ROLE OF ARYL HYDROCARBON RECEPTOR (AhR) POLYMORPHISMS AND TRANSCRIPTIONAL ACTIVITY IN TCDD-INDUCED SUPPRESSION OF THE B CELL IgM RESPONSE  By Natalia Kovalova       A DISSERTATION  Submitted to Michigan State University in partial fulfillment of the requirements  for the degree of  Pharmacology and Toxicology - Environmental Toxicology - Doctor of Philosophy   2016!!ABSTRACT ROLE OF ARYL HYDROCARBON RECEPTOR (AhR) POLYMORPHISMS AND TRANSCRIPTIONAL ACTIVITY IN TCDD-INDUCED SUPPRESSION OF THE B CELL IgM RESPONSE  By Natalia Kovalova Previous studies have demonstrated that most of the intraspecies variation in sensitivity to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including suppression of antibody responses, in murine models is due to single nucleotide polymorphisms (SNPs) within the aryl hydrocarbon receptor (AhR) gene. The underlying reason for the variation in sensitivity to TCDD-induced suppression of IgM responses among humans is not well understood but is thought, in part, to be a result of various polymorphic forms of the AhR expressed by different individuals. Additionally, it is well known that AhR mediates toxic effects of TCDD by, most likely, deregulating gene expression. However, the exact mechanism, as wells as transcriptional targets of TCDD are not well understood. Therefore, this dissertation research had two primary long-term goals. The first goal was to identify and characterize a core set of evolutionarily conserved gene expression responses across mouse, rat and human primary B cells focusing on the role of AhR in mediating immunotoxic effects of TCDD. The second goal was to comprehensively evaluate the correlation between B cell sensitivity to TCDD-mediated suppression of the IgM response and previously identified SNPs within the human AhR.  I investigated the functional properties of six (P517S, R554K, V570I, V570I+P517S, R554K+V570I and P517S+R554K+V570I) human AhR variants !!stably expressed in the human B cell line, SKW 6.4. I have demonstrated that the R554K human AhR SNP alone altered sensitivity of human B cells to TCDD-mediated induction of Cyp1B1 and Cyp1A2 metabolizing enzymes. By contrast, attenuation of TCDD-induced IgM suppression required a combination of all three SNPs P517S, R554K, and V570I.  The second part of my dissertation research focused on identification of the B cell-specific molecular targets of TCDD. Based on preliminary findings, we hypothesized that TCDD treatment across three different species (mouse, rat and human) triggers a conserved, B cell-specific mechanism that is involved in TCDD-induced immunosuppression. RNA sequencing (RNA-Seq) was used to identify B cell-specific orthologous genes that are differentially expressed in response to TCDD in primary mouse, rat and human B cells. Time course studies identified TCDD-elicited differential expression of 544 human, 2527 mouse and 772 rat genes over the 24-h period. Only 28 orthologs were differentially expressed in response to TCDD in all three species. Overrepresented pathways enriched in all three species included cytokine-cytokine receptor interaction, ECM-receptor interaction, focal adhesion, regulation of actin cytoskeleton and pathways in cancer. Differentially expressed genes functionally associated with calcium ion binding, cell adhesion and inflammatory response were overrepresented in all three species. Collectively, these results suggest that species-specific gene expression profiles mediate the species-specific effects of TCDD despite the conservation of the AhR and its signaling mechanism.  !iv ACKNOWLEDGEMENTS  First, I would like to thank my mentor, Dr. Norb Kaminski for his support, patience and guidance. I would also like to express my gratitude to the members of my graduate committee, Drs. Bryan Copple, Keith Lookingland and John LaPres for their time and interest in my work. I would also like to thank Drs. Anne Dorrance, Bill Atchison and Barb Kaplan for their helpful comments and unyielding support and I deeply appreciate their time and effort. Thanks are due to members of the extended AhR and THC groups: to Jose Suarez and Brian Zhou for comic relief during everyday laboratory life and for regularly suffering my lame jokes; to Dr. Sky Pike for teaching me the nuts and bolts of molecular biology techniques; to Bob Crawford who taught me most of the lab techniques I used in my work and for patiently enduring through my long Skype conversations with mom; to Dr. Ashwini Phadnis for all the thoughtful discussions, moral support and precious advice and to Kim Hambleton for her priceless help with anything I needed including tea and a sympathetic ear. And, collectively, many thanks go out to all members of the AhR and THC groups who have generously provided me with their valuable friendship (for free) and made my time as a graduate student an enjoyable one.  I would like to thank my pals from the department and beyond: Dr. Paulo Pires for treating me like one of the guys; Gabriela Lopez-Zeron for keeping it real and all the wine; Dr. Pavlo Kovalenko for staying on top of the latest stuff in science/politics, so that I wouldn't have to; Dr. Eileen Rodriges for giving me some of !v her determination; Dr. Nikita Joshi for unlimited hugs and encouragement; Dasha Bond for not letting me forget my roots, and all those others who have been there to make things better. To my ÒAmerican familyÓ, Ann, Dave, Nathan, Emma and Jenna BeBeau my heartfelt appreciation for being there for me, giving me a home away from home and all the countless camping trips and card games (especially ÒCards Against HumanityÓ). I would also like to thank SpartyÕs for all the coffee, Subway for providing my comfort food (tuna sandwiches) and YouTube for keeping a versatile library of cat videos available whenever I need them.   Financial support from the Michigan State University, the SOT and National Institute of Health for the duration of my Ph.D. degree is greatly appreciated. I would like to convey my profound gratitude to my family, to my mother and father for their unconditional love, support and for taking care of me so well and making me feel so loved.         !vi TABLE OF CONTENTS  LIST OF TABLES  ix  LIST OF FIGURES  xi  KEY TO ABBREVIATIONS  xiv  CHAPTER 1: LITERATURE REVIEW 1 1.1  Purpose of this research 1 1.2  Dioxins and AhR-mediated toxicity 2 1.2.1 Dioxins and dioxin toxicity in humans 2 1.2.2 AhR protein structure and polymorphisms 9 1.2.3 AhR signaling 16 1.3 Immune system and humoral immunity 24 1.3.1 Immune system overview 24 1.3.2 B cell activation 27 1.3.3 Regulation of B cell differentiation 30 1.4 TCDD-mediated effects on immune system 33 1.4.1 Immunotoxicity of TCDD 33 1.4.2 TCDD-induced suppression of B cell effector function 35 1.4.3 Rationale 40  CHAPTER 2: Materials and Methods 42 2.1 Chemicals and reagents 42 2.2 Cell lines 42 2.3 Animals 43 2.4 Isolation of rat and mouse primary B cells 43 2.5 Preparation of luciferase reporter constructs 44 2.6 Luciferase assays 44 2.7 Preparation of cell lines that stably express AhR 45 2.8 Cloning by limiting dilution 46 2.9 pTRIPZ-AHR-GFP lentiviral vector production 46 2.10 Site-directed mutagenesis 47 2.11 Quantitative real-time PCR 47 2.12 Western blot analysis 48 2.13 IgM enzyme-linked immunosorbent assay (ELISA)  49 2.14 Isolation of the naŁve human B cells 49 2.15 Whole transcriptome expression profiling via RNA-Seq 50 2.16 RNA-Sequencing, alignment, and analysis 50 2.17 Network and functional ontology enrichment analysis 51 2.18 Identification of putative dioxin response elements 52 2.19 Statistical analysis 52  !vii CHAPTER 3: EXPERIMENTAL RESULTS 53 3.1 Role of Aryl Hydrocarbon Receptor Polymorphisms on TCDD- mediated CYP1B1 Induction and IgM Suppression by Human B cells 53 3.1.1 Human SKW-AHR+ B cell line characterization 53 3.1.2 TCDD suppressed immune function in human SKW-AHR+ but not SKW 6.4 B cells 65 3.1.3 Temporal effects of TCDD on IgM antibody response in the SKW-AHR+, mouse and rat primary B cells 65 3.1.4 TCDD-mediated effects on the expression of critical regulators of plasmacytic differentiation 72 3.1.5 Effects of the AhR SNPs on the TCDD-mediated induction of Cyp1A2 and Cyp1B1 mRNA expression 75 3.1.6 Effects of the AhR SNPs on the Cyp1B1- and DRE-regulated reporter gene activity  79 3.1.7 Effects of AhR SNPs on sensitivity of the human B cells to TCDD-mediated suppression of the IgM secretion 82 3.2 Genome-wide responses of the naŁve human, mouse and rat primary B cells to TCDD treatment 84 3.2.1 Gene expression changes in naive primary mouse, rat and human B cells following TCDD treatment 84 3.2.2 Functional annotation and pathway enrichment of genes differentially expressed in response to TCDD in naive mouse, human and rat primary B cells 92 3.2.3 Temporal gene expression changes in the naive primary mouse, human and rat B cells following TCDD exposure 102 3.3 Genome-wide responses of the PWM-activated human, mouse and rat primary B cells to TCDD treatment 103 3.3.1 Gene expression changes in PWM-activated primary mouse, rat and human B cells following TCDD treatment 103 3.3.2 Functional annotation and pathway enrichment of genes differentially expressed in response to TCDD in activated mouse, human and rat primary B cells 112 3.3.3 Temporal gene expression changes in the PWM-activated primary  mouse, human and rat B cells following TCDD exposure 115 3.3.4 Identification of putative DREs in responsive genes 122 3.3.5 Gene network analysis 122 3.3.6 Validation of differentially expressed genes at the mRNA level 126 3.4 Comparison of TCDD-induced genome-wide gene expression  changes in naŁve and PWM-activated primary mouse, human and rat  B cells and CH12.LX mouse cell line 129  CHAPTER 4: DISCUSSION 139 4.1 Role of the AhR SNPs in human B cell sensitivity to the toxic  effects of TCDD 139 !viii 4.2 Role of the newly identified prospective B cell-specific molecular  targets of TCDD in AhR-mediated suppression of the B cell function 146  CHAPTER 5: FINAL CONCLUDING REMARKS 155  BIBLIOGRAPHY 160    !ix LIST OF TABLES  Table 1. Top 10 up- and down-regulated genes in the mouse, human and rat datasets.   90  Table 2. GO biological process terms associated with the 94 orthologs  deregulated by TCDD in all three species. 93  Table 3. GO terms associated with genes deregulated by TCDD in the mouse primary B cells. 95  Table 4. GO terms associated with genes deregulated by TCDD in the rat  primary B cells. 97  Table 5. GO terms associated with genes deregulated by TCDD in the mouse primary B cells. 99  Table 6. KEGG pathways associated with genes differentially expressed by  TCDD treatment in primary mouse, human and rat B cells.  101  Table 7. Top ten genes differentially up- or down-regulated in response to  TCDD treatment in mouse, human and rat primary B lymphocytes activated  by PWM.  106  Table 8. Conserved, TCDD-Induced ortholog differential expression.  107  Table 9. Gene ontology analysis of the 86 common human-mouse DEGs. 111  Table 10. KEGG pathways enriched in PWM-activated human, mouse and  rat primary B cells treated with TCDD. 117  Table 11. Number of putative DREs (pDREs) identified in the promoter region  of the 27 common orthologs differentially expressed in all three species. 123  Table 12. Genes differentially regulated by TCDD in naŁve and PWM-activated primary human B cells. 132  Table 13. Biological processes GO terms and KEGG pathways associated  with the 25 genes deregulated by TCDD in naŁve and PWM-activated  primary human B cells.  133  Table 14. Biological processes GO terms and KEGG pathways associated  with the 550 genes deregulated by TCDD in naŁve and PWM-activated  primary mouse B cells. 136 !x  Table 15. Biological processes GO terms and KEGG pathways associated  with the 138 genes deregulated by TCDD in naŁve and PWM-activated  primary rat B cells. 138    !  !xi LIST OF FIGURES  Figure 1. Chemical structure of polychlorinated dioxins.       3  Figure 2. A schematic representation of the functional and structural domains  of the aryl hydrocarbon receptor (AhR).  11  Figure 3. A schematic representation of the core unit of BCR/ immunoglobulin (antibody). 29  Figure 4. A schematic representation of pTRIPZ vector for doxycycline- inducible transgene expression. 54  Figure 5. Induction of the IgM response by different PAMPs in the  SKW-AHR+ cells. 57  Figure 6. Concentration-dependent induction of the IgM response by  different PAMPs in the SKW-AHR+ cells. 58  Figure 7. Induction of the IgM response with cytokines in the SKW-AHR+ cells. 59  Figure 8. AhR mRNA expression in SKW 6.4, SKW-AHR+ and HepG2 cell lines. 60  Figure 9. AhR expression levels in the newly generated SKW-AHR+  human B cell line. 62  Figure 10. Time-dependent induction of the Cyp1B1 gene expression by TCDD. 64  Figure 11. Effects of TCDD on LPS- and PWM-induced IgM secretion in the   SKW 6.4 and SKW-AHR+ cells. 66  Figure 12.  Effects of TCDD treatment on IgJ and Igµ chain mRNA levels in  LPS-activated SKW-AHR+ cell line. 68  Figure 13. Relationship between the time of TCDD addition and in vitro IgM response. 70  Figure 14. Effects of the TCDD treatment on Blimp1, BCL-6 and Pax-5 gene expression in the SKW-AhR+ cell line. 73  Figure 15.  Protein expression of the AhR variants in SKW clones. 76  Figure 16. Time-dependent induction of the Cyp1A2 (A) and Cyp1B1 (B) gene expression by TCDD in SKW clones. 77 !xii  Figure 17. Ability of the WT AhR and AhR variants to mediate TCDD-induced Cyp1B1 (A) and DRE (B) reporter gene activity. 80  Figure 18. Effects of TCDD on the LPS-induced IgM secretion in SKW clones. 83  Figure 19. A. Number of species-specific genes exhibiting TCDD-induced  changes in mRNA expression in naive primary mouse, human and rat B lymphocytes. 86  Figure 20. Bi-plot of Principal Component Analysis (PCA) of TCDD-induced differential gene expression in treated mouse, human and rat B cells at  4, 8, and 12h. 88  Figure 21. Comparison of TCDD-induced differential ortholog expression in  mouse, human and rat primary B cells. 91  Figure 22. Heat map representing fold-change of gene expression of the  suite of genes associated with top over-represented GO terms. 100  Figure 23. Number of mouse, rat and human orthologs differentially expressed  in response to TCDD. 104  Figure 24. Pairwise analysis of differentially expressed orthologs. 109  Figure 25. Functional annotation of the differentially expressed genes in  response to TCDD in primary human, mouse and rat B cells activated  with PWM. 113  Figure 26. List of KEGG biological pathways associated with genes  differentially expressed in response to TCDD in primary mouse, rat and  human B cells. 114  Figure 27. Effect of TCDD on the PWM-induced IgM response in mouse,  human and rat primary B cells. 116  Figure 28. Time-dependent, TCDD-elicited induction of the AhR ÒbatteryÓ genes. 121  Figure 29. Top implicated biological network containing common human-mouse orthologs significantly deregulated by TCDD exposure. 125  Figure 30. PCR verification of the expression of select genes.  127  Figure 31. Venn diagram of the differentially regulated genes in response to  TCDD in naŁve and PWM-activated human primary B cells and CH12.LX  cells.  131 !xiii  Figure 32. Venn diagram of the differentially regulated genes in response to  TCDD in naŁve and PWM-activated mouse primary B cells and CH12.LX  cells. 135  Figure 33. Venn diagram of the differentially regulated genes in response to  TCDD in naŁve and PWM-activated rat primary B cells and CH12.LX cells. 137    !xiv KEY TO ABBREVIATIONS !Ah   Aromatic hydrocarbon AHH   Aryl hydrocarbon hydroxylase AhR   Aryl hydrocarbon receptor AIP   AhR-interacting protein AP-1   Activator protein-1 ARA9   Ah receptor-associated protein ARNT  Aryl hydrocarbon receptor nuclear translocator AFC   Antibody forming cell ASC   Antibody secreting cell Bach2  BTB And CNC homology 1 BCL-6  B-cell lymphoma 6 BCR   B cell receptor BLIMP-1  B-Lymphocyte-induced maturation protein 1 CD   Cluster of differentiation CD40L  CD40 ligand ChIP   Chromatin immunoprecipitation DLC   Dioxin-like compounds DMSO  Dimethyl sulfoxide DRE   Dioxin response element EMSA  Electrophoretic mobility shift assay ERK   Extracellular regulated kinase EROD  7-ethoxy-resorufin-O-deethylase !xv FACS   Fluorescence-activated cell sorting GC   Germinal center h   hour HAH   Halogenated aromatic hydrocarbon HIC1  Hypermethylated in cancer 1 HSC   Hematopoietic stem cell Hsp90  Heat shock protein 90 ICAM-1  Intercellular adhesion molecule 1 IgM   Immunoglobulin M IL   Interleukin IRF   Interferon regulatory factor ITAM   Immunoreceptor tyrosine-based activation motif ITIM   Immunoreceptor tyrosine-based inhibition motif ITGB3  Integrin beta 3 JNK   Janus kinase LPS   Lipopolysaccharides MAPK  Mitogen-activated protein kinase MHC   Major histocompatibility complex mRNA  messenger RNA MYO6  Myosin VI NA   NaŁve NF!B   Nuclear factor kappa B NHL   non-HodgkinÕs lymphoma !xvi NQO1  NAD(P)H Quinone Dehydrogenase 1 PAX-5  Paired box-5 PBMC  Peripheral blood mononuclear cell PCB   Polychlorinated biphenyls PCDD  Polychlorinated dibenzodioxins PCDF  Polychlorinated dibenzofurans pERK   phosphorylated extracellular signal-regulated kinase PI3K   Phosphoinositide 3-kinase PMA   Phorbol 12-myristate 13-acetate  PWM   Pokeweed mitogen qRT-PCR  quantitative real time polymerase chain reaction RND2  Rho family GTPase 2 SNP   Single nucleotide polymorphism TCDD  2,3,7,8-tetrachlorodibenzo-p-dioxin Tfh   T follicular helper Th   T helper TIPARP TCDD inducible poly(ADP-Ribose) polymerase TLR   Toll-like receptor TRAF   TNF receptor associated factor VH   Vehicle XBP-1  X-box protein-1 !1 CHAPTER 1: LITERATURE REVIEW  1.1 Purpose of this research Exposure to the most toxic HAH congener, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), leads to significant suppression of the IgM antibody response in virtually all species tested. Animals, including human, differ in their sensitivity to the immunosuppressive and other biological and toxic effects of TCDD. Intra-species differential sensitivities of laboratory rodents to these compounds are well documented and result from single nucleotide polymorphisms (SNPs) of the AhR (Poland & Knutson, 1982; Poland & Glover, 1987; Hahn, 1998; Okey, 2007). It is conceivable that, within the human population, AhR polymorphisms could influence the level of individual sensitivity to TCDD and related HAHs (Atlas et al., 1976). Additionally, the exact mechanism of TCDD-induced suppression of the IgM response in humans and major B cell-specific molecular targets of TCDD are currently unknown. This dissertation research aims to establish the role of multiple human AhR SNPs in sensitivity to the immunotoxic effects of TCDD and identify novel molecular targets of TCDD in the primary human B cells.   !2 1.2 Dioxins and AhR-mediated toxicity 1.2.1 Dioxins and dioxin toxicity in humans Dioxins were first produced in 1848 in Germany as an unintentional byproduct of sodium carbonate production using Leblanc process (Weber et al., 2008). Dioxins and related HAHs are a large group of tasteless, odorless and highly lipophilic persistent environmental pollutants that are structurally and chemically related and consist of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like polychlorinated biphenyls (PCBs) (Olli et al., 2013). Dioxin-like compounds (DLCs) contain 10 of the polychlorinated dibenzofurans, 7 of the polychlorinated dibenzodioxins, and 12 of the polychlorinated biphenyls, which are of toxicological concern (Figure 1) (Van den Berg et al., 2006; Ahlborg et al., 1994).  Dioxins are resistant to degradation and have biological half-lives on the scale of years. Additionally, due to their high lipophilicity, they tend to bioaccumulate in the food chain and increase in concentration in bigger long-living organisms (Mochida et al., 2007; Van den Berg et al., 1994). Interestingly, the half-lives of dioxins in humans seem to be concentration-dependent, with faster rates of elimination observed at higher blood concentrations and slower rates at lower blood concentrations (Leung et al., 2007). The underlying reason for the variability of half-lives of dioxins in humans is not well understood but has been partially attributed to smoking status, body fat content, and breastfeeding (Flesch-Janys et al., 1996; Milbrath et al., 2009). The primary identified sources of dioxins and DLCs in the environment are combustion/incineration of municipal and medical waste, chemical manufacturing (production of polychlorinated and polybrominated biphenyls), and  !3  Figure 1. Chemical structure of polychlorinated dioxins. A. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); B. 1,2,3,7,8-PeCDD; C. 1,2,3,4,7,8-HxCDD; D. 1,2,3,6,7,8-HxCDD; E. 1,2,3,7,8,9-HxCDD; F. 1,2,3,4,6,7,8-HpCDD; G. octachlorodibenzodioxin (OCDD)  !4 chlorine bleaching in the pulp and paper industry (Urban et al., 2014). Since most of the dioxins are highly lipophilic and tend to bioconcentrate in the food chain, the majority of human exposure is through the dietary routes via ingestion of contaminated fatty foods (Schecter et al., 2003; Schecter et al., 2006). Therefore, most if not all of the human population have a "background" dioxin exposure that varies depending on whether people are living in industrialized regions (higher body burdens of dioxins) or non-industrialized areas with little herbicide exposure (lower burden) (Birnbaum, 1994; Hoffman et al., 1986). Additionally, a continuous accumulation of dioxins in the body to levels of potential adverse effects remains a concern (Warner et al., 2014; LaKind et al., 2001). Notably, some human populations have higher levels of exposure to HAHs due to dietary preferences. For example, Great Lakes fishermen are found to have elevated HAH levels of exposure resulting from greater than average consumption of contaminated fish (Svensson et al., 1991); breastfeeding babies also are exposed to higher HAHs levels due to the accumulation of these compounds in breast milk (Mead, 2008). Significantly higher levels of dioxins are found in individuals exposed to a large amount of dioxins due to industrial accidents or through exposure at work (Manz et al., 1991; Saracci et al., 1991; Thiess et al., 1982; Mastroiacovo et al., 1988).  The vast number of HAHÕs isomers and congeners present in the environment and the variation in their respective potencies for inducing toxicological responses requires a system to evaluate the relative risk from each compound. Risk assessment of dioxin-like chemicals uses a toxic equivalency factor (TEF) approach, comparing the potency of the individual dioxin-like compounds to that of TCDD, the !5 most toxic congener. TEF approach is based on a number of parameters such as congenerÕs structural similarity to TCDD, binding affinity and strength of activation of the AhR-mediated biochemical and toxic responses, and persistence and bioaccumulation in the food chain (Parvez et al., 2013; Van den Berg et al., 2006). The TEF value for TCDD, the most toxic HAH congener, is set to 1.0 (Safe, 1998; Van den Berg et al., 2006). TEFs are frequently used to estimate toxic equivalents (TEQs) of the TCDD-like activity for an HAH mixture by calculating the sum of the products of the concentration of each dioxin-like chemical multiplied by its TEF. However, assumptions associated with the use of TEQ carry certain weaknesses and uncertainties. For example, the TEQ approach assumes an identical mechanism of action, suggesting a common additive effect of all HAHs in a mixture and ignoring the possibility of a synergistic and/or antagonistic mechanism of action. However, multiple reports show that some HAHs antagonize the effects of TCDD indicating that TEQ calculation might overestimate or underestimate the toxicity of HAH mixtures (Safe, 1998; Safe, 1997; Birnbaum et al., 1995). Moreover, since different HAHs are metabolized to varying extents, their pharmacokinetic properties should be included in the calculation of TEQs. Regardless, the TEQ approach is one of the most widely accepted and used measure in HAH mixture research, human risk assessment and epidemiological studies (Van den Berg et al., 2006).  As mentioned above, TCDD is the most toxic congener among the HAH family of compounds and is often referred to as the "most toxic compound known to man" (Birnbaum, 1994). TCDD has been shown to induce a broad range of toxicities in wild and laboratory animals, have an extremely high potency, and cause !6 detrimental health effects even in near undetectable concentrations (Vandenberg et al., 2012; Birnbaum et al., 1991). In laboratory animals such as rats and mice, acute, high-dose exposure to TCDD results in death. Wasting syndrome is a common manifestation of TCDD exposure at non-lethal doses; it is presented as substantial weight loss and reduction in the amount of adipose tissue. Other adverse health effects of sub-lethal TCDD exposure include suppression of the humoral immune response, reduction of lymphoid tissue in the thymus, spleen and lymph nodes, hepatic hyperplasia and various cancers (Holladay et al., 1999; Patterson et al., 2016; Mann, 1997; Scott et al., 2001; Birnbaum et al., 2000; Poland & Knutson, 1982). Additionally, teratogenicity in rats, mice, birds, hamsters, guinea pigs and fish with symptoms ranging from cleft palate and kidney malformation to fetal death and desorption, and endocrine disruption in rodents and fish were reported (Birnbaum et al., 2000; Peters et al., 1999; Kransler et al., 2007; Bruggeman et al., 2003; Heiden et al., 2006).  The induction of metabolizing enzymes Cyp1A1 and Cyp1B1 mRNA and protein activity was demonstrated in all species tested and is frequently used as a biomarker of dioxin exposure. Interestingly, the lethal dose of TCDD (LD50) varies over a thousand-fold range across different species with guinea pig being approximately a 1000 fold as sensitive as a hamster. Inter-species differences in sensitivity to various non-lethal biological and toxic effects of TCDD are also commonly seen (Okey, 2007).  In a few early studies conducted during 1965 through 1968, human volunteers were intentionally exposed to up to 7,500 µg of TCDD by cutaneous !7 routes but due to poor study design and loss of records the study results were inconclusive (Hilberman, 1999). Since then, due to highly toxic nature of dioxins, most toxicities of TCDD in human are obtained from epidemiological studies of individuals who were exposed to high levels of HAHs by accident (Sany et al., 2015; Marinkovi" et al., 2010) or due to workplace contamination (Saracci et al., 1991; Johnson, 1992; Calvert et al., 1992). The most consistent toxic effect of TCDD and a hallmark of human exposure to dioxins is the occurrence of a skin condition known as chloracne (Bock, 2016). Chloracne is manifested as uninflamed nodules and cysts developed due to the hyperplasia and hyperkeratosis of the epidermis (Williams et al., 1995).  There were multiple accidents resulting in a release of the large amounts of dioxin and DLCs in the environment and subsequent human exposure to dioxin. For example, during the Yusho incident in Japan in 1968, and a Yucheng incident in Taiwan in 1979, a mass food poisoning occurred due to accidental ingestion of rice bran oil contaminated with polychlorinated biphenyls (PCBs) and various dioxins and dioxin-like compounds (Chen et al., 1981). The victims of both incidents have suffered from characteristic skin manifestations (chloracne, comedones, acneiform eruptions, and dark-brownish pigmentation), ophthalmological (increased cheese-like discharge from the meibomian glands, pigmentation of the conjunctiva, and swelling of the upper eyelids), and mucosal symptoms (pigmentation of gingiva) for a long period (Mitoma et al., 2015; Guo et al., 1999). Additionally, some of the exposed individuals reported instances of nonspecific symptoms, such as headache, general fatigue, numbness in the limbs, cough, and sputa (Kuratsune et al., 1971; !8 Okumura, 1984). Some of the babies exposed to dioxins in utero exhibited diffuse grayish dark-brown pigmented skin, called fetal Yusho disease (FYD) or fetal PCB syndrome (Kuratsune et al., 1971; Yamashita and Hayashi, 1985; Ikeda et al., 1996).   