PLACE IN RETURN BOX to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE MAY 0 5 2007 :x. 6/01 c*/CIRC/DateDue.p65-p.15 , . _ ——_— MECHANISMS OF (N-3) POLYUNSATURATED FATTY ACID INHIBITION ON EXPERIMENTAL IMMUNOGLOBULIN A NEPHROPATHY By QUNSHAN JIA A DISSERTATION Submitted to Michigan State University in partially fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition Center for Integrative Toxicology 2004 ABSTRACT MECHANISMS OF (N-3) POLYUNSATURATED FATTY ACID INHIBITION ON EXPERIMENTAL IMMUNOGLOBULIN A NEPHROPATHY By QUNSHAN JIA IgA nephropathy (IgAN) is the most common form of human glomerulonephritis in the world. Clinical studies have demonstrated that (n-3) polyunsaturated fatty acids (PUFAs) found in fish oil are beneficial in treating inflammatory diseases including IgAN. Consumption of mycotoxin deoxynivalenol (DON) elevates serum IgA, IgA immune-complex and IgA deposition in the kidney thus mimicking the early stages of IgAN. The goal of this study was to elucidate the molecular mechanisms by which (n-3) PUFAs attenuate DON-induced IgAN. Initially, it was determined that fish oil significantly decreased serum IgA, serum immune complex and kidney mesangial IgA deposition. Two major components of fish oil, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), were found to retard DON-induced IgAN with DHA enriched oil (60g/kg diet) being most efficacious. DHA’s effects were dose-dependent with 60 g/kg DHA enriched oil in diet determined to be optimal. Both cyclooxygenase-2 (COX-2) and IL-6 genes expression were inhibited significantly by DHA. However, COX-2 knockout mice and COX-2 specific inhibitor did not support a role for this enzyme in DON-induced IgAN. IL-6, which has been previously shown to play an essential role in DON-induced IgA elevation, might be down-regulated by DHA both at transcriptional level or post-transcription level. Dietary DHA did not affect IL-6 mRNA degradation in thioglycollate-elicited peritoneal macrophages, but significantly inhibited IL-6 gene transcription by blocking CAMP response element binding protein (CREB) phosphorylation and its binding to the IL-6 promoter in vivo. Although mitogen—activated protein kinases (MAPKs) p38 and ERK are the most important kinases for CREB phosphorylation, DON-induced phosphorylation of these two kinases were not affected in macrophages from mice fed DHA, nor were their downstream kinases MSKl and p9ORSK, which mainly mediate CREB phosphorylation. Taken together, the results suggest that the (n-3) PUFA, DHA potentially retards development of DON-induced IgAN by blocking IL-6 gene expression at the transcriptional stage with CREB being most important target. To my grandma in law. iv ACKNOWLEGMENTS My greatest gratitude goes to my supervisor, Dr. James J. Pestka, for his support, guidance and encouragement in my PhD study. I got a lot of benefits from his energetic and attentive enthusiasm in science. Without his help this thesis would not be what it is today. Thanks also to my committee Dr Dale Romsos, Dr Donald Jump and Dr Venugopal Gangur for their generous help and invaluable suggestions in my research. My thanks also extend to Dr Maurie Bennink for his support in lipid analysis. To the people in our lab, thanks for your impressive tolerance and support during the past years and I cannot go ahead without your hands and ideas. I cannot thank Dr Hui-Ren Zhou enough for the huge amounts of help given on this project during the last 4 years. Thanks for Dr Linz and his group members for their support and guidance in my experiments. I am extremely grateful to Dr Colin Barrow of Ocean Nutrition for providing (n-3) PUFA enriched fish oils for these studies. I would also like to thank everyone else who has helped me in my living and studying in this great school. TABLE OF CONTENTS List of tables .................................................................................. viii List of figures ................................................................................ ix Abbreviations ............................................................................... xiv Introduction ................................................................................... 1 Chapter 1 ....................................................................................... 3 Deoxynivalenol ............................................................................. 4 IgA nephropathy ........................................................................... 6 (n-3) PUFA and IgAN ................................................................... 9 IL-6 gene expression .. ................................................................. 17 COX-2 ........................................................................................... 22 Rationale ........................................................................................ 24 Chapter 2 ...................................................................................... 26 Dietary Fish Oil Suppresses Experimental Immunoglobulin A Nephropathy in Mice. Abstract ........................................................................................... 27 Introduction ..................................................................................... 28 Materials and methods .................................................................... 30 Results ............................................................................................. 35 Discussion ...................................................................................... 43 Chapter 3 ...................................................................................... 45 Docosahexaenic Acid and Eicosapentaenoic Suppress Deoxynivalenol Induced Experimental Immunoglobulin A Nephropathy in Mice. Abstract ........................................................................................... 46 Introduction ..................................................................................... 47 Materials and methods .................................................................... 49 Results ............................................................................................. 58 Discussion ....................................................................................... 70 Chapter 4 ...................................................................................... 74 Dose-Dependent Suppression of Experimental Immunoglobulin A Nephropathy by Docosahexaenoic Acid: Relation to Interleukin-6 Expression, Cyclooxygnease-Z and Mitogen-Activated Protein Kinase Phosphorylation. Abstract ........................................................................................... 75 vi Introduction ..................................................................................... 76 Materials and methods .................................................................... 79 Results ............................................................................................. 86 Discussion ....................................................................................... 1 03 Chapter 5 ........................................................................................ 111 Role of Cyclooxygenase-2 Gene In Deoxynivalenol-Induced Immunoglobulin A Nephropathy. Abstract ............................................................................................ 112 Introduction ...................................................................................... 1 13 Materials and methods ..................................................................... 115 Results .............................................................................................. 119 Discussion ........................................................................................ 130 Chapter 6 ....................................................................................... 133 Docosahexaenoic Acid Inhibits CREB Activation and Interleukin- 6 Gene Transcription Induced By the Ribotoxic Stressor Deoxynivalenol In Macrophage. Abstract ........................................................................................... 134 Introduction ..................................................................................... 1 3 5 Materials and methods .................................................................... 137 Results ............................................................................................. 146 Discussion ....................................................................................... 167 Chapter 7 ................................................................... 174 Summary and conclusions Appendix A DHA Effects on DON induced PPAR Binding Activities .............. 178 Appendix B Induction of hematuria by CD89, TNP-BSA and IgG anti-IgA ..... 182 Appendix C Protocol for ChIP assay .................................................................... 199 References ........................................................................................ 209 vii Table 2.1 Table 3.1 Table 3.2 Table 3.3 Table 4.1 Table 4.2. Table 4.3 Table 4.4 . Table 6.1 Table ABl Table AB2 LIST OF TABLES Fatty acid composition of oils used for experimental diets. Experimental groups for assessing DHA and EPA on DON- induced IgAN ( Study 1). Fatty acid composition of the diets in feeding study 1. Experimental groups for assessing effects of DHA /EPA on DON induced IL-6 production (Study 2). Experimental groups for assessing DHA enriched oil on DON- induced IgAN ( Study 1). Experimental groups for assessing DHA enriched oil on DON- induced IgAN ( Study 2). Fatty acid composition of liver phospholipid in study 1. Fatty acid composition of liver phospholipid in feeding study 2. Fatty acid composition of macrophage phospholipid in study 1(n=3). The hematuria and proteinuria induced by CD89 crude extract. The hematuria induced by TNP-BSA in B6C3F1 and in BALB/C induced by IgG anti IgA. viii 32 52 53 54 81 82 89 99 147 195 198 Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Figure 2.7 Figure 3.1 Figure 3.2 Figure 3.3 LIST OF FIGURES Structure of deoxynivalenol. Structure of (n-3) and (n-6) PUFAs. De novo synthesis pathways of PUFAs in plant and their metabolism in human and animal. The mechanisms by which PPARs block gene expression. Mouse IL-6 promoter region. Body weight changes in mice in control, control +DON and fish oil +DON group. Serum IgA levels in mice in control, control +DON and fish oil +DON group. Serum IgA-1C levels in control, control +DON and fish oil +DON group. Cell culture supernatant IgA levels in mice in control, control +DON and fish oil +DON group. Mean glomerular immunofluorescence intensity of kidney Serum IgG levels in mice in control, control +DON and fish oil +DON group. Serum IgM levels in mice in control, control +DON and fish oil +DON group. Feed intake in male B6C3F1 mice fed modified AIN93G diets containing DON (IOppm) and (n-3) PUFA from 8-18 wk. Body weight changes in male B6C3F 1 mice. Serum IgA level in male B6C3F1 mice fed AIN93G pure rodent diets containing DON and different kinds of (n-3) PUFA. ix 10 11 16 19 36 37 38 39 40 41 42 59 60 62 Figure 3.4 Figure 3.5 Figure 3.6. Figure 3.7 Figure 3.8 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure.4.7 Serum IgA-1C level in male B6C3F1mice fed AIN93G pure rodent diets containing DON and different kinds of (n-3) PUFA. Supernatant immunoglobulin (Ig)A concentrations from cultures of spleen and Peyer’s patch cells from male B6C3F1 mice fed modified AIN-93G diets containing DON and (n-3) PUFA. Mesangial immunoglobulin (Ig)A deposition in male B6C3F1 mice fed modified AIN-93G diets containing DON and (n-3) PUFA. DHA/EPA effects on DON induced serum IL-6. IL-6 mRNA in female B6C3F1 mice fed modified AIN- 93G diets containing DHA and EPA lipid mix (Study 2). Effects of dietary DHA on body weight changes in mice induced by AIN-93G diets containing DON (20 mg/kg) over 16 wk ( Study 1). Effects of dietary DHA on food intake changes in mice induced by AIN93G diets containing DON (20 mg /kg ) over 16 wk (Study 1). Effect of dietary DHA on serum IgA in mice fed AIN- 93G diets containing DON (20 mg/kg) over 16 wk (Study 1). Effect of dietary DHA on serum IgA-1C in mice fed modified AIN-93G diets containing DON (20 mg/kg) over 16 wk (Studyl). Effect of dietary DHA on ex vivo IgA production in mice fed modified AIN-93G diets containing DON (20 mg/kg) over 16 wk (studyl). Effect of dietary DHA on mesangial IgA deposition in mice fed modified AIN-93G diets containing DON (20 mg/kg) over 16 wk(Study1). Effects of dietary DHA on spleen IL—6 mRNA and hnRNA expression in mice fed with AIN93G diet containing DON (20 mg/kg) over 16 wk (Study 1). 63 66 67 68 69 87 88 9O 92 93 94 95 Figure 4.8 Figure 4.9 Figure 4.10 Figure 4.11 Figure 5.1 Figure 5.2 Figure 5.3 Figure 5.4 Figure 5.5 Figure 5.6 Figure 5.7 Figure 5.8 Figure 5.9 Effect of dietary DHA on spleen COX-2 mRNA expression in mice fed with AIN93G diet containing DON (20 mg/kg) over 16 wk (Study 1). Effects of dietary DHA on IL-6 mRNA and hnRNA expression in spleen of mice subjected to DON. Effect of DHA on COX-2 mRNA expression in spleen induced by acute oral exposure to DON (Study 2). Effects of dietary DHA on MAPK phosphorylation in spleen induced by acute oral exposure to DON (Study 3). Kinetics of body weight increase in COX-2 knockout mice (mt) and wild-type (wt) mice fed modified AlN-93G diets (Study 1) over 16-wk feeding period. Effects of DON (10ppm) on serum IgA in COX-2 knockout mice (mt) and wild type (wt) over 16 wk. Effects of DON (10ppm) on serum IgA-1C in COX-2 knockout mice (mt) and wild type (wt) over 16 wk. Effects of dietary DON (10ppm) on mesangial IgA deposition in COX-2 knockout mice (mt) and wild type (wt) mice afier16 wk. Effects of DON on ex vivo IgA production in COX-2 knockout mice (mt) and wild type (wt) mice. Body weight in B6C3F1 female mice fed with modified AIN-93G diets containing DON (25 mg/kg) and VIOXX (0.0075% w/w) for 16 wk. .Serum IgA in B6C3F1 female mice fed with modified AIN-93G diets containing DON (25 mg/kg) and VIOXX (0.0075% w/w) over16 wk. Serum IgA-1C in B6C3F1 female mice fed with DON (25 mg/kg) and VIOXX (0.0075% w/w) over16 wk. Effects of dietary DON (25 mg/kg) on mesangial IgA deposition in B6C3F1 female mice fed with DON (25 mg/kg) and VIOXX (0.0075% w/w) over16 wk. xi 100 101 102 119 120 121 123 124 125 126 127 128 Figure 5.10 Figure 6.1 Figure 6.2 Figure 6.3 Figure 6.4 A Figure 6.4 B Figure 6.4 C Figure 6.5 Figure 6.6 Figure 6.7 Figure 6.8 Figure 6.9 Figure 6.10 Figure 6.11 Figure 6.12 Effects of DON (25 mg/kg) on ex vivo IgA production in B6C3F1 female mice fed with VIOXX (0.0075% w/w) over16 wk. The position of the primers in ChIP assay. Effects of DHA on DON-induced IL-6 gene expression in thioglycollate elicited peritoneal macrophages. Effect of DHA on DON-induced IL—6 mRNA stability in LPS-treated thioglycollate elicited macrophage. Characterization of effects of DHA on DON—induced transcriptional factor binding to their consensus sequence in thioglycollate elicited peritoneal macrophage. EMSA competition experiments to identify the specific binding. Characterization of effects of DHA on DON-induced transcriptional factor binding to their consensus sequence in splenic nuclear extract. ChIP analysis of DHA effects on DON-induced transcriptional factor binding to IL-6 prompter in thioglycollate elicited peritoneal macrophage in vivo. DHA effects on DON induced transcriptional factor phosphorylation. Multiple inhibitors on DON-induced IL-6 gene expression in thioglycollate elicited peritoneal macrophage. DHA effects on DON induced MAPK phosphorylation. DHA effects on DON induced MSKl and p90RSK phosphorylation. Intracellular [Ca2+] modulation by DON. Intracellular CAMP modulation by DON. The pathways involved in DON induced IL-6 gene expression. xii 129 142 148 149 151 153 154 156 158 161 162 164 165 166 172 Figure AAl Figure AA2 Figure ABl Figure AB2 Figure AB3 Figure AB4 Figure ABS Figure AB6 Figure AB7 Figure AB8 Characterization of effects of DHA on DON-induced transcriptional factor binding to their consensus sequence in splenic nuclear extract. Characterization of effects of DHA on DON-induced transcriptional factor binding to their consensus sequence in macrophage nuclear extract. PCR the transcript of CD89 cDNA following the reverse transcription. The CD89 gene with its signal peptide deleted was sequenced and confirmed. PCR screening of GS] 15 Pichia clones. Optimization of recombinant Pichia strain expression. Indentifaction of CD 89 and its glycosylation. The expressed protein in the cell lysate injected into the mice. Serum IgA and IgA-1C level in male B6C3F1 mice. The red blood cell in urine under the high power microscope field 18 h after injection of CD89 crude extract. xiii 181 180 188 189 190 191 192 193 194 196 C/EBP CaMKII CAMP COX-2 CRE CREB DON ERK IgAN IL-6 LPS LTB4 MAPK MSKI NFKB p90RSK PGEz PKA PKC ABBREVIATIONS Activate protein 1 CCAAT/ enhancer binding protein Camodulin dependent kinase II cyclic adenosine monophosphate Cyclooxygnease-Z CREB response element CAMP response element binding protein Deoxynivalenol Extracellular signal-regulated kinase Immuno globulin A nephropathy Interleukin-6 N-terminal kinase of c-Jun Lipopolysaccharide Leukotrienes B4 Mitogen activated protein kinase mitogen- and stress—activated protein kinase 1 Nuclear factor kappa B ribosomal S6 kinase with MW 90 kDa Prostaglandin E2 Protein kinase A Protein kinase C xiv PLA PDGF PAF PPAR STAT JAK bZIP ATF TXA Phospholipase A Platelet derived growth factor receptor Platelet activated factor Proliferator activated receptor Interferon Signal transducer and activator of transcription Janus kinase Dimmeric basic region—leucine zipper Activating transcription factor Thromboxane XV INTRODUCTION Primary IgA nephropathy (IgAN) is an immune complex disease, which mainly affects children and young adults (Endo, 1997). Elevation of serum IgA is an important etiological factor for the development of IgAN (Feehally, 1997; Endo, 1997), which can deposit in the kidney and induce cytokine production, complement activation and ultimately causing renal injury (Wyatt and Julian, 1988; Ibels and Gyory, 1994; Feehally, 1997; Endo 1997). Diet supplementation with (n-3) polyunsaturated fatty acids (PUFAs) might be a promising treatment for IgAN due to their multiple effects on inflammation and safety record (Ng, 2003). Epidemiological studies reveal that dietary (n-3) PUFAs are negatively associated with the risk of IgAN (Wakai et al., 1999) whereas high (n-6) PUFA intake is associated with increased risk of IgAN (Wakai et al., 2002). Consistent with these findings, several clinical trials have shown that fish oil might be safe and beneficial for long-term treatment of progressive IgAN (Grande et al., 2001). Benefits of (n-3) PUFAs on IgAN might be explained by their inhibition on inflammatory gene expression (Endres et al., 1995; Watanabe