.0E-03 0.29 [0.06 , 0.53] 9.4E-02 0.06 [0.01 , 0.12] 1.9E-01 Axl 1.04 [0.56 , 1.51] 3.8E-03 0.29 [0.08 , 0.49] 7.1E-02 0.05 [0.02 , 0.09] 1.3E-01 Ikbke 1.04 [0.54 , 1.53] 4.3E-03 0.24 [0.02 , 0.45] 1.4E-01 0.05 [-0.02 , 0.11] 4.3E-01 Ctla4 1.04 [0.23 , 1.85] 4.0E-02 0.11 [-0.19 , 0.41] 6.0E-01 n.d [n.d , n.d] n.d Itga2 1.04 [0.19 , 1.90] 4.9E-02 0.17 [-0.22 , 0.55] 5.6E-01 -0.05 [-0.15 , 0.05] 5.8E-01 542 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ada 1.03 [0.59 , 1.47] 2.7E-03 0.31 [0.19 , 0.42] 1.5E-02 0.05 [-0.01 , 0.11] 3.8E-01 Pparg 1.03 [0.40 , 1.66] 1.4E-02 0.20 [-0.07 , 0.46] 3.0E-01 0.05 [-0.06 , 0.15] 6.4E-01 Rel 1.01 [0.66 , 1.36] 9.4E-04 0.23 [0.10 , 0.36] 3.3E-02 0.03 [-0.03 , 0.08] 6.0E-01 Irf8 1.01 [0.63 , 1.39] 1.4E-03 0.27 [0.13 , 0.41] 3.3E-02 0.04 [0.00 , 0.08] 3.3E-01 Ifi44 1.01 [0.60 , 1.43] 2.2E-03 0.22 [0.06 , 0.38] 7.1E-02 -0.01 [-0.08 , 0.06] 9.1E-01 Tlr6 1.01 [0.59 , 1.42] 2.3E-03 0.26 [0.10 , 0.42] 4.7E-02 0.04 [0.00 , 0.08] 3.3E-01 Tnfrsf4 1.01 [0.53 , 1.48] 4.3E-03 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d C1s1 1.01 [0.51 , 1.52] 5.3E-03 0.31 [0.09 , 0.53] 6.5E-02 0.06 [0.02 , 0.10] 1.5E-01 Ccr7 1.00 [0.39 , 1.62] 1.4E-02 0.12 [-0.19 , 0.42] 5.9E-01 -0.04 [-0.15 , 0.08] 7.3E-01 Myc 1.00 [0.44 , 1.56] 9.4E-03 0.30 [-0.02 , 0.63] 2.0E-01 0.00 [-0.05 , 0.04] 9.5E-01 Tgfb3 1.00 [0.30 , 1.70] 2.6E-02 0.22 [-0.08 , 0.53] 3.1E-01 0.04 [-0.05 , 0.12] 6.6E-01 Cd80 1.00 [0.39 , 1.60] 1.4E-02 n.d [n.d , n.d] n.d -0.07 [-0.19 , 0.06] 5.7E-01 C1ra 0.99 [0.52 , 1.46] 4.3E-03 0.25 [0.09 , 0.42] 5.6E-02 0.03 [0.00 , 0.07] 2.8E-01 Cd79b 0.99 [-0.22 , 2.19] 1.7E-01 0.04 [-0.50 , 0.58] 9.2E-01 -0.11 [-0.29 , 0.07] 5.0E-01 Il3ra 0.99 [0.54 , 1.43] 3.4E-03 0.27 [0.03 , 0.50] 1.2E-01 -0.02 [-0.08 , 0.04] 6.7E-01 Ccl19 0.97 [0.41 , 1.53] 1.1E-02 0.04 [-0.19 , 0.28] 8.1E-01 0.00 [-0.10 , 0.09] 9.7E-01 Tlr2 0.97 [0.36 , 1.59] 1.7E-02 0.33 [0.04 , 0.62] 1.3E-01 0.03 [-0.05 , 0.10] 7.0E-01 543 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ifi27 0.96 [0.56 , 1.36] 2.4E-03 0.34 [0.15 , 0.52] 3.3E-02 0.03 [-0.02 , 0.08] 5.2E-01 Cd274 0.96 [0.58 , 1.33] 1.9E-03 0.22 [0.05 , 0.38] 8.4E-02 0.03 [-0.05 , 0.11] 7.2E-01 Tlr1 0.95 [0.48 , 1.41] 5.1E-03 0.16 [-0.06 , 0.39] 3.1E-01 0.01 [-0.06 , 0.08] 9.2E-01 Il34 0.94 [0.10 , 1.78] 6.6E-02 0.35 [0.02 , 0.68] 1.5E-01 0.02 [-0.03 , 0.07] 6.8E-01 Il16 0.94 [0.37 , 1.51] 1.4E-02 0.11 [-0.14 , 0.36] 5.6E-01 -0.06 [-0.13 , 0.01] 3.8E-01 Tnfrsf11a 0.93 [0.38 , 1.48] 1.2E-02 0.13 [-0.05 , 0.30] 3.2E-01 -0.02 [-0.07 , 0.03] 7.5E-01 Itga5 0.93 [0.46 , 1.40] 6.0E-03 0.29 [0.01 , 0.57] 1.6E-01 0.02 [-0.03 , 0.07] 6.6E-01 Lyn 0.93 [0.52 , 1.33] 2.8E-03 0.25 [0.12 , 0.37] 3.3E-02 0.03 [-0.01 , 0.06] 3.6E-01 Cmpk2 0.92 [0.52 , 1.31] 2.7E-03 0.29 [0.07 , 0.50] 8.2E-02 0.06 [-0.03 , 0.15] 4.8E-01 Tap2 0.92 [0.44 , 1.39] 6.8E-03 0.25 [0.07 , 0.43] 7.3E-02 0.02 [-0.03 , 0.07] 6.8E-01 Tgfb1 0.91 [0.51 , 1.31] 3.0E-03 0.26 [0.03 , 0.49] 1.3E-01 0.02 [-0.03 , 0.06] 6.8E-01 Cebpb 0.90 [0.49 , 1.31] 3.6E-03 0.34 [0.22 , 0.46] 1.5E-02 0.15 [0.05 , 0.25] 1.4E-01 Lbp 0.90 [0.36 , 1.43] 1.3E-02 0.18 [-0.08 , 0.45] 3.3E-01 -0.01 [-0.07 , 0.06] 9.3E-01 Klrd1 0.89 [0.28 , 1.50] 2.3E-02 -0.06 [-0.26 , 0.14] 6.8E-01 -0.05 [-0.11 , 0.00] 2.8E-01 Isg20 0.89 [0.29 , 1.49] 2.2E-02 0.23 [0.04 , 0.42] 9.9E-02 0.01 [-0.06 , 0.09] 8.5E-01 Il6ra 0.88 [0.55 , 1.22] 1.6E-03 0.20 [0.04 , 0.35] 8.7E-02 0.01 [-0.03 , 0.05] 8.4E-01 Cmklr1 0.88 [0.50 , 1.26] 2.7E-03 0.18 [-0.01 , 0.37] 1.9E-01 0.03 [-0.03 , 0.09] 6.3E-01 544 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Sbno2 0.86 [0.44 , 1.28] 5.0E-03 0.19 [-0.05 , 0.42] 2.6E-01 -0.01 [-0.04 , 0.02] 6.7E-01 Il18r1 0.85 [-0.06 , 1.76] 1.2E-01 0.03 [-0.26 , 0.33] 8.9E-01 -0.12 [-0.23 , -0.01] 2.5E-01 Cd40 0.85 [0.52 , 1.18] 1.8E-03 0.18 [-0.02 , 0.38] 2.2E-01 -0.01 [-0.05 , 0.04] 8.6E-01 Ccl11 0.84 [0.23 , 1.45] 3.0E-02 0.09 [-0.09 , 0.27] 5.1E-01 -0.07 [-0.16 , 0.03] 4.5E-01 Slc7a11 0.84 [0.01 , 1.67] 9.2E-02 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d S100a8 0.84 [0.26 , 1.41] 2.4E-02 0.50 [0.10 , 0.90] 9.6E-02 0.17 [0.04 , 0.29] 1.7E-01 Ifitm2 0.83 [0.38 , 1.28] 8.3E-03 0.21 [0.01 , 0.41] 1.6E-01 0.03 [0.00 , 0.06] 1.9E-01 Col4a1 0.83 [0.30 , 1.35] 1.7E-02 0.16 [-0.13 , 0.46] 4.3E-01 -0.03 [-0.05 , 0.00] 3.1E-01 Cx3cl1 0.82 [0.37 , 1.28] 9.0E-03 0.21 [0.01 , 0.40] 1.6E-01 0.01 [-0.02 , 0.04] 8.1E-01 Nod2 0.82 [0.46 , 1.18] 2.8E-03 0.31 [0.16 , 0.46] 3.0E-02 0.06 [0.00 , 0.13] 3.1E-01 Rsad2 0.82 [0.47 , 1.17] 2.7E-03 0.28 [0.12 , 0.43] 3.5E-02 0.05 [-0.03 , 0.12] 4.9E-01 Rrad 0.80 [0.17 , 1.44] 4.2E-02 0.16 [-0.09 , 0.40] 3.6E-01 -0.03 [-0.07 , 0.00] 2.9E-01 Masp1 0.80 [0.17 , 1.43] 4.2E-02 0.27 [0.09 , 0.44] 5.8E-02 0.07 [0.00 , 0.13] 2.5E-01 Lgals3 0.80 [0.22 , 1.38] 3.0E-02 0.09 [-0.12 , 0.31] 5.6E-01 -0.01 [-0.03 , 0.02] 8.1E-01 Sh2d1a 0.79 [0.12 , 1.47] 5.5E-02 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Plaur 0.79 [0.42 , 1.17] 4.3E-03 0.24 [-0.02 , 0.49] 2.0E-01 0.01 [-0.04 , 0.05] 9.1E-01 Nfkb2 0.79 [0.41 , 1.17] 4.3E-03 0.22 [0.04 , 0.40] 9.7E-02 0.03 [0.00 , 0.06] 2.8E-01 545 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Klrc1 0.78 [0.20 , 1.37] 3.4E-02 0.20 [0.00 , 0.40] 1.6E-01 -0.04 [-0.08 , 0.00] 3.3E-01 Tcf7 0.78 [0.02 , 1.54] 8.8E-02 -0.06 [-0.39 , 0.28] 8.3E-01 -0.08 [-0.21 , 0.05] 5.2E-01 Cd99 0.78 [0.31 , 1.25] 1.4E-02 0.25 [0.02 , 0.48] 1.4E-01 0.02 [-0.02 , 0.05] 6.2E-01 Ifih1 0.78 [0.37 , 1.18] 6.8E-03 0.27 [0.14 , 0.39] 3.0E-02 0.05 [0.00 , 0.11] 3.2E-01 Mcam 0.77 [0.32 , 1.22] 1.2E-02 0.12 [-0.06 , 0.30] 3.5E-01 0.03 [-0.01 , 0.07] 4.1E-01 Ddx58 0.77 [0.33 , 1.21] 1.1E-02 0.25 [0.09 , 0.40] 5.0E-02 0.05 [0.01 , 0.10] 1.9E-01 Ifi35 0.76 [0.35 , 1.17] 8.4E-03 0.22 [0.07 , 0.37] 5.8E-02 0.03 [-0.01 , 0.07] 4.5E-01 Sell 0.76 [-0.27 , 1.79] 2.1E-01 -0.13 [-0.53 , 0.28] 6.7E-01 -0.10 [-0.28 , 0.09] 5.8E-01 Abca1 0.75 [0.22 , 1.28] 2.7E-02 0.21 [0.04 , 0.38] 1.0E-01 0.05 [0.00 , 0.09] 2.5E-01 Cfh 0.73 [0.30 , 1.16] 1.2E-02 0.20 [0.00 , 0.40] 1.7E-01 0.04 [0.00 , 0.08] 3.3E-01 Fap 0.73 [0.21 , 1.24] 2.7E-02 0.33 [0.07 , 0.59] 9.3E-02 0.03 [-0.09 , 0.15] 8.1E-01 Cd37 0.72 [0.08 , 1.36] 6.5E-02 -0.05 [-0.29 , 0.20] 8.1E-01 -0.03 [-0.13 , 0.06] 7.0E-01 Icosl 0.70 [0.33 , 1.08] 7.3E-03 0.17 [0.06 , 0.28] 5.4E-02 0.06 [0.01 , 0.10] 1.9E-01 Lrp1 0.70 [0.29 , 1.12] 1.3E-02 0.25 [0.07 , 0.42] 7.1E-02 0.07 [0.03 , 0.11] 9.3E-02 Tgfb2 0.70 [0.28 , 1.13] 1.3E-02 0.19 [-0.03 , 0.41] 2.3E-01 0.05 [0.00 , 0.10] 2.8E-01 Mapk11 0.70 [0.24 , 1.17] 2.0E-02 0.24 [0.02 , 0.46] 1.5E-01 -0.02 [-0.07 , 0.04] 7.6E-01 Tapbp 0.70 [0.37 , 1.04] 4.3E-03 0.18 [0.03 , 0.33] 1.1E-01 0.01 [-0.02 , 0.04] 8.0E-01 546 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cfi 0.70 [0.12 , 1.28] 4.9E-02 0.02 [-0.27 , 0.30] 9.4E-01 -0.06 [-0.09 , -0.03] 5.8E-02 Cd28 0.70 [0.20 , 1.20] 2.9E-02 0.28 [-0.04 , 0.60] 2.2E-01 -0.06 [-0.17 , 0.05] 5.4E-01 Tnfsf13 0.69 [0.31 , 1.08] 9.7E-03 0.20 [0.09 , 0.32] 3.6E-02 0.00 [-0.07 , 0.06] 9.9E-01 Itgb4 0.69 [0.11 , 1.26] 5.2E-02 0.22 [-0.04 , 0.48] 2.4E-01 0.00 [-0.06 , 0.06] 9.9E-01 Tgfbr2 0.69 [0.35 , 1.02] 4.9E-03 0.18 [0.03 , 0.33] 1.0E-01 0.01 [-0.01 , 0.03] 5.0E-01 Stat3 0.69 [0.28 , 1.10] 1.3E-02 0.12 [-0.04 , 0.29] 3.1E-01 0.01 [0.00 , 0.03] 3.7E-01 Ccr1 0.68 [0.04 , 1.32] 7.7E-02 0.14 [-0.30 , 0.57] 6.7E-01 -0.10 [-0.17 , -0.03] 1.3E-01 Thbd 0.68 [0.45 , 0.91] 9.4E-04 0.13 [0.06 , 0.21] 3.6E-02 0.05 [0.03 , 0.08] 2.6E-02 Il1r1 0.68 [0.24 , 1.12] 1.8E-02 0.17 [-0.01 , 0.35] 1.9E-01 -0.02 [-0.05 , 0.02] 6.4E-01 Litaf 0.68 [0.12 , 1.24] 4.9E-02 0.20 [-0.06 , 0.47] 2.9E-01 -0.03 [-0.09 , 0.04] 6.6E-01 Ifngr1 0.68 [0.31 , 1.04] 8.2E-03 0.14 [-0.05 , 0.33] 3.1E-01 0.00 [-0.02 , 0.02] 8.8E-01 Nfatc1 0.68 [0.44 , 0.92] 1.0E-03 0.16 [0.06 , 0.26] 4.5E-02 0.05 [0.00 , 0.09] 2.5E-01 Tnfrsf10b 0.67 [0.28 , 1.06] 1.2E-02 0.17 [-0.07 , 0.40] 3.1E-01 0.01 [-0.02 , 0.04] 8.1E-01 Flt3l 0.67 [0.24 , 1.10] 1.9E-02 0.11 [-0.01 , 0.24] 2.2E-01 0.00 [-0.05 , 0.05] 9.9E-01 Smpd3 0.67 [0.22 , 1.11] 2.1E-02 0.18 [-0.03 , 0.39] 2.3E-01 0.01 [-0.08 , 0.10] 8.9E-01 H2-T23 0.66 [0.32 , 0.99] 6.4E-03 0.16 [0.07 , 0.25] 3.3E-02 0.01 [-0.02 , 0.04] 7.5E-01 Cd97 0.65 [0.43 , 0.87] 9.4E-04 0.20 [0.11 , 0.30] 3.0E-02 0.04 [0.00 , 0.07] 2.4E-01 547 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Pdgfrb 0.65 [0.31 , 0.99] 7.4E-03 0.21 [0.05 , 0.37] 8.0E-02 0.00 [-0.03 , 0.03] 9.1E-01 Cxcr4 0.64 [0.17 , 1.12] 3.3E-02 -0.06 [-0.25 , 0.13] 6.7E-01 -0.08 [-0.16 , -0.01] 2.3E-01 Cd38 0.64 [0.42 , 0.85] 9.4E-04 0.17 [0.06 , 0.27] 5.4E-02 0.00 [-0.04 , 0.04] 9.5E-01 Myd88 0.62 [0.33 , 0.91] 4.1E-03 0.15 [0.04 , 0.26] 8.2E-02 0.02 [-0.01 , 0.05] 5.0E-01 Apoe 0.61 [0.24 , 0.98] 1.4E-02 0.11 [-0.02 , 0.23] 2.3E-01 0.03 [-0.01 , 0.08] 4.6E-01 Cd47 0.60 [0.26 , 0.94] 1.0E-02 0.19 [0.06 , 0.32] 6.5E-02 0.04 [0.02 , 0.06] 3.1E-02 Muc1 0.60 [-0.04 , 1.25] 1.2E-01 0.17 [-0.07 , 0.40] 3.2E-01 -0.03 [-0.11 , 0.04] 6.6E-01 St6gal1 0.60 [0.27 , 0.93] 9.2E-03 0.14 [-0.02 , 0.30] 2.2E-01 -0.01 [-0.05 , 0.02] 6.6E-01 Il1rl1 0.60 [0.14 , 1.06] 3.8E-02 0.14 [-0.12 , 0.40] 4.6E-01 -0.01 [-0.09 , 0.08] 9.1E-01 Nfatc4 0.59 [0.28 , 0.91] 7.5E-03 0.16 [-0.03 , 0.36] 2.3E-01 0.01 [-0.03 , 0.05] 8.5E-01 Csf1r 0.59 [0.27 , 0.91] 8.2E-03 0.07 [-0.04 , 0.19] 3.8E-01 -0.03 [-0.07 , 0.01] 3.8E-01 Fyn 0.58 [0.36 , 0.81] 1.8E-03 0.09 [0.00 , 0.18] 1.8E-01 -0.02 [-0.06 , 0.01] 5.0E-01 Ifitm1 0.58 [0.05 , 1.12] 7.0E-02 0.17 [-0.10 , 0.44] 3.8E-01 -0.04 [-0.15 , 0.06] 6.6E-01 Bcl6 0.58 [0.20 , 0.95] 1.9E-02 0.04 [-0.12 , 0.19] 7.5E-01 0.01 [-0.08 , 0.10] 9.0E-01 Il18 0.58 [0.14 , 1.02] 3.7E-02 0.14 [-0.01 , 0.29] 2.0E-01 0.08 [0.03 , 0.13] 7.7E-02 Hcst 0.56 [-0.01 , 1.13] 1.0E-01 0.24 [-0.01 , 0.48] 1.9E-01 n.d [n.d , n.d] n.d Cxcl16 0.56 [0.11 , 1.00] 4.3E-02 0.18 [0.01 , 0.35] 1.5E-01 0.04 [0.01 , 0.07] 1.7E-01 548 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Map3k1 0.56 [0.17 , 0.94] 2.4E-02 0.10 [-0.05 , 0.25] 3.4E-01 -0.03 [-0.06 , -0.01] 1.9E-01 Il4ra 0.55 [0.25 , 0.86] 9.2E-03 0.13 [0.01 , 0.25] 1.5E-01 0.00 [-0.03 , 0.03] 9.9E-01 Ripk2 0.54 [0.38 , 0.70] 9.4E-04 0.15 [0.07 , 0.23] 3.3E-02 0.05 [0.01 , 0.09] 1.9E-01 Pecam1 0.54 [0.36 , 0.72] 9.4E-04 0.12 [0.05 , 0.19] 4.7E-02 0.04 [0.01 , 0.07] 1.9E-01 Colec12 0.54 [0.19 , 0.88] 1.8E-02 0.17 [0.01 , 0.34] 1.6E-01 -0.01 [-0.05 , 0.04] 9.0E-01 Pvr 0.54 [0.15 , 0.93] 3.0E-02 0.12 [-0.06 , 0.29] 3.5E-01 0.01 [-0.03 , 0.04] 8.8E-01 Adora2a 0.53 [0.11 , 0.96] 4.3E-02 0.20 [-0.03 , 0.42] 2.2E-01 -0.02 [-0.09 , 0.06] 8.5E-01 Vwf 0.53 [-0.26 , 1.31] 2.5E-01 0.02 [-0.19 , 0.22] 9.1E-01 -0.06 [-0.16 , 0.04] 5.0E-01 Cd160 0.52 [0.04 , 1.01] 7.6E-02 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Cd19 0.52 [-0.85 , 1.88] 5.2E-01 n.d [n.d , n.d] n.d n.d [n.d , n.d] n.d Irak4 0.51 [0.22 , 0.81] 1.1E-02 0.14 [0.04 , 0.24] 6.5E-02 0.01 [-0.01 , 0.04] 6.6E-01 Tnfrsf1a 0.51 [0.24 , 0.78] 7.8E-03 0.16 [0.05 , 0.27] 6.4E-02 0.02 [0.00 , 0.04] 2.9E-01 Tal1 0.51 [0.28 , 0.74] 3.7E-03 0.14 [0.03 , 0.25] 8.5E-02 0.06 [0.01 , 0.11] 1.9E-01 Ncam1 0.51 [0.02 , 1.00] 8.6E-02 0.14 [-0.15 , 0.43] 5.0E-01 -0.04 [-0.08 , 0.01] 3.6E-01 Akt3 0.50 [0.23 , 0.77] 8.0E-03 0.17 [0.07 , 0.27] 3.6E-02 0.03 [0.00 , 0.06] 2.4E-01 Ccrl2 0.50 [0.02 , 0.98] 8.4E-02 0.09 [-0.14 , 0.31] 5.9E-01 0.01 [-0.07 , 0.09] 8.8E-01 Pvrl2 0.50 [0.16 , 0.84] 2.2E-02 0.23 [0.11 , 0.36] 3.3E-02 0.03 [0.01 , 0.06] 8.6E-02 549 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mfge8 0.49 [0.23 , 0.75] 7.5E-03 0.18 [0.07 , 0.30] 4.7E-02 0.04 [0.02 , 0.07] 9.6E-02 Cd34 0.48 [0.28 , 0.68] 2.4E-03 0.14 [0.06 , 0.23] 4.4E-02 0.02 [-0.01 , 0.06] 4.0E-01 Ets1 0.48 [0.26 , 0.70] 3.8E-03 0.11 [0.01 , 0.21] 1.3E-01 0.02 [-0.02 , 0.05] 6.3E-01 Cxcl14 0.48 [0.13 , 0.83] 3.1E-02 -0.01 [-0.23 , 0.20] 9.3E-01 -0.03 [-0.07 , 0.02] 5.0E-01 Abcg1 0.48 [0.01 , 0.95] 9.2E-02 0.11 [-0.08 , 0.31] 4.0E-01 -0.05 [-0.12 , 0.02] 4.0E-01 Nfkb1 0.47 [0.23 , 0.72] 7.1E-03 0.09 [-0.03 , 0.21] 2.8E-01 -0.01 [-0.03 , 0.01] 6.0E-01 Jak3 0.47 [0.14 , 0.81] 2.8E-02 0.18 [0.08 , 0.28] 3.3E-02 0.01 [-0.04 , 0.05] 8.8E-01 Cd55 0.47 [0.12 , 0.83] 3.5E-02 0.04 [-0.09 , 0.16] 6.9E-01 0.01 [-0.05 , 0.07] 8.5E-01 Fos 0.47 [-0.25 , 1.20] 2.7E-01 -0.20 [-0.47 , 0.07] 3.0E-01 -0.14 [-0.24 , -0.04] 1.5E-01 Mef2c 0.47 [0.27 , 0.67] 2.7E-03 0.09 [0.00 , 0.18] 1.8E-01 0.00 [-0.02 , 0.03] 8.9E-01 Cdh5 0.47 [0.31 , 0.62] 9.4E-04 0.13 [0.05 , 0.20] 3.5E-02 0.02 [0.00 , 0.04] 3.6E-01 Hsd11b1 0.47 [0.13 , 0.81] 2.9E-02 0.24 [0.10 , 0.38] 4.0E-02 0.06 [0.01 , 0.11] 2.1E-01 Snai1 0.47 [-0.06 , 0.99] 1.3E-01 0.14 [-0.09 , 0.37] 4.0E-01 0.00 [-0.08 , 0.08] 9.9E-01 Tlr3 0.47 [0.17 , 0.77] 1.8E-02 0.15 [0.08 , 0.22] 3.0E-02 0.01 [-0.03 , 0.05] 8.1E-01 Prdm1 0.46 [0.14 , 0.78] 2.5E-02 0.04 [-0.09 , 0.17] 6.8E-01 -0.04 [-0.08 , 0.00] 3.1E-01 F2rl1 0.46 [-0.04 , 0.95] 1.2E-01 0.11 [-0.11 , 0.33] 4.8E-01 -0.01 [-0.05 , 0.02] 7.2E-01 Hras 0.45 [0.18 , 0.73] 1.5E-02 0.06 [-0.10 , 0.22] 5.9E-01 -0.01 [-0.05 , 0.03] 8.1E-01 550 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value C2 0.45 [0.16 , 0.75] 1.9E-02 0.16 [0.03 , 0.29] 1.1E-01 0.06 [0.03 , 0.09] 4.1E-02 Cd1d1 0.45 [-0.01 , 0.91] 1.0E-01 0.18 [0.01 , 0.36] 1.5E-01 0.09 [0.04 , 0.14] 8.4E-02 Cd9 0.45 [0.07 , 0.83] 5.5E-02 0.07 [-0.10 , 0.24] 5.8E-01 -0.02 [-0.04 , 0.00] 1.9E-01 Ms4a1 0.43 [-1.02 , 1.89] 6.1E-01 -0.20 [-0.72 , 0.33] 6.0E-01 -0.21 [-0.44 , 0.01] 3.0E-01 Fas 0.43 [0.18 , 0.68] 1.2E-02 0.10 [0.02 , 0.19] 1.0E-01 0.03 [-0.01 , 0.07] 4.4E-01 Tnfsf10 0.41 [0.08 , 0.75] 4.5E-02 0.11 [0.00 , 0.21] 1.6E-01 -0.02 [-0.06 , 0.03] 6.9E-01 Nfkbia 0.41 [0.13 , 0.69] 2.3E-02 0.17 [0.06 , 0.28] 5.8E-02 0.02 [-0.05 , 0.08] 8.1E-01 Arg2 0.41 [0.10 , 0.72] 3.5E-02 -0.02 [-0.23 , 0.18] 8.9E-01 -0.02 [-0.09 , 0.04] 6.8E-01 Cd59b 0.40 [0.21 , 0.59] 4.3E-03 0.16 [0.03 , 0.29] 9.4E-02 -0.04 [-0.09 , 0.00] 3.1E-01 Il7r 0.40 [-0.47 , 1.27] 4.3E-01 n.d [n.d , n.d] n.d -0.11 [-0.24 , 0.02] 3.6E-01 Cklf 0.40 [0.16 , 0.63] 1.4E-02 0.02 [-0.10 , 0.15] 8.1E-01 -0.01 [-0.06 , 0.03] 8.1E-01 Il6st 0.39 [0.19 , 0.60] 6.7E-03 0.09 [-0.01 , 0.19] 2.3E-01 0.00 [-0.01 , 0.02] 8.1E-01 Elk1 0.38 [0.13 , 0.63] 2.0E-02 0.08 [-0.06 , 0.22] 4.0E-01 0.02 [-0.02 , 0.06] 6.0E-01 Ctsl 0.38 [0.06 , 0.70] 5.3E-02 0.12 [-0.05 , 0.28] 3.3E-01 0.00 [-0.02 , 0.01] 9.0E-01 Notch1 0.38 [0.16 , 0.60] 1.2E-02 0.06 [-0.02 , 0.14] 3.1E-01 0.00 [-0.03 , 0.02] 9.1E-01 Nfatc2 0.38 [0.02 , 0.74] 8.3E-02 0.05 [-0.07 , 0.18] 5.7E-01 -0.02 [-0.06 , 0.03] 7.0E-01 Tgfbr1 0.38 [0.13 , 0.62] 1.8E-02 0.04 [-0.05 , 0.13] 5.7E-01 0.00 [-0.01 , 0.02] 8.0E-01 551 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd1d2 0.38 [-0.05 , 0.80] 1.4E-01 0.16 [-0.03 , 0.35] 2.3E-01 -0.02 [-0.11 , 0.07] 8.0E-01 Jun 0.37 [-0.07 , 0.81] 1.5E-01 -0.04 [-0.25 , 0.18] 8.2E-01 -0.07 [-0.11 , -0.03] 7.7E-02 Rela 0.37 [0.09 , 0.65] 3.5E-02 0.10 [-0.05 , 0.26] 3.3E-01 0.01 [-0.01 , 0.03] 8.0E-01 Tyk2 0.37 [0.11 , 0.62] 2.7E-02 0.09 [-0.02 , 0.21] 2.6E-01 -0.02 [-0.06 , 0.02] 6.5E-01 Bcl10 0.36 [0.12 , 0.61] 2.2E-02 0.09 [-0.04 , 0.22] 3.2E-01 -0.01 [-0.03 , 0.01] 6.9E-01 Msln 0.36 [-0.57 , 1.28] 5.1E-01 0.26 [-0.09 , 0.60] 3.0E-01 -0.05 [-0.19 , 0.10] 7.5E-01 Casp8 0.35 [0.20 , 0.51] 3.0E-03 0.08 [-0.01 , 0.17] 2.0E-01 0.03 [0.02 , 0.04] 1.6E-02 Trp53 0.35 [0.15 , 0.55] 1.0E-02 0.09 [-0.01 , 0.20] 2.2E-01 -0.03 [-0.06 , 0.00] 3.0E-01 Irf5 0.34 [0.09 , 0.60] 3.5E-02 0.02 [-0.07 , 0.11] 8.1E-01 -0.02 [-0.06 , 0.02] 5.2E-01 Itga2b 0.34 [0.07 , 0.62] 4.5E-02 0.27 [0.12 , 0.41] 3.3E-02 0.11 [0.06 , 0.17] 3.1E-02 Itgb1 0.34 [0.13 , 0.55] 1.6E-02 0.09 [-0.05 , 0.22] 3.5E-01 0.01 [-0.01 , 0.02] 5.8E-01 Pml 0.34 [0.14 , 0.54] 1.1E-02 0.08 [0.02 , 0.13] 7.7E-02 0.00 [-0.03 , 0.03] 9.8E-01 Mr1 0.34 [0.02 , 0.66] 8.3E-02 0.12 [0.03 , 0.22] 9.2E-02 0.02 [-0.02 , 0.05] 6.4E-01 Ptgs2 0.33 [-0.46 , 1.12] 4.8E-01 0.09 [-0.28 , 0.46] 7.5E-01 -0.07 [-0.17 , 0.04] 4.9E-01 Ly96 0.33 [0.13 , 0.52] 1.4E-02 0.08 [-0.03 , 0.19] 3.1E-01 -0.02 [-0.05 , 0.01] 4.4E-01 Pin1 0.32 [0.14 , 0.51] 1.1E-02 0.00 [-0.12 , 0.11] 9.7E-01 0.01 [-0.03 , 0.04] 8.4E-01 Eng 0.32 [0.14 , 0.51] 1.2E-02 0.07 [0.02 , 0.12] 7.1E-02 0.00 [-0.03 , 0.02] 8.8E-01 552 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ifnar2 0.32 [0.13 , 0.51] 1.2E-02 0.08 [0.04 , 0.13] 3.4E-02 0.01 [0.00 , 0.03] 4.6E-01 Blnk 0.32 [0.00 , 0.63] 9.4E-02 0.12 [0.03 , 0.20] 8.4E-02 0.00 [-0.03 , 0.02] 9.3E-01 Bst1 0.32 [-0.37 , 1.00] 4.3E-01 0.05 [-0.34 , 0.44] 8.7E-01 -0.10 [-0.24 , 0.04] 4.2E-01 Tfe3 0.31 [0.10 , 0.51] 2.1E-02 0.15 [0.04 , 0.25] 6.6E-02 0.01 [-0.02 , 0.04] 6.4E-01 Alcam 0.31 [0.09 , 0.53] 2.9E-02 0.05 [-0.04 , 0.13] 4.3E-01 0.01 [-0.02 , 0.04] 8.0E-01 Ccl21a 0.31 [-0.55 , 1.17] 5.4E-01 -0.22 [-0.47 , 0.03] 2.2E-01 -0.20 [-0.32 , -0.09] 7.6E-02 Nod1 0.30 [0.15 , 0.46] 6.4E-03 0.02 [-0.02 , 0.06] 5.2E-01 -0.04 [-0.06 , -0.02] 5.9E-02 Ifnar1 0.30 [0.09 , 0.51] 2.6E-02 0.07 [-0.01 , 0.15] 2.2E-01 0.00 [-0.01 , 0.01] 9.4E-01 Cd200 0.30 [0.15 , 0.45] 6.3E-03 0.05 [0.00 , 0.10] 2.0E-01 0.00 [-0.03 , 0.02] 9.1E-01 Traf2 0.29 [0.10 , 0.48] 2.0E-02 0.05 [-0.03 , 0.13] 3.8E-01 -0.01 [-0.03 , 0.01] 6.0E-01 Tank 0.28 [0.08 , 0.49] 3.0E-02 0.07 [-0.02 , 0.17] 3.1E-01 -0.01 [-0.04 , 0.02] 8.1E-01 Traf3 0.28 [0.13 , 0.42] 8.0E-03 -0.01 [-0.08 , 0.07] 9.2E-01 -0.01 [-0.04 , 0.01] 6.0E-01 Stat6 0.27 [0.13 , 0.42] 6.9E-03 0.06 [-0.01 , 0.13] 2.2E-01 0.02 [0.00 , 0.03] 1.7E-01 Ppbp 0.27 [-0.13 , 0.67] 2.4E-01 0.36 [0.10 , 0.62] 7.1E-02 0.11 [-0.04 , 0.26] 4.4E-01 Ikzf2 0.27 [0.00 , 0.54] 9.8E-02 -0.02 [-0.10 , 0.06] 7.0E-01 -0.02 [-0.06 , 0.01] 4.3E-01 Xbp1 0.26 [0.12 , 0.41] 8.9E-03 0.02 [-0.03 , 0.07] 5.9E-01 -0.02 [-0.03 , 0.00] 1.9E-01 Thbs1 0.26 [-0.08 , 0.61] 2.0E-01 -0.07 [-0.24 , 0.10] 5.8E-01 -0.13 [-0.18 , -0.07] 2.3E-02 553 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mill2 0.26 [0.08 , 0.44] 2.2E-02 0.01 [-0.09 , 0.11] 9.0E-01 -0.03 [-0.07 , 0.01] 3.8E-01 Icam2 0.26 [0.14 , 0.37] 3.5E-03 0.00 [-0.13 , 0.13] 9.7E-01 0.00 [-0.04 , 0.03] 9.1E-01 Il11ra1 0.25 [0.04 , 0.47] 5.8E-02 0.07 [-0.05 , 0.19] 3.9E-01 0.00 [-0.04 , 0.05] 9.1E-01 Mapkapk2 0.25 [0.03 , 0.48] 6.5E-02 0.05 [-0.02 , 0.11] 3.3E-01 -0.01 [-0.03 , 0.01] 4.7E-01 Nrp1 0.25 [0.03 , 0.47] 6.1E-02 0.09 [0.02 , 0.16] 7.4E-02 0.02 [0.00 , 0.03] 1.9E-01 Psen2 0.25 [0.01 , 0.49] 8.8E-02 0.07 [0.03 , 0.12] 4.7E-02 0.01 [-0.02 , 0.03] 8.3E-01 Itga1 0.22 [-0.02 , 0.47] 1.3E-01 0.05 [-0.02 , 0.13] 3.3E-01 0.01 [-0.01 , 0.02] 7.1E-01 Il1rap 0.22 [-0.04 , 0.48] 1.5E-01 0.05 [-0.06 , 0.16] 5.4E-01 0.03 [0.00 , 0.07] 3.3E-01 Gata3 0.22 [0.01 , 0.42] 8.4E-02 -0.01 [-0.06 , 0.04] 7.5E-01 -0.03 [-0.05 , -0.01] 1.7E-01 Anp32b 0.20 [0.07 , 0.33] 1.8E-02 0.02 [-0.05 , 0.10] 6.8E-01 0.00 [-0.01 , 0.01] 