In a different incident during the 1960s and early 1970s, chemical waste oil contaminated with dioxin was sprayed at several horse-riding arenas and unpaved streets of Times Beach, MO for dust control resulting in soil contamination, death of horses and small wildlife, and eventual disincorporation of the city (Carter et al., 1975). Studies of potentially exposed individuals from Times Beach did not reveal any adverse health effects that could be directly linked to dioxin exposure (White and Birnbaum, 2009).  Another accidental release of a large quantity of dioxins and DLCs occurred in the mid-1970s, at a small chemical production plant in the northern Italy. The accident resulted in contamination of the town of Seveso with TCDD. Within a couple of weeks, more than 1000 chickens and rabbits had died, and approximately a 1000 people were evacuated from the most contaminated zones (Mocarelli, 2001). It has been reported that human effect of dioxin exposure in Seveso was limited by approximately 200 cases of chloracne. The long-term epidemiological studies reported a skewed sex ratio in the children born to exposed fathers toward female babies (Mocarelli et al., 2000).  Epidemiological studies of cancer mortality in different groups of individuals exposed to TCDD provide evidence for contradictory conclusions (Bailar, 1991; Sany et al., 2015). Additionally, epidemiological studies indicate that TCDD !9 exposure might lead to prevalence of metabolic syndrome, birth of more girls to exposed fathers, adverse reproductive outcomes, increase in insulin resistance, increased level of cholesterol and triglycerides, neurological effects, dental defects in children and increased incidence of non HodgkinÕs lymphoma (Alaluusua et al., 2004; Nishijo et al., 2014; Kim et al., 2003; Pelclova et al., 2002; Greene et al., 2003; Cranmer et al., 2000; Revich et al., 2001; Ryan et al., 2002). Studies discussed above indicate that TCDD, in addition to immediate adverse health outcomes, might cause subtle biological and toxic effects that could become apparent only years after the exposure.   1.2.2 AhR protein structure and polymorphisms Multiple lines of evidence support the view that the aryl hydrocarbon receptor (AhR) mediates virtually all toxic effects of dioxin and DLCs and that the key mechanism of dioxin toxicity lies at the level of altered gene expression (Okey et al., 1994). The AhR has a modular structure in which specific domains mediate particular functions of the receptor (Figure 2). Specifically, PAS-B domain contains ligand-binding pocket and is responsible for ligand binding. Interestingly, TCDD-binding function of the AhR protein requires a number of evolutionary conserved amino acids such as alanine 375, phenylalanine 345, isoleucine 319, tyrosine 316, phenylalanine 289, histidine 285 and threonine 283 located within the ligand-binding pocket of the AhR to mediate high-affinity TCDD binding (Pandini et al., 2009; Bisson et al., 2009). Both PAS-B and the bHLH domains are necessary for Hsp90 binding. Heterodimerization with ARNT requires the bHLH domain and both PAS-A !10 and PAS-B domains. Basic region of the bHLH domain harbors DNA binding and the nuclear localization sequences. The carboxyl part of the AhR protein is responsible for the transactivation function (Ko et al., 1997; Reen et al., 2002; Sogawa et al., 1995; Gu et al., 2001; Kewley et al., 2004; Okey et al., 2005). Interestingly, murine and human AhR demonstrate remarkable inter-species sequence similarity especially in the N-terminal regions: bHLH domain share 100% sequence homology, PAS domain is approximately 87% homologous and homology for the Q-rich region is about 60% (Dolwick et al., 1993). Multiple laboratories documented great inter- and intra-species differences in the susceptibility to the TCDD-induced lethal effects and other toxicities (Poland & Knutson, 1982; Pohjanvirta & Tuomisto, 1994; Hahn, 1998; Okey, 2007). For example, the most susceptible species, guinea pig (LD, of 1 pg/kg) and one of the most resistant, the Han-Wistar Kuopio rat, (LD, of over 9600 pg/kg) differ in TCDD tolerance by approximately four orders of magnitude. Multiple biological responses to TCDD exposure are even more variable. For example, chloracne, a common human end-point of dioxin exposure, is not seen in rat, guinea pig or hamster.  Some of the intra-species variability in sensitivity to dioxin-induced toxic and biological responses can be attributed to single nucleotide polymorphisms (SNPs). The term single nucleotide polymorphism refers to a variation in a single nucleotide that occurs at a particular position in the genome, with the rare allele occurring in at least 1% of individuals (Nachman, 2001; Sachidanandam et al., 2001). For example, a ten-fold difference in sensitivity to TCDD-induced Cyp1A1 mRNA levels and aryl hydrocarbon hydroxylase (AHH) activity in C57BL/6 and DBA/2 mice is attributed to  !11   Figure 2. A schematic representation of the functional and structural domains of the aryl hydrocarbon receptor (AhR). The basic Helix-Loop-Helix (bHLH) domain mediates the interactions of the receptor with DNA and is involved in DNA binding, dimerization with ARNT and Hsp90 binding. The Per-Arnt-Sim (PAS) domain mediates interactions with chaperone and co-chaperone proteins and ligand and DNA binding. The transactivation domain, located at the COOH half of the receptor, mediates transactivation of gene transcription.    Basic helix-loop-helixPAS-APAS-BTranscriptional activation domainDNA binding domainLigand binding domainHsp-90 binding domain!12 a single SNP (Nebert and Gielen, 1972). The DBA/2 mice express AhR with decreased affinity for ligands, diminished response to high-affinity ligands such as TCDD, and an abolished response to weaker ligands such as 3-methylcholanthrene (3-MC) (Poland & Knutson 1982; Okey et al., 1989). Site-directed mutagenesis in combination with ligand-binding studies demonstrated that differences in TCDD affinity for different polymorphic forms of the AhR could be mapped to a single amino acid substitution at codon 375. Alanine at codon 375 in the high-affinity receptors is substituted by a Valine in the low-affinity receptor (Poland et al. 1994). Thus, AhR polymorphism in the mouse model is a classic example of genetic variability resulting in a functional change of the gene product.  Likewise, genetic breeding studies demonstrated that in the rat, Ah locus is the major genetic factor that determines the sensitivity to dioxin-induced lethality (Tuomisto et al., 1999). The Han/Wistar rat is extremely resistant to TCDD-induced lethality demonstrating LD50 value of 9600 pg TCDD/kg body weight, approximately three log units higher than the LD50 value for a sensitive strain of rat, the Long Evans (LE), which has an LD50 of only 10 pg TCDD/kg body weight (Pohjanvina et al., 1993). Despite the large difference in sensitivity to TCDD-induced acute lethality, the H/W and L-E rats have similar sensitivities for multiple other biological responses to TCDD such as the induction of Cyp1A1, thymic atrophy, and fetotoxicity (Pohjanvirta & Tuomisto, 1994).  Interestingly, resistance to TCDD toxic effects is inherited as a recessive trait in mouse whereas it is a dominant trait in rats. Moreover, no observable inter-strain variation in binding affinity for TCDD was reported in the rat (Pohjanvirta and !13 Tuomisto, 1994; Tuomisto et al., 1999). However, the AhR protein in the highly resistant H/W rat has a lower molecular mass than the wild-type AhR found in L-E rats and other dioxin-sensitive strains. Cloning of the AhR cDNA revealed a point mutation in the AhR gene in H/W rats in the intron ten-exon eleven junction at the nucleotide position 2454. This substitution disrupts the normal RNA splice site and reveals three cryptic splice sites that lead to splice variants in the mRNA and, ultimately, deletion of either 38 or 43 amino acids from the transactivation domain of the AhR protein (Pohjanvirta et al., 1998; Okey, 2005). However, no direct evidence connecting these sequence modifications to changes in susceptibility to acute lethality exists, but it is tempting to speculate that such a profound alterations to the H/W AhR structure could significantly alter receptor function. The fact that biochemical and toxic effects of TCDD do not differ by the same magnitude between sensitive and resistant strains suggests a complex combination of molecular and cellular pathways that regulate different responses.  AhR polymorphisms in the mouse and the rat exert a strong effect on biochemical and toxic responses to dioxin-like chemicals. Thus, the knowledge of the genetic variation at the human Ah locus is instrumental for assessing human mechanisms of toxicity and human risk from dioxin exposure. Kawajiri et al. (1995) first reported the presence of an SNP at the position 554 in the human AhR. Since then, multiple studies investigated the effects of this SNP on AhR function making it the most studied human AhR polymorphism. Interestingly, most of the AhR SNPs are concentrated within the exon 10, a region that encodes the transactivation domain of the receptor. Experimental evidence suggests that SNPs in !14 transactivation domain may result in differentially recruited coactivator/corepressor complexes, and consequently, differentially regulate gene expression (Flaveny et al., 2008; Flaveny et al., 2010). Indeed, previous studies characterizing polymorphisms at the codon 517, 554 and 570 led to a complicated and conflicting interpretation of the functional effects of these polymorphisms (Wong et al., 2001b; Celius and Matthews, 2010). For instance, the codon 554 variant correlated with induced Cyp1A1 activity in one study (Smart and Daly, 2000) but not in the others (Cauchi et al., 2001; Kawajiri et al., 1995). The combined occurrence of polymorphisms at codons 554 and 570, or 554 and 517 produced an AhR that failed to induce Cyp1A1 mRNA expression in vitro even though the ligand- and DRE-binding capacity of this variant is unaffected by these polymorphisms (Wong et al., 2001b).  The polymorphism at codon 554, alone, does not to exert a significant effect on transactivation function of the human AhR (Harper et al., 2002) and does not influence the risk of lung cancer in Japanese smokers (Kawajiri et al., 1995). Additionally, Wong et al. (2001a) reported that P517S is in linkage disequilibrium (a nonrandom association of alleles at two or more loci) with R554K and V570I in individuals of African decent. It is conceivable that the combined expression of these three polymorphisms might result in an AhR with distinct intrinsic transactivation potentials.   Multiple epidemiological studies have investigated the effects of the R554K polymorphism on susceptibility to disease in different human populations. The R554K variant was found to be associated with a decreased risk of developing male infertility (Safarinejad et al., 2013) and significantly lower levels of AhR, ARNT and !15 Cyp1B1 mRNA expressed in white blood cells from the Caucasian population (Helmig et al., 2011). No association was found between R554K and increased risk of non-HodgkinÕs lymphoma (Ng et al., 2010). The R554K variant was found to be associated with a higher risk of coronary arterial disease in the Chinese population (Huang et al., 2015). Additionally, polymorphisms in AhR-interacting genes AHRR, Hsp90, and AIP have been identified and could potentially contribute to physiological and health outcomes associated with TCDD-induced AhR activation (Cavaco et al., 2013; Rowlands et al., 2011; Urban et al., 2012).  However, no studies to date investigated the effects of the AhR SNPs on a direct toxicological end-point, for example, suppression of the IgM response.   None of the identified polymorphisms alter human Ah receptor expression levels or receptor function.  However, within the human population, there is greater than a 10-fold range of variation in the affinity with which TCDD binds to the AhR, but no polymorphisms have been identified that account for this variation in binding affinity (Harper et al., 2002; Nebert et al., 2004). Humans are resistant to most of the adverse health effects of dioxin-like chemicals due to the relatively low affinity of the human AhR to TCDD compared to high affinities in the most susceptible mammals (Harper et al., 2002; Okey, 2007). Additionally, mice that express the human AhR in vivo are less susceptible to TCDD-induced cleft palate formation than mice homozygous for either the mouse high-affinity allele or the mouse low-affinity allele highlighting species-specific manner of the AhR function (Flaveny et al., 2009).    !16 1.2.3 AhR signaling Studies in AhR null rodents and cell lines suggest that most, if not all, of the toxic effects mediated by TCDD are mediated by the AhR (Poland and Glover, 1974; Okey et al., 1989; Sulentic et al., 1998). The results of studies using transgenic mice in which the AhR, or selected functions of the AhR, or ARNT have been disrupted demonstrate an absolute requirement of the AhR nuclear translocation, DRE binding and ARNT dimerization in the ability of TCDD to produce its major toxic and biological effects as well as a role for AhR-associated co-chaperones in selected adverse effects (Bunger et al., 2003 and 2008; Gonzalez and Fernandez-Salguero, 1998; Nukaya et al., 2010).   The AhR was identified by Poland, Glower and Kende who demonstrated [3H]-TCDD bound with high affinity (KD of 0.27nM) to protein isolated from hepatic cytosol of the C57BL/6 mice in a reversible, saturable, and stereospecific manner at concentrations needed to induce enzymatic activity of the cytochrome P4501A1 (CYP1A1) (Poland et al., 1976). Subsequently, details of the molecular mechanism of the AhR action were determined in studies that investigated the ability of TCDD to bind to and activate the AhR-dependent Cyp1A1 gene expression (Okey, 2007).   Multiple protein chaperones are important regulators of the AhR pathway. AhR is associated with heat shock protein 90 (Hsp90) via basic helix-loop-helix (bHLH) and Per-AHR-Sim (PAS) domains, which are involved in DNA and ligand binding, respectively (Perdew, 1988; Denis et al., 1988; Antonsson et al., 1995; Perdew et al., 1996). Another identified member of the receptor complex is the AhR-interacting protein (also known as AIP, XAP2, ARA9), which interacts with the PAS !17 domain of the AhR and Hsp90 (Meyer and Perdew, 1999; Carver et al., 1997; Meyer et al., 1998). Additionally, the co-chaperone protein p23 was identified to interact with the Hsp90 and AhR proteins as a part of AhR complex (Grenert et al., 1997; Nair et al., 1996). The Hsp90 protein plays multiple roles in the normal AhR function including inactive form maintenance and potentiation of activation by a ligand (Pongratz et al., 1992).  Ligand binding does not displace Hsp90 (McGuire et al., 1994), but leads to a conformational change of the AhR that allows importin # to bind to a nuclear localization signal and regulate AhR nucleo-cytoplasmic shuttling. Additionally, p23 ensures AhR cytoplasmic localization by preventing AhR nuclear translocation and nonspecific interaction with ARNT in the absence of a ligand (Kazlauskas et al., 1999; Kazlauskas et al., 2001; Soshilov and Denison, 2008). Thus, Hsp90 chaperone complex functions to regulate the abundance and cellular localization of the AhR at the posttranslational level.  The AIP was shown to inhibit ubiquitination of the AhR and it is required for the maintenance of sufficient AhR protein levels (Kazlauskas et al., 2000; Lees et al., 2003). Heterodimerization of the AhR with nuclear ARNT is required for ligand-induced release of the chaperone complex from the AhR. Protein tyrosine kinase, c-Src, was shown to associate with the cytosolic AhR-Hsp90 complex and activate by AhR ligand binding, providing an additional layer of AhR regulation through a protein phosphorylation cascades (Enan and Matsumura, 1996). The AhR-ARNT-ligand complex binds to a specific DNA sequence known as dioxin response elements (DREs, AHREs and XREs all refer to the same sequence) !18 usually located in the promoter region of the responsive genes. It was proposed that the AhR-ARNT complex binding disrupts chromatin structure around the DREs, allowing other transcription factors to bind to their respective sites within the promoter region initiating transcription (Wu and Whitlock, 1992; Okino and Whitlock, 1995). AhR has been shown to interact with multiple transcription factors interdependently and cooperatively to regulate gene transcription. For example, AhR interacts with basal transcription factors such as the TATA-binding protein, transcription factor TFIIF and transcription factor TFZIB recruiting them to their prospective cis-acting elements and exhibiting either activating or inhibiting net effect on gene regulation (Rowlands et al., 1996; Swanson and Yang 1998). AhR-dependent gene transcription is terminated via multiple mechanisms that contribute to down-regulation of the AhR signaling by facilitating nuclear export of the AhR and its subsequent ubiquitin-mediated proteasomal degradation. Additionally AhR regulates its own activity by induction of the AhR repressor (AHRR) protein expression that antagonizes its own activity by competing with AhR for ARNT dimerization and binding to DRE sequences thus attenuating AhR activity (Baba et al., 2001; Pollenz, 2002; Giannone et al., 1995; Mimura et al., 1999).  Although the canonical pathway of AhR activation, which involves ligand binding and recruitment to DRE sites on chromatin has been studied extensively, multiple reports suggest that AhR can exert its action via diverse nonclassical genomic and nongenomic mechanisms. The majority of these nonclassical AhR mechanisms are the direct result of its activity as a nuclear protein- and DNA-binding !19 transcription factor and thus are directly related to its ability to interact and cross-talk with other nuclear proteins and signaling factors. For example, AhR disrupts the endocrine signaling pathways mediated by the steroid hormones and the endocrine systems. TCDD has been shown to regulate cell cycle, mitogen-activated protein kinase cascades, immediate-early gene induction, and cross-talk with estrogen receptor (ER) in AhR-dependent but DRE-independent manner (Guyot et al. 2013; Sorg 2013). Several studies have reported a crosstalk between the AhR and ER that leads to the activation of ER-dependent genes in the absence of ER agonists by AhR:ARNT complex (Ohtake et al. 2003; Swedenborg and Pongratz 2010). Moreover, recent finding shows that TCDD is able to alter the amount of steroid receptors with the aid of the proteasome (Ohtake et al. 2007; Sorg 2013).  Additionally, recent studies reported that AhR modulates the immune system by inducing regulatory T cells (Treg) expansion, by activating the NF-KB transcription factor, or by enhancing IL-22 production and simultaneously repressing IL-17 producing cells (Guyot et al., 2013; Harper, 2007; Ohtake et al., 2007; Sorg, 2013). There is increasing evidence that AhR interacts with multiple signaling cascades such as MAPK, cytokine growth factor and Src kinase (Matsumura, 2009; Puga et al., 2009; Tian et al., 2002). A study in human and mouse cancer cell lines showed that TCDD-mediated AhR activation led to its recruitment to the loci of E2F-regulated S phaseÐspecific genes with subsequent repression through a mechanism implicating displacement of the HAT p300 from these loci (Marlowe et al., 2004). Thus, AhR may indirectly regulate gene expression by disrupting binding of other transcription factors.  !20 A different study conducted in a human breast cancer cell line revealed that, in the absence of a ligand, AhR is part of a complex along with CDK4/CCND1 (cyclin D1) that regulates retinoblastoma protein phosphorylation. Upon activation with TCDD, AhR dissociates from this complex, allowing for reduced retinoblastoma protein phosphorylation and increased activity to bring about the G1/S arrest (Barhoover et al., 2010). Moreover, AhR-dependent induction of Cox-2 expression was shown to be mediated by nonclassical and nongenomic mechanisms in transgenic mice that contained a mutant AhR that was unable to translocate into the nucleus (Li et al., 2010).  It has been previously demonstrated that some of the inflammatory effects induced by TCDD are mediated via nongenomic pathway including events, such as the increase in intracellular Ca(2+) concentration, enzymatic activation of cytosolic phospholipase A2 (cPLA2) and Cox-2 that require ligand-activated AhR but not ARNT (Matsumura, 2008).  Moreover, AhR-mediated promoter hypermethylation, deregulation of micro-RNAs and activation of mobile genetic elements add an additional level of complexity to AhR-dependent regulation of biological and toxic responses. For example, it has been recently demonstrated that AhR functions to regulate activation of transposable elements. AhR activation resulted in expression of long interspersed nuclear element-1 (LINE-1) retrotransposons in human cell lines linking carcinogens, LINE-1 elements and AhR (Teneng et al., 2007; Okudaira et al., 2013). Additionally, AhR was shown to indirectly suppress differentiation-related genes Lpp (cell-adhesion related lipoma preferred partner), Tbc1d1 (obesity risk gene), and Dad !21 (defender against cell death 1) by initiating transcription of the non-coding RNA from a novel B1-SINE retrotransposon, B1-X35S, which was proposed to act as an insulator and heterochromatin barrier (Roman et al., 2011; Mulero-Navarro and Fernandez-Salguero, 2016). It was previously demonstrated that in MCF7 human breast cancer cells, AhR activation results in BRCA1 silencing via the recruitment of DNA methyltransferases to its promoter region (Papoutsis et al., 2015). Interestingly, the AhR-inducible miR-132/212 cluster was demonstrated to promote inflammatory responses by inducing Th17 cells and suppressing the development of IL-10-producing T cells (Chinen et al., 2015). This research highlights the complexity of the ligand-dependent AhR-mediated signaling and regulation of the cellular responses by the AhR. The abnormal phenotypes of AhR-deficient rodents, the evolutionary conservation of the AhR among species, and the expression of multiple AhR-regulated genes during fetal development indicate a potential physiological role for the AhR and encouraged the search for an endogenous ligand of the AhR. Numerous molecules including environmental contaminants, drugs, dietary compounds and metabolites isolated from mammalian tissues have been shown to bind to and activate the AhR (Bjeldanes et al., 1991; Miller, 1997; Thatcher et al., 2016; Lowe et al., 2014; Kawasaki et al., 2014; Denison and Nagy, 2003; Denison et al., 1999). For example, indigo and indirubin, plant compounds frequently used for textile coloring, have been suggested to be endogenous, specific AhR agonists (Adachi et al., 2001). Indigo and indirubin were shown to compete with TCDD for AhR occupancy and induce Cyp1A1 activity (Guengerich et al., 2004; Sugihara et !22 al., 2004). However, indigoids are quickly metabolized by Cyp1A1, are present in small amounts in mammalian tissues and their synthesis in the vertebrate animals has not been demonstrated. Nevertheless, indigo and indirubin should be considered as possible endogenous AhR ligands since local concentrations of the indigo and indirubin may reach physiologically relevant levels at individual sites in vivo. An in-depth knowledge of the spatial and temporal distribution of these compounds will help to determine the ability of indigoids to activate the AhR in vivo. Another AhR agonist, 2-(1ÕH-indole-3Õ-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), was isolated from porcine lung tissue. Multiple lines of evidence support the conclusion that ITE is an AhR agonist. Specifically, ITE was shown to compete with [3H]-TCDD for binding to the AhR, ITE demonstrated high affinity for AhR derived from Hepa cell cytosol, and ITE induced Cyp1A1 protein expression and DRE-dependent luciferase activity in a concentration-dependent manner (Song et al., 2002; Henry et al., 2006). However, it is not clear whether ITE is a byproduct of the isolation process or indeed exists in mammalian tissues since the method used for ITE isolation utilized high temperature and acidic conditions. Thus a direct measurement of ITE in biological samples will provide additional evidence in support of a physiological role for ITE. The arachidonic acid metabolite, lipoxin 4A, is potential endogenous ligands of the AhR (Schaldach et al., 1999). Lipoxin A has been demonstrated to compete with TCDD for AhR occupancy, induce Cyp1A1 and Cyp1A2 mediated enzyme activity and DRE-regulated luciferase reporter activity (Schaldach et al., 1999). Additionally, six prostaglandins (Prostaglandin B2, D2, F3$, G2, H1 and H2) have been !23 shown to induce AhR activity weakly. Since prostaglandins and lipoxins are rather weak, low-affinity AhR agonists, it seems unlikely that these particular compounds are biologically relevant.  Metabolites of heme (biliverdin, bilirubin, and hemin) are also capable of activating the AhR as demonstrated by time and dose-dependent induction of Cyp1A1 mRNA, EROD and DRE-regulated reporter activity (Sinal and Bend, 1997; Phelan et al., 1997). Additionally, bilirubin and biliverdin have been shown to compete with [3H]-TCDD for receptor binding, indicating a potential role of these compounds as AhR endogenous ligands. However, under physiological conditions, most of the heme metabolites are bound to serum proteins restricting the amount of bioavailable bilirubin indicating that these components might be physiologically relevant only under some pathological conditions.  The tryptophan photoproduct FICZ is believed to be an endogenous AhR agonist. It is generated by both UVB radiation and reactive oxygen species (ROS) via indole-3-acetaldehyde pathway suggesting that FICZ is likely to be produced systemically. Cyp1A1 efficiently metabolize FICZ, resulting in transient AhR activation (Heath-Pagliuso et al., 1998; Bock, 2016). Tryptophan (Trp) catabolite kynurenine (Kyn) is another potential endogenous ligand of the human AhR. Kyn is constitutively generated by human tumor cells via tryptophan-2,3-dioxygenase (TDO) and has been shown to suppress antitumor immune responses and promote tumor-cell survival in AhR-dependent manner. It is tempting to speculate that activation of Trp catabolism and subsequent Kyn !24 production represents an endogenous feedback loop to restrict inflammation and provide local immune suppression via the AhR activation (Opitz et al., 2011). These observations, taken together, emphasize a substantive role for the AhR in cellular development and normal physiology. Yet, to date, an endogenous ligand capable of AhR activation at physiological concentrations and physiological function of the AhR remain a mystery.   1.3 Immune system and humoral immunity 1.3.1 Immune system overview The immune system is complex and pervasive and functions to prevent or limit infection by distinguishing between normal, healthy cells and unhealthy cells or pathogens. Unhealthy cells include cells damaged by infection or non-infectious agents like sunburn or cancer. Infectious agents such as viruses and bacteria release a set of signals recognized by the immune system known as pathogen-associated molecular patterns (PAMPs) (Wu, 2016; Oth et al., 2016; Uthaisangsook et al., 2002; H−cker et al., 2002). The skin, cornea, and mucosa of the respiratory, gastrointestinal (GI), and genitourinary (GU) tracts form a physical barrier that is the body's first line of defense. Additionally, numerous immune system cell types play unique roles (such as pathogen recognition, communication with other cell types and effector function) and circulate throughout the body or reside in a particular tissue. For example, skin cells produce and secrete multiple antimicrobial proteins, and harbor immune cells within skin layers.  !25 The bone marrow contains stem cells that develop into primary initial responders to infection, innate immune cells (such as neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells, and macrophages). The common lymphoid progenitor stem cells give rise to the B and T lymphocytes, that are a part of the adaptive immune response and mount a robust immune response against previously encountered pathogens and have immunological memory (Hofer et al., 2016; Cortez et al., 2015). Bone marrow, thymus, and spleen are all organs of the immune system (Kuper et al., 2016;). Bone marrow is a primary location of hematopoiesis; it produces B cells, natural killer cells, granulocytes and immature thymocytes, in addition to red blood cells and platelets.  Thymus functions to generate mature T cells through a process of positive and negative selection resulting in a release of mature non-auto-responsive T cells into the bloodstream. Immune cells constantly circulate throughout the bloodstream and lymphatic system in order to quickly and efficiently locate pathogens. The spleen contains multiple types of immune cells including B cells, T cells, macrophages, dendritic cells, natural killer cells and red blood cells that capture foreign antigens and initiate an immune response.  The lymph nodes are found throughout the body and function to capture antigens and initiate an immune response. Among the cells of the immune system are T and B cells, NK cells, macrophages, granulocytes, and dendritic cells. The major function of T lymphocytes is to augment or potentiate immune responses by the secretion of specific factors that activate other white blood cells to fight off infection. T killer (CD8+) subset of T cells is involved in the direct killing of the certain !26 tumor cells, viral infected cells and parasites and down-regulation of immune responses. Natural killer cells function as effector cells that directly kill tumor or infected cells (Suck et al., 2016; Vacca et al., 2016). The major function of B lymphocytes is the production of large amount of antibodies that specifically recognize and bind to one particular antigen. Neutrophils, eosinophils and basophils engulf and degrade bacteria and parasites thus removing them from the body. Macrophages are important antigen-presenting cells (APC) that regulate immune responses by presenting antigens to the T and B cells (Kzhyshkowska et al., 2016). Dendritic cells also function as potent antigen presenting cells (APC) that are mostly found in lymphoid organs (Cook and MacDonald 2016; Macri et al., 2016).  Two major types of immune response exist - innate and acquired. Innate immunity is the first line of defense against pathogens and does not require prior exposure to the antigen. It recognizes a wide range of PAMPs such as pieces of viral DNA or components of bacterial cell wall. Cellular elements of the innate immune system include neutrophils, monocytes, macrophages, natural killer cells, eosinophils, basophils and mast cells. On the other hand, acquired (also known as adaptive) immunity has immunologic memory to an antigen and it requires a longer period of time to mount an effective immune response against a new pathogen. T cells and B cells are major components of the acquired immune response. T cells provide cell-mediated adaptive immune response and B cells provide humoral immunity (Crisan et al., 2016; DiNardo et al., 2016).  Successful immune response requires three steps: activation, regulation, and resolution. Activation of the immune system involves antigen presentation and !27 recognition by the immune system cells. A foreign antigen is recognized by the B- or T-cell receptors and pattern-recognition receptors, such as Toll-like, mannose, and scavenger receptors. The immune response is tightly regulated by multiple mechanisms to prevent unnecessary damage to the host. For example, regulatory T cells secrete immunosuppressive cytokines, such as interleukin 10 (IL-10) and transforming growth factor-# (TGF-#), which control the immune response. An immune response is resolved when the antigen is eliminated from the body resulting in abrogation of cytokine secretion and apoptosis of the activated cytotoxic T cells (Bahadar et al., 2015; Yatim and Lakkis, 2015).    1.3.2 B cell activation B cells develop from hematopoietic precursors in bone marrow and are essential elements of the humoral part of the adaptive immune system. The B cell development and function is substantially dependent on the B cell antigen receptor (BCR). The BCR is a heterotetramer of two identical heavy chains, two identical light chains and Ig$/# chains located at the plasma membrane and involved in diverse signaling events (Figure 3) (Surova and Jumaa, 2014; Treanor, 2012). BCRs and their soluble counterparts, antibodies, are capable of recognizing an enormous repertoire of foreign and self-antigens (Burnet, 1959; Wardemann et al., 2003). BCRs function to transmit B cell activation and differentiation signals and process and present antigen to the T helper (Th) cells. Antigen binding to the variable region of the BCR triggers phosphorylation of the immunoreceptor tyrosine activation motifs (ITAM) associated with the Ig$/# chains of the BCR by the Src-family tyrosine kinase !28 Lyn and spleen tyrosine kinase (Syk) (Reth, 1989; DeFranco, 1997). Disruption of the primary kinase signaling, Lyn and Syk, disrupts BCR signaling and B cell development in mice (Xu et al., 2005). Activation of other signaling molecules and pathways such as PI3K, AKT, MAPK pathway, PKC and ERK are instrumental for the proper BCR signaling and B cell function. For example, ERK activation triggers NFkB translocation into the nucleus and subsequent degradation of the B cell repressor, BCL-6 (Niu et al., 1998; Kubo et al., 2016). Additionally, multiple molecular regulators control BCR signaling to prevent excessive B cell activation, proliferation and effector function. For example, paired immunoglobulin-like receptors (PIRs) contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that can associate with different phosphatases to inhibit B cell receptor signaling (Nakamura et al., 2004; Yamashita et al., 1998).   Multiple antigens such as LPS, CpG and PWM activate B cells in a T cell-independent manner via ligation and activation of the Toll-like receptors. In contrast,  T cell-dependent B cell activation requires the antigen to be processed and presented as a part of MHCII complex. MHCII:antigen complex is then recognized by the T helper (Th) cells and, with the involvement of co-stimulatory signals such as CD28 and CTLA-4 engagement, activate antigen-specific T cells. Importantly, the cells secrete crucial signaling molecules that enhance B cell function such as IL-2, IL-4, IL-6 and IL-21 (Jamin et al., 2014; Jawa et al., 2013; Bishop et al., 2003). Additionally, T cells express CD40 ligand on the cell surface that binds to the CD40 receptor located on the B cell surface. CD40 receptor is essential for B cell development and function and has been shown to play an important role in B cell  !29  Figure 3. A schematic representation of the core unit of BCR/immunoglobulin (antibody). 1) Fragment antigen-binding (Fab) region; responsible for antigen binding and consists of one constant and one variable domain of both heavy and light chains. 2) Fragment crystallizable region (Fc) is composed of two identical protein fragments and functions to activate immune system. 3) Heavy chain (blue) with one variable (VH) domain followed by a constant domain (CH1), a hinge region, and two more constant (CH2 and CH3) domains. 4) Light chain (green) with one variable (VL) and one constant (CL) domain. 5) Antigen binding site (paratope). 6) Hinge regions.  !30 proliferation and class-switch recombination (Banchereau et al., 1994; Bishop and Hostager, 2003). CD40 receptor engagement results in recruitment of TNFR-associated factors (TRAFs) that in turn activates NFkB, MAPK and PI3K signaling pathways and results in activation of the multiple transcription factors such as AP-1 and NFAT (Bishop and Hostager, 2001; Francis et al., 1995; Elgueta et al., 2009; Kehry, 1996).   Signaling events described above result in B cell activation that is phenotypically characterized by elevated expression of the CD80, CD86, CD69, MHCII and ICAM-1 molecules (Bishop and Hostager, 2001). The importance of the CD40-CD40L signaling was demonstrated in the CD40 null mice, which develop severe B cell function defects and autoimmune disorders (Bishop and Hostager, 2001). Regulation of the BCR signaling and B cell activation is complex and shaped by a balance of negative and positive co-receptor stimuli (Kuokkanen et al., 2015).  1.3.3 Regulation of B cell differentiation The development of functional B cells is greatly dependent on the correct maturation of progenitor cells in the bone marrow that includes activation of B cell lineage genes and restriction of alternative cell fates (Lou et al., 2015). Use of the genetically modified mice allowed for the identification of the major transcription factors such as PAX6, IKAROS, E2A and EBF1 crucial for the regulation of the normal B cell differentiation (Zhang et al., 2016; Basso and Dalla-Favera, 2012). Plasma cells function as antibody production factories and play a critical role in the humoral immune response. Differentiation of the B cell into a plasma cell is an !31 irreversible process controlled by a complex transcriptional network. The process of terminal differentiation gives rise to two types of antibody-secreting cells: short-lived and long-lived plasma cells. Terminal differentiation is preceded by multiple rounds of proliferation culminating in the cessation of cell division and significant changes in the morphology and gene expression profiles of the differentiated cells. Specifically, plasma cells are characterized by increased cytoplasmic to nuclear ratio to accommodate for increased steady-state mRNA levels and immunoglobulin synthesis (Jack and Wabl, 1988; Calame et al., 2003). Plasmacytic differentiation is also associated with a decrease of the MHCII, B220 and CD19 expression on the cell surface; expression of plasma cell markers CD38 and Syndecan-1 gradually increases on the cell surface of plasma cells. Additionally, plasma cells downregulate CXCR5 and CCL7 and up-regulate CXCR4, thus losing their capacity to stay in the GC and, instead, migrating to the bone marrow (Wehrli et al., 2001; Hauser et al., 2002; Pereira et al., 2010). Plasmablasts lacking CXCR4, the receptor for CXCL12, fail to enter the bone marrow from blood (Nagasawa et al., 1996; Hargreaves et al., 2001).  The details of the mechanism underlying B cell to plasma cell differentiation were illuminated by using CD40 ligand, LPS and CpG to stimulate mature B cells in vitro. The critical transcription factor regulators of B cell differentiation include repressors of B cell differentiation Ð Pax-5, BCL-6 and Bach2. Pax5 is the earliest identified transcription factor in the complex regulatory network for terminal differentiation of the B cells into antibody secreting cells (Nera et al., 2006; Nera and Lassila, 2006; Kallies et al., 2007). Pax5 is a transcriptional repressor required for !32 the maintenance of mature B cell identity (Cobaleda et al., 2007).  Pax5 has been shown to directly repress genes involved in the antibody secretion such as Igh and Igk transcription, and expression of J chain and XBP-1 (Linderson et al., 2004; Roque et al., 1996; Reimold et al., 1996; Rinkenberger et al., 1996). Loss of Pax5 alone is sufficient to induce IgM secretion (Delogu et al., 2006). Additionally, Pax5 promotes expression of another B cell differentiation repressor - Bach2 (Muto et al., 2004; Muto et al., 2010).   Bach2 and BCL-6 prevent B cell differentiation by suppression of the Blimp-1 expression and function (Muto et al., 2010). Bach2 deficient mice show an inability to produce IgM in response to antigen challenge (Muto et al., 2004). Blimp-1, known as a master regulator of plasma cell differentiation, functions to down-regulate genes expressed in mature B cells such as Pax-5, Bcl-6 and CXCR5 (Lin et al., 2002; Nera et al., 2006; Delogu et al., 2006; Calame et al., 2003). On the other hand, Blimp-1 induces the expression of genes involved in the regulation of protein synthesis and secretion of large amounts of antibodies such as XBP-1 (Crotty et al., 2010).  Expression of Blimp-1 is positively regulated by AP-1 and IRF4 and guarantees the irreversibility of plasmacytic differentiation Bcl-6 and Pax-5 in a bi-stable switch manner (Calame et al., 2003; Vasanwala et al., 2002; Bhattacharya et al., 2010; Zhang et al., 2013). Additionally, Blimp-1 is required for the maintenance of the long-lived bone marrow plasma cells, but it is not expressed in memory B cells (Kallies et al., 2004; Shapiro-Shelef et al., 2005; Angelin-Duclos et al., 2000).    !33 1.4 TCDD-mediated effects on immune system 1.4.1 Immunotoxicity of TCDD Studies performed in multiple laboratory animals demonstrate that the immune system is an incredibly sensitive target of TCDD-induced toxicity (Holsapple et al., 1991). One of the earliest reports published by Vos et al. (1973) described TCDD-induced suppression of the cell-mediated immune responses in mice and guinea pigs. Subsequent studies reported that TCDD treatment induced lymphopenia and thymic atrophy in rodents (Harris et al., 1973). A common rodent TCDD-induced disorder, thymic atrophy, is developed due to suppression of the thymic epithelial cell differentiation and a subsequent sharp decrease in T cell maturation and a number of the CD4+ and CD8+ T cells in the thymus (Greenlee et al., 1985; Kerkvliet and Brauner, 1990). Direct addition of TCDD to mature T cells in vivo resulted in diminished CTL activity (Kerkvliet, 2002; Kerkvliet et al., 2002).  Studies first performed in the 1980s demonstrated that TCDD suppressed humoral immune responses by cultured lymphocytes. Specifically, TCDD suppressed both primary and secondary responses to T cell-dependent and independent activators (Vecchi et al., 1983). TCDD suppressed formation of the AFCs and IgM secretion after ship red blood cell, LPS, and dinitrophenyl-Ficoll (DNP-Ficoll) or trinitrophenyl-LPS (TNP-LPS) stimulation in mice (Dooley and Holsapple, 1998; Tucker et al., 1986). Moreover, direct addition of TCDD to LPS-activated splenocytes resulted in a concentration-dependent decrease of the IgM secretion (Holsapple et al., 1986). Subsequent separation-reconstitution experiments demonstrated that the suppression of the IgM response was a direct !34 effect of TCDD on the B cells and not due to any possible effect of TCDD on other cell types present in splenocytes homogenate (Dooley and Holsapple, 1988). Additionally, experiments using purified mouse B cells confirmed that the B cell is a sensitive target of TCDD toxicity (Morris et al., 1993).  Immunotoxic effects of TCDD are AhR-dependent as demonstrated by AhR ligand structure-activity relationship and the use of AhR null animals and cell lines. Structure-activity relationship studies using PCDD, PCDF and PCB congeners with differing affinity to the AhR demonstrate a strong correlation between the intensity of the suppression of the B cell IgM response and ligand affinity to AhR (Tucker et al., 1986). Studies performed in the AhR null mice and B cell lines provide conclusive evidence for AhR involvement in TCDD-induced suppression of the IgM response. Sulentic et al. (1998) demonstrated that the AhR null mouse B cells, BCL-1, were resistant to TCDD-induced suppression of the IgM response even at TCDD concentrations as high as 100 nM, while AhR expressing mouse B cell line, CH12.LX, was highly sensitive to TCDD demonstrating suppression of the IgM response at 0.03 nM TCDD. Experiments performed in AhR null mice demonstrate a strict requirement for AhR expression in the TCDD-mediated suppression of the primary IgM response (Vorderstrasse et al., 2001). Importantly, B lymphocytes have a narrow window of sensitivity to the toxic effects of TCDD. Multiple studies report that TCDD must be added to the activated B cells within the initial 24 hours post-activation in order to suppress IgM response (Tucker et al., 1986; Holsapple et al., 1986). These experiments suggest that TCDD interferes with critical, early post-activation signaling events thus deregulating B cell to plasma cell differentiation.  !35 Hematopoietic stem cells (HSCs) express high levels of the AhR and are also a sensitive target of TCDD-induced, AhR-mediated toxicity. Long-term reconstitution ability of the mouse stem cells was also impaired by TCDD (Singh et al., 2009). Interestingly, AhR null mice presented an increase in the Lin-, Sca-1+, c-Kit+ (LSK) population in the bone marrow, indicating a potential endogenous role of the AhR in stem cell biology (Singh et al., 2011). Moreover, AhR antagonists have been shown to promote the expansion of the human HSCs (Boitano et al., 2010). Epidemiological data indicate that TCDD exposure leads to an elevated risk of contracting bacterial and viral infections (Burleson et al., 1996; Van Den Heuvel et al., 2002). Few significant effects of TCDD have been reported in resting T cells, but an activated T cell is a sensitive target of TCDD toxicity. It was demonstrated that TCDD suppressed cytotoxic T lymphocyte (CTL) response of the mouse T lymphocytes by approximately 90% (Kerkvliet et al., 2002). Adverse effects of TCDD on dendritic cell development and function have also been demonstrated. TCDD has been shown to disturb homeostasis and decrease the frequency and number of splenic dendritic cells, the professional antigen-presenting cells in the immune system (Bankoti et al., 2010).  1.4.2 TCDD-induced suppression of the B cell effector function In the past decades improved understanding of the molecular mechanisms of BCR signaling, as well as transcriptional and signaling events during normal B cell activation and differentiation helped improve our understanding of a potential mechanism underlying TCDD-induced B cell toxicity. Multiple cell-line-based tools !36 and knockout animal models proved to be instrumental in identifying specific transcriptional events taking place during B cell to plasma cell differentiation. Early studies of TCDD-induced suppression of B cell differentiation aimed to identify and characterize the effects of TCDD on components of B cell signaling pathways. For example, reports indicate that TCDD treatment can enhance phosphorylation of kinases and increase basal kinase activity in activated mouse B lymphocytes (Kramer et al., 1987; Snyder et al., 1993). Membrane protein phosphorylation was also elevated by TCDD treatment indicating that deregulation of kinase activity could play a significant role in the regulation of B cell activation and differentiation (Clark et al., 1991). Ligation of BCR triggers numerous signaling events that culminate in B cell activation and differentiation. Mobilization of calcium, an important signaling event preceding B cell differentiation, was demonstrated to be affected by TCDD treatment (Karras and Holsapple, 1994). IgM secretion could also be affected by TCDD-induced disruption of calcium homeostasis and subsequent delay in elevation of intracellular calcium levels (Karras et al., 1996). More recently, studies performed by North et al. (2010) demonstrated the involvement of multiple kinases (for example, AKT, ERK, JNK) in the regulation of the BCL-6 levels and TCDD-induced B cell toxicity in LPS-activated primary mouse B cells. The exact mechanism of TCDD-induced, kinase-mediated impairment of B cell function is not well understood and requires additional investigation.   Multiple findings illuminating the mechanism of TCDD-induced suppression of the B cell activation, differentiation and effector function come from studies performed in the mouse B cell line, CH12.LX. The CH12.LX cell line was derived !37 from a mouse CH12 lymphoma cells using cloning by limiting dilution (Bishop and Haughton, 1986). Importantly, polyclonal activators such as LPS and PWM could be used to induce CH12.LX cells differentiation into plasma cells and IgM secretion (Sulentic et al., 1998). The basal levels of AhR and ARNT expression were slightly higher in CH12.LX cells as compared to primary mouse B cells. AhR levels in these cells increased in response to activating stimuli and CH12.LX proved to be extremely sensitive to TCDD-induced suppression of the IgM response (Sulentic et al., 1998; Crawford et al., 1997). Sulentic et al. (2000, 2004) demonstrated AhR:ARNT:Ligand complex binding within the Ig3Õ$ enhancer region and regulatory region of Ig heavy chain leading to a decrease in IgM protein secretion. Subsequent studies investigated the involvement of upstream regulators of the IgM response Pax-5, Blimp-1, AP-1 and NFkB in TCDD-induced suppression of the B cell function. Suh et al. (2002) demonstrated that TCDD inhibited AP-1 DNA binding in the LPS-activated B cells in AhR-dependent manner. Additionally, Pax-5, IgH, Ihk, IgJ and XBP-1 expression patterns were deregulated by TCDD (Yoo et al., 2004).  Moreover, TCDD-induced AP-1 binding within the Blimp-1 promoter, as well as a decrease in Blipm-1 expression, indicate that TCDD affected IgM secretion via Blimp-1 and Pax-5 signaling deregulation in the mouse B cells (Schneider et al., 2009).  Studies performed in mouse primary B cells confirm findings from the CH12.LX model.  Specifically, in primary mouse splenocytes, TCDD increased BCL-6 levels but suppressed the expression of Blimp-1, XBP-1, Igµ, IgJ,  and Ig! chains resulting in a net decrease of plasma cell numbers (North et al., 2009 and 2010).  !38 Additionally, a genome-wide study performed in LPS-activated, TCDD-treated CH12.LX cell line was used to identify novel, direct, B cell-specific molecular targets of TCDD. A combination of microarray and chromatin immunoprecipitation on whole genome tiling arrays (ChIP-on-chip) analysis was used to identify genes directly regulated by the AhR. In the activated CH12.LX cells a total of 78 genes were deregulated by TCDD treatment and showed AhR binding in the promoter region (De Abrew et al., 2010). This study identified multiple signaling nodes and possible mechanisms of TCDD-induced, AhR-mediated suppression of the IgM response that require further investigation. Studies described above help to illuminate molecular mechanisms underlying TCDD-induced suppression of B cell IgM response in the mouse models.  Traditionally rodents are used as a model for human disease and risk assessment. However, significant differences in development, activation and regulation of the immune system and immune responses between rodents and human have been described (Mestas and Hughes, 2004; Seok et al., 2013; Shanks et al., 2009). From the point of human risk assessment, it is critical to know the human-specific effects and sensitive end-points of toxicant exposure. Human risk assessment decisions are usually based on data obtained from animal models and epidemiological studies and carry particular uncertainties (Nurminen et al., 1999). Use of primary cells or tissues obtained from healthy human donors can help reduce these uncertainties. Concerning the AhR research, initial studies performed in tonsil tissue led to identification and characterization of the human AhR and establishment of the human B cells as a sensitive target of TCDD toxicity (Lorenzen and Okey, !39 1991; Wood et al., 1992; Wood and Holsapple, 1993). Additionally, AhR has been shown to bind within the CD19 promoter and down-regulate CD19 expression indicating a direct transcriptional role of the AhR in human B cells (Masten and Shiverick, 1995). Recently, Lu et al. (2009) established a primary human B cell model for investigation of the IgM antibody-forming cell response in human donors. Using this model Lu et al. demonstrated that TCDD treatment of primary human B cells led to induction of the CYP1A1, AHRR and TIPARP genes and concentration-dependent suppression of the IgM response. Interestingly, a fraction of human donors (one in 5) was resistant to TCDD-induced suppression of the IgM response, likely, due to a variation in the AhR gene sequence (Lu et al., 2010). Notably, human B cells show a lower magnitude of induction of the AhR battery genes as compared to mouse primary B cells likely reflecting a lower TCDD binding affinity between the mouse and human AhRs. Previous studies indicate that human and mouse AhRs differ in their ability to regulate gene expression as human and mouse AhR expressed in the same genetic background deregulated different sets of genes (Flaveny et al., 2010). The divergence between mouse and human responses was also observed in primary B cell responses to TCDD treatment. For example, TCDD deregulated expression of Blimp-1 and Pax-5 in mouse B cells but not human. Additionally, in human B cells, TCDD significantly suppressed CD80, CD86 and CD69 B cell activation marker expression resulting in a decrease of activation and differentiation into a plasma cell (Lu et al., 2011). Species-specific effects of TCDD on the mouse and human B cells indicate potential mechanistic differences of !40 TCDD-induced B cell IgM suppression and highlight the need for improving our understanding of the mechanisms underlying TCDD toxicity in human B cells.  1.4.3 Rationale TCDD is an omnipresent environmental toxicant and the most toxic congener within the chemical family of halogenated aromatic hydrocarbons. Multiple species exhibit immunosuppressive phenotype shortly after TCDD exposure. Specifically, TCDD suppresses B cell activation, differentiation and antibody secreting function in many species tested, including humans. Despite scientific advancement during the last two decades, specific molecular targets of TCDD and itÕs mechanism of action remain unknown. Additionally, specific molecular players and qualities of the AhR that contribute to the great level of variability in sensitivity to toxic effects of dioxin in the human population remain poorly understood. Therefore, the overarching goal of my dissertation research was two-fold: 1) to characterize effects of select AhR polymorphisms on the variability of the human B cells to TCDD-induced suppression of the IgM response; and 2) to identify novel, B cell-specific molecular targets of TCDD. In the first part of my dissertation, IÕm specifically testing the hypothesis that SNPs within the transactivation domain of the AhR influence human B cell sensitivity to TCDD-induced suppression of the IgM response. The aim of the second part of my thesis is to test the hypothesis that TCDD acts via a conserved mechanism by deregulating expression levels of a set of B cell-specific genes.  Understanding to what degree AhR SNPs influence human sensitivity to the immunosuppressive effect of TCDD could help identify vulnerable and resistant !41 human populations and contribute to a development of improved risk assessment framework for dioxin and dioxin-like compounds. Importantly, identification of novel transcriptional B cell-specific TCDD targets will enhance our understanding of the mechanisms underlying TCDD-induced AhR-mediated suppression of the IgM response.    !42 CHAPTER 2: MATERIALS AND METHODS  2.1 Chemicals and reagents TCDD in dimethyl sulfoxide (DMSO) (purity 99.1%) was purchased from AccuStandard Inc (New Haven, CT). DMSO and lipopolysaccharide (LPS) (Escherichia coli, catalog no.  L2755-10MG) were purchased from Sigma-Aldrich (St. Louis, MO). The anti-human AhR antibody was purchased from eBioscience (catalog no. 14-9854-82, San Diego, CA). The anti-mouse immunoglobulin capture antibody and the horseradish peroxidase anti-mouse IgM detection antibody were purchased from Boehringer Mannheim (Indianapolis, IN) and Sigma-Aldrich, respectively. The pokeweed mitogen (PWM) and puromycin were purchased from Sigma-Aldrich (St. Louis, MO, Lot. O L8777-5MG and P8833).   