et al., 2000). Deoxynivalenol is a type B trichothecene mycotoxin that is produced by the fungi of Fusarium genus, and that is very stable in the environment and detrimental to both livestock and human health (Rotter et al., 1996). Irnmunotoxicological studies have shown that chronic exposure to DON can upregulate IgA production in mice which mimics the early stages of human IgAN (Pestka et al., 1989; Dong et al., 1991; Dong and Pestka, 1993; Pestka, 2003). DON might destroy mucosa] tolerance by induction of macrophage and T cell cytokines, most notably, IL-6, that promote IgA production and contribute to the systemic compartment polymeric IgA elevation and ultimately IgAN (Pestka, 2003). DON-induced IgAN provides a unique model to investigate potential therapeutic mechanisms of (n-3) PUFA. In vitro and in vivo experiments indicate that IL-6 is essential for IgA production (Bertolini and Benson, 1990; Kono et al., 1991; Xu-Amano et al., 1992). Ex vivo cell reconstitution (Y an et al., 1998), antibody neutralization (Yan et al., 1997) and IL-6 deficient (Pestka and Zhou, 2000) mice studies indicate that IL-6 is also required in DON-induced IgAN. The aims of this research were to verify and optimize the regimen of (n-3) PUFAs for prevention of DON induced IgAN and uncover molecular mechanisms by which (n-3) PUFAs inhibit IL-6 gene expression. Chapter 1 is a review of the key literature pertinent to this thesis. Chapter 2 confirms the inhibitory effects of fish oil on DON-induced IgAN in mice. Chapter 3 compares the effects of the two main components of fish oil, DHA and EPA, on DON- induced IgAN. Chapter 4 assesses the optimal DHA feeding regimen and the potentential mechanisms for DHA suppression of DON-induced IgAN. In chapter 5, both COX-2 knockout mice and COX-2 specific inhibitors (VIOXX) were employed to assess the role of COX-2 gene in DON-induced IgAN model. Finally, Chapter 6 provides insight into possible mechanisms of transcriptional regulation of DON-induced IL-6 by DHA. CHAPTER 1 Literature Review Deoxynivalenol. Deoxynivalenol (DON, Vomitoxin, dehydronivalenol, RD- Toxin, Figure 1.1) [12,13-epoxy—3, 4,15-trihydroxytrichotec-9en-8-one, C15H2006, MW 296.32] is a type B trichothecene produced by the fungi of F usarr'um genus, i.e. F usarium culmorum and Fusarium gramz'nearum. These fungi are important plant pathogens and cause F usarz'um head blight in wheat and Gibberella ear rot in maize. DON is very stable in the environment and detrimental to both livestock and human health (Rotter et al., 1996). DON can bind readily to eukaryotic 6OS ribosomal subunit and prevent polypeptide initiation and elongation. An acute dose of DON can induce vomiting in pigs, while chronic exposure to it will cause anorexia, weight loss and nutrient utilization problems. Immunotoxicological studies have shown that DON can be immunosuppressive or immunostimulatory (Pestka, 2003). On the one side, it can depress antibody plaque-forming and delayed hypersensitivity responses and suppresses normal immune response to pathogens. On the other side, DON upregulates IgA production, which is similar to human immunoglobulin A nephropathy (IgAN) by superinduction of cytokines from T helper cells and macrophages. The mitogen-activated protein kinase (MAPK) is a family of serine/threonine protein kinases, which transduces extracellular stimuli into intracellular post-translational and transcriptional responses. The involvement of MAPKs in DON-induced proinflammatory gene expression in vivo has been documented extensively. As little as 5 mg/kg BW DON induces phosphorylation of extracellular signal regulated kinase (ERK), p38 and N-terminal kinase of c-Jun (JNK) in mice spleen within 15 min and these effects can be reconstituted in vitro in macrophage (Zhou et al., 2003; Moon and Pestka, .2003). A ~33 32 eoamcuv BEEESDEO case; 2i 2 as»; IO Iofo IO Two possible DON targets upstream of the MAPKs have been identified. One is an Src family kinase called hematopoietic cell kinase (Hck), and the other is double stranded RNA-dependent protein kinase (PKR) (Zhou et al., 2003; Pestka et al, 2004). DON can activate these two kinases as early as 5 min in RAW267.4 macrophage cells. Inclusion of Hck and PKR inhibitors impairs. DON-induced MAPK phosphorylation. Activated MAPKs mediate phosphorylation of different transcription factors such as activated protein 1(AP-1), CCAAT enhancer binding protein (C/EBPB), NFch and CAMP response element binding protein (CREB) which, in turn, upregulate expression of proinflammatory genes (Wong et al., 2001; Zhou et al., 2003; Sugita-Konishi and Pestka, 2001) IgA nephropathy. IgAN is the most common form of glomerular nephritis worldwide. It mainly affects children and young adults of which 20-40 % of which will develop the end stage of renal disease (Endo, 1997). Clinical diagnosis of IgAN is based on the presence of mesangial IgA deposition, which can induce renal injuries varying from segmental or diffused glomerular hypercellularity, tubular atrophy and interstitial fibrosis to renal failure (Ibels and Gyory, 1994). Although a high serum IgA concentration alone is not sufficient to induce IgAN (Kilgore et al., 1985), it can be an important etiological factor (Feehally, 1997; Endo 1997). IgA can exist in polymeric and monomeric forms. Polymeric IgA is believed to originate from the mucosal compartments and is secreted into the gut, while monomeric IgA is released into the serum by bone marrow (Floege and Feehally, 2000). Polymeric IgA production by the mucosal immune system is decreased in patients with IgA nephropathy, whereas polymeric IgA production in the bone marrow was increased (Feehally, 1997). The observations that IgAN is frequently associated with upper respiratory tract infections and that increased permeability of the gastrointestinal mucosa will increase IgA production in IgAN patients suggests that IgA nephropathy is related to a hypetactivated mucosal immune system (Suzuki et al., 2000; Kovacs et al., 1996). Aberrant IgA structure is another etiological factor for IgAN. In some human IgAN patients, the IgAl hinge region O-link glycan is deficient of galactose, which makes it difficult to be cleared through the liver IgA receptor (ASGP-R) (Rifai and Mannik, 1984). Galactose-deficient IgAl is also prone to form self-aggregates and immune complexes with anti -glycan IgG and glomerular deposits (Novak and Julian, 2001). Mice have only one isotype of IgA, which was similar to the IgA2 in human, . however, the Th2 cytokines (IL-4 and IL-5) can decrease the galactosylation of IgA in BALB/c mice and may facilitate the IgA deposition (Chintalacharuvu et al., 2001). Elevated levels of IgA and IgA immune complexes can bind to receptors on mesangial cells and induce proliferation and cytokine production. Dimeric and polymeric IgA can activate complement via the alternative pathway and cause glomerular damage (Wyatt and Julian, 1988). At the genetic level, IgAN has been related to race, family, HLA polymorphisms, cytokine (IL-1, IL-6) polymorphisms, renin-angiotensin system and TCR polymorphisms. However cause-effect relationship remains ill-defined (Galla et al., 2001) Treatment of IgAN is still not disease-specific. The role of immunosuppressive therapy in IgAN remains controversial (Harmankaya et al., 2002). Treatments being investigated include angiogenesis inhibition, glucocorticoids, cyclophosphamide, tonsillectomy and (n-3) PUFA (Julian et al, 1999). Future therapies will likely focus on 1) decreasing IgA-IC level, thus limiting the binding of IgA to mesangial cells; 2) antagonizing the effects of platelet-derived growth factors (PDGF) and transforming ~ growth factors beta (TGF-B); 3) reducing noxious glomerular injuries due to infiltrating neutrophils (Lai et al., 2002); and 4) reducing inflammatory mediators including thromboxanes, leukotrienes and platelet-activating factors (PAF) (Wardle er al., 2000). Due to its multiple effects on inflammation and safety record (n-3) PUFA might be a promising treatment for IgAN (N g, 2003). Multiple IgAN animal models have been developed in the past. Rifai et al., (1979) reported a model in mice with injection of immune-complex of IgA and bovine serum albumin (BSA) conjugated with DNP (DNP-BSA). A spontaneous glomerulonephritis resembling human IgA nephritis was developed in ddY (W akui et al., 1989). Uteroglobin (UG) is a potent endogenous immunomodulatory and anti-inflammatory protein. UG .knockout mice also represent a new model for IgAN (Pouria and Challacombe, 2000). CD89 is a human IgA receptor, which can form complexes with mouse IgA. CD89 transgenic mice can develop IgAN in six-month (Launay et al., 2000) DON consumption induces IgAN in mice and this might result from stimulation of the mucosal immune response (Pestka, 2003). It has been shown that mice fed 25 mg/kg DON for 12 wk develop IgAN with high concentrations of serum polymeric IgA (pIgA) (Pestka et a/., 1989; Dong et al., 1991; Dong and Pestka 1993; Pestka, 2003). Although the gut acts as a portal of entry to a vast array of foreign antigens in food, lymphocytes in the gut-associated lymphoid tissues (GALT) are seldom activated because of mucosal tolerance. DON might destroy this tolerance by induction of macrophage and T cell cytokines, most notably, E-6, that promote IgA production and contribute to the systemic compartment polymeric IgA elevation and ultimately IgAN (Pestka, 2003). (n-3) PUFA and IgAN. Mammals lack the ability to synthesize PUFA with double bonds distal to the ninth carbon atom. Therefore, linoleic acid and a-linolenic acid are two essential PUFAs in diet. Linoleic acid (C18: 2 n-6) is the precursor of n-6 PUFA found in corn, soy and safflower oil. Once consumed, linoleic acid is elongated and desaturated to yield arachidonic acid (C20: 4 n-6), the usual precursor of the 2 and 4 series eicosanoids, synthesized by the cyclooxygenase pathway and 5-lipoxygenase pathway (Donadio and Grande 2004; Figure 1.2, 1.3). a-Linolenic acid (C18:3 n-3) is another essential fatty acid, which is mainly found in the chloroplasts of green leafy vegetables, plant oils (canola, flaxseed, and soy), and nuts (walnut oil and walnuts) (Donadio and Grande, 2004). In mammals, once consumed, a—linolenic acid only slowly elongates and desaturates to eicosapentaenoic acid (EPA, C2025 n-3) and docosahexaenoic acid (DHA, C2226 n-3),, the parent of 3 and 5 series eicosanoids. EPA and DHA compete with arachidonic acid as substrate for cyclooxygenase, for the sn-2 position in membrane phospholipids and for elongase and desaturase enzymes, thereby reducing synthesis of arachidonic acid from linoleic acid (Donadio and Grande, 2004). DHA and EPA are the (n-3) PUFA with most biological activities. However, conversion of a-linolenic acid by the human to the more active longer-chain metabolites is inefficient: < 5-10% for EPA and 2-5% for DHA (Davis and Kris-Etherton, 2003). Since .922 6-5 as 2-5% Danae one 2 2:3... 9mm 0 Bow 2059858800 On: You 0 28 282582 OmI NUS 0 28 228: IO8\/\/\/\/H\/n\/>\ 8: «é o 28 2865.5 1000 On: 5.3.8.5 5.28.5 10 .935,” 3-5 em a 583 8m muoom 82 8m :0 5mm 28 $55 .1235 98 SEE: E 833308 :05 can :83 5 EEE no @3358 mmmofiam 96: on m4 Bantu 1m."— mmhd < 0.. 26¢ on 0 0:3 con—«moo as: Tamed 2 _ . a 3.5.3 A 52.5... VP...— 0.. wan. g Eon 3:02:32 3... + Eon o_o:m~xw:amoooo t Eon 0.0:ouuconmm02m a Bow u_:e_o:_._.mcn_a 4 £8 222.: + Eon £20 <00 _»~00< ._.z<..n_ 11 marine algae are rich in and marine fish can bioaccumulate the (n-3) PUFA, fish oil is an ideal food for humans to obtain DHA and EPA. During the past 30 years, the ratio of (n-6) to (n-3) PUFA in diets has increased in industrialized societies because of (1) increased consumption of vegetable oils rich in (n- 6) PUFA, (2) increased use of intensive, cereal-based livestock, and (3) reduced consumption of foods rich in (n-3) PUFA (Sanders, 2000). In the United States, the intake of (n-3) PUFA is 1.4 g for linolenic acid (ALA; 18:3) and 0.1—0.2 g for DHA and EPA. The recent estimated ratio of (n—6) to (n-3) in the diet is about 9.8:1, higher than that suggested to be optimized (i.e., 2.3:1), thus a four-fold increase in fish consumption ' has been recommended (Kris-Etherton et al, 2000). The Food and Nutrition Board raised the acceptable range for alpha-linolenic acid from 0.6% to 1.2% of energy, or 1.3—2.7 g per day on the basis. of a 2000 calorie diet for healthy people (Donadio and Grande, 2004). (n-3) PUFA beneficial effects in IgAN are based on epidemiologic studies and randomized clinical trials. Epidemiologic studies reveal that dietary (n-3) PUFAs are negatively associated the risk of IgAN (Wakai et al., 1999) and high intake of (n-6) PUFAs was associated with increased risk of IgAN (Wakai et al., 2002). Holman et al, (1994) also found that some IgAN patients were deficient in a-linolenic acid [18:3 (n-3)], and supplementation with EPA and DHA will decrease proteinuria and improved glomerular filtration rate in these persons. Consistent with these results, several clinical trials have shown that fish oil might be safe and beneficial for long-term treatment of progressive IgAN (Grande et al., 2001). The largest long-term clinical trial in high-risk patients with IgAN has shown that fish oil could retard renal disease progression by reducing inflammation and glomerulosclerosis (Donadio, 2001a). Two 4-year prospective 12 studies have been conducted in the United States to test the high dose (n-3) PUFA effects on IgAN (Donadio, 2000). The results show that both high dose (EPA 3.76g, DHA 2.94g /day) and low dose (EPA l.88g,DHA 1.47g/day) have the same benefits on IgAN. (n-3) PUFA benefits in IgAN might depend on their downregulation of multiple inflammatory genes. Kaminski et a1. (1993) showed that consumption of 7g/day of 85% pure (n-3) PUFA for 7 wk increases (n-3) PUFA content in human monocyte phospholipids and down-regulates PDGF-A and PDGF-B expression by 70%. Intake of 18 g/day (n-3) PUFA by healthy volunteers without altering their normal Western diet for 6 wk suppressed IL-1 and TNF-or production by stimulated peripheral-blood mononuclear cells in vitro. These effects persist more than 10 wk after (n-3) PUFA supplementation (Endres et al., 1995). LPS-induced spleen IL-lB mRNA expression was inhibited in mice fed a diet containing 4% DHA but not EPA (Watanabe et al., 2000). (n- 3) PUFA also inhibited inflammation by downregulating endothelial cell activation. Preincubation of endothelial cells with DHA (1-25 umol) reduces endothelial expression of vascular cell adhesion molecule 1(VCAM-l)‘, E-selectin, intercellular adhesion molecule 1(ICAM-1), IL-6 and IL-8 in response to IL-1, IL-4, TNF or bacterial endotoxin (De Caterina et al., 2000). (n-3) PUFA might affect gene transcription through cell membrane phospholipid modification and altered cell signaling. Dietary supplementation with (n-3) PUFA increases DHA and EPA while decreasing the AA level in the cell membrane phospholipids (Palombo et al., 1994). This modification reduces prostaglandin E2 (PGEZ), leukotriene B4 (LTB4) and platelet activated factor (PAF) production by decreasing the ratio of (n-6)/(n-3) in cell membrane (Calder 2002; Simopoulos, 2002; 13 Whelan, 1996). Reduced membrane arachidonic acid level can further reduce Ras protein activation (Sermon et al., 1996). Lipid rafis, membrane microdomains enriched in cholesterol and glycosphingolipids, are central to cell signaling and have been implicated in processes as diverse as signal transduction, endocytosis and cholesterol trafficking (Pike, 2004). Both in vivo and in vitro studies have shown that sphingomyelin, which facilitates rafi formation, is significantly decreased in T cells by (n-3) PUFA (Fan et al., 2003). This modification might block protein tyrosine phosphorylation and calcium response in T cells and thus contribute to the inhibitory effects of (n-3) PUFA on cell signaling (Stulnig et al., 2001). In addition, Src family protein-tyrosine kinases are highly concentrated in lipid rafts due to post-translational palmitoylation. It has been shown that (n-3) PUFA can selectively displace signaling proteins such as Src family kinases from lipid rafts, which are generally attached to the cytoplasmic membrane lipid leaflet by means of acyl moieties under physiological conditions (Stuhiig et al., 2001). Several studies have reported that (n-3) PUFAs inhibit MAPK activation in vitro (Denys et al., 2001), which are important for lL-6 gene expression (Koranteng et al., 2004; Kim et,al., 2004). With an anti-thymocyte (ATS) model of mesangial proliferative glomerulonephritis, it was recently demonstrated that DHA, but not EPA, decreased ERK activation by 30%. (Yusufi et al., 2003). In the human pulmonary microvascular endothelial cells (HPMECs), EPA suppressed IL-l stimulated p38 phosphorylation (Ait- Said et al., 2003). When treated with a sterile, commercially available, pharmaceutical grade (n-3) PUFA emulsion, LPS-induced ERK and INK phosphorylation in the RAW 264.7 macrophage is inhibited, however, p38 remains unchanged. Also, (n-3) PUFA can bind directly to the different recombinant activation domains of PKC, CaMKII and PKA 14 and inhibit their activation in vitro (Mimikjoo et al., 2001). Phosphorylation of ERK, JNK and p38 are the immediate prior steps in AP—l, CREB, and NFKB activation (Cho et al., 2004; Wadgaonkar et al., 2004; J ang et al., 2004). Attenuated transcriptional factor activation and subsequent depression of proinflammatory cytokine gene expression would thus be anticipated after inhibition of these MAPKs (Babcock et al., 2004). Peroxisomal proliferator activated receptor alpha (PPARor) and gamma (PPARy) might be important transcriptional factors that can mediate (n-3) PUFA effects on gene transcription (Jump et al., 2004; Jump et al., 1997). When PPARs bind to their ligands, they physically interfere with transcriptional factors binding to the cis-element and thus downregulating proinflammatory gene expression (Ren et al., 1996; Figure 1.4). For example, interferon gamma (IFN-y), IL-6 and TNF-a production are impaired in spleens from mice fed the PPARor ligand, WY14, 643 (Cunard et al., 2002). When WY14, 643 binds to PPARa, it can sharply reduce IL-6 and COX-2 gene expression by physically interfering with p65, c-jun and CBP interaction with DNA in vitro (Delerive et al., 1999). The PPARy ligands, troglitazone, pioglitazone and lS-deoxy—Delta (12,1‘4)-prostaglandin J (2) also inhibit IL-lB-induced IL-6 expression at transcriptional level in Vascular smooth muscle cells by interfering NFKB and C/EBP binding to the DNA (Takata et al., 2002). Arachidonic acid and LTB4 can also bind to the PPARor and 7, but the binding will elicit their degradation by increasing B-oxidation of PUFA (Devchand et al., 1996). Thus, PPARs are important negative feedback molecules in vivo for control of inflammation responses (Delerive et al., 1999; Pointer and Daynes, 1998; Berger and Moller, 2002; Delerive et al,, 2000; Clark, 2002). Since DHA and EPA are now recognized to be natural ligands for PPARot and PPARy, DHA and/or EPA might inhibit 15 34:8 .825 Em a 88: a a 3:25 33% ...... b23298 25m :38 Sam can uefiofioa 0-..: 8 mamas 805 £83 £88m Raougtomqmb c938 £302 53 32mm 62%: :05 8 wfiufin B¢< dommmoaxo 28w x83 mmiadm new?» 3 "55:88 2E. v.