IMMUNO-ENDOCRINE INTERACTIONS IN INTESTINAL INFLAMMATION PhD Thesis- Shajib, MS; McMaster University-Medical Sciences Immuno-endocrine interactions in intestinal inflammation By Md. Sharif Shajib, BSc. (Hons) A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy McMaster University © Copyright by Md. Sharif Shajib, 2017. PhD Thesis- Shajib, MS; McMaster University-Medical Sciences Descriptive notes Doctor of Philosophy (2017) McMaster University, Hamilton, Ontario (Medical Sciences) TITLE Immuno-endocrine interactions in intestinal inflammation AUTHOR Md. Sharif Shajib, BSc. (Hons) SUPERVISOR Dr. Waliul I. Khan NUMBER OF PAGES: XX, 292. II PhD Thesis- Shajib, MS; McMaster University-Medical Sciences Lay abstract The gut produces most of the serotonin found in our body, where it regulates many normal functions. A group of special cells, named enterochromaffin cells, produces nearly all of the serotonin in the gut. In diseases of the gut, especially ones that involve inflammation resulting in symptoms like abdominal pain, diarrhea and bleeding, the number of these cells and serotonin concentration are different from that in the normal gut. I found that these changes are controlled by a particular protein produced by immune cells, called interleukin-13, and alteration in serotonin levels, in turn, contributes to the inflammatory process. Our laboratory experiments with cells and animals establish this connection between interleukin-13 and serotonin in gut inflammation. We further confirm this association between interleukin-13 and serotonin in human inflammatory bowel disease. Moreover, we identify a potential genetic cause of these changes in serotonin concentrations which may ultimately result in inflammatory bowel disease. III PhD Thesis- Shajib, MS; McMaster University-Medical Sciences Abstract Mucosal inflammation in conditions ranging from infective acute enteritis or colitis to inflammatory bowel disease (IBD) is accompanied by alteration in enterochromaffin (EC) cell numbers and serotonin (5-hydroxytryptamine; 5-HT) content in the gut. Previously we had shown that CD4+ T cells, via production of T helper (Th)2 cytokines, regulate EC cell biology in the Trichuris muris- infectious colitis model. I further examined the mechanisms of immuno-endocrine interactions in the context of intestinal inflammation. In chapter 3, utilizing human EC cell line and Trichuris muris-mouse model of infectious colitis we identified a critical role of interleukin (IL)-13, a key Th2 cytokine, in increasing EC cell numbers, tryptophan hydroxylase (TPH)1 expression (rate-limiting enzyme of mucosal 5-HT bio-synthesis), and 5-HT production. In chapter 4, we show that IL-13 driven intestinal inflammation is critically dependent on increased 5-HT production using dextran sulfate sodium (DSS) and dinitrobenzene sulphonic acid (DNBS) models of colitis. In DSS-induced colitis, we were the first to identify the increased production of IL-13 and its pathogenic role as IL-13 knockout (IL-13-KO) mice had less severe inflammation compared to wild-type, which was exacerbated following replenishment of 5-HT in IL-13- KO mice. In chapter 5, biopsy examination revealed, higher mucosal IL-13 expression accompanied inflammation in Crohn's disease (CD), which was additionally associated with increased TPH1, 5-HT receptor (5-HTR)3A, 5-HTR7 IV PhD Thesis- Shajib, MS; McMaster University-Medical Sciences and decreased 5-HT transporter (5-HTT) expressions. Moreover, CD patients had elevated plasma and platelet-poor plasma 5-HT levels compared to healthy controls (HCs). Furthermore, 5-HTT polymorphism associated genotypes causing inefficiency in 5-HT re-uptake were more common in our patient cohort than HCs. The findings included in this thesis further emphasize the role of immuno- endocrine interactions in intestinal inflammation, which may be a step toward a better diagnosis or management or even a cure for a disease that is of growing concern, and in understanding IBD pathogenesis. V PhD Thesis- Shajib, MS; McMaster University-Medical Sciences Acknowledgements I have been fortunate enough to have the time, support and resources to attempt to add new knowledge to the understanding of a disease, and in the process, also develop a great interest in GI immunology and inflammation. For which I owe a great debt of gratitude to a number of individuals: Dr. Waliul Khan, thank you for taking a chance on someone who is not particularly talented and emphasizing the learning process, conceptualization, and promoting independent thinking. Thank you for editing all the documents that I have sent you throughout the course of this degree, I can only hope to be as good a writer/story teller as you. Huqaing and Jean-Eric, for