2.2 Cell lines The SKW 6.4 cell line has been previously characterized by Ralph et al. (1984) and was obtained from ATCC (American Type Culture Collection, Rockville,MD). Cells were maintained under standard conditions (5% CO2/95% air, 98% humidity, 37oC) in RPMI-1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% bovine calf serum (HyClone Laboratories, Logan, UT), 13.5 mM HEPES, 23.8 mM sodium bicarbonate, 100 units/ml penicillin, 100 µg/ml streptomycin, 0.1 mM nonessential amino acids, 1.0 mM sodium pyruvate and 2.5 g/l dextrose. Cells were cultured in suspension in 25-cm2 flasks (Becton Dickinson, Plymouth, GB) at density of 1< l05 cells/ml. !43  HEK293T cells were used in the co-culture experiments to generate SKW-based cell lines that stably express different polymorphic forms of the AhR and during transient transfections with DRE- or Cyp1B1-regulated luciferase reporter constructs.  HEK293T cells were cultured in DMEM supplemented with 10% bovine calf serum (HyClone Laboratories, Logan, UT), 100 units/ml penicillin, 100 µg/ml streptomycin, 0.1 mM nonessential amino acids and 1.0 mM sodium pyruvate.  Cells were kept in a thermo-regulated incubator at 37%C, 5% CO2 and had 80% confluence when used.   2.3 Animals Pathogen-free, female C57BL/6 mice and Sprague-Dawley rats (5-8 weeks of age) were purchased from Charles River (Portage, MI). Animals were randomized, transferred to plastic cages containing sawdust bedding (five mice/3 rats per cage), and quarantined for 1 week. Animals were provided food (Purina certified laboratory chow) and water ad libitum. Animal holding rooms were kept at 21Ð24 ¡C and 40Ð60% humidity with a 12-h light/dark cycle. The Michigan State University Institutional Animal Care & Use Committee approved all experiments involving the use of animals.  2.4 Isolation of rat and mouse primary B cells Mouse or rat B cells were isolated from spleens of female C57BL/6 mice or Sprague-Dawley rats, and were made into single-cell suspensions by passage !44 through a 40 µm cell strainer (BD Biosciences, San Jose, CA). Negative selection of rat or mouse B cells was conducted using MACS Naive Rat B cell, or Mouse B Cell Isolation Kits following the manufacturer's protocols (Miltenyi Biotec, Auburn, CA) and as previously described (Lu et al., 2009). In all cases, the purity of isolated B cells was & 95%.  2.5 Preparation of luciferase reporter constructs pGL3-basic firefly luciferase reporter vector containing the 5Õ-flanking region from -1523 to +20 of the human Cyp1B1 gene was kindly provided by Dr. Weiguo Han of the Albert Einstein College of Medicine. The 5Õ-flanking region of the human Cyp1B1 and luciferase were transferred into the pLEX-MCS lentiviral vector (Thermo Scientific, Waltham, MA). pGudLuc6.1 expression vector containing a 480-bp fragment isolated from the 5Õ-flanking region of the mouse cytochrome P450A1 gene containing four consecutive DREs was kindly provided by Dr. Michael S. Denison of the University of California (Davis, CA). The 480-bp fragment containing DREs was transferred into the pLEX-MCS lentiviral vector.  Nucleotide sequences were confirmed by DNA sequencing analyses.   2.6 Luciferase assays For transient transfections, HEK293T cells were plated at 1.2 ' 106 cells/well in 6-well plates and transfected with 6 µg of the Cyp1B1- or DRE-luciferase constructs using lentiviral packing mix (Open Biosystems, Huntsville, AL) according to the manufacturerÕs instructions. After a 16 h incubation, HEK293T cell were !45 overlaid with 2 ' 106 SKW cells/well and co-cultured for an additional 24h. After 24-h of co-culture, SKW cells were separated from HEK293T cells and treated with vehicle (0.01% DMSO) or TCDD (30nM) for 24 h. The cells were then washed with 1X phosphate-buffered saline and lysed with 1X reporter lysis buffer (Promega, Madison, WI). Samples were immediately frozen at -80¡C. To measure luciferase enzyme activity, samples were thawed, and 20 µl of sample lysate was mixed with 100 µl of luciferase assay reagent (Promega, Madison, WI) using an autoinjector.  Luciferase activity was measured by KC-4 automated microplate reader (Bio-Tek, Winooski, VT) and represented as relative light units. Luciferase activity was normalized to the amount of protein determined by Bradford reaction (Protein Assay Kit, Pierce). Results are shown as fold induction determined by normalizing activation of different groups against VH control.  2.7 Preparation of cell lines that stably express AhR  HEK293T cells were used for the transfection of the recombinant control and polymorphic human AhR fused to GFP as described above. After a 48h co-culture incubation media was replaced, target cells were separated and cultured at 37 ¡C to approximately 80% confluence. Cells were then passaged and selected using RPMI media containing 0.5 µg/mL puromycin. Clones of the SKW cells were established using cloning by limiting dilution for 2 weeks in culture RPMI media containing 0.5 µg/mL puromycin.   !46 2.8 Cloning by limiting dilution Stably transduced SKW cells were diluted to a density of 3 cells/ml in RPMI media containing 0.5 µg/mL puromycin and a 100 µL of media, approximately 1/3 of a cell, was added to each well of a round bottom 96-well plate. An average of approximately 10 individual clones were obtained from each plate using this method.  2.9 pTRIPZ-AHR-GFP lentiviral vector production  Human AhR cDNA (cloned from HepG2 cells) was amplified by PCR from a previously described pSV-Sport1 vector (Dolwick et al., 1993) kindly donated by C. Bradfield. To generate the human AhR fused to GFP, AhR was amplified from pSV-Sport1 and sub-cloned into phCMV-C-GFP vector (Genlantis, San Diego, CA) using primers 5Õ-CCG TCG TCG ACT TAACCGG TCT GGG CAC CAT GA and 3Õ-CCC GGG CCC GCG GACGCGT CGA CTG CAG A. To generate a positive control vector containing only GFP, GFP cDNA was amplified by PCR from phCMV-C-GFP vector using the following primers 5Õ-CCG TCG TCG ACT TA ACCGGT CTC GAG CTC AAG CT and 3Õ- CCC GGG CCC GCG G ACGCGT CGA CTG CAG A. Underlined nucleotides denote restriction sites AgeI and MluI respectively. Each cDNA was amplified by PCR using Phusion Pfu enzyme (Takara, Mountain Viev, CA) following manufacturerÕs recommendations and sub-cloned in pZerO-blunt vector (Invitrogen, Grand Island, NY). After enzymatic restriction of pZerO-blunt vectors with AgeI and MluI, fragments were gel purified and extracted using the QIAquick gel extraction kit (QIAGEN, Valencia, CA) and ligated into the lentiviral inducible transfer vector pTRIPZ Tet-OnR (Open Biosystems, Lafayette, CO) using !47 T4 ligase (NEB, Ipswich, MA). Sanger sequencing was used to verify nucleotide sequences.   2.10 Site-directed mutagenesis  Site-directed mutagenesis was performed using the Quik-Change II XL kit (Stratagene, Santa Clara, CA) as previously described (Scott et al., 2002). Briefly, a total of 125 ng of two degenerate complementary primers with mutant sequences (517 sense 5'-CAT GAG CAA ATT GAC CAG TCT CAG GAT GTG AAC TCA T-3', antisense 5'-ATG AGT TCA CAT CCT GAG ACT GGT CAA TTT GCT CAT G-3'; 554 sense 5'-CCT AGG CAT TGA TTT TGA AGA CAT CAA ACA CAT GCA GAA TG-3', antisense 5'-CAT TCT GCA TGT GTT TGA TGT CTT CAA AAT CAA TGC CTA GG-3'; 570 sense 5'-CAG AAA TGA TTT TTC TGG TGA GAT TGA CTT CAG AGA CAT TGA CTT-3', antisense 5'-AAG TCA ATG TCT CTG AAG TCA ATC TCA CCA GAA AAA TCA TTT CTG-3') and 80 ng of template pTRIPZ-AHR-GFP was used for PCR amplification. PCR conditions were as follows: 95¡C for 30 s, 58¡C for 1 min, and 68¡C for 27min.  After15 cycles, the PCR product was digested with 10 U of DpnI to cleave template DNA at 37¡C for 2 h. PCR reaction was precipitated with ethanol and resuspended in 10ul of water. Half of the PCR product was used for transfections. Sanger sequencing was used to verify nucleotide sequences of the constructs.  2.11 Quantitative real-time PCR Total RNA was isolated using the SV40 Total RNA Isolation System !48  (Promega Corporation, Madison, WI) and RNA concentrations were quantified using a Nanodrop ND-1000 spectrophotometer (Wilmington, DE). Double stranded cDNA was synthesized using 1000 ng of total RNA using the Applied Biosystems high capacity cDNA reverse transcription kit (Foster City, CA). qRT-PCR was performed according to manufacturer's instructions using the Taqman Universal PCR Master Mix and Taqman gene expression assays for human Cyp1A2 (Hs01070374_m1), Cyp1B1 (Hs00164383_m1) and AhR (Hs00169233_m1), CD27 (Hs00386811_m1), CD93 (Hs00362607_m1), LCK (Hs00178427_m1), SERPINB2 (Hs01010736_m1), FOSB (Hs00171851_m1), MTSS1L (Hs00416413_m1), ITGB3 (Hs01001469_m1), RND2 (Hs00183269_m1), HIC1 (Hs00359611_s1), SHF (Hs00403125_m1), BEND7 (Hs00381740_m1). All qRT-PCR measurements were made on an ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). The change in gene expression was calculated using the ((Ct method using 18S ribosomal RNA (4319413E) as an internal control (Livak et al., 2001). For statistical analysis, unpaired two-tailed Student's t-tests were performed between treatments and their corresponding controls.  2.12 Western blot analysis Total protein samples were prepared and concentrations determined as previously described (Bradford et al., 1976; Lowry et al., 1951). Proteins were separated on 4Ð20% Nu-Page Bis-Tris gels (Bio-Rad Laboratories, Hercules, CA), transferred to nitrocellulose membranes, and probed with anti-AhR antibody !49 (eBioscience, San Diego,CA). Western blots were visualized using ECL Femto Western blotting substrate (Pierce, Rockford, IL).  2.13 IgM enzyme-linked immunosorbent assay (ELISA) A detailed procedure for determination of human, mouse and rat IgM concentration can be found in Sulentic et al. (1998). In brief, cells were activated with 150 µg/ml lipopolysaccharide (LPS) or pokeweed mitogen (PWM), cell culture supernatants were collected 120h post-activation and analyzed by a kinetic colorimetric sandwich ELISA specific for human, mouse and rat IgM. The IgM concentration (ng/ml) in each sample was calculated based on the standard curve using the KC4 software (BioTek, Winooski, VT). The concentration of IgM/106 cells was calculated by dividing the concentration (ng/ml) by the number of viable cells.  2.14 Isolation of the naŁve human B cells Human naŁve (CD19+ CD27-) B cells were isolated from peripheral blood mononuclear cells (PBMCs) enriched from each leukocyte pack by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Piscataway, NJ). Negative selection of human B cells was conducted using MACS Naive human B-cell Isolation Kit following the manufacturerÕs protocols (Miltenyi Biotec, Auburn, CA) and as described previously (Lu et al., 2009a). In all cases, the purity of isolated B cells was above 95%.   !50 2.15 Whole transcriptome expression profiling via RNA-Seq Total RNA was isolated from mouse, human (only ÒresponderÓ donors were considered for RNA-Seq profiling) and rat primary B cells activated with PWM and treated with vehicle (0.02% DMSO) or TCDD (30 nM) for 4, 8 and 24 h using RNeasy Kit (Qiagen, Valencia, CA) with gDNA eliminator columns.  RNA sample quality was assessed using Bioanalyzer RNA Nano chips (Agilent); all RNA samples had an RNA Integrity Number greater than or equal to eight.   2.16 RNA-Sequencing, alignment, and analysis RNA-Sequencing was performed at the Michigan State University Research Technology Support Facility Genomics Core (RTSF, https://rtsf.natsci.msu.edu/genomics). In summary, libraries from three human donors (N)=)3) and a pool of 20 mice and 5 rats were prepared using the Illumina TrueSeq RNA Sample Preparation Kit (Illumina, San Diego, CA) according to manufacturerÕs instructions. Library sizes were confirmed using Caliper GX (Perkin Elmer, Waltham, MA), and quantified by qPCR using the Kapa Biosystems quantification kit (Wilmington, MA). Sequencing of libraries was performed at an average depth of 30M each on an Illumina HiSeq 2500. The Michigan State High Performance Computer (MSU HPCC; https://icer.msu.edu/hpcc) was used for read processing and analysis. Reads, 1'50 bp with a seven-base index, were demultiplexed and quality was determined using FASTQC v0.11.2 (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Adaptor sequences were removed using Cutadapt v1.4.1 and low-complexity reads were !51 cleaned using FASTX v0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/index.html). Reads were mapped to the mouse (GRCm38 release 81), rat (Rn5 release 76), and human (GRch38 release 76) reference genomes using Bowtie 1.0.0 and TopHat v1.4.1 using default parameters and a minimum and maximum intron length of 10 and 15000, respectively. Alignments were converted to SAM format using SAMTools v0.1.19. (samtools.sourceforge.net/). Gene counts were determined using HTSeq v0.6.1 in intersection-nonempty mode (*m intersection-nonempty). RNA-Seq data is deposited in GEO. Counts were transformed through variance stabilizing transformation (VST) using the DESeq package in R (www.r-project.org) according to the DESeq reference manual. Data was normalized using a semi-parametric approach in SAS v9.3 (SAS Institute Inc., Cary, NC). Posterior probabilities P1(t) values were calculated for human datasets using an empirical Bayes method based on a per gene and dose basis using model-based t values as previously described (Eckel et al., 2004; Nault et al., 2015).  2.17 Network and functional ontology enrichment analysis  Functional enrichment analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID, http://david.abcc.ncifcrf.gov) filtered for gene ontology biological processes (BP), molecular functions (MF), and cellular component (CC). Functional categories were considered enriched when the Ðlog scale geometric mean p-value)+)0.05 (enrichment score)&)1.3).   !52 Signaling networks were constructed to connect 114 common orthologs deregulated by TCDD in human and mouse dataset.  The network was constructed using GeneGo Metacore database (GeneGo, Inc., St Joseph, MI) with a maximum number of steps set to 2.   2.18 Identification of putative dioxin response elements Identification and scoring of putative DREs (pDREs) core in mice was previously published (Nault et al., 2015b). For rat and human pDREs, identification and scoring was performed as previously described (Dere et al., 2011; Nault et al., 2015b). Briefly, bona fide DREs identified in Nault et al., 2015 were used to generate a position weight matrix (PWM). Using the UCSC genome database and the UCSC tool findMotif, all DREs were extracted from the rat (Rn5) and human (hg38) genomes. The UCSC tool twoBitToFa was then used to extract sequences and flanking 5Õ and 3Õ regions which were then used to calculate a matrix similarity score (MSS) as previously described (Dere et al., 2011)  2.19 Statistical analysis The mean ± S.E. was determined for each treatment group.  Differences between means were determined with a parametric analysis of variance.  When significant differences were detected, treatment groups were compared to the appropriate control using DunnettÕs two-tailed t test. Statistical analyses were performed using GraphPad Prism version 4.0a for Macintosh OS X, GraphPad Software, San Diego, CA.  !53 CHAPTER 3: EXPERIMENTAL RESULTS  3.1 Role of Aryl Hydrocarbon Receptor Polymorphisms on TCDD-mediated CYP1B1 Induction and IgM Suppression by Human B cells  3.1.1 Human SKW-AHR+ B cell line characterization  The AhR null human B cell line, SKW 6.4 was used to develop a series of clones that stably express either the control AhR (SKW-AHR+) or one of the known human AhR variants: P517S, V570I, and R554K. We included the combined R554K+V570I, V570I+ P517S double SNPs and V570I+ P517S+ R554K triple SNP, since it have been previously shown that each failed to mediate TCDD induction of Cyp1A1 mRNA. In addition, polymorphisms at the codon 517 were found only in individuals who carried alleles encoding lysine at codon 554 plus isoleucine at codon 570 (Wong et al., 2001a). Cell lines of the lymphoid lineage are difficult to transfect efficiently and to establish long-lasting transgene expression using retroviruses, diethylaminoethyl dextran, or liposomes (Guven et al., 2005). Therefore, a doxycycline-inducible lentiviral vector system, pTRIPZ was used to establish all SKW clones (Figure 4). The SKW-AHR+ clone was chosen for preliminary investigations. Different methods of B cell activation have been shown to activate different signaling programs and subsequent responses resulting in variation in levels of IgM production by the B cells (Donahue and Fruman, 2007). It have been previously demonstrated that IL-6 mediates differentiation and IgM production, but not proliferation in the human !54   Figure 4. A schematic representation of pTRIPZ vector for doxycycline-inducible transgene expression. In this vector, the expression of transgenic human aryl hydrocarbon receptor fused to green fluorescent protein (AhR-GFP) is under the control of human cytomegalovirus (CMV) constitutive promoter plus tetracycline response element (TRE) promoter, which can be activated by reverse tetracycline transactivator 3 (rtTA3) in the presence of doxycycline. pTRIPZ vector contains the 5Õ and 3Õ long terminal repeats (LTRs), packaging signal (!), internal ribosomal entry site (IRES) and puromycin selectable marker (Puro).   !55 mediates differentiation and IgM production, but not proliferation in the human lymphoblastoid B cell line, SKW 6.4 (Goldstein et al., 1990; Korholz et al., 1992). Additionally, it has been shown that phorbol 12-myristate 13-acetate (PMA) stimulated approximately a 3-fold increase of IgM secretion by SKW 6.4 cells (Cheng et al., 1992).   In order to identify the most efficient way to stimulate SKW-AHR+ cell differentiation and IgM production multiple polyclonal B cell activators and cytokines were investigated. TLR4 activator, LPS, was more efficient in inducing an IgM response in the SKW-AHR+ cells compared to TLR7/TLR8/TLR9 ligands such as R848 and CPG (Figure 5). As shown in figure 6 both LPS and PWM induce highest, approximately 3-fold, induction of the IgM secretion in the SKW-AHR+ cells. Additionally, cytokines such as IL-2, IL-6 and IL-10 have been previously demonstrated to play a critical role in B cell activation, proliferation and antibody secretion. As demonstrated in figure 7 IL-6 alone induced a maximum IgM response and a combination of various cytokines did not have an additive effect on antibody secretion by SKW-AHR+ cells. These data are in agreement with previous findings that the SKW 6.4 cells can be induced to produce IgM and that the maximum level of induction is approximately 3-fold (Goldstein et al., 1990; Korholz et al., 1992; Cheng et al., 1992).  In order to confirm that the full-length AhR mRNA product was transcribed we used custom PCR primers that span exons 3 and 4, 8 and 9 and exon 10. As shown in figure 8, SKW-AHR+ but not SKW 6.4 cells express a full-length AhR mRNA. Additionally, RT-PCR was used to compare AhR mRNA expression levels between !56 SKW 6.4, SKW-AHR+, HEPG2 and human primary B cells (Figure 9). SKW 6.4 cells express no detectable AhR mRNA, while SKW-AHR+, HEPG2 and human primary B cells express comparable levels of AhR mRNA. Western blot analysis was used to quantify approximate AhR protein levels in whole cell lysates obtained from the SKW 6.4, SKW-AHR+, HEPG2 and human primary B cells. AhR protein was expressed at the expected molecular weight of AhR-GFP fusion protein (approximately 130 KDa) in the SKW-AHR+ and not in the SKW 6.4 cell line (negative control). HEPG2 and primary human B cells were used as positive controls and AhR protein was identified as expected at 95kDa (Figure 9B).  To test the stability of the AhR mRNA expression upon induction of AhR levels with doxycycline and TCDD treatment, SKW-AHR+ cells were treated with doxycycline (0.2ug/ml) and either vehicle (0.02% DMSO) or TCDD (30 nM). Samples for mRNA isolation were collected at 7h and 96h post-treatment and AhR mRNA levels were measured by real time RT-PCR. There was no significant difference in the AhR mRNA levels between VH and TCDD treated samples. As expected, doxycycline treatment resulted in approximately 3.5 fold increase of the AhR mRNA (Figure 9C).   To investigate the transcriptional activity of the expressed AhR, SKW-AHR+ cells were treated with 30 nM TCDD or VH and incubated for 1, 2, 3, 4, 5, 6, 7, and 8h to obtain an expression kinetics profile for Cyp1B1 mRNA. As shown in figure 10, TCDD treatment induced a rapid and marked increase in Cyp1B1 mRNA levels in the SKW-AHR+ cells. TCDD-induced Cyp1B1 expression occurred as early as 1 h post TCDD treatment and remained elevated throughout the time course. Cyp1B1  !57   Figure 5. Induction of the IgM response by different PAMPs in the SKW-AHR+ cells. SKW cells (1"105 cells/ml) were activated with LPS (150 µg/ml), R848 (5 µg/ml) and C\pG (10 µg/ml) alone or in combination. Supernatants were harvested on day 5 post-activation and the amount of secreted IgM was measured by sandwich ELISA. The data are representative of two separate experiments with 4 experimental replicates per group. Statistical signiÞcance was determined using DunnettÕs two-tailed t test; * represents values that are signiÞcantly different from naive at p < 0.05.   Activation/PAMPsIgM (ng/106cells)NaLPSR848CPGLPS+CPGLPS+R848CPG+R848LPS+CPG+R8480200400600800****!58    Figure 6. Concentration-dependent induction of the IgM response by different PAMPs in the SKW-AHR+ cells. SKW-AHR+ cells (1"105 cells/ml) were activated with LPS or PWM at increasing concentrations. Supernatants were harvested on day 5 post activation and the amount of secreted IgM was measured by sandwich ELISA. The data are representative of two separate experiments with 4 experimental replicates per group. Statistical signiÞcance was determined using DunnettÕs two-tailed t test; * represents values that are signiÞcantly different from naive at p < 0.05.   !59   Figure 7. Induction of the IgM response with cytokines in the SKW-AHR+ cells. SKW-AHR+ cells (1"105 cells/ml) were activated with recombinant human IL-2 (10 U/ml), IL-6 (50 U/ml) and IL-10 (20 ng/ml) alone or in combination. Supernatants were harvested on day 5 post activation and the amount of secreted IgM was measured by sandwich ELISA. The data are representative of two separate experiments with 4 experimental replicates per group. Statistical signiÞcance was determined using DunnettÕs two-tailed t test; * represents values that are signiÞcantly different from naive at p < 0.05.   IgM (ng/106cells)NaIL-2IL-6IL-10IL-2+6IL-2+10IL-6+10IL-2+6+10050100150200250****!60    Figure 8. AhR mRNA expression in SKW 6.4, SKW-AHR+ and HepG2 cell lines. The SKW 6.4, SKW-AHR+(E8), and HepG2 cells (1"106 cells/ml) were used. Total RNA was isolated, and steady-state AhR mRNA levels were measured by SYBR Green qRT-PCR using custom primers designed against exon 3, exon 8-9 junction, and exon 10. Data are presented as CT values for each corresponding primer pair. The data are representative of three separate experiments with three experimental replicates per group.   "!#!$"!$#!%"!%#!&"!&#!'"!'#!()*+!&!,*!-.!()*+/01!,*!-.!()*+!$"!,*!-.!234!(/!5678%!!61 mRNA was not induced in TCDD-treated AhR null SKW 6.4 cell line (data not shown). Our findings indicate that WT AhR retained its ability to transactivate Cyp1B1 gene expression in SKW-AHR+ cells with no interference in function due to the GFP tag. Moreover, the magnitude of induction by TCDD of Cyp1B1 mRNA levels in SKW-AHR+ cells was relatively modest, compared to liver derived cells, and closely paralleled what we have previously reported in primary human peripheral blood B cells (Lu et al., 2010). AhR is known to mediate TCDD toxicity by binding to the dioxin response element (DRE) within the promoter region of multiple target genes and deregulating gene expression (Okey, 2007). To determine whether the AHR-GFP fusion protein expressed by the SKW-AHR+ cells can bind the DRE, electrophoretic mobility shift assays were performed using a radiolabeled human DRE probe harboring the AHR-ARNT binding core sequence (GCGTG). Nuclear proteins were isolated from SKW-AHR+ treated with TCDD (30 nM) or VH (0.02% DMSO) at various time points. We were unable to show AhR DRE binding in the SKW-AHR+ cells despite all of the technique optimization attempts. This could be due to inability of the AHR-GFP fusion protein to bind the DREs. However, it is unlikely, because we demonstrated the hallmark functional outcomes of the AhR activation such as induction of the metabolizing enzymes. Alternatively, the negative results could be due to inadequate levels of expressed AhR, displacement of the AhR receptor by other transcription factors under our EMSA conditions. Accordingly, binding sites for a number of transcription factors such as TF2/1,Foxp3, C/EBP, GR-alfa, and AP2 were all found within the DRE probes used.   !62 A                                                              B                       C  Figure 9. AhR expression levels in the newly generated SKW-AHR+ human B cell line. A. Levels of the AhR gene expression in the SKW 6.4, SKW-AHR+, HepG2 cell lines and human primary B cells. Total RNA was extracted from untreated SKW 6.4, SKW-AHR+, HepG2, human primary B cells (1"106 cells/ml) and 500 ng of total RNA were analyzed by RT-PCR for AhR mRNA. Steady-state mRNA levels of the AhR were normalized to the endogenous 18S ribosomal RNA. AhR mRNA in the SKW 6.4 cells was not detected (ND). Representative of 3 independent experiments. Data are presented as the mean ± SD. B. Western blot analysis of the AhR protein expression.  Whole cell lysates from the SKW 6.4 (80 µg), SKW-AHR+ (80 µg), HepG2 (5 µg) and human primary B cells (5 µg) were subjected to electrophoresis, blotted, and stained as described under Materials !63 Figure 9 (contÕd) and Methods. Lane 1: SKW 6.4; lane 2: SKW-AHR+; lane 3: human primary B lymphocytes; lane 4: HepG2. One representative result from three independent experiments is shown. C. Inducibility and stability of the AhR mRNA in the absence and presence of doxycycline. SKW-AHR+ cells (1"106 cells/ml) were treated with vehicle (0.02 % DMSO) or TCDD (30 nM) in the presence of doxycycline (0.2 µg/ml) harvested at 7 h and 96 h of culture and analyzed for levels of AhR mRNA expression. AhR mRNA levels were normalized to the endogenous 18S ribosomal RNA. Data are presented as the mean ± SD from 3 experiments.   !64  Figure 10. Time-dependent induction of the Cyp1B1 gene expression by TCDD.  The SKW-AHR+ cells (1"106 cells/ml) were treated with 30 nM of TCDD or 0.02% DMSO (VH) for indicated periods of time.  Total RNA was isolated, and steady-state Cyp1B1 mRNA levels were measured by TaqMan qRT-PCR and normalized to the endogenous 18S ribosomal RNA.  Data are presented as fold change compared to the VH control group at corresponding time point.  The data are representative of two separate experiments with three experimental replicates per group.  Statistical signiÞcance was determined using a two-way ANOVA and DunnettÕs two-tailed t test; * represents values that are signiÞcantly different from naive at p < 0.05.    !65 3.1.2 TCDD suppressed immune function in human SKW-AHR+ but not SKW 6.4 B cells In primary human and mouse B cells, TCDD is well established to markedly suppress IgM secretory responses to lipopolysaccharide (LPS) and CD40 ligand activation (Dooley and Holsapple, 1988; Lu et al., 2010). To evaluate the sensitivity of SKW-AHR+ and SKW 6.4 cells to TCDD-mediated suppression of the IgM response, supernatant IgM post LPS- and pokeweed mitogen (PWM) activation, was measured by ELISA in the absence and presence of TCDD treatment. TCDD (0, 3, 10, 30 nM) treatment resulted in a marked, concentration-related, suppression of LPS- and PWM-induced IgM secretion in the SKW-AHR+ (Figures 11B and 11D) but not in the AhR-deficient SKW 6.4 cells (Figures 11A and 11C). The ability of the B cells to secrete IgM depends on the continued production of immunoglobulin (Ig) components required for the assembly of IgM pentamer. In order to determine whether TCDD interfered with the production of Ig components, SKW 6.4 and SKW-AHR+ cells were activated with LPS and incubated with 30 nM TCDD for 5 days. Quantification of mRNA expression for the IgJ and Igµ immunoglobulin components by RT-PCR demonstrated that in SKW-AHR+ cells, both IgJ and Igµ mRNA levels were suppressed by TCDD (Figure 12). TCDD treatment did not affect IgJ and Igµ mRNA levels in SKW 6.4 cells (data not shown).  