“ 95”....— oh: macaw: xxx 0 min. B u_._. meH<>_._.O<.OO '16 lL-6 gene expression by blocking transcription factors p65, c-jun binding to the IL-6 promoter. IL-6 gene expression. IL-6 is a pleiotropic cytokine produced by monocytes/macrophages, fibroblasts, endothelial cells and astrocytes in response to infections and toxins (Horn et al., 2000). As a major mediator of acute phase responses, IL-6 is involved in the regulation of differentiation, proliferation and survival of lymphocytes, astrocytes, endothelial and hematopoietic cells, bone marrow turnover and liver regeneration. Both in vitro and in vivo experiments suggest that IL-6 is an important factor for the development of IgA producing B cells. IL-6 was found to induce IgA production in Epstein Barr virus (EBV) transformed B lymphoblasts (Bertolini and Benson, 1990). Addition of human rIL-6 to peripheral blood mononuclear cell (PBMC) increases IgA-subclass spot-forming cell (SFC) responses (Kono et al., 1991) and IL-6 produced by Th2-type cells has been shown to induce Peyer’s Patches sIgA+ B cells to secrete IgA (Xu-Amano et al., 1992). Anti IL-6 antibody can block B cell differentiation, immunoglobulin isotype class-switch and antibodies production induced by anti-CD40 antibodies (Labara et al., 1990). IL-6 deficient mice show defects in inflammatory and immune responses, impaired macrophage and neutrophile functions and are resistant to experimental autoimmune encephalomyelitis (Friedmann et al., 2000). Ex vivo cell reconstitution, antibody neutralization (Y an et al., 1997; 1998) and IL-6 deficient mice studies (Pestka and Zhou, 2000) indicate that IL-6 is essential in DON-induced IgAN. IL-6 binds to receptor glycoprotein 80 and the IL-6/gp80 complex will then interact with the transmembrane protein gpl30 to trigger gp130 dimerization (Montero- Julian, 2001). This recruits and activates the non-receptor cytoplasmic protein tyrosine l7 kinase Janus kinases (JAK) via phosphorylation. JAKl/2 activate transcription factors C/EBPB and 6. The cis-elements for these two factors have been found on the promoter region of immunoglobulin light and heavy chain, suggesting that IL-6 signal might be directly involved in immunoglobulin production. Another unique transcription factor activated by JAK1/2 is signal transducer and activator of transcription (STAT3), which also plays a crucial role in cell proliferation, differentiation and antibody production (Friedmann et al., 2000). IL-6 can act synergistically with CD40. Cross-linking CD40 with CD40 ligand (CD40L) will promote B cell proliferation, immunoglobulin class-switching and prevent B apoptosis in germinal centers. STAT3 also mediates CD40 signal transduction. So IL- 6/JAK1/2/STAT3 and CD40/JAK3/STAT3 signaling pathways are important for B cell survival and antibody production. Two lineages of murine B cells mediate immune defense at mucosal surfaces, designated as B1 and B2 cells, identified by their origins, anatomical distribution, cell surface markers, antibody repertoire. Both contribute to the IgA plasma cells found in the intestine. The majority of intestinal IgA plasma cells derive from B2 cell precursors originating from Peyer's patches (Bao et al., 1998; Husband et al., 1978; Su et al., 2000). In Bl B cells, which contribute almost half of the IgA production in the mucosal compartments, STAT3 is constitutively activated (Feehally, 1997), which might explain why IgA production in B cell from the peritoneal cavity is IL-6-independent (Beagley et al., 1995). Normally IL—6 transcription is tightly controlled despite its potent induction during acute phase responses. Multiple transcriptional factors are involved in IL-6 transcription (Figure 1.5). Electrophoresis mobility shift assay (EMSA) and point 18 omooommuou ouomuuouuu omommmmoum mmummmoouo wouoouuomm mumuowfiaa :2on .88an on: 8:02 m." 953% 92H momoouummo ummumoommo mmoomooomm oououoouou umouommumu oumumommmu mxmz Doommumumm mummuoouom mpummmmmmp ummmmououm mmumooouuu ummooumumm Hmm\mmm Hmm wmz moumuuuuum ommmommoou uoomooooum oooomououo mooomoooomrmoummouuuu mmm\o \ mmmo mmmmummupo ummomumuum omouoommom mmuomumoou uuopuMODmu pompoouuoo Hm¢\mmmu ouuuuomumo mmmuuommwm mommouoomm uommmmuuou pooumouoou mmmmmmmmmm Hmm mmmmmmmuuu uomoummouo mummmouomu mommmmoumo oouuuouuuu mmouupmwmu x¢2\0%2 ouumommmmu muuoououup ououuoomup uoomuumumm ummmuuuoom uoumumomom omumououou monoumuouo umumumuouo umpououopm umuouououo wouououmum HNMH HmNH HONH HvHH Hmoa HNOH Hem Hom va l9 mutation analysis revealed that APl, CREB, C/EBPB and NFicB are important for IL-6 transcription regulation (Matsusaka et al., 1993; Robb et al., 2002). Generally, after APl, C/EBPB, CREB and NFKB are sequentially arranged along the promoter region of IL-6, CBP/P300 will be recruited onto the multiple-protein complex called enhancesome by protein-protein interaction, which will then interact with the general transcription factors and RNA polymerase (RNP) H to turn on IL-6 expression. However, the exact mechanism of IL—6 regulation is different between different cell lines and different kinds 4 of stimulation. The AP-l transcription factor family is dimeric basic region—leucine zipper (bZIP) proteins, which belong to Jun (c-Jun, JunB, JunD), Fos (c-Fos, FosB, Fra-l and Fra2), Maf, and ATP subfamilies. AP-l recognizes 5’-TGAG/CTCA-3’or cAMP response element (CRE, 5’- TGACGTCA-B’). c-Jun has the most potential transacting activity in Jun family (Shaulian and Karin, 2001). AP-l can mediate the induction of pro- inflarnmatory cytokines triggered by MAPKs. Activated JNK can phosphorylate c-jun, which then will bind to the AP-l cis-element and drive gene expression (Y oon et al., 2004). p38 can directly activate ATF2, monocyte specific enhancer binding factors 20 (MEFZC) and ternary complex factors (TCFs) by phosphorylation. Activated ERK translocates into the nucleus and activates Fral and/or Fra2, which enhance c-Jun DNA binding. AP-l plays an important role in cell proliferation, transformation, cell survival and death (Shaulian and Karin, 2001, 2002). In B cells AP-l family (c-Jun, JunB, JunD, and c-Fos) are involved in the IL-6 expression via CD40 ligation (Mann et al., 2002). The C/EBP proteins, including or,B,y,8,e,§, belong to the basic leucine zipper class transcription factor family, all of which contain a conserved bZIP domain at C-terrninus 20 which can interact with CCAAT box motif. The transactivation properties of C/EBP members might come from its recruitment of Co-activators. C/EBPB can form dimmers with p50, CREB/ATP, API and retinoblastoma protein. Different heterodimmers might have different transactivation potentials. In P388 murine B-lymphocytes, C/EBP bZIP region can confer LPS-inducible IL-6 expression and this effect is completely dependent on NFicB binding sites, suggesting an interaction between C/EBP and NFKB (Hu et al., 2000). In the human enterocyte Caco-2 cell line, C/EBP beta and delta are important for IL-6 expression induced by lL-10 (Robb et al, 2002; Hungness et al., 2002). CREB belongs to the bZIP superfamily. The C-terminal domain of CREB mediates DNA binding and the leucine zipper domain facilitates its dimmerization. Usually, CREB will bind to the palindromic cis-regulatory elements (CRE): 5’- TGACGTCA-3’ and organize the transcription machine (Bonnie et al., 2002). Deletion and mutation studies within IL—6 promoter region have revealed that CAMP response element (CRE) plays a crucial role in the regulation of IL-6 transcription (Tokunou et al., 2001). p3 8-activated MSKl, Ca/CAMKII and PKA all can phosphorylate CREB at serine 133, which is required for CREB transactivation (Shaywitz and Greenberg, 1999; Rosenberg et al., 2002). NFKB contains several subunits including p50, p65 (RelA), C-Rel, p52, and RelB. All of these proteins share a conserved Rel homology region (RHR), which is responsible for dimerization, DNA binding and interaction with IKB. These form homodimers or heterodimers composed of several of combination of members. In mammalian cells, the p65 homodimer is the most potent and abundant transcriptional activator found in most cell types (Dixit and Mak, 2002). NFKB stays in an inactivated form in cytoplasm by 21 interaction with the protein IKBS. TNFot, LPS and IL-1, potent activators of NFkB, rapidly induces phosphorylation of lchs and degradation by 26S proteasome (Dixit and Mak, 2002). The p50/p65 will then translocate into the nucleus and tum on a large number of genes, which are involved in the immune and inflammation responses. During the development and in response to injury and infection, IL-1 and TNF-or are considered the most potent factors for NFKB activation in vivo (Bowie and O'Neill, 2000). It has been firmly established that IKB phosphorylation at Ser32 and Ser36 are critical for NFKB activation. Protein kinases specific for IKBa and B are called Ich kinases (IKK). These are considered to be key convergence points for many NFKB signaling pathways. Three IKKs (a, B, 7) have been identified of which IKKB is the most critical for inflammation. Termination of NFicB activation is complicated and is thought to involve new synthesis of IKBa, which enable NFKB to be transported back into the cytoplasm. In murine monocytic PU5-1.8 cells, U937 and HeLa cell lines, NFKB is critical for LPS- induced IL-6 expression (Endorser et al., 1994). An acute dose of DON (25 mg/kg BW) induces serum IL-6 and lL-6 mRNA in spleen and Peyer’s patches of mice (Moon and Pestka, 2003). In RAW 264.7 macrophages, DON can increase DNA binding of junD, junB, phosphorylated C-Jun, c- Fos, Fra—2, the NFKB family of p50, C-Rel subunits, and C/EBPB (Wong et al., 2002). EMSA has also revealed that DON could also induce AP-l, CREB, C/EBPB and NFIcB binding in mouse spleen (Zhou et al., 2003). One or more of these transcription factors might be involved in DON induced the IL-6 gene regulation. CyclooxygenaseJ. Cyclooxygenase (COX) is the rate-limiting enzyme in the conversion of arachidonic acid to prostanoids after arachidonic acid is released from 22 membrane phospholipids by phospholipase A2 (PLAz). There are two isoforrns of COX. COX-l is a constitutive enzyme and is associated with the endoplasmic reticulum (ER). Prostaglandins (PG) synthesized by COX-1 mediate cell homeostasis functions. In contrast, COX-2, expressed by macrophages and monocytes, is an inducible, immediate- early gene product and primarily responsible for increased PG production during inflammation, reproduction and carcinogenesis (Ristimaki, 2004). Expression of COX-2 is low or nondetectable in most tissues, but can be readily induced in response to cell activation by cytokines, growth factors and Chemicals. The COX-2 products, PGs, are generally considered to be proinflammatory agents, but in vitro studies show that they play anti-inflammatory roles during certain situations (Takayama et al., 2002). For example, PGE2 can behave as a pro- or an anti-inflammatory lipid depends on which PGE2 receptor (EP) subtype is stimulated. Four subtypes of PGE2 receptor (EP), designated EPl, EP2, EP3, and EP4, have been found (Nataraj et al., 2001). EPl is coupled with the Gq protein, which signals through the phospholipase C (PLC) pathway, increasing intracellular Ca2+ concentration. EP2 and EP4 are coupled with the Gs protein and activate adenylyl cyclase, increasing cyclic adenosine monophosphate (CAMP) levels and signaling through the protein kinase A (PKA) pathway. EP3 or and [3 decrease CAMP by inhibiting adenylyl cyclase and increase intracellular Ca2+ concentration via Gi-mediated PLC activation, whereas EP3 y signaling increases CAMP via the Gs protein (Akaogi et al., 2004). PGE2 and EP4/2 agonists can modulate macrophage E-6 production via EP2 or EP4 receptor. (Akaogi et al., 2004). EP4/EP2-mediated IL-6 induction was PKA-dependent in an early human T’ cell line, whereas PKC and p38 MAPK dependence is reported in human astroglioma 23 cells suggesting that different signaling pathways mediated IL-6 induction. EPs may regulate IL-6 differentially depending on the mode of stimulation, species, cell types (Akaogi et al., 2004). DON-induced ERK and p38 activation mediates COX-2 transcription, while p38 mediate COX-2 mRNA stability (Moon and Pestka, 2002). Furthermore, DON upregulation of LPS-induced IL-6 production is significantly reduced by the COX-2 inhibitors indomethacin and NS-398. COX-2 knockout mice also produce significantly reduced splenic IL-6 mRNA and serum IL-6 responses to oral DON exposure compared to their parental wild type (Moon and Pestka, 2003). These in vitro and in vivo data suggest that DON-induced COX-2 gene expression and resultant COX-2 metabolites contribute, in part, to subsequent upregulation of IL-6 gene expression (Moon and Pestka, 2003). Recent studies suggest that (n-3) PUFA might reduce the concentrations of 2- series PG and increase the synthesis of 3-series PG by competing with arachidonic acid for the COX-2 (Bagga et al., 2003; Obata et al., 1999; Dommels eta1., 2003). Rationale. To summarize, primary IgA nephropathy is an immue complex disease, which mainly affects young adults, of which 20-40% will develop the end stage renal disease. The etiology of IgAN is still not clear and different animal models have been constructed to study the development and treatment of this disease. Mice exposed to the mycotoxin DON develop high serum IgA and kidney mesangial IgA deposition that mimics the early stages of IgAN and provides a unique IgAN model to investigate potential therapeutic mechanisms. Treatment of IgAN is still not disease-specific, but consumption of (n-3) PUFA holds promise for retarding the disease progression (Donadio, 2001a; Donadio et al., 2001; Prokopiuk et al., 2001; Grande and Donadio, 24 1998; Donadio et al., 1994; Pettersson et al., 1994) by reducing renal inflammation. Although (n-3) PUFAs inhibit inflammation, the mechanisms remain unclear. In this thesis, the effects of (n-3) PUFA on DON-induced IgAN will be confirmed and an optimized regimen determined. Next (n-3) PUFA effects on regulation of IL-6 gene expression will be investigated. A long term outcome of this work will be to uncover new cellular regulatory pathways that may be exploited in the control of IgAN. This research is important for the following reasons. First, it will provide the basis and regimen for the prevention and treatment of IgAN with (n-3) PUFA. Second, this research will reveal specific intervention targets for treatment of IgAN and potentially other inflammatory diseases. Third, this work will uncover the molecular basis of (n-3) PUFA on IL-6 gene regulation, which may help to understand (n-3) PUFA anti- inflammatory effects. Fourth, this study will provide insight into different signal transduction pathways, which regulate upstream lL-6 gene expression and how these might be affected by (n-3) PUFA in diet. 25 CHAPTER 2 Dietary Fish Oil Suppresses Experimental Immunoglobulin A Nephropathy in Mice 26 ABSTRACT Dietary fish oil supplementation reportedly retards the progression of renal disease in patients with immunoglobulin A nephropathy (IgAN), the most common glomerulonephritis worldwide. Here the effects of fish oil were assessed with mycotoxin deoxynivalenol (DON) induced experimental IgAN. DON significantly increased serum IgA, serum IgA immune complexes and kidney mesangial IgA deposition compared with the control group, whereas all three variables were significantly attenuated in mice fed DON and fish oil. In addition, spleen cell cultures from the DON plus fish oil group produced markedly less IgA than those cultures from mice fed DON plus corn oil. Taken together, the results suggested that diets containing fish oil might impair early immunopathogenesis in DON-induced IgAN. 27 INTRODUCTION Human IgA nephropathy (IgAN) is a primary autoimmune disease with diffuse mesangial IgA deposition in the kidney glomerulus, which accounts for up to 50% glomerulopathies in Japan (Hunley and Kon, 1999; Emancipator and Lamm, 1989). Approximately 150,000 people in the United States have been diagnosed with IgAN with 4000 new cases occurring each year (Hellegers et al., 1993) and 30% of them will develop progressive renal failure over a 25 y period (Donadio et al, 1994; Harper et al, 1993). Mucosal infections (D’Amico, 1987), genetic predisposition (Schena et al., 2001), diet (Coppo et al., 1986) and environmental agents such as mycotoxins (Coppo et al., 1988; Hinoshita et al., 1997) have been related to with IgAN. Currently no disease specific treatments are available for IgAN. Human studies have demonstrated that fish oil consumption was correlated with low incidence of autoimmune and inflammatory disorders (Kromhout et al., 1985). In several mouse models fish oil also showed potential to reduce inflammation (Cathcart et al., 1991; Robinson et al., 1986; Robinson et al., 1993; Empey et al., 1991). Several studies have proposed that fish oil may benefit patients with immune-related renal diseases including IgAN, lupus nephritis and cyclosporine-induced nephrotoxicity (Donadio, 1991; Holman et al., 1994; Wakai et al., 1999; Hamazaki et al., 1984; Donadio et al., 2001). A randomized trial of fish oil in IgAN (Donadio et al., 1994; Donadio et al., 1999) also reported that fish oil can significantly retard the development of renal failure. Since dietary fish oil supplementation appears to be a promising therapeutic regimen for IgAN, further research is required both to establish the mechanistic basis for these effects and to determine the optimal dosing regimens of fish oil. 28 It was found that mice exposed experimental diets containing mycotoxin deoxynivalenol (DON) deve10ps the early characteristic features of human IgAN including elevated serum polymeric IgA and IgA-1C as well as mesangial IgA deposition (Pestka et al., 1989) which last for up to 3 months after removal of DON from diet (Dong and Pestka, 1993). DON-exposed mice also exhibits increased numbers of membrane IgA+ cells and IgA-secreting cells in Peyer’s patches and spleens (Pestka et al., 19903, b; Bondy and Pestka, 1991). The mechanisms underlying this model appear to involve dysregulation of cytokine gene expression, which promote differentiation of IgA- secreting cells and systemic over production of IgA (Pestka, 2003). The purpose of this study was to test the hypothesis that fish can suppress serum IgA, serum IgA-1C elevation and kidney mesangial IgA deposition in DON induced experimental IgAN in mice. The results suggest that diets containing fish oil attenuated IgA production in this model. 