THE ROLE OF VITAMIN D IN INFLAMMATORY BOWEL DISEASE PATHOGENESIS AND SEVERITY by Berkeley Limketkai A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland March, 2017 ABSTRACT Vitamin D has traditionally been known for its role in bone metabolism, but more recently been implicated in immune function. Emerging evidence has further suggested that vitamin D may be involved in inflammatory bowel disease (IBD) pathogenesis and activity. Studies found lower vitamin D levels among IBD patients, particularly in the setting of increased disease activity, implying that vitamin D influenced IBD. However, interpretation of these findings is challenged by the concept of reverse causation, where intestinal inflammation is already known to reduce vitamin D levels. These studies have therefore not established vitamin D deficiency as a cause or effect of IBD in humans. The principal objective of this thesis was to clarify the causal role of vitamin D in IBD pathogenesis and severity, while employing diverse methodologic approaches to overcome the issue of reverse causation. The first study was a case-control comparison of vitamin D levels from sera that were prospectively collected before and around the time of diagnosis of Crohn’s disease (CD). This longitudinal analysis permitted the evaluation of vitamin D levels as precursors to CD pathogenesis, while reducing interference by reverse causation. Vitamin D levels were similar between cases and controls prior to diagnosis, but significantly lower among cases around the time of diagnosis. The second study was an ecologic study that evaluated the association of ultraviolet (UV) exposure – a surrogate marker of vitamin D that was not influenced by IBD – and IBD hospitalization severity. Low UV exposures were associated with increased rates of hospitalization, prolonged hospitalization, and need for bowel surgeries. The third study was a systematic review of randomized trials that evaluated the effect of vitamin D supplementation on IBD activity. ii Randomization in these trials would have theoretically permitted assessment of the effect of vitamin D on IBD independent of disease activity. The meta-analysis of 4 studies with 12-month follow-up showed a trend toward fewer clinical relapses, but this was not statistically significant. Nonetheless, most studies had small sample sizes, significant risk of bias, and substantial methodologic heterogeneity. In conclusion, this thesis investigation did not find vitamin D to significantly influence CD pathogenesis. There was nonetheless some evidence to suggest that vitamin D may influence IBD activity. Further investigation with high-quality and larger randomized trials of vitamin D intervention in IBD is warranted. Committee Chair James A. Tonascia, PhD, Professor of Biostatistics and Epidemiology Committee Chair, Emeritus Theodore M. Bayless, MD, Professor Emeritus of Medicine Research Advisor Steven R. Brant, MD, Professor of Medicine Academic Advisor Craig W. Hendrix, MD, Professor of Medicine Thesis Readers N. Franklin Adkinson, MD, Professor of Medicine David M. Levine, MD, Professor of Medicine Eliseo Guallar, MD, DrPH, Professor of Epidemiology Susan M. Hutfless, PhD, Assistant Professor of Medicine iii ACKNOWLEDGEMENTS This dissertation is the culmination of the selfless contributions of the whole “village” that has helped me along my path of clinical and research training. I express my deepest gratitude to Dr. Theodore Bayless who has shown immense altruism in mentorship since my early internship days through my junior faculty years. He has served as a preeminent role model whose life and passion for inflammatory bowel disease (IBD) inspired me to eventually subspecialize in the field. As he has played a strongly defining role in my budding medical career, the opportunity to pursue the Theodore M. Bayless Fellowship in Inflammatory Bowel Diseases was an incredible honor that I will cherish throughout my career. Dr. Francis Giardiello was formally my gastroenterology fellowship director, although he also served as a career mentor during my residency and fellowship training. I truly appreciate his interest in my personal and professional development over the years. My early conversations with him while still an Osler resident solidified my desire to someday pursue further studies through the Graduate Training Programs in Clinical Investigation (GTPCI). Subsequently, as a gastroenterology fellow, Dr. Giardiello guided me through the intricacies of balancing gastroenterology fellowship, my Ph.D. studies, and in my last year at Johns Hopkins, the fellowship in IBD. Drs. N. Franklin Adkinson and Craig Hendrix are pillars of my GTPCI studies. I am indebted to them for their great generosity in time and advice, particularly as I navigated through my Ph.D. studies and thesis research. They have also provided invaluable advice on a diversity of academic matters, such as grant funding, promotions, politics, and building a research career. The foundations of this dissertation and my early IBD research career are credited to Drs. Steven Brant and Susan Hutfless. They served as my primary research mentors on this and other research endeavors throughout fellowship. Despite their myriad of responsibilities and very busy schedules, they have invested innumerable hours, days, and weeks over the past six years in teaching me how to conduct research. While serving as the chair of my Ph.D. thesis committee, Dr. James Tonascia has demonstrated admirable leadership, humor, and kindness. He shepherded me along the path leading up to the final defense with a genuine interest in my success. Despite our nascent interactions over the past few months, I feel as though he had already mentored me for years. I hope to continue learning from him well beyond my graduate education. iv Finally, the strongest drivers behind the person who I am … To my parents, there is no measure for how I can repay you. Your unconditional love and sacrifice throughout my childhood have molded me into the person I have become. I took risks and steadfastly pursued my passions when you helped me overcome internal doubts. I learned to rise from failure when you pulled me out of the pit of discouragement or defeat. I aimed for the skies when you reminded me that I could dream bigger than my limited imagination. As such, I dedicate all my successes to you. To my two sons, Aidan and Ethan, you are still too young to appreciate the inexpressible joy (and stress) you have brought your mother and me. When the days buried in work may feel endless at times, you remind me how rich my life truly is. Thank you for your patience during the many moments I could not play with you, while I focused on my thesis research and this dissertation. I hope to someday inspire you to far exceed what I have done. To my wife, DanDan, just as I have repeated over our 16 years of marriage, I feel greatly blessed to have you in my life. You never cease to amaze me with your incredible love, patience, and selflessness. Throughout my impossible balancing act of life as a husband, father, son, physician, and scientific explorer, you have helped me pick up the pieces and have encouraged me to persevere. While I worked on this dissertation, you minimized any distractions, entertained our sons when I was not available, kept the house in order, and repaired any disorder. This would not have been possible without you. Thank you. v TABLE OF CONTENTS ABSTRACT ..................................................................................................................................................II ACKNOWLEDGEMENTS ....................................................................................................................... IV TABLE OF CONTENTS ........................................................................................................................... VI LIST OF TABLES .................................................................................................................................. VIII LIST OF FIGURES ................................................................................................................................... IX LIST OF ABBREVIATIONS ...................................................................................................................... X CHAPTER I. INTRODUCTION ................................................................................................................. 1 VITAMIN D SYNTHESIS AND ACTION........................................................................................................... 2 VITAMIN D AND THE IMMUNE SYSTEM ....................................................................................................... 3 VITAMIN D AND AUTOIMMUNE DISEASE .................................................................................................... 5 VITAMIN D AND INFLAMMATORY BOWEL DISEASE .................................................................................... 7 Animal Models ....................................................................................................................................... 7 Genetic Evidence ................................................................................................................................... 8 Epidemiologic Evidence ...................................................................................................................... 10 Intervention