3.1.3 Temporal effects of TCDD on IgM antibody response in the SKW-AHR+, mouse and rat primary B cells Previous studies have demonstrated that suppression of the antibody-forming  !66   Figure 11. Effects of TCDD on LPS- and PWM-induced IgM secretion in the SKW 6.4 and SKW-AHR+ cells.  A. SKW 6.4 cells (1"106 cells/ml) activated with PWM.  B. SKW-AHR+ cells activated with PWM.  C. SKW 6.4 cells activated with LPS and D. SKW-AHR+ cells activated with LPS.  SKW cells (1"106 cells/ml) were activated with LPS or PWM (150 #g/ml) and treated with 3, 10, 30 nM TCDD or vehicle (0.02% DMSO). Supernatants were harvested on day 5 post LPS activation and the amount of secreted IgM was measured by sandwich ELISA.  The data are representative of three separate experiments with 4 experimental replicates per  !67 Figure 11 (contÕd) group. Statistical signiÞcance was determined using DunnettÕs two-tailed t test; * represents values that are signiÞcantly different from naive at p < 0.05.    !68   Figure 12.  Effects of TCDD treatment on IgJ and Igµ chain mRNA levels in LPS-activated SKW-AHR+ cell line.  The SKW-AHR+ cells (5x105/ml) were activated with LPS (150 µg/ml) and cultured for 5 days in the presence of TCDD (30 nM) or vehicle (0.02% DMSO).  Total RNA was isolated, and steady-state IgJ and Igµ mRNA levels were measured by TaqMan qRT-PCR and normalized to the endogenous 18S ribosomal RNA.  Data are presented as fold change compared to the VH control group.  Results represent the mean ± S.E. of triplicate determinations in each group from two separate experiments.  *, values significantly different (p<0.05) from time-matched vehicle controls.    !69 cell response in splenocytes from C57BL/6 mice by TCDD occurred only when TCDD was added within the initial 24 h post activation (Tucker et al., 1986).  In order to determine whether a window of sensitivity existed for the suppressive effects of TCDD on IgM secretion in the SKW-AHR+ cells, time of addition studies were conducted. Additionally, studies were conducted to compare SKW-AHR+ cells to well-characterized primary mouse and rat B lymphocytes. SKW-AHR+ cells were activated with pokeweed mitogen (PWM) and treated with 30 nM TCDD at various times after activation (2, 4, 6, 8, 12 and 24 h).    In concordance with previous studies conducted in mouse primary splenocytes, TCDD was unable to suppress IgM production if added after the initial 24 h post activation in SKW-AHR+ (Figure 13A). Moreover, TCDD must be added to the activated SKW-AHR+ cells within the initial 12 h of PWM stimulation to suppress the IgM secretion.  Additionally, the window of sensitivity during which TCDD can suppress IgM secretion in primary isolated mouse and rat B lymphocytes is similar to that of SKW-AHR+.  As shown in figures 13B and 13C, TCDD-induced suppression of the IgM secretion in primary mouse and rat B cells is greatest during the initial 12 h period post activation. Taken together, the above findings confirm that SKW-AHR+, mouse and rat primary B cells exhibit a similar and narrow period of susceptibility for TCDD-mediated suppression of the IgM response. In addition, these findings suggest a common mechanism of action of IgM suppression by TCDD across all three species.    !70  Figure 13. Relationship between the time of TCDD addition and in vitro IgM response. SKW-AHR+ (A), mouse (B), and rat (C) primary B cells were activated with PWM (150 #g/ml and 15 #g/ml respectively) and treated with 30 nM TCDD  !71 Figure 13 (contÕd) and/or vehicle (0.01% DMSO) at indicated time-points after activation. Supernatants were harvested at 120 h post PWM activation and the amount of secreted IgM was measured by sandwich ELISA. The data are representative of three separate experiments with 4 experimental replicates per group. Statistical signiÞcance was determined using DunnettÕs two-tailed t test; * represents values that are signiÞcantly different from naive at p < 0.05.   !72 3.1.4 TCDD-mediated effects on the expression of critical regulators of plasmacytic differentiation. Activation of the B cells with polyclonal B cell activators and multiple other stimuli lead to B cell proliferation and differentiation into the antibody secreting plasma cells, a process that requires coordinated action of multiple transcription factors, including, Blimp-1, Pax5, and BCL-6. Previously, it has been shown that TCDD induced Pax5 and suppressed Blimp-1 mRNA and protein expression in mouse (North et al., 2009; Schneider et al., 2009). Additionally, in the primary human B cells activated with CD-40L and cytokines, TCDD treatment resulted in elevated levels of the BCL-6 protein and BCL-6-dependent suppression of the CD80 and CD69 expression indicating B cell activation impairment. Based on the sensitivity of LPS- and PWM-induced SKW-AHR+ cells to the suppression by TCDD as described above, studies were conducted to investigate whether the suppressive effect of TCDD on IgM secretion in the SKW-AHR+ was also due to the deregulated expression of Blimp-1, Pax5 and BCL-6. A series of experiments aimed at obtaining the expression kinetics of Blimp-1, Pax5 and BCL-6 were performed in the SKW-AHR+. As shown in figure 14, TCDD at concentrations that suppressed IgM response (30 nM) of the SKW-AHR+ did not exhibit significant effects on the mRNA expression of Blimp-1, Pax5 and BCL-6 at any of the time points assessed. These data is in agreement with Lu et al. (2011) who demonstrated that TCDD did not alter Blimp-1 or Pax5 in the primary human B cells activated with CD-40L and cytokines. !73   Figure 14. Effects of the TCDD treatment on Blimp1, BCL-6 and Pax-5 gene expression in the SKW-AhR+ cell line. The SKW-AHR+ cells (5x105/ml) were activated with PWM (150 µg/ml) and cultured for 5 days in the presence of TCDD (30 nM) or vehicle (0.01% DMSO). Total RNA was isolated, and steady- mRNA  Blimp1051015dCTVHTCDD (30 nM)    24h         48h        72h         96h      120hPax-5051015dCT    24h        48h         72h        96h       120hBCL-6010203040dCT    24h        48h        72h         96h      120h!74 Figure 14 (contÕd) levels were measured by TaqMan qRT-PCR and normalized to the endogenous 18S ribosomal RNA. Results represent the mean ± S.E. of triplicate determinations in each group from two separate experiments. *, values significantly different (p<0.05) from time-matched vehicle controls.    !75 3.1.5 Effects of the AhR SNPs on the TCDD-mediated induction of Cyp1A2 and Cyp1B1 mRNA expression   SKW 6.4 cells were transfected with AhR expression vectors to generate SKW clones that stably express P517S, R554K, I570V, P517S+I570V, R554K+I570V and P517S+R554K+I570V AhR variants. Western blot analysis showed that WT and variant AhR proteins are expressed at comparable levels across the SKW clones (Figure 15A). It has been shown that TCDD treatment induced rapid degradation of AhR in certain cell preparations (Ma and Baldwin, 2002). To evaluate the effects of SNPs on AhR protein degradation, AhR protein was measured in the SKW-AHR+ and R554K cell lines at 0, 4, 8 and 24h post TCDD treatment. No difference in AhR protein levels or evidence of AhR degradation was observed after TCDD treatment in either of the two clones (Figure 15B).             To investigate the transcriptional activity of the P517S, R554K, I570V, P517S+I570V, R554K+I570V and P517S+R554K+I570V variant AhRs, TCDD-mediated induction of Cyp1B1 and Cyp1A2 mRNA expression was measured by PCR. As shown in figure 16A, TCDD treatment resulted in 2-5-fold induction of the Cyp1A2 mRNA in WT, P517S, I570V and I570V+P517S clones at all time points.  In contrast, R554K, R554K+I570V and P517S+R554K+I570V AhR variants had a decreased ability to mediate sustained activation of the Cyp1A2 mRNA at the time-points assessed when compared to the WT control. As expected, Cyp1A2 was not induced in the SKW 6.4 cells at any time-point. Similar findings were observed with R554K, R554K+I570V, and P517S+R554K+I570V AhR variants which displayed a decreased ability to up-regulate Cyp1B1 transcript levels (Figure 16B). The data  !76  Figure 15. Protein expression of the AhR variants in SKW clones. A. Equal amounts of whole cell lysates from the 1-SKW 6.4, 2-SKW-AHR+, 3-P517S, 4-V570I, 5-R554K, 6-R554K+V570I, 7-V570I+P517S and 8-V570I+P517S+R554K SKW cells were examined for AhR expression levels by Western Blotting. Cell lysate protein (40 #g) was loaded in each lane, resolved on 4-20% SDS-PAGE gel and probed with anti-AhR antibody. Results are representative of more than three separate experiments. B. Effects of the R554K SNP on the AhR protein stability. Time-dependent AhR protein degradation in SKW-AHR+ and R554K cells at 0, 4, 8 and 24h post TCDD treatment was examined by Western Blotting. Cell lysate protein (80 #g) was loaded in each lane, resolved on 4-20% SDS-PAGE gel and probed with anti-AhR antibody. Results are representative of more than three separate experiments.    !77  Figure 16. Time-dependent induction of the Cyp1A2 (A) and Cyp1B1 (B) gene expression by TCDD in SKW clones. The SKW cells (5$106/ml) were treated with 30 nM of TCDD or 0.01% DMSO (VH) for the indicated periods of time.  Total RNA was isolated, and steady-state Cyp1A2 and Cyp1B1 mRNA levels were measured by TaqMan qRT-PCR. Samples were normalized to the endogenous 18S ribosomal  !78 Figure 16 (contÕd) RNA, which served as loading control. Fold induction values were calculated relative to corresponding time-matched vehicle controls. The data are representative of two separate experiments with three experimental replicates per group. *, values significantly different (p<0.05) from time-matched vehicle controls.    !79 indicate that R554K AhR SNPs influence the transactivational activity of the AhR in human B lymphocytes, as suggested by a previous report (Wong et al., 2001b).  3.1.6 Effects of the AhR SNPs on the Cyp1B1- and DRE-regulated reporter gene activity   TCDD-mediated deregulation of gene expression involves the ability of AhR/ARNT complex to bind DRE elements in the regulatory regions of target genes (Okey, 2007). To determine whether any of the identified SNPs influence the ability of AhR to regulate Cyp1B1 levels or other DRE-regulated genes, the SKW cell lines were transiently transfected with either a Cyp1B1- or DRE-regulated reporter and treated with TCDD (30 nM) for 24h. A modest increase in Cyp1B1-regulated reporter gene activity was observed in SKW-AHR+, P517S, I570V and I570V+P517S but not in the R554K, R554K+I570V, and P517S+R554K+I570V (Figure 17A). Transfection of SKW cells with DRE-regulated reporter resulted in a 2Ð2.5-fold increase in TCDD-induced DRE-regulated reporter gene activity following 24 h of treatment (Figure 17B). Interestingly, no significant differences in DRE-regulated reporter gene activity were observed among the different AhR variants compared to wild-type receptor (Figure 17B). Induction of luciferase activity was lower for Cyp1B1 reporter compared with the induction observed for the DRE reporter. Induction of luciferase activity was not observed in the SKW 6.4 cells for either the Cyp1B1 or DRE reporter genes, indicating that these responses require a functional AhR/ARNT signaling pathway.    !80    Figure 17. Ability of the WT AhR and AhR variants to mediate TCDD-induced Cyp1B1 (A) and DRE (B) reporter gene activity. SKW cells were transiently transfected with DRE or Cyp1B1 reporter plasmids and treated with 30 nM TCDD or SKW 6.4SKW-AHR+P517SR554KV570IV570I+P517SV570I+R554KV570I+P517S+R554K012345Normalized LuciferaseActivityEmpty VectorVHTCDD (30 nM)****SKW 6.4SKW-AHR+P517SR554KV570IV570I+P517SV570I+R554KV570I+P517S+R554K012345Normalized LuciferaseActivity*******AB!81 VH (0.01% DMSO) for 24h. Luciferase enzyme activity was measured in relative light units (RLUs) then normalized to background and transformed to fold change as shown on the y-axis. n=4 for each treatment; results are combined from 4 independent experiments. Significance was determined by a one-way ANOVA followed by a DunnettÕs post hoc test. A Ò*Ó denotes significance compared with the corresponding vehicle (VH) control at p < 0.05.    !82 3.1.7 Effects of AhR SNPs on sensitivity of the human B cells to TCDD-mediated suppression of the IgM secretion To investigate the effects of a single P517S, R554K, I570V or a combination of P517S+I570V, R554K+I570V and P517S+R554K+I570V AhR SNPs on human B cell sensitivity to TCDD-mediated suppression of the IgM response, LPS-induced IgM secretion was measured by ELISA. To minimize the variability in background IgM levels among the SKW clones, the IgM response in all treatment groups was normalized to percent of the VH-treated control group for each SKW clone. The combined results from 6 independent experiments are presented in figure 18. TCDD at 1 nM significantly suppressed IgM secretion in SKW-AHR+, P517S, R554K, I570V, P517S+I570V and R554K+I570V SKW clones. Additionally, in all clones except for P517S+R554K+I570V, 30 and 100 nM TCDD suppressed the LPS-induced IgM response to approximately 50-60% of the VH control response. By contrast, only 10, 30 and 100 nM TCDD significantly suppressed IgM response in P517S+R554K+I570V clone, with maximum suppression of approximately 30% of VH control. No suppression of LPS-induced IgM secretion was observed in AhR-deficient SKW 6.4 cells at concentrations as high as 100 nM TCDD. These findings indicate that a combination of all three AhR SNPs attenuate B cell sensitivity to TCDD-mediated suppression of the IgM response.  !83  Figure 18. Effects of TCDD on the LPS-induced IgM secretion in SKW clones. SKW cells (1"105/ml) were activated with LPS (150 #g/ml) and treated with 0.3, 1, 3, 10, 30 and 100 nM TCDD or vehicle (0.01% DMSO). Supernatants were harvested on day 5 of culture and analyzed for IgM by sandwich ELISA. Data were normalized to VH control (100%) and presented as percentage of control. The data are combined from six separate experiments with four experimental replicates per group. Statistical signiÞcance was determined using DunnettÕs two-tailed t test; * represents values that are signiÞcantly different from VH control at p < 0.05.   !84 3.2 Genome-wide responses of the naŁve human, mouse and rat primary B cells to TCDD treatment  3.2.1 Gene expression changes in naŁve primary mouse, rat and human B cells following TCDD treatment 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a persistent environmental pollutant that activates the aryl hydrocarbon receptor (AhR) resulting in altered gene expression. In vivo, in vitro, and ex vivo studies have demonstrated B cells are directly affected by TCDD, and are a sensitive target of TCDD-induced immunosuppression. The window of sensitivity to TCDD-induced suppression of IgM secretion among mouse, rat and human B cells is similar. Specifically, TCDD must be added to the culture within the initial 12 h post activation, indicating that TCDD disrupts early signaling network(s) necessary for B lymphocyte activation and differentiation. Therefore, we hypothesized that TCDD treatment across three different species (mouse, rat and human) will trigger an actively conserved, B cell-specific mechanism that is involved in TCDD-induced immunosuppression. RNA sequencing (RNA-Seq) was used to identify B cell-specific orthologous genes that are differentially expressed in response to TCDD in primary mouse, rat and human B cells. Primary human, mouse and rat B-lymphocytes were isolated using Miltenyi B cell isolation kits for corresponding species and treated with vehicle (DMSO 0.02%) or TCDD (30 nM). Samples for the RNA-seq analysis were collected at 2, 4, 6, 8, and 12 h post-treatment. Total mRNA was isolated using Qiagen RNeasy Mini Kit. !85 RNA-seq was conducted using lluminaÕs HiSeq 2500 next-generation sequencer on samples from 4, 8 and 12h time points at the RTSF facility (MSU campus East Lansing MI). All datasets were filtered using a |fold-change| &1.5 criterion.   Analysis of the time-course RNA-Seq data identified TCDD-elicited differential expression of 536 human, 3069 mouse, and 3536 rat genes over the 12-h time period (Figure 19A). In the mouse dataset slightly higher number of genes were up-regulated as compared to down-regulated in response to TCDD (Figure 19B). In the human dataset approximately twice as many genes were down-regulated as up-regulated in response to TCDD. By contrast, in the rat dataset significantly more genes were down-regulated than up-regulated at 8 and 12h. Specifically, up-regulation was observed for 1792 mouse, 1114 rat, and 187 human genes; down regulation was observed for 1373 mouse, 2184 rat and 358 human genes (Figure 19B).   Principal component analysis (PCA) indicates that early responses to TCDD are the strongest and most divergent among the species. Additionally, the differentially expressed genes appear to converge at later time points suggesting that TCDD-mediated expression changes are less pronounced (Figure 20). Mapped orthologs were analyzed using HID (HomoloGene ID) in order to compare TCDD-induced differential expression of equivalent genes across human, rat and mouse primary activated B cells. The HomoloGene database contains 18,981 human, 21,766 mouse and 19,229 rat unique HIDs. Comparison of the top ten differentially expressed genes among the three species, revealed one common up-regulated gene - CYP1A1 but no common down-regulated genes (Table 1). !86 A   B  Figure 19. A. Number of species-specific genes exhibiting TCDD-induced changes in mRNA expression in naive primary mouse, human and rat B lymphocytes. The temporal profile of the number of differentially expressed genes across time points  4h8h12h 0100200300200030004000Time pointsNumber of Active GenesMouseHumanRat05001000150020002500Number of DEGs Up-regulatedDown-regulatedMouseRatHuman!87 Figure 19 (contÕd) suggests a rapid increase in number of responding genes in mouse and rat B cell at 4h, followed by sharp decrease at later time points. In human primary B cells the kinetics of the response to TCDD were dramatically different: slow and steady increase in the number of responding genes. B. Number of deferentially expressed genes (DEGs) that were either up- or down-regulated by TCDD in mouse, rat and human primary B cells. In the human and rat datasets more genes were down-regulated than up-regulated while mouse dataset contained more up-regulated genes.   !88  Figure 20. Bi-plot of Principal Component Analysis (PCA) of TCDD-induced differential gene expression in treated mouse, human and rat B cells at 4, 8, and 12h. Mouse, human and rat samples at 4h appear to group separately. However all samples group together at the later time points. PCA was performed in Rv2.15.0 using all detected genes with available data across all species and time points.    !89 Additionally, gene that codes for eukaryotic translation initiation factor 4E binding protein, Eif4ebp3, that is involved in regulation of translation was up-regulated in mouse and rat. PSD ortholog, codes guanine nucleotide exchange factor, involved in regulation of signal transduction by activation of ADP-ribosylation factor 6 and induction of cytoskeletal remodeling was up-regulated in mouse and human. Neuropilin 1 (Nrp1), known to regulate cell migration was down-regulated in mouse and rat. Mitochondrial ribosomal protein (MRPS24), involved in mitochondrial translation, and prostaglandin reductase PTGR1, involved in metabolic inactivation of leukotriene B4, were down-regulated in both mouse and human (Table 1).  Only 94 orthologs were identified as differentially expressed in response to TCDD in all three species. The majority of mouse and rat genes exhibited species-specific expression. Specifically, 28% (149/536) of human, 62% (1896/3069) of mouse, and 67% (2376/3536) of rat orthologs exhibited species-specific differential expression (Figure 21). 24 of the 94 orthologs differentially expressed in all three species exhibited a comparable expression pattern. This included the induction of prototypical ÒAhR batteryÓ genes such as CYP1A1, CYP1B1, TIPARP, and CYB5RL, HYI and HAAO metabolizing enzymes as well as the induction of EIF4EBP3, involved in translation repressor activity, peptidase inhibitor SERPINB2, member of the TNF-receptor superfamily TNFRSF9, and ALKBH6 gene involved in oxidative reduction. Repression of the B3GNT9, involved in glycoprotein metabolic process, transcriptional regulators SCAND1 and PROX1, genes involved in oxidation reduction TET1 and PTGR1, and RSAD2 involved in immune response. However, several orthologs exhibited divergent expression. !90  Table 1. Top 10 up- and down-regulated genes in the mouse, human and rat datasets. The maximum fold of induction/suppression is indicated in brackets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igure 21. Comparison of TCDD-induced differential ortholog expression in mouse, human and rat primary B cells. Venn diagram of the human, mouse and rat genes differentially regulated by TCDD at least at one time point (DEGs; |fold-change| %1.5).     !92 For example, ATP6V0C gene that encodes a component of vacuolar ATPase (V-ATPase), was repressed in human but induced in rodents, while the E2F transcription factor was repressed in rodents and induced in human primary B lymphocytes. The chemokine (C-C motif) receptor 5 (CCR5) was repressed in human and rat but induced in the mouse.  Functional analysis of the 94 orthologs deregulated by TCDD in human, mouse and rat primary B cells identified several enriched gene ontology biological processes: regulation of cell proliferation, oxidation reduction, and immune response. Pathways associated with the 94 common genes include tryptophan metabolism, cytokine-cytokine receptor interaction and endocytosis (Table 2).  3.2.2 Functional annotation and pathway enrichment of genes differentially expressed in response to TCDD in naive mouse, human and rat primary B cells In order to determine functional relevance of DEGs the 3069 mouse, 3536 rat, and 536 human significantly altered genes were analyzed for overrepresentation within specific functional categories and canonical pathways using the DAVID analysis tool. The top enriched pathways and biological functions and processes specific to mouse, rat and human primary B cells are presented in tables 3, 4 and 5. The enriched categories were compared between species identifying mitochondrion, immune response and regulation of transcription as common enriched categories (enrichment score &1.3). Species-specific enriched functions included RNA splicing and histone modification in mouse primary B cells (Table 3A and B), protein transport and JNK cascade in rat primary B cells (Table 4A and B), and cell  !93  Table 2. GO biological process terms associated with the 94 orthologs deregulated by TCDD in all three species. All GO biological processes identified for 94 common orthologs deregulated by TCDD treatment (DEGs; |fold-change| %1.5).   GO TermP*regulation of cell proliferation1.20E-03oxidation reduction4.80E-03dibenzo-p-dioxin metabolic process1.20E-02negative regulation of cell proliferation1.20E-02organic ether metabolic process1.60E-02hemopoiesis1.70E-02toxin metabolic process2.00E-02benzene and derivative metabolic process2.40E-02hemopoietic or lymphoid organ development2.50E-02immune system development2.90E-02muscle organ development3.30E-02lens fiber cell differentiation3.50E-02T cell differentiation3.70E-02immune response3.90E-02xenobiotic metabolic process5.00E-02smooth muscle tissue development5.00E-02positive regulation of developmental process5.30E-02regulation of cell cycle5.30E-02thymic T cell selection5.80E-02inflammatory response6.00E-02response to xenobiotic stimulus6.20E-02lymphocyte differentiation7.50E-02T cell activation7.80E-02T cell selection8.00E-02cellular ion homeostasis8.50E-02cellular chemical homeostasis9.00E-02!94 adhesion and Golgi apparatus in human primary B cells (Table 5A and B). Importantly, the number of genes within functional categories varied among species and direction of gene changes was frequently discordant between species (Figure 22). For example, mitochondrial genes Cox5e and Ndufa11 were suppressed in mouse and human but up-regulated in the rat. Conversely, genes involved in regulation of transcription E2F3, ATF7 and SOX12 were up-regulated in all three species. TCDD-induced differential gene expression mapped to 88 mouse, 61 rat and 63 human KEGG canonical pathways, among which 18 were common between mouse, human and rat (Table 6). Pathways significantly enriched in all three species included B cell receptor signaling pathway, cell cycle, and MAPK signaling pathway among others. 36 out of 87 pathways were specific for mouse-specific differential gene expression and were associated with ABC transporters, SNARE interactions in vesicular transport, and citrate cycle (TCA). In the rat primary B cells differentially expressed genes mapped to chronic myeloid leukemia, oxidative phosphorylation, and proteasome; 18 out of 61 pathways were rat-specific. In human primary B cells the specific significantly enriched canonical pathways (19 out of 63) included calcium signaling, cell adhesion, ECM receptor interaction, and TGF-beta signaling pathways. Importantly, genes within common enriched pathways frequently displayed divergent pattern of regulation.    !95    Table 3. GO terms associated with genes deregulated by TCDD in the mouse primary B cells. A. Significantly enriched (enrichment score&1.3) GO biological processes identified for genes up-regulated by TCDD treatment. B. Significantly enriched (enrichment score&1.3) GO biological processes identified for genes down-regulated by TCDD treatment (DEGs; |fold-change| %1.5).     GO TermGene NameEnrichment Scoreion bindingHaao, Nt5e, Atp9b, Acap3, Bspry, Bcl6b, Brf1, Cd93,Klf12, Lonrf3,  Luc7l11.82ATP bindingFancm, Fastk, Grk6, Ino80, Jak3, Khsrp, Mapkapk5, Mknk1, Mark4, Nat10, Nek87.66protein kinase activityAak1, Blk, Clk4, Clk2, Clk3, Fastk, Grk6, Jak3, Mapkapk5, Mknk1, Mark47.66RNA splicingDdx41, Ddx46, Ddx5, Khsrp, Lsm5, Prpf39, Prpf4b, Prpf40b, Wdr83, Luc7l3, Rbm396.27regulation of transcriptionSetd1b, Spen, Smarcc2, Sfmbt2, Tbx6, Tarbp2, Taf1a, Taf1c, Taf1d, Tmf1, Tgif14.7regulation of GTPase activityAcap3, Git1, Rictor, Arhgap27, Arhgap6, Tbc1d2, Tbc1d24, Tbc1d8, Tbc1d9, Acap14.12tRNA metabolic processMto1, Farsa, Polg2, Pop1, Pusl1, Qtrt1, Qtrtd1, Trmu, Trpt1, Yars2, Vars23.59helicase activityDdx55, Ddx5, Dhx16, Dqx1, Fancm, Ino80, Recql4, Upf1, Atrx, Chd7, Eif4a13.46structural constituent of ribosomeFlt3l, Mrpl24, Mrps10, Mrps18b, Rpl9, Rpl11, Rpl36, Mrpl23, Rpl5, Rps27a, Uba522.62histone modificationHuwe1, Kdm6b, RNF8, Baz2a, Brd8, Carm1, Ezh2, Hdac10, Ing4, Irf4, Map3k122.49tRNA metabolic processCog3, Cdk5, Exoc1, Gga3, Gdi1, Hook2, Htt, Sft2d1, Ipo4, Lax1, Macf12.15transcription repressor activityZglp1, Ikzf4, Nab2, Phf12, Scai, Spen, Tgif1, Atf3, Ahrr, Atxn1, Foxp41.85activation of protein kinase activityTraf7, Cerk, Cish, Dgka, Dgkh, Dgkq, Ercc6, Irak2, Pick11.45A !96 Table 3 (contÕd) B    GO TermGene NameEnrichment ScoremitochondrionOxct1, Oxsm ,Ogg1 ,Acly, Atpif1, Atp6v1a, Bad, Cmc1, Cox11, Cox16, Rab3d6.8mitochondrial inner membrane Cpt2, Cox5a, Glud1, Hadh, Mpst, Mgst1, Nnt, Tomm22, Vdac3, Sfxn2, Ucp26.8apoptosisArf6, Bcl7c, Bcl2a1a, Bcap31, Bmf, Bad, Bag3, C1d, E2f1, E2f2, Htatip25.87induction of apoptosisBad, Cd3e, Cd5, Fcgr3, Ndufa13, Pycard, Rab27a, Casp7, Casp9, Dapk14.57ribosomeItgam, Mrp63, Mrpl14, Mrpl27, Mrpl46, Mrpl48, Mrpl51, Rsp3, Rsp9, Rpl7, Uba523.93translationEef1b2, Eif2ak2, Itgam, Lars2, Mrp63, Mrpl14, Rps3, Rps9, Rpl38, Tarsl2, Rps233.93cofactor metabolic processOxsm, Pgls, Acly, Acot10, Alas1, Blvra, Ehhadh, Fh1, Ggt7, G6pdx, Gclm3.19positive regulation of lymphocyte activationBad, Cd3e, Cd38, Cd5, Cd74, Cd80, Hlx, Ikzf1, Ada, Bloc1s3, Cdkn1a3.13positive regulation of lymphocyte proliferationCd38, Cd80, Ada, Cdkn1a, Itgal, Il12b, Il15, Il18, Spn, Pnp, Tlr43.13electron transport chainNdufa11, Ndufa5, Ndufa7, Ndufc2, Ndufs5, Cyba, Cybb, Cyb5, Enox2, Fads3, Fdx12.62lymphocyte activationIl12b, Il15, Lcp1, Ms4a1, Msh2, Prdx2, Pik3r1, Ptpn22, Spn, Ccnd3, Slc11a12.4B cell activationCd79a, Gapt, Ikzf1, Aicda, Ada, Igbp1, Lrrc8a, Ms4a1, Msh2, Pik3r1, Skap22.4positive regulation of cytokine productionCd14, Cd3e, Ddx58, Fcer1g, Nlrp3, Pycard, Il18, Panx1, Slc11a1, Tlr4, Tlr72.28regulation of protein transportArfip1, Nlrp3, Pycard, Gsk3b, Lst1, Lcp1, Ltb, Panx1, Prdx1, Txn1, Trip62.18GTPase activityRab19, Rab20, Rab27a, Rab34, Rab3d, Rab8b, Rap2b, Rap2a, Rac2, Rit11.98immune responseCd14, Fcgr3, Nlrp3, Pycard, Acvr1, Chst2, Ccl22, Ccl24, Ccl5, Cxcl3, Tlr41.9regulation of protein kinase cascadeFgd4, Adrb2, Cat, Cav1, Fgfr1, Gab1, Grb2, Hcls1, Lst1, Ltb, Socs21.47!97 A  Table 4. GO terms associated with genes deregulated by TCDD in the rat primary B cells. A. Significantly enriched (enrichment score&1.3) GO biological processes identified for genes up-regulated by TCDD treatment. B. Significantly enriched (enrichment score&1.3) GO biological processes identified for genes down-regulated by TCDD treatment (DEGs; |fold-change| %1.5).      