29 MATERIALS AND METHODS Materials. All chemicals (reagent grade or better) were purchased from Sigma Chemical (St. Louis, MO) unless otherwise noted. The DON used in this study was produced in F usarium graminearum R6576 cultures and purified by the water-saturated silica gel Chromatography method of Witt et al., (1985). Purity of DON was verified by a single HPLC peak occurring at 224 nm. Concentrated toxin solutions were handled in a fume hood. Labware that was contaminated with mycotoxin was detoxified by soaking for >1 h in 100 mL/L sodium hypochlorite (Thompson & Wannemacher, 1986). Purified DON was added to powdered diets as described previously (Dong et al. , 1991). Animals. Male B6C3F1 mice (7 wk old) were obtained from Charles River (Portage, MI). Mice were housed singly in environmentally protected cages, which consisted of a transparent polycarbonate body with a filter bonnet, stainless steel wire lid and a layer of heat-treated hardwood chips. The mice were allowed to acclimate for at least 7 cl to their new housing, regulated temperature (25°C), feed, 12-h lightzdark cycle and to a negative-pressure ventilated area before feeding regimens began. All animal handling was conducted in accordance with guidelines established by the National Institutes of Health. Experiments were designed to minimize numbers of animals required to adequately test the proposed hypothesis and were approved by Michigan State University Laboratory Animal Research Committee. Diets and experimental design. The diet was based on the AIN—93G formulation (Reeves et al., 1993) and consisted of the following ingredients (per kg): 35 g AIN-93G mineral mix, 10 g AIN-93 vitamin mix, 200 g casein, 397.5 g cornstarch, 132 g Dyetrose (dextrinized cornstarch), 50 g cellulose, 3 g L-cystine, 2.5 g Choline bitartrate, 14 mg 30 TBHQ, which were purchased from Dyets, and 100 g sucrose, which was obtained from a local commercial source. Control and menhaden fish oil (each already containing 200 mg/kg of TBHQ) from Dyets, were used to amend the basal diet to yield three experimental diet groups containing the following finer kg): 1) 70 g corn oil; 2) 70 g corn oil and 10 mg DON; and 3) 10 g corn oil, 60 g fish oil and 10 mg DON. The PUFA compositions of these corn oil and fish oil-containing diets are shown in Table 2.1. Mice were fed for 20 wk and body weight were measured every wk. Animals were bled at 4 wk intervals. Serum was analyzed for IgA, IgG and IgM. After bleeding at wk 20, mice were killed by cervical dislocation. Spleens and Peyer’s patches were removed aseptically for preparing cell cultures. Kidneys of each mouse were removed for immunohistochemical examination. Measurement of immunoglobulin and IgA-1C. Senun IgA, IgG and IgM were measured by capture ELISA (Dong et al, 1991) using mouse immunoglobulin reference serum (Bethyl Laboratories, Montgomery, TX), goat anti-mouse IgA, G and M (heavy chain specific) and peroxidase-conjugated goat IgG fraction to mouse IgA, IgG, IgM (Organon Teknika, West Chester, PA). For detection of IgA-1C, diluted serum samples were precipitated using 70 g/L polyethylene glycol (PEG 6000; Sigma) (Irnai et al., 1987). IgA in precipitate was redissolved in PBS and quantified by ELISA. Assessment of kidney mesangial IgA deposition. At experiment termination, kidneys of each euthanized mouse were removed, cut in half and immediately frozen in liquid nitrogen. Each kidney was sectioned to 7 pm with a cryostat (Reichert-Jung, . Cambridge Instruments, Buffalo, NY) and stained for IgA deposition with fluorescein 31 .93? won wocfiucoo «0% BE. .8523 0.8 m28 been 8.88 05 >30 ms - 9.: 3m 3 - $592 32 - 8-598 no - 6-518 3 - 9.5me 3 OS 6-32: nfl 3N 8-53: 2 Z on: «.2 M2: . 98 on 38 82$ E E A E 32 woozwv :0 68 28 9am 30% 3595.695 8m new: $6 mo :oEmon—Eoo 28 .93 mm 2an 32 isthiocyanate—labeled goat anti-mouse IgA (Sigma) as previously described (Valenzuela & Deodhar 1980). Sections from each animal were viewed under a Nikon Labophot epifluorescence microscope through a Sony (Tokyo, Japan) imaging system consisting of CCD Video Camera DXC-lSlA and a PVM 13442Q Trinitron Video Monitor. Ten glomeruli from each section were randomly selected and the image captured on a microcomputer using a Snappy Video System (Play Incorporated, Rancho Cordova, CA). Mean fluorescence intensity was determined in polygons encircling the glomeruli using UTHSCSA Image Tool Software V 1.2 (available via anonymous FTP at fip://maxrad6.uthscsa.edu). This system generates a quantitative value from an encircled immunofluorescent stained glomerulus in a frozen frame and calculates the average brightness for the circled area based on each pixel of the screen included in the circle. The pixels in the circled area were measured on a grayness scale that ranged from 0 (black) to 255 (white). Cell culture. Spleen and Peyer’s patches were teased apart in harvest buffer consisting of 0.01 mol/L PBS, pH 7.4 containing 20 mL/L heat inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY), 1 x 105 U/L penicillin and 100 mg/L streptomycin. Tissues were passed through a sterile lOO-mesh stainless screen in the same buffer and cell suspensions held on ice for 10 min to allow settling of tissue particles. Supernatant was removed following centrifugation at 450 x g for 10 min. Erythrocytes were lysed for 3 min at room temperature in 0.02 mol/L Tris buffer (pH 7.65) containing 0.14 mol/L ammonium chloride. Cells were centrifuged, resuspended in RPMI-1640 medium supplemented with 100 mL/L FBS, 1 mmol/L sodium pyruvate, l x 105 U/L penicillin, 100 mg/L streptomycin, 0.1 mmol/L nonessential amino acid and 50 umol/L 2- 33 mercaptoethanol and then counted using a hemacytometer (American Optical, Buffalo, NY). Cells (2 x lOg/L) from individual mice were cultured separately in 1 mL of medium in flat-bottomed 24-well tissue culture plates (Fisher Scientific, Corning, NY) at 37°C under 70% CO; in a humidified incubator. Supematants were collected at 5 d and stored in aliquots at -20°C until analysis for IgA. Statistics. Data were analyzed using the Sigma Stat for Windows (Jandel Scientific, San Rafael, CA). Data were subjected to one-way ANOVA and pairwise comparisons made by Bonferroni or Student-Newman-Keuls methods. If data did not meet the normality assumption, they were subjected to Kruskal-Wallace ANOVA on Ranks and pairwise comparisons made by Dunn’s or Student-Newman Student-Newman- Keuls methods. Differences were considered significant at P < 0.05. 34 RESULTS Dietary DON retarded the weight gain and fish oil did not alter the inhibition on body weight gain induced by DON (Figure 2.1). DON induced IgA elevation at wk 4, 8, 12, 16 and 20 from 0.3 to 15 fold, respectively (Figure 2.2). Serum IgA increased only from 0.7 to 6-fold, respectively in mice exposed to fish oil and DON. Serum IgA in the DON and fish oil + DON groups did not differ until after 12 wks (Figure 2.2). Serum IgA-1C followed similar trends with IgA-1C concentrations in the DON group being consistently.( p = 0.05) higher than fish oil plus DON group (Figure 2.3). IgA production ex vivo by spleen and Peyer’s cells cultured was assessed. Spleen and Peyer’s patch cultures from DON groups produced more IgA than did their corresponding control groups (Figure 2.4). Supernatant IgA levels in spleen cultures from the fish oil + DON group were also higher than control group but were lower than DON group. Supernatant IgA levels in Peyer’s patch cultures from the fish oil + DON group were not significantly different from the control group or the control + DON group. Irnmunofluorescence microscopy revealed strong mesangial deposition of IgA in mice fed DON compared with mice fed corn oil only (Figure 2.5). IgA deposition was lower in mice fed fish oil and DON. Mean glomerular fluorescent intensities for DON and fish oil + DON groups were 3 and 1.3 fold greater than that of the control group (P < 0.05). DON did not alter sermn IgG (Figure 2.6) and IgM (Figure 2.7) concentration at wk 4, 8, l6 and 20. The IgG and IgM level in fish oil + DON groups were also not different from that of DON group. 35 .wu: 4E3 one EobbE “a 3on :80 mo :38 2a 823/ dozen mambo.“ 33 ON .8>o 85%“: Emma...» men we 88:9 28+ .6 an e8 209.838 4288 a 8E a sweat Ewe; boom 3 2:3... (5) iufiieM Apoa $0 9: our 2: cm om ow om o _ _ _ p L b ON I mm 1 on r on r ov 209.6 5... 111.1 209.928 II. in .928 IIOII me 36 .Amodvmv 5&6 $32 “:80me 2:3 9332: Pam .wna 42mm H 508 Pa QEEEV 323/ .> ON or N? w v fin o m m m m n n m I N o 0 D l V n a u o o I w n .. or -- I NF . 200 + :0 cm: § 3 zOO+_o.=coo I .928 D .. 3. (|w/6w) Lumes u! v6| 37 Amodvnc 8th .832 Bobbmu 53, 8me8 mam .wnc .EMm H 588 Pa QEEHV 833/ 494mm an 856on mm? 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I our Iovw (Iw/Bfi) W5l 42 DISCUSSION It has been previously shown that mice fed DON exhibited a high level of polyclonal serum IgA, IgA-1C, mesangial IgA deposition, systemic and mucosal compartment IgA-bearing and IgA-secreting lymphocytes (Pestka et al., 1989; Dong et al., 1991; Dong & Pestka, 1993; Greene et al., 1994b; Rasooly & Pestka, 1994), which mimic the early stage of human IgAN. Our data here were consistent with previous results. At the same time, diet containing fish oil attenuated DON-induced IgA elevation, mesangial IgA deposition and ex vivo IgA production. These results provided a unique model for understanding the development of IgA nephropathy and how (n-3) might affect early development of IgAN. It should be noted that this model only mimics the early stage of IgAN with high serum IgA and kidney IgA deposition without hematuria and proteinuria during the 20- wk period. Although fish oil has been used for IgAN patients due to its multiple anti- inflammatory effects during the later stage of the disease (Donadio et al., 1999), our results nevertheless suggested that fish oil might also block IgA production. As the fundamental abnormality in IgAN lies within the IgA system (Harper et al., 1996), the observation that fish oil inhibited overactive IgA production, IgA-1C development and mesangial IgA deposition indicated that this nutritional regimen could have prophylactic value for IgAN at the onset of very early clinical signs. DON retarded weight gain throughout the feeding study, which confirmed the previous results of DON-feeding studies (Rotter et al., 1996). The effects are likely to be related to DON-induced inhibition of protein translation (Rotter et al., 1996) and 43 depressed nutrient absorption (Maresca, 2002). Fish oil treatment did not alter DON effect on body weight loss. Previous studies indicate that DON specifically elevates IgA concentration but decreases or has no effect on serum IgG and IgM concentration and kidney deposition in different feeding studies (Banotai et al., 1999; Greene et al., 1994b; Rasooly et al., 1992; Dong et al., 1993). This suggests that DON’s effects are isotype-specific (Pestka et al., 1990b). In this study, the serum IgG level was not changed and IgM level was decreased at 16 wk, which is consistent with previous data. Since IgA is mainly formed in the mucosal compartment, DON might target IgA production at the level of the intestinal tract (Pestka, 2003). IL-6 plays a critical role in B cell terminal differentiation, antibody-secreting cell accumulation and IgA production in the gut (Bao et al, 1998). Recently, ex vivo cell reconstitution (Y an et al., 1988), antibody neutralization (Y an et al., 1997) and IL-6 deficient mice study indicated that IL-6 appears to be the most important cytokine in DON-induced IgAN (Pestka & Zhou, 2000). In the future, fish oil effects on DON- induced IL-6 gene expression should be examined. 44 CHAPTER 3 Docosahexaenoic Acid and Eicosapentaenoic Suppress Deoxynivalenol Induced Experimental Immunoglobulin A N ephropathy in Mice 45 ABSTRACT Primary IgA nephropathy (IgAN) is an immune complex disease with elevated serum IgA and kidney mesangial IgA deposition as its hallmaks. (n-3) polyunsaturated fatty acids (PUFAs) can retard the progression of IgAN in humans and in a mouse model induced by trichothecene mycotoxin deoxynivalenol (DON). In order to assess the efficiency of two major (n-3) PUFAs found in fish oil, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) effects on DON-induced IgAN were evaluated. Mice were fed for 18 wk with AIN-93G diets containing: 1) 10 g/kg corn oil plus 60 g/kg oleic acid (control); 2) 10 g/kg corn oil plus 35 g/kg oleic acid and 25 g/kg DHA-enriched fish oil (DHA); 3) 10 g/kg corn oil plus 33 g/kg oleic acid and 27 g/kg EPA-enriched fish oil (EPA); and 4) 10 g/kg corn oil plus 37 g/kg oleic acid and 23 g/kg DHA + EPA (1:1) enriched fish oil (DHA + EPA). DON significantly increased serum IgA, IgA immune complexes and kidney mesangial IgA deposition. DHA, EPA and DHA/EPA significantly attenuated all three immunopathological parameters as well as IgA secretion by spleen cells. Pre-feeding of DHA/EPA significantly reduced serum interleukin 6 (IL- 6) induced by acute oral exposure to DON. Taken together, both DHA and EPA were effective at ameliorating DON-induced IgAN and E-6 gene expression, which is required for IgA production. 46 INTRODUCTION Primary IgA nephropathy (IgAN) is an autoimmune disease, which mainly affects children and young adults. About 20-40 % of IgAN patients develop end stage renal disease. High serum IgA and IgA immune complexes (IgA-1C) are considered to be important etiologic factors for IgAN (Endoh et al., 1984 Layward et al., 1993; Suga 1985; van den Wall Bake et al., 1989). Serum IgA-1C, dimeric and polymeric IgA might deposit in the kidney and cause inflammation by activating complement via the alternative pathway (Floege and Feehally, 2000). Renal injury in IgAN ranges from glomerular hypercellularity, tubular atrophy, and interstitial fibrosis to renal failure in the end. Deoxynivalenol (DON) is a trichothecene mycotoxin produced by the fungus of F usarium genus, which can bind readily to eukaryotic 6OS ribosome and prevent polypeptide initiation and elongation (Rotter et al., 1996). Chronic exposure to DON can induce IgAN in mice, characterized by high serum IgA, IgA-1C and IgA deposition in the kidney (Greene et al., 1994a,b; Rasooly et al., 1994; Yan et al., 1998). Antigenic stimulation of the gut mucosal immune system apparently contributes to DON induced IgA dysregulation (Pestka, 2003). Although the gut acts as an entry to a vast array of foreign antigens, antibody responses are seldom induced due to the mucosal tolerance. Acute oral exposure of DON can induce proinflammatory cytokines IL-1 , IL-6 and TNF- a (Dong et al., 1994; Wong et al., 2001; Moon and Pestka 2003), which might overide the mucosal tolerance and promote IgA production (Pestka, 2003). Therapeutic targets for IgAN include decreasing the synthesis of IgA-1C, inhibition of IgA binding to the mesangial cell, antagonizing the effects of platelet 47 derived growth factor receptor (PDGF) and transform growth factor (TGF)[3, reducing infiltrating neutrophiles (Lai et al., 2002) and blocking the production of lipid inflammation mediators (Wardle et al., 2000). Based on these considerations (n-3) PUFA might be a promising treatment because of their anti-inflammatory effects (Cheng et al., 1990; Donadio, 1991; Pettersson et al., 1994; Endres et al., 1995; Whelan 1996). Clinical trials in high-risk patients with IgAN have shown that fish oil retards the IgAN progression by reducing the inflammation and glomerulosclerosis (Donadio, 2001a,b). Recently, feeding studies in our lab have revealed that supplementation with fish oil (4- 6g/ 100g diet) can also block IgA dysregulation and deposition in the kidney of DON-fed mice (Pestka et al., 2002). DHA and EPA are primary (n-3) PUFAs found in fish oil. Here, it was hypothesized that both DHA and EPA can inhibit DON-induced IgAN. This hypothesis was confirmed and correlated with inhibition of cytokine IL-6 expression in vivo in response to DON stimulation. 48 MATERIALS AND METHODS Materials. All chemicals (reagent grade or better) were purchased from Sigma Chemical (St. Louis, MO) unless otherwise noted. The DON used in this study was produced in Fusarium graminearum R6576 cultures and purified by the water-saturated silica gel chromatography method of Witt et al. (1985). Purity of DON was verified by a single HPLC peak occurring at 224 nm. Concentrated toxin solutions were handled in a fume hood. Labware that was contaminated with mycotoxin was detoxified by soaking for >1 h in 100 mL/L sodium hypochlorite. Purified DON was added to powdered diets as described previously (Dong et al., 1991). Animals. Male B6C3F1 mice (7 wk) were obtained fiom Charles River (Portage, MI). Mice were housed singly in environmentally protected cages, which consisted of a transparent polycarbonate body with a filter bonnet, stainless steel wire lid and a layer of heat-treated hardwood chips. The mice were allowed to acclimate for at least 7 d to their new housing, regulated temperature (25°C), feed, 12-h light: dark cycle and to a negative- pressure ventilated area before feeding regimens began. All animal handling was conducted in accordance with guidelines established by the National Institutes of Health. Experiments were designed to minimize numbers of animals required to adequately test the proposed hypothesis and were approved by Michigan State University Laboratory Animal Research Committee. Diets and experimental design. Experimental diets were based upon purified AIN-93G formulation (Reeves et al., 1993), which consisted of the following ingredients (per kg): 35 g AIN-93G mineral mix, 10 g AIN-93 vitamin mix, 200 g casein, 397.5 g 49 cornstarch, 132 g dyetrose (dextrinized cornstarch), 50 g cellulose, 3 g L-cystine, 2.5 g chlorine bitartrate, 14 mg TBHQ, 100 g sucrose and 70 g oil. In study 1, corn oil (Dyets), oleic acids (Dyets), Dim-enriched oil (containing 604 g/kg DHA, 71 g/kg EPA, Ocean Nutrition Canada Ltd), EPA-enriched oil (540 g/kg EPA, 71 g/kg DHA, Ocean Nutrition Canada Ltd.) and DHA/EPA lipid mix (402 g/kg DHA, 341 g/kg EPA, Ocean Nutrition Canada Ltd) were used to amend the basal diet to yield five experimental groups: control, control+DON, DHA+DON, EPA+DON and DHA/EPA + DON (Table 3.1). Diets were prepared every two wk, stored at -20°C and provided fresh to the mice each day. Final lipid composition of experimental diets is shown in table 3.2. Mice (n=10) were fed diets for 18 wk. Food intake was measured daily and body weight was monitored weekly. Animals were bled at 4 wk intervals and blood samples were used for plasma IgA, IgA-1C measurement. At Week 18, mice were anesthetized with methoxyfluorane and killed by cervical dislocation. Spleens and Peyer’s patches were removed aseptically for preparing cell cultures. Kidneys from each mouse were removed for immunohistochemical examination. Study 2, corn oil, oleic acids and DHA/EPA lipid mix enriched (n-3) PUFA were used to amend the basal diet to yield 2 diet groups (n=10) (Table 3.3): control and DHA/EPA. Mice (n=5) were fed control or DHA/EPA lipid mix for 8 wk. At experiment termination, one half of the mice were exposed to DON (25mg/kg. body weight) by gavage and the other half was exposed to vehicle. After 3 hrs mice were anesthetized, bled and their spleen and Peyer’s patches were removed. Serum IL-6 and IL-6 mRNA in I spleen and Peyer’s patches were measured. 