UNIVERSITY OF CALIFORNIA Los Angeles Inflammatory Bowel Disease-associated Dysbiosis and Immune Gardening of the Intestinal Microbiome A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Cellular and Molecular Pathology by Jonathan Patrick Jacobs 2015 ABSTRACT OF THE DISSERTATION Inflammatory Bowel Disease-associated Dysbiosis and Immune Gardening of the Intestinal Microbiome by Jonathan Patrick Jacobs Doctor of Philosophy in Cellular and Molecular Pathology University of California, Los Angeles, 2015 Professor Jonathan Braun, Chair Inflammatory bowel disease (IBD) – comprised of Crohn’s disease (CD) and ulcerative colitis (UC) – is believed to arise from a combination of genetic susceptibility and environmental factors that trigger an inappropriate mucosal immune response to constituents of the intestinal microbiome. There is now an extensive literature demonstrating that the microbiome has profound effects on immune function and, conversely, that the immune system can shape the microbiome. I hypothesized that genetic variation in mucosal immune gardening of the intestinal microbiome can result in pro-inflammatory dysbiosis, which acts as a risk factor for overt IBD. To evaluate whether individuals at risk for IBD develop dysbiosis prior to the onset of disease, a family based study was performed to characterize the microbiome and metabolome of pediatric IBD patients and their first degree relatives. These relatives are at higher risk for dysbiosis than the general population due to shared genetic and environmental factors with the IBD proband. A ii subset of healthy relatives in this cohort had dysbiosis with fecal metabolomic profiles (metabotypes) shared with IBD patients. The effect of the transcription factor RORγt on the intestinal microbiome was then investigated as a model of how perturbation of immune gardening could result in dysbiosis. Mice deficient in RORγt had an altered small intestinal and colonic mucosa-associated microbiome characterized by overgrowth of segmented filamentous bacteria (SFB), a microbe previously shown to promote colitis. Further knockout and cell engraftment experiments demonstrated that small intestinal gardening of SFB was mediated by RORγt-dependent T cells in a manner independent of IL-17A. The protective rs4845604 polymorphism in the RORC gene encoding RORγt was associated with altered microbial composition in mucosal wash samples from IBD patients and healthy individuals. These findings demonstrated that RORγt-dependent T cells garden the intestinal microbiome and suggest that genetic variation in this process could influence susceptibility to IBD. iii The dissertation of Jonathan Patrick Jacobs is approved. Linda Baum James Borneman Charalabos Pothoulakis Jonathan Braun, Committee Chair University of California, Los Angeles 2015 iv TABLE OF CONTENTS CHAPTER 1 THE MUCOSAL MICROBIOME: IMPRINTING THE IMMUNE SYSTEM OF THE INTESTINAL TRACT..……………………………………………………...…………….1 CHAPTER 2 IMMUNE AND GENETIC GARDENING OF THE INTESTINAL MICROBIOME ..……………………………………………………...………………………...48 CHAPTER 3 DISEASE-ASSOCIATED METABOTYPES AND ENTEROTYPES IN FAMILIES WITH PEDIATRIC INFLAMMATORY BOWEL DISEASE………….…………88 CHAPTER 4 RORγT-DEPENDENT CD4+ T CELLS GARDEN THE MUCOSA- ASSOCIATED MICROBIOME OF THE SMALL INTESTINE AND COLON…….……….139 LIST OF FIGURES Figure 1-1 Factors that shape the intestinal microbiome..……………………………………….25 Figure 1-2 Immune pattern recognition receptors and their compartments.……….…………….26 Figure 2-1 Putative mechanisms for immune gardening of the intestinal microbiome...……......70 Figure 3-1 Reduced diversity and altered microbial composition in IBD patients. ...…….........109 Figure 3-2 A low-diversity enterotype is associated with IBD but also is present in healthy relatives....………………………………………..……………...………………….………..…111 Figure 3-3 Microbial modules are associated with enterotype and influence the predicted metagenome.......………………………………………..……...……...……...………….……..112 v Figure 3-4 KEGG modules enriched in E1 and EC1/EC3………………………….…….…….114 Figure 3-5 CD patients have distinct metabolomics features also seen in a subset of healthy relatives…..........………………………………………..……………...……...………….…….115 Figure 3-6 Individuals divide into metabotypes related to IBD status…………….…….……..117 Figure 3-7 Negative ESI mode metabotypes map to positive ESI mode metabotypes……..….118 Figure 3-8 Androstenedione and estradiol are increased in metabotype 1 in both males and females…..........………………………………………..……………...……...…….…….…….119 Figure 3-9 Microbe-metabolite interactions between the IBD-associated enterotype and metabotype…..........………………………………………..……………...……...…….………120 Figure 3-10 Association of enterotype, metabotype, and genetic risk score with IBD….