GO TermGene NameEnrichment Scorecell cycleFbxo31, Mad2l2, Ruvbl1, Taf10, E2f1, Anp32b, Apbb1, Avpi1, Bod1, Cdkn1a, Cdkn3, Ing4, Mapk3, Tgfb15.62ribosome biogenesisEif6, Mina, Rrp1, Rpl11, Rpl26, Rpl5, Psmb6, Psmb9, Ppp1cc, RPL24, Siah2, Psmc4, Phgdh, Nudc, Pes1, Psmb34.25protein complex assemblyDecr1, Atpif1, Cd74, Clpp, Ndufaf3, Ndufs5, Ndufs7, Ndufs8, Taf10, Aldoc, Calr, Cuta, Cyba, Gsn, Hba-a2, Hsd17b10, Ipo13, Mif, Med16, Mapk3, Pak13.64DNA repairOgg1, Mus81, Mpg, Rad23a, Apbb1, Ercc1, Kif22, Neil1, Nsmce1, Nhej1, Nthl1, Nudt1, Pttg1, Pold2, Pold4, Pnkp, Rfc4, Gtf2h5, Hmgn1, Hmgb13.27oxidative phosphorylationAtp5g1, Atp5g3, Atp5g3, Atp5d, Atp5e, Atp6v0c, Atp6v0b, Ndufa7, Ndufb8, Fxn, Atp5h, Atp5g2, Atp5j, Uqcrb, Uqcrc1 3.06response to bacteriumBcl3, Cd14, Jund, Akirin2, Adh5, Aldh2, Aldoa, Ccl5, Ccr1, C5ar1, Cyp1a1, Gpx1, Hsf1, Hist1h2bc, Il1b, Il1rn, Lyz2, Mif, Mapk3, Prdx2, Socs1, Rela3.04apoptosisBak1, Bag1, Bag3, E2f1, Ndufa13, Pycard, Siva1, Taf10, Aldoc, Apbb1, Aph1b, Cib1, Dad1, Dnase2a, Diablo, Fis1, Gsn, Gpx1, Gadd45g, Mtch12.65response to oxidative stressAtox1, Btg1, Ndufa6, Ndufs2, Ndufs8, Park7, Ada, Gpx1, Hmox1, Ldha, Mmp14, Neil1, Prdx2, Prdx5, Pebp1, Pnkp, Psmb5, Gpx4, Txn2, Rela 2.5chronic inflammatory responseCcl5, Il1b, Il1rn, Ahcy, Unc13d2.19carboxylic acid catabolic processKlf4, Ada, Alad, Asl, Bckdhb, Dad1, Fads1, Gsn, Gpx1, Hmox1, Il1b, Ldha, Mif, Rara, Scamp3, Ahcy, Kpna2, Slc27a4, Txn2, Tgfb1, Rela2.17mitochondrial respiratory chainNdufaf3, Ndufs5, Ndufs7, Ndufs81.92protein transportArf5, Arfgap1, Atg4b, Atg4d, Bcl3, Cd3g, Cd74, Gipc1, Grpel1, Kdelr1, Gnptg, Rab13, Rab34, Sec61g, Snf8, Ap2s1, Calr, Chml, Chmp2a1.9DNA catabolic processOgg1, Mpg, Dnase2a, Ercc1, Nthl1, Bax, Xpa1.62immune system developmentBcl3, Bak1, Cd3d, Cd74, Ada, Ccr1, Dnase2a, Ercc1, Hba-a2, Rps19, Il15, Nhej1, Prdx2, Rogdi, Bax, Rps14, Tgfb1, Zap701.32negative regulation of cell sizeNdufa13, Apbb1, Cfl1, Cdkn1a, Eno1, Ei24, Mylpf, Ntn1, Osgin1, Plxna3, Tgfb11.3!98 Table 4 (contÕd) B    GO TermGene NameEnrichment ScorephosphorylationPdpk1, Adam10, Adam9, Aak1, Atp6v1a, Bcl2, Bmp2k, Cdc42bpa, Erc1, Fgd4, Jak1, Jak210.9regulation of transcriptionArid4a, Bcl6b, Bclaf1, Brip1, Bach1, Bmi1, Ctcf, Cnot2, Cnot6, Crebbp, Dnmt3a, Ep300, E2f3, Elf1, Elf4, Elk4, Esf1, Fli19.39negative regulation of gene expressionGzf1, Klf12, Mdm4, Nkrf, Rest, Rybp, Satb1, Setd8, Skil, Smad3, Smad4, Atxn1, Brca1, Cenpf, Cbx1, Dicer1, Epc18.85MAPKKK cascadeJak2, Traf6, Arrb1, Frs2, Itpkb, Insr, Lrrk2, Lpar1, Lpar2, Met, Mapk1, Map2k1, Map2k4, Map2k7, Map3k1, Map3k2, Mapk87B cell differentiationBcl11a, Bcl2, Dclre1c, Pou2f2, Sp3, Malt1, Pik3r1, Prkdc, Ptprc, Rbpj, Tpd526.88regulation of Rho protein signal transductionFgd4m, Fgd6, Arhgef6, Arhgef12, Sos1, Als2, Bcr, Dnmbp, Itsn2, Lpar1, Lpar2, Rictor, Sos2, Spata13, Tsc15.5histone acetylationBmi1, Crebbp, Ep400, Phf15, Sap130, Taf1, Taf5l, Brpf3, Epc1, Ing5, Mecp2, Mettl85.31regulation of locomotionAdam10, Adam9, Abhd2, Apc, Amot, Ccr2, Cxcl10, Col18a1, Igf1r, Itgb3, Mmp9, Mapk1, Nf1, Pten, Pik3r1, Prkca, Rbpj,4.93regulation of apoptosisAtp7a, Bcl2, Bard1, Bmi1, Cflar, Cd28, Dnajc5, Jak2, Notch1, Rasa1, Rrn3, Rock1, Smad3, Stil, Traf6, Acvr1b, Apc, Apaf13.91positive regulation of protein kinase cascadeCflar, Jak2, Taok3, Traf6, Casp8, Ccr2, Chuk, Hipk2, Igf1r, Itgav, Itgb3, Lif, Lpar1, Mier1, Malt1, Ptprc, Rictor, Tlr43.72protein ubiquitinationBmi1, G2e3, Hectd1, Traf6, Ubxn7, Cdc73, Cul5, Itch, Map3k1, Ubr5, Rbbp6, Rnf144b, Socs7, Trim33, Ube2d13.38regulation of cytoskeleton organizationEp300, Mycbp2, Rasa1, Rock2, Smad3, Apc, Ccdc88a, Clasp2, Dlg1, Edn1, Xpo1, Prox1, P2rx7, Rictor, Fmn1, Sorbs33.38protein transportAbca1, Erc1, Jak2, Rabif, Rab14, Sdad1, Sft2d2, Sec23a, Cenpf, Cep290, Cxcl10, Ccdc91, Exoc2, Xpo1, Xpo7, Gsk3b3.05regulation of MAPKKK cascade Apc, Ccr2, Edn1, Hipk3, Insr, Igf1r, Itgav, Itgb3, Lif, Lpar1, Map2k4, Map3k1, Nf1, Ncor1, Ptprc, P2rx7, Sorbs3, Tlr42.88proteolysisLnpep, Mmp9, Mbtps2, Map3k1, Malt1, Phka2, Herc6, Pja2, Psen1, Ubr5, Pias2, Cyld, Usp12, Socs2, Tbl1x, Usp252.54protein deubiquitinationBrcc3, Atxn7, Mysm1, Otud7b, Usp12, Usp7, Usp9x, Uck22.36JNK cascadeFgd4, Rb1cc1, Traf6, Dusp19, Mapkbp1, Map2k4, Map2k7, Map3k1, Mapk8, Map3k5, Trib12.13immune effector processBcl2, Pou2f2, Samhd1, Dlg1, Lig4, Lyst, Prkdc, Ptprc, Rsad2, Tlr4, Tlr7, Tlr92.12RNA transportKhsrp, Casc3, Ckap5, Xpo1, Xpot, Hnrnpa3, Nxt2, Nup155, Nup160, Nup214, Nup35, Thoc2, Tsc12.07DNA methylationCtcf, Dnmt3a, Gramd4, Atf7ip, Baz2a, Mbd4, Piwil2, Trdmt12.06!99    Table 5. GO terms associated with genes deregulated by TCDD in the mouse primary B cells. A. Significantly enriched (enrichment score&1.3) GO biological processes identified for genes up-regulated by TCDD treatment. B. Significantly enriched (enrichment score&1.3) GO biological processes identified for genes down-regulated by TCDD treatment (DEGs; |fold-change| %1.5).  GO TermGene NameEnrichmet Scorecation bindingNt5c2, Atp7b, Atp1b2, Fxyd2, Zglp1, Glis2, Tiparp, Adam15, Bco2, Catsper2, Cit3.73regulation of transcriptionE2f2, Zglp1, Glis2, Cand2, Ccna2, Etv5, Nfia, Gm14325, Gm6712, Etohi1, Zfp3453.73membrane fractionAtp7b, H2-Ke6, Ccnb2, Cyp1a1, Cyp1b1, Jag2, Pstpip2, Ptgs2, Slc26a6, Slc8a1, Vamp12.26cell cycle processAnln, Cit, Ccna2, Ccnb2, Ccnd1, Ccnf, Fam83d, Mns11.98!"#$%&'$()*+*),Gbp9, Gbp4, Gbp6, Gbp8, Gbp101.94cell adhesionCd226, Arhgap6, Amica1, Itgal, Itgb3, Pxn, Pstpip1, Thbs1, tp1b21.52-,./01(,)&'$()*+$)*12Cd27, Cd3d, Itgal, Jag2, Shb1.3GO TermGene NameEnrichmet ScoremitochondrionAtp5d, Rab24, Amacr, Clpp, Ctsd, Cox5a, Dhrs4, Fth1, Glrx2, Gpx1, Mpst2.83immune responseCcl3, Ccr2, Ccr5, Cxcl10, Fth1, Lif, Prg4, Rsad2, Sbno2, Ticam2, Tgfb12.31cytokine bindingCcr5, Ccr2, Csf1r, Il12rb2, Tnfrsf1a, Tnfrsf42.02proteolysisFbxl8, Isg15, Pycard, Senp8, Bace1, Clpp, Casp4, Ctsd, Cbx4, C1ra, Cand21.55protein dimerization activityJund, Junb, Pycard, ATF7, Bhlha15, Csf1r, Klrb1b, Pvrl2, Klrb1c, Slc27a1, Ticam21.32vesicle-mediated transportRab34, Arf6, Sh3bp4, Clta, Chic2, Epn1, Ldlr, P2rx7, Rabgef1, Stx16, Trappc5 1.3Golgi apparatusB3gnt9, Acer2, Bace1, Chst2, Clta, Chic2, Asap2, Golm1, Atl1, Stx16, Rab341.3A                B   !100  A                                           B                                                      C  Figure 22. Heat map representing fold-change of gene expression of the suite of genes associated with top over-represented GO terms. A. Orthologous genes differentially expressed in response to TCDD within the ÒImmune ResponseÓ GO term. B. Differentially expressed orthologous genes within the ÒMitochondrionÓ GO term. C. Orthologs differentially expressed within the ÒRegulation of transcriptionÓ GO annotation term. Overexpressed functions within differentially expressed datasets were investigated using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v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able 6. KEGG pathways associated with genes differentially expressed by TCDD treatment in primary mouse, human and rat B cells. A. Pairwise comparison of canonical KEGG pathways enriched in human, mouse and rat B cells. B. KEGG canonical pathways enriched in all three species.   Human-Mouse (16)Mouse-Rat (13)Human-Rat (5)Cytokine-cytokine receptor interactionAdherens junctionErbB signaling pathwayCytosolic DNA sensing pathwayFc gamma R mediated phagocytosisGap junctionEndocytosisHomologous recombinationGnRH signaling pathwayEther lipid metabolismLong term potentiationRegulation of actin cytoskeletonGlycerolipid metabolismN glycan biosynthesisWnt signaling pathwayGlycerophospholipid metabolismNeurotrophin signaling pathwayHematopoietic cell lineageNon homologous end joiningLeukocyte transendothelial migrationNotch signaling pathwayLysosomePhosphatidylinositol signaling systemMetabolism of xenobiotics by Cyp450RNA degradationPPAR signaling pathwayRibosomePyrimidine metabolismType II diabetes mellitusSphingolipid metabolismmTOR signaling pathwaySystemic lupus erythematosusTryptophan metabolismTyrosine metabolismPathways common in three species (18)ApoptosisB cell receptor signaling pathwayCell cycleChemokine signaling pathwayFc epsilon RI signaling pathwayFocal adhesionInsulin signaling pathwayJak/STAT signaling pathwayMAPK signaling pathwayNOD-like receptor signaling pathwayNatural killer cell mediated cytotoxicityPathways in cancerPurine metabolismRIGI-like receptor signaling pathwayT cell receptor signaling pathwayToll-like receptor signaling pathwayVEGF signaling pathwayp53 signaling pathwayA !102 3.2.3 Temporal gene expression changes in the naive primary mouse, human and rat B cells following TCDD exposure  Enrichment analysis was performed on the differentially expressed gene sets at each time point to access TCDD-mediated time-dependent changes in biological and molecular functions. In the human dataset, biological processes related to fatty acid metabolic process, regulation of small GTPase mediated signal transduction, regulation of JNK cascade and Fc epsilon RI signaling pathway showed enrichment at 4h followed by enrichment in chemotaxis, immune response and cytokine-cytokine receptor interaction processes at 8h post-treatment. At 12h regulation of cell cycle and oxidative phosphorylation were enriched.   In the mouse, biological processes involved in apoptosis, RNA processing, phosphorylation, chronic myeloid leukemia, pathways in cancer and acute myeloid leukemia KEGG pathway were enriched at 4h post TCDD treatment. Interestingly, regulation of cell proliferation, regulation of lymphocyte activation and cytokine-cytokine receptor interaction were enriched at 8h time point. KEGG pathways associated with tryptophan metabolism and keratan sulfate biosynthesis were enriched at 12h but not at the earlier time points.   In the rat, biological functions associated with phosphorylation, regulation of transcription, and B cell differentiation and activation demonstrated peak enrichment at 4h; KEGG pathways associated with ribosome, oxidative phosphorylation and chronic myeloid leukemia were enriched only at the earliest time point. At 8 hours regulation of cell development, positive regulation of cell cycle, immune response and pathways in cancer were significantly enriched. At 12h, regulation of Rho !103 protein signal transduction and intracellular signaling cascade were significantly enriched. None of the biological processes remained enriched during the entire time-course, indicating the gene expression changes and changes in cell function associated with them are transient in nature.   3.3 Genome-wide responses of the PWM-activated human, mouse and rat primary B cells to TCDD treatment 3.3.1 Gene expression changes in PWM-activated primary mouse, rat and human B cells following TCDD treatment Human datasets were filtered using a |fold-change| &1.5 and P1(t) & 0.8 criteria while mouse and rat data sets were filtered using |fold-change| & 1.5 criterion. No P(t) value was calculated for the mouse and rat data since the samples were pooled from 20 mice and 5 rats. Analysis of the time-course RNA-Seq results identified TCDD-elicited differential expression of 544 human, 2527 mouse, and 772 rat genes over the 24-h time period.  In the mouse dataset approximately the same number of genes were up- or down- regulated in response to TCDD (Figure 23A). In the human dataset slightly more genes were up-regulated in response to TCDD at 8h and twice as many genes were up-regulated than down-regulated at 24h. By contrast, in the rat dataset significantly more genes were down-regulated than up-regulated at 8 and 24h. Specifically, up-regulation was observed for 1493 mouse, 308 rat, and 338 human genes; down regulation was observed for 1451 mouse, 523 rat and 217 human genes (Figure 23A). Comparison of the top ten differentially expressed genes !104 A   Figure 23. Number of mouse, rat and human orthologs differentially expressed in response to TCDD. (A) Total number of mouse, rat and human orthologs differentially expressed at least at one time-point during the time-course. (B) Total number of orthologs up- or down-regulated at each time point in response to TCDD treatment. Human datasets were filtered using a |fold-change| %1.5 and P1(t) % 0.8 criteria while mouse and rat data sets were filtered using |fold-change| % 1.5 criterion.      Number of DEGs (Mouse)4h Up4h Down8h Up8h Down24h Up24h Down02004006008004h Up4h Down8h Up8h Down24h Up24h Down0100200300Number of DEGs (Rat)Number of DEGs (Human)4h Up4h Down8h Up8h Down24h Up24h Down050100150200!105 among the three species, revealed one common up-regulated gene - CYP1A1 but no common down-regulated genes (Table 7). Additionally, immunoregulatory genes Il22 and Pde4c were up-regulated in mouse and rat, AHRR and CYP1B1 orthologs in mouse and human, and PACSIN3 in the human and rat datasets. Conversely, only human and mouse datasets shared a down-regulated ortholog, TMEM40 (Table 7). All data is deposited on Gene Expression Omnibus (GEO; accession number GSE80953).  Mapped orthologs were analyzed using HID (HomoloGene ID; HID build 67) in order to compare TCDD-induced differential expression of equivalent genes across human, rat and mouse primary activated B cells. The HomoloGene database contains 18,981 human, 21,766 mouse and 19,229 rat unique HIDs, of which 15,816, 16,461 and 14,145 HIDs were expressed in primary B cells. In the time course study, 30 nM TCDD elicited the differential expression of 515 human, 2371 mouse, and 712 rat orthologs. 28 orthologs were identified as differentially expressed in response to TCDD in all 3 species. The majority of orthologs exhibited species-specific expression. Specifically, 73% (379/515) of human, 81% (1935/2371) of mouse, and 50% (350/712) of rat orthologs exhibited species-specific differential expression (Figure 23B). Ten of the 28 orthologs differentially expressed in all 3 species, exhibited a comparable expression pattern (Table 8). This included the induction of prototypical AhR battery genes such as CYP1A1, TIPARP, AHRR, as well as induction of CD27, a memory B cell marker, the actin binding gene, MTSS1L, the MYO6 gene that plays a role in intracellular vesicle and organelle transport, and the potassium-dependent sodium/calcium exchanger, SLC24A3. !106  Table 7. Top ten genes differentially up- or down-regulated in response to TCDD treatment in mouse, human and rat primary B lymphocytes activated by PWM. The maximum fold of induction/suppression is indicated in brackets.   UP-REGULATEDDOWN-REGULATED4h (Fold)8h (Fold)24h (Fold)4h (Fold)8h (Fold)24h (Fold)MouseCyp1a1 (31.54)Cyp1a1 (20.3)Cyp1a1 (14.8)Arhgef5 (-3.12)Cldn15 (-3.15)Sh2d1a (-3.15)Cyp1b1 (7.39)Ahrr (5.36)Unc5b (5.56)Rab13 (-3.12)Stk30 (-3.15)Styx (-3.15Serpinb2 (5.68)Nap1l3 (4.78)Vdr (4.7)Trim46 (-3.12)Sfmbt2 (-3.15)Eva1b (-3.2)Ahrr (4.8)Cyp1b1 (4.39)Efcab12 (4.7)Zfp957 (-3.12)Ccdc92 (-3.15)Fasl (-3.55)Gpnmb (4.54)Tvp23a (3.99)Apol10b (4.26)Myo5b (-3.12)Gm5814 (-3.15)H2-Bl (-3.55)Sh2d1a (4.39)Rtn4rl1 (3.91)Pde4c (3.53)Sirpb1c (-3.12)Ckb (-3.15)Gnb5 (-3.55)Thbs1 (4.39)Fam229b (3.59)Il22 (3.46)Gm7102 (-3.12)Spon1 (-3.15)Cd70 (-3.95)Col7a1 (4.12)Jag2 (3.59)Cd8b1 (3.36)Pkp2 (-3.12)Astl (-3.15)Msrb3 (-3.95)Rab4a (3.7)Psd2 (3.59)Trim34b (3.36)Slc38a8 (-3.12)Sgms2 (-3.15)Tmem151b (-3.95)Smox (3.6)Sec31b (3.59)Mcf2l (3.36)Tmem40 (-3.84)Ccne1 (-2.85)Gnaz (-3.95)RatCyp1a1 (2.71)Acoxl (2.39)Spred3 (1.87)Tmem14a (-1.82)Gstcd (-2.3)Cebpb (-1.8)Pik3r3 (2.36)Hey2 (2.19)Stc2 (1.87)Tbx6 (-1.82)Fam222a (-2.45)Itgb3 (-1.8)Itgb1bp2 (2.24)Lrrc25 (2.09)Cubn (1.75)Cldn7 (-1.82)Chchd10 (-2.62)Dnlz (-1.8)Fcnb (2.24)Pex19 (2.09)Il22 (1.75)Hhat (-1.95)Scand1 (-2.7)Efcab4b (-1.82)Rab12 (2.2)Dna2 (1.92)Pde4c (1.75)Rps19 (-1.95)Socs1 (-2.93)Fscn3 (-1.82)Lst1 (2.05)Cuedc1 (1.87)Bricd5 (1.75)Clvs1 (-1.95)Spr (-2.97)Bola1 (-1.92)Garnl3 (1.99)CLIC5 (1.87)Dcn (1.75)Cmklr1 (-2.07)Rpl13 (-3.01)Rpl13 (-1.96)Ifnb1 (1.99)Spred3 (1.87)Itgb5 (1.75)Ctbp2 (-2.07)Bloc1s3 (-3.36)Card9 (-2)Mras (1.91)Dyrk3 (1.87)Prrt1 (1.75)Ppm1m (-2.09)Nudc (-3.56)Rimbp3 (-2.16)Prkar1b (1.86)Scn4a (1.84)Pacsin3 (1.74)Mxd3 (-2.31)Cox8a (-4.33)Cox8a (-2.24)HumanCYP1B1 (8.49)ARL4C (4.05)AHRR (14.24)SLC13A3 (-2.5)CATSPER2 (-2.9)PEG3 (-3)CD93 (5.41)C15orf26 (3.97)CYP1A1 (10.17)GREM1 (-2.56)CCDC171 (-2.94)CCNA2 (-3.02)STEAP4 (4.11)ITGA1 (3.73)ADGRB2 (5.73)HUNK (-2.56)SLC28A2 (-3.03)KLC3 (-3.08)EREG (3.47)KIF5A (3.67)ASB2 (4.88)COL15A1 (-2.56)CD300A (-3.05)ATP10A (-3.14)SLC35G5 (3.19)BIK (3.62)RARRES2 (4.7)TSACC (-2.63)XYLB (-3.12)CACNB4 (-3.23)SFN (2.83)SHISA8 (3.34)GRIN2D (4.61)ZC3HAV1L (-2.63)SLC5A9 (-3.19)UBE2QL1 (-3.25)SNORA5A (2.97)ADGRB2 (3.33)GPR68 (4.22)MCEMP1 (-2.61)PLEKHH2 (-3.13)C2orf74 (-3.27)TMPRSS9 (2.90)MTFR2 (3.19)PACSIN3 (4.18)MST1 (-2.74)FPR2 (-3.14)DUSP15 (-3.39)CLNK (2.87)MYO7B (3.18)CLCN2 (4.08)PROKR1 (-2.81)NRTN (-3.56)SPNS3 (-3.61)SHISA4 (2.78)SATB2 (3.15)ADAM11 (4.04)SLC16A14 (-3)TAS2R4 (3.62)TMEM40 (-4.04)!107  Table 8. Conserved, TCDD-Induced ortholog differential expression. The time point of the maximum fold change is indicated in brackets. Mouse, rat and human data sets were filtered using fold change cutoff of 1.5 and, for human dataset, P1(t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alcium-binding protein, CABYR, CHRM4 involved in regulation of the actin skeleton, and a G protein-coupled receptor, P2RY13, genes were down-regulated in all 3 species. However, most of the orthologs (64%) differentially regulated by TCDD in all three species, exhibited divergent expression. For example, the transcriptional repressor, Hic1, Rnd2 involved in disassembly of actin and cell surface signaling, and Itgb3 were induced in mouse and human but suppressed in rat primary B cells. The actin binding protein, Fscn2, and Pacsin, involved in endocytosis, were induced in human and rat but suppressed in the mouse.  Additionally, transcriptional repressor, Hey2, and Map6, involved in microtubule stabilization, were induced in rodents but suppressed in human.  Pairwise comparisons identified 86 common differentially regulated human-mouse orthologs and 12 common human-rat orthologs. Interestingly, the kinetics of gene expression change in response to TCDD was different between mouse and human resulting in 45 common up-regulated and 21 down-regulated orthologs at least at one time point. Among the 12 common human-rat orthologs 6 were down-regulated, 4 up-regulated and 2 displayed divergent gene expression changes (Figure 24 A and B). No significant correlation between any two species was identified by pairwise comparison (Figure 24C).  The functional relevance of 86 common mouse and human significantly changed orthologs was examined using Database for Annotation, Visualization and Integrated Discovery (DAVID). Results for the top 25 gene ontology (GO) biological functions (which includes 3 classes: molecular function, biological processes and cellular components) are displayed in  table 9. The three top GO terms include extracellular matrix, integrin-mediated  !109  Figure 24. Pairwise analysis of differentially expressed orthologs.  Heat maps of the 86 common human-mouse (A) and 12 human-rat (B)orthologs deregulated by TCDD at one or more time-points. (C) Correlation of time-dependent gene   !110 Figure 24 (contÕd) expression changes between mouse and human and human and rat. Genes were filtered using fold change cutoff of 1.5 and, for human dataset, P1(t) > 0.8.   !111  Table 9. Gene ontology analysis of the 86 common human-mouse DEGs. The functional relevance of 86 common mouse and human significantly changed genes was examined using Database for Annotation, Visualization and Integrated Discovery (DAVID).  Results for the top 25 gene ontology (GO) biological functions (which includes 3 classes: molecular function, biological processes and cellular components) are shown. Genes were filtered using fold change cutoff of 1.5 and, for human dataset, P1(t) > 0.8.    GO IDGO TermFisher p-valueGO:0031012extracellular matrix1.50E-04GO:0044421extracellular region part2.90E-04GO:0007229integrin-mediated signaling pathway3.30E-04GO:0005509calcium ion binding7.40E-04GO:0043394proteoglycan binding1.50E-03GO:0005178integrin binding2.40E-03GO:0005578proteinaceous extracellular matrix3.20E-03GO:0007155cell adhesion5.40E-03GO:0008201heparin binding1.10E-02GO:0005518collagen binding1.20E-02GO:0001948glycoprotein binding1.20E-02GO:0044420extracellular matrix part1.60E-02GO:0030246carbohydrate binding2.20E-02GO:0005539glycosaminoglycan binding2.60E-02GO:0030247polysaccharide binding3.30E-02GO:0001871pattern binding3.30E-02GO:0032989cellular component morphogenesis3.90E-02GO:0032403protein complex binding6.00E-02GO:0009986cell surface7.30E-02GO:0043066anti-apoptosis7.40E-02GO:0005871kinesin complex7.90E-02GO:0007050cell cycle arrest8.50E-02GO:0009968negative regulation of signal transduction8.70E-02GO:0019200carbohydrate kinase activity8.70E-02GO:0000902cell morphogenesis8.80E-02!112 signaling pathway and calcium ion binding.  3.3.2 Functional annotation and pathway enrichment of genes differentially expressed in response to TCDD in activated mouse, human and rat primary B cells In order to determine functional relevance of DEGs the 2527 mouse, 772 rat, and 544 human significantly changed genes were analyzed for overrepresentation within specific functional categories and canonical pathways using the DAVID analysis tool. The top enriched pathways and biological functions and processes specific to mouse, rat and human primary B cells are presented in figures 25 and 26.   The number of overrepresented GO terms (DAVID) was comparable (311 mouse, 294 rat and 184 human enriched functional categories) in the time course data sets. The enriched categories were compared between species identifying 41 common enriched categories including cell adhesion, extracellular region and the inflammatory response. Species-specific enriched functions included chemotaxis and positive regulation of MAP kinase activity in mouse primary B cells, mitochondrion and oxidation reduction in rat primary B cells, and integrin binding and cell-cell signaling in human primary B cells. Importantly, the number of genes within functional categories varied among species and direction of gene changes was frequently discordant between species despite the consistent phenotypic consequence of TCDD exposure Ð suppression of the IgM response (Figure 27). For example, genes involved in cell adhesion, CCR1 and DST, were induced in human but suppressed in mouse and rat.  Conversely, genes involved in cell differentiation   !113  Figure 25. Functional annotation of the differentially expressed genes in response to TCDD in primary human, mouse and rat B cells activated with PWM. (A) Enriched GO terms in response to TCDD treatment in primary mouse, rat and human B cells. (B) Heat map of DEGs associated with immune response differentially expressed in response to TCDD in mouse, rat and human B cells.   A B !114   Figure 26. List of KEGG biological pathways associated with genes differentially expressed in response to TCDD in primary mouse, rat and human B cells. A. Top pathways associated with mouse, human and rat DEGs. B. Heat map displays differential expression of genes within common human, mouse and rat pathways in response to TCDD. Time of the highest fold change is indicated.   !115 BOC, RUNX2, COL18A1, and MYO6 were up-regulated in all three species (Figure 25B).  TCDD-induced differential gene expression mapped to 34 mouse, 25 rat and 27 human KEGG canonical pathways, among which 5 were common between mouse, human and rat (Table 10A, B and C). Top ranking mouse, rat and human pathways are presented in figure 26. Pathways significantly enriched in all three species included ECM-receptor interaction, focal adhesion and regulation of actin cytoskeleton. Mouse-specific differential gene expression was associated with acute myeloid leukemia and toll-like receptor signaling pathway. In the rat primary B cells differentially expressed genes mapped to oxidative phosphorylation and mTOR signaling pathway. Majority of the genes within enriched rat-specific pathways were down-regulated including mitochondrial ATP synthase subunit Atp5d, subunits of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) Ndufab1, Ndufb2 and Ndufv3 and nuclear-coded polypeptide chains of cytochrome c oxidase Cox8a, Cox6a1 and Cox5a. In human primary B cells the specific significantly enriched canonical pathways included ribosome and metabolism of xenobiotics by cytochrome pathway (Table 10A, B and C).  Importantly, genes within common enriched pathways frequently displayed divergent pattern of deregulation.  3.3.3 Temporal gene expression changes in the PWM-activated primary mouse, human and rat B cells following TCDD exposure  Time course analysis of the data revealed that the standard AhR battery genes (i.e., CYP1A1, CYP1B1, NQO1, TIPARP) showed unusual and divergent !116    Figure 27. Effect of TCDD on the PWM-induced IgM response in primary B cells. Primary human, mouse and rat B cells (1"105/ml) were activated with PWM (15 #g/ml) and treated with 30 nM TCDD or vehicle (0.02% DMSO).  Supernatants were harvested on day 5 (mouse rat) or 7 (human) of culture and analyzed for IgM by sandwich ELISA. Data from five human donors and five rats/mice were normalized to VH control (100%) and presented as percentage of control. Statistical signiÞcance was determined using DunnettÕs two-tailed t test; * represents values that are signiÞcantly different from VH control at p < 0.05.    