50 Lipid extraction and fatty acids analysis. To confirm tissue incorporation of (n-3) PUFA after 18 wk of feeding the experimental diets, liver phospholipid contents were measured by a modification of the method of Hasler et al. (1991) with the assistance of Dr M Bennink (Michigan State University) and Sherry Shi (Michigan State University). This organ was selected as a tissue surrogate because the two immune organs of concern, spleen and Peyer’s patches, were completely utilized for cell culture studies. Briefly, mouse livers were homogenized with a chloroformzmethanol (2:1) solution. Total phospholipids were extracted, separated, and collected using a silica column. Phospholipid samples were dried and esterified with methanolzacetonitrilezboron trifluoride (11:4:5, by vol). The resulting fatty acid methyl ester (FAME) was extracted with hexane. After centrifugation at 1200 x g for 5 min, the hexane supernatant was decanted, dried, and redissolved in chloroform and then analyzed by GC utilizing a Varian 3700 GLC. Fatty acids profiles were identified by comparing the retention times with those of appropriate standard FAME (N u-Check-Prep). IgA and IgA-1C measurement. 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A m. 0.0 0 ,, 0 m _ , 4.0 0.. ._ . , 000 .. .H .0 n H 0 0 Na 5.20 Fxmw NOLLV‘IAHOHdSOHd BALLV‘IEH 102 DISCUSSION Potential effects of fish oil and (n-3) PUFA supplementation for human IgAN patients have been demonstrated by clinical studies which report marked retarded renal disease progression in association with reduced inflammation and glomerulosclerosis (Grande and Donadio, 2001; Donadio, 2001b). As shown here and previously (Pestka et al., 2002; Jia et al., 2004), (n—3) PUFAs impair production and accumulation of serum IgA potentially in a chemical-induced IgAN model. These preclinical findings are valuable because they suggest that consumption (n-3) PUFAs might also have potential benefits for early interventionand prophylaxis in persons with a familial history of IgAN or in patients diagnosed to be at an early stage of IgAN. The results presented here indicate that DHA-enriched oil at 60 g/kg completely abrogated increases in serum IgA as early as 4 wk, whereas, at 2 and 10g lkg DHA, efficacy was detectable only at wk 16. The highest DHA concentration used here was also the most effective at inhibiting serum IgA-IC elevation and mesangial IgA deposition. In our prior study (Jia et al., 2004), DHA-enriched oil at 20 g/kg, partially inhibited DON-induced IgAN beginning at 12 wk of toxin exposure. Thus, a correlation exists between the dietary concentration of DHA and its inhibitory effects on DON- induced IgAN. The cytokine IL-6 drives IgA-committed B cells to terminally differentiate to IgA-secreting plasma cells (Beagley et al., 1989). Differentiated IgA-secreting B cells can migrate to distal mucosal and systemic sites, survive for prolonged periods and produce IgA. IL—6 deficient mice are resistant to DON-induced IgA elevation (. Pestka et al., 2000). Ex vivo studies also implicate a role for IL-6 in DON-induced IgAN (Yan et 103 al., 1997; 1998). A critical observation made here was that consumption of 10 and 60 g/kg DHA diet significantly reduced splenic IL-6 mRNA levels to below those of mice fed control or control + DON diets. Since hnRNA is a precursor species observed in cells prior to RNA splicing to mRNA, its abundance can be used as a surrogate for the run-on assay in detection of gene transcriptional activity (Johnson et al., 2003). The finding that DHA-enriched oil consumption significantly blocked accumulation of IL-6 hnRNA as well as IL-6 mRNA suggests that DHA blocks IL-6 gene expression at the transcriptional level. This contention is further supported by the observation that mice consuming 60 g/kg DHA for 4 wk exhibited markedly less induction of IL-6 mRNA and hnRNA expression following acute DON exposure. These results have potential clinical significance because: IL—6 is believed to play a contributory role in human IgAN (Donadio and Grande, 2002) and because IL-6 production by peripheral blood mononuclear cells is decreased in persons consuming (n-3) PUFA which corresponds to increased plasma and cell membrane (n-3) PUFA incorporation (Trebble et al., 2003). Differences between lL-6 mRNA expression following chronic and acute DON exposure (ie. no induction vs. induction) might arise from two factors. First, DON—fed mice (20 mg/kg of diet) ingest approximately 2 g/d of food, an exposure level of 3 mg/kg body weight DON per (1 can be estimated. This is much lower than the acute dose (25 mg/kg body weight) used. Second, in this study, experiments were terminated in the morning. However, mice typically will eat most food when daily light period ends and then consume smaller amounts sporadically throughout the day. Since E-6 is an early response gene with rapid turnover (Ross, 1995), its induction at the local sites in DON- fed mice might have escaped detection in our model but was, nevertheless, sufficient to 104 chronically promote B cell differentiation to IgA secretion with attendant cumulative effects. These possibilities are consistent with our previous observations that DON enhances immunoglobulin response to commensal and self antigens (Rasooly and Pestka, 1992; Pestka et al., 19903) and that IgA-secreting cells, serum IgA, serum IgAalC and mesangial IgA remain elevated for at least 4 months after withdrawal of DON from diet (Dong and Pestka, 1993; Pestka et al., 1990b). The effects of DHA-enriched oil on IL-6 expression might be partially mitigated by attenuated expression of COX-2, an essential enzyme known to mediate functions of (n-3) and (n—6) fatty acids (Lee et al., 2003a). Moon and Pestka (2003a) found that COX- 2-deficient mice have significantly reduced capacity to respond to DON with increased IL-6 mRNA and serum IL—6 expression. The acute DON exposure data presented here suggest that DHA can reduce COX-2 mRNA expression with similar trends observed following DON feeding. DHA’s capacity to suppress COX-2 gene expression might thus be one upstream mechanism by which IL-6 expression is impaired and, ultimately, IgA production is attenuated. Another finding of this study was that AA concentrations in liver phospholipid were significantly depressed by DHA consumption. AA is considered to be a risk factor in IgAN (Holman et al., 1994; Leflmwith and Klahr, 1996). Decreasing AA concentration corresponded to reduction in serum IgA, serum IgA-1C, mesangial IgA and expression of IL-6 and COX-2. Reduced AA in phospholipids might decrease generation of inflammatory mediators such as PGE2 (Trebble et al., 2003) thereby attenuating mucosal inflammation and IgA production. In support of this contention, Bagga et al., (2003) demonstrated that (n-3) PUFAs impair PGE2 release but enhance PGE3 release by 105 macrophages in vitro. PGE3 is less efficient in inducing COX-2 and IL-6 gene expression compared to PGE2. Since DON induces PGE2 production in mice (Moon and Pestka, 2003a), it is reasonable to suggest that decreased AA tissue concentration could reduce IL-6 gene expression and attenuate overall IgA production in DON-exposed mice. This suppression could be synergistically enhanced by reduced COX-2 expression. The DHA-enriched oil used here also contained EPA (113 g/kg) and it thus is not surprising that the'tissue phospholipids from mice fed this oil also contained elevated EPA concentrations. Since the inhibitory effect of this (n—3) PUFA on COX-2 enzyme activity is reportedly greater than DHA (Ringbom et al., 2001), EPA might be another key factor in suppression of DON-induced IgA dysregulation. However, this interpretation is tempered by previous observations in our model that DHA-enriched oil was somewhat more effective than EPA-enriched oil attenuating plasma IgA elevation (J ia et al., 2004a). Although it is not yet clear how DHA impairs IL~6 and COX-2 expression, in vitro studies have provided evidence that (n-3) PUFAs inhibit kinase activities in several cell phenotypes (Denys et al., 2001). Particularly noteworthy are the MAPKs, which couple cell-surface receptors to critical regulatory targets and transcription of genes including COX-2 and IL-6 (Hedges et al., 2000). Yusufi et al. (2003) found that DHA, but not EPA, decreased ERK activation in mesangial cells, whereas JNK activity was increased and p38 activity was not significantly affected. DON can activate ERK, JNK and p38 both in macrophages and mice and this contributes to transcriptional and post- transcriptional up-regulation of proinflammatory genes (Zhou et al., 2003; Moon and Pestka, 2002; Fan et al., 2000). Recently, Moon and Pestka (2003b) found that 106 consumption of 60 g/kg menhaden fish oil by mice suppressed DON-induced ERK 1/2 and JNK 1/2 phosphorylation but not p38 in spleen. In contrast, results presented herein demonstrated that prior consumption of 60 g/kg DHA for 4 wk not only impaired DON- induced ERK 1/2 and JNK 1/2 phosphorylation but p38 phosphorylation as well. There is one difference between these two investigations. Since mice here consumed 36 g/kg (n-3) PUFA in DHA-enriched oil whereas the previous study’s mice consumed 16 g/kg (n-3) in menhaden oil PDFA (Moon and Pestka, 2003b), it is possible that the lack of significant p38 effect in the latter study might be dose-related. Further investigation of these possibilities should involve assessing effect of DHA dose on DON-induced p38 activation as well as the effects of concurrent ingestion of AA or other (n-6) PUFA. It was interesting to note that while DHA consumption suppressed DON-induced phosphorylation of all three MAPK families, these inhibitions were quite modest relative to rather extensive IL-6 inhibition. Two considerations are preeminent here. First, Western analysis, provides a qualitative picture of total MAPK phosphorylation status in a tissue. In reality, MAPK signaling modules interact via a series of sequential binary interactions to create protein kinase cascade (Morrison and Davis, 2003). These modules are likely to be associated with scaffold proteins which can allow sorting of relevant and irrelevant stimuli and provide spatial and temporal control of MAPK signaling. Thus, DHA might affect one or more specific modules that control induction of IL-6 by DON but this cannot be resolved by Western analysis from other less relevant or irrelevant modules. Second, the analysis conducted here represents the entire spleen cell population. While we speculate DHA’s effects on MAPKs and IL-6 are mediated in macrophages and dendritic cells, Western analysis does not allow discrimination of such effects from 107 those of B cells, T cells, epithelial and endothelial cells. Thus, future studies of DHA’s effects in this model require consideration of the spatio-temporal context of MAPK targets as well as cell phenotypes affected. Other mechanisms besides interference with MAPK activation might explain DHA inhibition of IL-6 and COX-2 gene expression. For example, a recent study suggests that DHA inhibits COX-2 mRNA expression though toll-like receptors (Lee et al., 2003b). Also, lipid rafis are important signaling platforms for T cell activation. Sphingomyelin, which facilitates rafi formation, is significantly decreased in T cells from (n-3) PUFA-fed mice (Fan et al., 2003). In vitro studies using the Jurkat T cell line also indicate that (n-3) PUFAs selectively modify lipid rafts and suppress signal transduction. Therefore, DHA might alter surface receptor protein function and lymphocyte signal transduction by altering raft phospholipid composition. These and other signaling mechanisms require further exploration. Typical (n-3) PUFA intake recommendations for healthy people are 0.3 to 0.5 g/d for DHA plus EPA along with 0.8 to 1.1 g/d for (it-linolenic acid (Institute of Medicine, 2002). However, consumption of more DHA and EPA might be required for disease prophylaxis and therapy. The combined DHA and EPA concentrations in the diets employed here ranged from 1 to 36 g/kg, which would account for 0.2 to 7.2% of total caloric intake. Upon extrapolation, a human consuming 2000 kCal/day would need to ingest 0.5 tol6 g/d DHA to correlate with that amount consumed by the mouse per (1 per kg body weight in this experiment. Relative to prophylaxis and therapy for immune diseases, the upper level of safe intake of (n-3) PUFA is a critical consideration. Although the FDA has ruled that intakes 108 of up to 3 g/d of marine (n-3) PUFAs in diet are generally recognized as safe (Department of Health and Human Services, 1997), higher doses have been used in clinical trials. Two 4-year prospective studies have demonstrated that high dose (n-3) PUFA (3.76g EPA plus 2.94g DHA per (1) and low dose (1.88g EPA, 1.47g DHA per (1) are equivalent in treating IgAN (Donadio et al., 2001). In another fish oil therapy study, doses of (n-3) PUFA (4.3g EPA, 2.8 g DHA/d) were used for 5 years without any observed side effects (N g, 2003). In a 6-month trial, 6.9 g/d EPA plus DHA was provided to 275 patients, no side effects were found (Leaf et al., 1994). No adverse effects were reported when subjects consumed up to 1.8 g/d of EPA and DHA and 9.0 g/d ALA over a 4 wk period (Mantzioris et al., 2000). Further study is needed to determine the requisite amounts of (n-3) PUFA in diet to achieve sufficient tissue phospholipid concentrations for optimal, efficacious prevention and treatment of IgAN and other immune-related diseases. The approach described herein offers one animal model for obtaining such preclinical data. In summary, the results presented here suggest that consumption of diets containing DHA—enriched oil significantly inhibited DON-induced IgAN in dose- dependent fashion. These effects correlated with impairment of IL-6 and COX-2 expression as well as MAPK activation. It might be speculated that DHA consumption will have prophylactic value in suppressing elevation of IgA and IgA-1C among persons with genetic predisposition for IgAN or who have been diagnosed with the disease. Future perspectives should include improved understanding of the mechanistic basis for DHA’s effects in this IgAN model and determination of the required intake to establish 109 prophylactic and therapeutic tissue concentrations that modulate critical molecular targets. 110 CHAPTER 5 Role of Cyclooxygenase-2 Gene In Deoxynivalenol-Induced Immunoglobulin A Nephropathy 111 ABSTRACT Ingestion of the trechothecene mycotoxin deoxynivalnenol (DON) induces serum IgA elevation and kidney mesangial deposition in a manner that mimics human IgA nephrophathy (IgAN). Previous studies indicate that interleukin-6 (IL-6) is crucial for DON—IgAN and that DON-induced cyclooxygenase-2 (COX-2) might contribute to E-6 upregulation. (N-3) Polyunsaturated fatty acids (PUFAs) inhibit IL-6 and COX-2 gene expression as well as DON-induced IgAN. It was therefore hypothesized that COX-2 and its metabolites thus are essential for DON-IgAN. In this study, COX-2 knockout mice and COX-2 specific inhibitor, VIOXX, were employed in order to test the contribution of COX-2 to this model. The results demonstrated that neither COX-2 deficiency nor dietary exposure to COX-2 enzyme activity inhibitor, VIOXX, could block DON-induced serum IgA, IgA-1C accumulation, IgA kidney deposition and spleen IgA secretion. Rather, these treatments promoted DON-induced serum IgA elevation. These results suggest that COX-2 might not be required for DON-induced IgAN and fiirther, that COX-2 inhibitor, VIOXX, would be contraindicated for the prevention of early stages of IgAN. 112 INTRODUCTION Cyclooxygenases (COX) convert arachidonic acid (AA) released from membrane phospholipids to prostanoids. COX-1 is a constitutive enzyme, which mediates cell homeostasis. COX-2 is an inducible, immediate-early gene product, which is primarily responsible for increased prostaglandin (PG) production during inflammation, reproduction and carcinogenesis. Expression of COX-2 is low in most tissues, but can be readily induced by cytokines, growth factors and chemicals (Ristimaki, 2004). The trichothecene mycotoxin, deoxynivalenol (DON), induces COX-2 gene expression by promoting transcriptional activity and mRNA stability via mitogen activated protein kinase (MAPK) signaling pathways in vitro and in vivo (Moon and Pestka, 2002; Moon et al., 2003). The observations that DON upregulation of IL—6 production is significantly reduced by COX-2 inhibitors indomethacin and NS-398 (Moon and Pestka, 2003) and that COX-2 knockout mice significantly reduced splenic IL-6 mRNA and serum IL-6 level in response to oral DON exposure (Moon and Pestka, 2003) suggest that DON-induced COX-2 gene expression and resultant COX-2 metabolites might contribute, in part, to subsequent upregulation of IL-6 gene expression. IgA nephropathy (IgAN) is the most common form of glomerular nephritis, which mainly affects children and young adults (Endo, 1997). Clinical diagnosis of IgAN is based on the presence of kidney mesangial IgA deposition (Ibels and Gyory, 1994). Serum IgA concentration is considered to be an important etiological factor for IgAN (Feehally et al.,1997; Endo, 1997). It has been shown that mice exposed to a diet containing DON developed IgAN with high serum IgA (Pestka et al., 1989; Dong et al., 1991; Dong and Pestka, 1993 and Pestka, 2003). IL-6 gene plays an important role in the 113 development of IgAN induced by DON (Pestka and Zhou, 2000). DON might destroy mucosal tolerance by induction of IL-6 and promote IgA production, theraby contributing to systemic compartment IgA elevation (Pestka, 2003). Since (n-3) polyunsaturated fatty acids (PUFAs), such as DHA, inhibit DON-induced IgAN as well as COX-2 and IL-6 gene expression (J ia et al, 2004), it was hypothesized that direct suppression of COX-2 by (n-3) PUFAs might contribute to their inhibition on IL-6 gene expression and IgAN. Therefore, COX-2 knockout mice and COX-2 enzyme inhibitor, VIOXX, were employed in this study to assess the role of COX-2 in DON-induced experimental IgAN. The results indicated that both COX-2 knockout mice and mice fed with COX-2 specific inhibitors, VIOXX, were not resistant to DON-induced IgAN but rather exhibited even higher serum IgA levels than mice fed DON alone. 