9.1E-01 Smad2 0.19 [-0.10 , 0.48] 2.7E-01 0.05 [-0.06 , 0.17] 5.5E-01 -0.01 [-0.03 , 0.01] 6.6E-01 Tie1 0.18 [0.14 , 0.23] 5.1E-04 0.02 [-0.05 , 0.09] 7.3E-01 -0.01 [-0.02 , 0.00] 5.4E-01 Irf2 0.18 [0.06 , 0.30] 2.1E-02 0.01 [-0.03 , 0.06] 6.1E-01 0.01 [-0.01 , 0.03] 7.6E-01 Map3k5 0.18 [0.10 , 0.26] 3.3E-03 0.02 [-0.03 , 0.07] 5.7E-01 0.01 [-0.02 , 0.04] 7.4E-01 Ccr9 0.18 [-0.32 , 0.67] 5.4E-01 -0.07 [-0.30 , 0.15] 6.6E-01 -0.03 [-0.10 , 0.04] 6.6E-01 Nos2 0.18 [-0.26 , 0.61] 4.9E-01 0.06 [-0.11 , 0.24] 6.1E-01 -0.07 [-0.15 , 0.02] 3.8E-01 Txnip 0.17 [-0.08 , 0.42] 2.4E-01 0.06 [-0.08 , 0.19] 5.6E-01 0.00 [-0.06 , 0.06] 9.9E-01 554 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Igf1r 0.17 [0.09 , 0.24] 2.7E-03 0.11 [0.07 , 0.15] 1.5E-02 0.05 [0.03 , 0.06] 3.4E-03 Cdh1 0.16 [-0.07 , 0.39] 2.4E-01 0.00 [-0.09 , 0.09] 9.7E-01 -0.03 [-0.06 , -0.01] 1.2E-01 Cspg4 0.16 [-0.01 , 0.33] 1.2E-01 0.09 [0.02 , 0.16] 8.2E-02 0.04 [0.01 , 0.06] 1.4E-01 Il17rb 0.16 [-0.14 , 0.45] 3.7E-01 0.14 [0.05 , 0.22] 5.5E-02 0.07 [0.04 , 0.11] 3.1E-02 Angpt2 0.15 [-0.30 , 0.61] 5.6E-01 0.09 [-0.23 , 0.42] 6.9E-01 -0.08 [-0.19 , 0.04] 4.9E-01 Vegfc 0.15 [-0.03 , 0.33] 1.6E-01 -0.09 [-0.18 , 0.00] 1.9E-01 -0.06 [-0.11 , -0.02] 1.3E-01 Ilf3 0.15 [0.06 , 0.24] 1.2E-02 0.00 [-0.04 , 0.04] 9.5E-01 -0.01 [-0.04 , 0.01] 4.8E-01 Rora 0.15 [0.07 , 0.22] 7.0E-03 0.08 [0.03 , 0.12] 3.7E-02 0.03 [0.02 , 0.05] 4.9E-02 Irak2 0.14 [0.04 , 0.24] 2.4E-02 0.05 [-0.03 , 0.12] 3.9E-01 0.02 [0.00 , 0.04] 3.8E-01 Dusp4 0.14 [-0.09 , 0.37] 2.9E-01 0.15 [0.01 , 0.28] 1.4E-01 0.06 [0.02 , 0.10] 1.6E-01 Tfeb 0.14 [0.02 , 0.26] 4.9E-02 0.01 [-0.03 , 0.06] 6.9E-01 -0.03 [-0.05 , -0.01] 1.6E-01 Creb1 0.14 [0.02 , 0.26] 5.8E-02 -0.02 [-0.07 , 0.02] 4.7E-01 -0.04 [-0.07 , -0.01] 1.8E-01 Mapk3 0.14 [0.04 , 0.23] 2.5E-02 0.07 [0.03 , 0.11] 3.8E-02 0.00 [-0.01 , 0.01] 8.3E-01 Bcl2 0.14 [-0.10 , 0.37] 3.3E-01 0.00 [-0.11 , 0.10] 1.0E+00 -0.05 [-0.09 , -0.01] 1.8E-01 Cd36 0.13 [-0.08 , 0.34] 2.9E-01 -0.08 [-0.20 , 0.04] 3.3E-01 0.01 [-0.03 , 0.05] 7.6E-01 Kit 0.13 [-0.19 , 0.45] 4.9E-01 0.01 [-0.09 , 0.11] 9.0E-01 -0.04 [-0.08 , 0.00] 3.1E-01 Tek 0.13 [0.04 , 0.21] 2.1E-02 -0.01 [-0.11 , 0.08] 8.7E-01 0.00 [-0.02 , 0.02] 9.3E-01 555 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ticam1 0.12 [0.05 , 0.19] 1.4E-02 0.00 [-0.04 , 0.05] 9.0E-01 0.01 [-0.01 , 0.03] 6.1E-01 Ep300 0.12 [0.02 , 0.22] 4.9E-02 0.06 [0.02 , 0.09] 4.5E-02 0.00 [-0.02 , 0.01] 8.7E-01 Masp2 0.12 [-0.26 , 0.50] 6.0E-01 0.08 [-0.15 , 0.31] 6.4E-01 -0.02 [-0.10 , 0.05] 7.6E-01 Nup107 0.12 [-0.06 , 0.29] 2.5E-01 0.03 [-0.04 , 0.10] 6.0E-01 0.00 [-0.02 , 0.01] 9.1E-01 Bax 0.12 [-0.05 , 0.28] 2.3E-01 0.01 [-0.08 , 0.11] 8.6E-01 -0.01 [-0.03 , 0.01] 5.2E-01 Ythdf2 0.11 [0.01 , 0.22] 7.9E-02 0.04 [-0.02 , 0.10] 3.2E-01 0.00 [-0.01 , 0.02] 8.1E-01 Tbk1 0.11 [0.00 , 0.21] 8.9E-02 0.04 [-0.02 , 0.09] 3.9E-01 -0.02 [-0.03 , 0.00] 3.7E-01 Smad3 0.11 [-0.01 , 0.22] 1.3E-01 -0.01 [-0.06 , 0.04] 8.4E-01 -0.03 [-0.05 , -0.01] 1.7E-01 Epcam 0.11 [-0.16 , 0.38] 5.0E-01 0.02 [-0.13 , 0.17] 8.7E-01 -0.04 [-0.10 , 0.01] 4.1E-01 Prkcd 0.10 [-0.04 , 0.25] 2.3E-01 0.02 [-0.02 , 0.07] 4.6E-01 0.01 [-0.01 , 0.02] 5.6E-01 Atg7 0.10 [-0.03 , 0.23] 1.9E-01 0.03 [-0.02 , 0.08] 4.1E-01 0.01 [0.00 , 0.02] 5.2E-01 Prkce 0.10 [-0.03 , 0.22] 2.0E-01 0.01 [-0.04 , 0.07] 7.5E-01 0.00 [-0.02 , 0.02] 9.4E-01 Nfatc3 0.09 [0.00 , 0.18] 1.0E-01 0.01 [-0.03 , 0.06] 6.5E-01 0.01 [-0.01 , 0.03] 5.2E-01 Abl1 0.09 [-0.03 , 0.21] 1.9E-01 0.05 [0.02 , 0.07] 3.3E-02 0.00 [-0.02 , 0.01] 9.4E-01 Smad4 0.09 [-0.03 , 0.20] 1.9E-01 0.00 [-0.03 , 0.04] 8.7E-01 -0.01 [-0.02 , 0.01] 6.6E-01 Zfp13 0.09 [-0.15 , 0.33] 5.4E-01 0.05 [-0.04 , 0.13] 4.6E-01 0.00 [-0.03 , 0.03] 9.1E-01 Atm 0.08 [-0.02 , 0.19] 1.9E-01 0.02 [-0.04 , 0.08] 6.0E-01 0.00 [-0.02 , 0.03] 8.8E-01 556 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Hif1a 0.08 [-0.08 , 0.24] 3.8E-01 0.00 [-0.07 , 0.07] 9.5E-01 0.00 [-0.03 , 0.02] 9.2E-01 Map2k1 0.08 [-0.01 , 0.17] 1.3E-01 0.04 [0.01 , 0.08] 8.2E-02 0.00 [-0.01 , 0.01] 9.8E-01 Reps1 0.08 [-0.04 , 0.19] 2.6E-01 0.02 [-0.03 , 0.07] 6.1E-01 -0.02 [-0.04 , 0.00] 3.7E-01 Dpp4 0.07 [-0.12 , 0.26] 5.1E-01 0.03 [-0.03 , 0.08] 4.9E-01 0.01 [-0.01 , 0.03] 5.3E-01 Nt5e 0.07 [-0.08 , 0.22] 4.2E-01 -0.13 [-0.23 , -0.03] 8.7E-02 -0.09 [-0.11 , -0.07] 2.5E-04 Ikbkb 0.07 [-0.02 , 0.16] 1.7E-01 -0.01 [-0.05 , 0.04] 8.7E-01 -0.01 [-0.03 , 0.01] 5.2E-01 Psen1 0.07 [0.00 , 0.14] 9.4E-02 0.01 [-0.02 , 0.03] 7.5E-01 0.00 [-0.01 , 0.01] 9.0E-01 Tnfsf15 0.07 [-0.06 , 0.20] 3.5E-01 0.12 [0.02 , 0.23] 1.1E-01 0.05 [0.01 , 0.10] 1.7E-01 Egfr 0.07 [-0.04 , 0.17] 2.6E-01 0.00 [-0.07 , 0.06] 9.2E-01 -0.01 [-0.03 , 0.02] 8.3E-01 Cd3eap 0.07 [-0.12 , 0.25] 5.4E-01 -0.01 [-0.14 , 0.12] 9.0E-01 -0.02 [-0.05 , 0.02] 6.6E-01 Cxcl12 0.07 [-0.16 , 0.29] 6.2E-01 -0.09 [-0.25 , 0.08] 4.6E-01 -0.01 [-0.05 , 0.03] 7.6E-01 Ikbkg 0.06 [-0.05 , 0.18] 3.5E-01 0.00 [-0.06 , 0.06] 9.3E-01 -0.01 [-0.03 , 0.01] 6.6E-01 Smn1 0.06 [-0.04 , 0.17] 2.9E-01 0.02 [-0.03 , 0.07] 5.9E-01 0.00 [-0.02 , 0.02] 8.8E-01 Cyld 0.06 [-0.11 , 0.23] 5.2E-01 0.02 [-0.07 , 0.11] 8.1E-01 -0.01 [-0.06 , 0.03] 7.9E-01 Ltbr 0.06 [-0.07 , 0.20] 4.3E-01 -0.01 [-0.04 , 0.03] 8.1E-01 0.01 [0.00 , 0.02] 4.9E-01 Tirap 0.06 [-0.09 , 0.21] 4.8E-01 0.04 [-0.04 , 0.13] 4.7E-01 -0.01 [-0.04 , 0.02] 6.2E-01 Tnfsf12 0.05 [-0.08 , 0.18] 4.8E-01 0.06 [0.00 , 0.12] 1.8E-01 -0.02 [-0.04 , 0.00] 3.2E-01 557 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il13ra1 0.05 [-0.12 , 0.22] 6.2E-01 -0.05 [-0.12 , 0.03] 3.6E-01 -0.05 [-0.07 , -0.03] 2.5E-02 Itga6 0.04 [-0.19 , 0.27] 7.6E-01 -0.16 [-0.29 , -0.03] 1.1E-01 -0.14 [-0.17 , -0.11] 2.5E-04 Irf3 0.04 [-0.03 , 0.11] 3.1E-01 0.00 [-0.03 , 0.04] 8.9E-01 -0.01 [-0.02 , 0.00] 3.8E-01 Ewsr1 0.04 [-0.03 , 0.10] 3.1E-01 0.01 [-0.02 , 0.04] 6.4E-01 0.00 [-0.01 , 0.01] 6.8E-01 Hspb2 0.04 [-0.27 , 0.35] 8.4E-01 0.08 [-0.10 , 0.26] 5.4E-01 -0.05 [-0.10 , 0.01] 3.8E-01 App 0.04 [-0.01 , 0.08] 1.7E-01 0.02 [-0.01 , 0.05] 2.5E-01 0.00 [-0.01 , 0.01] 9.1E-01 Mertk 0.04 [-0.08 , 0.15] 6.1E-01 0.07 [-0.09 , 0.22] 5.7E-01 0.06 [0.03 , 0.08] 2.3E-02 Jak1 0.03 [-0.07 , 0.14] 5.7E-01 0.02 [-0.03 , 0.06] 6.4E-01 0.01 [-0.01 , 0.02] 5.9E-01 Bmi1 0.03 [-0.03 , 0.10] 3.5E-01 0.01 [-0.03 , 0.06] 6.9E-01 0.00 [-0.02 , 0.01] 8.2E-01 Ccl25 0.03 [-0.15 , 0.21] 7.5E-01 0.08 [0.00 , 0.17] 1.6E-01 -0.01 [-0.04 , 0.03] 8.5E-01 Psmd7 0.03 [-0.03 , 0.09] 3.9E-01 -0.01 [-0.04 , 0.03] 7.7E-01 -0.01 [-0.02 , 0.00] 1.9E-01 Map3k7 0.03 [-0.08 , 0.14] 6.7E-01 0.01 [-0.04 , 0.05] 8.9E-01 0.00 [-0.01 , 0.02] 8.5E-01 Syt17 0.03 [-0.13 , 0.18] 7.7E-01 -0.03 [-0.09 , 0.04] 5.9E-01 -0.01 [-0.04 , 0.01] 5.8E-01 Egr1 0.02 [-0.80 , 0.84] 9.7E-01 -0.18 [-0.50 , 0.14] 4.4E-01 -0.13 [-0.24 , -0.02] 2.0E-01 Map4k2 0.02 [-0.10 , 0.14] 7.6E-01 0.02 [-0.07 , 0.11] 8.1E-01 0.02 [0.01 , 0.03] 3.1E-02 Yy1 0.02 [-0.04 , 0.07] 6.0E-01 0.02 [-0.01 , 0.05] 3.9E-01 0.00 [-0.01 , 0.00] 5.8E-01 Il17ra 0.01 [-0.06 , 0.08] 8.1E-01 -0.02 [-0.07 , 0.04] 6.7E-01 0.00 [-0.01 , 0.02] 8.1E-01 558 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il22ra1 0.01 [-0.27 , 0.28] 9.7E-01 0.12 [0.01 , 0.23] 1.4E-01 -0.05 [-0.11 , 0.01] 3.7E-01 Cfb 0.00 [-0.20 , 0.21] 9.7E-01 0.04 [-0.05 , 0.14] 5.6E-01 0.03 [0.00 , 0.06] 2.4E-01 Itch 0.00 [-0.14 , 0.15] 9.7E-01 0.06 [-0.07 , 0.18] 5.4E-01 0.07 [0.04 , 0.11] 2.3E-02 Angpt1 0.00 [-0.16 , 0.16] 9.7E-01 -0.06 [-0.16 , 0.03] 3.4E-01 -0.02 [-0.06 , 0.01] 4.9E-01 Tab1 0.00 [-0.09 , 0.09] 9.7E-01 0.03 [-0.04 , 0.11] 5.6E-01 0.03 [0.02 , 0.05] 3.1E-02 Crebbp 0.00 [-0.08 , 0.08] 9.9E-01 -0.04 [-0.08 , 0.00] 1.9E-01 -0.01 [-0.02 , 0.00] 3.3E-01 Tmed1 0.00 [-0.10 , 0.10] 9.9E-01 -0.01 [-0.06 , 0.04] 7.9E-01 -0.01 [-0.02 , 0.01] 6.0E-01 Casp3 0.00 [-0.12 , 0.12] 9.7E-01 0.00 [-0.04 , 0.03] 9.5E-01 -0.01 [-0.03 , 0.00] 3.4E-01 Stat5b -0.01 [-0.14 , 0.12] 9.2E-01 0.01 [-0.07 , 0.08] 9.0E-01 0.02 [0.00 , 0.04] 3.8E-01 Atf1 -0.01 [-0.20 , 0.18] 9.4E-01 -0.06 [-0.12 , 0.01] 2.2E-01 -0.01 [-0.04 , 0.02] 7.0E-01 Cd81 -0.01 [-0.07 , 0.05] 7.8E-01 -0.01 [-0.06 , 0.03] 6.8E-01 0.00 [-0.01 , 0.01] 9.9E-01 Cd63 -0.01 [-0.11 , 0.09] 8.4E-01 -0.03 [-0.06 , 0.00] 1.9E-01 -0.02 [-0.03 , 0.00] 1.4E-01 Lamp1 -0.01 [-0.12 , 0.09] 8.3E-01 0.00 [-0.05 , 0.05] 9.6E-01 0.01 [0.00 , 0.02] 3.1E-01 Atg16l1 -0.02 [-0.11 , 0.08] 7.8E-01 -0.04 [-0.08 , 0.00] 1.4E-01 -0.01 [-0.03 , 0.00] 3.2E-01 Traf6 -0.02 [-0.11 , 0.08] 7.8E-01 -0.01 [-0.04 , 0.03] 8.1E-01 0.00 [-0.02 , 0.01] 8.7E-01 Mapk1 -0.02 [-0.08 , 0.05] 6.5E-01 -0.01 [-0.04 , 0.03] 8.1E-01 0.00 [-0.01 , 0.01] 8.8E-01 Mavs -0.02 [-0.11 , 0.07] 7.5E-01 0.08 [0.04 , 0.13] 3.3E-02 0.03 [0.01 , 0.04] 1.3E-01 559 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Map2k4 -0.02 [-0.14 , 0.09] 7.4E-01 -0.02 [-0.06 , 0.02] 5.8E-01 0.00 [-0.01 , 0.01] 7.9E-01 Lamp2 -0.03 [-0.13 , 0.07] 6.5E-01 -0.01 [-0.06 , 0.04] 7.6E-01 0.01 [-0.01 , 0.02] 5.4E-01 Irak1 -0.03 [-0.10 , 0.04] 5.0E-01 -0.01 [-0.05 , 0.03] 7.4E-01 -0.01 [-0.02 , 0.01] 6.6E-01 Jam3 -0.03 [-0.17 , 0.11] 7.2E-01 -0.01 [-0.12 , 0.09] 8.7E-01 0.01 [-0.01 , 0.04] 6.4E-01 Il15ra -0.03 [-0.19 , 0.13] 7.5E-01 0.08 [-0.02 , 0.17] 2.5E-01 0.01 [-0.01 , 0.04] 5.7E-01 Atg12 -0.03 [-0.13 , 0.07] 6.0E-01 0.02 [-0.03 , 0.07] 6.1E-01 0.00 [-0.02 , 0.01] 8.8E-01 Tollip -0.03 [-0.09 , 0.03] 3.9E-01 -0.01 [-0.07 , 0.05] 8.0E-01 0.01 [0.00 , 0.02] 4.1E-01 Igf2r -0.03 [-0.13 , 0.07] 5.8E-01 0.06 [0.01 , 0.12] 1.2E-01 0.06 [0.04 , 0.08] 5.0E-03 Fcgr2b -0.03 [-0.20 , 0.13] 7.3E-01 -0.03 [-0.11 , 0.05] 5.9E-01 -0.02 [-0.05 , 0.01] 4.4E-01 Erbb2 -0.04 [-0.12 , 0.04] 4.1E-01 0.00 [-0.07 , 0.06] 9.6E-01 -0.01 [-0.03 , 0.01] 4.5E-01 Vhl -0.04 [-0.23 , 0.15] 7.4E-01 0.06 [-0.01 , 0.13] 2.6E-01 -0.02 [-0.05 , 0.02] 6.4E-01 Atf2 -0.04 [-0.13 , 0.05] 4.3E-01 -0.01 [-0.07 , 0.05] 8.1E-01 0.01 [-0.01 , 0.02] 6.0E-01 Jak2 -0.04 [-0.13 , 0.05] 4.4E-01 -0.01 [-0.05 , 0.04] 8.2E-01 0.00 [-0.01 , 0.02] 8.1E-01 Ubc -0.06 [-0.15 , 0.04] 2.9E-01 -0.04 [-0.11 , 0.03] 3.9E-01 -0.01 [-0.03 , 0.00] 3.8E-01 Cd164 -0.06 [-0.12 , 0.00] 9.5E-02 -0.06 [-0.12 , -0.01] 9.9E-02 -0.02 [-0.03 , -0.01] 1.4E-03 Ccl27a -0.06 [-0.26 , 0.14] 6.0E-01 -0.02 [-0.09 , 0.04] 6.0E-01 -0.05 [-0.07 , -0.02] 7.6E-02 Bcl2l1 -0.06 [-0.17 , 0.04] 2.9E-01 -0.03 [-0.07 , 0.02] 4.0E-01 0.00 [-0.04 , 0.04] 9.4E-01 560 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Psma2 -0.07 [-0.12 , -0.01] 6.4E-02 -0.04 [-0.08 , -0.01] 7.3E-02 0.00 [-0.01 , 0.01] 7.6E-01 Atg10 -0.07 [-0.18 , 0.04] 2.8E-01 -0.03 [-0.08 , 0.03] 5.4E-01 -0.02 [-0.04 , 0.00] 3.1E-01 Mapk14 -0.09 [-0.20 , 0.02] 1.7E-01 -0.01 [-0.08 , 0.07] 9.2E-01 0.02 [0.00 , 0.04] 1.9E-01 Map2k2 -0.09 [-0.18 , 0.00] 9.8E-02 -0.03 [-0.08 , 0.03] 5.1E-01 0.01 [0.00 , 0.02] 3.7E-01 Mif -0.09 [-0.28 , 0.10] 4.2E-01 -0.04 [-0.19 , 0.10] 6.9E-01 0.01 [-0.01 , 0.03] 6.6E-01 Rps6 -0.09 [-0.19 , 0.01] 1.4E-01 -0.04 [-0.09 , 0.01] 2.3E-01 -0.03 [-0.05 , -0.01] 1.7E-01 Gpi1 -0.09 [-0.16 , -0.03] 2.7E-02 -0.01 [-0.06 , 0.04] 8.6E-01 0.01 [0.00 , 0.02] 2.1E-01 Mapk8 -0.10 [-0.21 , 0.01] 1.4E-01 -0.02 [-0.05 , 0.02] 5.1E-01 -0.01 [-0.02 , 0.00] 3.7E-01 Fadd -0.10 [-0.23 , 0.03] 1.9E-01 -0.03 [-0.13 , 0.06] 6.1E-01 -0.02 [-0.04 , 0.01] 4.1E-01 Ctsh -0.10 [-0.20 , -0.01] 7.2E-02 -0.07 [-0.18 , 0.04] 3.4E-01 -0.02 [-0.03 , -0.01] 9.3E-02 Hmgb1 -0.11 [-0.20 , -0.01] 6.5E-02 -0.02 [-0.08 , 0.04] 6.1E-01 0.00 [-0.01 , 0.01] 9.9E-01 Dock9 -0.11 [-0.22 , 0.00] 9.2E-02 -0.01 [-0.12 , 0.09] 8.8E-01 0.01 [0.00 , 0.03] 4.1E-01 H2-Q10 -0.11 [-1.02 , 0.80] 8.4E-01 0.01 [-0.64 , 0.65] 9.9E-01 -0.01 [-0.25 , 0.22] 9.4E-01 Chuk -0.11 [-0.21 , -0.01] 6.8E-02 -0.06 [-0.13 , 0.00] 1.7E-01 0.02 [0.00 , 0.03] 1.5E-01 Gtf3c1 -0.12 [-0.20 , -0.04] 1.8E-02 -0.06 [-0.14 , 0.02] 2.8E-01 -0.02 [-0.04 , -0.01] 1.7E-01 Atg5 -0.13 [-0.23 , -0.03] 4.3E-02 -0.04 [-0.11 , 0.02] 3.1E-01 -0.02 [-0.05 , 0.00] 1.9E-01 Psmb7 -0.15 [-0.22 , -0.07] 5.8E-03 -0.06 [-0.12 , 0.00] 1.8E-01 0.00 [-0.01 , 0.02] 6.6E-01 561 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccnd3 -0.15 [-0.25 , -0.05] 2.0E-02 0.00 [-0.10 , 0.09] 9.6E-01 0.01 [0.00 , 0.02] 5.0E-01 Cmah -0.16 [-0.35 , 0.03] 1.7E-01 -0.14 [-0.34 , 0.07] 3.4E-01 -0.04 [-0.06 , -0.01] 1.7E-01 Pla2g6 -0.17 [-0.33 , -0.01] 7.2E-02 -0.06 [-0.14 , 0.03] 3.4E-01 -0.03 [-0.05 , 0.00] 2.0E-01 Il15 -0.17 [-0.37 , 0.02] 1.3E-01 -0.07 [-0.20 , 0.06] 4.2E-01 0.02 [-0.02 , 0.06] 5.4E-01 Kdr -0.18 [-0.40 , 0.05] 1.9E-01 -0.07 [-0.22 , 0.07] 4.8E-01 -0.01 [-0.03 , 0.01] 5.6E-01 Ms4a2 -0.19 [-0.60 , 0.23] 4.4E-01 -0.15 [-0.37 , 0.08] 3.6E-01 0.02 [-0.03 , 0.07] 6.7E-01 Plau -0.19 [-0.44 , 0.06] 2.0E-01 -0.08 [-0.17 , 0.01] 2.2E-01 -0.09 [-0.13 , -0.05] 2.6E-02 Ceacam1 -0.19 [-0.42 , 0.03] 1.5E-01 -0.08 [-0.22 , 0.05] 3.9E-01 0.02 [0.00 , 0.04] 2.0E-01 Mme -0.20 [-0.43 , 0.04] 1.6E-01 -0.12 [-0.24 , 0.00] 1.8E-01 -0.04 [-0.06 , -0.01] 8.7E-02 Dusp6 -0.23 [-0.54 , 0.07] 2.0E-01 0.03 [-0.11 , 0.17] 8.0E-01 -0.02 [-0.08 , 0.04] 6.8E-01 C1qbp -0.24 [-0.40 , -0.09] 1.8E-02 -0.07 [-0.18 , 0.03] 3.4E-01 0.01 [0.00 , 0.02] 2.1E-01 Sigirr -0.25 [-0.45 , -0.06] 3.8E-02 -0.09 [-0.22 , 0.04] 3.1E-01 -0.03 [-0.05 , -0.02] 5.8E-03 Bid -0.26 [-0.44 , -0.07] 3.1E-02 -0.03 [-0.17 , 0.11] 8.1E-01 0.02 [-0.02 , 0.05] 6.0E-01 Ecsit -0.26 [-0.41 , -0.11] 1.1E-02 -0.03 [-0.15 , 0.09] 7.1E-01 0.00 [-0.02 , 0.02] 8.7E-01 Pdgfc -0.27 [-0.46 , -0.08] 2.8E-02 -0.04 [-0.12 , 0.04] 4.6E-01 0.01 [-0.02 , 0.04] 7.9E-01 Defb1 -0.29 [-0.53 , -0.04] 5.4E-02 -0.09 [-0.19 , 0.02] 2.5E-01 -0.02 [-0.05 , 0.01] 5.3E-01 Vegfa -0.30 [-0.60 , 0.00] 9.5E-02 -0.09 [-0.34 , 0.15] 6.0E-01 0.00 [-0.01 , 0.02] 6.6E-01 562 Table 36 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in kidney at 13 wk post final exposure cSiO2 + CON mRNA cSiO2 + 0.4% DHA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Rorc -0.30 [-0.56 , -0.05] 5.1E-02 -0.01 [-0.17 , 0.14] 9.1E-01 0.05 [0.02 , 0.08] 1.3E-01 Abcb1a -0.32 [-0.71 , 0.07] 1.6E-01 -0.08 [-0.25 , 0.08] 4.7E-01 -0.02 [-0.05 , 0.01] 4.0E-01 Maf -0.34 [-0.56 , -0.12] 1.9E-02 -0.06 [-0.22 , 0.11] 6.3E-01 0.01 [-0.03 , 0.05] 8.0E-01 C8g -0.34 [-0.63 , -0.06] 5.1E-02 -0.16 [-0.44 , 0.13] 4.3E-01 0.02 [0.00 , 0.04] 2.2E-01 Cyfip2 -0.36 [-0.67 , -0.04] 6.5E-02 -0.10 [-0.32 , 0.13] 5.7E-01 0.01 [-0.03 , 0.04] 8.7E-01 Dll4 -0.46 [-0.77 , -0.15] 2.3E-02 -0.21 [-0.32 , -0.10] 3.3E-02 -0.13 [-0.18 , -0.08] 1.1E-02 Ambp -0.51 [-1.14 , 0.12] 1.7E-01 0.09 [-0.40 , 0.59] 8.1E-01 -0.04 [-0.19 , 0.12] 8.2E-01 C8a -0.60 [-1.14 , -0.07] 6.4E-02 -0.33 [-0.79 , 0.12] 3.1E-01 -0.06 [-0.11 , -0.01] 2.0E-01 Il17f -0.61 [-1.16 , -0.06] 6.8E-02 -0.32 [-0.61 , -0.03] 1.4E-01 -0.12 [-0.19 , -0.05] 7.6E-02 Tfrc -0.78 [-1.25 , -0.30] 1.4E-02 -0.27 [-0.55 , 0.02] 1.9E-01 0.01 [-0.02 , 0.04] 7.3E-01 Mbl2 -0.82 [-1.34 , -0.31] 1.5E-02 -0.30 [-0.72 , 0.12] 3.2E-01 -0.09 [-0.20 , 0.02] 3.8E-01 563 REFERENCES 564 REFERENCES 1. 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The latter is evidenced by the downregulation of multiple pathways that contribute to the pathological sequalae that drive cSiO 2-triggered autoimmunity. DHA attenuates inflammation induced by airway cSiO2 exposure from the early stages of initiation of autoimmunity in the lung to exacerbation of systemic autoimmunity in the kidney. The results further indicate that cSiO2 -triggered ELT in the lung is a major precursor of this response. Given that cSiO2-induced cellular infiltration in the lungs is suppressed by DHA within 1 wk after the final cSiO2 exposure, a likely primary target of DHA is blocking the recruitment of autoreactive lymphocytes that infiltrate the lung after cSiO2 exposure. A number of aberrant immune pathways known to contribute to human lupus were induced here in the lupus-prone NZBWF1 mouse following intranasal cSiO2 exposure; remarkably, these were suppressed by DHA. Importantly, multiple endpoints targeted by DHA are individually targeted by expensive immunotherapies approved for or in clinical trials for lupus and other AD. Thus, dietary supplementation with DHA could be employed as a broad approach to block multiple pathways that collectively influence the progression to autoimmunity in susceptible individuals. Workers employed in occupations with high exposure to cSiO 2 and potentially other toxicants 588 could reduce their risk for developing lupus or other autoimmune diseases by consuming DHA. Other at-risk individuals for autoimmunity, including people with a known genetic predisposition or those in remission, could potentially employ dietary supplementation with DHA to prevent onset of active autoimmunity or progression of existing autoimmunity. To summarize, using lupus-prone NZBWF1 mice, we have established a model that permits dissection of the countervailing roles of cSiO 2 (potentiation) and DHA (attenuation) in initiation and progression of autoimmunity. The findings presented in this dissertation provide putative avenues by how dietary modification with the ω-3 PUFA, DHA, can be exploited to counter environmental triggers, such as cSiO2, that elicit autoimmunity. However, several questions remain to be answered that will further our understanding of the dynamic interplay between countervailing environmental factors and autoimmunity. Future Directions Dietary DHA as an intervention after cSiO2 exposure The studies in this dissertation focused on a prophylactic approach with DHA following toxicant exposure, thus empowering at-risk individuals with a safe, low-cost, protective approach to reduce their risk of lupus or potentially other autoimmune diseases. An additional study of interest is employing DHA as an intervention following airway toxicant exposure and assessing its effectiveness to halt the exacerbated progression of autoimmunity. Preliminary results from our laboratory (Akbari, P., unpublished observations) support such an approach. This opens the door to number of other studies that could be employed to assess the effectiveness of a dietary approach with DHA compared to conventional therapeutics. 