Rat Primary B cells IgM response(percent of control)050100150VHTCDD*Human Primary B cellsIgM response(percent of control)050100150VHTCDD*Mouse Primary B cellsIgM response(percent of control)050100150VHTCDD*!117  Table 10. KEGG pathways enriched in PWM-activated human, mouse and rat primary B cells treated with TCDD. KEGG pathways associated with   KEGG PathwayP*Cytokine-cytokine receptor interaction1.90E-17Hematopoietic cell lineage1.90E-06Pathways in cancer1.20E-05ECM-receptor interaction1.70E-05Focal adhesion8.00E-05Dilated cardiomyopathy1.10E-04Chemokine signaling pathway2.50E-04Axon guidance4.80E-04Jak-STAT signaling pathway6.40E-04Adherens junction9.70E-04Basal cell carcinoma3.70E-03Regulation of actin cytoskeleton4.10E-03Complement and coagulation cascades5.30E-03Glycine, serine and threonine metabolism9.40E-03Colorectal cancer1.00E-02Cell adhesion molecules (CAMs)1.20E-02Phosphatidylinositol signaling system1.20E-02Small cell lung cancer1.90E-02Intestinal immune network for IgA production2.10E-02TGF-beta signaling pathway2.30E-02Natural killer cell mediated cytotoxicity2.60E-02Acute myeloid leukemia3.10E-02ABC transporters3.30E-02Cardiac muscle contraction3.60E-02Melanogenesis4.20E-02Glycerophospholipid metabolism4.60E-02Inositol phosphate metabolism4.70E-02NOD-like receptor signaling pathway5.50E-02Toll-like receptor signaling pathway6.90E-02Arginine and proline metabolism8.70E-02Biosynthesis of unsaturated fatty acids8.90E-02Primary bile acid biosynthesis9.00E-02Tryptophan metabolism9.00E-02T cell receptor signaling pathway9.50E-02A !118 Table 10 (contÕd) mouse (A), human (B) and rat (C) DEGs as identified by DAVID analysis. *p-value & 0.05 were considered significantly enriched     KEGG PathwayP*ECM-receptor interaction1.80E-03Regulation of actin cytoskeleton5.20E-03Focal adhesion9.20E-03ABC transporters1.30E-02Glutathione metabolism2.00E-02Metabolism of xenobiotics by cytochrome P4503.70E-02Neuroactive ligand-receptor interaction4.20E-02TGF-beta signaling pathway1.10E-01Pathogenic Escherichia coli infection1.20E-01p53 signaling pathway1.70E-01Tryptophan metabolism2.10E-01Bladder cancer2.20E-01Arachidonic acid metabolism3.30E-01Drug metabolism3.80E-01NOD-like receptor signaling pathway3.80E-01Melanoma4.40E-01Leukocyte transendothelial migration4.50E-01Cytokine-cytokine receptor interaction4.80E-01MAPK signaling pathway5.00E-01Calcium signaling pathway5.10E-01Small cell lung cancer5.30E-01Pathways in cancer5.40E-01Ribosome5.50E-01Chemokine signaling pathway5.60E-01Cell cycle7.50E-01Purine metabolism8.40E-01Huntington's disease9.00E-01B !119 Table 10 (contÕd)     KEGG PathwayP*Regulation of actin cytoskeleton2.70E-05Focal adhesion3.40E-04Renal cell carcinoma2.30E-03Cytokine-cytokine receptor interaction3.00E-03ECM-receptor interaction6.30E-03Bladder cancer7.20E-03Jak-STAT signaling pathway8.00E-03Cell adhesion molecules (CAMs)1.20E-02Tight junction1.30E-02Parkinson's disease2.40E-02mTOR signaling pathway3.40E-02Insulin signaling pathway3.70E-02Vascular smooth muscle contraction4.00E-02VEGF signaling pathway4.10E-02Pathways in cancer4.40E-02Oxidative phosphorylation4.50E-02Hematopoietic cell lineage5.10E-02Glycosphingolipid biosynthesis5.30E-02Aldosterone-regulated sodium reabsorption5.90E-02Huntington's disease6.20E-02Axon guidance7.00E-02ErbB signaling pathway7.10E-02Alzheimer's disease7.60E-02Type II diabetes mellitus8.60E-02Leukocyte transendothelial migration1.00E-01C !120 gene expression pattern (Figure 28). In many well-characterized cell models, for example HEPG2, activation of metabolizing enzymes CYP1A1 and CYP1B1 is robust and persistent. However, in primary human and mouse B lymphocytes CYP1B1 mRNA expression peaks as soon as 4h post-TCDD treatment and gradually decreases over time. Human CYP1A1 showed gradual time-dependent increase while in mouse and rat primary B cells Cyp1a1 expression peaked at 4h. Enrichment analysis was performed on the differentially expressed gene sets at each time point to access TCDD-mediated time-dependent changes in biological and molecular functions. In human, biological processes related to cell adhesion and cell-cell signaling showed high enrichment at 4h followed by a decline at 24h. Molecular functions associated with pattern binding and KEGG pathway associated with focal adhesion remained enriched during the time-course. In the mouse, biological processes involved in locomotory behavior were highly enriched at 4 and 8h post TCDD treatment while inflammatory response associated biological processes became enriched at later time points; KEGG pathway associated with cytokine-cytokine receptor interaction was enriched at all time points. In the rat, biological functions associated with cell adhesion and response to extracellular stimulus demonstrated peak enrichment at 4h; KEGG pathway associated with adhesion was enriched at all time points while Jak-STAT signaling pathway was significantly enriched only at the earliest time-point.  !121  Figure 28. Time-dependent, TCDD-elicited induction of the AhR ÒbatteryÓ genes. RNA-Seq evaluation of TCDD-mediated induction of the AhR responsive genes in primary human, mouse and rat B cells activated with PWM.   4h8h24h051015AHRR mRNA (Fold Change)HumanMouseRatCYP1B1 mRNA Levels(Fold Change)4h8h24h0246810HumanMouseRatNQO1 mRNA Levels (Fold Change)4h8h24h01234HumanMouseRatCYP1A1 mRNA Levels (Fold Change)4h8h24h010203040HumanMouseRatTIPARP mRNA Levels (Fold Change)4h8h24h01234HumanMouseRat!122 3.3.4 Identification of putative DREs in responsive genes Genomic sequences for genes presented in table 6 and figure 24 were examined for presence of putative DREs, which were identified by computational scanning. Of the 86 common mouse-human orthologs 85 mouse and 86 human orthologs contained a pDRE with an MSS & 0.8 (high scoring pDRE) while 29 mouse and 34 human orthologs contained pDREs with an MSS & 0.9 (very high scoring pDRE) within its genomic region or 10 kb upstream of the transcription start site. Between 12 human-rat orthologs 11 rat and 12 human contained ÒhighÓ scoring pDREs but only 3 rat and 6 human orthologs contained Òvery highÓ scoring pDREs. 27 out of 28 human and all mouse and rat common orthologs deregulated in the three species contained ÒhighÓ scoring pDREs; Òvery highÓ pDREs were mapped to 13 mouse, 10 rat and 10 human orthologs (Table 11). CD27, HIC1 and MTSS1L all possessed DREs within their promoter region and displayed an early induction response to TCDD treatment, suggesting that they are primary AhR-responsive genes.    3.3.5 Gene network analysis To discover potential mechanisms of TCDD-mediated suppression of B cell function, a subgroup of 114 common mouse and human orthologs deregulated by TCDD was evaluated with MetaCore. Thresholds of 1.5 (fold change) and P1(t) 0.8 were applied and dataset was analyzed with Compare Experiments followed by network building option ÒAnalyze Network (Transcription Factor)Ó, which incorporates  !123  Table 11. Number of putative DREs (pDREs) identified in the promoter region of the 27 common orthologs differentially expressed in all three species. 27 out of 28 human and all mouse and rat orthologs commonly deregulated in the three species contained ÒhighÓ scoring pDREs; Òvery highÓ pDREs  !"##"$%"&'(")"*+%,#"$*%-%+./01/+2))31*(4/&5%31*(2))31*(4/&5%31*(2))31*(4/&5%31*(!"##-6789-:7:77969;6:!<76:=>9=8>98:>$%&'!'799:?9799=799-=@ABB9C968>7?7>>-:7;>@DE?6:;:>=?-=>?97=>BC!7;2-8===7969>9=>77>AFG2HG7;9?96;>98977!2IDH7>:>7:9:>9:8>$"#()7-9;79>=7->777G7HD9-:?>?->9>>"*$'--969-?7-7-7989FAJI-?--6999789>-89?>IKL<6;-7-7:=;:9=;7:><CJ=6==9>-?7-99==:>;GCM<!789;7>977?:>9?==:7GHH9:9-99>9;9>>9=6>HL<77-9-79-9>9-997>B3N7-99>:;>->9;>BA2G7969>>:=>?-9:>NB!L79-6>9=:>;6-7>HKKG79:->9>=>9:8>G2!BFL-99;>998>989>>+,(!-.;778776989:--8=3KD77;9?>7798>9-8>@2G?=>-9>??-:>;=79>JGH9?>=:;:9:=>?;>G3C<29?99=7>:;9/012:==>99-98==99-?9-(3456#78"497:!124 Table 11 (contÕd) were mapped to 13 mouse, 10 rat and 10 human orthologs. Highlighted genes contained Òvery highÓ pDREs in all 3 species.    !125  Figure 29. Top implicated biological network containing common human-mouse orthologs significantly deregulated by TCDD exposure. A subgroup of 114 common mouse and human orthologs deregulated by TCDD was evaluated with MetaCore. Thresholds of 1.5 (fold change) and P1(t) 0.8 were applied and dataset was analyzed with Compare Experiments followed by network building option ÒAnalyze Network (Transcription Factor)Ó, which incorporates canonical pathways in its algorithm. The seed data is marked with red circles. For conventions and symbols, refer to MetaCore website legend description (https://ntp.niehs.nih.gov/ntp/ohat/diabetesobesity/wkshp/mc_legend.pdf).   !126 canonical pathways in its algorithm. The resulting most significant network is presented in figure 29. This network shows interaction of two key transcriptional regulators CREB1 that, if activated in B cells, promotes proliferation and survival, and AP-1 a transcription factor that had been shown to suppress B cell to plasma cell differentiation in primary mouse B cells by directly binding to and inhibiting PRDM1 (Grotsc et al., 2014).    3.3.6 Validation of differentially expressed genes at the mRNA level Differential expression of 13 genes in human primary B cells was validated by quantitative real-time PCR (Figure 30). In order to strengthen the comparison between RNA-Seq and qRT-PCR, PCR was carried out using the RNA samples that were used for RNA-Seq experiments plus two additional human donors. Two genes from AhR battery of genes (TIPARP and CYP1A1) and 11 genes deregulated in more than one species were chosen for confirmation. Differential expression was confirmed for all genes and the fold change calculated with the qRT-PCR data showed mostly a similar magnitude of change compared to the fold change calculated with the RNA-Seq data. qRT-PCR fold change was significantly greater than RNA-Seq fold change only for CYP1A1 gene; however, the direction of gene expression change was the same.     !127  Figure 30. PCR verification of the expression of select genes. Thirteen genes up-regulated by TCDD in the human primary B cells were validated by qRT PCR using the same set of RNA samples used in RNA-seq analysis plus samples from two additional donors. PWM-activated primary human B cells (1"106 cells/ml) were activated with PWM and treated with 30 nM of TCDD or 0.02% DMSO (VH) for indicated periods of time. Total RNA was isolated, and steady-state mRNA levels of  4 hFold ChangeSERPINB2LCKCD27Cyp1A1TIPARPMTSS1LRND2HIC1CD93ITGB3SHFFOSBBEND70246810qRT-PCRRNA-Seq8 hSERPINB2LCKCD27Cyp1A1TIPARPMTSS1LRND2HIC1CD93ITGB3SHFFOSBBEND702468102550Fold Change24 hSERPINB2LCKCD27Cyp1A1TIPARPMTSS1LRND2HIC1CD93ITGB3SHFFOSBBEND7048100200Fold Change!128 Figure 30 (contÕd) select genes were measured by TaqMan qRT-PCR and normalized to the endogenous 18S ribosomal RNA. Data are presented as fold change compared to the VH control group at corresponding time point. Human DEGs were validated by qRT-PCR using the same set of RNA samples used in RNA-Seq analysis plus two additional donors.    !129 3.4 Comparison of TCDD-induced genome-wide gene expression changes in naŁve and PWM-activated primary mouse, human and rat B cells and CH12.LX mouse cell line  We have previously reported the effect of TCDD on genome-wide changes in gene expression in the mouse B cell line CH12.LX identifying 78 genes as possible direct targets of TCDD (De Abrew et al., 2010). Here, comparative analysis was extended to include naŁve and PWM-activated mouse primary B cells and a set of 78 genes previously identified in the LPS-activated CH12.LX cells. Additionally, naŁve and PWM-activated human and rat primary B cell gene expression profiles were compared to the 78 genes identified in the CH12.LX cells.   A total of 25 genes were identified as differentially expressed in naŁve and PWM-activated primary human B cells by TCDD (Table 12). Only CYP1A1, CYP1B1 and TIPARP were deregulated in primary human B cells and CH12.LX cells (Figure 31). Interestingly a number of cell surface adhesion molecules such as ITGA3, ITGB3, ITGAL, THBS1 and AMICA1 were deregulated by TCDD in both naŁve and PWM-activated primary human B cells, indicating that TCDD might affect B cell migration, adhesion and co-stimulatory signaling.   Energy metabolism gene XYLB, adapter protein SHF, serpin peptidase inhibitor SERPINB2, peroxisomal membrane protein PXNP4, mitochondrial translational activator MSS51, serine/threonine protein kinase MAP3K6, immunoregulatory chemokine CXCL2, member of the TNF-receptor superfamily CD27, and cell cycle regulator CCNA2 were deregulated by TCDD in naŁve and !130 PWM-induced primary human B cells. Functional annotation of the 25 genes deregulated in both naŁve and activated human primary B cells identified over-represented functions associated with integrin-mediated signaling pathway, cell adhesion, regulation of cell migration and immune response (Table 13).   Comparison of the primary naive and PWM-activated mouse B cells and CH12.LX cell line identified 11 genes in common deregulated by TCDD (Figure 32). These included typical ÒAhR batteryÓ genes Ahrr, Cyp1A1, Cyp1B1, and Tiparp. The rest of the genes deregulated by TCDD in mouse B cell included Cbr3, a monomeric NADPH-dependent oxidoreductase that catalyzes the reduction of carbonyl compounds to their corresponding alcohols (Watanabe, 1998). Hivep3 (human immunodeficiency virus type 1 enhancer-binding protein 3) acts as a transcription factor and is able to regulate NF!B-mediated transcription by binding the kappaB motif in target genes involved in immunity, inflammation, and growth (Allen and Wu, 2004). This protein also binds the recombination signal sequence that flanks the V, D, and J regions of immunoglobulin and T-cell receptors and inhibits TNF$-induced NF!B activation. Ptgs2 cyclooxygenase (Cox2) the key enzyme in prostaglandin biosynthesis, which plays important roles in modulating motility, proliferation and resistance to apoptosis and associated with biologic events such as injury, inflammation, and proliferation was deregulated in both naŁve and activated primary mouse B cells and CH12.LX cells (Jones et al., 1993; Tazawa et al. 1994). Spint1, a serine protease inhibitor, involved in degradation of the extracellular matrix was also deregulated between three different experimental settings (Oberst et al., 2001;  !131   Figure 31. Venn diagram of the differentially regulated genes in response to TCDD in naŁve and PWM-activated human primary B cells and CH12.LX cells. Total number of human naŁve, human PWM-activated and mouse CH12.LX orthologs differentially expressed at least at one time-point during the time-course. Human datasets were filtered using a |fold-change| %1.5 and P1(t) % 0.8 criteria while mouse data set contained genes that are deregulated by TCDD and showed AhR binding within their promoter region.    !132  Table 12. Genes differentially regulated by TCDD in naŁve and PWM-activated primary human B cells. Highest fold-change of gene expression is indicated.    Gene SymbolGene NameNaŁvePWM-activatedALDH3B1aldehyde dehydrogenase 3 family, member B1-1.562.58AMICA1adhesion molecule, interacts with CXADR antigen 11.993.67C15orf61chromosome 15 open reading frame 61-1.961.90CCNA2cyclin A21.581.63CD27CD27 molecule1.722.81CXCL2chemokine (C-X-C motif) ligand 21.501.82F3coagulation factor III1.662.50FGL2fibrinogen-like 2-1.53-1.53HAAO3-hydroxyanthranilate 3,4-dioxygenase1.65-2.27HCAR3hydroxycarboxylic acid receptor 31.912.53HSPA4Lheat shock 70kDa protein 4-like1.972.03ITGA3integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor)-1.611.97ITGALintegrin, alpha L (antigen CD11A (p180)1.531.83ITGB3integrin, beta 3 (antigen CD61)1.541.98MAP3K6mitogen-activated protein kinase kinase kinase 6-2.122.10MNS1meiosis-specific nuclear structural 11.531.54MSS51mitochondrial translational activator1.562.14NEURL2neuralized homolog 2 (Drosophila)-1.61-2.56OSGIN1oxidative stress induced growth inhibitor 1-1.56-1.63PXMP4peroxisomal membrane protein 41.59-2.07REEP2receptor accessory protein 2-1.522.59SERPINB2serpin peptidase inhibitor 21.592.13SHFSrc homology 2 domain containing F1.531.78THBS1thrombospondin 11.702.22XYLBxylulokinase-1.53-3.07Fold Change!133   Table 13. Biological processes GO terms and KEGG pathways associated with the 25 genes deregulated by TCDD in naŁve and PWM-activated primary human B cells.     GO Biological ProcessGene NameP-ValueEnrichment Scoreintegrin-mediated signalingITGA3, ITGAL, ITGB33.80E-032.08cell adhesionAMICA1, ITGA3, ITGAL, ITGB3, THBS11.20E-022.08response to woundingCXCL2, F3, ITGAL, ITGB3, SERPINB2, THBS15.10E-042.03regulation of cell migrationF3, ITGB3, THBS12.10E-022.03immune responseCD27, CXCL2, ITGAL, THBS16.10E-021.98anti-apoptosisCD27, F3, SERPINB2, THBS12.40E-031.45intracellular signaling cascadeCD27, CCNA2, MAP3K6, NEURL2, THBS17.90E-021.45KEGG PathwayECM-receptor interaction1.30E-02Focal adhesion6.80E-02Regulation of actin cytoskeleton7.60E-02!134 Shimomura et al., 1997). Interestingly, Spint1 -/- mice presented with overt hyperkeratosis of the forestomach, hyperkeratosis of the epidermis, and hypotrichosis associated with abnormal cuticle development (Kosa, 2009). Tnfrsf18 gene belongs to the TNFR family and had been shown to regulate diverse biologic functions, including cell proliferation, differentiation, and survival (Nocentini et al., 1997; Kwon et al., 1999; Gurney et al., 1999). Upb1 gene that encodes beta-ureidopropionase, which catalyzes the last step in pyrimidine degradation and Zfp28, involved in modulation of the core circadian network were deregulated by TCDD in naŁve and activated mouse primary B cells and CH12.LX cells (Vreken et al., 1999; Schick et al., 2016). Functional annotation of the 550 genes deregulated in both naŁve and activated mouse primary B cells identified over-represented functions associated with cytokine-cytokine receptor interaction, NOD-like receptor signaling pathway, and Jak-STAT signaling pathway (Table 14).   A total of 138 genes were identified as differentially expressed in naŁve and PWM-activated primary rat B cells in response to TCDD treatment. Not surprisingly, these include genes commonly used as markers of AhR activation. Only Cyp1A1, Cyp1B1 and Tiparp were deregulated in primary rat B cells and CH12LX cells (Figure 33). Functional annotation of the 138 genes deregulated in both naŁve and activated rat primary B cells identified over-represented functions associated with cell morphogenesis, oxidative phosphorylation, regulation of actin cytoskeleton, and focal adhesion (Table 15).  !135   Figure 32. Venn diagram of the differentially regulated genes in response to TCDD in naŁve and PWM-activated mouse primary B cells and CH12.LX cells. Total number of genes differentially expressed at least at one time-point during the time-course. Mouse primary B cell datasets were filtered using a |fold-change| %1.5 criterion while CH12.LX data set contained genes that are deregulated by TCDD and showed AhR binding within their promoter region.   !136  Table 14. Biological processes GO terms and KEGG pathways associated with the 550 genes deregulated by TCDD in naŁve and PWM-activated primary mouse B cells.   GO Biological ProcessGene NameP-ValueEnrichment Scorenegative regulation of cell growthApbb2, Caprin2, Dcbld2, Nrp1, Ndufs3, Ntn17.40E-031.37JAK-STAT cascadeNotch4, Ager, Ppt2, Socs13.60E-021.21regulation of cytokine productionCd27, Cd28, Cd3e, Ido1, Il10, Il18, Lrrc32, Tnf, Tnfrsf42.90E-021.06protein transportRab13, Rab24, Rab 34, Rab4b, Hook2, Myo6, Slc15a, Snx32, Stx3, Vps26b 3.50E-021.03KEGG PathwayCytokine-cytokine receptor interactionCd27, Flt3l, Acvr1, Ccl24, Ccr9, Csf1r, Csf2ra, Csf2rb, Inhba, Il1r1, Il10, Il12b, Il18, Il3ra, Ltbr, Pdgfa, Tnf, Tnfsf4, Vegfb1.20E-04Hematopoietic cell lineageCd3e, Cd3g, Cd9, Flt3l, Csf1r, Csf2ra, Daf2, Il1r1, Il3ra, Tnf4.80E-04NOD-like receptor signaling pathwayNaip2, Naip6, Naip5, Il18, Trip6, Tnf2.80E-02Glycerophospholipid metabolismCds1, Chkb, Dgkh, Dgkg, Ppap2a, Pld23.70E-02Phosphatidylinositol signaling systemCds1, Dgkh, Dgkg, Itpr3, Pik3r2, Plcb35.60E-02Jak-STAT signaling pathwayCsf2ra, Csf2rb, Ccnd1, Il10, Il12b, Il3ra, Pik3r2, Cbl, Socs16.00E-02Intestinal immune network for IgA productionCd28, Aicda, Ccr9, Il10, Ltbr6.20E-02PPAR signaling pathwayCpt1b, Cyp27a1, Ehhadh, Lpl, Nr1h3, Gm78086.70E-02Allograft rejectionCd28, Gzmb, Il10, Il12b, Tnf7.60E-02!137  Figure 33. Venn diagram of the differentially regulated genes in response to TCDD in naŁve and PWM-activated rat primary B cells and CH12.LX cells. Total number of genes differentially expressed at least at one time-point during the time-course. Rat naŁve and PWM-activated datasets were filtered using a |fold-change| %1.5 criterion while CH12.LX data set contained genes that are deregulated by TCDD and showed AhR binding within their promoter region.        !138  Table 15. Biological processes GO terms and KEGG pathways associated with the 138 genes deregulated by TCDD in naŁve and PWM-activated primary rat B cells.         GO Biological ProcessGene NameP-ValueEnrichment Scorecell morphogenesisL1cam, Apbb1, Celsr2, Pak1, Plxna3, Prox1, Spr6.60E-031.78response to bacteriumJund, Gpx1, Selp, Sox18.80E-021.34regulation of growthApbb1, Fxn, Plxna3, Prox1, Spr, Socs11.60E-021.29regulation of cell adhesionL1cam, Celsr2, Dpp4, Pik3cb, Selp2.90E-031.19regulation of actin cytoskeletonPak1, Prox1, Fmn14.00E-021.11KEGG PathwayErbB signaling pathwayEif4ebp1, Map2k2, Pak1, Pak2, Pik3cb, Crkl4.40E-04Oxidative phosphorylationAtp5d, Cox11, Ndufb2, Ndufab1, Cox8a, Cox6a13.70E-03Regulation of actin cytoskeletonItgb3, Map2k2, Myl9, Pak1, Pak2, Pik3cb, Crkl4.90E-03Focal adhesionItgb3, Myl9, Pak1, Pak2, Pik3cb, Crkl1.60E-02T cell receptor signaling pathwayMap2k2, Pak1, Pak2, Pik3cb5.10E-02mTOR signaling pathwayEif4ebp1, Pik3cb, Eif4b6.20E-02Acute myeloid leukemiaEif4ebp1, Map2k2, Pik3cb6.60E-02!139 CHAPTER 4: DISCUSSION 4.1. Role of the AhR SNPs in human B cell sensitivity to the toxic effects of TCDD AhR genetic polymorphisms are associated with differences in sensitivity to toxic effects of dioxins in animal models (Moffat et al., 2007; Okey et al., 2005).  Lu and colleagues (2010) reported that approximately one in six human donors showed no TCDD-induced suppression of the B cell effector function. Subsequent sequencing of the AhR genes revealed that two out of the three non-responsive human donors had an R554K polymorphism within their AhR, indicating that AhR SNPs may play a role in human B cell sensitivity to immunotoxic effects of TCDD.  To determine the influence of AhR SNPs on human B cell sensitivity to TCDD-induced suppression of the IgM response, the functional activity of three known (P517S, R554K, V570I) AhR variants, alone or in combination (V570I+P517S, V570I+R554K, and P517S+R554K+V570I), were examined in the human AhR null B lymphoblastoid cell line, SKW 6.4. In a series of preliminary experiments, the control AhR, derived from HepG2 human hepatoma cells, was transduced into SKW 6.4 cells for the purpose of establishing an AhR expressing SKW-AHR+ cells that would serve as a comparative control for investigating specific AhR SNPs. Initial experiments were aimed to select SKW-AHR+ clones that were readily activated to produce IgM. Multiple PAMPs including LPS, PWM, CPG, and resiquimod (R848) and cytokines such as IL-2, IL-6 and IL-10 have been previously demonstrated to activate murine and human B cells to produce IgM.  !140 LPS activates B cells by engaging both BCR and TLR4 receptors. Interestingly, LPS from different pathogens has been demonstrated to induce distinct classes of immune responses (Pulendran et al., 2013). Moreover, previous studies conducted in our laboratory show that different strains and lots of LPS vary in their ability to induce IgM response. We demonstrate that LPS derived from E.coli is the most potent activator of the IgM production in the SKW-AHR+ cells. IL-2, IL-6 and IL10 are critical cytokines that promote human B cell activation, differentiation and IgM secretion (Arpin et al., 1995). Additionally, it has been previously demonstrated that SKW 6.4 cells secrete IgM in response to IL-6 treatment (Korholz et al., 1992). Thus we evaluated the ability of select cytokines (IL-2, IL-6 and IL-10) to induce IgM secretion in the SKW-AHR+ cells. Between the three different cytokines, IL-6 induced maximum IgM response. Notably, a combination of the various cytokines did not increase the fold of IgM induction indicating that the effect of the different cytokines on SKW-AHR+ cells is not additive. R848 is a synthetic imidazoquinoline compound that activates a diversity of immune competent cells by binding to the TLR7/TLR8 and initiating the MyD88-dependent signaling pathway (Tomai et al., 2007). CpG motifs are short single-stranded DNA molecules that induce proliferation, differentiation and cytokine production in B cells by binding to the TLR9 receptors (Avalos et al., 2009). Both R848 and CpG were ineffective in the induction of the IgM response in SKW-AHR+ cells indicating that R848 and CpG might not be readily internalized by SKW cells, TLR7/TLR8/TLR9 are not sufficiently expressed, or R848 and CpG are not delivered to the TLR7/TLR8/TLR9 containing compartments. Overall, our experiments !141 demonstrate that PWM and LPS E.coli at comparatively high concentrations are potent activators of the IgM response in the SKW-AHR+ cells. Functional characterization of the control SKW-AHR+ cell line showed the following critical features. First, activation of SKW-AHR+ cells using the polyclonal activators, lipopolysaccharide (LPS) and pokeweed mitogen (PWM), readily induced IgM secretion of similar magnitude in SKW 6.4 and SKW-AHR+ cells demonstrating that introduction of AhR expression did not suppress or augment production of IgM by SKW cells.  Second, in spite of adding GFP to the C-terminus of the AhR, the AhR expressed by SKW-AHR+ was functional as evidenced by TCDD-mediated induction of metabolizing enzymes Cyp1B1 and Cyp1A2, and TCDD-induced suppression of the IgM response all of which did not occur in SKW 6.4 cells. Third, in the time of addition experiments, the SKW-AHR+ cell line exhibited a discrete window of sensitivity to TCDD-induced suppression of the IgM response similar to that previously observed in primary mouse B lymphocytes and in the mouse B cell line, CH12.LX (Crawford et al., 2003; Tucker et al., 1986).  Specifically, TCDD must be added to the cultured cells within the initial 12 h post activation to suppress IgM secretion.  Taken together, the above findings confirm that the human AhR expressed in the SKW-AHR+ control line responds to TCDD in a manner similar to primary mouse and rat B cells as well as a previously characterized mouse B cell line. In murine models, AhR polymorphisms contribute to differences in intraspecies sensitivity to the toxic effects produced by TCDD (Okey et al., 2005).  Previously, it was reported that differences in sensitivity to TCDD toxicity and !142 induction of metabolizing enzymes in humans might be due to AhR polymorphisms (Micka et al., 1997).  R554K is one of the most frequent (frequency of 21.7 %) and studied AhR polymorphism (Exome Variant Server, http://evs.gs.washington.edu/EVS/).  Wong et al. (2001a) reported that P517S is in linkage disequilibrium (a nonrandom association of alleles at two or more loci) with R554K and V570I in individuals of African decent.  All three polymorphisms are located in the transactivation domain of the AhR, and even though P517S and V570I are comparatively rare, frequencies of 0.24 % and 2.3 %, respectively, their combined expression might result in an AhR with distinct intrinsic transactivation potentials.  Current experimental findings suggest that the effects of the R554K AhR variant are controversial and require additional investigation.  For example, Smart and Daly (2000) showed that the R554K SNP resulted in increased ability of the AhR to induce Cyp1A1 mRNA levels in human lymphocytes compared with the control AhR in response to 3-methylcholantrene (3MC) treatment. Conversely, Celius and Matthews (2010) found that R554K does not alter the ability of AhR to transactivate Cyp1A1 and Cyp1B1 expression in Hepa1, MCF-7 or AHR100 cells after TCDD treatment.  Koyano and coworkers (2005) reported that R554K SNP did not affect transactivation properties of AhR with beta-naphthoflavone (BNF), 3MC or omeprazole (OME) treatment in transiently transfected HeLa cells. However, a different study reported that the AhR variants with two SNPs R554K+V570I as well as the three SNPs K554R+V570I+P517S concomitantly expressed, failed to mediate TCDD-dependent induction of the Cyp1A1 mRNA in Hepa 1 cells (Wong et al., 2001b).  The underlying reason for the differences among the studies is not well !143 understood but might be due, in part, to intrinsic differences associated with assay conditions and cell models used. It is also noteworthy that the magnitude of induction by TCDD of Cyp1A2 and Cyp1B1 mRNA levels in SKW-AHR+ cell line, which was relatively modest compared to liver cells, closely paralleled what is typically observed in the primary human peripheral blood B cells (Lu et al., 2010).  Our studies are in agreement with Wong and coworkers (2001b) in that the R554K, R554K+V570I and K554R+V570I+P517S variants all failed to maintain sustained induction of the Cyp1A2, and Cyp1B1 mRNA levels and Cyp1B1-driven luciferase reporter in SKW cells.  Interestingly, DRE-driven luciferase reporter activity was unaffected by either polymorphism, indicating that not all, but some of the AhR transactivation activity was affected by R554K. It is possible that the inability of R554K polymorphic AhRs to induce Cyp1A2 and Cyp1B1 expression is due to a decrease in protein and mRNA stability; however, previous studies showed that this variant did not affect AhR mRNA and/or protein levels (Koyano et al., 2005).  