114 MATERIALS AND METHODS Materials. All chemicals (reagent grade or better) were purchased from Sigma Chemical (St. Louis, MO) unless otherwise noted. VIOXX (Merck & Co Inc, Whitehouse Station, NJ) was obtained through Michigan State University Clinical Center. The DON used in this study was produced in F usarium graminearum R6576 cultures and purified by the water-saturated silica gel chromatography method of Clifford et al. (2003). Purity of DON was verified by a single HPLC peak occurring at 224 nm. Concentrated toxin solutions were handled in a fume hood. Labware that was contaminated with mycotoxin was detoxified by soaking for >1 h in 100 mI/L sodium hypochlorite. Purified DON was added to powdered diets as described previously (Dong and Pestka, 1993). Animals 36,129P2—Ptgs2’m’Sm" (002181-M; cox-2 knockout) mice those are homozygous for a targeted disruption of COX-2 gene (Morham et al, 1995), and B6, 129P2-Ptg52'm’5m (002181-W) mice which served as wild-type controls, were obtained at 7 wk of age from Taconic (Germantown, NY USA). Mice were housed in environmentally protected cages, which consisted of a transparent polycarbonate body with a filter bonnet, stainless steel wire lid and a layer of heat-treated hardwood chips. The mice were allowed to acclimate for at least 7 d to their new housing, regulated temperature (25°C), feed, 12-h light and dark cycle and to a negative-pressure ventilated area before feeding regimens began. All animal handling was conducted in accordance with guidelines established by the National Institutes of Health. Experiments were designed to minimize numbers of animals required to adequately test the proposed hypothesis and were approved by Michigan State University Laboratory Animal Research Committee. 115 Diets and experimental design. For studies with COX-2 knockout mice, diet was based on the AlN-93G formulation of Reeves et al. (1993) which consisted of the following ingredients (per kg): 35 g AIN-93G mineral mix, 10 g AIN-93 vitamin mix, 200 g casein, 397.5 g cornstarch, 132 g dyetrose (dextrinized cornstarch), 50 g cellulose, 3 g L-cystine, 2.5 g choline bitartrate, 14 mg TBHQ, 100 g sucrose, 10 g corn oil (Dyets) and 60g high oleic acid sunflower oil (Hain pure food, Garden City NY). DON (10 mg/kg) was used to amend the basal diet to yield 3 experimental groups (n=5): wild type (WT) control, wild type (wt) + DON, mutant type (KO) + DON. For studies with COX-2 inhibitor, VIOXX was mixed in the above diet (0.0075% w/w) (Hegazi et al., 2003) and the B6C3F1 female mice were divided into three groups (n=6): control, control+DON and VIOXX + DON. Mice were fed for 16 wk, bled at 8 wk intervals and serum analyzed for IgA and IgA-1C. At wk 16, mice were killed by cervical dislocation. Spleens and Peyer’s patches were removed aseptically for preparing cell cultures. Detection of IgA and IgA-1C. IgA and IgA-1C was measured in serum by capture ELISA (Dong and Pestka, 1993) using mouse irnmunoglobin reference serum (Bethyl Laboratories, Montgomery, TX), goat anti-mouse IgA (heavy chain specific) and peroxidase-conjugated goat IgG fraction to mouse IgA (Organon Teknika, West Chester, PA). For detection of IgA-1C, diluted serum samples were precipitated using 70 g/L polyethylene glycol (PEG 6000; Sigma) (Irnai et al., 1987) and IgA in precipitate redissolved in PBS and quantified by IgA ELISA. Assessment of kidney IgA deposition. At experiment termination, kidneys of each euthanized mouse were removed, cut in half and immediately frozen in liquid nitrogen. 116 Each kidney was sectioned to 7 pm with a cryostat (Reichert-Jung, Cambridge Instruments, Buffalo, NY) and stained for IgA deposition with fluorescein isthiocyanate— labeled goat anti-mouse IgA (Sigma) as previously described (Jia et al., 2004). IgA immunofluorescence was assessed with a Nikon Labophot microscope (Mager Scientific, Dexter, MI). Three glomeruli from each section were randomly selected and mean fluorescence intensity was determined in polygons encircling the glomeruli using UTHSCSA Image Tool Software . V 1.2. (http://www.scioncorp.com/frames/fr_download_now.htrn). Pixels in the circled area were measured on a grayness scale that ranged from 0 (black) to 255 (white). Cell culture. Spleen and Peyer’s patches were teased apart in harvest buffer consisting of 0.01 mol/L PBS, pH 7.4 containing 20 ml/L heat inactivated fetal bovine serum (FBS, Gibco, Grand Island, NY), 1x105 U/L penicillin and 100 mg/L streptomycin. Tissues were passed through a sterile 100—mesh stainless screen in the same buffer and cell suspensions held on ice for 10 min to allow settling of tissue particles. Supernatant was removed following centrifugation at 450 g for 10 min. Erythrocytes were lysed for 3 min at room temperature in 0.02 mol/L Tris buffer (pH 7.65) containing 0.14 mol/L ammonium chloride. Cells were centrifuged, resuspended in RPMI-1640 medium supplemented with 100 mL/L FBS, 1 mmol/L sodium pyruvate, l x 105 U/L penicillin, 100 mg/L streptomycin, 0.1 mmol/L nonessential amino acid and 50 umol/L 2- mercaptoethanol and then counted using a hemacytometer (American Optical, Buffalo, NY). Cells (2x108/L) from individual mice were cultured separately in 1 mL of medium in flat-bottomed 24-well tissue culture plates (Fisher Scientific, Corning, NY) at 37°C 117 under 70 g/L C02 in a humidified incubator. Supematants were collected after 5 d and stored in aliquots at -20°C until analysis for IgA. Statistics. Data were analyzed using the Sigma Stat for Windows (Jandel Scientific, San Rafael, CA). Data were subjected to one-way ANOVA and pairwise comparisons made by Bonferroni or Student-Newman-Keuls methods. If data were not normally distributed, they were subjected to Kruskal-Wallace ANOVA on Ranks and pairwise comparisons made by Dunn’s or Student-Newman-Keuls methods. Differences were considered significant at P < 0.05. 118 RESULTS In the first study, dietary DON retarded weight gain in both wild type and COX—2 knockout mice (Figure 5.1). Mice fed DON exhibited significant elevation of serum IgA. COX-2 deficiency did not impair but rather instead promoted IgA concentration (Figure 5.2). DON also significantly induced serum IgA-1C elevation in mice, which was not suppressed in COX-2 knockout mice (Figure 5.3). DON treatment significantly induced IgA secretion from spleen (Figure 5.4) while COX-2 knockout mice are not resistant to DON-induced spleen IgA production. Significant mesangial IgA deposition was also observed in kidney from DON-fed wild type mice as compared to wild type control (Figure 5.5). Mesangial IgA deposition in DON-fed COX-2 knockout mice was also higher than wild type fed control diet but was at the same level with DON-fed wild-type mice. Thus COX-2 deficiency in mice could not attenuate induction of DON-induced IgAN. A second study was conducted in which the effects of specific COX-2 inhibition were evaluated in DON-induced IgAN. Similar with first study, DON impaired body weight increase (Figure 5.6). No differences in terminate body weight were found between DON and VIOXX plus DON group after 16 wk. At wk 16, DON also caused serum IgA to increase significantly (Figure 5.7). VIOXX did not inhibit but rather promoted DON-induced IgA elevation. DON also significantly induced serum IgA-1C, IgA deposition and spleen IgA secretion, but VIOXX treatment did not reduce these effects (Figure 5.8, 5.9, 5 .10). Thus, mice treated with COX-2 inhibitor behaved similarly to COX-2 knockout mice in their lack of resistance to DON-induced IgAN. 119 .EoEflomxo mo ES 05 we Emmoa boom Amc .08: “skunk—V S 96% come we Em“; >609 508 .«o 0822: can mosfi>fi>+ 9. (5) aseenu! mam/loos 120 £3 v 5 more Hoes 08mm 05 023 “on on 85 mam .G-m n 5 2mm “m 338 Be 822$ {mim— 3 @83on was» <3 880m 33 2 ~26 A fl 2: as 2m? can AOMV 8:: 38.8.3 $80 5 EH eaom oo 938 o: zoo co 38$ «a tow:— 2335: or NF v -o m m n .F m m .N S m n -m w n m a ....N w m :0 w ZOD+O¥I u ZOQ+P>>I r- ._.>>l -o n a \ 121 Amod v 5 comma 832 05am 2: 0236: at $5 mam .G-m u 5 2mm um 88.: 0.8 825» . .>v mEE. . . o a N - w - o ZOD+XXO_> I 20019500 0 or (ILU/Bw)vfi| wnJeS 126 Amod v .3 £56 5:2 25m 05 96: 8a at 65 mam .G-m u 5 SEE u” wanna 8a 333/ . Ed Amie 3 zoo as) 8% 8? oaaa E82 5 2&3 Beam 3 2:»:— . CON . oov cow ( Iw/fifi) OI-vfil wmes 127 Amod v .5 5&6 532 25m 2: 023 go: ow 85 mam .G-mn 5 2mm H :88 can 823/ .mmmbfia 0325 3 wogotoq mm? nowfiquw <3 E3332 .<3 umsoEéGn c23~72~§>8§£ £8883.“ 53> 38.8w Ea 3:288 33on 203 3289 33 c :96 923 $38.8 NXO; £5, we.“ 038 225m EmUom E noEmoqow <3 3388 8 Rama cu zom base 08 38mm 3 2:»:— o /4 o 0 +0 0,, o ..+ x/ 00 00 . - o m V . m D. 9 d w . o? m 0 u .2 M. a u S. . ow .M. d/ a m. R W 128 Amod v a: 9&6 632 2:3 05 PE: 8: cu 35 mam .G-mH5 2mm H 338 2a mo=_m> {mim— NE 8.5988 8? <3 d m 8m 35:3 20>» 3&3 x 3 £3 52% 4.3 £53 3? §8q8 585 as, 3 8:: ease ESE E 830308 <3 03> xo :0 waEE mmv ZOO mo Eoobm ofim 95w?— 0/4 000 ++ 09 x x/ 00 oo 00 90 xy Ax xo . 90 m H m m , Fe m. R F m n m. n T «O V 0‘", Rh w. «:0 ( 129 DISCUSSION COX-2 is the rate-limiting enzyme in the conversion of AA to prostanoids after AA is released from membrane phospholipids by phospholipase A2 (PLAz) (Ristimaki, 2004 ). The COX-2 products, PGs, are generally considered to be proinflammatory agents, but in vitro studies show that they could also play anti-inflammatory roles during certain situations (Takayama et al., 2000). Since high levels of IL-6 accompanies over- expression of COX-2 and non-steroidal anti-inflammatory agent (NSAIDS), COX-2 inhibitors, might be useful in treating chronic inflammatory conditions in which IL-6 is . abnormally elevated (Joy and Emily, 1997). IL-6 is a pleiotropic cytokine produced by monocytes/macrophages, fibroblasts, endothelial cells and astrocytes in response to infections and toxins. Since IL-6 plays an important role in DON-induced IgAN (Pestka and Zhou, 2000) and COX-2 gene deficiency partially blocks induction of IL-6 gene expression by acute DON exposure both in vitro and in vivo (Moon and Pestka, 2003) suggest COX-2 might mediate DON-induced IgAN by enhancing IL-6 gene expression (Moon and Pestka, 2003). However, the data presented here demonstrated that interference with COX-2 function did not prevent DON-induced IgAN but rather enhanced DON’s capacity to promote IgA elevation. COX-2 knockout mice completely lack normal lipopolysaccharide (LPS) induction of COX-2 mRNA and protein (Morham et al, 1995). Here we found that COX- 2 knockout mice could not resist DON-induced serum IgA, IgA-immune complex (IC) elevation, spleen IgA secretion and IgA deposition. As described above, COX-2 initiates the conversion of AA into PGs, whereas 5-1ipoxygenase (S-LO) generates leukotrienes (LT) from AA. Knocking out COX-2 gene might disturb AA metabolism by reducing 130 PGs but could result in increased LTB4 production (Tuo et al., 2004). Since LTB4 is also a potential inducer of IL-6 (Stankova and Rola-Pleszczynski, 1992), knocking out COX-2 must be insufficient to remove IL-6 production completely (Moon and Pestka, 2003). Such a disturbance in AA metabolism might account for the higher serum IgA in COX-2 knockout mice. Further studies on DON’s effects on S-LO gene expression and LTB4 production were therefore warranted. VIOXX is an NSAID that selectively inhibits COX-2 enzyme activity but not COX-2 gene transcription (Evans, 2003). Although selective COX-2 inhibitors have anti- inflammatory activity and can reduce proteinuria in experimental membranous glomerulonephritis, they also possess several side effects such as 1) impairing glomerular capillary-repair in rat anti-Thy 1.1-induced glomerulonephritis (Kitahara et al., 2002), 2) increasing the risk for serious cardiac and/or cerebrovascular events (Zhao et al., 2001) and 3) causing acute renal failure (Brater, 1999). In the DON-induced IgAN model, VOIXX did not reduce serum IgA, IgA-1C, IgA deposition and spleen IgA secretion but rather, promoted DON-induced serum IgA which is an early pathogenic factor for the development of IgAN (Feehally et al.,1997). These data are consistent with that of the COX-2 knockout study described above. Thus blocking COX-2 enzyme activity might not be effective reducing IL-6 gene expression and ultimately serum IgA production in mouse chronically exposed to DON. It should be therefore be further noted that VIOXX might cause damage of intestine duct (Leite et al., 2004), which could promote IgA production by exposing lymphoid tissues to food antigens. Since high serum IgA is an early etiologic factor for the development of IgAN, the observation that COX-2 gene knockout and VIOXX treatment did not reduce but 131 promote DON-induced IgA production suggested that COX-2 gene is not required for the development of DON-induced IgAN. Furthermore, regardless of mechanism, these preclinical data suggest that the COX-2 inhibitor, VIOXX, might not be good for the prophylaxis of IgAN due to its induction on IgA production. 132 CHAPTER 6 Docosahexaenoic Acid Inhibits CREB Activation and Interleukin-6 Gene Transcription Induced By the Ribotoxic Stressor Deoxynivalenol In Macrophage 133 ABSTRACT The trichothecene mycotoxin, deoxynivalenol (DON), induces experimental IgA nephropathy (IgAN) in mice by upregulating interleukin-6 (IL-6) gene expression. Consumption of (n-3) polyunsaturated fatty acids (PUFAs), including docosahexaenoic acid (DHA), retards DON-induced IL-6 and IgAN. The purpose of this study was to determine the effects of DHA consumption on DON-induced IL-6 mRNA transcription. DON significantly induced binding of CAMP response element binding protein (CREB), active protein (AP-l), but not nuclear factor K B (NFKB) and CCAAT/enhancer binding protein (C/EBPB) to their respective consensus sequences in thioglycollate—elicited peritoneal macrophages and these effects were suppressed in macrophages similarly elicited from DHA-fed mice. These findings were consistent with the results found in splenic nuclear extracts. Chromosome immunoprecipitation (ChIP) assay confirmed that DON increased CREB binding to the cis element of the IL-6 promoter in macrophages and that this was inhibited by DHA feeding. DON also induced phosphorylation of p3 8, extracellular signal-regulated kinase (BRKl/Z) and their downstream kinases, mitogen and stress-activated protein kinase 1 (MSKI), ribosomal S6 kinase (p90RSK) which are important for CREB activation. DON-induced CREB phosphorylation was blocked in macrophage form DHA—fed mice, but analogous effects were not observed for p3 8, ERK, MSKl and p90RSK. At the post-transcriptional level, DON enhanced IL-6 mRNA stability in macrophages from control mice but this was not affected in macrophages from DHA fed mice. Taken together, these data suggest that DHA might suppress transcriptional activation of IL-6 gene by blocking CREB phosphorylation and subsequently binding to the IL-6 promoter. 134 INTRODUCTION Deoxynivalenol (DON) induced IgA nephropathy (IgAN) in mice provides a unique preclinical window for studying prophylaxis and treatment of IgAN at its early stages (Jia et al., 2004a,b). Ex Vvivo reconstitution (Yan et al., 1998), antibody neutralization (Y an et al., 1997) and interleukin-6 (IL-6) knockout mice studies (Pestka and Zhou, 2000) have revealed induction of IL-6 gene expression to be crucial to DON- induced IgAN. (n-3) Polyunsaturated fatty acids (PUFAs) were efficacious in treatment of IgAN in several clinical studies (Donadio, 2001b). In our previous studies, consumption of DHA, EPA was found to retard the progress of IgAN in mouse and this correlated with its inhibition on IL-6 gene expression and MAPK activation (Jia et al, 2004b). However the mechanisms by which DHA downregulates IL-6 gene expression and the upstream signal transduction remain unclear. Regulation of IL-6 gene expression can potentially involve several signal transduction pathways and multiple transcriptional factors (Hershko et al., 2002; Mainiero et al., 2003). CAMP Response element binding protein (CREB), activating protein-1 (AP-1), CCAAT/enhancer binding protein beta (C/EBPB) and nuclear factor K B (NFKB) all contribute to IL-6 transcriptional upregulation based on electrophoresis mobility shift assay (EMSA) and point mutation analyses (Matsusaka et 01.1993; Robb et al. 2002). Acute oral exposure to DON induces serum IL-6 as well as IL—6 mRNA expression in spleen and Peyer’s patches and this correlates withboth MAPK activation and increased AP-l, CREB, C/EBPB and NFKB binding activity both in mice and in RAW-264.7 macrophage cells (Zhou et al., 2003; Moon and Pestka, 2003). 135 The purpose of this study was to identify molecular mechanisms by which DHA inhibits IL-6 gene expression. Multiple transcriptional factors involved in IL-6 regulation and their relative upstream pathways were examined. It was found that DON significantly induced transcriptional factor CREB and AP-l activation and CREB binding to the IL-6 promoter. DHA antagonized DON-induced CREB phoshorylation and subsequent binding to IL-6 gene promoter both in vitro and iv vivo. 136 MATERIALS AND METHODS Materials. All chemicals (reagent grade or better) were purchased from Sigma Chemical (St. Louis, MO) unless otherwise noted. Fluo-3, F125, ionomycin, SB203580, SP600125, PD98059, MDL—1233OA, KN-62, KT5720 were purchased form EMD Biosciences, Inc (San Diego, CA). DON used in the cell culture was purchased from Sigma Chemical (St. Louis). DON used in feeding study was produced in Fusarium graminearum R6576 cultures and purified by the improved water-saturated silica gel chromatography method (Clifford et al., 2003). Purity of DON was verified by a single HPLC peak occurring at 224 nm. Concentrated toxin solutions were handled in a fume hood. Labware contaminated with mycotoxin were detoxified by soaking for at least 1 hr in 100 m1/L sodium hypochlorite. Purified DON was added to powdered diets as detailed by Pestka et al. (1989). Animals. Female B6C3F1 mice (7 wk old), weighing between 20 to 25 g were obtained from Charles River (Portage, MI). Mice were housed in environmentally protected transparent polypropylene cages with stainless steel wire tops for 1 wk prior to introduction of different treatments. Mice were given free access to water and food. Experimental diets were placed in feed jars containing stainless steel top and mesh cylinder to minimize spillage. The environmental conditions included 23—25°C, 45 to 55% relative humidity, and 12: 12 hr artificial photoperiod. Housing, handling and sample collection procedures conformed to the policies and recommendations of Michigan State University. All University Committees on Animals were in accordance with guidelines established by the National Institute of Health. 