589 Early events after cSiO2 exposure that exacerbate systemic autoimmunity in lupus-prone mice The findings here demonstrate that inflammation initially localized to the lung following intranasal cSiO2 exposure ultimately affects systemic and distal tissues. Findings in Chapter 4 suggest that the primary effect of cSiO2 occurs early in the pathogenesis of cSiO2-triggered autoimmunity (within the 4 wk cSiO2 exposure period). Preliminary data in Appendix A support this contention and a suppressive role for DHA at these early time points. It is possible that DHA influences cSiO2 phagocytosis by alveolar macrophages and thus modulates resultant cell death. While findings in Appendix B suggest that DHA supplementation in vitro does not impair phagocytosis of cSiO2 particles, DHA may influence cSiO2-induced macrophage cell death or phagocytosis of resultant cell corpses. Cell death in alveolar macrophages by phagocytosis of cSiO2 in vitro and in vivo occurs by two principal mechanisms – pyroptosis and apoptosis [1–7]. Pyroptosis occurs in macrophages following inflammasome-mediated activation of caspase-1 and is concurrent with IL-1β secretion [8,9]. Aberrant inflammasome activation has been linked to lupus [10], and importantly, is known to be suppressed by DHA and its metabolites [11,12]. In contrast, apoptosis following cSiO2 exposure is a consequence of intracellular proteases [13]. Defective phagocytosis of cell corpses (efferocytosis) may result in release of nuclear antigens that trigger autoimmunity. It is well known that a pathological feature in lupus is defective efferocytosis [14–16]. Importantly, DHA and its metabolites have been shown to enhance efferocytosis, which could prevent the release of nuclear antigens elicited by aberrant cSiO 2-induced cell death that trigger autoimmunity [17–19]. Future in vivo and in vitro experiments should address DHA modulation of cell death following cSiO2 exposure. BALF from cSiO2-exposed NZBWF1 mice fed with or without DHA 590 could be analyzed for the presence of extracellular nucleosomes (complexes of DNA and histones) using commercial assays such as the Roche Cell Death ELISAPLUS (catalog #11774425001). A decrease of nucleosomes in cSiO2-exposed, DHA fed mice would suggest that DHA either reduces cell death and/or enhances phagocytosis of cell corpses. Thereafter, in vitro experiments can be conducted to address how cSiO2 induces cell death in alveolar macrophages from NZBWF1 mice and if DHA alters cell death and/or its consequences. Possible avenues to pursue include identifying if DHA supplementation 1) protects alveolar macrophages from cSiO 2-induced cell death or 2) modulates cell death to a less inflammatory phenotype. In the latter scenario, it will be critical to first identify the mode of cell death following cSiO 2 exposure in vitro and if DHA is capable of shifting cell death to a different mechanism. Once the mechanism of cell death is elucidated, it can then be determined if DHA impacts the consequences of this mode of cell death (e.g. decreased inflammasome activation/IL-1β secretion if pyroptosis is the predominate mode of cell death, enhanced efferocytosis of cell corpses if pyroptosis and/or apoptosis is the predominate form of cell death). Towards this end, an in vitro assay to measure efferocytosis is discussed in Appendix B. Furthermore, understanding the role of DHA or its metabolites by these putative mechanisms could be addressed using cyclooxygenase/lipoxygenase inhibitors in vitro that block the formation of lipid metabolites following cSiO2 exposure. It would be of further interest to investigate if this mode of cell death is unique to lupus-prone NZBWF1 mice or if this strain is more sensitive to cell death compared to non-autoimmune mouse strains. An additional analysis that could be performed is to determine if DHA supplementation decreases the immunogenicity of nuclear components released during pyroptosis and/or aberrant apoptosis. Differences in the immunoreactivity of nuclear components attributed to DHA could be preliminarily assessed with conditioned media from the aforementioned in vitro experiments by 591 culturing immature dendritic cells and then measuring dendritic cell activation markers (e.g. CD80 and CD86) by flow cytometry. Susceptibility of male NZBWF1 mice to autoimmune triggering by cSiO2 exposure An additional study of interest is to expose male NZBWF1 mice to intranasal cSiO2 exposure. Unlike female NZBWF1 mice, male NZBWF1 mice do not develop autoimmunity. Thus, if cSiO2 exposure in male NZBWF1 mice can incite autoimmunity, this finding would open the door to future investigations that may identify at-risk people to environmental-toxicant triggers of autoimmunity. Effect of cSiO2 on otherAD in murine models of autoimmunity cSiO2 exposure has been linked to not only lupus, but several other AD including rheumatoid arthritis, Sjӧgren’s syndrome, systemic sclerosis, and systemic vasculitis [20–25]. While cSiO2 is speculated to trigger other autoimmune diseases, solid conclusions are inhibited by a lack of sufficiently powered human epidemiological studies and murine studies that reinforce these associations. Murine models that spontaneously develop features of human autoimmune diseases are an invaluable tool to study the effects of environmental triggers, such as cSiO 2, on development of autoimmunity [26,27]. To determine if airway cSiO2 exposure triggers other autoimmune diseases, various murine models can be exploited for this purpose. Experimental autoimmune encephalomyelitis is a commonly used murine model of multiple sclerosis that is induced in various strains of mice following immunization with myelin peptides or by adoptive transfer of myelin-specific CD4+ T cells [28]. Features of rheumatoid arthritis can be induced in murine models following collagen injection and is dependent on TNF-α and IL-1β [29]. Given that these cytokines are induced 592 following cSiO2 exposure [1,3,30,31], it should be expected that cSiO2 exposure exacerbates collagen-induced arthritis. However, considering that the aforementioned models of autoimmunity require induction of autoimmunity by administration of exogenous agents, dissecting the individual contribution of an additional exogenous treatment, cSiO 2, may be difficult. Towards this end, lupus-prone mouse strains that spontaneously develop neurological and arthritic manifestations of lupus, such as the MRL/lpr mouse, may be useful as a “screening tool” to identify tissue-specific pathologies outside of the lung and kidneys impacted by cSiO 2 exposure [32]. However, most spontaneous murine models of lupus develop severe glomerulonephritis [32], therefore, mice could succumb to glomerulonephritis prior to the appearance of other tissue pathologies. In that case, mouse models that develop more mild glomerulonephritis, such as the pristane-induced murine model of lupus, may be advantageous [33]. Global gene deletion of adhesion molecules have been demonstrated to be protective against lupus nephritis in spontaneous models of murine lupus [34,35]. However, global deletion of these molecules would be expected to decrease cellular infiltration and resultant pathology in all tissues, thus limiting their application to identifying extra-renal tissue pathologies following cSiO2 exposure. Particularly useful would be the development of conditional knock out strains that specifically block cellular infiltration into the kidney, yet permit infiltration into other tissues. However, conditional knockouts in murine models that spontaneously develop lupus are unavailable at this time. Additional environmental factors that trigger autoimmunity The ambiguity of the role of environmental factors and autoimmunity is highlighted in several publications [21–23,36,37,37–39]. The results presented here are one step in filling in critical gaps in knowledge, however, a number of significant questions remain: how do 593 environmental toxicants trigger autoimmunity? What other toxicants trigger autoimmunity? Is DHA effective in all these toxic triggers? To address these questions, it would be of interest to compare other airborne toxicants suspected to contribute to autoimmunity using the NZBWF1 mouse. Potential toxicants include ozone [37,39], cigarette smoke [40,41], as well as airborne particles collected from urban or rural areas with autoimmune disease incidences above what would be expected in a comparable population [36,42–44]. If it is found that pathological features of autoimmune diseases triggered by these putative factors are similar to cSiO 2, this might suggest a common mechanism is induced by these toxicants that mediate the progression to autoimmunity. Disparate findings would indicate unique mechanisms of each toxicant, thus prompting further investigation into related environmental factors with similar toxic mechanisms of action. Ultimately, these findings could be applied to classify environmental triggers of autoimmunity and lead to approaches applied to protect exposed individuals. Utility of the plasma lipidome to identify individuals at-risk for SLE Analytical and computational advances in “-omics” technologies have led to the emergence of lipidomics, the qualitative representation and quantification of all lipids present in the human ‘lipidome’[45,46]. Altered or impaired lipid metabolism occurs in several known diseases, including SLE [47–50], cardiovascular disease [51–53], and colorectal cancer [54,55]. Human prospective studies that have defined patterns of a ‘normal’ lipidome vs one uniquely expressed in a given disease are limited [56–59]. Therefore, application of DHA in cSiO2 -exposed mice could uncover unique lipid biomarkers that could be exploited to 1) predict human susceptibility to toxicant-triggered AD and 2) determine thresholds for protective effects of ω-3 PUFAs in susceptible populations. These findings could be validated with archived, serial blood samples 594 obtained from individuals with SLE and analyzed for lipid biomarkers in plasma and/or ω-3:ω-6 in RBCs that correlate to disease severity or indicate impending disease onset. 595 APPENDICES 596 APPENDIX A Short-term in vivo experiments with cSiO2 in NZBWF1 mice Introduction The findings in this dissertation have well established that dietary DHA is effective at preventing the infiltration of autoreactive lymphocytes into the lung following cSiO2 exposure. The results presented in Chapter 4 indicate that the ability of DHA to counter ELT in the lung were evident within 1 wk post final exposure to cSiO2. Therefore, we still have not fully elucidated the early triggers induced by cSiO2 exposure in the lung. Short-term in vivo experiments will contribute substantially towards this effort. With these experiments, it will be possible to address the initial trigger that sets off the cascade of events following cSiO2 exposure and how DHA specifically counteracts this trigger. Experimental design At 6 wks of age, NZBWF1 mice were assigned either CON or 1.0% DHA and maintained on experimental diets until termination. At 8 wk of age, mice received VEH or 1.0 mg cSiO2. One cohort of mice was sacrificed 24 hr after a single exposure to cSiO2 (acute exposure). The second cohort of mice received 1.0 mg cSiO2 once per wk, for 4 wk, and were sacrificed 24 hr after the final exposure to cSiO2. RNA was isolated from lung tissue stored in RNAlater and then asssed for inflammatory gene expression with the Mouse PanCancer Immune Profiling Panel. Preliminary data While at first glance undramatic, the results presented in Figure 61A, B are important for future studies. 24 hr after a single exposure of cSiO2 is insufficient to induce rampant proinflammatory gene expression (Figure 61A), thus likely indicating that either 1) the cSiO2 particles have not yet been phagocytosed within 24 hr, thus there is no overt toxic consequence at 597 this time. Alternatively, a single 1.0 mg instillation may not be sufficient to elicit inflammation. However, the cSiO2 dose-response study in Chapter 2 suggests that a cumulative dose of 1.0 mg of cSiO2 can also exacerbate systemic autoimmunity. Indeed, further observations with birefringent microscopy to visualize cSiO2 particles putatively suggest that 24 hr is an insufficient amount of time to elicit an overt inflammatory response following intranasal exposure to 1.0 mg cSiO2. On the contrary, the results presented in Fig 61 (B) and Table 31 suggest that many of the same inflammatory gene expression patterns are present with 24 hr after a final exposure to cSiO2 as there are present 1 wk post final exposure (presented in Chapter 4). Impressively, DHA also dampened inflammatory gene expression at this time, which may suggest that DHA can resolve cSiO2-triggered acute inflammation. However, it is difficult to dissect if the gene expression induced by cSiO2 here is specific to an inflammatory response towards the latest cSiO2 insult, or if the pathological sequalae following cSiO2 exposure in the lung have already been initiated. Nevertheless, these preliminary studies provide a benchmark for future studies to investigate these endpoints and others in further detail. 598 Figure 58. Volcano plots depicting immune-related mRNA gene expression data at 24 hr post single exposure (A) or 24 hr post final exposure (B) to cSiO2 in lung of NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log2(fold change) relative to VEH + CON mice. Data are included in Table 37 for all treatment groups for genes log2(fold change) ± > 1.2 and BH p-value < 0.05 in cSiO2 + CON treated NZBWF1 mice as indicated in Fig. 57B. 599 Table 37. mRNA signatures upregulated in lung of cSiO2-exposed NZBWF1 mice compared to VEH + CON mice 24 hrs post final exposure. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log2(fold change) relative to VEH + CON mice. Data are included for all treatment groups for those genes which were log2(fold change) ± 1.2 and BH p-value < 0.05 in cSiO2 + CON treated NZBWF1 mice. Table 37 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hrs post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl5 3.89 [2.95 , 4.83] 1.1E-05 0.68 [0.51 , 0.85] 2.3E-05 Cxcl1 2.79 [2.14 , 3.44] 8.1E-06 0.48 [0.36 , 0.61] 2.7E-05 Lcn2 2.49 [1.91 , 3.07] 8.1E-06 0.48 [0.37 , 0.60] 1.2E-05 Ccl2 2.45 [1.88 , 3.02] 7.9E-06 0.39 [0.28 , 0.50] 8.3E-05 Cxcl10 2.44 [1.58 , 3.30] 2.9E-04 0.13 [0.00 , 0.25] 1.3E-01 Ctla4 2.41 [1.97 , 2.86] 1.8E-06 0.44 [0.35 , 0.52] 2.6E-06 Pdcd1 2.20 [1.83 , 2.57] 1.1E-06 0.41 [0.32 , 0.49] 3.8E-06 Foxp3 2.18 [1.88 , 2.47] 2.2E-07 0.41 [0.36 , 0.46] 1.3E-07 Ccl3 2.10 [1.52 , 2.69] 4.0E-05 0.41 [0.30 , 0.52] 3.4E-05 Irf7 2.02 [1.38 , 2.66] 1.2E-04 0.01 [-0.04 , 0.06] 7.0E-01 Oasl1 1.98 [1.36 , 2.60] 1.1E-04 0.02 [-0.05 , 0.10] 6.6E-01 Mx2 1.97 [1.40 , 2.54] 5.5E-05 0.09 [0.03 , 0.15] 2.9E-02 600 Table 37 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hrs post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ifi44 1.94 [1.34 , 2.54] 9.5E-05 0.02 [-0.02 , 0.06] 4.6E-01 Oas2 1.91 [1.36 , 2.47] 5.5E-05 0.08 [0.03 , 0.13] 2.0E-02 Cxcl2 1.91 [1.29 , 2.53] 1.5E-04 0.32 [0.20 , 0.45] 9.7E-04 Fcer2a 1.89 [1.50 , 2.28] 3.5E-06 0.23 [0.16 , 0.31] 2.3E-04 Ccl4 1.88 [1.27 , 2.48] 1.4E-04 0.26 [0.14 , 0.38] 2.7E-03 Ccl7 1.87 [1.40 , 2.34] 1.6E-05 0.23 [0.13 , 0.34] 2.3E-03 Isg15 1.83 [1.25 , 2.40] 1.2E-04 0.00 [-0.05 , 0.05] 1.0E+00 Cxcl3 1.81 [0.78 , 2.85] 9.0E-03 0.22 [0.01 , 0.43] 1.0E-01 Il1rn 1.78 [1.25 , 2.31] 7.2E-05 0.29 [0.18 , 0.40] 7.0E-04 Csf2 1.76 [1.44 , 2.08] 1.8E-06 0.34 [0.28 , 0.41] 1.3E-06 Il12b 1.76 [1.25 , 2.26] 5.4E-05 0.34 [0.26 , 0.41] 8.1E-06 Clec5a 1.73 [1.45 , 2.00] 8.3E-07 0.26 [0.20 , 0.31] 4.5E-06 Marco 1.71 [1.33 , 2.10] 6.5E-06 0.36 [0.29 , 0.43] 3.1E-06 Trem2 1.68 [1.34 , 2.02] 3.5E-06 0.30 [0.24 , 0.36] 3.1E-06 Cxcl9 1.68 [0.69 , 2.67] 1.1E-02 0.18 [0.00 , 0.35] 1.3E-01 Ccl20 1.68 [0.21 , 3.14] 6.9E-02 0.19 [-0.09 , 0.47] 2.9E-01 C3ar1 1.67 [1.31 , 2.02] 4.0E-06 0.29 [0.23 , 0.35] 4.0E-06 Ccl8 1.66 [0.67 , 2.66] 1.2E-02 0.25 [0.05 , 0.45] 5.6E-02 601 Table 37 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hrs post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mmp9 1.64 [1.16 , 2.12] 5.7E-05 0.33 [0.25 , 0.40] 1.2E-05 Cfb 1.63 [1.28 , 1.98] 4.0E-06 0.24 [0.17 , 0.32] 1.4E-04 Oas3 1.59 [1.03 , 2.15] 2.9E-04 n.d n.d n.d Il2ra 1.57 [1.29 , 1.84] 1.6E-06 0.29 [0.25 , 0.33] 2.6E-07 C1qa 1.54 [1.33 , 1.75] 2.2E-07 0.24 [0.21 , 0.28] 4.6E-07 Ifit1 1.54 [0.98 , 2.10] 3.8E-04 0.02 [-0.03 , 0.07] 4.6E-01 Zbp1 1.53 [1.02 , 2.03] 1.6E-04 0.03 [-0.02 , 0.09] 3.6E-01 Tigit 1.52 [1.20 , 1.84] 3.8E-06 0.22 [0.16 , 0.28] 5.1E-05 Ccl12 1.47 [1.09 , 1.86] 2.3E-05 0.16 [0.09 , 0.23] 2.0E-03 Msr1 1.45 [1.12 , 1.77] 6.5E-06 0.19 [0.12 , 0.26] 4.5E-04 C1qb 1.40 [1.16 , 1.65] 1.4E-06 0.22 [0.18 , 0.26] 9.0E-07 Ccl9 1.38 [1.10 , 1.65] 3.2E-06 0.23 [0.17 , 0.28] 1.8E-05 Clec4n 1.36 [1.06 , 1.66] 5.2E-06 0.20 [0.14 , 0.26] 8.4E-05 Cd207 1.28 [0.89 , 1.68] 1.0E-04 0.34 [0.29 , 0.38] 1.3E-07 Tnfsf11 1.26 [0.95 , 1.57] 1.3E-05 0.18 [0.12 , 0.24] 3.5E-04 Usp18 1.26 [0.80 , 1.73] 3.8E-04 0.04 [0.00 , 0.09] 1.6E-01 Siglec1 1.23 [0.89 , 1.58] 4.4E-05 0.16 [0.10 , 0.21] 4.8E-04 Fcgr1 1.20 [0.97 , 1.44] 2.7E-06 0.07 [0.04 , 0.10] 2.3E-03 602 Table 37 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hrs post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Rsad2 1.20 [0.60 , 1.81] 4.0E-03 0.06 [-0.02 , 0.14] 2.2E-01 603 Table 38. Complete mRNA transcript profile in lung 24 hr post single exposure to cSiO 2 in NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log2(fold change) relative to VEH + CON mice. Expression data are included for all mRNA transcripts that were above background threshold. Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl5 2.34 [1.32 , 3.37] 7.9E-02 0.45 [0.32 , 0.59] 4.7E-03 Dmbt1 1.64 [0.60 , 2.67] 1.4E-01 n.d [n.d , n.d] n.d Chil3 1.57 [0.33 , 2.81] 2.0E-01 0.19 [0.05 , 0.33] 1.4E-01 Lcn2 1.52 [0.65 , 2.39] 1.2E-01 0.36 [0.22 , 0.50] 1.9E-02 Arg1 1.43 [0.06 , 2.81] 2.6E-01 0.06 [-0.06 , 0.17] 6.2E-01 Ccl24 1.40 [-0.22 , 3.02] 3.3E-01 0.07 [-0.03 , 0.16] 4.7E-01 Ccl4 1.39 [0.27 , 2.51] 2.1E-01 0.19 [0.06 , 0.32] 1.1E-01 Ccl8 1.33 [0.05 , 2.62] 2.6E-01 0.13 [-0.02 , 0.28] 3.6E-01 Il1r2 1.21 [0.29 , 2.14] 1.9E-01 0.31 [0.19 , 0.43] 1.9E-02 Mefv 1.19 [0.46 , 1.93] 1.3E-01 0.13 [0.03 , 0.22] 1.6E-01 Ccl20 1.15 [-0.25 , 2.55] 3.5E-01 0.42 [0.16 , 0.68] 8.6E-02 Cxcl1 1.10 [0.18 , 2.01] 2.2E-01 0.23 [0.10 , 0.36] 6.3E-02 604 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Saa1 1.00 [0.09 , 1.91] 2.5E-01 n.d [n.d , n.d] n.d Il1rn 0.98 [0.04 , 1.93] 2.6E-01 0.11 [0.00 , 0.21] 2.4E-01 Cfb 0.98 [0.35 , 1.60] 1.4E-01 0.19 [0.10 , 0.27] 3.5E-02 Ccl3 0.97 [0.04 , 1.89] 2.6E-01 0.18 [0.04 , 0.31] 1.4E-01 Ms4a2 0.93 [0.37 , 1.48] 1.3E-01 n.d [n.d , n.d] n.d Csf3r 0.91 [0.40 , 1.43] 1.2E-01 0.15 [0.07 , 0.23] 5.8E-02 Cxcr2 0.87 [0.27 , 1.47] 1.6E-01 0.16 [0.08 , 0.24] 3.9E-02 Ifitm1 0.83 [0.32 , 1.34] 1.3E-01 0.14 [0.08 , 0.19] 2.3E-02 Ccr3 0.83 [0.18 , 1.47] 2.0E-01 0.05 [-0.04 , 0.13] 5.8E-01 Ccl2 0.75 [-0.18 , 1.68] 3.6E-01 0.07 [-0.07 , 0.21] 6.1E-01 Ccl11 0.73 [-0.10 , 1.57] 3.3E-01 0.01 [-0.08 , 0.09] 9.6E-01 H2-Q10 0.73 [0.23 , 1.23] 1.6E-01 0.09 [0.01 , 0.18] 2.3E-01 Ccl7 0.72 [-0.25 , 1.68] 3.9E-01 0.02 [-0.08 , 0.12] 8.8E-01 Mmp9 0.71 [0.34 , 1.08] 8.7E-02 0.12 [0.07 , 0.16] 1.4E-02 Gzmk 0.70 [-0.13 , 1.53] 3.4E-01 n.d [n.d , n.d] n.d Il1b 0.68 [0.08 , 1.29] 2.4E-01 0.11 [0.04 , 0.18] 1.1E-01 Cxcl10 0.68 [-0.79 , 2.15] 6.3E-01 -0.10 [-0.26 , 0.05] 4.7E-01 605 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfsf14 0.67 [0.20 , 1.14] 1.6E-01 0.12 [0.07 , 0.18] 2.9E-02 Ccr1 0.65 [0.04 , 1.27] 2.6E-01 0.11 [0.04 , 0.17] 8.6E-02 Fcgr2b 0.64 [-0.10 , 1.38] 3.3E-01 0.09 [0.02 , 0.16] 1.6E-01 Ctla4 0.63 [0.03 , 1.23] 2.6E-01 0.12 [0.01 , 0.23] 2.2E-01 Cd244 0.62 [0.10 , 1.15] 2.2E-01 0.09 [0.01 , 0.18] 2.4E-01 Csf2 0.62 [0.25 , 0.99] 1.3E-01 0.18 [0.11 , 0.25] 2.0E-02 Cfi 0.62 [0.11 , 1.12] 2.2E-01 0.10 [0.02 , 0.19] 1.7E-01 Slc11a1 0.61 [0.15 , 1.06] 1.9E-01 0.06 [0.00 , 0.12] 2.4E-01 Zbp1 0.60 [-0.12 , 1.31] 3.4E-01 -0.03 [-0.09 , 0.03] 6.6E-01 Oasl1 0.58 [-0.21 , 1.37] 3.9E-01 -0.10 [-0.15 , -0.05] 4.9E-02 Gbp2b 0.58 [-0.04 , 1.19] 3.1E-01 0.02 [-0.07 , 0.11] 8.4E-01 Ccl17 0.58 [-0.06 , 1.22] 3.3E-01 0.12 [-0.01 , 0.25] 2.9E-01 C3ar1 0.56 [-0.07 , 1.18] 3.3E-01 0.06 [-0.01 , 0.13] 3.6E-01 Fcgr1 0.55 [0.02 , 1.07] 2.6E-01 -0.01 [-0.06 , 0.04] 8.4E-01 C3 0.55 [0.14 , 0.96] 1.9E-01 0.09 [0.03 , 0.16] 1.3E-01 Fcer1a 0.54 [0.09 , 1.00] 2.2E-01 0.10 [0.02 , 0.17] 1.7E-01 Ambp 0.54 [-0.19 , 1.27] 3.9E-01 0.02 [-0.11 , 0.15] 9.2E-01 606 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ulbp1 0.52 [0.13 , 0.91] 1.9E-01 0.09 [0.03 , 0.14] 9.6E-02 Nlrp3 0.51 [0.16 , 0.87] 1.6E-01 0.07 [0.00 , 0.13] 2.7E-01 Cxcr4 0.51 [0.28 , 0.74] 7.9E-02 0.07 [0.02 , 0.13] 1.3E-01 Irf7 0.51 [-0.06 , 1.08] 3.3E-01 -0.02 [-0.07 , 0.03] 6.4E-01 Xcl1 0.51 [-0.20 , 1.21] 4.1E-01 -0.03 [-0.10 , 0.05] 7.6E-01 Cd6 0.50 [0.20 , 0.81] 1.3E-01 0.06 [-0.01 , 0.13] 3.6E-01 Ccr9 0.50 [0.14 , 0.86] 1.8E-01 0.07 [0.01 , 0.13] 2.3E-01 Clec5a 0.50 [-0.01 , 1.00] 2.9E-01 0.07 [0.01 , 0.14] 2.3E-01 Cxcl2 0.49 [-0.53 , 1.52] 6.0E-01 0.17 [0.05 , 0.30] 1.4E-01 Tnfsf15 0.49 [0.24 , 0.74] 8.7E-02 0.09 [0.05 , 0.13] 3.9E-02 Clec4a2 0.49 [0.11 , 0.87] 2.0E-01 0.05 [0.00 , 0.10] 3.0E-01 Tnfrsf18 0.48 [0.08 , 0.88] 2.2E-01 0.06 [0.00 , 0.12] 2.9E-01 Msr1 0.48 [-0.09 , 1.04] 3.4E-01 0.03 [-0.05 , 0.10] 7.1E-01 Icos 0.47 [0.05 , 0.89] 2.4E-01 0.06 [0.01 , 0.12] 2.0E-01 S100a8 0.46 [0.03 , 0.90] 2.6E-01 0.10 [0.01 , 0.20] 2.4E-01 Itgam 0.46 [0.06 , 0.86] 2.3E-01 0.05 [-0.01 , 0.10] 3.5E-01 Cxcr6 0.46 [0.05 , 0.86] 2.4E-01 0.06 [0.01 , 0.10] 1.6E-01 607 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Foxp3 0.45 [-0.07 , 0.97] 3.3E-01 n.d [n.d , n.d] n.d Isg15 0.45 [-0.29 , 1.19] 5.0E-01 -0.06 [-0.11 , -0.01] 1.7E-01 Irf4 0.45 [0.09 , 0.80] 2.0E-01 0.07 [0.02 , 0.11] 1.3E-01 Cd14 0.44 [-0.07 , 0.95] 3.3E-01 0.08 [0.02 , 0.13] 1.3E-01 Tnf 0.44 [-0.11 , 0.98] 3.6E-01 0.09 [0.01 , 0.17] 2.0E-01 Col3a1 0.44 [0.06 , 0.81] 2.4E-01 0.03 [-0.02 , 0.07] 5.1E-01 Cd7 0.43 [0.23 , 0.63] 7.9E-02 0.04 [0.01 , 0.06] 1.3E-01 Ccr2 0.43 [-0.01 , 0.86] 2.9E-01 0.04 [-0.03 , 0.12] 5.6E-01 Ccr5 0.43 [-0.11 , 0.97] 3.7E-01 0.03 [-0.04 , 0.09] 7.2E-01 C1ra 0.42 [0.16 , 0.69] 1.3E-01 0.07 [0.03 , 0.11] 8.6E-02 Usp18 0.42 [0.02 , 0.82] 2.6E-01 -0.02 [-0.06 , 0.02] 5.6E-01 Runx3 0.42 [-0.08 , 0.93] 3.4E-01 0.00 [-0.08 , 0.09] 9.6E-01 Ifi44 0.42 [-0.17 , 1.01] 4.1E-01 -0.09 [-0.15 , -0.04] 8.6E-02 Trem2 0.41 [0.02 , 0.81] 2.6E-01 0.02 [-0.01 , 0.06] 5.3E-01 Ikbke 0.41 [0.13 , 0.68] 1.6E-01 0.05 [0.01 , 0.09] 1.4E-01 Birc5 0.41 [-0.27 , 1.08] 5.0E-01 0.12 [-0.01 , 0.26] 3.0E-01 Cd27 0.40 [0.16 , 0.64] 1.3E-01 0.03 [-0.02 , 0.09] 5.5E-01 608 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il2ra 0.40 [-0.29 , 1.08] 5.3E-01 0.02 [-0.04 , 0.08] 7.6E-01 Fcer2a 0.40 [-0.14 , 0.93] 3.9E-01 0.07 [-0.03 , 0.17] 4.3E-01 Il10ra 0.40 [0.15 , 0.64] 1.3E-01 0.02 [-0.03 , 0.07] 7.5E-01 Cd80 0.39 [-0.05 , 0.84] 3.3E-01 0.07 [0.00 , 0.14] 2.4E-01 Mx2 0.39 [-0.30 , 1.08] 5.3E-01 -0.06 [-0.12 , 0.01] 3.6E-01 Tnfsf13b 0.39 [0.24 , 0.54] 6.4E-02 0.02 [-0.01 , 0.05] 4.5E-01 Trem1 0.38 [-0.05 , 0.82] 3.3E-01 0.05 [-0.01 , 0.12] 3.4E-01 Tnfrsf4 0.38 [-0.03 , 0.79] 3.2E-01 0.05 [-0.02 , 0.12] 4.1E-01 Cd59b 0.38 [-0.06 , 0.81] 3.3E-01 n.d [n.d , n.d] n.d Fcer1g 0.37 [0.11 , 0.64] 1.6E-01 0.02 [-0.02 , 0.05] 6.2E-01 Il2rb 0.37 [0.08 , 0.66] 2.0E-01 0.01 [-0.03 , 0.05] 8.4E-01 Havcr2 0.37 [0.07 , 0.67] 2.1E-01 0.05 [0.03 , 0.08] 5.8E-02 Nlrc5 0.37 [-0.04 , 0.78] 3.3E-01 -0.01 [-0.07 , 0.04] 8.2E-01 Ifit1 0.37 [-0.23 , 0.96] 4.9E-01 -0.04 [-0.08 , 0.01] 3.6E-01 Ctsw 0.36 [0.11 , 0.61] 1.6E-01 0.02 [-0.03 , 0.06] 7.4E-01 Hcst 0.36 [-0.06 , 0.78] 3.3E-01 0.11 [0.04 , 0.18] 8.6E-02 Osm 0.36 [-0.27 , 0.99] 5.3E-01 0.03 [-0.04 , 0.09] 6.7E-01 609 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cfp 0.35 [-0.16 , 0.86] 4.3E-01 0.05 [-0.01 , 0.12] 3.6E-01 Ccl22 0.35 [-0.33 , 1.04] 5.8E-01 -0.03 [-0.12 , 0.05] 7.1E-01 C7 0.35 [0.23 , 0.47] 5.1E-02 0.10 [0.07 , 0.13] 4.7E-03 Selplg 0.35 [0.14 , 0.55] 1.3E-01 0.05 [0.02 , 0.08] 5.8E-02 Cd5 0.35 [0.03 , 0.66] 2.5E-01 0.05 [0.00 , 0.10] 2.4E-01 Csf2rb 0.34 [0.00 , 0.69] 2.9E-01 0.03 [-0.01 , 0.08] 3.6E-01 Cd3d 0.34 [0.08 , 0.60] 1.9E-01 0.07 [0.03 , 0.11] 7.2E-02 Fasl 0.34 [-0.02 , 0.69] 3.0E-01 0.01 [-0.06 , 0.08] 9.3E-01 Cd8a 0.34 [-0.09 , 0.77] 3.7E-01 0.04 [-0.04 , 0.12] 6.1E-01 Socs1 0.34 [-0.12 , 0.79] 3.9E-01 -0.01 [-0.05 , 0.02] 7.5E-01 Thy1 0.33 [0.13 , 0.54] 1.3E-01 0.03 [0.00 , 0.06] 2.6E-01 Irgm2 0.33 [-0.05 , 0.71] 3.3E-01 -0.03 [-0.08 , 0.01] 4.5E-01 Tnfrsf11b 0.33 [-0.01 , 0.68] 3.0E-01 0.02 [-0.05 , 0.08] 8.0E-01 Tnfrsf1b 0.33 [0.11 , 0.55] 1.5E-01 0.02 [-0.01 , 0.06] 5.2E-01 Ccl19 0.33 [-0.11 , 0.76] 3.9E-01 0.00 [-0.07 , 0.07] 9.6E-01 Cxcl9 0.33 [-1.41 , 2.06] 8.5E-01 -0.18 [-0.37 , 0.01] 2.9E-01 Il12rb2 0.33 [0.02 , 0.63] 2.5E-01 0.05 [0.00 , 0.09] 2.4E-01 610 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd3g 0.32 [0.07 , 0.58] 2.0E-01 0.05 [-0.01 , 0.11] 4.1E-01 Cd3e 0.32 [0.07 , 0.58] 2.0E-01 0.05 [0.01 , 0.09] 1.7E-01 Klrg1 0.32 [0.03 , 0.62] 2.5E-01 -0.06 [-0.12 , -0.01] 2.3E-01 Il7r 0.32 [0.04 , 0.61] 2.4E-01 0.08 [0.04 , 0.13] 6.9E-02 Stat1 0.32 [-0.07 , 0.71] 3.5E-01 -0.03 [-0.07 , 0.01] 4.1E-01 Cd69 0.32 [0.01 , 0.63] 2.6E-01 0.07 [-0.01 , 0.16] 3.7E-01 Ltb 0.32 [0.00 , 0.64] 2.8E-01 0.07 [0.02 , 0.12] 1.3E-01 Itgal 0.32 [0.15 , 0.48] 8.7E-02 0.01 [-0.03 , 0.04] 8.6E-01 Ifit3 0.31 [-0.08 , 0.71] 3.6E-01 0.00 [-0.04 , 0.05] 9.4E-01 Psmb9 0.31 [0.05 , 0.57] 2.2E-01 0.00 [-0.04 , 0.03] 1.0E+00 Ncf4 0.31 [0.02 , 0.60] 2.6E-01 0.02 [-0.02 , 0.05] 6.4E-01 Spn 0.31 [0.07 , 0.55] 2.0E-01 0.01 [-0.03 , 0.06] 7.5E-01 Itk 0.31 [0.01 , 0.60] 2.6E-01 0.04 [-0.01 , 0.10] 4.3E-01 Arg2 0.31 [-0.13 , 0.74] 4.2E-01 0.09 [0.04 , 0.15] 8.6E-02 Cd163 0.30 [-0.29 , 0.89] 5.8E-01 0.04 [-0.01 , 0.10] 4.1E-01 Ifit2 0.30 [-0.01 , 0.61] 3.0E-01 0.00 [-0.05 , 0.05] 9.9E-01 Ddx60 0.30 [0.00 , 0.59] 2.7E-01 -0.02 [-0.05 , 0.02] 6.8E-01 611 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Clu 0.30 [0.04 , 0.55] 2.4E-01 0.05 [0.01 , 0.08] 1.4E-01 Psmb8 0.29 [0.06 , 0.53] 2.1E-01 0.00 [-0.03 , 0.03] 9.5E-01 Pik3cd 0.29 [0.10 , 0.49] 1.5E-01 0.05 [0.02 , 0.08] 9.3E-02 Tnfrsf13b 0.29 [0.08 , 0.50] 1.7E-01 0.05 [0.02 , 0.09] 1.1E-01 Tlr6 0.29 [0.05 , 0.53] 2.2E-01 0.03 [-0.01 , 0.07] 3.6E-01 Cd160 0.29 [-0.11 , 0.70] 4.1E-01 0.07 [0.02 , 0.12] 1.4E-01 Ccl12 0.29 [-0.44 , 1.03] 6.8E-01 -0.01 [-0.11 , 0.08] 9.2E-01 Cxcl12 0.29 [0.01 , 0.57] 2.6E-01 0.01 [-0.04 , 0.05] 9.3E-01 Tbx21 0.29 [0.01 , 0.57] 2.6E-01 0.00 [-0.06 , 0.05] 9.6E-01 Zap70 0.28 [0.06 , 0.50] 2.0E-01 0.03 [0.00 , 0.07] 2.9E-01 Lck 0.28 [0.04 , 0.53] 2.3E-01 0.02 [-0.02 , 0.06] 6.6E-01 Cd33 0.28 [0.08 , 0.48] 1.7E-01 0.05 [0.01 , 0.09] 1.7E-01 Gzmb 0.28 [-0.04 , 0.60] 3.3E-01 -0.05 [-0.10 , -0.01] 1.7E-01 Angpt2 0.28 [-0.05 , 0.61] 3.4E-01 0.04 [-0.02 , 0.11] 4.3E-01 Ptprc 0.28 [0.11 , 0.45] 1.3E-01 0.03 [-0.01 , 0.06] 3.7E-01 Ncam1 0.28 [-0.12 , 0.68] 4.3E-01 -0.01 [-0.07 , 0.06] 9.5E-01 Pdcd1lg2 0.28 [-0.36 , 0.92] 6.5E-01 0.02 [-0.04 , 0.07] 7.4E-01 612 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Clec7a 0.28 [0.04 , 0.51] 2.3E-01 0.01 [-0.03 , 0.06] 8.1E-01 Sele 0.27 [-0.36 , 0.91] 6.5E-01 -0.04 [-0.13 , 0.04] 5.9E-01 Il18rap 0.27 [0.04 , 0.50] 2.2E-01 0.03 [0.00 , 0.07] 2.9E-01 Tnfsf13 0.27 [0.02 , 0.53] 2.6E-01 0.03 [0.00 , 0.06] 3.3E-01 Clec4n 0.27 [-0.01 , 0.55] 3.0E-01 0.04 [0.00 , 0.08] 2.7E-01 H2-T23 0.27 [0.12 , 0.42] 1.2E-01 0.01 [-0.02 , 0.04] 7.1E-01 Itgae 0.27 [0.03 , 0.50] 2.4E-01 0.07 [0.02 , 0.11] 1.0E-01 Slamf7 0.27 [0.10 , 0.43] 1.3E-01 0.03 [0.00 , 0.06] 2.7E-01 Lag3 0.27 [-0.19 , 0.72] 5.2E-01 0.03 [-0.06 , 0.11] 7.8E-01 Gbp5 0.27 [-0.25 , 0.78] 5.8E-01 -0.05 [-0.11 , 0.01] 3.8E-01 Csf1r 0.26 [0.12 , 0.40] 1.1E-01 0.00 [-0.03 , 0.03] 9.6E-01 Irf5 0.26 [0.07 , 0.45] 1.8E-01 0.01 [-0.02 , 0.05] 6.9E-01 Syk 0.26 [0.12 , 0.39] 8.7E-02 0.03 [0.01 , 0.05] 5.8E-02 Klra17 0.26 [0.02 , 0.49] 2.5E-01 0.03 [-0.02 , 0.08] 5.8E-01 Cd38 0.26 [0.12 , 0.39] 9.5E-02 0.01 [-0.02 , 0.04] 6.6E-01 Il11ra1 0.26 [-0.16 , 0.68] 5.0E-01 0.09 [0.01 , 0.16] 1.9E-01 Tapbp 0.25 [0.14 , 0.37] 7.9E-02 0.01 [-0.01 , 0.03] 7.8E-01 613 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tlr9 0.25 [-0.04 , 0.54] 3.3E-01 0.03 [-0.01 , 0.06] 4.7E-01 Cd247 0.25 [0.08 , 0.42] 1.6E-01 0.03 [-0.02 , 0.07] 5.5E-01 Nfatc2 0.25 [-0.01 , 0.51] 3.1E-01 0.04 [0.00 , 0.08] 3.2E-01 Tap1 0.25 [-0.06 , 0.55] 3.5E-01 -0.02 [-0.05 , 0.01] 5.0E-01 Il21r 0.25 [-0.20 , 0.70] 5.5E-01 -0.02 [-0.08 , 0.05] 8.4E-01 Ikzf1 0.25 [0.07 , 0.43] 1.8E-01 0.03 [0.00 , 0.05] 2.2E-01 Amica1 0.24 [0.07 , 0.42] 1.6E-01 0.05 [0.02 , 0.08] 9.6E-02 Bst2 0.24 [0.01 , 0.48] 2.6E-01 -0.01 [-0.03 , 0.02] 8.4E-01 Cd200r1 0.24 [-0.04 , 0.53] 3.4E-01 0.05 [0.02 , 0.08] 1.1E-01 Fcgr3 0.24 [-0.07 , 0.56] 3.7E-01 0.02 [-0.01 , 0.06] 4.7E-01 Itga4 0.24 [0.08 , 0.40] 1.5E-01 0.02 [-0.02 , 0.05] 6.6E-01 Pik3cg 0.24 [-0.02 , 0.50] 3.1E-01 0.06 [0.02 , 0.10] 1.1E-01 Cd53 0.24 [0.01 , 0.48] 2.6E-01 0.01 [-0.02 , 0.05] 7.6E-01 Il1rl2 0.24 [0.02 , 0.46] 2.5E-01 0.05 [0.02 , 0.08] 9.6E-02 Cd86 0.24 [-0.07 , 0.55] 3.7E-01 0.04 [0.00 , 0.08] 2.6E-01 Cxcr3 0.24 [0.02 , 0.46] 2.5E-01 0.00 [-0.04 , 0.04] 9.7E-01 Itgax 0.24 [0.03 , 0.45] 2.4E-01 0.03 [-0.01 , 0.07] 4.5E-01 614 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd48 0.24 [0.08 , 0.39] 1.4E-01 0.02 [-0.01 , 0.05] 4.5E-01 Cmpk2 0.24 [-0.14 , 0.61] 4.9E-01 -0.03 [-0.06 , 0.00] 2.0E-01 H2-DMb1 0.23 [-0.05 , 0.52] 3.5E-01 0.04 [-0.01 , 0.10] 3.8E-01 H2-Q2 0.23 [-0.03 , 0.49] 3.3E-01 0.03 [-0.01 , 0.08] 4.4E-01 Tnfrsf14 0.23 [0.11 , 0.35] 8.7E-02 -0.01 [-0.03 , 0.01] 4.9E-01 Gpr183 0.23 [-0.02 , 0.48] 3.2E-01 0.03 [-0.02 , 0.09] 4.9E-01 Klrc1 0.23 [0.02 , 0.44] 2.5E-01 0.00 [-0.04 , 0.04] 9.7E-01 Klra4 0.23 [-0.03 , 0.48] 3.3E-01 0.00 [-0.06 , 0.05] 9.6E-01 Emr1 0.23 [0.01 , 0.44] 2.6E-01 0.02 [-0.02 , 0.06] 5.4E-01 Psmb10 0.22 [0.04 , 0.41] 2.2E-01 -0.01 [-0.04 , 0.02] 7.5E-01 Csf1 0.22 [0.01 , 0.44] 2.6E-01 0.00 [-0.03 , 0.04] 9.6E-01 Klrk1 0.22 [-0.03 , 0.48] 3.3E-01 0.00 [-0.05 , 0.04] 9.4E-01 Cxcl16 0.22 [0.04 , 0.41] 2.2E-01 0.04 [0.00 , 0.07] 2.2E-01 Txk 0.22 [-0.10 , 0.54] 4.3E-01 0.02 [-0.04 , 0.08] 7.9E-01 Slamf1 0.22 [-0.11 , 0.55] 4.5E-01 0.05 [-0.03 , 0.12] 4.9E-01 C8g 0.22 [-0.18 , 0.62] 5.6E-01 0.00 [-0.10 , 0.10] 1.0E+00 Tlr1 0.22 [-0.21 , 0.64] 5.8E-01 0.01 [-0.05 , 0.07] 8.4E-01 615 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tlr2 0.22 [0.03 , 0.40] 2.4E-01 0.02 [-0.01 , 0.06] 5.3E-01 C1s1 0.22 [0.01 , 0.42] 2.6E-01 0.05 [0.02 , 0.07] 8.7E-02 Eomes 0.21 [-0.10 , 0.53] 4.4E-01 0.00 [-0.05 , 0.05] 9.6E-01 Rsad2 0.21 [-0.62 , 1.05] 7.9E-01 -0.09 [-0.17 , -0.02] 1.7E-01 Ctss 0.21 [0.02 , 0.39] 2.4E-01 0.01 [-0.02 , 0.05] 6.6E-01 C5ar1 0.21 [-0.06 , 0.48] 3.7E-01 0.01 [-0.02 , 0.05] 6.6E-01 Ly9 0.21 [0.05 , 0.36] 1.9E-01 0.02 [0.00 , 0.05] 3.6E-01 Il1rl1 0.21 [-0.04 , 0.46] 3.5E-01 0.07 [0.05 , 0.09] 6.7E-03 Nfkbia 0.20 [-0.02 , 0.43] 3.2E-01 0.06 [0.01 , 0.10] 1.7E-01 Il17ra 0.20 [0.07 , 0.33] 1.4E-01 0.02 [0.00 , 0.04] 2.4E-01 Hck 0.20 [0.05 , 0.35] 1.8E-01 0.03 [0.00 , 0.05] 2.4E-01 Card9 0.20 [-0.08 , 0.48] 4.1E-01 -0.02 [-0.07 , 0.02] 5.8E-01 Xaf1 0.20 [-0.03 , 0.42] 3.3E-01 -0.02 [-0.06 , 0.01] 5.0E-01 Tlr7 0.20 [-0.03 , 0.43] 3.3E-01 0.02 [-0.02 , 0.06] 6.0E-01 Il1a 0.20 [-0.19 , 0.59] 5.8E-01 -0.06 [-0.12 , 0.00] 2.9E-01 Tdo2 0.20 [-0.37 , 0.76] 7.2E-01 -0.02 [-0.13 , 0.08] 8.5E-01 Xcr1 0.20 [-0.13 , 0.52] 5.0E-01 0.03 [-0.01 , 0.08] 3.9E-01 616 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd274 0.20 [-0.15 , 0.54] 5.3E-01 -0.04 [-0.07 , -0.01] 1.3E-01 H2-DMa 0.20 [0.04 , 0.35] 2.1E-01 0.02 [-0.01 , 0.05] 4.4E-01 Cd8b1 0.19 [-0.26 , 0.65] 6.5E-01 0.02 [-0.05 , 0.10] 7.8E-01 Klra2 0.19 [-0.05 , 0.44] 3.7E-01 -0.01 [-0.07 , 0.04] 8.4E-01 Il5ra 0.19 [-0.28 , 0.66] 6.6E-01 0.05 [-0.04 , 0.13] 5.7E-01 Flt3l 0.19 [-0.05 , 0.43] 3.6E-01 0.05 [0.03 , 0.08] 5.8E-02 Ccrl2 0.19 [0.04 , 0.34] 2.1E-01 0.04 [0.01 , 0.06] 1.3E-01 Ccr7 0.19 [-0.04 , 0.41] 3.4E-01 0.06 [0.02 , 0.11] 1.4E-01 Lcp1 0.18 [0.03 , 0.34] 2.3E-01 0.04 [0.01 , 0.06] 1.1E-01 Sell 0.18 [-0.03 , 0.40] 3.3E-01 0.02 [-0.02 , 0.06] 6.3E-01 Traf3 0.18 [0.05 , 0.31] 1.7E-01 0.03 [0.01 , 0.05] 1.6E-01 Blnk 0.18 [0.01 , 0.36] 2.6E-01 0.05 [0.02 , 0.07] 5.8E-02 Cd44 0.18 [0.06 , 0.31] 1.6E-01 0.03 [0.01 , 0.05] 1.7E-01 Ticam2 0.18 [-0.06 , 0.42] 3.9E-01 0.01 [-0.04 , 0.05] 9.3E-01 Ccl5 0.18 [-0.13 , 0.50] 5.3E-01 -0.04 [-0.09 , 0.00] 2.7E-01 Foxj1 0.18 [-0.26 , 0.61] 6.6E-01 0.02 [-0.06 , 0.10] 7.9E-01 Cd74 0.18 [-0.02 , 0.37] 3.3E-01 0.03 [0.00 , 0.06] 2.9E-01 617 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Inpp5d 0.18 [0.02 , 0.33] 2.5E-01 0.04 [0.01 , 0.07] 1.7E-01 H2-Ab1 0.17 [-0.05 , 0.39] 3.7E-01 0.03 [-0.01 , 0.07] 4.1E-01 Cd84 0.17 [0.02 , 0.32] 2.4E-01 0.01 [-0.02 , 0.04] 7.9E-01 Casp3 0.17 [0.01 , 0.33] 2.6E-01 0.04 [0.01 , 0.06] 1.7E-01 Egr2 0.17 [-0.17 , 0.51] 5.8E-01 0.01 [-0.08 , 0.10] 9.3E-01 Ly86 0.17 [-0.08 , 0.41] 4.4E-01 0.00 [-0.05 , 0.04] 9.6E-01 C2 0.17 [-0.09 , 0.42] 4.7E-01 0.01 [-0.05 , 0.07] 8.4E-01 Cd4 0.17 [-0.05 , 0.38] 3.8E-01 0.03 [0.00 , 0.06] 2.9E-01 Muc1 0.17 [0.06 , 0.27] 1.4E-01 0.03 [0.01 , 0.05] 1.6E-01 Tap2 0.16 [-0.02 , 0.35] 3.3E-01 -0.01 [-0.04 , 0.03] 8.5E-01 Ncr1 0.16 [-0.15 , 0.47] 5.8E-01 -0.01 [-0.06 , 0.04] 8.7E-01 Ccl9 0.16 [-0.41 , 0.73] 7.6E-01 -0.01 [-0.07 , 0.06] 9.5E-01 Cklf 0.16 [-0.02 , 0.33] 3.3E-01 0.06 [0.04 , 0.09] 2.9E-02 Tnfrsf11a 0.15 [-0.07 , 0.38] 4.3E-01 0.05 [0.01 , 0.08] 1.4E-01 Lyn 0.15 [0.07 , 0.23] 8.7E-02 0.02 [0.00 , 0.03] 1.9E-01 Prkcd 0.15 [0.06 , 0.24] 1.2E-01 0.01 [-0.02 , 0.03] 7.5E-01 Slamf6 0.15 [-0.07 , 0.37] 4.3E-01 0.03 [-0.03 , 0.08] 6.2E-01 618 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfaip3 0.15 [-0.12 , 0.42] 5.3E-01 0.03 [-0.02 , 0.08] 5.6E-01 Cd68 0.15 [-0.02 , 0.32] 3.3E-01 0.01 [-0.02 , 0.04] 7.0E-01 Il2rg 0.15 [-0.05 , 0.35] 3.9E-01 -0.01 [-0.04 , 0.03] 8.0E-01 H2-M3 0.15 [0.00 , 0.30] 2.9E-01 0.00 [-0.02 , 0.03] 9.6E-01 Il16 0.15 [-0.01 , 0.30] 3.1E-01 0.02 [-0.01 , 0.05] 4.3E-01 Stat4 0.15 [-0.14 , 0.43] 5.8E-01 0.02 [-0.04 , 0.08] 7.2E-01 Prf1 0.15 [-0.21 , 0.50] 6.6E-01 -0.05 [-0.11 , 0.01] 3.7E-01 Ifnar2 0.14 [0.05 , 0.24] 1.4E-01 0.03 [0.01 , 0.05] 1.4E-01 Fcgr4 0.14 [-0.08 , 0.36] 4.7E-01 -0.07 [-0.11 , -0.02] 1.4E-01 Lrrn3 0.14 [-0.17 , 0.45] 6.4E-01 0.06 [0.01 , 0.10] 1.5E-01 Ceacam1 0.14 [0.04 , 0.23] 1.6E-01 0.01 [-0.01 , 0.03] 5.8E-01 Ddx58 0.14 [-0.02 , 0.29] 3.3E-01 0.00 [-0.02 , 0.02] 1.0E+00 Pou2f2 0.14 [-0.08 , 0.36] 5.0E-01 -0.01 [-0.05 , 0.04] 9.3E-01 Lbp 0.14 [-0.10 , 0.37] 5.3E-01 -0.01 [-0.04 , 0.02] 7.9E-01 Fn1 0.13 [-0.16 , 0.43] 6.3E-01 0.00 [-0.04 , 0.05] 9.4E-01 Btk 0.13 [-0.08 , 0.34] 4.9E-01 0.02 [-0.01 , 0.05] 5.4E-01 Ifi27 0.13 [0.06 , 0.20] 1.1E-01 -0.01 [-0.02 , 0.01] 6.3E-01 619 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Jak2 0.13 [-0.02 , 0.28] 3.4E-01 0.01 [0.00 , 0.03] 2.3E-01 Cd180 0.13 [-0.10 , 0.35] 5.3E-01 0.00 [-0.04 , 0.05] 9.6E-01 Sh2d1a 0.13 [-0.18 , 0.44] 6.6E-01 0.00 [-0.05 , 0.06] 9.6E-01 Rel 0.13 [-0.04 , 0.29] 3.9E-01 0.02 [-0.01 , 0.06] 4.8E-01 Syt17 0.13 [-0.21 , 0.46] 7.0E-01 0.03 [-0.02 , 0.09] 5.3E-01 Tyk2 0.12 [-0.03 , 0.28] 3.6E-01 0.00 [-0.02 , 0.02] 8.8E-01 Fap 0.12 [-0.19 , 0.44] 6.8E-01 -0.05 [-0.09 , -0.01] 2.2E-01 Irak2 0.12 [0.02 , 0.22] 2.1E-01 0.03 [0.02 , 0.04] 3.9E-02 Cfh 0.12 [0.00 , 0.25] 3.0E-01 0.01 [-0.02 , 0.03] 7.3E-01 Ripk2 0.12 [-0.04 , 0.28] 3.9E-01 0.01 [-0.02 , 0.04] 8.0E-01 H2-K1 0.12 [-0.06 , 0.30] 4.5E-01 -0.02 [-0.05 , 0.01] 5.5E-01 Hspb2 0.12 [-0.06 , 0.30] 4.5E-01 0.05 [0.02 , 0.07] 4.6E-02 Cd40lg 0.12 [-0.23 , 0.47] 7.2E-01 0.05 [-0.01 , 0.11] 3.7E-01 Cd37 0.12 [-0.03 , 0.27] 3.7E-01 0.01 [-0.02 , 0.04] 6.9E-01 Abca1 0.12 [-0.19 , 0.43] 6.8E-01 0.05 [0.00 , 0.10] 2.3E-01 Icam1 0.12 [-0.02 , 0.26] 3.3E-01 0.01 [-0.02 , 0.03] 8.4E-01 Cd83 0.12 [-0.05 , 0.29] 4.3E-01 0.00 [-0.03 , 0.04] 9.4E-01 620 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Klrc2 0.12 [-0.17 , 0.40] 6.6E-01 0.00 [-0.05 , 0.05] 9.8E-01 Tcf7 0.11 [-0.10 , 0.32] 5.6E-01 0.04 [0.02 , 0.07] 8.6E-02 Ciita 0.11 [-0.19 , 0.42] 7.1E-01 0.00 [-0.05 , 0.04] 9.4E-01 Lyve1 0.11 [-0.12 , 0.34] 6.0E-01 0.01 [-0.04 , 0.05] 9.0E-01 Ifih1 0.11 [-0.03 , 0.25] 3.8E-01 -0.01 [-0.03 , 0.00] 3.8E-01 Mrc1 0.11 [-0.09 , 0.30] 5.6E-01 0.01 [-0.01 , 0.02] 7.2E-01 Vcam1 0.11 [-0.13 , 0.34] 6.4E-01 -0.03 [-0.07 , 0.01] 4.1E-01 Stat2 0.10 [-0.15 , 0.35] 6.6E-01 -0.03 [-0.06 , 0.00] 2.3E-01 Raet1c 0.10 [-0.10 , 0.30] 5.8E-01 0.03 [0.00 , 0.07] 2.9E-01 Cyfip2 0.10 [-0.08 , 0.29] 5.5E-01 0.01 [-0.02 , 0.04] 6.3E-01 Klrd1 0.10 [-0.14 , 0.34] 6.5E-01 -0.01 [-0.05 , 0.03] 8.4E-01 Abl1 0.10 [-0.01 , 0.21] 3.3E-01 0.02 [0.01 , 0.04] 1.4E-01 Cd22 0.10 [-0.26 , 0.46] 7.7E-01 0.01 [-0.05 , 0.07] 8.8E-01 Herc6 0.10 [-0.23 , 0.43] 7.6E-01 -0.05 [-0.09 , 0.00] 2.7E-01 St6gal1 0.10 [-0.02 , 0.21] 3.3E-01 0.03 [0.00 , 0.05] 1.9E-01 Ifitm2 0.10 [-0.04 , 0.23] 4.1E-01 0.01 [-0.01 , 0.04] 5.0E-01 Cd1d1 0.10 [-0.09 , 0.28] 5.7E-01 0.01 [-0.02 , 0.03] 8.0E-01 621 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Lta 0.09 [-0.47 , 0.66] 8.7E-01 0.01 [-0.05 , 0.07] 8.4E-01 Sh2b2 0.09 [-0.23 , 0.42] 7.6E-01 -0.04 [-0.09 , 0.01] 4.1E-01 Hif1a 0.09 [-0.08 , 0.26] 5.7E-01 0.00 [-0.02 , 0.01] 8.4E-01 Nfkb1 0.09 [0.02 , 0.16] 2.0E-01 0.00 [-0.01 , 0.01] 1.0E+00 Cd276 0.09 [-0.06 , 0.24] 5.3E-01 -0.01 [-0.05 , 0.02] 7.4E-01 H2-Aa 0.09 [-0.07 , 0.24] 5.4E-01 0.03 [0.00 , 0.05] 2.4E-01 Irf8 0.09 [-0.02 , 0.19] 3.7E-01 0.00 [-0.02 , 0.03] 8.4E-01 Adora2a 0.08 [-0.24 , 0.41] 7.9E-01 0.00 [-0.06 , 0.07] 9.6E-01 Egr3 0.08 [-0.40 , 0.57] 8.7E-01 -0.01 [-0.10 , 0.09] 9.6E-01 Irf1 0.08 [-0.07 , 0.23] 5.5E-01 0.00 [-0.02 , 0.02] 9.7E-01 Itga2 0.08 [-0.10 , 0.27] 6.4E-01 0.02 [0.00 , 0.04] 3.8E-01 Il6ra 0.08 [-0.12 , 0.28] 6.6E-01 0.03 [0.00 , 0.06] 2.4E-01 Map3k5 0.08 [-0.09 , 0.26] 6.3E-01 0.02 [-0.01 , 0.05] 4.0E-01 H2-Ea-ps 0.08 [-0.05 , 0.21] 5.0E-01 0.02 [0.00 , 0.05] 2.7E-01 Ptgdr2 0.08 [-0.38 , 0.54] 8.7E-01 0.06 [-0.02 , 0.14] 4.5E-01 Nfkb2 0.08 [-0.17 , 0.32] 7.4E-01 0.01 [-0.03 , 0.05] 8.9E-01 Col1a1 0.08 [-0.12 , 0.28] 6.8E-01 -0.01 [-0.05 , 0.03] 7.8E-01 622 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Itgb2 0.08 [-0.04 , 0.20] 4.8E-01 -0.02 [-0.05 , 0.00] 3.5E-01 Lamp3 0.08 [-0.05 , 0.20] 4.9E-01 0.02 [-0.01 , 0.05] 4.4E-01 Maf 0.08 [-0.13 , 0.28] 7.0E-01 0.02 [-0.03 , 0.06] 6.9E-01 Rorc 0.08 [-0.19 , 0.34] 7.6E-01 0.04 [-0.01 , 0.08] 3.5E-01 Cd3eap 0.08 [-0.15 , 0.30] 7.3E-01 0.00 [-0.04 , 0.04] 9.3E-01 Ilf3 0.07 [-0.01 , 0.16] 3.3E-01 0.02 [0.00 , 0.03] 2.3E-01 Ikzf2 0.07 [-0.14 , 0.29] 7.2E-01 0.02 [-0.02 , 0.06] 6.2E-01 Gzma 0.07 [-0.27 , 0.42] 8.3E-01 -0.05 [-0.11 , 0.00] 2.3E-01 H2-D1 0.07 [-0.03 , 0.18] 4.2E-01 -0.01 [-0.03 , 0.02] 7.5E-01 Ccnd3 0.07 [-0.07 , 0.22] 5.9E-01 0.00 [-0.02 , 0.02] 8.9E-01 Cxcl3 0.07 [-1.33 , 1.47] 9.7E-01 -0.01 [-0.21 , 0.19] 9.7E-01 C4b 0.07 [-0.15 , 0.28] 7.4E-01 0.00 [-0.04 , 0.05] 9.6E-01 Nfatc1 0.07 [-0.07 , 0.20] 5.8E-01 0.02 [-0.01 , 0.04] 4.8E-01 H2-DMb2 0.07 [-0.17 , 0.30] 7.6E-01 0.05 [0.00 , 0.10] 2.9E-01 Pycard 0.07 [-0.28 , 0.41] 8.5E-01 n.d [n.d , n.d] n.d Klra7 0.07 [-0.61 , 0.75] 9.4E-01 0.02 [-0.08 , 0.12] 8.4E-01 Il15 0.07 [-0.23 , 0.37] 8.3E-01 0.03 [-0.01 , 0.06] 4.1E-01 623 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Flt3 0.07 [-0.25 , 0.38] 8.4E-01 0.03 [-0.02 , 0.08] 5.7E-01 Casp8 0.07 [-0.02 , 0.15] 3.8E-01 -0.01 [-0.02 , 0.01] 7.1E-01 Oas2 0.07 [-0.55 , 0.68] 9.3E-01 -0.13 [-0.20 , -0.06] 5.8E-02 Bid 0.06 [-0.13 , 0.25] 7.4E-01 -0.01 [-0.04 , 0.02] 8.0E-01 Il34 0.06 [-0.05 , 0.17] 5.3E-01 -0.01 [-0.03 , 0.02] 7.4E-01 Cd63 0.06 [-0.14 , 0.27] 7.6E-01 0.00 [-0.03 , 0.02] 8.9E-01 Il6 0.06 [-1.04 , 1.17] 9.7E-01 -0.12 [-0.33 , 0.09] 5.6E-01 Mavs 0.06 [-0.04 , 0.16] 5.3E-01 0.02 [0.00 , 0.04] 2.7E-01 Pdgfc 0.06 [-0.22 , 0.34] 8.3E-01 0.01 [-0.04 , 0.06] 8.9E-01 Tgfb1 0.06 [-0.03 , 0.15] 4.7E-01 -0.01 [-0.02 , 0.00] 4.8E-01 C1qbp 0.06 [-0.03 , 0.15] 4.5E-01 0.01 [-0.01 , 0.03] 6.9E-01 Jak3 0.06 [-0.15 , 0.27] 7.6E-01 -0.04 [-0.08 , 0.00] 3.0E-01 Ifi35 0.06 [-0.02 , 0.14] 4.1E-01 0.00 [-0.02 , 0.01] 9.4E-01 Pla2g1b 0.06 [-0.22 , 0.34] 8.4E-01 0.00 [-0.04 , 0.05] 9.6E-01 Il23r 0.05 [-0.33 , 0.44] 9.0E-01 0.05 [-0.02 , 0.12] 4.3E-01 Tnfsf10 0.05 [-0.16 , 0.27] 7.9E-01 -0.05 [-0.09 , -0.01] 1.7E-01 Lrp1 0.05 [-0.05 , 0.16] 5.7E-01 0.01 [-0.01 , 0.03] 7.1E-01 624 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mme 0.05 [-0.24 , 0.35] 8.6E-01 0.02 [-0.01 , 0.05] 5.6E-01 Plau 0.05 [-0.23 , 0.34] 8.5E-01 -0.02 [-0.06 , 0.02] 6.6E-01 Traf2 0.05 [-0.04 , 0.15] 5.5E-01 0.02 [0.00 , 0.04] 2.3E-01 Ly96 0.05 [-0.06 , 0.17] 6.1E-01 -0.01 [-0.02 , 0.01] 8.0E-01 Isg20 0.05 [-0.09 , 0.20] 7.1E-01 -0.04 [-0.05 , -0.02] 2.9E-02 Sh2d1b1 0.05 [-0.41 , 0.52] 9.2E-01 0.03 [-0.03 , 0.10] 5.8E-01 Tlr5 0.05 [-0.18 , 0.28] 8.2E-01 0.02 [-0.02 , 0.07] 5.9E-01 Il13ra1 0.05 [-0.11 , 0.21] 7.5E-01 -0.01 [-0.03 , 0.01] 6.6E-01 Tgfbr1 0.05 [0.00 , 0.10] 3.1E-01 0.01 [-0.01 , 0.02] 5.1E-01 Icam4 0.05 [-0.25 , 0.35] 8.7E-01 0.03 [-0.03 , 0.09] 6.6E-01 Mif 0.05 [-0.18 , 0.28] 8.4E-01 -0.01 [-0.03 , 0.02] 8.3E-01 Vim 0.05 [-0.06 , 0.15] 6.4E-01 0.01 [-0.01 , 0.04] 4.5E-01 Lamp1 0.05 [-0.04 , 0.14] 5.8E-01 0.01 [0.00 , 0.02] 1.4E-01 Ep300 0.05 [-0.06 , 0.15] 6.6E-01 0.02 [0.00 , 0.04] 2.0E-01 Klrb1c 0.05 [-0.19 , 0.28] 8.5E-01 -0.01 [-0.05 , 0.02] 6.6E-01 Nfatc3 0.04 [-0.06 , 0.15] 6.5E-01 0.02 [0.01 , 0.04] 1.4E-01 Cd47 0.04 [-0.05 , 0.14] 6.4E-01 0.02 [0.00 , 0.04] 2.3E-01 625 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Colec12 0.04 [-0.11 , 0.20] 7.7E-01 0.01 [-0.01 , 0.03] 6.4E-01 Atg10 0.04 [-0.13 , 0.21] 8.0E-01 0.03 [0.01 , 0.06] 1.3E-01 Atf1 0.04 [-0.09 , 0.17] 7.5E-01 0.00 [-0.02 , 0.03] 9.5E-01 H2-Eb1 0.04 [-0.14 , 0.22] 8.2E-01 0.00 [-0.03 , 0.03] 9.4E-01 Stat5b 0.04 [-0.09 , 0.18] 7.5E-01 0.02 [0.01 , 0.04] 1.4E-01 Relb 0.04 [-0.22 , 0.30] 8.9E-01 -0.01 [-0.06 , 0.04] 8.5E-01 Abcb1a 0.04 [-0.27 , 0.34] 9.1E-01 0.02 [-0.02 , 0.05] 6.6E-01 Tigit 0.04 [-0.49 , 0.56] 9.6E-01 0.05 [-0.02 , 0.12] 4.7E-01 Pml 0.04 [-0.12 , 0.19] 8.3E-01 -0.02 [-0.04 , -0.01] 7.4E-02 Ccl27a 0.03 [-0.31 , 0.37] 9.4E-01 0.01 [-0.04 , 0.05] 9.2E-01 Gata3 0.03 [-0.21 , 0.27] 9.1E-01 0.00 [-0.03 , 0.02] 9.6E-01 Irak1 0.03 [-0.04 , 0.10] 6.6E-01 0.01 [-0.01 , 0.03] 5.7E-01 Nod1 0.03 [-0.11 , 0.16] 8.3E-01 -0.01 [-0.04 , 0.01] 5.1E-01 Pnma1 0.03 [-0.30 , 0.36] 9.5E-01 0.03 [-0.04 , 0.10] 6.6E-01 Nup107 0.03 [-0.04 , 0.10] 6.8E-01 0.01 [-0.01 , 0.03] 5.7E-01 Serping1 0.03 [-0.07 , 0.12] 7.8E-01 0.01 [-0.01 , 0.02] 5.8E-01 Prkce 0.02 [-0.24 , 0.29] 9.4E-01 0.01 [-0.02 , 0.04] 7.8E-01 626 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mapk14 0.02 [-0.07 , 0.12] 8.0E-01 0.01 [-0.01 , 0.03] 5.8E-01 Yy1 0.02 [-0.07 , 0.12] 8.0E-01 0.01 [-0.01 , 0.02] 5.3E-01 Itgb3 0.02 [-0.19 , 0.24] 9.2E-01 -0.01 [-0.05 , 0.03] 8.0E-01 Tlr4 0.02 [-0.11 , 0.16] 8.7E-01 -0.02 [-0.05 , 0.01] 5.4E-01 Il3ra 0.02 [-0.15 , 0.20] 9.1E-01 0.01 [-0.02 , 0.04] 8.0E-01 Tlr8 0.02 [-0.15 , 0.19] 9.2E-01 -0.02 [-0.07 , 0.03] 7.4E-01 Ifnar1 0.02 [-0.07 , 0.11] 8.2E-01 0.00 [-0.01 , 0.02] 8.4E-01 Fas 0.02 [-0.16 , 0.20] 9.2E-01 0.02 [-0.02 , 0.06] 6.0E-01 Psen1 0.02 [-0.05 , 0.09] 7.6E-01 0.00 [-0.01 , 0.02] 8.6E-01 Bcl2 0.02 [-0.20 , 0.23] 9.5E-01 0.01 [-0.04 , 0.05] 9.3E-01 Ewsr1 0.02 [-0.07 , 0.11] 8.5E-01 0.01 [0.00 , 0.02] 4.8E-01 Il18r1 0.01 [-0.17 , 0.20] 9.6E-01 0.00 [-0.03 , 0.03] 1.0E+00 Ada 0.01 [-0.27 , 0.30] 9.7E-01 0.02 [-0.02 , 0.07] 5.5E-01 Igf2r 0.01 [-0.07 , 0.10] 8.7E-01 -0.01 [-0.02 , 0.01] 5.5E-01 C1qb 0.01 [-0.34 , 0.37] 9.8E-01 -0.01 [-0.05 , 0.02] 7.1E-01 Mill2 0.01 [-0.25 , 0.28] 9.7E-01 0.00 [-0.03 , 0.02] 9.3E-01 Smad2 0.01 [-0.09 , 0.11] 9.1E-01 0.01 [0.00 , 0.02] 4.7E-01 627 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il6st 0.01 [-0.10 , 0.13] 9.2E-01 -0.01 [-0.04 , 0.01] 4.4E-01 Dll4 0.01 [-0.11 , 0.14] 9.3E-01 -0.03 [-0.06 , 0.00] 2.7E-01 Pvrl2 0.01 [-0.11 , 0.14] 9.3E-01 0.00 [-0.03 , 0.02] 9.7E-01 App 0.01 [-0.14 , 0.17] 9.6E-01 0.00 [-0.01 , 0.01] 9.7E-01 Map2k4 0.01 [-0.06 , 0.08] 9.1E-01 0.01 [-0.01 , 0.02] 7.1E-01 Cyld 0.01 [-0.14 , 0.15] 9.7E-01 0.00 [-0.03 , 0.04] 9.3E-01 Spp1 0.01 [-0.36 , 0.37] 9.9E-01 -0.05 [-0.09 , -0.02] 1.3E-01 Cebpb 0.01 [-0.16 , 0.17] 9.9E-01 -0.01 [-0.06 , 0.04] 8.9E-01 Card11 0.01 [-0.21 , 0.22] 9.9E-01 0.00 [-0.05 , 0.05] 9.6E-01 Dpp4 0.01 [-0.19 , 0.20] 9.9E-01 0.02 [-0.01 , 0.04] 4.5E-01 Tgfb2 0.00 [-0.22 , 0.23] 9.9E-01 0.02 [0.00 , 0.05] 3.6E-01 Myc 0.00 [-0.23 , 0.23] 9.9E-01 -0.02 [-0.05 , 0.01] 4.8E-01 Ctsl 0.00 [-0.10 , 0.10] 9.9E-01 0.01 [-0.01 , 0.03] 5.6E-01 Gtf3c1 0.00 [-0.15 , 0.16] 9.9E-01 0.00 [-0.02 , 0.02] 9.9E-01 Tbk1 0.00 [-0.09 , 0.09] 9.9E-01 0.01 [-0.01 , 0.03] 5.5E-01 Trp53 0.00 [-0.16 , 0.16] 9.9E-01 -0.02 [-0.05 , 0.02] 6.8E-01 Fadd 0.00 [-0.14 , 0.14] 9.9E-01 0.01 [-0.01 , 0.04] 6.7E-01 628 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Axl 0.00 [-0.13 , 0.13] 9.9E-01 0.00 [-0.01 , 0.02] 8.5E-01 Siglec1 0.00 [-0.23 , 0.23] 9.9E-01 -0.04 [-0.10 , 0.01] 3.8E-01 Il1r1 0.00 [-0.14 , 0.15] 9.9E-01 -0.03 [-0.06 , 0.00] 2.4E-01 Cmah 0.00 [-0.19 , 0.19] 9.9E-01 -0.01 [-0.05 , 0.02] 7.4E-01 Tgfb3 0.00 [-0.24 , 0.25] 9.9E-01 0.05 [0.01 , 0.08] 1.7E-01 Bcl10 0.00 [-0.11 , 0.12] 9.9E-01 -0.01 [-0.04 , 0.02] 7.5E-01 Zfp13 0.00 [-0.27 , 0.27] 1.0E+00 0.02 [-0.01 , 0.05] 4.1E-01 Anp32b 0.00 [-0.06 , 0.06] 9.9E-01 0.00 [-0.01 , 0.01] 8.4E-01 Cd99 0.00 [-0.14 , 0.14] 9.9E-01 0.01 [-0.01 , 0.03] 7.5E-01 Tfrc 0.00 [-0.12 , 0.11] 9.9E-01 0.03 [0.01 , 0.04] 8.6E-02 Mapk3 0.00 [-0.17 , 0.16] 9.9E-01 0.01 [-0.01 , 0.02] 7.1E-01 Itga1 0.00 [-0.30 , 0.29] 9.9E-01 0.00 [-0.04 , 0.03] 9.0E-01 Mr1 -0.01 [-0.14 , 0.13] 9.8E-01 0.01 [-0.01 , 0.03] 6.9E-01 Mertk -0.01 [-0.31 , 0.30] 9.9E-01 0.01 [-0.03 , 0.06] 7.8E-01 Cxcl15 -0.01 [-0.21 , 0.20] 9.9E-01 -0.01 [-0.04 , 0.01] 5.6E-01 Irak4 -0.01 [-0.10 , 0.09] 9.6E-01 0.00 [-0.02 , 0.02] 9.4E-01 Kit -0.01 [-0.34 , 0.33] 9.9E-01 0.01 [-0.06 , 0.07] 9.3E-01 629 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tie1 -0.01 [-0.25 , 0.24] 9.9E-01 0.00 [-0.02 , 0.02] 9.6E-01 Ets1 -0.01 [-0.22 , 0.20] 9.8E-01 0.00 [-0.02 , 0.03] 8.9E-01 Ecsit -0.01 [-0.16 , 0.14] 9.7E-01 -0.01 [-0.03 , 0.02] 7.4E-01 Cx3cr1 -0.01 [-0.20 , 0.18] 9.8E-01 -0.04 [-0.09 , 0.01] 4.4E-01 Cd1d2 -0.01 [-0.43 , 0.41] 9.9E-01 0.02 [-0.03 , 0.07] 7.2E-01 Il7 -0.01 [-0.19 , 0.16] 9.6E-01 -0.06 [-0.13 , 0.00] 2.4E-01 Casp1 -0.01 [-0.19 , 0.16] 9.6E-01 -0.02 [-0.04 , 0.01] 4.4E-01 Mapk8 -0.01 [-0.13 , 0.11] 9.2E-01 0.01 [-0.01 , 0.03] 5.8E-01 Map4k2 -0.01 [-0.25 , 0.22] 9.7E-01 0.00 [-0.02 , 0.03] 9.3E-01 Tgfbr2 -0.01 [-0.10 , 0.07] 8.7E-01 0.00 [-0.01 , 0.01] 9.4E-01 C6 -0.01 [-0.17 , 0.14] 9.4E-01 0.00 [-0.03 , 0.02] 9.6E-01 Pin1 -0.02 [-0.26 , 0.22] 9.6E-01 -0.01 [-0.05 , 0.03] 8.5E-01 Atg12 -0.02 [-0.14 , 0.11] 9.1E-01 -0.01 [-0.02 , 0.01] 7.9E-01 Pla2g6 -0.02 [-0.12 , 0.08] 8.7E-01 0.01 [-0.01 , 0.02] 7.6E-01 Ikbkb -0.02 [-0.09 , 0.06] 8.2E-01 0.00 [-0.01 , 0.01] 9.6E-01 Rps6 -0.02 [-0.17 , 0.14] 9.2E-01 0.02 [-0.01 , 0.05] 5.8E-01 Thbd -0.02 [-0.34 , 0.31] 9.7E-01 0.02 [0.00 , 0.04] 3.9E-01 630 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Psen2 -0.02 [-0.16 , 0.13] 9.2E-01 0.00 [-0.02 , 0.02] 9.6E-01 Map2k1 -0.02 [-0.07 , 0.03] 7.1E-01 -0.01 [-0.02 , 0.00] 3.5E-01 Atm -0.02 [-0.15 , 0.11] 9.0E-01 0.01 [-0.01 , 0.04] 5.8E-01 Cxcr5 -0.02 [-0.51 , 0.47] 9.8E-01 0.07 [0.00 , 0.14] 2.6E-01 Cd19 -0.02 [-0.33 , 0.29] 9.6E-01 0.02 [-0.04 , 0.08] 8.0E-01 Jam3 -0.02 [-0.18 , 0.14] 9.1E-01 0.01 [-0.01 , 0.04] 5.9E-01 Icosl -0.02 [-0.15 , 0.11] 8.7E-01 -0.01 [-0.04 , 0.02] 8.1E-01 Cspg4 -0.02 [-0.27 , 0.22] 9.5E-01 0.01 [-0.03 , 0.04] 9.0E-01 Egfr -0.02 [-0.11 , 0.06] 7.7E-01 -0.02 [-0.04 , 0.00] 2.3E-01 Nrp1 -0.02 [-0.23 , 0.18] 9.2E-01 0.00 [-0.03 , 0.03] 9.6E-01 Masp1 -0.02 [-0.37 , 0.33] 9.6E-01 0.00 [-0.08 , 0.08] 9.9E-01 Plaur -0.02 [-0.21 , 0.16] 9.1E-01 -0.01 [-0.04 , 0.02] 7.9E-01 C1qa -0.02 [-0.35 , 0.31] 9.6E-01 -0.02 [-0.05 , 0.01] 5.7E-01 Bst1 -0.02 [-0.22 , 0.17] 9.2E-01 -0.03 [-0.08 , 0.01] 4.1E-01 Abcg1 -0.03 [-0.37 , 0.32] 9.6E-01 0.00 [-0.06 , 0.06] 9.6E-01 Mef2c -0.03 [-0.15 , 0.10] 8.4E-01 -0.01 [-0.04 , 0.02] 7.9E-01 Ythdf2 -0.03 [-0.09 , 0.03] 6.5E-01 0.01 [0.00 , 0.02] 2.9E-01 631 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Jak1 -0.03 [-0.12 , 0.06] 7.5E-01 -0.01 [-0.03 , 0.01] 5.6E-01 Atf2 -0.03 [-0.13 , 0.07] 7.6E-01 0.01 [0.00 , 0.03] 2.6E-01 Irak3 -0.03 [-0.12 , 0.06] 7.3E-01 0.00 [-0.02 , 0.02] 9.4E-01 Hmgb1 -0.03 [-0.13 , 0.07] 7.5E-01 0.00 [-0.01 , 0.02] 8.0E-01 Irf2 -0.03 [-0.14 , 0.08] 7.6E-01 0.00 [-0.01 , 0.02] 8.8E-01 Irf3 -0.03 [-0.14 , 0.08] 7.6E-01 -0.01 [-0.02 , 0.01] 6.8E-01 Sigirr -0.03 [-0.17 , 0.11] 8.2E-01 0.00 [-0.02 , 0.01] 8.4E-01 Cdh1 -0.03 [-0.14 , 0.08] 7.6E-01 0.00 [-0.02 , 0.02] 9.6E-01 Crebbp -0.03 [-0.14 , 0.07] 7.5E-01 0.00 [-0.02 , 0.01] 8.8E-01 Mapkapk2 -0.03 [-0.14 , 0.07] 7.4E-01 -0.01 [-0.04 , 0.01] 5.5E-01 Creb1 -0.03 [-0.15 , 0.08] 7.6E-01 0.01 [-0.01 , 0.03] 6.9E-01 Tfeb -0.03 [-0.15 , 0.08] 7.6E-01 0.00 [-0.02 , 0.03] 8.8E-01 Stat6 -0.03 [-0.13 , 0.07] 7.2E-01 0.00 [-0.02 , 0.02] 1.0E+00 Atg7 -0.03 [-0.14 , 0.07] 7.3E-01 -0.01 [-0.02 , 0.00] 4.2E-01 Blk -0.04 [-0.30 , 0.22] 9.1E-01 0.02 [-0.05 , 0.09] 8.3E-01 Tnfrsf13c -0.04 [-0.56 , 0.48] 9.6E-01 0.01 [-0.09 , 0.10] 9.6E-01 Itga6 -0.04 [-0.11 , 0.04] 5.8E-01 0.01 [-0.01 , 0.03] 4.4E-01 632 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Erbb2 -0.04 [-0.13 , 0.05] 6.4E-01 0.00 [-0.02 , 0.01] 8.3E-01 Nos2 -0.04 [-0.49 , 0.41] 9.5E-01 -0.02 [-0.10 , 0.06] 8.4E-01 Ebi3 -0.04 [-0.21 , 0.14] 8.2E-01 0.01 [-0.02 , 0.04] 7.9E-01 Dock9 -0.04 [-0.25 , 0.17] 8.5E-01 0.00 [-0.02 , 0.02] 9.3E-01 Alcam -0.04 [-0.20 , 0.12] 8.0E-01 0.00 [-0.02 , 0.02] 9.6E-01 Bax -0.04 [-0.18 , 0.09] 7.5E-01 0.00 [-0.02 , 0.02] 8.6E-01 Hamp -0.04 [-1.21 , 1.13] 9.9E-01 0.06 [-0.14 , 0.26] 7.9E-01 Cd2 -0.04 [-0.37 , 0.29] 9.1E-01 0.02 [-0.02 , 0.06] 6.2E-01 Itch -0.04 [-0.14 , 0.05] 6.2E-01 0.00 [-0.02 , 0.02] 9.7E-01 Il15ra -0.04 [-0.22 , 0.13] 8.0E-01 0.01 [-0.02 , 0.04] 8.4E-01 Traf6 -0.04 [-0.16 , 0.07] 6.8E-01 -0.01 [-0.03 , 0.01] 6.9E-01 Mapk1 -0.04 [-0.11 , 0.02] 4.5E-01 0.00 [-0.01 , 0.00] 6.0E-01 Apoe -0.04 [-0.27 , 0.18] 8.4E-01 0.01 [-0.02 , 0.05] 6.9E-01 Cd200 -0.05 [-0.16 , 0.07] 6.6E-01 -0.04 [-0.06 , -0.01] 1.1E-01 Cma1 -0.05 [-0.48 , 0.39] 9.2E-01 -0.02 [-0.11 , 0.07] 8.5E-01 Cd164 -0.05 [-0.15 , 0.05] 6.1E-01 -0.02 [-0.03 , 0.00] 1.9E-01 Tlr3 -0.05 [-0.19 , 0.09] 7.2E-01 -0.02 [-0.05 , 0.01] 4.8E-01 633 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tollip -0.05 [-0.18 , 0.09] 7.1E-01 0.00 [-0.02 , 0.01] 8.5E-01 Il1rap -0.05 [-0.22 , 0.12] 7.6E-01 -0.01 [-0.03 , 0.02] 8.0E-01 Atg16l1 -0.05 [-0.19 , 0.09] 7.1E-01 0.00 [-0.02 , 0.01] 8.0E-01 Smn1 -0.05 [-0.14 , 0.04] 5.2E-01 -0.01 [-0.02 , 0.01] 7.5E-01 Epcam -0.05 [-0.20 , 0.10] 7.3E-01 0.02 [-0.01 , 0.04] 4.7E-01 Tmem173 -0.05 [-0.45 , 0.34] 9.1E-01 -0.03 [-0.08 , 0.02] 4.7E-01 Bcl6 -0.05 [-0.23 , 0.13] 7.6E-01 0.01 [-0.02 , 0.05] 6.6E-01 Smad4 -0.05 [-0.17 , 0.06] 6.3E-01 0.00 [-0.02 , 0.02] 8.9E-01 Cybb -0.05 [-0.20 , 0.09] 7.0E-01 -0.04 [-0.09 , 0.00] 2.7E-01 Mfge8 -0.05 [-0.26 , 0.16] 7.9E-01 0.01 [-0.01 , 0.03] 6.8E-01 Angpt1 -0.05 [-0.41 , 0.30] 8.9E-01 0.03 [-0.01 , 0.08] 4.5E-01 Vhl -0.05 [-0.23 , 0.12] 7.5E-01 -0.02 [-0.05 , 0.02] 6.6E-01 Cd96 -0.06 [-0.35 , 0.24] 8.5E-01 -0.01 [-0.05 , 0.04] 8.8E-01 Map2k2 -0.06 [-0.12 , 0.01] 3.3E-01 -0.01 [-0.02 , 0.00] 1.1E-01 Vwf -0.06 [-0.24 , 0.13] 7.5E-01 -0.02 [-0.04 , 0.01] 4.7E-01 Tfe3 -0.06 [-0.17 , 0.05] 5.8E-01 -0.02 [-0.05 , 0.00] 2.7E-01 Notch1 -0.06 [-0.21 , 0.10] 6.9E-01 -0.01 [-0.03 , 0.01] 4.6E-01 634 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Entpd1 -0.06 [-0.17 , 0.05] 5.6E-01 -0.02 [-0.05 , 0.01] 4.6E-01 Myd88 -0.06 [-0.26 , 0.14] 7.5E-01 -0.03 [-0.05 , -0.01] 1.7E-01 Tmed1 -0.06 [-0.12 , 0.00] 2.6E-01 -0.03 [-0.04 , -0.01] 6.5E-02 Runx1 -0.06 [-0.14 , 0.02] 3.8E-01 -0.02 [-0.03 , 0.00] 2.5E-01 Atg5 -0.06 [-0.20 , 0.07] 6.2E-01 -0.01 [-0.04 , 0.01] 5.7E-01 Tnfsf12 -0.06 [-0.25 , 0.12] 7.2E-01 0.00 [-0.02 , 0.02] 9.9E-01 Txnip -0.06 [-0.35 , 0.22] 8.2E-01 -0.03 [-0.06 , 0.01] 3.8E-01 Cmklr1 -0.06 [-0.25 , 0.12] 7.2E-01 0.00 [-0.03 , 0.02] 9.9E-01 Fpr2 -0.07 [-0.28 , 0.15] 7.5E-01 -0.04 [-0.09 , 0.02] 5.3E-01 Lamp2 -0.07 [-0.13 , -0.01] 2.5E-01 -0.01 [-0.02 , 0.00] 6.0E-01 Batf -0.07 [-0.27 , 0.14] 7.4E-01 -0.02 [-0.05 , 0.02] 5.8E-01 Psma2 -0.07 [-0.12 , -0.02] 1.9E-01 -0.01 [-0.02 , 0.00] 1.7E-01 Ccl21a -0.07 [-0.31 , 0.18] 7.6E-01 -0.06 [-0.11 , -0.01] 1.7E-01 Vegfa -0.07 [-0.28 , 0.14] 7.4E-01 -0.01 [-0.04 , 0.01] 5.9E-01 Il17b -0.07 [-0.46 , 0.32] 8.7E-01 -0.06 [-0.14 , 0.03] 4.8E-01 Igf1r -0.07 [-0.22 , 0.08] 6.3E-01 0.00 [-0.02 , 0.03] 9.3E-01 Psmb7 -0.07 [-0.13 , -0.02] 1.7E-01 -0.01 [-0.02 , 0.00] 2.6E-01 635 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tank -0.07 [-0.16 , 0.01] 3.3E-01 -0.02 [-0.04 , -0.01] 9.6E-02 Reps1 -0.07 [-0.19 , 0.04] 4.8E-01 0.00 [-0.02 , 0.02] 1.0E+00 Map3k1 -0.07 [-0.26 , 0.11] 6.7E-01 0.00 [-0.04 , 0.03] 9.6E-01 Itga5 -0.07 [-0.21 , 0.06] 5.4E-01 -0.01 [-0.03 , 0.01] 6.4E-01 Ubc -0.08 [-0.20 , 0.05] 5.2E-01 -0.01 [-0.04 , 0.02] 6.6E-01 H2-Ob -0.08 [-0.35 , 0.19] 7.6E-01 0.02 [-0.03 , 0.06] 7.1E-01 Dusp6 -0.08 [-0.43 , 0.28] 8.3E-01 -0.02 [-0.10 , 0.07] 8.6E-01 Chuk -0.08 [-0.16 , 0.00] 2.9E-01 -0.01 [-0.03 , 0.01] 6.2E-01 Msln -0.08 [-0.46 , 0.30] 8.4E-01 -0.04 [-0.12 , 0.04] 6.2E-01 Bmi1 -0.08 [-0.24 , 0.08] 5.8E-01 -0.01 [-0.03 , 0.02] 8.4E-01 Kdr -0.08 [-0.30 , 0.14] 7.0E-01 -0.01 [-0.04 , 0.01] 5.9E-01 Cx3cl1 -0.09 [-0.43 , 0.26] 8.0E-01 -0.04 [-0.08 , 0.01] 3.8E-01 Ccl6 -0.09 [-0.28 , 0.11] 6.5E-01 0.00 [-0.03 , 0.04] 9.9E-01 Cd97 -0.09 [-0.37 , 0.19] 7.5E-01 0.00 [-0.03 , 0.03] 9.1E-01 Eng -0.09 [-0.27 , 0.10] 6.1E-01 -0.04 [-0.06 , -0.02] 8.3E-02 Socs3 -0.09 [-0.64 , 0.47] 8.8E-01 -0.09 [-0.18 , -0.01] 2.4E-01 Col4a1 -0.09 [-0.27 , 0.09] 5.9E-01 -0.02 [-0.05 , 0.01] 4.1E-01 636 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tal1 -0.09 [-0.30 , 0.13] 6.6E-01 -0.01 [-0.04 , 0.02] 8.0E-01 Lgals3 -0.09 [-0.33 , 0.15] 7.0E-01 -0.03 [-0.06 , 0.00] 2.9E-01 Pdgfrb -0.09 [-0.34 , 0.16] 7.1E-01 0.01 [-0.01 , 0.04] 6.7E-01 Creb5 -0.09 [-0.37 , 0.19] 7.4E-01 -0.01 [-0.06 , 0.05] 8.9E-01 Cd81 -0.09 [-0.26 , 0.07] 5.5E-01 -0.02 [-0.04 , 0.00] 2.0E-01 Vegfc -0.09 [-0.41 , 0.23] 7.6E-01 0.02 [0.00 , 0.05] 2.6E-01 Cd79b -0.09 [-0.44 , 0.25] 7.7E-01 0.02 [-0.05 , 0.08] 8.0E-01 Cd28 -0.09 [-0.60 , 0.41] 8.5E-01 0.03 [-0.03 , 0.09] 6.2E-01 Il22ra1 -0.09 [-0.41 , 0.22] 7.5E-01 0.01 [-0.03 , 0.06] 8.0E-01 Pparg -0.10 [-0.32 , 0.13] 6.6E-01 -0.02 [-0.06 , 0.03] 6.9E-01 Ifngr1 -0.10 [-0.30 , 0.11] 6.1E-01 0.01 [-0.04 , 0.05] 8.9E-01 Ms4a1 -0.10 [-0.48 , 0.28] 7.9E-01 0.01 [-0.07 , 0.09] 9.2E-01 Fyn -0.10 [-0.22 , 0.02] 3.5E-01 -0.01 [-0.03 , 0.02] 7.9E-01 Cdh5 -0.10 [-0.33 , 0.13] 6.5E-01 -0.01 [-0.03 , 0.01] 5.9E-01 Ticam1 -0.10 [-0.23 , 0.03] 3.9E-01 -0.01 [-0.03 , 0.01] 5.6E-01 Ltk -0.10 [-0.36 , 0.16] 6.9E-01 -0.01 [-0.08 , 0.07] 9.5E-01 Akt3 -0.10 [-0.29 , 0.08] 5.5E-01 0.01 [-0.02 , 0.03] 8.0E-01 637 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Gzmm -0.11 [-0.73 , 0.52] 8.7E-01 0.07 [-0.03 , 0.16] 4.7E-01 Nod2 -0.11 [-0.31 , 0.09] 5.6E-01 -0.07 [-0.11 , -0.03] 5.8E-02 Ltbr -0.11 [-0.18 , -0.04] 1.3E-01 -0.02 [-0.03 , -0.01] 3.9E-02 Fez1 -0.11 [-0.45 , 0.23] 7.4E-01 -0.04 [-0.12 , 0.04] 6.6E-01 Gpi1 -0.11 [-0.17 , -0.06] 8.7E-02 -0.01 [-0.02 , 0.01] 5.9E-01 Jun -0.12 [-0.38 , 0.15] 6.5E-01 -0.01 [-0.05 , 0.03] 9.1E-01 Rela -0.13 [-0.28 , 0.03] 3.5E-01 -0.03 [-0.06 , -0.01] 1.7E-01 Masp2 -0.13 [-0.49 , 0.24] 7.2E-01 0.03 [-0.02 , 0.08] 5.8E-01 Xbp1 -0.13 [-0.27 , 0.01] 3.2E-01 -0.03 [-0.06 , 0.00] 2.0E-01 Pecam1 -0.13 [-0.28 , 0.02] 3.4E-01 -0.02 [-0.04 , 0.01] 4.5E-01 Anxa1 -0.13 [-0.26 , 0.00] 2.8E-01 -0.03 [-0.06 , -0.01] 1.4E-01 Lyz2 -0.13 [-0.34 , 0.08] 4.8E-01 -0.01 [-0.04 , 0.02] 6.3E-01 Tnfrsf1a -0.13 [-0.22 , -0.04] 1.6E-01 -0.03 [-0.05 , -0.02] 4.6E-02 Itgb1 -0.13 [-0.26 , -0.01] 2.6E-01 -0.01 [-0.02 , 0.00] 4.4E-01 Rora -0.13 [-0.34 , 0.08] 4.8E-01 -0.01 [-0.04 , 0.03] 8.9E-01 Cd55 -0.13 [-0.29 , 0.02] 3.4E-01 -0.02 [-0.04 , 0.01] 4.1E-01 Psmd7 -0.14 [-0.20 , -0.07] 7.9E-02 -0.02 [-0.03 , -0.01] 1.4E-01 