Additionally, it had been demonstrated that polymorphisms in exon 10 of the AhR do not affect the ability of the receptor to bind ligands or DREs in vitro (Wong et al., 2001b). One possible interpretation for the lack of Cyp1B1 inducibility in SKW cells that express R554K AhR is that SNPs within the transactivation domain interfere with the ability of the variant receptors to maintain necessary protein-protein interactions. To induce CYP1 gene expression, the AhR/Arnt heterodimer must bind DRE sequences, recruit and interact (via carboxyl terminus) with a large number of coactivators and mediators. For example, CREB binding protein (CBP), steroid receptor coactivator (SRC-1), the retinoblastoma protein Rb and multiple other !144 protein coactivators have been shown to interact with the AhR/Arnt complex to mediate TCDD-induced reporter gene expression (Fujii-Kuriyama and Mimura, 2005). Considering a large number of protein-protein interactions are necessary to mediate upregulation of CYP1 metabolizing enzymes, it is plausible to speculate that R554K AhR variant might fail to recruit coactivators as efficiently as the control AhR. Additionally, R554K SNP may impact the stability of the AHR/ARNT heterodimerÕs interaction with DNA or decrease the AHR affinity for the necessary coactivators. Multiple epidemiological studies have investigated the effects of the R554K polymorphism on susceptibility to disease in different human populations. The R554K variant was found to be associated with a decreased risk of developing male infertility (Safarinejad et al., 2013) and significantly lower levels of AhR, Arnt and Cyp1B1 mRNA expressed in white blood cells from the Caucasian population (Helmig et.al., 2011). No association was found between R554K and increased risk of non-HodgkinÕs lymphoma (Ng et al., 2010). The R554K variant was found to be associated with a higher risk of coronary arterial disease in the Chinese population (Huang et al., 2015). Additionally, polymorphisms within AhR-interacting genes Ahrr, Hsp90, and AIP have been identified and could potentially contribute to physiological and health outcomes associated with TCDD-induced AhR activation (Cavaco et al., 2013; Rowlands et al., 2011; Urban et al., 2012). In order to identify the effects of the investigated AhR SNPs on the sensitivity of the human B cells to TCDD-mediated IgM suppression, extensive concentration-response studies were conducted. Interestingly, TCDD suppressed IgM secretion in all SKW-based cell lines in a concentration-dependent manner.  With 100nM TCDD !145 suppressing the IgM response to approximately 50% of the VH control group. However, SKW-K554R+V570I+P517S expressing cells had a decreased sensitivity to TCDD-mediated IgM suppression as evidenced by a higher IC50 and an attenuated maximum level of suppression as demonstrated by only a 30% decrease in IgM secretion at 100nM TCDD, compared with vehicle control. The precise mechanism that causes receptors possessing multiple polymorphisms to partially loose the ability to mediate TCDD-induced suppression of the IgM secretion is unknown. One possibility is that the SNPs in the transactivation domain have a greater cumulative effect on the recruitment of co-activators and transactivation of the effector genes. Our study has several limitations. Unlike previous studies, we used lentiviral transfection to establish clones that stably express different AhR variants. Genome-wide studies have shown that lentivirus randomly integrates into actively transcribed genes, possibly resulting in insertional mutagenesis potentially leading to changes in gene expression.  Therefore, for each AhR SNP, at least three clones were evaluated all of which exhibited a similar profile of activity. Additionally, we used the parental SKW 6.4 B cell line that, unlike naŁve primary B cells, is intrinsically in the early stages of differentiation. Moreover, B cells have been previously reported to have a lower magnitude of TCDD-driven induction of Cyp1A1 and Cyp1B1 mRNA making it harder to differentiate between small differences in AhR variant activity (Lu et al., 2010). Overall, this study demonstrates that AhR SNPs within the transactivation domain decreased transactivation potency of the AhR and, when expressed !146 concomitantly, has an attenuating effect on TCDD-mediated suppression of IgM production by human B cells. Until now, we have lacked an appropriate model to study the effects of different polymorphic forms of the human AhR on one of the most sensitive endpoints of TCDD exposure, suppression of B cell function. The expression of different polymorphic forms of the human AhR in the human B cell line SKW 6.4 represents the first direct functional comparison between polymorphic receptor forms in a cell type- and species-specific manner yielding insights into aspects of the AhR biology that may contribute to differences in receptor function within and across species.   4.2 Role of the newly identified prospective B cell-specific molecular targets of TCDD in AhR-mediated suppression of the B cell function Studies in mouse, rat and human B lymphocytes show a similar magnitude of TCDD-induced IgM suppression and time-ofÐaddition kinetics leading to the assumption of a common mode of action. TCDD-mediated, AhR-dependent deregulation of gene expression is assumed to be a primary mode of action for TCDD-induced effects across species (Bunger et al., 2003; Birnbaum, 1994). Therefore the underlying hypothesis of this series of studies was that TCDD deregulates a core set of orthologous genes in mouse, rat and human primary B cells at early time points after B cell activation. To evaluate this hypothesis, comprehensive genome-wide time course gene expression was measured in naŁve and pokeweed mitogen (PWM)-activated primary mouse, human and rat B cells treated with a single dose of TCDD. Previous B cell gene expression studies !147 examining TCDD-induced suppression of the IgM response focused on a limited number of genes such as Pax5, Blimp1, BCL-6 and Bach2 or used transformed cell lines that may not accurately represent normal B cell responses (North et al., 2010; Lu et al., 2010; DeAbrew et al., 2011, Phadnis-Moghe et al., 2015). Therefore, use of primary B cells provided a unique opportunity to investigate and compare gene expression changes in response to TCDD treatment across different species and to identify additional B cell-specific targets of TCDD.  It has been previously demonstrated that B lymphocytes pretreated with TCDD have a similar magnitude of IgM secretion suppression as compared to B cells treated with TCDD at the time of activation. In order to keep the experimental conditions simple and reduce the background gene expression changes associated with activation and differentiation program we first used naŁve primary human, mouse and rat B cells. Comparative genome-wide gene expression analysis identified a large number of divergent and species-specific responses elicited by TCDD across naive human, mouse and rat primary B cells. Not surprisingly, only a limited number of candidate orthologs deregulated by TCDD in all three species were identified. Additionally, data generated during this experiment indicate that TCDD, likely, acts in a species-specific manner to suppress IgM response. Multiple stimuli such as LPS, PWM, and a combination of cytokines and CD-40L have been previously used to activate B lymphocytes. Importantly, human naŁve primary B cells do not express TLR4, the receptor for LPS and do not become activated or secrete IgM upon LPS treatment. Additionally, due to the lack of prior research, CD-40L and cytokine-mediated B cell activation model is not available for !148 the primary rat B cells. However, PWM had been previously demonstrated to trigger activation and proliferation in mouse, human and rat B cells. Thus, in order to keep experimental conditions between the species as similar as possible, we used PWM as a polyclonal B cell activator for all three species. Additionally, it has been previously demonstrated that differences in the AhR properties, one of which is the AhR's ligand affinity, contribute to the variability in sensitivity to TCDD among species (Ramadoss and Perdew, 2004). Thus, we used a high dose of TCDD (30 nM) to strongly activate the AhR signaling and subsequent induction of the responsive genes in all three species. Comparative genome-wide gene expression analysis identified a limited number of candidate orthologs deregulated by TCDD in all three species and a large number of divergent and species-specific responses elicited by TCDD across human, mouse and rat primary B cells activated with PWM. In agreement with previous comparative analysis studies performed in hepatocytes, most of the TCDD-mediated gene expression changes showed species-specific deregulation (Forgacs et al., 2013; Forgacs et al., 2012; Dere et al., 2011; Nault et al., 2013). For example, only 16 orthologs were differentially expressed in human, mouse and rat primary hepatocytes following treatment with 10 nM TCCD for 48 h (Forgacs et. al., 2013). In the current study, TCDD elicited limited overlap between human, mouse and rat primary B cell differential gene expression with only 28 orthologs in common between all three species. This included the induction of ÒAhR battery Ó genes such as Cyp1a1, Tiparp and Ahrr. The promoter analysis showed that all common orthologs have AhR binding sites in their promoter region. Interestingly, CD27, HIC1 !149 and MTSS1L genes all have high scoring pDREs and are activated in response to TCDD in all three species indicating that these genes are direct targets of AhR. CD27 encodes a member of the TNF-receptor superfamily that activates NF-kB and MAPK8/JNK signaling and plays a role in B cell activation and immunoglobulin synthesis (Akiba et al., 1998; Agematsu et al., 1995; Lens et al., 1995). HIC1 encodes a sequence-specific transcriptional repressor that directly targets ATOH1 (a pro-neuronal transcription factor), CXCR7 (a receptor for the chemokine CXCL12), and ephrin-A1 (a cell surface ligand for Eph receptors) and regulates p53- and E2F1-dependent cell survival and growth control (Briggs et al., 2008; Van Rechem et al., 2009; Zhang et. al., 2010; Dehennaut and Leprince, 2009). HIC1 transcription factor recruits multiple co-repressors, including CtBP (a co-repressor for BCL6 autoregulation), possibly targeting genes involved in B cell proliferation and survival, or preventing B cell differentiation by targeting PRDM1 via MTA3/NuRD co-regulators (Ci et al., 2008; Van Rechem et al., 2010). MTSS1L (ABBA) is an actin dynamics regulator that controls actin and plasma membrane dynamics in radial glial cells, plays a critical role in Rac1-mediated cell spreading in NIH3T3 cells, and is essential for Rac1 activation (Saarikangas et al., 2008; Zeng et al., 2013). Rac1, a small signaling GTPase, is ubiquitously expressed and regulates multiple processes including cell adhesion, transcription, proliferation, and differentiation (Benninger et al., 2007; Rose et al., 2007; Fritz and Kaina, 2006; Chae et al., 2008; Bosco et al., 2009). The exact role of the TCDD-induced deregulation of CD27, HIC1 and MTSS1L genes in the suppression of the IgM response should be further evaluated.  !150 Interestingly, most of the genes that were differentially regulated by TCDD were so in a species-specific manner. For example, in the mouse dataset, the most up-regulated transcript, not including drug metabolizing enzyme genes, was Serpinb2, a plasminogen activator inhibitor type 2 (PAI-2).  Serpinb2 is up-regulated under multiple inflammatory conditions and might be involved in regulation of the adaptive immune response since it had been demonstrated that Serpinb2-/- mice generate increased Th1 responses (Schroder et al., 2011). The most down-regulated transcript was methionine sulfoxide reductase B3 (MsrB3), an endoplasmic reticulum oxidoreductase that regulates cell growth through the p53Ðp21 and p27 pathways (Lee et al., 2014). A number of genes involved in regulation of signal transduction (Il22, Mcf2l, Psd2 and Thbs1) were among the top 10 up-regulated, and genes associated with the immune response (Cd70, Fasl, H2-Bl, Sh2d1a) were among the most down-regulated transcripts in the mouse dataset.  In the rat dataset, genes associated with Jak-STAT signaling pathway (Ifnb1, Il22, Pik3r3, Spred3) were among the top up-regulated, and genes associated with cell differentiation (Ctbp2, Cebpb, Tbx6, Fscn3, Rps19, Spr, Socs1) were among the most down-regulated. The most strongly expressed genes in human primary B cells were associated with signal transduction (ARL4C, GPR68, ASB2, CLNK, GRIN2D); the transmembrane transport genes (SLC16A14, SLC13A3, SLC28A2, SLC5A9, SPNS3) were among the most down-regulated. The most up-regulated gene, CD93 (encodes for the CD93 molecule), is important for the maintenance of plasma cells in the bone marrow; CD93-deficient mice fail to maintain bone-marrow plasma-cell numbers and antibody secretion (Chevrier et al., 2009). The most down-regulated !151 gene, TMEM40, encodes for uncharacterized protein TMEM40 that has been associated with arthritis in mice, suggesting a potential role in inflammation (Macaulay et al., 2007; Yu et al., 2009). Gene ontology and pathway enrichment analyses were performed to compare, group and functionally interpret conserved and species-specific gene expression changes elicited by TCDD in primary mouse, human and rat B cells.  Functional analysis identified cytokine activity, cell adhesion and inflammatory response among the biological processes that were enriched in human, mouse and rat primary B cells. Additionally, multiple KEGG pathways including focal adhesion, ECM-receptor interaction, and regulation of actin cytoskeleton were enriched in all three species. Interestingly, even thought comparable functions and pathways were affected between the species, different orthologs within common pathways and biological processes were differentially regulated indicating that TCDD may use species-specific mechanisms to suppress the IgM response. For example, among the KEGG pathways enriched in the rat were oxidative phosphorylation and the Jak-STAT signaling pathway. The Jak-STAT pathway plays a role in the initiation of B cell differentiation through activation of Prdm1 and down-regulation of Bcl6 (Desrivieres et al., 2006; Diehl et al., 2008; Klein et al., 2003; Riley et al., 2005). AhR affects Jak-STAT signaling at the early time points and may play a role in suppression of B-cell activation and differentiation in the rat primary B cells. In the human primary B cells, TGF-# and calcium signaling KEGG pathways were enriched. TGF-# is a well-known potent immunosuppressor. It has previously been demonstrated that TGF-# inhibits proliferation and survival of B cells, blocks !152 progression through the cell cycle and inhibits secretion of IgM (Lebman and Edmiston, 1999; Kehrl et al., 1991). Additionally, it has been previously demonstrated that lowering intracellular calcium levels inhibits IgM secretion (Tartakoff et al., 1977). In the mouse primary B cells, pathways associated with chemokine signaling and ABC transporters were enriched. The pattern of expression and function of ABC transporters on B cells is not well understood, but recent reports have demonstrated that ABC transporters play important roles in the differentiation, and maturation of multiple types of immune cells (Van de Ven et al., 2009). Collectively, gene ontology and pathway enrichment analyses demonstrate that TCDD might act via different species-specific pathways to suppress IgM response.  Additionally, using MetaCore analysis, a cAMP-dependent transcription factor CREB1 and an activator protein 1 (AP1) were identified as top upstream transcriptional regulators. CREB1 involvement in the cAMP/PKA signaling and in the AhR signaling pathways had been previously demonstrated in multiple cell types, including in mouse B cells (Feng et al., 2016; Landers and Bunce, 1991). Similarly, it has been previously demonstrated that in mouse primary B cells, a member of AP1 transcription factors Fra1 blocks plasma cell differentiation and IgM production by directly repressing Blimp1 expression (Grıtsch et al., 2014). Together, these data indicate that TCDD, likely, acts via multiple and species-specific pathways to produce IgM secretion suppression in mouse, human and rat B cells.   Importantly, study design limitations such as quality of the genome builds, accuracy and number of annotated genes, media composition, use of PWM as an activating stimulus may have limited the overall interspecies comparison and biased !153 data interpretation. Specifically, human and mouse genomes are more complete and have a higher number of annotated genes as compared with the rat. Furthermore, the exact mechanism of PWM-induced B cell proliferation and IgM secretion is not well understood and exact early signaling events may vary between species (96:6;6<=>?+0@>+A!6B!?CDE!%"$%). Additionally, this study included B cells from female human donors of undisclosed age; mouse and rat primary B cells were isolated from mature female rodents. Despite these biases, the hallmark measure of AhR activation, induction of metabolizing enzymes Cyp1a1 and Cyp1b1 was prompt and robust among species, indicating that the study design limitations likely did not have a detrimental effect on data interpretation. Notably, in human primary B cells, independent of culture conditions, cellular functions associated with cell adhesion, cytokine signaling, regulation of actin and immune response were altered by TCDD, indicating that TCDD might impair B cellÕs ability to detect and respond to external stimuli. Comparison of the genes deregulated by TCDD in naŁve and PWM-activated B cells identified, surprisingly, a very limited number of genes deregulated by TCDD under both culture conditions. The fact that multiple high-throughput RNA-Seq and microarray studies and years of research have failed to identify specific target genes of AhR that directly lead to TCDD-induced toxicity indicate that AhR, likely, functions via diverse mechanisms including non-genomic pathways. In our datasets approximately an equal number of genes were up- or down-regulated, suggesting that AhR might prevent critical transcription factor binding or compete for binding sites within the promoter region of numerous genes. AhR-mediated sequestration of !154 ARNT and other co-regulators might also contribute to the indirect non-genomic effects of AhR activation. Additionally, recent research indicates that AhR interacts with mitochondrial proteins to shift energy homeostasis in murine cells (Hwang et al., 2016). Moreover, multiple new studies show that AhR regulates a large number of genes by processes involving genomic insulators, chromatin dynamics, transcription of the mobile genetic elements and epigenetics (Mulero-Navarro and Fernandez-Salguero, 2016). This functional interaction of the AhR with mitochondrial proteins, cellular signaling cascades, and mobile genetic elements represents a novel mechanism of AhR-mediated regulation of gene expression programs. In this study, a comprehensive analysis of TCDD-induced transcriptomic changes in mouse, human and rat primary B cells was performed to compare and characterize common and species-specific gene expression and identify B cell molecular targets of TCDD. Importantly, this is the first study to investigate genome-wide responses in human, mouse and rat primary B cells treated with TCDD. Collectively, time-course comparison of mouse, human and rat genomic responses presented in this study suggest that despite the evolutionary conservation of the AhR and its signaling pathway (Hahn, 2002), there are significant differences in TCDD-regulated molecular mechanisms between species that disrupt early signaling during B cell activation and differentiation and ultimately lead to the suppression of the IgM response. Further studies will be needed to confirm the specific roles of deregulated genes and molecular mechanisms responsible for impaired B cell activation and plasmacytoid differentiation.    !155 CHAPTER 5: FINAL CONCLUDING REMARKS  Toxic environmental pollutants such as TCDD are a continuous concern to human and animal health. The results from this dissertation provide novel insights into the molecular mechanisms mediating TCDD-induced adverse effects on B cell activation, differentiation and effector function. The first part of this thesis research aimed at elucidating the effects of the most frequent AhR SNP, R554K, and two additional SNPs I570V and P517S SNPs, shown to be in linkage disequilibrium with R554K, on B cell sensitivity to TCDD-mediated suppression of the IgM response in the human population. Specifically, the study demonstrates that the presence of the most frequent SNP R554K alone is sufficient to attenuate TCDD-mediated induction of the metabolizing genes Cyp1A2 and Cyp1B1 as well as the activity of a Cyp1B1-regulated luciferase reporter gene. However, attenuation of TCDD-induced suppression of B cell IgM secretion requires the combined expression of all three polymorphisms: R554K, I570V, P517S.  It is well known that SNPs within the AhR gene are mainly responsible for the rodent inter-species resistance to TCDD-induced lethality and underlie interspecies variability in sensitivity to toxic and biological effects of TCDD. It is also known that the level of TCDD-mediated induction of metabolizing enzymes varies approximately 30 fold between humans (Kremers et al., 1999; Heuval et al., 1993). Additionally, it has been previously demonstrated that approximately one in six human donors is resistant to TCDD-induced suppression of B cell effector function (Lu et al., 2010). Taken together these data suggest that AhR polymorphisms within the human !156 population might render at least some of the human variability in sensitivity to biological and toxic effects of TCDD. My research indicates that such a scenario is possible, however, considering the overall low frequency of the 570 and 517 polymorphisms within the human population, the investigated polymorphisms cannot fully account for the variability in sensitivity discussed above.  To date, at least 53 polymorphisms resulting in amino acid substitutions within the AhR gene have been identified posing a question whether SNPs in different domains of the receptor can influence human B cell sensitivity to TCDD (Exome Variant Server, http://evs.gs.washington.edu/EVS/). One of the recently identified AhR polymorphisms, C130T, had been shown to modulate AhR expression and expression levels of IL-24 and IL-1#, suggesting an association of this SNP with the susceptibility to TCDD (Liu et al., 2015).  Additionally, polymorphisms within the AhR co-chaperone proteins such as Hsp90, AIP, and p23 and AhR dimerization partner ARNT have been identified. Specifically, at least nine ARNT, eight Hsp90, and fourteen AIP unique SNPs have been identified (Swapan et al., 2008; Urban et al., 2011; Urban et al., 2012; Forst et al., 2015; Rowlands et al., 2011). Hsp90 and AIP function to stabilize AhR in the most favorable conformation and ensure optimal ligand binding. It is tempting to speculate that polymorphisms within this protein could alter AhR affinity for TCDD and related ligands. It is also likely that single nucleotide polymorphisms within the genes associated with AHR-regulated pathways may result in greater susceptibility to TCDD toxicity. However, to date, there are no data directly showing the effects of Hsp90 and AIP polymorphisms, alone or in combination with AhR polymorphisms, !157 on any of the toxic end-points associated with dioxin exposure in humans. Moreover, none of these SNPs were investigated in a context of TCDD-induced suppression of IgM secretion in human B cells and are avenues for the future research. Whether the combination of the most frequent polymorphisms of the AhR, co-chaperons, ARNT and co-stimulators have an additive effect and result in a decrease of B cell sensitivity to immunosuppressive effects is also an impending research question. Our studies investigating the window of sensitivity of B cells to TCDD immunosuppressive effects demonstrated that human, mouse and rat B lymphocytes share a narrow window of sensitivity to TCDD. Specifically, TCDD must be present in the culture within the initial 12 h post-activation to suppress the IgM response, indicating that TCDD disrupts an early signaling event necessary for successful B cell activation, differentiation and effector function. Considering that AhR mediates most if not all of the toxic effects of TCDD, and functions via deregulation of gene expression, we hypothesized that TCDD deregulates a common B cell-specific set of genes in all three species resulting in a similar profile of IgM suppression. The second part of this dissertation research utilized RNA-Seq to identify a common subset of genes deregulated by TCDD in human, mouse and rat primary B cells in an attempt to identify novel, B cell-specific molecular targets of TCDD. We demonstrate that TCDD elicited unique temporal and species-specific gene expression changes indicating a possibility of a species-specific mechanism of TCDD toxicity. !158 A comprehensive investigation of TCDD effects in models of B cell activation other than the one presented in this dissertation might further improve our understanding of the TCDD mode of action in the suppression of B cell function. Additionally considering that AhR-mediated suppression of the IgM response might be elicited by secondary, indirect effects of the AhR on gene expression, later time points such as day 5, 6 and 7 should be investigated using an RNA-Seq strategy. Furthermore, considering recent research implicating AhR-induced micro-RNA-mediated gene regulation, TCDD-induced changes in the B cell micro-RNA profiles should be investigated. Future cross-species comparisons should consider the incompleteness of rat genome annotation that presents a challenge during functional comparison between species. Additionally, considering the level of genetic diversity within the human population subsequent RNA-Seq studies will benefit from using a higher number of human donors. Furthermore, TCDD has been reported to act in a gender-specific manner; thus, an inclusion of both sexes in genome-wide studies of TCDD-elicited primary B cell responses might prove beneficial and improve our understanding of mechanisms underlying IgM response suppression. In summary, this research has addressed two questions: 1) do AhR polymorphisms influence human B cell sensitivity to TCDD? and 2) what are the B cell-specific molecular targets of dioxin that produce impaired B cell function? The findings presented in this thesis have highlighted the role of the AhR SNPs R554K, V570I and P517S in B cell variability in sensitivity to TCDD toxic effects and led to improved understanding of AhR SNPÕs influence on B cell sensitivity to immunosuppression within the human population. The RNA-Seq analysis led to an !159 identification of a set of potential direct molecular targets of TCDD that needs further investigation. To date, this is the first study to investigate the influence of AhR SNPs on a specific human TCDD-mediated toxic end-point: suppression of the IgM response. Additionally, this is the first study to compare genome-wide TCDD-induced gene expression changes in naŁve and PWM-induced mouse, human and rat primary B lymphocytes. Both aspects of this work reveal insights into the mechanism of TCDD-mediated, AhR-dependent disruption of primary humoral immunity and broaden the knowledge on the diversity of gene expression responses mediated by TCDD resulting in reduced uncertainty associated with the health hazard posed by TCDD and dioxin-like compounds.     !160            BIBLIOGRAPHY           !!!161 BIBLIOGRAPHY  Adachi, J., Mori, Y., Matsui, S., Takigami, H., Fujino, J., Kitagawa, H., Miller, C. A., 3rd, Kato, T., Saeki, K., and Matsuda, T. (2001). 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