137 Diets and experimental design. Experimental diets were derived from a purified AlN-93G formulation (Reeves et al., 1993). The basal diet consists of the following ingredients (per kg): 10 g AIN-9BG mineral mix, 10 g AIN-93 vitamin mix, 200 g casein, 397.5 g cornstarch, 132 g dyetrose, 50 g cellulose, 3 g L—cystine, 2.5 g choline bitartrate, 14 mg TBHQ, and 100 g sucrose (Dyets Inc, Bethelehem, PA). Corn oil (Dyets Inc), oleic acids (Dyets Inc) and MEG-3TM DHA enriched oil (containing DHA 483g/kg and 113g/kg EPA), a gift from Ocean Nutrition Canada Ltd, were used to amend to yield two experimental diets: Control (10 g corn oil and 60 oleic acid/ kg diet) and DHA (10 g corn oil and 60 g DHA enriched oil/ kg diet). Diets were prepared every 2 wk, stored at -20°C and provided to mice daily. Previous studies demonstrated that consumption of this DHA diet increased liver DHA from 3% to 11% and EPA from 0.11 to 4.8% of total phospholipid within 4 wk (J ia et al., 2004b). Mice were kept on diets for 4-5 wk before experiments. Macrophage were collected as described below. For spleen nuclear protein extraction, mice were anesthetized with methoxyfluorane and killed by cervical dislocation. Spleen were removed for nuclear protein extraction. Peritoneal macrophage cultures. Macrophages were harvested as described by (Conrad, 1981). Briefly, after kept on diets for 4 wk, mice were injected (ip) with 1 ml 9% thioglycollate. After 3 d, macrophages were collected by peritoneal lavage with cold Hanks buffer (Invitrogen Corporation, CA) and pelleted at 1200 rpm for 5 min. Cells were resuspended in DMEM (Invitrogen Corporation) containing 10% heat inactivated FBS, and 0.025% Penicillin-Streptomycin solution (Sigma) and settled down at 37°C under 7% C02 in a humidified incubator for 24 h before addition of DON. 138 Lipid extraction and analysis. Fatty acids were analyzed by a modification of the method of Hasler et al. (1991) by gas chromatography (GC) utilizing GC-2010 gas Chromatograph (Shimadzu Scientific Instruments, Inc, IL) and standard fatty acid methyl ester (Nu-Check Prep.Inc. Elysian, MN). IL-6 gene expression. DON (250 ng/ml) was added to the macrophage cell culture (1x106/ml) for 24 hrs. Cell supernatant was analyzed for IL-6 by ELISA (Moon and Pestka, 2003) using purified rat anti-mouse 1L-6 (Pharmingen, San Diego, CA) and biotinylated rat anti-mouse-IL-6 (Pharmingen, San Diego, CA). IL-6 hnRNA and mRNA were measured by Real-time PCR as described by J ia et al. (2004b). 18S RNA was used to normalize target gene expression. Target gene expression levels were calculated relative to the control group (PE Applied Biosystems user bulletin number 2). Probe and primers for mouse IL-6 mRNA and endogenous control (188 RNA) were purchased as TaqMan assay reagents (PE Applied Biosystems, Foster City, CA). Real-time Polymerase Chain Reaction Primer Express software (PE Applied Biosystems, Foster City, CA) was employed to design primer pairs for mouse IL-6 hnRNA (forward primer: GTC CAA CTG TGC TAT CTG CTC ACT; backward primer: AGA AGG CAA CTGG ATG GAA GTC T). Nuclear extraction. After keeping mice on diets for 4 wk, macrophages were prepared as described above. Cells were treated with DON (250 ng/ml) for 30 min before nuclear protein extraction. For splenic nuclear extracts, mice were gavaged with DON (25 mg/kg BW), 30 min later spleen cells were prepared as described previously (Pestka et al., 2002). Nuclear extracts were prepared by lysing 1x107 cells in buffer A (20 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, containing 1.5 139 microgram/ml aprotinin, pepstatin, leupeptin and chymostatin) for 15 min on ice. After the addition of Nonidet P-40 (0.5% final concentration), cell lysates were centrifuged at lOOOxg for 10 min at 4 C. Pelleted material was incubated with 20 mM HEPES pH 7.9, 400mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, containing 1.5 microgram/ml aprotinin, pepstatin, leupeptin and chymostatin for 60 min on ice. Insoluble material was removed by centrifugation at 14 000 x g for 15 min at 4 °C. The protein concentration of the supernatant was determined using a Bio-Rad protein assay (Bio-Rad Laboratories Inc, Melville, NY). Electrophoretic mobility shift assay (EMSA). EMSAs (Zhou et al, 2003) were performed as using NFKB consensus sequence 5' AGT TGA GGG GAC TTT CCC AGG C 3', mutant 5’AGT TGA GG_C_ GAC TTT CCC AGG C 3’; C/EBP consensus sequence 5' TGC AGA TTG CGC AAT CTG CA 3', mutant 5’ TGC AGA G_A§_ mg ET CTG CA 3’; AP-l consensus sequence 5' CGC TTG ATG ACT CAG CCG GAA 3', mutant 5’ CGC TTG ATG ACT 19G CCG GAA 3’ and CRE consensus sequence 5' AGA GAT TGC CTG ACG TCA GAC AGC TAG 3', mutant 5’-AGA GAT TGC CTG mG TCA GAG AGC TAG-3’ from Santa Cruz Laboratories (Santa Cruz, CA). Probes were end- labeled with [y—32P] ATP using polynucleotide kinase T4 (GIBCO BRL, Grand Island, NY) and purified from unincorporated [7-32P] ATP using a purification column (Bio-Rad Laboratories). After purification using NucTrap probe column (Stratagene, CA), binding was achieved by incubating 20 ug of cell nuclear extracts with 2 ul of labeled fragment at room temperature for 30 min. For competition experiments, synthetic wild type and mutant type oligonucleotides were used in 150-fold molar excess and incubated with nuclear extract for 15 min before the radiolabelled probe was loaded. Samples were then 140 subjected to electrophoretic separation on a nondenaturing 5% polyacrylamide gel at 30 mA using Tris borate-glycine buffer (0.45 M Tris borate, 0.001 M EDTA, pH 8.3). Blots were dried at 80°C for 3 h and analyzed by autoradiography. Chromosome immunoprecipitation (ChIP) assay. ChIP-IT Kit (Active motif, Carlsbad, Ca1ifornia).was used according to manufacturer’s instruction. Briefly, peritoneal macrophage cells (2x107) were fixed using 1% (v/v) formaldehyde to cross link protein and DNA, 30 min after DON (250 ng/ml) stimulation. DNA was sonicated into small uniform fragments form 200-500 bp. DNA/protein complexes were immunoprecipitated with rabbit polyclonal antibodies directed against the phospho- CREB (Cell signaling), c-jun, C/EBP and p65 (Santa Cruz Biotech, CA). Following immunoprecipitation, cross-linking was reversed, proteins were removed by proteinase K treatment and the DNA purified on column provided. The DNA fragments were then screened by SYBR green Real-time PCR to determine which transcriptional factor was bound to the IL-6 promoter. Primers that flanked the different transcriptional factors binding sites were designed using Primer express software (AppliedBiosystems, Inc) and were listed as follows: CREB binding site: Forward: GGC TAG CCT CAA GGA TGA CTT AAG Backward: TTG CAC AAT GTG ACG TCG TTT; AP-l binding site : Forward : CCT TAT AAA ACA TTG TGA ATT TCA GTT TTC , Backward : CAT GAG CAC TCT TCT TTT TTT CTT TAA A; NFKB : Forward: CTT TCG ATG CTA AAC GAC GTC ACA Backward : AAA TCT TTG TTG GAG GGT GGG (Figure 6.1). Western analysis. Macrophages were washed with ice-cold phosphate buffer, lysed in 1% (w/v) SDS buffer containing 1.0 mM sodium ortho-vanadate and 10 mM Tris (pH 7.4) and sonicated for 10 3. After protein concentration determination, equal amounts 141 .mfiz é a :42 5 Na mama 9a mmeo é a saw a5 a 208E as .8 858a .5 3 25E umooommuop Doomuuoouu mmooommmuo mmummmoouo uuuoopummm oumuomoomm 92H momoopummo pomummommm mmoomooomm monouomuou umouommumu oumummmmmu mxhz ommmmumumm mummuoouom mpuomommmu ummmmououm mmumooouup umoomumumm ma .V moumpuuuum mmomommoou ooomooooom ooooomuouo mooomoooom moummoouuu #9 $50 \ mmmu V. mmmmummuuo ummoouODum omflumommom mmuumpmmou uuuuummuou poopoopuoo mmmo mm ouuuuomomo mmMuuommum ommmouoomm uomommuuou uomumouomu mmommmmmmm a. a a mmmmmmmuuu uomoummmuo mummmouomu momommoumo oouuuouuuu mmopuummmu V. .4 ouumommmmu muuUUDUuuu ouuupmomuu pommoumomm ummmuuumuo uooououooo Na HNMH HmNH HONH HvHH Hmoa HNOH Hom Mom 142 of protein were fractionated by SDS-PAGE with 10%(w/v) acrylamide separation gel, and subjected to Western analysis using specific antibodies for p3 8, phospho-p3 8, ERK, phospho-ERK, JNK, phospho-JNK antibodies (New England Biolabs, Beverly, MA), phospho-CREB, CREB (cell signalling CA), phospho c-jun, c-jun, phospho p65, p65, phospho C/EBPB and C/EBPB, phospho MSKl and phospho p90RSK (Santa Cruz, CA). Inhibitor studies. Macrophages were harvested and cultured in 1 ml of DMEM in 6 well plate (1x106 cells /well) at 37 °C under 7% C02 in a humidified incubator for 24 h as described above. Inhibitors for different kinases, PKA inhibitor (KT5720, luM), CaMkII inhibitor (KN62, luM), adenylyl cyclase inhibitor (MDL13322, 1 uM), p38 inhibitor (SB-203580, 20 uM), ERK inhibitor (PD-98059, 100 uM) and JNK inhibitor (SP600125, 10 uM) were added to the medium for 30 min before DON (250 ng/ml) stimulation. Three hours after DON treatment, total RNA was extracted and IL-6 mRNA measured. Following treatment described in inhibitor assay, cytotoxicity was measured by MTT assay. Briefly, 20 ul of a 5 mg/ml solution of 3-(4,5 dimethylthiazol-Z-ul)-2,5- diphenyl tetrazolium bromide (MTT) in 0.01 M PBS was added to each well for the final 3 h incubation period (Uzarski et al, 2003). 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O ........ .....O \ noon GSBGJOU! eoueseJomd >\b\ com 165 - oood - mood - 90.0 . mwod - owed (Iw/Iowd) dwvo mwod . .890 50000. 0000000 000000.000 00.03 000m .mu0 .0508 000 $030M 00000000000 0.0000 05 w0030=0.0 000000000 003 .00/006 000 000.0 Z06 .3 000.: 0003 :00 .008 8 80 080.. 000 ZOO 00003 0000000000 0003 0090000000000 0000000000 00000000 2008002.: .200 .3 80000200 .025 0002082000 00.0 2:000 166 DISCUSSION (n-3) PUFA effects on proinflammatory gene expression (Endres et al, 1995) and transcriptional factor, AP-l , NFKB and C/EBP, activation have been previously described (Babcock et al., 2003; Zhao et al., 2004; Weber et al., 1995; Bousserouel et al., 2003). Although the interaction between the (n-3) PUFA and the CREB has been proposed by Logan (2003), this is the first study to report that DHA potentially inhibit expression of the proinflammatory gene IL-6 by blocking transcriptional factor CREB activation and its binding to the IL-6 promoter. Changes in macrophage phospholipid profile might modulate to IL-6 gene expression. Here, in macrophages DHA was increased from 0.9 to 3.9%, and arachidonic acid was decreased from 4.2% to 2%. These modifications can potentially reduce prostaglandin E2 (PGE2), leukotriene B4 (LTB4) and platelet activated factor (PAF) production (Calder, 2002; Simopoulos, 2002;Whelan, 1996) as well as disrupt lipid raft on cell membrane, which are important for cell signaling (Stulnig et al., 2001). Kaminski et al. (1993), in a human study, reported that consumption of 7g/day of 85% pure (n-3) PUFA for 7 wk increases (n-3) PUFA content in human monocyte phospholipids and down-regulates by 70% both PDGF-A and PDGF-B expression. Jia et al. (2004b) previously found that DHA consumption significantly increase DHA percentage from 3 to 11% while decreasing arachidonic acid from 12 to 3% in mouse liver after 4 wk DHA consumption of 60 g/kg DHA enriched oil. However, since the liver acts a storage organ for phospholipid of DHA and EPA (Sekine, 1995), the phospholipid profile in liver can not be directly compared to concentrations of these fatty acids in lymphoid tissue after DHA feeding. 167 Levels of hnRNA, the precursor of mRNA, can be utilized as a substitute method for run-on assay (Johnson et al., 2003), to measure gene transcriptional activity. Because DON induces both IL-6 mRNA and hnRNA in macrophages and because DON-induced IL-6 mRNA expression is blocked by transcriptional inhibitor DRB, DON appeared to activate IL-6 gene transcription in the thioglycollate elicited macrophages. The observation that DHA significantly reduced IL-6 protein, IL-6 hnRNA and mRNA level in DON-treated macrophage, was highly consistent with the previous in vivo study using spleens of DHA-fed mice and‘exposed acutely to DON (J ia et al., 2004b). Control of mRNA stability facilitates a rapid adjustment of mRNA levels at posttranscriptional stage. p38 mediates the stability of mRNA with clusters of the AUUUA motif in their 3' untranslated regions (Saklatvala et al., 2003). Wong et al (2001) found that post-transcriptional control via enhancement of mRNA stability was likely to contribute to IL-6 superinduction in RAW 264.7 macrophages by DON. DON- induced p38 and ERK activation contributes to transcriptional upregulation of IL-6 whereas p38 plays a role in increasing mRNA stability in cloned macrophage cells (Chung et al., 2003). It had been found that DON could superinduce IL-2 by enhancing mRNA stability (Li et al., 1997). Moon et al (2003) showed that DON enhanced both COX-2 transcriptional activation and mRNA stabilization with luciferase reporter vectors. As found previously, addition of DON did enhance IL-6 mRNA stability in peritoneal macrophage. The observation that DHA did not affect IL-6 mRN A degradation suggests that DHA acts primarily by impairing DON-induced 1L-6 gene transcriptional activity. 168 DON induced IL-6 expression correlated with significantly increased CREB, AP- 1 binding activities 30 min after stimulation both in spleen and in macrophage revealed by EMSA experiments. NFKB binding activity was also detected in spleen but not in macrophage and C/EBP binding activities were only weakly induced by DON. These results suggested among the transcriptional factors, CREB and AP-l were initially and potently induced by DON both in thioglycollate-elicited peritoneal macrophages and in spleen. The DNA in cell nucleus is condensed and packaged by being wrapped around histone octamers called nucleosomes which suppress transcription by imposing a barrier to the access of transcription factors and basal transcriptional machinery to DNA. Chromatin structural remodeling plays fundamental roles in eukaryotic gene regulation (Kadonaga, 1998.). Thus EMSA assay does not always reflect in vivo interaction between transcriptional factors and DNA. However, this endpoint can be measured with ChIP, which revealed that DON significantly induced phosphorylated-CREB binding to the native IL—6 promoter, and this was blocked by DHA treatment. Thus, ChIP was indeed consisted with EMSA results, suggesting CREB might be the most important transcriptional factor involved in DON-induced IL-6 transactivation in elicited peritoneal macrophage, and, furthermore, that DHA consumption blocks CREB binding to IL-6 promoter in vivo. CREB is a nuclear target of signaling pathways activated by multiple stimuli. Evidence suggested that CREB binding to adapter protein CBP that can recruit and stabilize the RNA polymerase H (Pol II) transcription complex at the TATA box. CBP possesses an intrinsic histone acetyltransferase (HAT) activity and contributes to 169 chromatin structure modification, which can make the DNA template more accessible to the transcriptional machinery (Shaywitz and Greenberg, 1999). DON has been previously proved to drive phosphorylation of multiple transcriptional factors (Zhou et al., 2003). Phosphorylation of CREB, c-Jun, NFKB, will then enhance their affinity to recruit CBP to the IL-6 promoter thus enhancing their transactivtion (Theresia, 2002; Quinn, 2002; Shaywitz and Greenberg 1999 ). DON induces CREB, c-jun and p65 phosphorylation 30 min after stimulation, which was impaired by the DHA treatment. The finding that, phosphorylation of CREB was most affected by DON and impaired by DHA was consistent with EMSA and ChIP data, suggesting that DHA only affected the signal transduction elicited by DON. ChIP data did not support involvement of c-jun, C/EBP and NFKB in intranuclear ' IL-6 promoter. It should be noted here that the inability to detect the interaction between c-jun, C/EBP, p65 and IL—6 promoter region might be because the antibodies used in this experiment were not suitable for ChIP assay. Forrnadehyde fixation might change the epitope of the protein or the protein complex formed in vivo might also block the antibody-binding site (Kuo and Allis, 1999). PKA, CaMKII, p38 and ERK all reportly can contribute phosphorylation of CREB at serine 133 (Ser133) which is required for CREB-mediated gene transcription (Shaywitz and Greenberg, 1999; Figure 6.10). The p38 inhibitor, SB20219O and the inhibitor of the upstream activator of ERK1/2, MEK (ERK kinase), PD 98059 showed the most potent inhibition on IL-6 gene expression whereas JNK inhibitor, SP-600125, showed no effect. The failure of JNK inhibitor, SP-600124, on IL-6 gene expression can be explained by the fact that it stimulates CREB phosphorylation (Vaishnav et al., 2003). 170 Since dominant negative-JNK do not block DON induced PGE2 and COX-2 in macrophage cells (Moon and Pestka, 2003), JNK is likely to be less important than p38 and ERK in IL-6 production. The observation that PKA and CaMKII inhibitor also contributed partially to DON-induced IL-6 mRNA while adenylyl cyclase did not suggests the [Ca2+] and CAMP might be induced by DON. However, we failed to detected DON effects on [Ca2+] and cAMP (Data are not shown). Thus the role of PKA and CaMKII in DON-induced IL-6 gene expression need to be further investigated Although DHA blocked DON-induced CREB phosphorylation, it failed to suppress p38 and ERK phosphorylation. MSKl is widely distributed in mammalian cells and can be activated by grth factors, cell-damaging stimuli and proinflammatory cytokines through both ERK3 and p38 pathway) while p90 RSK (also called MAPKAP- Kl) can only be activated through ERK pathway. Both MSKl and p90RSK can phoshorylate CHEB at serine 133 (Song et al, 2003). MSKl knockout mouse showed that it was required for the stress-induced phosphorylation of transcription factors CREB and ATFl in primary embryonic fibroblasts (Wiggin et al., 2002). The activation of MSKl and CREB by LPS, adenosine, membrane disruption, angiotensin 11, Na, lysophosphatidic acid can be similiarly prevented by preincubating the cells with PD 98059 and/or SB 203580 (Caivano and Cohen, 2000; Nemeth et al., 2003; Togo, 2004; Cammarota et al., 2001; Gustin et al., 2004; Lee et al., 2003c; Vaishnav et al., 2003). However, DHA failed to impair DON induced MSKl and p90RSK phosphorylation suggesting other mechanisms might mediate DHA inhibition on CREB phosphorylation and, ultimately IL-6 gene expression. Similar results were also found in a recent study in which DHA failed to block phorbol lZ-tetradecanoate 171 dosflbofimofi 858 2a Em: Ea ma 8 38% 2:: a; 233 cefibofimofi mmmo wagooi 3 countomawb 28w 0.5 0268 mugomawmm :3 «in domatomawb 25w 0%: 55 new aouflbonamofi mph—MU 05 9 .858 cores—ugh 3:me 3265-20Q 0862: 523 momma EwtanM 508 on: on Eme Mam 98 wmm demmoaxo 8% wk: 3265 ZOO E 339:: mmmafimm oEMmmom 36 0.5»:— m..= Lilli 35 .i $5 55% 55: Z «in. .