……..122 Figure 4-1 Mice deficient in RORγt or adaptive immunity have SFB overgrowth in the small intestinal mucosa…..........………………………………………..……………...……...………155 Figure 4-2 Increased SFB is seen throughout the small intestine and colon of mice deficient in RORγt…..........………………………………………..……………...……...………………….157 Figure 4-3 T cell deficiency promotes SFB overgrowth in the small intestine and colon….…..159 Figure 4-4 IL-17 deficiency and antibody neutralization increases colonic SFB without affecting SFB in the small intestine…………………………..……………...……...………………...….161 Figure 4-5 Lamina propria Th17 cells suppress SFB in the small intestine in a RORγt-dependent and IL-17-independent manner……………………..……………...……...………………...….162 Figure 4-6 The rs4845604 polymorphism in RORC is associated with shifts in the colonic mucosal microbiome of Crohn’s disease patients and healthy controls...…………………..….164 vi LIST OF TABLES Table 1-1 Effects of the intestinal microbiome on immune cell function....…………………….27 Table 3-1 Cohort demographics…………………………..……………...……...…….…….…123 Table 3-2 Metabotypes and enterotypes of non-IBD individuals are associated with family….125 Table 3-3 Taxa contained within IBD- and enterotype-associated microbial modules…...........126 Table 3-4 Validated metabolites associated with metabotypes 1 and 3…...................................129 vii ACKNOWLEDGEMENTS Chapter One is a version of: Jacobs J, Braun J, “The Mucosal Microbiome: Imprinting the Immune System of the Intestinal Tract,” in Mucosal Immunology, Mestecky J and Strober W, Eds. (Academic Press, Waltham, ed. 4, 2015). Jonathan Braun served as the PI. Chapter Two is a version of: Jacobs JP, Braun J. Immune and genetic gardening of the intestinal microbiome. FEBS Letters. 2014 Mar 5; pii: S0014. Jonathan Braun served as the PI. Chapter Three is a version of: Jacobs JP, Goudarzi M, Singh N, Tong M, McHardy I, Ruegger P, Huang H, Asadourian M, Ayson A, Borneman J, McGovern D, Fornace A, Braun J, Dubinsky M. Disease-associated Metabotypes and Enterotypes in Families with Pediatric Inflammatory Bowel Disease. This manuscript is in preparation for publication. Jonathan Braun and Marla Dubinsky served as co-PIs. Maryam Goudarzi and Al Fornace performed metabolomics. Maomeng Tong, Ian McHardy, Miro Asadourian, and Allyson Ayson processed fecal samples. Paul Ruegger and James Borneman performed 16S rRNA sequencing. Hailiang Huang and Dermot McGovern provided genetic risk scores. Chapter Four has the following co-authors. Jonathan Braun served as the PI. Lin Lin and Venu Lagishetty assisted with mouse dissection and microbial collections. Paul Ruegger and James Borneman performed 16S rRNA sequencing. This research was supported by NIH/NIDDK T32 DK007180-36. viii VITA/BIOGRAPHICAL SKETCH EDUCATION: A.B., Harvard University, 2003 M.D., Harvard Medical School, 2008 PROFESSIONAL EXPERIENCE: Resident, Stanford University Hospital, Stanford, CA 7/2008-6/2010 Fellow, UCLA Division of Digestive Diseases, Los Angeles, CA 7/2010-present HONORS AND SPECIAL AWARDS: Phi Beta Kappa, Harvard University, 2003 Magna cum laude with highest honors, Harvard University, 2003 Magna cum laude in a special field, Harvard Medical School, 2008 Best Overall Abstract, CURE Annual Research Meeting, 2014 AGA/AGA-GRG Fellow Abstract Prize, 2015 RESEARCH GRANTS AND FELLOWSHIPS RECEIVED: NIH/NIDDK T32 DK007180-36, 7/01/2011-6/30/2014 RESEARCH PAPERS - PEER REVIEWED 1. Solomon DH, Katz JN, Jacobs JP, La Tourette AM, Coblyn J. Management of glucocorticoid-induced osteoporosis in patients with rheumatoid arthritis: rates and predictors of care in an academic rheumatology practice. Arthritis and Rheumatism. 2002 Dec; 46(12):3136-42. [PMID:12483716] 2. Jacobs JP, Pettit AR, Shinohara ML, Jansson M, Cantor H, Gravallese EM, Mathis D, Benoist C. Lack of requirement of osteopontin for inflammation, bone erosion, and ix cartilage damage in the K/BxN model of autoantibody-mediated arthritis. Arthritis and Rheumatism. 2004 Aug; 50(8):2685-94. [PMID:15334485] 3. Hyatt G, Melamed R, Park R, Seguritan R, Laplace C, Poirot L, Zucchelli S, Obst R, Matos M, Venanzi E, Goldrath A, Nguyen L, Luckey J, Yamagata T, Herman A, Jacobs J, Mathis D, Benoist C. Gene expression microarrays: glimpses of the immunological genome. Nature Immunology. 2006 Jul; 7(7):686-91. [PMID:16785882] 4. Nguyen LT, Jacobs J, Mathis D, Benoist C. Where FoxP3-dependent regulatory T cells impinge on the development of inflammatory arthritis. Arthritis and Rheumatism. 2007 Feb; 56(2):509-20. [PMID:17265486] 5. Monach PA, Verschoor A, Jacobs JP, Carroll MC, Wagers AJ, Benoist C, Mathis D. Circulating C3 is necessary and sufficient for induction of autoantibody-mediated arthritis in a mouse model. Arthritis and Rheumatism.