638 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tek -0.14 [-0.45 , 0.18] 6.5E-01 -0.01 [-0.04 , 0.02] 7.3E-01 Tab1 -0.14 [-0.28 , 0.01] 3.2E-01 -0.02 [-0.05 , 0.02] 5.8E-01 Litaf -0.14 [-0.37 , 0.09] 5.2E-01 -0.05 [-0.09 , -0.02] 9.5E-02 Sbno2 -0.14 [-0.45 , 0.17] 6.4E-01 -0.07 [-0.13 , -0.02] 1.4E-01 Mst1r -0.14 [-0.37 , 0.09] 5.0E-01 -0.05 [-0.12 , 0.01] 3.8E-01 Prdm1 -0.14 [-0.44 , 0.16] 6.1E-01 -0.01 [-0.05 , 0.03] 7.5E-01 Bcl2l1 -0.14 [-0.43 , 0.15] 5.9E-01 0.00 [-0.06 , 0.05] 9.7E-01 Cd34 -0.15 [-0.30 , 0.01] 3.1E-01 -0.04 [-0.07 , -0.02] 7.2E-02 Fos -0.16 [-0.64 , 0.32] 7.4E-01 -0.03 [-0.19 , 0.12] 8.4E-01 Ikbkg -0.16 [-0.29 , -0.03] 2.1E-01 -0.01 [-0.03 , 0.00] 4.3E-01 Mapk11 -0.16 [-0.38 , 0.06] 3.9E-01 -0.03 [-0.07 , 0.01] 3.6E-01 Tnfrsf10b -0.16 [-0.47 , 0.14] 5.6E-01 -0.05 [-0.09 , 0.00] 2.3E-01 Itgb4 -0.17 [-0.35 , 0.02] 3.3E-01 -0.06 [-0.10 , -0.01] 1.4E-01 Nt5e -0.17 [-0.41 , 0.08] 4.4E-01 -0.04 [-0.09 , 0.01] 4.5E-01 Cd36 -0.17 [-0.29 , -0.05] 1.8E-01 -0.03 [-0.04 , -0.01] 6.3E-02 Btla -0.17 [-0.42 , 0.08] 4.4E-01 0.01 [-0.04 , 0.06] 8.8E-01 Smad3 -0.17 [-0.30 , -0.04] 1.9E-01 -0.03 [-0.06 , -0.01] 1.9E-01 639 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il4ra -0.17 [-0.37 , 0.03] 3.4E-01 -0.07 [-0.11 , -0.04] 3.9E-02 Map3k7 -0.17 [-0.44 , 0.10] 4.8E-01 0.01 [-0.02 , 0.03] 7.9E-01 Ccl25 -0.17 [-0.45 , 0.11] 5.0E-01 -0.06 [-0.11 , -0.01] 1.5E-01 F2rl1 -0.17 [-0.48 , 0.13] 5.3E-01 0.00 [-0.04 , 0.04] 9.7E-01 Il12a -0.17 [-0.56 , 0.22] 6.4E-01 0.06 [-0.01 , 0.13] 3.8E-01 Hras -0.18 [-0.39 , 0.04] 3.4E-01 0.02 [-0.04 , 0.08] 7.4E-01 Lif -0.18 [-0.58 , 0.22] 6.4E-01 -0.06 [-0.14 , 0.03] 4.7E-01 Stat3 -0.18 [-0.30 , -0.06] 1.5E-01 -0.04 [-0.06 , -0.02] 4.9E-02 Cd9 -0.18 [-0.36 , 0.00] 2.9E-01 -0.02 [-0.05 , 0.00] 2.6E-01 Il18 -0.19 [-0.63 , 0.26] 6.6E-01 -0.01 [-0.06 , 0.04] 8.8E-01 Gfi1 -0.19 [-0.43 , 0.06] 3.8E-01 -0.04 [-0.09 , 0.01] 4.3E-01 Rrad -0.19 [-0.49 , 0.11] 4.8E-01 0.02 [-0.03 , 0.06] 7.6E-01 Il22ra2 -0.19 [-0.68 , 0.29] 6.8E-01 0.00 [-0.08 , 0.07] 9.7E-01 Nfatc4 -0.19 [-0.40 , 0.01] 3.1E-01 -0.04 [-0.07 , -0.01] 1.9E-01 Itga2b -0.19 [-0.44 , 0.05] 3.7E-01 0.03 [-0.03 , 0.08] 6.1E-01 Pdcd1 -0.20 [-0.64 , 0.25] 6.4E-01 n.d [n.d , n.d] n.d Ctsh -0.20 [-0.36 , -0.03] 2.2E-01 -0.01 [-0.04 , 0.03] 8.8E-01 640 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mcam -0.20 [-0.43 , 0.03] 3.3E-01 -0.01 [-0.05 , 0.02] 7.4E-01 Icam2 -0.20 [-0.42 , 0.02] 3.3E-01 -0.02 [-0.06 , 0.01] 4.0E-01 Crp -0.20 [-0.82 , 0.41] 7.4E-01 -0.15 [-0.30 , -0.01] 2.4E-01 Cxcl13 -0.21 [-1.51 , 1.10] 8.8E-01 -0.06 [-0.24 , 0.11] 7.1E-01 Ccr6 -0.22 [-0.59 , 0.15] 5.2E-01 0.00 [-0.07 , 0.07] 9.9E-01 Tirap -0.23 [-0.41 , -0.05] 2.0E-01 -0.03 [-0.06 , 0.00] 2.4E-01 Snai1 -0.23 [-0.44 , -0.02] 2.5E-01 -0.03 [-0.07 , 0.01] 3.8E-01 Ppbp -0.24 [-0.59 , 0.11] 4.4E-01 -0.06 [-0.12 , 0.00] 2.6E-01 Pvr -0.24 [-0.46 , -0.02] 2.5E-01 -0.05 [-0.09 , 0.00] 2.7E-01 Pax5 -0.25 [-0.61 , 0.12] 4.5E-01 0.01 [-0.09 , 0.10] 9.6E-01 Tnfsf11 -0.25 [-0.73 , 0.24] 5.8E-01 0.03 [-0.04 , 0.10] 6.7E-01 Cxcl14 -0.25 [-0.48 , -0.02] 2.5E-01 -0.08 [-0.13 , -0.03] 8.6E-02 Hsd11b1 -0.25 [-0.60 , 0.10] 4.1E-01 -0.01 [-0.05 , 0.03] 8.0E-01 Fut7 -0.26 [-0.55 , 0.03] 3.3E-01 -0.04 [-0.09 , 0.01] 3.8E-01 Smpd3 -0.27 [-0.81 , 0.28] 6.0E-01 -0.12 [-0.23 , -0.01] 2.4E-01 Pou2af1 -0.27 [-0.78 , 0.25] 5.8E-01 0.00 [-0.10 , 0.10] 1.0E+00 Elk1 -0.27 [-0.49 , -0.04] 2.3E-01 0.01 [-0.05 , 0.06] 9.3E-01 641 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cd40 -0.28 [-0.41 , -0.14] 8.7E-02 -0.04 [-0.06 , -0.01] 1.7E-01 Timd4 -0.28 [-0.71 , 0.15] 4.6E-01 -0.10 [-0.17 , -0.03] 1.3E-01 F13a1 -0.28 [-0.67 , 0.10] 4.0E-01 -0.01 [-0.08 , 0.05] 8.4E-01 Dusp4 -0.30 [-0.61 , 0.02] 3.1E-01 -0.04 [-0.10 , 0.02] 4.3E-01 Hc -0.30 [-0.63 , 0.02] 3.1E-01 -0.09 [-0.14 , -0.04] 8.2E-02 Egr1 -0.32 [-0.73 , 0.10] 3.8E-01 -0.08 [-0.21 , 0.06] 5.5E-01 Cdk1 -0.32 [-0.80 , 0.15] 4.4E-01 n.d [n.d , n.d] n.d Cd207 -0.33 [-0.78 , 0.12] 3.9E-01 -0.02 [-0.11 , 0.07] 8.4E-01 Marco -0.36 [-0.68 , -0.03] 2.5E-01 -0.02 [-0.09 , 0.06] 8.1E-01 Lilra5 -0.36 [-0.91 , 0.19] 4.7E-01 -0.05 [-0.13 , 0.03] 4.7E-01 Ptgs2 -0.37 [-0.71 , -0.02] 2.6E-01 -0.05 [-0.12 , 0.02] 4.7E-01 Tnfrsf12a -0.41 [-0.85 , 0.02] 3.1E-01 -0.09 [-0.18 , 0.00] 2.7E-01 Thbs1 -0.42 [-0.87 , 0.02] 3.0E-01 -0.08 [-0.18 , 0.02] 3.8E-01 Cr2 -0.44 [-1.01 , 0.14] 3.8E-01 -0.04 [-0.13 , 0.05] 6.7E-01 Cdkn1a -0.47 [-0.86 , -0.08] 2.2E-01 -0.06 [-0.15 , 0.03] 4.6E-01 Camp -0.51 [-0.97 , -0.06] 2.4E-01 -0.04 [-0.13 , 0.05] 6.6E-01 Serpinb2 -0.52 [-1.42 , 0.38] 5.3E-01 -0.06 [-0.19 , 0.08] 6.8E-01 642 Table 38 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post single exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcr1 -0.82 [-1.48 , -0.15] 2.1E-01 -0.15 [-0.24 , -0.06] 8.6E-02 Tnfsf18 -0.85 [-1.39 , -0.32] 1.3E-01 -0.17 [-0.27 , -0.06] 9.3E-02 Slc7a11 -0.89 [-1.31 , -0.48] 7.9E-02 -0.16 [-0.21 , -0.10] 1.5E-02 Cfd -1.68 [-3.79 , 0.44] 3.7E-01 -0.30 [-0.67 , 0.08] 4.0E-01 643 Table 39. Complete mRNA transcript profile in lung 24 hr post final exposure to cSiO2 in NZBWF1 mice. mRNA was measured using the nCounter Mouse PanCancer Immune Panel. Data are displayed for each treatment group as log2(fold change) relative to VEH + CON mice. Expression data are included for all mRNA transcripts that were above background threshold. Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Cxcl5 3.89 [2.95 , 4.83] 1.1E-05 0.68 [0.51 , 0.85] 2.3E-05 Cxcl1 2.79 [2.14 , 3.44] 8.1E-06 0.48 [0.36 , 0.61] 2.7E-05 Lcn2 2.49 [1.91 , 3.07] 8.1E-06 0.48 [0.37 , 0.60] 1.2E-05 Ccl2 2.45 [1.88 , 3.02] 7.9E-06 0.39 [0.28 , 0.50] 8.3E-05 Cxcl10 2.44 [1.58 , 3.30] 2.9E-04 0.13 [0.00 , 0.25] 1.3E-01 Ctla4 2.41 [1.97 , 2.86] 1.8E-06 0.44 [0.35 , 0.52] 2.6E-06 Pdcd1 2.20 [1.83 , 2.57] 1.1E-06 0.41 [0.32 , 0.49] 3.8E-06 Foxp3 2.18 [1.88 , 2.47] 2.2E-07 0.41 [0.36 , 0.46] 1.3E-07 Ccl3 2.10 [1.52 , 2.69] 4.0E-05 0.41 [0.30 , 0.52] 3.4E-05 Irf7 2.02 [1.38 , 2.66] 1.2E-04 0.01 [-0.04 , 0.06] 7.0E-01 Oasl1 1.98 [1.36 , 2.60] 1.1E-04 0.02 [-0.05 , 0.10] 6.6E-01 Mx2 1.97 [1.40 , 2.54] 5.5E-05 0.09 [0.03 , 0.15] 2.9E-02 644 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ifi44 1.94 [1.34 , 2.54] 9.5E-05 0.02 [-0.02 , 0.06] 4.6E-01 Oas2 1.91 [1.36 , 2.47] 5.5E-05 0.08 [0.03 , 0.13] 2.0E-02 Cxcl2 1.91 [1.29 , 2.53] 1.5E-04 0.32 [0.20 , 0.45] 9.7E-04 Fcer2a 1.89 [1.50 , 2.28] 3.5E-06 0.23 [0.16 , 0.31] 2.3E-04 Ccl4 1.88 [1.27 , 2.48] 1.4E-04 0.26 [0.14 , 0.38] 2.7E-03 Ccl7 1.87 [1.40 , 2.34] 1.6E-05 0.23 [0.13 , 0.34] 2.3E-03 Isg15 1.83 [1.25 , 2.40] 1.2E-04 0.00 [-0.05 , 0.05] 1.0E+00 Cxcl3 1.81 [0.78 , 2.85] 9.0E-03 0.22 [0.01 , 0.43] 1.0E-01 Il1rn 1.78 [1.25 , 2.31] 7.2E-05 0.29 [0.18 , 0.40] 7.0E-04 Csf2 1.76 [1.44 , 2.08] 1.8E-06 0.34 [0.28 , 0.41] 1.3E-06 Il12b 1.76 [1.25 , 2.26] 5.4E-05 0.34 [0.26 , 0.41] 8.1E-06 Clec5a 1.73 [1.45 , 2.00] 8.3E-07 0.26 [0.20 , 0.31] 4.5E-06 Marco 1.71 [1.33 , 2.10] 6.5E-06 0.36 [0.29 , 0.43] 3.1E-06 Trem2 1.68 [1.34 , 2.02] 3.5E-06 0.30 [0.24 , 0.36] 3.1E-06 Cxcl9 1.68 [0.69 , 2.67] 1.1E-02 0.18 [0.00 , 0.35] 1.3E-01 Ccl20 1.68 [0.21 , 3.14] 6.9E-02 0.19 [-0.09 , 0.47] 2.9E-01 C3ar1 1.67 [1.31 , 2.02] 4.0E-06 0.29 [0.23 , 0.35] 4.0E-06 645 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccl8 1.66 [0.67 , 2.66] 1.2E-02 0.25 [0.05 , 0.45] 5.6E-02 Mmp9 1.64 [1.16 , 2.12] 5.7E-05 0.33 [0.25 , 0.40] 1.2E-05 Cfb 1.63 [1.28 , 1.98] 4.0E-06 0.24 [0.17 , 0.32] 1.4E-04 Oas3 1.59 [1.03 , 2.15] 2.9E-04 n.d [n.d , n.d] n.d Il2ra 1.57 [1.29 , 1.84] 1.6E-06 0.29 [0.25 , 0.33] 2.6E-07 C1qa 1.54 [1.33 , 1.75] 2.2E-07 0.24 [0.21 , 0.28] 4.6E-07 Ifit1 1.54 [0.98 , 2.10] 3.8E-04 0.02 [-0.03 , 0.07] 4.6E-01 Zbp1 1.53 [1.02 , 2.03] 1.6E-04 0.03 [-0.02 , 0.09] 3.6E-01 Tigit 1.52 [1.20 , 1.84] 3.8E-06 0.22 [0.16 , 0.28] 5.1E-05 Ccl12 1.47 [1.09 , 1.86] 2.3E-05 0.16 [0.09 , 0.23] 2.0E-03 Msr1 1.45 [1.12 , 1.77] 6.5E-06 0.19 [0.12 , 0.26] 4.5E-04 C1qb 1.40 [1.16 , 1.65] 1.4E-06 0.22 [0.18 , 0.26] 9.0E-07 Ccl9 1.38 [1.10 , 1.65] 3.2E-06 0.23 [0.17 , 0.28] 1.8E-05 Clec4n 1.36 [1.06 , 1.66] 5.2E-06 0.20 [0.14 , 0.26] 8.4E-05 Cd207 1.28 [0.89 , 1.68] 1.0E-04 0.34 [0.29 , 0.38] 1.3E-07 Tnfsf11 1.26 [0.95 , 1.57] 1.3E-05 0.18 [0.12 , 0.24] 3.5E-04 Usp18 1.26 [0.80 , 1.73] 3.8E-04 0.04 [0.00 , 0.09] 1.6E-01 646 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Siglec1 1.23 [0.89 , 1.58] 4.4E-05 0.16 [0.10 , 0.21] 4.8E-04 Fcgr1 1.20 [0.97 , 1.44] 2.7E-06 0.07 [0.04 , 0.10] 2.3E-03 Rsad2 1.20 [0.60 , 1.81] 4.0E-03 0.06 [-0.02 , 0.14] 2.2E-01 Ccr5 1.18 [0.86 , 1.49] 2.8E-05 0.19 [0.13 , 0.25] 1.9E-04 Ifit3 1.18 [0.72 , 1.65] 6.5E-04 0.00 [-0.05 , 0.04] 9.0E-01 Icos 1.16 [0.91 , 1.40] 4.0E-06 0.18 [0.13 , 0.23] 8.3E-05 Ccr8 1.15 [0.78 , 1.53] 1.5E-04 0.19 [0.13 , 0.26] 1.9E-04 Slc11a1 1.14 [0.73 , 1.54] 3.4E-04 0.19 [0.10 , 0.27] 3.0E-03 Cd83 1.12 [0.89 , 1.36] 3.9E-06 0.17 [0.13 , 0.22] 2.3E-05 Cd14 1.12 [0.79 , 1.44] 5.5E-05 0.21 [0.14 , 0.27] 1.1E-04 Cx3cr1 1.11 [0.93 , 1.29] 8.3E-07 0.17 [0.13 , 0.22] 1.4E-05 Fcgr2b 1.11 [0.85 , 1.38] 8.8E-06 0.24 [0.18 , 0.29] 1.0E-05 Tnfrsf11a 1.10 [0.89 , 1.31] 2.4E-06 0.20 [0.17 , 0.24] 8.2E-07 Cd80 1.09 [0.84 , 1.34] 6.6E-06 0.23 [0.17 , 0.28] 1.0E-05 Cxcr1 1.09 [0.69 , 1.50] 4.1E-04 0.12 [0.04 , 0.21] 2.6E-02 Tnfrsf4 1.06 [0.87 , 1.25] 1.7E-06 0.16 [0.12 , 0.21] 8.4E-05 Ccl22 1.05 [0.67 , 1.44] 3.8E-04 0.24 [0.17 , 0.30] 6.7E-05 647 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il1a 1.05 [0.65 , 1.45] 5.1E-04 0.12 [0.04 , 0.21] 2.9E-02 Cd86 1.03 [0.77 , 1.29] 1.6E-05 0.18 [0.13 , 0.23] 3.0E-05 Ccr6 1.02 [0.68 , 1.36] 2.0E-04 0.16 [0.09 , 0.23] 1.4E-03 Mefv 1.02 [0.60 , 1.44] 9.4E-04 0.16 [0.09 , 0.24] 3.7E-03 Birc5 1.02 [0.46 , 1.57] 6.7E-03 0.18 [0.08 , 0.28] 9.2E-03 Chil3 1.02 [-0.23 , 2.28] 1.9E-01 0.04 [-0.18 , 0.25] 8.0E-01 Ccl6 1.01 [0.80 , 1.22] 3.9E-06 0.14 [0.09 , 0.19] 5.6E-04 C1ra 1.00 [0.82 , 1.18] 1.6E-06 0.17 [0.14 , 0.20] 1.9E-06 C3 0.99 [0.73 , 1.25] 2.3E-05 0.20 [0.15 , 0.24] 2.1E-05 Arg2 0.97 [0.75 , 1.18] 6.5E-06 0.22 [0.17 , 0.26] 5.9E-06 Tlr2 0.96 [0.75 , 1.16] 4.0E-06 0.12 [0.07 , 0.17] 1.1E-03 Cxcl13 0.95 [0.29 , 1.62] 2.7E-02 0.21 [0.05 , 0.36] 4.2E-02 Il6 0.95 [0.23 , 1.67] 3.9E-02 0.04 [-0.12 , 0.19] 7.3E-01 Gbp2b 0.95 [0.49 , 1.40] 3.1E-03 0.06 [-0.01 , 0.12] 1.6E-01 Ccr1 0.94 [0.61 , 1.27] 2.9E-04 0.16 [0.10 , 0.22] 5.2E-04 Itgae 0.93 [0.61 , 1.25] 2.5E-04 0.25 [0.21 , 0.30] 6.9E-07 Cd68 0.93 [0.75 , 1.11] 2.7E-06 0.14 [0.10 , 0.17] 2.6E-05 648 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il10ra 0.92 [0.67 , 1.17] 3.5E-05 0.14 [0.09 , 0.19] 2.5E-04 Cfi 0.91 [0.48 , 1.34] 2.6E-03 0.15 [0.05 , 0.25] 2.5E-02 Ncf4 0.91 [0.76 , 1.05] 8.3E-07 0.14 [0.11 , 0.18] 6.7E-06 Tnfrsf18 0.90 [0.67 , 1.14] 2.1E-05 0.10 [0.03 , 0.18] 3.2E-02 Clec7a 0.90 [0.64 , 1.16] 5.5E-05 0.08 [0.02 , 0.14] 5.6E-02 Ikzf2 0.88 [0.70 , 1.07] 4.0E-06 0.18 [0.15 , 0.21] 1.2E-06 Xcr1 0.87 [0.49 , 1.26] 1.7E-03 0.22 [0.18 , 0.26] 3.1E-06 Havcr2 0.87 [0.70 , 1.04] 2.7E-06 0.14 [0.10 , 0.18] 6.5E-05 Tnfaip3 0.86 [0.67 , 1.06] 6.8E-06 0.18 [0.15 , 0.21] 1.9E-06 Cmpk2 0.86 [0.53 , 1.19] 5.6E-04 0.00 [-0.03 , 0.02] 9.0E-01 Cd74 0.86 [0.69 , 1.03] 2.7E-06 0.14 [0.11 , 0.17] 3.2E-06 Fcer1g 0.85 [0.68 , 1.03] 3.6E-06 0.13 [0.09 , 0.16] 6.5E-05 Ddx60 0.84 [0.49 , 1.19] 1.1E-03 0.00 [-0.03 , 0.03] 9.0E-01 Tnf 0.84 [0.51 , 1.16] 6.2E-04 0.17 [0.09 , 0.24] 3.3E-03 Ctss 0.84 [0.62 , 1.05] 1.9E-05 0.06 [0.01 , 0.11] 6.0E-02 Il1b 0.83 [0.51 , 1.16] 6.0E-04 0.13 [0.06 , 0.19] 6.1E-03 Ifitm1 0.83 [0.47 , 1.19] 1.4E-03 0.18 [0.11 , 0.25] 1.1E-03 649 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Csf2rb 0.82 [0.64 , 1.00] 5.1E-06 0.16 [0.12 , 0.20] 1.9E-05 Ccr2 0.82 [0.60 , 1.03] 2.4E-05 0.13 [0.08 , 0.18] 5.2E-04 Blnk 0.81 [0.48 , 1.14] 8.6E-04 0.13 [0.07 , 0.20] 4.1E-03 Irf5 0.81 [0.69 , 0.93] 3.9E-07 0.14 [0.12 , 0.16] 3.0E-07 Bst2 0.80 [0.51 , 1.10] 3.8E-04 -0.01 [-0.03 , 0.01] 4.7E-01 Osm 0.80 [0.45 , 1.15] 1.5E-03 0.11 [0.04 , 0.17] 1.3E-02 Slamf7 0.78 [0.61 , 0.96] 6.6E-06 0.14 [0.11 , 0.17] 1.3E-05 Fcgr3 0.78 [0.60 , 0.96] 8.1E-06 0.11 [0.06 , 0.15] 1.6E-03 Emr1 0.78 [0.55 , 1.01] 5.7E-05 0.07 [0.01 , 0.13] 6.4E-02 Stat1 0.78 [0.51 , 1.05] 2.6E-04 0.01 [-0.01 , 0.04] 4.6E-01 Xaf1 0.77 [0.47 , 1.07] 6.1E-04 0.00 [-0.02 , 0.02] 9.9E-01 Tap1 0.77 [0.51 , 1.03] 2.0E-04 0.06 [0.04 , 0.08] 7.4E-04 H2-Ab1 0.76 [0.61 , 0.92] 3.2E-06 0.12 [0.09 , 0.14] 4.4E-06 Relb 0.76 [0.59 , 0.93] 5.7E-06 0.13 [0.10 , 0.15] 3.0E-06 Ebi3 0.76 [0.51 , 1.00] 1.4E-04 0.15 [0.10 , 0.19] 1.4E-04 Spp1 0.75 [0.48 , 1.03] 3.5E-04 0.11 [0.05 , 0.18] 1.1E-02 H2-Ea-ps 0.75 [0.60 , 0.90] 3.3E-06 0.14 [0.11 , 0.17] 2.8E-06 650 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tlr9 0.75 [0.48 , 1.02] 3.8E-04 0.12 [0.08 , 0.16] 1.3E-04 Nlrc5 0.75 [0.44 , 1.05] 8.9E-04 0.05 [0.02 , 0.09] 2.5E-02 Il23r 0.75 [0.40 , 1.09] 2.2E-03 0.16 [0.11 , 0.22] 3.2E-04 C1s1 0.74 [0.59 , 0.89] 3.5E-06 0.14 [0.11 , 0.17] 2.6E-06 Cd33 0.74 [0.59 , 0.89] 3.5E-06 0.15 [0.11 , 0.19] 3.2E-05 H2-Aa 0.74 [0.58 , 0.89] 3.5E-06 0.12 [0.09 , 0.14] 1.2E-05 Il21r 0.73 [0.42 , 1.05] 1.3E-03 0.07 [0.00 , 0.13] 9.8E-02 Cd5 0.73 [0.43 , 1.03] 1.0E-03 0.10 [0.04 , 0.16] 1.8E-02 Clec4a2 0.73 [0.47 , 0.98] 2.9E-04 0.09 [0.04 , 0.14] 1.0E-02 Klrg1 0.72 [0.53 , 0.90] 2.0E-05 0.13 [0.09 , 0.17] 1.0E-04 Il1r2 0.72 [0.20 , 1.23] 3.0E-02 0.19 [0.11 , 0.28] 1.8E-03 Cd4 0.72 [0.47 , 0.96] 2.6E-04 0.08 [0.03 , 0.14] 2.4E-02 Ccl17 0.71 [0.36 , 1.07] 3.6E-03 0.23 [0.17 , 0.28] 1.4E-05 Csf1r 0.71 [0.62 , 0.81] 2.2E-07 0.12 [0.10 , 0.15] 1.2E-05 Nlrp3 0.71 [0.51 , 0.92] 5.7E-05 0.13 [0.08 , 0.18] 1.1E-03 H2-T23 0.71 [0.51 , 0.90] 4.0E-05 0.04 [0.02 , 0.06] 8.3E-03 Stat2 0.71 [0.42 , 0.99] 8.0E-04 0.03 [-0.01 , 0.07] 1.8E-01 651 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Itgax 0.70 [0.49 , 0.91] 7.9E-05 0.11 [0.07 , 0.14] 3.0E-04 H2-Eb1 0.70 [0.51 , 0.88] 2.5E-05 0.13 [0.10 , 0.17] 3.4E-05 Ly86 0.70 [0.47 , 0.93] 1.7E-04 0.05 [0.02 , 0.09] 3.0E-02 Cxcr3 0.69 [0.44 , 0.95] 4.0E-04 0.06 [0.02 , 0.11] 3.3E-02 H2-Q2 0.69 [0.30 , 1.09] 8.9E-03 0.09 [0.02 , 0.16] 4.5E-02 Cybb 0.69 [0.46 , 0.92] 1.6E-04 0.03 [-0.03 , 0.11] 4.4E-01 Pdcd1lg2 0.68 [0.39 , 0.98] 1.3E-03 0.16 [0.11 , 0.20] 1.1E-04 Fap 0.68 [0.33 , 1.03] 4.5E-03 0.09 [0.02 , 0.17] 5.6E-02 Pik3cg 0.68 [0.52 , 0.84] 7.6E-06 0.12 [0.08 , 0.15] 1.3E-04 Gbp5 0.68 [0.45 , 0.90] 1.7E-04 0.04 [-0.01 , 0.08] 1.8E-01 Herc6 0.68 [0.35 , 1.01] 3.1E-03 0.00 [-0.04 , 0.03] 8.6E-01 H2-DMb1 0.67 [0.54 , 0.80] 2.7E-06 0.13 [0.11 , 0.16] 2.8E-06 Amica1 0.67 [0.48 , 0.86] 5.1E-05 0.15 [0.12 , 0.18] 3.3E-06 Cd6 0.67 [0.38 , 0.95] 1.2E-03 0.08 [0.02 , 0.13] 3.9E-02 Psmb9 0.66 [0.50 , 0.82] 9.3E-06 0.03 [0.01 , 0.06] 3.8E-02 Lif 0.66 [0.08 , 1.24] 6.9E-02 0.10 [-0.02 , 0.22] 2.0E-01 Ikbke 0.66 [0.40 , 0.91] 6.6E-04 0.15 [0.10 , 0.20] 1.4E-04 652 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value C6 0.65 [0.49 , 0.82] 2.1E-05 0.14 [0.11 , 0.17] 4.8E-06 Tnfrsf1b 0.65 [0.52 , 0.78] 3.5E-06 0.08 [0.05 , 0.12] 1.1E-03 Cxcr6 0.65 [0.38 , 0.93] 1.2E-03 0.10 [0.04 , 0.16] 1.2E-02 Cd200r1 0.65 [0.43 , 0.87] 2.4E-04 0.13 [0.08 , 0.18] 7.3E-04 H2-K1 0.64 [0.48 , 0.81] 1.9E-05 0.02 [0.00 , 0.04] 1.2E-01 Runx3 0.64 [0.33 , 0.95] 3.1E-03 0.08 [0.01 , 0.14] 6.2E-02 H2-DMa 0.64 [0.53 , 0.75] 1.0E-06 0.11 [0.09 , 0.14] 3.1E-06 Tlr7 0.64 [0.43 , 0.84] 1.3E-04 0.04 [-0.01 , 0.09] 2.2E-01 Cd276 0.63 [0.49 , 0.77] 4.8E-06 0.10 [0.07 , 0.13] 1.5E-04 Lcp1 0.63 [0.49 , 0.77] 5.1E-06 0.09 [0.06 , 0.12] 1.6E-04 Csf1 0.63 [0.43 , 0.83] 1.2E-04 0.10 [0.06 , 0.13] 9.9E-04 Ccl19 0.63 [0.38 , 0.87] 6.4E-04 0.11 [0.06 , 0.15] 3.1E-03 Gpr183 0.63 [0.38 , 0.87] 6.4E-04 0.08 [0.03 , 0.13] 2.5E-02 Psmb8 0.62 [0.47 , 0.78] 1.5E-05 0.04 [0.02 , 0.07] 8.5E-03 Tnfsf15 0.62 [0.43 , 0.81] 1.1E-04 0.08 [0.04 , 0.12] 2.7E-03 Slc7a11 0.62 [0.22 , 1.02] 1.8E-02 0.11 [0.04 , 0.18] 1.7E-02 Lag3 0.62 [0.21 , 1.03] 2.1E-02 0.09 [0.00 , 0.17] 1.4E-01 653 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Gzmk 0.62 [0.19 , 1.04] 2.6E-02 n.d [n.d , n.d] n.d Lgals3 0.61 [0.40 , 0.81] 2.2E-04 0.07 [0.03 , 0.11] 1.3E-02 Ticam2 0.60 [0.44 , 0.76] 2.4E-05 0.10 [0.05 , 0.15] 4.3E-03 H2-DMb2 0.60 [0.23 , 0.97] 1.4E-02 0.10 [0.04 , 0.17] 1.6E-02 Il17rb 0.59 [0.23 , 0.96] 1.3E-02 0.16 [0.09 , 0.22] 1.5E-03 Muc1 0.59 [0.43 , 0.76] 3.2E-05 0.11 [0.08 , 0.14] 8.4E-05 C4b 0.59 [0.31 , 0.88] 2.8E-03 0.08 [0.04 , 0.13] 7.9E-03 Plau 0.59 [0.28 , 0.91] 5.5E-03 0.07 [0.02 , 0.13] 4.4E-02 Nfkb2 0.59 [0.45 , 0.73] 8.8E-06 0.11 [0.09 , 0.14] 4.4E-06 Nod2 0.59 [0.28 , 0.90] 5.5E-03 0.11 [0.03 , 0.20] 4.9E-02 Sh2b2 0.59 [0.37 , 0.81] 4.6E-04 0.14 [0.09 , 0.19] 2.5E-04 Cd53 0.58 [0.43 , 0.73] 1.9E-05 0.09 [0.05 , 0.13] 1.4E-03 Il1rl2 0.58 [0.37 , 0.79] 3.5E-04 0.15 [0.12 , 0.18] 4.0E-06 Ddx58 0.58 [0.40 , 0.76] 1.1E-04 0.04 [0.02 , 0.05] 4.8E-03 Cd274 0.57 [0.41 , 0.74] 5.7E-05 0.06 [0.03 , 0.10] 5.6E-03 Lyz2 0.57 [0.42 , 0.73] 3.2E-05 0.07 [0.04 , 0.10] 3.5E-03 Sh2d1a 0.57 [0.36 , 0.78] 4.5E-04 0.04 [0.00 , 0.09] 1.1E-01 654 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Flt3 0.57 [0.33 , 0.81] 1.3E-03 0.12 [0.08 , 0.16] 4.6E-04 Batf 0.55 [0.29 , 0.81] 2.6E-03 0.07 [0.03 , 0.12] 2.1E-02 Vcam1 0.55 [0.32 , 0.78] 1.0E-03 0.05 [0.01 , 0.10] 5.1E-02 Cd3g 0.55 [0.37 , 0.73] 1.6E-04 0.05 [0.01 , 0.09] 4.9E-02 Ciita 0.55 [0.30 , 0.80] 1.9E-03 0.12 [0.09 , 0.15] 2.6E-05 Cd84 0.55 [0.36 , 0.74] 2.2E-04 0.07 [0.04 , 0.11] 5.2E-03 S100a8 0.55 [-0.10 , 1.19] 1.7E-01 0.08 [-0.04 , 0.20] 3.1E-01 Casp1 0.55 [0.39 , 0.70] 5.4E-05 0.04 [0.00 , 0.07] 1.0E-01 Socs1 0.55 [0.28 , 0.81] 3.2E-03 0.06 [0.01 , 0.11] 6.2E-02 Cd180 0.55 [0.25 , 0.84] 6.8E-03 0.05 [0.00 , 0.11] 9.6E-02 Fos 0.54 [-0.15 , 1.23] 2.0E-01 0.02 [-0.07 , 0.11] 7.8E-01 H2-D1 0.54 [0.43 , 0.66] 3.6E-06 0.04 [0.03 , 0.05] 2.0E-04 Pou2f2 0.54 [0.29 , 0.79] 2.1E-03 0.08 [0.02 , 0.14] 3.5E-02 Irf4 0.54 [0.18 , 0.91] 2.2E-02 0.10 [0.03 , 0.18] 3.4E-02 Cxcr4 0.53 [0.37 , 0.69] 8.8E-05 0.09 [0.05 , 0.12] 2.4E-03 Lamp3 0.53 [0.37 , 0.69] 1.0E-04 0.08 [0.04 , 0.11] 1.3E-03 Selplg 0.53 [0.42 , 0.63] 3.3E-06 0.07 [0.05 , 0.09] 1.6E-04 655 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il1rl1 0.53 [0.31 , 0.75] 1.1E-03 0.18 [0.14 , 0.22] 1.0E-05 Ifih1 0.53 [0.34 , 0.71] 2.9E-04 0.02 [0.01 , 0.03] 4.9E-03 Ly9 0.52 [0.23 , 0.82] 8.3E-03 0.04 [-0.01 , 0.09] 2.4E-01 Ulbp1 0.52 [0.19 , 0.86] 1.7E-02 0.06 [0.00 , 0.12] 1.1E-01 Btk 0.52 [0.39 , 0.65] 1.9E-05 0.09 [0.06 , 0.11] 9.5E-05 Trem1 0.52 [0.26 , 0.78] 3.7E-03 0.13 [0.07 , 0.19] 2.7E-03 Tnfsf13 0.52 [0.26 , 0.78] 4.3E-03 0.15 [0.09 , 0.20] 4.8E-04 Csf3r 0.52 [0.12 , 0.91] 4.0E-02 0.14 [0.07 , 0.21] 5.0E-03 Cxcl16 0.52 [0.35 , 0.68] 1.4E-04 0.07 [0.04 , 0.10] 1.3E-03 