=v=2m0 xmm .wnn /200 ‘ 172 13-acetate (TPA) or epidermal growth factor (EGF) -induced phosphorylation of ERK or p38 kinase and JNK kinase activity in JB6 C1 4lcells (Liu et al., 2001). The JNK signal is crucial for AP-l transcription factor activation in cells (Ventura et al., 2003). Neff et al., (2001) found that cells lack JNK exhibited decreased c-Jun, phosphorylation of c-Jun and AP-l DNA binding activity. Since AP-l activation is associated with the production of IL-6 (Cuschieri et al., 2004), mice deficient in the JNK pathway had decreased serum levels of IL-6 in response to LPS compared (Morse et al., 2003). Furthermore DN-c-Jun, DN-JNK as well as AP-l inhibitor, curcumin, can significantly impeded TGF-betal induced IL-6 (Park et al., 2003) and JNK/APl pathway has also been approved to play an important role in IL-6 promoter activation. (An et al., 2003). So DHA’s inhibition on JNK phosphorylation might contribute to its inhibition on c-jun phosphorylation and IL-6 gene expression. In summary, DON activated multiple transcriptional factors, especially CREB, through p38 and ERK kinase pathways. DHA treatment likely inhibited IL—6 gene transcription by blocking multiple transcription factor activation, among which CREB was likely to be most important. The failure of DHA to inhibit phophorylation of p38 and ERK or their down stream kinases MSKl and p90RSK, suggests that other mechanisms might exist for DHA inhibition on CREB phosphorylation and IL-6 gene transcription or CREB might also be a target for prophylaxis against IgAN. 173 CHAPTER 7 Summary and Conclusions 174 IgA nephropathy (IgAN) is the most common form of nephritis but treatment of this disease remains non-specific. Based on clinical studies, (n-3) pOIyunsaturated fatty acids (PUFAs) have been showing promising beneficial effects due to its anti- inflammatory effects and safety. The capacity of DON to induce the early stage of IgAN provides a unique window for testing our guiding hypothesis that (n-3) PUFAs might retard development of IgAN by impairing the proinflammatory cytokine production. The first investigation for this dissertation was to determine if fish oil could attenuate DON-induced IgAN. DON significantly increased serum IgA, serum IgA immune complexes and kidney mesangial IgA deposition compared with the control group, whereas all three variables were significantly attenuated in mice fed 60 g/kg fish oil. In addition, spleen cell cultures fiom the DON +fish oil group produced markedly less IgA than those cultures from mice fed DON and fish oil. The results confirmed that diets containing fish oil might impair early immunopathogenesis in DON-induced IgAN. DHA and EPA are two major components of (n-3) PUFAs in fish oil. The second investigation compared their efficacy in preventing DON-induced IgAN. Mice were fed for 18 wk with AIN-93G diets containing EPA or DHA enriched oil. It was found that while DHA and EPA significantly attenuated serum IgA, serum IgA immune complexes and kidney mesangial IgA deposition as well as IgA secretion by spleen cells. DHA was. slightly more efficacious than EPA. Pre-feeding of DHA/EPA also significantly reduced serum IL-6 concentration induced by acute oral exposure to DON, which is required for DON induced IgAN. In the third investigation, the dose response effects of DHA on DON-induced IgAN were measured. DHA dose-dependently inhibited elevated serum IgA and IgA 175 immune complex (IC) as well as IgA deposition in the kidney. At the same time, spleen IL-6 mRNA and heterogeneous nuclear RNA (hnRNA) concentrations were significantly reduced by DHA. In an acute study, DHA consumption inhibited DON-induced COX-2 mRNA, IL-6 mRNA and hnRNA expression. Phosphorylation of p3 8, ERK and JNK was also attenuated by DHA. Together, the results indicated that DHA enriched oil at 60 g/kg significantly inhibited DON-induced IgAN and this correlated with impaired IL-6, cyclooxygenase-2 (COX-2) gene expression and mitogen active protein kinase (MAPK) activation. The inhibition of IL-6 hnRNA suggested that DHA might inhibit IL-6 gene expression at transcriptional stage. To clarify the role of COX-2 gene in development of IgAN, both COX-2 knockout mice and COX-2 specific inhibitor, VIOXX were employed in the fourth investigation. The results demonstrated that knocking out COX-2 and VIOXX treatment failed to block DON induced IgAN, but rather enhanced DON induced serum IgA elevation. These results suggested that COX-2 was not required for DON induced IgAN and the COX-2 inhibitor, VIOXX, might not be suitable for prophylaxis of IgAN. Finally, IL-6 mRNA regulation at post transcriptional and transcriptional stages were investigated in thioglycollate-elicited macrophages from mice fed control and DHA diet. It was found that dietary DHA did not affect IL-6 mRNA stability but significantly inhibited IL-6 gene transcription. Binding of CAMP response element binding protein (CREB), active protein-1 (AP-1), nuclear factor K B (NFKB) and CCAAT/enhancer binding protein (C/EBP) in vitro and in vivo was inhibited by DHA consumption. Subsequent demonstration of inhibition on in vivo interaction between CREB and IL-6 176 promoter by DHA suggested CREB to be particularly critical in DON-induced IL-6 gene transcription. Taken together, these studies confirmed prophylactic effects of (n-3) PUFA in this chemical-induced IgAN and provided the insight into molecular mechanisms of DHA on proinflammatory gene, IL-6, expression, which can be exploited for disease prevention. The following experiments might be considered in the future to elucidate mechanisms by. which DHA blocks IL-6 gene expression and CREB activation. 1) In vitro study to measure DHA effects on MSKl enzyme activity. 2) In vitro study to detect if DHA can bind CREB and block its phosphorylation 3) ChIP study to investigate DHA effects on chromatin acetylation. 4) Irnmunostaining to detect if DHA can block transcription factor nuclear translocation. 5) Immunoprecipitationstudy to detect if DHA can bind to PPARs and block IL- 6 gene expression. 177 APPENDIX A 178 DHA EFFECTS ON DON INDUCED PPAR BINDING ACIVITIES Peroxisomal proliferator activated receptor alpha (PPAROL) and gamma (PPARy) might be important transcriptional factors that can mediate (n-3) PUFA effects on gene transcription (Jump et al., 2004; Jump et al., 1997). PPARs can bind to their ligands and physically interfere with transcriptional factors binding to their cis-element and thus downregulating proinflammatory gene expression (Ren et al., 1996). For example, interferon gamma (IFN-y), IL-6 and TNF-O. production are impaired in spleen from mice fed with PPAROL ligand, WY14, 643 (Cunard et al., 2002). When WY14, 643 binds to PPARa, it can sharply reduce IL-6 and COX-2 gene expression by physically interfering with p65, c-jun and CBP interaction with DNA in vitro (Delerive et al., 1999). The PPARy ligands, troglitazone, pioglitazone and lS-deoxy-Delta (12,14)-prostaglandin J (2) also inhibit IL-lB-induced 1L-6 expression at transcriptional level in vascular smooth muscle cells by interfering NFKB and C/EBP binding to the DNA (Takata et al., 2002). Arachidonic acid and LTB4 can also bind to the PPAROL and 7, but the binding will elicit their degradation by increasing B-oxidation of PUFA (Devchand et al., 1996). Thus, PPARs are important negative feedback molecules in vivo for control of inflammation responses (Delerive et al., 1999; Pointer and Daynes, 1998; Berger and Moller, 2002; Delerive et al., 2000; Clark, 2002). 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The B6C3F1 mice were injected with TNP-BSA (1mg/mouse) for 3 consecutive days and the BALB/C mice were injected with IgG anti IgA (SOpg/mouse) for 3 consecutive days. Urine was collected at the 5th day after the first injection. The urine sample was centrifuged at 500g for 10 min. The sediments were used to check hematuria (Table 1). Multistix was also employed to check hematuria and proteinuria but the results for all sample are negative. 197 00:00:00 003 08:0 00 08008 2... 0E. .800: 00:0: :0300 :8: 0: 0:05 00 008000 0:03 0:00 000:0. 00: 0:0. .:800 80:00::0 :0 0000 0:000:08 8 00:00:00 003 08:3 A0038 :00 \ 01 on: <0: 0:0 00: 5:3 028.8: 203 8:8 00:00 :0:0 0808: $0-02: 5:3 028.8: 0:03 008: Emocm 0:3 N: :00 A 800 on 6:: ZOQ m880800 :0:0 :0 :000: 0:03 008: 0:0. - - - ow on 00 200 8%: om mm - :0 00 N: N: 200 5:0 om :m - S on 0: 0: 7:00 50:: on 00 - - - - o: 200 8:: c: 3. - :.: 00 mm :: 7:00 8:: 0: 00 a. 0: 00 Na 00 200 80: c: - - m a: - 0 30:8 N: m: m an - a 388 t : : m: s N 6008 :0:0 h :8 0 :8 0 0.0 s be 0 :03. 00.20 :..:800 <0: 0:0 00: 0: 8260: 000.00 a: 20 £800 0: «00.02.: 3 326:: 0.5.082: 2:. ~00 2%.: 198 APPENDIX C 199 PROTOCOL FOR CHIP ASSAY Preparing chromatin 1. 0\ Culture 5 x106 thioglycollate—elicited macrophages in 10 cm plates for 48 hours and four dish cells are good for one treatment group. Prepare fresh fixation solution, ice-cold 1X PBS, glycine stop-fix solution and cell scraping solution as follows: a.Fixation solution: Add 1.62 ml of 37% formaldehyde to 60 ml minimal cell culture medium and mix thoroughly. Leave at room temperature. b.1X PBS: Add 7 ml 10X PBS to 63 ml dHZO, mix and place on ice. c.Glycine stop-fix solution: Combine 3 m1 10X glycine buffer, 3 m1 10X PBS and 24 ml dHZO. (1. Cell scraping solution: Add 600 pl 10X PBS to 5.4 ml dH2 0, mix and place on ice. just before usel(in step 7 below) add 30 pl 100 mM PMSF. Pour medium off the plates and add 10 m1 fixation solution to each plate. Incubate on a shaking platform for 10 minutes at room temperature. Pour fixation solution off the plates and wash by adding 10 ml ice-cold PBS to each plate, rocking the plate for 5 seconds and then pouring off the PBS. Stop the fixation reaction by adding 5 ml glycine stop-fix solution to each of the plates, swirling to cover and then rocking at room temperature for 5 minutes. . Wash each plate by pouring off the glycine stop-fix solution and adding 5 ml ice-cold PBS, rocking the plate for 5 seconds, and then pouring off the PBS. 200 7. Add 1 m1 ice cold cell scraping solution to each of the plates and scrape cells with a rubber policeman. Hold the plate at an angle and scrape the cells down to collect them at the bottom edge of the plate. Use a 1 ml pipette to transfer the cells to a 15 ml conical tube on ice. Do the same for the other 3 plates and pool cells from the three plates in one 10 ml conical tube. 8. Pellet the pooled cells by centrifugation for 10 minutes at 2500 rpm (720 RCF) at 4°C. 9. Remove the supernatant. At this point the protocol can be continued or the pellet can be fi'ozen. If freezing the pellet, add 1 pl 100 mM PMSF and 1 p1 PIC and freeze at - 80°C. When you are ready, continue with step 10. 10. Thaw pellet (if necessary) and resuspend cells in 1.5 ml ice-cold Lysis Buffer (supplemented with 7.5 p1 PIC + 7 .5 pl PMSF). Incubate on ice for 30 minutes. 11. Transfer the cells to an ice-cold dounce homogenizer. Gently dounce on ice with 10 strokes to aid in nuclei release. Transfer cells to a 15 m1 conical tube and centrifuge at 5000 rpm (approximately RCF 2400) for 10 minutes at 4°C to pellet the nuclei. 12. Carefully remove the supernatant. Resuspend the nuclei pellet in 1.0 ml shearing buffer (supplemented with 5 pl PIC) and aliquot into 2 1.7 ml microcentrifuge tubes. Each aliquot should be approximately 400 pl. 13. Shear the three aliquots of DNA using your optimized conditions 25% of power 30 second pulse with 30 5 rest on ice and repeat for 5 times. 14. Centrifuge the sheared DNA samples at 10,000 to 15,000 rpm in a 4°C microcentrifuge for 12 minutes. Pool the supematants by transferring each to the same fresh tube. This contains the sheared chromatin. It can be used right away or aliquoted and stored at ~80°C. Remove 25 p1 for use in checking the DNA shearing 201 efficiency and DNA concentration. Aliquot the remainder into 2 equal aliquots (usually about 200 pl each). Each aliquot can then be used for 4 ChIP reactions (i.e. each aliquot can be tested with four different antibodies). Pre-clearing of Chromatin (Day 2) Chromatin is pre-cleared with Protein G beads to reduce non-specific background. Normally, each chromatin preparation will be used for several ChIPs (e.g. a negative control ChIP, a positive control ChIP and ChIP with antibody of interest). In these cases, the chromatin for the ChIPs can be pre-cleared in the same tube (see example below). The volumes below assume that the chromatin was prepared as described in the preceding sections. Reagent p1 for one ChIP rxn pl for 3 ChIP rxns; Chromatin 50 pl 150 pl; Resuspend Protein G beads 100 pl 300 pl; ChIP IP Buffer 59 pl 177 pl ; PIC 1 pl 3 pl; Total Volume 210 pl 630 pl 1. Use the above table to calculate the amount of chromatin, resuspended Protein G beads, ChIP 1P Buffer and PIC required for pre—clearing reactions. Combine the reagents in a 1.7 ml microcentrifuge tube. 2. Rotate the tube at 4°C for 1 to 2 hours. 3. Place tube in a microcentrifuge for 2 minutes at 4,000 rpm. 4. After centrifugation is complete, place tube on ice for 2 minutes to let the beads settle. 5. Transfer the supernatant (chromatin) to a fresh tube. Do not disturb the beads. 6. Repeat steps 3 through 5 to ensure that all beads are removed from the chromatin. Addition of Antibodies to Pre-cleared Chromatin 202 Note: Some volume (~15%) is lost during pre-clearing. Assume that 170 pl of pre- cleared chromatin is available for each ChIP reaction. . Transfer 10 pl of the pre-cleared chromatin to a microcentrifuge tube and store at - 20°C. This sample is the “Input DNA” and will be used in PCR analysis. It will be treated to remove cross-links at a later stage (see Section H. Reverse Cross-links and Remove RNA). . Perform the antibody incubations in the provided 0.65 ml siliconized tubes. To begin, label the appropriate number of tubes. . Add 170 pl pre-blocked chromatin to each of the labeled 0.65 ml tubes. Add the appropriate antibody to each of the labeled tubes. We recommend using between 1 and 3 pg of antibody for each ChIP reaction. The kit’s Negative Control IgG should be used at 9 pl (1.8 pg) per ChIP reaction.( 3 pl for p-CREB) Incubate the tubes overnight on a rotator at 4°C (sensitivity may be improved by overnight incubation) Addition of Protein G to Antibody/Chromatin Mixture (Day 3) 1. Resuspend the blocked Protein G beads fully by inverting the tubes several times. 2. Aliquot 100 pl of the fully resuspended beads into each of the antibody/chromatin incubations performed in the preceding step. 3. Incubate the tubes on a rotator for 1.5 hours at 4°C. 4. During this incubation, prepare the ChIP IP and Wash Buffers as described below. Each ChIP reaction will be washed once with ChIP IP Buffer + PIC, four times with 203 -~I m Wash Buffer 1 + PIC, once with Wash Buffer 2 + PIC and twice with Wash Buffer 3. The quantities listed below are sufficient for one ChIP reaction. ChIP IP Buffer + PIC: Add 2 pl Protease Inhibitor Cocktail (PIC) to 400 pl ChIP IP Buffer, mix and place on ice. Wash Buffer 1 + PIC: Add 1.6 pl PIC to 1.6 ml Wash Buffer 1, mix and place on ice. Wash Buffer 2 + PIC: Add 0.4 pl PIC to 400 pl Wash Buffer 2, mix and place on ice. Wash Buffer 3: Place on ice (supplied ready-to-use). Washing ChIP Reactions 1. (It .0) Following incubation of the beads with the antibody/chromatin mixture, pellet the beads by centrifuging each ChIP reaction for 2 minutes at 4000 rpm. Place tubes in a rack and allow 30 seconds for the beads to fully settle. Note: All subsequent bead pelleting steps should be performed in this manner. Remove the supernatant. Use a 200 pl pipette to withdraw 200 pl twice (discard supematants). Avoid disturbing the beads. Add 400 pl ChIP [P Buffer + PIC to each tube of beads and cap the tubes. Flick tubes to fully resuspend beads and incubate on rotator for 1-3 minutes. Pellet beads and remove supernatant. Add 400 pl Wash Buffer 1 and cap tubes. Resuspend beads and incubate on rotator for 1-3 minutes. Pellet beads and remove supernatant. . Repeat Step 4 three times. Add 400 pl Wash Buffer 2 + PIC and cap tubes. Resuspend beads and incubate on rotator for 1-3 minutes. Pellet beads and remove supernatant. 204 7. Add 400 pl Wash Buffer 3 and cap tubes. Resuspend beads and incubate on rotator for 1-3 minutes. Pellet beads and remove supernatant. 8. Repeat Step 7 once. This is the final wash. Remove as much buffer as possible without disturbing the bead DNA Elution from Protein G In this section, irrnnunoprecipitated DNA will be collected from the washed Protein G beads using two elutions with 50 pl ChIP Elution Buffer. 1. Freshly prepare 105 p1 of ChIP Elution Buffer for each ChIP reaction by adding 5 pl 1M NaHCO3 to 100 pl 1% SDS and mixing thoroughly. 2. Add 50 pl ChIP Elution Buffer to each of the washed Protein G bead pellets in the 0.65 ml tubes. Cap tubes, vortex briefly and incubate for 15 minutes at room temperature with gentle rotation. 3. Centrifuge tubes for 2 minutes at 4000 rpm to pellet beads. Remove tubes from centrifuge, place in tube holder in vertical position and wait several seconds for the beads to settle completely. 4. Use a 200 pl pipette to transfer each supernatant to an appropriately labeled 1.5 or 1.7 m1 microcentrifuge tube. 5. Repeat steps 2 to 4 and pool the appropriate elutions. Reverse Cross-links and Remove RNA Note: The reserved Input DNA (from Step 1 of Section D on page 13) must also be taken through the following steps. Remove the reserved Input DNA from the freezer and add 205 90 pl dH2 O to bring the volume to 100 pl and treat this sample along with the ChIP elutions below. 1. Add 4 pl 5 M NaCl and 1 pl RNase A to each ChIP elution and to the sample of Input DNA. 2. Vortex to mix completely and centrifuge the tubes briefly to remove liquid from the sides of the tubes. Place tubes in a 65°C incubator or water bath for 4 hours to overnight. (The experiment can be stopped here and tubes stored at -20°C until use.) Treat with Proteinase K (Day 4) 1. Remove tubes from 65°C incubator and centrifuge for 1 minute to collect liquid from the sides of the tubes. 2. Add the following three components to each tube: 2 pl 0.5 M EDTA, 2 pl 1 M Tris-Cl pH 6.5 and 2 pl Proteinase K solution. 3. Vortex to mix, centrifuge briefly to collect liquid from the sides of the tubes and incubate at 42°C for 1.5 to 2 hours to digest the proteins. During this incubation, prepare the reagents that will be needed for DNA purification in the next section. 206 Purify Eluted DNA 1 'J'I O\ 00 Label and place the required number of DNA purification mini-columns in their provided collection tubes in a rack. The DNA will be eluted from the columns into microcentrifiige tubes (1.5 ml or 1.7 ml). Label and set aside the appropriate number of tubes Remove the Proteinase K—treated samples fi'om the 42°C incubator and centrifuge briefly to collect the liquid condensed on the sides of the tubes. Add 500 pl of DNA Binding Buffer to each DNA sample and vortex to mix completely. Transfer each sample into a labeled DNA purification mini-column and centrifuge for 30 seconds at 10,000 to 15,000 rpm. Remove the mini-column from the collection tube, discard the flow-through and replace the mini-column in the tube. . Add 600 pl of DNA Wash Buffer to each mini-column. . Centrifuge for 30 seconds at 10,000 to 15,000 rpm. Remove the mini-column from the collection tube, discard the flow-through and replace the mini-column in the tube. . Add 300 pl of DNA Wash Buffer to each mini-column. 9. Centrifuge for 2 minutes at 10,000 to 15,000 rpm. 10. Place each dry mini-column into the appropriately labeled 1.5 ml or 1.7 ml tube prepared in Step 2. Add 50 pl dH2 0 directly to the resin at the bottom of each mini- column. Incubate for 3 minutes at room temperature. 11. Spin for 1 minute at 10,000 to 15,000 rpm. 207 12. Remove the mini-columns and collection tubes from the centrifuge and place in a tube rack. 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