teaching me the basics of the techniques that I have used throughout my degree. Marcus and Musfeque, for reminding me that science is supposed to be fun. Janice, for being an amazing labmate, I could not have asked for a better peer to learn with or to learn from. All the Khan lab trainees past and present for their support and help in the lab, especially most recently, Eric. Thank you especially to Dr. Manel Jordana for IL-13 knockout mice, and Dr. John Marshall, without whom we would not be able to peruse our basic findings into the clinical realm. Likewise, my committee members, Drs. Jan Huizinga and Zhou Xing for your invaluable input and guidance as well as shared enthusiasm. I would like to thank all of our collaborators. Our work and conference presentations would not have been possible without the generous support from CIHR, CCC, NSERC, and the McMaster Department of Pathology and Molecular Medicine. I would also like to thank my institute (Farncombe Family Digestive Health Research Institute), for providing the environment to guide its trainees into their future. I would like to thank my graduate life friends, D'andra and Zainab, for being understanding and supportive and my friends from other walks of life for your kind words. You all make me I feel blessed. I am so very thankful to my family for their support and encouragement. Especially my parents, who up-rooted themselves to move to Canada for a better future for their children, and my sister (and her family) for her unwavering support for her younger sibling. Lastly, I want to thank Allah, for my faith to keep plugging along, even when the challenges feel insurmountable. VI PhD Thesis- Shajib, MS; McMaster University-Medical Sciences Table of contents PRELIMINARIES Immuno-endocrine interactions in intestinal inflammation I Descriptive notes II Lay abstract III Abstract IV Acknowledgements VI Table of contents VII List of figures X List of tables XI List of abbreviations and symbols XII List of peer-reviewed publications and submitted manuscripts XVIII CHAPTER 1: LITERATURE OVERVIEW 1 Serotonin 2 EC cells 3 Synthesis and metabolism of 5-HT 6 5-HTRs and GI functions 8 5-HTT 12 5-HT and the immune system 14 5-HT and innate immune system 14 5-HT and adaptive immune system 18 IBD: A multifactorial disease 20 Environmental factors 23 Gut microbiota 26 VII PhD Thesis- Shajib, MS; McMaster University-Medical Sciences Genetic susceptibilities 28 Intestinal immune system 29 The epithelium 31 DCs and macrophages 36 T lymphocytes 44 IL-13 and gut inflammation 51 5-HT in gut inflammation 58 Experimental approaches to studying gut inflammation 67 T. muris model of infectious colitis 68 Chemical models of intestinal inflammation 68 DSS-induced colitis 69 DNBS-induced colitis 70 CHAPTER 2: HYPOTHESIS AND AIMS 72 AIM 1: Investigate the role of IL-13 in EC cell biology 75 AIM 2: Elucidate the influence of IL-13 on the severity of 75 experimental colitis as mediated by 5-HT AIM 3: To characterize mucosal 5-HT signaling components in 76 IBD patients and its relationship with IL-13, as well as to explore the association between 5-HTTLPR and IBD. CHAPTER 3: IL-13-MEDIATED IMMUNOLOGICAL 77 CONTROL OF ENTEROCHROMAFFIN CELL HYPERPLASIA AND SEROTONIN PRODUCTION IN THE GUT CHAPTER 4: INTERLEUKIN 13 AND SEROTONIN: LINKING 91 THE IMMUNE AND ENDOCRINE SYSTEMS IN MURINE MODELS OF INTESTINAL INFLAMMATION CHAPTER 5: LINKAGE BETWEEN 5-HT TRANSPORTER 109 PROMOTER POLYMORPHISM AND 5-HT SIGNALING MACHINERY WITH CROHN’S DISEASE VIII PhD Thesis- Shajib, MS; McMaster University-Medical Sciences CHAPTER 6: GENERAL DISCUSSION 147 Implication of our findings 148 IL-13/5-HT axis and other factors in intestinal inflammation 152 IL-13 and 5-HT in the management of intestinal inflammation 155 Concluding statements 159 APPENDIX I: THE ROLE OF SEROTONIN AND ITS 160 RECEPTORS IN ACTIVATION OF IMMUNE RESPONSES AND INFLAMMATION APPENDIX II: DIVERSE EFFECTS OF GUT DERIVED 181 SEROTONIN IN INTESTINAL INFLAMMATION References for chapters 1 and 6 195 IX PhD Thesis- Shajib, MS; McMaster University-Medical Sciences List of figures Figure I: Schematic representation of mucosal 5-HT production 8 and functions Figure II: A schematic representation of various layers of the gut 22 Figure III: Factors involved in IBD 23 Figure IV: Immune system associated genes whose mutations are 29 linked with increased risk of IBD Figure V: Intestinal permeability pathways and overview of 33 junctional proteins Figure VI: Immunological regulation of mucosal 5-HT signaling 62 Figure VII: Schematic representation of hypothesis and aims 74 X PhD Thesis- Shajib, MS; McMaster University-Medical Sciences List of tables Table I: Murine intestinal DC subsets and their locations in the 39 gut. Table