Nfatc2 0.52 [0.31 , 0.72] 6.9E-04 0.13 [0.09 , 0.17] 9.7E-05 Ltb 0.51 [0.25 , 0.77] 4.5E-03 0.07 [0.01 , 0.12] 7.2E-02 Itgam 0.51 [0.22 , 0.80] 9.5E-03 0.08 [0.02 , 0.14] 4.4E-02 Cd44 0.51 [0.32 , 0.70] 5.3E-04 0.08 [0.04 , 0.12] 2.6E-03 Slamf6 0.50 [0.19 , 0.81] 1.5E-02 0.03 [-0.03 , 0.09] 3.9E-01 Spn 0.49 [0.32 , 0.66] 2.5E-04 0.06 [0.02 , 0.11] 2.4E-02 Icosl 0.49 [0.25 , 0.73] 3.1E-03 0.10 [0.06 , 0.13] 5.6E-04 Col3a1 0.49 [0.25 , 0.72] 3.5E-03 0.10 [0.05 , 0.14] 3.6E-03 656 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Zap70 0.48 [0.31 , 0.66] 3.1E-04 0.08 [0.04 , 0.11] 3.3E-03 Xcl1 0.48 [0.22 , 0.75] 6.6E-03 -0.03 [-0.11 , 0.06] 6.1E-01 Jak3 0.48 [0.31 , 0.65] 3.7E-04 0.09 [0.06 , 0.13] 3.6E-04 Lta 0.48 [0.21 , 0.75] 8.9E-03 0.07 [0.02 , 0.13] 4.9E-02 Fcgr4 0.48 [0.27 , 0.69] 1.6E-03 -0.03 [-0.10 , 0.05] 5.8E-01 Cfp 0.48 [0.07 , 0.88] 6.5E-02 0.05 [-0.04 , 0.14] 3.6E-01 Cd48 0.47 [0.29 , 0.66] 5.8E-04 0.03 [0.00 , 0.07] 1.6E-01 Tlr1 0.47 [0.12 , 0.83] 3.9E-02 0.04 [-0.02 , 0.10] 3.2E-01 Syk 0.46 [0.30 , 0.62] 2.9E-04 0.08 [0.05 , 0.11] 1.1E-03 Irgm2 0.45 [0.21 , 0.69] 6.7E-03 -0.01 [-0.04 , 0.03] 7.8E-01 Raet1c 0.45 [0.23 , 0.67] 3.1E-03 0.07 [0.03 , 0.12] 1.5E-02 Rel 0.45 [0.28 , 0.61] 3.9E-04 0.09 [0.06 , 0.13] 3.4E-04 Itgb2 0.44 [0.29 , 0.59] 2.1E-04 0.04 [0.00 , 0.08] 1.2E-01 Hcst 0.44 [-0.01 , 0.88] 1.1E-01 0.06 [-0.02 , 0.14] 2.1E-01 Abcg1 0.43 [0.30 , 0.57] 1.0E-04 0.05 [0.01 , 0.09] 3.9E-02 Tlr6 0.43 [0.28 , 0.59] 3.2E-04 0.05 [0.03 , 0.08] 7.6E-03 Tap2 0.43 [0.22 , 0.65] 3.8E-03 0.05 [0.02 , 0.08] 5.9E-03 657 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfrsf11b 0.43 [0.03 , 0.83] 8.5E-02 0.08 [0.00 , 0.17] 1.4E-01 Il34 0.43 [0.30 , 0.56] 7.8E-05 0.10 [0.07 , 0.13] 8.3E-05 Psmb10 0.43 [0.29 , 0.57] 1.4E-04 -0.01 [-0.04 , 0.02] 6.2E-01 Ifit2 0.42 [0.10 , 0.75] 4.0E-02 -0.05 [-0.07 , -0.03] 3.2E-03 Ada 0.42 [0.21 , 0.63] 3.7E-03 0.06 [0.02 , 0.10] 2.8E-02 Pdgfc 0.41 [0.19 , 0.64] 7.4E-03 0.04 [-0.01 , 0.09] 1.8E-01 Cd3e 0.41 [0.21 , 0.62] 3.6E-03 0.03 [-0.01 , 0.07] 2.6E-01 Irf8 0.41 [0.24 , 0.58] 1.2E-03 0.07 [0.04 , 0.09] 1.4E-03 Egr3 0.41 [0.02 , 0.80] 9.6E-02 0.04 [-0.04 , 0.11] 4.8E-01 Ptgs2 0.40 [0.05 , 0.76] 7.3E-02 0.02 [-0.05 , 0.09] 7.0E-01 Itgal 0.40 [0.29 , 0.52] 6.2E-05 0.02 [-0.02 , 0.06] 3.9E-01 Pik3cd 0.40 [0.18 , 0.63] 8.1E-03 0.04 [0.00 , 0.09] 9.6E-02 Card9 0.40 [0.17 , 0.62] 8.7E-03 0.04 [-0.02 , 0.11] 2.9E-01 Pla2g1b 0.40 [0.24 , 0.56] 8.0E-04 0.05 [0.02 , 0.08] 3.0E-02 Tmem173 0.39 [0.19 , 0.60] 5.2E-03 0.04 [-0.01 , 0.09] 1.8E-01 Irak2 0.39 [0.30 , 0.48] 6.6E-06 0.09 [0.08 , 0.11] 8.2E-07 H2-M3 0.39 [0.25 , 0.52] 2.6E-04 0.02 [0.01 , 0.04] 4.9E-02 658 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Egr2 0.39 [-0.03 , 0.81] 1.4E-01 0.09 [0.02 , 0.16] 4.4E-02 Cxcr2 0.39 [-0.04 , 0.82] 1.5E-01 0.12 [0.04 , 0.20] 3.2E-02 Prkcd 0.39 [0.32 , 0.46] 1.7E-06 0.08 [0.06 , 0.09] 3.3E-06 Ptgdr2 0.39 [-0.07 , 0.84] 1.7E-01 0.06 [-0.05 , 0.16] 4.2E-01 H2-Ob 0.38 [0.07 , 0.70] 5.6E-02 0.06 [0.01 , 0.12] 6.6E-02 Ptprc 0.37 [0.22 , 0.52] 8.8E-04 0.04 [0.01 , 0.07] 6.8E-02 Abca1 0.37 [0.12 , 0.61] 2.1E-02 0.07 [0.02 , 0.12] 3.0E-02 Lck 0.37 [0.21 , 0.53] 1.5E-03 0.02 [-0.02 , 0.06] 4.9E-01 Ccr7 0.36 [0.10 , 0.62] 2.9E-02 0.10 [0.05 , 0.14] 6.7E-03 Tnfrsf13b 0.36 [0.13 , 0.59] 1.7E-02 0.05 [0.01 , 0.09] 8.1E-02 Klra2 0.36 [0.20 , 0.52] 1.9E-03 -0.04 [-0.08 , 0.01] 2.2E-01 Cd3d 0.35 [0.15 , 0.56] 1.1E-02 0.02 [-0.02 , 0.06] 4.6E-01 Fut7 0.35 [0.01 , 0.69] 1.0E-01 0.04 [-0.03 , 0.11] 4.2E-01 Cd69 0.35 [0.05 , 0.65] 6.5E-02 0.06 [-0.01 , 0.12] 1.8E-01 Flt3l 0.35 [0.11 , 0.59] 2.6E-02 0.07 [0.03 , 0.11] 1.8E-02 Tgfbr1 0.34 [0.24 , 0.44] 4.8E-05 0.07 [0.05 , 0.09] 2.0E-05 Ly96 0.34 [0.19 , 0.48] 1.6E-03 0.07 [0.05 , 0.09] 1.1E-04 659 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value C2 0.33 [0.12 , 0.55] 1.9E-02 -0.01 [-0.05 , 0.04] 8.2E-01 Stat4 0.33 [0.15 , 0.52] 8.0E-03 0.03 [0.00 , 0.07] 9.6E-02 Tapbp 0.33 [0.24 , 0.42] 4.6E-05 0.03 [0.02 , 0.05] 1.5E-03 Isg20 0.33 [0.19 , 0.47] 1.3E-03 0.01 [-0.02 , 0.04] 6.4E-01 Tgfb1 0.33 [0.26 , 0.39] 3.5E-06 0.05 [0.03 , 0.08] 6.8E-04 Ccl11 0.33 [-0.05 , 0.70] 1.7E-01 0.11 [0.04 , 0.17] 2.2E-02 Cd96 0.32 [-0.03 , 0.68] 1.5E-01 0.06 [-0.02 , 0.13] 2.2E-01 Pou2af1 0.32 [-0.19 , 0.83] 3.1E-01 0.02 [-0.09 , 0.12] 8.0E-01 H2-Q10 0.32 [-0.05 , 0.69] 1.7E-01 0.06 [-0.02 , 0.15] 2.6E-01 Traf3 0.32 [0.24 , 0.40] 1.6E-05 0.09 [0.07 , 0.10] 9.0E-07 Ifnar2 0.32 [0.24 , 0.39] 9.3E-06 0.05 [0.03 , 0.06] 3.1E-04 Cd27 0.32 [-0.02 , 0.66] 1.4E-01 0.00 [-0.08 , 0.07] 9.8E-01 Pml 0.32 [0.07 , 0.56] 4.5E-02 0.01 [-0.02 , 0.04] 6.3E-01 Tnfsf13b 0.31 [0.17 , 0.46] 2.7E-03 0.02 [-0.01 , 0.05] 2.5E-01 Ikzf1 0.31 [0.12 , 0.50] 1.3E-02 0.03 [-0.01 , 0.07] 2.4E-01 Cxcl15 0.31 [0.04 , 0.58] 6.4E-02 0.02 [-0.04 , 0.07] 6.4E-01 Nfkbia 0.31 [0.09 , 0.53] 2.8E-02 0.08 [0.04 , 0.12] 6.3E-03 660 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Bid 0.31 [0.15 , 0.46] 4.3E-03 0.03 [0.00 , 0.05] 6.9E-02 Klra17 0.30 [0.11 , 0.50] 1.9E-02 -0.02 [-0.06 , 0.02] 4.9E-01 Serping1 0.30 [0.22 , 0.38] 2.9E-05 0.06 [0.04 , 0.07] 3.4E-05 Nfkb1 0.30 [0.23 , 0.37] 1.5E-05 0.07 [0.07 , 0.08] 7.4E-09 Casp8 0.30 [0.21 , 0.38] 5.5E-05 0.03 [0.02 , 0.05] 8.4E-03 Apoe 0.30 [0.19 , 0.41] 3.7E-04 0.06 [0.04 , 0.09] 1.5E-03 Itga4 0.30 [0.12 , 0.47] 1.1E-02 0.03 [-0.01 , 0.07] 2.6E-01 Lilra5 0.30 [0.18 , 0.41] 5.6E-04 -0.02 [-0.07 , 0.03] 5.4E-01 Sbno2 0.30 [0.14 , 0.45] 6.2E-03 0.06 [0.03 , 0.10] 7.6E-03 Inpp5d 0.30 [0.13 , 0.47] 9.5E-03 0.08 [0.05 , 0.10] 2.0E-04 Il5ra 0.30 [-0.13 , 0.72] 2.6E-01 0.06 [-0.03 , 0.16] 2.9E-01 Lbp 0.29 [0.17 , 0.41] 8.8E-04 0.06 [0.03 , 0.09] 9.2E-03 Irak3 0.29 [0.16 , 0.42] 2.3E-03 0.07 [0.03 , 0.11] 7.3E-03 Tlr8 0.29 [0.17 , 0.41] 1.2E-03 -0.01 [-0.06 , 0.03] 6.5E-01 Il7r 0.28 [0.17 , 0.39] 6.6E-04 0.09 [0.06 , 0.13] 3.4E-04 Cyfip2 0.28 [0.08 , 0.48] 3.0E-02 0.05 [0.01 , 0.09] 5.2E-02 Ifi27 0.28 [0.20 , 0.36] 7.7E-05 0.01 [-0.01 , 0.03] 4.6E-01 661 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfsf14 0.28 [0.06 , 0.49] 4.6E-02 0.08 [0.04 , 0.13] 7.2E-03 Ccl21a 0.27 [0.11 , 0.43] 1.1E-02 0.03 [0.00 , 0.05] 1.2E-01 Il17ra 0.27 [0.12 , 0.42] 7.5E-03 0.01 [-0.02 , 0.04] 4.9E-01 Cd8a 0.27 [-0.05 , 0.59] 1.7E-01 -0.03 [-0.10 , 0.04] 4.9E-01 Jak2 0.27 [0.20 , 0.34] 1.3E-05 0.06 [0.05 , 0.08] 2.8E-06 Socs3 0.27 [-0.10 , 0.64] 2.4E-01 0.05 [-0.03 , 0.12] 3.3E-01 C5ar1 0.27 [0.07 , 0.47] 3.5E-02 0.02 [-0.02 , 0.06] 4.6E-01 Ccr9 0.27 [-0.08 , 0.61] 2.1E-01 -0.04 [-0.09 , 0.01] 2.5E-01 Tank 0.26 [0.18 , 0.35] 1.6E-04 0.04 [0.03 , 0.06] 9.0E-04 Sh2d1b1 0.26 [-0.06 , 0.59] 1.9E-01 0.00 [-0.06 , 0.06] 1.0E+00 Gfi1 0.26 [-0.05 , 0.58] 1.8E-01 -0.05 [-0.10 , 0.01] 1.7E-01 Il15 0.26 [0.04 , 0.48] 5.9E-02 0.03 [0.00 , 0.06] 1.5E-01 Vim 0.26 [0.17 , 0.35] 2.7E-04 0.04 [0.02 , 0.06] 1.4E-03 Lyn 0.26 [0.20 , 0.32] 7.9E-06 0.04 [0.03 , 0.05] 3.1E-04 Irf1 0.26 [0.19 , 0.33] 3.0E-05 0.04 [0.02 , 0.06] 4.8E-03 Ripk2 0.26 [0.13 , 0.38] 3.4E-03 0.03 [0.00 , 0.06] 9.6E-02 Myd88 0.25 [0.11 , 0.40] 8.9E-03 0.03 [0.00 , 0.06] 1.7E-01 662 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Il7 0.25 [-0.03 , 0.54] 1.6E-01 0.02 [-0.04 , 0.09] 5.5E-01 Icam4 0.25 [-0.05 , 0.55] 1.7E-01 0.05 [-0.02 , 0.13] 2.7E-01 Traf2 0.25 [0.17 , 0.33] 1.2E-04 0.05 [0.04 , 0.07] 8.4E-05 Il13ra1 0.24 [0.13 , 0.36] 2.1E-03 0.05 [0.03 , 0.07] 9.3E-04 Hck 0.24 [0.05 , 0.44] 4.8E-02 0.01 [-0.03 , 0.06] 6.8E-01 Il2rb 0.24 [0.07 , 0.42] 3.3E-02 0.01 [-0.03 , 0.04] 7.0E-01 Slamf1 0.24 [-0.02 , 0.51] 1.5E-01 0.04 [-0.02 , 0.10] 2.8E-01 Itk 0.24 [-0.01 , 0.49] 1.2E-01 0.04 [-0.01 , 0.09] 2.4E-01 Bst1 0.24 [0.11 , 0.37] 7.0E-03 -0.05 [-0.09 , -0.01] 7.5E-02 Casp3 0.24 [0.09 , 0.38] 1.6E-02 0.04 [0.02 , 0.07] 1.3E-02 Masp1 0.23 [0.09 , 0.38] 1.4E-02 0.04 [0.00 , 0.09] 1.0E-01 Cxcl12 0.23 [0.06 , 0.41] 3.7E-02 0.06 [0.01 , 0.10] 4.2E-02 Il2rg 0.23 [0.14 , 0.33] 9.3E-04 0.00 [-0.02 , 0.01] 6.6E-01 Ccrl2 0.23 [0.09 , 0.37] 1.1E-02 0.04 [0.01 , 0.07] 3.0E-02 Btla 0.23 [-0.05 , 0.51] 1.9E-01 0.05 [0.00 , 0.11] 1.5E-01 Cd244 0.23 [-0.15 , 0.61] 3.3E-01 0.05 [-0.01 , 0.11] 1.7E-01 Cd1d1 0.23 [0.14 , 0.31] 5.5E-04 0.03 [0.01 , 0.05] 1.3E-02 663 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tyk2 0.22 [0.09 , 0.35] 9.7E-03 0.07 [0.04 , 0.10] 1.2E-03 Foxj1 0.22 [-0.17 , 0.61] 3.6E-01 0.12 [0.06 , 0.19] 9.0E-03 Tlr4 0.22 [0.10 , 0.34] 6.8E-03 0.04 [0.01 , 0.06] 1.7E-02 F13a1 0.22 [-0.05 , 0.49] 1.9E-01 0.00 [-0.07 , 0.06] 9.1E-01 Cd8b1 0.22 [-0.08 , 0.52] 2.4E-01 0.00 [-0.07 , 0.07] 9.6E-01 Cd37 0.22 [-0.13 , 0.55] 3.1E-01 0.04 [-0.02 , 0.10] 3.1E-01 Ifi35 0.21 [0.10 , 0.33] 6.7E-03 -0.02 [-0.03 , 0.00] 7.0E-02 Trp53 0.21 [0.06 , 0.36] 2.7E-02 0.04 [0.01 , 0.08] 5.0E-02 Nfatc3 0.21 [0.01 , 0.41] 9.0E-02 0.01 [-0.01 , 0.02] 5.8E-01 Tnfsf10 0.21 [0.10 , 0.32] 5.5E-03 0.00 [-0.04 , 0.03] 8.4E-01 Litaf 0.21 [0.08 , 0.34] 1.5E-02 0.05 [0.02 , 0.08] 9.0E-03 Col1a1 0.20 [-0.03 , 0.44] 1.7E-01 0.07 [0.04 , 0.11] 6.6E-03 Cd247 0.20 [-0.04 , 0.44] 1.7E-01 0.02 [-0.03 , 0.07] 4.9E-01 Ctsw 0.20 [0.11 , 0.30] 2.1E-03 -0.02 [-0.04 , 0.01] 2.9E-01 Il18rap 0.20 [0.09 , 0.32] 8.9E-03 0.01 [-0.02 , 0.04] 5.4E-01 Pla2g6 0.20 [0.04 , 0.36] 4.3E-02 0.06 [0.03 , 0.09] 3.5E-03 Hc 0.20 [0.04 , 0.36] 4.7E-02 0.04 [0.02 , 0.07] 2.4E-02 664 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value C8g 0.20 [-0.24 , 0.64] 4.7E-01 0.05 [-0.05 , 0.14] 4.5E-01 Axl 0.20 [0.08 , 0.32] 1.2E-02 0.04 [0.02 , 0.06] 5.6E-03 Maf 0.20 [-0.02 , 0.41] 1.4E-01 0.04 [0.00 , 0.07] 8.3E-02 Il15ra 0.20 [-0.07 , 0.46] 2.3E-01 0.05 [-0.01 , 0.10] 1.6E-01 Icam1 0.19 [0.10 , 0.29] 3.8E-03 0.02 [-0.01 , 0.04] 2.4E-01 Cd55 0.19 [0.06 , 0.33] 2.8E-02 0.06 [0.03 , 0.08] 2.3E-03 Fpr2 0.19 [-0.03 , 0.41] 1.7E-01 -0.03 [-0.13 , 0.06] 6.2E-01 Il12rb2 0.19 [-0.02 , 0.40] 1.5E-01 0.03 [-0.02 , 0.07] 3.8E-01 Atg10 0.18 [0.09 , 0.27] 3.7E-03 0.03 [0.01 , 0.06] 2.2E-02 Runx1 0.18 [0.06 , 0.29] 1.9E-02 0.06 [0.03 , 0.08] 8.2E-04 Mrc1 0.18 [0.04 , 0.31] 4.2E-02 0.01 [-0.01 , 0.04] 5.3E-01 Il22ra1 0.18 [-0.11 , 0.46] 3.2E-01 0.00 [-0.07 , 0.07] 9.9E-01 Pnma1 0.18 [-0.08 , 0.43] 2.6E-01 0.10 [0.04 , 0.16] 2.0E-02 Ifnar1 0.17 [0.12 , 0.23] 1.6E-04 0.05 [0.03 , 0.06] 2.5E-04 Tnfrsf14 0.17 [0.04 , 0.31] 4.1E-02 0.02 [0.00 , 0.05] 1.4E-01 Cd59b 0.17 [-0.14 , 0.48] 3.7E-01 -0.03 [-0.08 , 0.02] 2.9E-01 Cd63 0.17 [0.04 , 0.31] 4.2E-02 0.02 [-0.01 , 0.05] 2.6E-01 665 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Mr1 0.17 [0.04 , 0.31] 4.7E-02 0.04 [0.01 , 0.06] 3.9E-02 Cklf 0.17 [0.00 , 0.34] 1.1E-01 0.02 [-0.02 , 0.06] 4.0E-01 Creb5 0.17 [-0.03 , 0.37] 1.7E-01 0.04 [-0.01 , 0.10] 2.1E-01 Ctsh 0.17 [-0.03 , 0.37] 1.7E-01 0.07 [0.03 , 0.11] 1.1E-02 Ifitm2 0.17 [0.07 , 0.26] 7.7E-03 0.05 [0.04 , 0.07] 1.3E-04 Cd2 0.17 [-0.22 , 0.56] 4.8E-01 0.05 [-0.01 , 0.11] 2.2E-01 C7 0.17 [0.00 , 0.33] 1.1E-01 0.05 [0.03 , 0.07] 4.7E-03 Thy1 0.17 [0.04 , 0.29] 3.4E-02 0.01 [-0.01 , 0.04] 4.2E-01 Il1rap 0.17 [0.04 , 0.30] 4.6E-02 0.05 [0.03 , 0.08] 2.1E-03 Lrp1 0.17 [-0.12 , 0.45] 3.5E-01 0.08 [0.04 , 0.12] 4.9E-03 Tfe3 0.16 [0.06 , 0.27] 1.7E-02 0.03 [0.01 , 0.05] 4.3E-02 Psen2 0.16 [0.05 , 0.27] 2.0E-02 0.03 [0.01 , 0.05] 2.7E-02 Plaur 0.16 [0.01 , 0.31] 9.2E-02 0.06 [0.02 , 0.09] 9.7E-03 Mef2c 0.16 [-0.01 , 0.32] 1.2E-01 0.02 [-0.01 , 0.05] 3.5E-01 Txnip 0.16 [-0.01 , 0.32] 1.3E-01 0.01 [-0.02 , 0.04] 7.0E-01 Map2k1 0.15 [0.11 , 0.20] 1.0E-04 0.02 [0.01 , 0.03] 1.8E-03 Cd40 0.15 [0.06 , 0.25] 1.4E-02 0.04 [0.01 , 0.06] 1.4E-02 666 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tfrc 0.15 [-0.04 , 0.35] 1.9E-01 0.02 [-0.02 , 0.06] 3.9E-01 Gzmb 0.15 [-0.14 , 0.44] 4.0E-01 -0.03 [-0.10 , 0.04] 4.7E-01 Cxcr5 0.15 [-0.42 , 0.72] 6.7E-01 0.03 [-0.07 , 0.13] 6.8E-01 Pvrl2 0.15 [0.03 , 0.27] 4.4E-02 0.02 [0.01 , 0.04] 3.7E-02 Map3k1 0.15 [0.02 , 0.29] 7.0E-02 0.04 [0.02 , 0.06] 9.9E-03 Il1r1 0.15 [0.02 , 0.28] 6.1E-02 0.05 [0.02 , 0.08] 6.4E-03 Ms4a2 0.15 [-0.18 , 0.48] 4.7E-01 0.00 [-0.07 , 0.08] 9.5E-01 Colec12 0.15 [0.05 , 0.24] 2.3E-02 0.02 [0.00 , 0.04] 6.2E-02 Cd1d2 0.15 [-0.09 , 0.38] 3.2E-01 0.02 [-0.03 , 0.07] 4.7E-01 Myc 0.14 [-0.05 , 0.34] 2.3E-01 0.04 [0.00 , 0.08] 1.2E-01 Nfatc1 0.14 [-0.03 , 0.31] 1.9E-01 0.05 [0.02 , 0.07] 1.3E-02 Itgb3 0.14 [-0.01 , 0.29] 1.3E-01 0.06 [0.03 , 0.09] 3.1E-03 Hif1a 0.14 [0.04 , 0.23] 2.8E-02 0.04 [0.02 , 0.06] 1.6E-03 Ikbkb 0.14 [0.00 , 0.27] 1.1E-01 0.04 [0.02 , 0.06] 4.1E-03 Rorc 0.14 [-0.13 , 0.40] 4.2E-01 0.05 [-0.02 , 0.12] 2.9E-01 Elk1 0.13 [-0.18 , 0.45] 5.0E-01 0.05 [-0.01 , 0.12] 1.9E-01 Itga5 0.13 [-0.04 , 0.31] 2.1E-01 0.06 [0.03 , 0.10] 9.7E-03 667 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Kit 0.13 [-0.04 , 0.30] 2.1E-01 0.04 [-0.01 , 0.08] 2.2E-01 Lamp1 0.13 [0.06 , 0.20] 7.3E-03 0.02 [0.01 , 0.04] 1.7E-02 Clu 0.13 [-0.15 , 0.40] 4.5E-01 0.00 [-0.05 , 0.06] 9.1E-01 Ccl5 0.13 [-0.01 , 0.26] 1.3E-01 -0.03 [-0.06 , 0.00] 9.2E-02 Irak4 0.13 [0.02 , 0.23] 6.1E-02 0.03 [0.02 , 0.05] 7.9E-03 Camp 0.13 [-0.22 , 0.47] 5.6E-01 0.06 [-0.03 , 0.15] 2.9E-01 Tbk1 0.12 [0.02 , 0.23] 7.0E-02 0.03 [0.01 , 0.05] 4.6E-02 Stat6 0.12 [0.05 , 0.20] 1.4E-02 0.03 [0.02 , 0.04] 1.2E-03 Abl1 0.12 [-0.04 , 0.28] 2.3E-01 0.05 [0.02 , 0.08] 1.8E-02 Cr2 0.12 [-0.36 , 0.60] 6.9E-01 -0.04 [-0.13 , 0.06] 5.3E-01 Tnfrsf13c 0.12 [-0.36 , 0.60] 6.9E-01 -0.01 [-0.09 , 0.07] 8.7E-01 Bcl6 0.12 [-0.05 , 0.29] 2.7E-01 0.05 [0.02 , 0.07] 1.4E-02 Tnfrsf10b 0.12 [-0.09 , 0.33] 3.7E-01 0.03 [-0.02 , 0.07] 3.5E-01 Il6st 0.11 [0.04 , 0.19] 1.7E-02 0.04 [0.02 , 0.06] 1.4E-03 Ccl25 0.11 [-0.13 , 0.35] 4.5E-01 0.04 [0.00 , 0.08] 9.6E-02 Cd22 0.11 [-0.25 , 0.48] 6.2E-01 -0.03 [-0.10 , 0.04] 5.3E-01 Il16 0.11 [-0.07 , 0.29] 3.4E-01 -0.01 [-0.05 , 0.02] 6.4E-01 668 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Nos2 0.11 [-0.17 , 0.39] 5.4E-01 0.01 [-0.05 , 0.07] 8.3E-01 Tfeb 0.11 [-0.05 , 0.27] 2.7E-01 0.04 [0.01 , 0.07] 6.2E-02 Atg7 0.11 [0.02 , 0.19] 5.2E-02 0.01 [-0.01 , 0.03] 3.9E-01 Tlr3 0.11 [0.01 , 0.21] 8.7E-02 0.01 [-0.01 , 0.03] 6.5E-01 Cd28 0.10 [-0.28 , 0.48] 6.7E-01 0.04 [-0.02 , 0.11] 2.9E-01 Irf2 0.10 [0.00 , 0.20] 1.1E-01 0.04 [0.03 , 0.06] 6.8E-04 Mst1r 0.10 [-0.23 , 0.42] 6.4E-01 0.02 [-0.07 , 0.11] 7.3E-01 Atm 0.10 [-0.02 , 0.22] 2.0E-01 0.02 [0.00 , 0.04] 9.5E-02 Ilf3 0.09 [0.00 , 0.19] 1.3E-01 0.01 [0.00 , 0.03] 2.9E-01 Tab1 0.09 [-0.05 , 0.23] 2.8E-01 0.04 [0.01 , 0.06] 1.4E-02 Fez1 0.09 [-0.15 , 0.33] 5.5E-01 -0.02 [-0.11 , 0.07] 7.5E-01 Cd19 0.09 [-0.45 , 0.63] 7.9E-01 0.02 [-0.10 , 0.13] 8.4E-01 Cmah 0.09 [-0.16 , 0.34] 5.6E-01 -0.02 [-0.06 , 0.02] 4.6E-01 Map3k5 0.09 [-0.06 , 0.23] 3.2E-01 0.03 [0.00 , 0.05] 1.4E-01 Card11 0.09 [-0.18 , 0.35] 6.0E-01 -0.02 [-0.07 , 0.02] 4.0E-01 Cx3cl1 0.08 [-0.14 , 0.30] 5.4E-01 0.03 [-0.02 , 0.08] 3.2E-01 Bcl10 0.08 [-0.01 , 0.18] 1.5E-01 0.03 [0.02 , 0.05] 9.2E-04 669 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ms4a1 0.08 [-0.43 , 0.59] 8.0E-01 -0.01 [-0.11 , 0.08] 8.7E-01 Alcam 0.08 [0.00 , 0.16] 1.0E-01 0.03 [0.01 , 0.04] 8.3E-03 Fadd 0.08 [-0.06 , 0.22] 3.6E-01 0.02 [-0.01 , 0.04] 2.2E-01 Cd79b 0.08 [-0.35 , 0.51] 7.7E-01 -0.01 [-0.09 , 0.06] 7.9E-01 Sell 0.08 [-0.13 , 0.29] 5.5E-01 -0.03 [-0.07 , 0.00] 1.6E-01 Ythdf2 0.08 [0.02 , 0.14] 3.9E-02 0.03 [0.01 , 0.04] 1.5E-02 Psen1 0.07 [0.00 , 0.15] 1.3E-01 0.02 [0.01 , 0.04] 9.7E-03 St6gal1 0.07 [-0.03 , 0.18] 2.5E-01 0.04 [0.03 , 0.06] 7.0E-04 Bax 0.07 [-0.03 , 0.18] 2.7E-01 0.02 [0.00 , 0.05] 8.1E-02 Ncam1 0.07 [-0.32 , 0.46] 7.7E-01 0.03 [-0.04 , 0.10] 4.9E-01 Egr1 0.07 [-0.34 , 0.48] 7.8E-01 -0.02 [-0.10 , 0.06] 7.2E-01 Vhl 0.07 [-0.07 , 0.21] 4.2E-01 0.07 [0.04 , 0.09] 1.2E-03 Ctsl 0.07 [-0.04 , 0.18] 3.1E-01 0.01 [-0.01 , 0.03] 5.4E-01 Ltbr 0.07 [-0.01 , 0.15] 1.7E-01 0.03 [0.02 , 0.04] 2.1E-03 Mavs 0.07 [-0.04 , 0.18] 3.3E-01 0.02 [0.00 , 0.04] 1.9E-01 Blk 0.07 [-0.43 , 0.56] 8.3E-01 0.04 [-0.08 , 0.15] 6.3E-01 Ltk 0.07 [-0.16 , 0.29] 6.5E-01 0.07 [0.02 , 0.13] 4.9E-02 670 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Stat3 0.06 [-0.02 , 0.15] 2.2E-01 0.02 [0.01 , 0.03] 7.5E-03 Angpt2 0.06 [-0.07 , 0.19] 4.5E-01 -0.01 [-0.05 , 0.02] 6.6E-01 Ubc 0.06 [-0.02 , 0.14] 2.1E-01 0.01 [0.00 , 0.03] 1.3E-01 Dll4 0.06 [-0.05 , 0.17] 3.8E-01 0.01 [-0.03 , 0.05] 7.2E-01 Stat5b 0.06 [-0.03 , 0.15] 2.9E-01 0.02 [0.00 , 0.04] 2.1E-01 Il3ra 0.06 [-0.07 , 0.19] 4.5E-01 0.02 [-0.01 , 0.05] 2.4E-01 Atg16l1 0.06 [-0.06 , 0.17] 4.3E-01 0.03 [0.01 , 0.04] 9.7E-03 Cebpb 0.05 [-0.08 , 0.19] 5.3E-01 0.04 [0.01 , 0.07] 5.0E-02 Cfh 0.05 [-0.02 , 0.13] 2.6E-01 0.03 [0.01 , 0.04] 1.6E-02 Ewsr1 0.05 [-0.02 , 0.12] 2.6E-01 0.02 [0.01 , 0.03] 3.4E-02 Ceacam1 0.05 [-0.07 , 0.17] 5.2E-01 0.01 [-0.01 , 0.04] 4.5E-01 Smad2 0.05 [-0.02 , 0.12] 2.5E-01 0.02 [0.01 , 0.04] 4.7E-03 Rela 0.05 [-0.04 , 0.14] 3.8E-01 0.02 [0.00 , 0.04] 9.1E-02 Cd38 0.05 [-0.09 , 0.19] 5.8E-01 0.00 [-0.02 , 0.02] 9.0E-01 Gata3 0.05 [-0.20 , 0.29] 7.7E-01 0.02 [-0.04 , 0.07] 6.2E-01 Lamp2 0.04 [-0.04 , 0.13] 4.2E-01 0.00 [-0.01 , 0.02] 6.7E-01 Mapk14 0.04 [-0.06 , 0.14] 5.1E-01 0.03 [0.01 , 0.04] 1.7E-02 671 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Map3k7 0.04 [-0.03 , 0.11] 3.8E-01 0.02 [0.01 , 0.03] 2.4E-02 Atg12 0.04 [-0.04 , 0.12] 4.2E-01 0.02 [0.01 , 0.03] 3.1E-02 Anp32b 0.04 [-0.03 , 0.10] 3.5E-01 0.02 [0.00 , 0.03] 9.5E-02 Cdh1 0.04 [-0.06 , 0.13] 5.2E-01 0.01 [-0.01 , 0.03] 4.0E-01 Klra7 0.04 [-0.29 , 0.37] 8.6E-01 -0.05 [-0.12 , 0.01] 2.2E-01 Ccl27a 0.04 [-0.21 , 0.28] 8.2E-01 0.02 [-0.03 , 0.08] 5.3E-01 Mapk11 0.03 [-0.15 , 0.22] 7.7E-01 0.05 [0.01 , 0.08] 7.6E-02 Pin1 0.03 [-0.11 , 0.18] 7.1E-01 0.04 [0.00 , 0.07] 1.5E-01 Atf1 0.03 [-0.11 , 0.17] 7.1E-01 0.02 [0.00 , 0.04] 2.1E-01 Fcer1a 0.03 [-0.20 , 0.25] 8.4E-01 0.03 [-0.02 , 0.09] 3.8E-01 Tgfbr2 0.03 [-0.05 , 0.11] 5.9E-01 0.02 [0.01 , 0.04] 3.5E-02 Bcl2 0.03 [-0.17 , 0.23] 8.3E-01 0.06 [0.03 , 0.08] 4.9E-03 Cd47 0.03 [-0.05 , 0.11] 5.9E-01 0.00 [-0.01 , 0.01] 9.2E-01 Ep300 0.03 [-0.10 , 0.16] 7.4E-01 0.03 [0.01 , 0.06] 4.9E-02 Zfp13 0.02 [-0.17 , 0.22] 8.4E-01 0.02 [-0.02 , 0.06] 4.9E-01 Cd40lg 0.02 [-0.22 , 0.27] 8.7E-01 0.02 [-0.03 , 0.07] 5.2E-01 Yy1 0.02 [-0.04 , 0.09] 5.5E-01 0.02 [0.01 , 0.03] 1.4E-03 672 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Fyn 0.02 [-0.07 , 0.12] 6.9E-01 0.01 [0.00 , 0.02] 1.5E-01 Pax5 0.02 [-0.43 , 0.47] 9.4E-01 -0.02 [-0.11 , 0.07] 7.2E-01 Rps6 0.02 [-0.10 , 0.14] 7.9E-01 0.00 [-0.02 , 0.02] 9.8E-01 Mapk1 0.02 [-0.02 , 0.06] 4.6E-01 0.01 [0.00 , 0.02] 4.0E-02 Map2k4 0.01 [-0.06 , 0.08] 8.5E-01 0.02 [0.01 , 0.03] 1.5E-02 Igf2r 0.01 [-0.17 , 0.18] 9.4E-01 0.04 [0.02 , 0.06] 5.2E-03 Cd3eap 0.01 [-0.31 , 0.32] 9.7E-01 -0.01 [-0.07 , 0.06] 9.0E-01 Cyld 0.01 [-0.14 , 0.16] 9.5E-01 -0.02 [-0.05 , 0.01] 3.9E-01 Smn1 0.01 [-0.08 , 0.10] 9.3E-01 0.01 [-0.01 , 0.03] 4.1E-01 Ticam1 0.01 [-0.12 , 0.13] 9.5E-01 0.03 [0.00 , 0.06] 1.3E-01 Bmi1 0.00 [-0.07 , 0.08] 9.4E-01 0.01 [0.00 , 0.03] 1.8E-01 Il6ra 0.00 [-0.17 , 0.17] 9.9E-01 0.05 [0.02 , 0.08] 1.0E-02 Mapk3 0.00 [-0.07 , 0.06] 9.6E-01 0.01 [-0.01 , 0.02] 6.1E-01 Sigirr 0.00 [-0.12 , 0.11] 9.6E-01 0.01 [0.00 , 0.03] 1.8E-01 Mapkapk2 0.00 [-0.09 , 0.08] 9.4E-01 0.00 [-0.02 , 0.02] 9.5E-01 Eomes -0.01 [-0.22 , 0.21] 9.7E-01 -0.03 [-0.07 , 0.01] 1.8E-01 Chuk -0.01 [-0.06 , 0.05] 8.6E-01 0.04 [0.03 , 0.04] 1.1E-05 673 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Irf3 -0.01 [-0.10 , 0.08] 8.7E-01 0.01 [-0.01 , 0.04] 3.8E-01 Itga6 -0.01 [-0.13 , 0.11] 8.9E-01 0.02 [0.00 , 0.04] 1.0E-01 Tbx21 -0.01 [-0.26 , 0.24] 9.5E-01 -0.08 [-0.13 , -0.04] 8.3E-03 Adora2a -0.01 [-0.17 , 0.14] 8.8E-01 0.01 [-0.04 , 0.06] 8.3E-01 Crebbp -0.02 [-0.14 , 0.11] 8.5E-01 0.02 [0.01 , 0.04] 2.3E-02 Jak1 -0.02 [-0.07 , 0.03] 5.3E-01 0.01 [0.00 , 0.02] 1.4E-01 Dock9 -0.02 [-0.19 , 0.15] 8.6E-01 0.04 [0.01 , 0.08] 6.0E-02 Psmb7 -0.02 [-0.12 , 0.08] 7.5E-01 -0.01 [-0.03 , 0.00] 1.5E-01 Psma2 -0.02 [-0.11 , 0.07] 7.2E-01 -0.02 [-0.03 , 0.00] 1.1E-01 Itch -0.02 [-0.09 , 0.05] 6.5E-01 0.02 [0.01 , 0.03] 5.9E-03 Nod1 -0.02 [-0.20 , 0.15] 8.4E-01 0.02 [-0.01 , 0.05] 3.1E-01 Jam3 -0.02 [-0.24 , 0.19] 8.6E-01 0.01 [-0.03 , 0.05] 7.6E-01 Atf2 -0.02 [-0.12 , 0.07] 6.7E-01 0.01 [0.00 , 0.03] 2.1E-01 Nup107 -0.02 [-0.11 , 0.06] 6.5E-01 -0.01 [-0.03 , 0.01] 6.2E-01 Epcam -0.03 [-0.19 , 0.14] 8.1E-01 0.00 [-0.03 , 0.03] 8.6E-01 Klra4 -0.03 [-0.22 , 0.17] 8.3E-01 -0.07 [-0.11 , -0.03] 1.2E-02 Map2k2 -0.03 [-0.10 , 0.05] 5.6E-01 0.01 [0.00 , 0.03] 1.2E-01 674 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Itga2 -0.03 [-0.16 , 0.11] 7.4E-01 0.00 [-0.03 , 0.02] 8.5E-01 Pparg -0.03 [-0.19 , 0.13] 7.8E-01 -0.03 [-0.07 , 0.01] 2.9E-01 Txk -0.03 [-0.27 , 0.21] 8.4E-01 -0.05 [-0.10 , 0.01] 1.8E-01 Tcf7 -0.03 [-0.19 , 0.13] 7.5E-01 -0.01 [-0.05 , 0.03] 6.6E-01 Traf6 -0.04 [-0.12 , 0.05] 4.8E-01 0.01 [-0.01 , 0.02] 5.2E-01 Atg5 -0.04 [-0.15 , 0.08] 6.0E-01 -0.02 [-0.04 , 0.00] 2.1E-01 Map4k2 -0.04 [-0.16 , 0.08] 6.2E-01 -0.01 [-0.04 , 0.02] 6.2E-01 Gpi1 -0.04 [-0.10 , 0.02] 2.7E-01 0.00 [-0.01 , 0.02] 4.8E-01 Ikbkg -0.04 [-0.22 , 0.14] 7.4E-01 0.02 [-0.01 , 0.06] 2.8E-01 Smad4 -0.04 [-0.11 , 0.03] 3.4E-01 0.01 [0.00 , 0.02] 9.6E-02 Prf1 -0.04 [-0.20 , 0.13] 7.0E-01 -0.09 [-0.14 , -0.04] 1.2E-02 Smad3 -0.04 [-0.13 , 0.05] 4.5E-01 0.01 [-0.01 , 0.03] 3.9E-01 Creb1 -0.04 [-0.14 , 0.06] 5.2E-01 0.01 [-0.01 , 0.02] 5.4E-01 Gtf3c1 -0.04 [-0.12 , 0.04] 4.2E-01 0.02 [0.01 , 0.04] 3.9E-02 Erbb2 -0.04 [-0.15 , 0.07] 5.4E-01 0.04 [0.01 , 0.07] 6.0E-02 Tlr5 -0.04 [-0.29 , 0.20] 7.8E-01 -0.04 [-0.08 , 0.01] 2.4E-01 Il18 -0.05 [-0.30 , 0.21] 7.8E-01 -0.08 [-0.13 , -0.03] 2.2E-02 675 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tnfsf12 -0.05 [-0.13 , 0.03] 3.3E-01 -0.02 [-0.04 , 0.00] 1.1E-01 Pdgfrb -0.05 [-0.30 , 0.19] 7.3E-01 0.03 [-0.01 , 0.08] 2.8E-01 Nrp1 -0.05 [-0.15 , 0.04] 3.7E-01 0.00 [-0.02 , 0.01] 6.8E-01 Gzma -0.06 [-0.19 , 0.07] 4.5E-01 -0.10 [-0.14 , -0.07] 1.9E-04 Lrrn3 -0.06 [-0.30 , 0.18] 6.9E-01 -0.03 [-0.08 , 0.02] 3.3E-01 Klrc1 -0.06 [-0.23 , 0.11] 5.6E-01 -0.06 [-0.09 , -0.03] 5.4E-03 Tnfrsf1a -0.06 [-0.13 , 0.01] 1.7E-01 0.00 [-0.02 , 0.01] 6.1E-01 Fn1 -0.06 [-0.31 , 0.18] 6.9E-01 0.03 [0.00 , 0.07] 1.5E-01 Hmgb1 -0.06 [-0.12 , -0.01] 6.5E-02 -0.01 [-0.03 , 0.00] 1.7E-01 Mapk8 -0.07 [-0.16 , 0.03] 2.7E-01 0.00 [-0.01 , 0.02] 7.6E-01 App -0.07 [-0.14 , 0.01] 1.7E-01 0.00 [-0.01 , 0.02] 7.2E-01 Il4ra -0.07 [-0.24 , 0.11] 5.4E-01 0.00 [-0.03 , 0.03] 9.9E-01 Abcb1a -0.07 [-0.20 , 0.06] 3.9E-01 -0.01 [-0.04 , 0.01] 4.6E-01 Reps1 -0.07 [-0.13 , -0.01] 6.3E-02 0.00 [-0.01 , 0.01] 7.7E-01 Mif -0.07 [-0.17 , 0.03] 2.4E-01 -0.02 [-0.04 , 0.00] 9.6E-02 Prkce -0.07 [-0.20 , 0.06] 3.7E-01 0.01 [-0.02 , 0.03] 6.3E-01 Cd200 -0.07 [-0.15 , 0.01] 1.4E-01 0.00 [-0.01 , 0.02] 6.7E-01 676 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Tdo2 -0.08 [-0.81 , 0.66] 8.6E-01 0.03 [-0.12 , 0.19] 7.5E-01 Cd99 -0.08 [-0.24 , 0.08] 4.2E-01 -0.01 [-0.05 , 0.03] 8.1E-01 Pvr -0.08 [-0.24 , 0.08] 4.2E-01 0.03 [0.00 , 0.06] 1.6E-01 Notch1 -0.08 [-0.28 , 0.11] 5.1E-01 0.03 [0.00 , 0.05] 1.6E-01 Syt17 -0.08 [-0.32 , 0.15] 5.8E-01 -0.03 [-0.10 , 0.04] 5.3E-01 Prdm1 -0.08 [-0.29 , 0.13] 5.2E-01 -0.02 [-0.06 , 0.01] 3.0E-01 Cd9 -0.09 [-0.18 , 0.01] 1.4E-01 -0.03 [-0.05 , -0.01] 3.7E-02 Psmd7 -0.09 [-0.15 , -0.02] 4.8E-02 0.00 [-0.02 , 0.01] 7.5E-01 C1qbp -0.09 [-0.16 , -0.02] 5.1E-02 -0.03 [-0.04 , -0.01] 5.9E-03 Tie1 -0.09 [-0.21 , 0.03] 2.2E-01 0.00 [-0.02 , 0.02] 8.6E-01 Ifngr1 -0.09 [-0.16 , -0.03] 3.2E-02 0.02 [0.00 , 0.04] 1.2E-01 Tollip -0.09 [-0.17 , -0.02] 5.4E-02 0.01 [0.00 , 0.03] 1.7E-01 Dusp6 -0.10 [-0.35 , 0.16] 5.5E-01 0.00 [-0.06 , 0.05] 9.8E-01 Klrk1 -0.10 [-0.26 , 0.06] 3.3E-01 -0.08 [-0.12 , -0.05] 9.9E-04 Cd164 -0.10 [-0.14 , -0.05] 1.9E-03 -0.03 [-0.04 , -0.01] 6.7E-03 Cd34 -0.10 [-0.17 , -0.03] 3.6E-02 -0.01 [-0.02 , 0.01] 6.2E-01 Dpp4 -0.10 [-0.17 , -0.03] 3.0E-02 -0.02 [-0.04 , 0.00] 1.9E-01 677 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Irak1 -0.10 [-0.17 , -0.03] 2.5E-02 -0.01 [-0.03 , 0.01] 3.1E-01 Ets1 -0.10 [-0.17 , -0.03] 2.1E-02 -0.01 [-0.02 , 0.00] 2.3E-01 Klrd1 -0.11 [-0.21 , 0.00] 1.1E-01 -0.07 [-0.09 , -0.05] 3.4E-04 Rora -0.11 [-0.27 , 0.06] 2.9E-01 0.02 [-0.01 , 0.04] 2.9E-01 Fasl -0.11 [-0.37 , 0.16] 5.2E-01 0.00 [-0.07 , 0.07] 9.8E-01 Entpd1 -0.11 [-0.22 , -0.01] 9.4E-02 -0.01 [-0.03 , 0.01] 6.5E-01 Il11ra1 -0.12 [-0.53 , 0.30] 6.5E-01 0.05 [-0.03 , 0.13] 3.0E-01 Cd7 -0.13 [-0.35 , 0.10] 3.7E-01 -0.09 [-0.14 , -0.05] 5.1E-03 Mill2 -0.13 [-0.28 , 0.02] 1.7E-01 -0.02 [-0.04 , 0.00] 1.6E-01 Nfatc4 -0.13 [-0.35 , 0.09] 3.5E-01 0.03 [0.00 , 0.05] 7.8E-02 Dusp4 -0.13 [-0.42 , 0.15] 4.6E-01 0.02 [-0.03 , 0.06] 5.8E-01 Ncr1 -0.15 [-0.27 , -0.03] 4.6E-02 -0.09 [-0.12 , -0.06] 2.8E-04 Cd97 -0.16 [-0.34 , 0.03] 1.7E-01 -0.01 [-0.04 , 0.03] 7.2E-01 Xbp1 -0.16 [-0.25 , -0.06] 1.5E-02 -0.02 [-0.05 , 0.01] 2.4E-01 Cd160 -0.16 [-0.44 , 0.13] 3.7E-01 -0.06 [-0.13 , 0.00] 1.4E-01 Anxa1 -0.16 [-0.34 , 0.02] 1.5E-01 -0.04 [-0.08 , 0.00] 1.0E-01 Cd36 -0.17 [-0.28 , -0.05] 2.5E-02 -0.01 [-0.03 , 0.01] 3.6E-01 678 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccr3 -0.17 [-0.59 , 0.26] 5.3E-01 -0.09 [-0.19 , 0.01] 1.6E-01 Tal1 -0.17 [-0.35 , 0.00] 1.2E-01 -0.01 [-0.04 , 0.03] 7.7E-01 Pecam1 -0.17 [-0.27 , -0.08] 7.0E-03 -0.02 [-0.04 , 0.00] 1.4E-01 Tmed1 -0.18 [-0.29 , -0.06] 1.6E-02 -0.03 [-0.05 , -0.01] 3.2E-02 Mfge8 -0.18 [-0.33 , -0.03] 5.6E-02 0.00 [-0.03 , 0.03] 9.7E-01 Il22ra2 -0.18 [-0.49 , 0.13] 3.5E-01 -0.01 [-0.07 , 0.05] 7.7E-01 Snai1 -0.18 [-0.39 , 0.03] 1.6E-01 -0.03 [-0.08 , 0.03] 4.2E-01 Vwf -0.18 [-0.35 , -0.02] 7.9E-02 0.02 [-0.02 , 0.05] 4.5E-01 Ccnd3 -0.19 [-0.28 , -0.10] 3.3E-03 -0.02 [-0.04 , 0.00] 1.9E-01 Il12a -0.19 [-0.46 , 0.08] 2.6E-01 -0.04 [-0.09 , 0.01] 2.2E-01 Kdr -0.19 [-0.28 , -0.10] 2.3E-03 -0.04 [-0.06 , -0.02] 1.3E-02 Ambp -0.19 [-1.01 , 0.63] 7.1E-01 -0.02 [-0.22 , 0.18] 8.9E-01 Jun -0.19 [-0.44 , 0.05] 2.0E-01 -0.01 [-0.03 , 0.01] 5.6E-01 Il18r1 -0.20 [-0.38 , -0.01] 9.1E-02 -0.02 [-0.05 , 0.02] 5.3E-01 Tgfb3 -0.21 [-0.34 , -0.07] 1.7E-02 0.00 [-0.03 , 0.04] 8.1E-01 Tirap -0.21 [-0.35 , -0.06] 3.1E-02 -0.02 [-0.05 , 0.01] 2.2E-01 Col4a1 -0.21 [-0.41 , -0.01] 9.1E-02 0.05 [0.01 , 0.10] 6.2E-02 679 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON mRNA cSiO2 + 1.0% DHA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ecsit -0.21 [-0.36 , -0.06] 3.0E-02 -0.02 [-0.05 , 0.02] 4.9E-01 Mertk -0.21 [-0.42 , 0.00] 1.1E-01 0.00 [-0.04 , 0.05] 8.8E-01 Cd81 -0.22 [-0.30 , -0.14] 2.7E-04 -0.03 [-0.05 , -0.01] 1.5E-02 Klrb1c -0.24 [-0.42 , -0.05] 4.9E-02 -0.10 [-0.13 , -0.07] 1.5E-04 Cdh5 -0.24 [-0.34 , -0.13] 1.6E-03 -0.02 [-0.05 , 0.00] 8.4E-02 Tnfsf18 -0.24 [-0.73 , 0.25] 4.2E-01 -0.04 [-0.12 , 0.04] 4.3E-01 Tek -0.24 [-0.34 , -0.14] 9.5E-04 -0.04 [-0.06 , -0.02] 1.4E-02 Egfr -0.24 [-0.40 , -0.09] 1.7E-02 -0.02 [-0.05 , 0.01] 2.8E-01 Eng -0.24 [-0.39 , -0.10] 1.2E-02 -0.04 [-0.07 , -0.01] 7.5E-02 Sele -0.24 [-0.84 , 0.35] 5.1E-01 -0.16 [-0.28 , -0.04] 5.0E-02 Igf1r -0.25 [-0.44 , -0.06] 3.7E-02 0.01 [-0.02 , 0.04] 5.9E-01 Thbd -0.25 [-0.44 , -0.06] 3.7E-02 -0.01 [-0.05 , 0.02] 6.2E-01 Hamp -0.25 [-1.03 , 0.53] 6.0E-01 0.04 [-0.13 , 0.22] 7.2E-01 Nt5e -0.26 [-0.46 , -0.06] 4.1E-02 -0.02 [-0.06 , 0.02] 3.8E-01 Itgb4 -0.27 [-0.43 , -0.10] 1.5E-02 -0.03 [-0.06 , 0.01] 2.1E-01 Cmklr1 -0.27 [-0.48 , -0.06] 4.1E-02 -0.01 [-0.05 , 0.03] 8.2E-01 Timd4 -0.27 [-0.93 , 0.39] 5.1E-01 -0.11 [-0.26 , 0.04] 2.6E-01 680 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Ccl24 -0.27 [-0.97 , 0.42] 5.3E-01 -0.10 [-0.26 , 0.06] 3.2E-01 Rrad -0.28 [-0.55 , 0.00] 1.1E-01 0.04 [-0.04 , 0.11] 4.5E-01 Akt3 -0.29 [-0.39 , -0.18] 5.6E-04 -0.01 [-0.04 , 0.01] 4.5E-01 Lyve1 -0.29 [-0.52 , -0.06] 4.9E-02 -0.07 [-0.11 , -0.03] 1.3E-02 Smpd3 -0.29 [-0.73 , 0.15] 2.8E-01 -0.09 [-0.19 , 0.01] 1.5E-01 Msln -0.29 [-0.62 , 0.03] 1.5E-01 -0.12 [-0.18 , -0.05] 7.6E-03 Cspg4 -0.30 [-0.65 , 0.06] 1.8E-01 0.05 [-0.01 , 0.12] 2.2E-01 Icam2 -0.30 [-0.39 , -0.20] 1.3E-04 -0.05 [-0.07 , -0.03] 7.9E-04 Cxcl14 -0.30 [-0.50 , -0.10] 2.2E-02 -0.02 [-0.06 , 0.03] 5.8E-01 Itga1 -0.30 [-0.46 , -0.15] 5.0E-03 -0.02 [-0.05 , 0.02] 4.1E-01 Mme -0.31 [-0.38 , -0.23] 1.6E-05 -0.06 [-0.08 , -0.03] 2.2E-03 Itgb1 -0.31 [-0.38 , -0.25] 3.3E-06 -0.03 [-0.05 , -0.01] 2.4E-02 Hspb2 -0.32 [-0.44 , -0.19] 7.5E-04 -0.03 [-0.05 , 0.00] 1.3E-01 Cma1 -0.32 [-0.71 , 0.07] 1.9E-01 -0.08 [-0.18 , 0.01] 1.7E-01 Vegfc -0.33 [-0.51 , -0.15] 6.4E-03 0.00 [-0.03 , 0.03] 9.5E-01 Fas -0.33 [-0.54 , -0.12] 1.5E-02 -0.01 [-0.05 , 0.04] 7.6E-01 Klrc2 -0.33 [-0.46 , -0.21] 5.0E-04 -0.09 [-0.12 , -0.06] 1.4E-04 681 Table 39 (cont’d) Gene expression of cSiO2-treated NZBWF1 relative to VEH + CON mice in lung 24 hr post final exposure cSiO2 + CON cSiO2 + 1.0% DHA mRNA log2(FC) 95% CI BH p-value log2(FC) 95% CI BH p-value Vegfa -0.33 [-0.42 , -0.24] 2.9E-05 -0.07 [-0.09 , -0.04] 3.1E-04 Hras -0.33 [-0.65 , -0.01] 9.6E-02 0.00 [-0.06 , 0.07] 9.1E-01 Tgfb2 -0.34 [-0.50 , -0.18] 2.6E-03 -0.03 [-0.07 , 0.01] 1.9E-01 Mcam -0.35 [-0.49 , -0.21] 8.0E-04 0.00 [-0.04 , 0.03] 8.8E-01 Cd163 -0.37 [-0.67 , -0.07] 5.3E-02 0.02 [-0.06 , 0.10] 6.7E-01 Angpt1 -0.38 [-0.53 , -0.22] 1.2E-03 -0.03 [-0.06 , 0.01] 1.9E-01 F2rl1 -0.43 [-0.59 , -0.26] 5.3E-04 -0.10 [-0.12 , -0.07] 6.5E-05 Thbs1 -0.43 [-0.93 , 0.08] 1.7E-01 -0.06 [-0.16 , 0.05] 4.0E-01 Cfd -0.44 [-1.99 , 1.11] 6.5E-01 0.03 [-0.29 , 0.35] 8.9E-01 Bcl2l1 -0.46 [-0.70 , -0.22] 5.0E-03 0.00 [-0.06 , 0.06] 1.0E+00 Arg1 -0.46 [-1.23 , 0.31] 3.3E-01 -0.19 [-0.36 , -0.02] 8.6E-02 Hsd11b1 -0.53 [-0.67 , -0.38] 4.4E-05 -0.07 [-0.10 , -0.04] 3.0E-03 Cdkn1a -0.54 [-1.00 , -0.08] 6.3E-02 -0.02 [-0.12 , 0.08] 7.9E-01 Itga2b -0.64 [-0.99 , -0.29] 6.6E-03 0.03 [-0.04 , 0.10] 5.4E-01 Ppbp -0.90 [-1.29 , -0.50] 1.6E-03 -0.07 [-0.15 , 0.02] 2.3E-01 Tnfrsf12a -0.94 [-1.34 , -0.55] 1.1E-03 -0.17 [-0.25 , -0.09] 2.9E-03 Serpinb2 -1.14 [-2.19 , -0.08] 8.5E-02 -0.15 [-0.40 , 0.09] 3.2E-01 682 APPENDIX B In vitro models of cSiO2 exposure Introduction The findings in this dissertation highlight the complexity of cSiO2-triggered autoimmunity and the global suppressive effects of DHA. With these considerations, elucidation of precise mechanism by which cSiO2 ultimately triggers autoimmunity will have to be explored with reductionist in vitro models. Much of the current evidence suggests that the alveolar macrophage is 1) responsible for cSiO2-triggered toxicity, 2) may die following cSiO2 phagocytosis in an inflammatory mode of cell death and 3) be a target of DHA. Cell death in alveolar macrophages by phagocytosis of cSiO2 in vitro and in vivo occurs by two principal mechanisms – pyroptosis and apoptosis. Pyroptosis occurs in macrophages following inflammasome-mediated activation of caspase-1 and results in IL-1β secretion. In contrast, apoptosis following cSiO2 exposure is a consequence of intracellular proteases, and defective efferocytosis may result in nuclear antigens that trigger autoimmunity. Herein, two models of macrophages, the conventional RAW 264.7 cell line and the recently described MPI cell, were utilized to identify an appropriate in vitro macrophage model that could test the effect of DHA on IL-1β and efferocytosis induced by cSiO2 exposure. Experimental design First, the immortalized murine cell line, RAW 264.7 cells, were utilized to test if cSiO2 particle phagocytosis is impaired by DHA. While this experiment was only performed once, the results shown in Figure 62 enforce future study into the role of DHA on cSiO2-triggered toxicity in macrophages. 683 Figure 59. Phagocytosis of cSiO2 particles is not affected in vitro by DHA. RAW 264.7 cells (n = 3/gp) were cultured for 24 hr with 25 μM of FA in ethanol, then exposed to 50 μg/ml of cSiO2 for 2 h and phagocytosis measured by flow cytometry using side scatter (SSC). This experiment was performed in singlet. 684 Since cSiO2 particles have a crystalline structure, they have a large capacity to reflect light. Therefore, an increase in mean side scatter is indicative of phagocytosis. Here, there is no difference in mean side scatter between groups treated with FA. Through later experiments, it became evident that RAW 264.7 cells lack the ASC protein, a protein that is critically involved in inflammasome activation and caspase-1 recruitment to the inflammasome. Therefore, innate RAW 264.7 cells do not secrete active IL-1β. Through a collaboration with Dr. Mikhail Gavrilin at the Ohio State University, we obtained RAW 264.7 cells stably transfected with ASC. The results shown in Fig. 60 demonstrate the application of this in vitro model of inflammasome activation following cSiO2 exposure. However, there is question about the relevance of in vitro models. The gold-standard is alveolar macrophages, however, these are cumbersome to isolate from the intact mouse and cannot be cultured for long periods of time ex vivo. While in vitro models are used to simulate alveolar macrophages, there are many faults with using in vitro monocyte/macrophages to model alveolar macrophages and cSiO2 toxicity. From our collaborator, Dr. Mikhail Gavrilin, we also obtained MPI cells, a recently described non-immortalized, self-renewing in vitro model of alveolar macrophages that share a developmental origin and phenotype with alveolar macrophages. The results shown in Fig. 61 demonstrate that MPI cells robustly secrete IL-1β following cSiO2 exposure. Taken together, MPI cells have tremendous potential as an in vitro model of alveolar macrophages to pursue the effects of environmental particles on inflammasome activation. Development of an in vitro model of efferocytosis Apoptosis is normally a non-lytic, non-inflammatory mode of cell death. However, if efferocytosis (i.e. phagocytosis of apoptotic cells by viable cells) is deficient, cells that have died by apoptosis can advance to the stage of secondary necrosis. This results in the lysis of the cell 685 Figure 60. IL-1β dose-response to cSiO2 in vitro in RAW 264.7 cells stably transfected with ASC. RAW 264.7 cells+ASC were cultured with increasing amounts of cSiO2 for 6 hr (n = 4/gp). Following completion, IL-1β was measured by ELISA in culture supernatant. Different letter indicates statistically significant difference by t-test. This experiment was performed in duplicate. 686 Figure 61. IL-1β response to cSiO2 in relation to time in vitro with MPI cells. MPI cells (n = 4/gp) were cultured with 10μg/cm2 of cSiO2 with or without LPS for 2, 6, 12, and 24 hr. Following completion, IL-1β was measured by ELISA in culture supernatant. Different letter indicates statistically significant difference by t-test. This experiment was performed in singlet. 687 membrane, releasing ‘danger signals’ and potentially nuclear antigens into the cytosol, which could have been proteolytically modified such that they are no longer recognized as self and as a result, could trigger an autoimmune response. Importantly, DHA has been shown to increase efferocytosis of apoptotic cells, which could serve to remove apoptotic cells before they progress to secondary necrosis, thus preserving self-tolerance. The early stages of a model for in vitro efferocytosis were conducted. Briefly, target cells were cultured until ~50% confluency, then labeled with 2.5μM CFSE and induced to undergo apoptosis by UV light exposure at 100mJ/s. This was done to avoid introduction of additional chemicals into cell culture. 2 hr later, cells were collected by scraping, and co-cultured 1:1 with phagocytic cells for 1 hr on glass coverslips in 24 well plates. After, viable cells were stained with 1μg/ml Hoescht 33342, washed 3X with PBS, fixed with 4% paraformaldehyde, and cover slips transferred to glass slides for immunofluorescent microscopy. An image of the results is depicted in Fig. 62. There are limitations to using this method. One, scraping cells from tissue culture plates can itself induce necrosis, thus causing conflicting results if the end point is apoptosis. Second, much of this method is subject to technical variance, such as scraping efficiency and the efficiency of transfer of glass cover slips from tissue culture plates to slides. Inadequate removal of air bubbles from cover slips can interfere with fluorescent image capture, and fluorescent image capture must be done manually or with automated equipment. While automated equipment has its advantages, they require a degree of skill. Calculation of an efferocytotic index (i.e. the number of apoptotic cells being phagocytosed) is done through overlay of images, which may have to be done manually, and is extremely labor intensive. Again, automated software can be employed, such as the use of ImageJ, however, this also requires a high degree of knowledge on the software in order 688 Figure 62. Development of an in vitro model of efferocytosis. Hoescht 33342 cells (blue) are viable phagocytes; CFSE cells (green) are apoptotic; cells from merged images captured in blue and green channels are viable phagocytes performing phagocytosis of apoptotic cells (efferocytosis). Image captured at 20X with a Nikon Ti-U2 fluorescent microscope. Efferocytosis Apoptotic cell Efferocytosis 689 to ensure that cells are being counted appropriately. Taken together, this method serves as a preliminary step towards an in vitro model of efferocytosis that can be applied. 690 REFERENCES 691 REFERENCES 1. 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