Bilirubin modulates leukocyte recruitment to sites of inflammation A dissertation presented by Megan Elizabeth Vogel B.S., Ohio University 2011 To The Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Internal Medicine, Division of Digestive Diseases of the College of Medicine March 2017 Committee Chair: Stephen D. Zucker, M.D. Abstract Background: Bilirubin is the principal end-product of heme catabolism. While generally thought to be little more than a metabolic by-product, there is accumulating epidemiological evidence that higher serum bilirubin levels are associated with a lower incidence of inflammatory disorders such as inflammatory bowel and cardiovascular disease. However, the mechanism(s) by which bilirubin may exert an anti-inflammatory effect remains poorly understood. The transendothelial migration of immune cells to sites of inflammation is a highly- ordered, multi-step process that is initiated when endothelial cells become activated to express adhesion molecules, including Vascular Cell Adhesion Molecule 1 (VCAM-1) and Intercellular Adhesion Molecule 1 (ICAM-1), on their luminal surface. The specific binding of leukocyte integrins to VCAM-1 and/or ICAM-1 triggers endothelial signaling cascades that result in the intracellular generation of superoxide and hydrogen peroxide. These reactive oxygen species (ROS) induce reorganization of the actin cytoskeleton, promoting leukocyte transmigration. There are many disease states in which VCAM-1 and ICAM-1 are believed to play an essential pathogenic role in mediating leukocyte trafficking. As bilirubin is a potent, chain-breaking antioxidant, our central hypothesis is that it exerts an anti-inflammatory effect by disrupting adhesion molecule-mediated leukocyte migration through the scavenging of ROS signaling intermediaries. Aim: To validate the key role played by VCAM-1 and ICAM-1 in the pathogenesis of inflammatory bowel disease and cardiovascular disease and, to elucidate the molecular mechanisms underlying the ameliorating effect of bilirubin on these disorders. ii Methods: In vitro analyses of the mechanisms by which bilirubin impedes the transmigration of human leukocyte cell lines across monolayers of isolated human umbilical vein endothelial cells (HUVEC) were performed using Boyden chamber assay and confocal microscopy to assess adhesion molecule-stimulated ROS generation. Murine models of inflammatory bowel disease (DSS-induced colitis) and cardiovascular disease (LDL receptor-deficient mice; Ldlr -/-) were employed to investigate the effect of bilirubin on local and systemic inflammation (immunohistochemistry, immunoassay), and tissue oxidation (immunofluorescence). Results & Conclusions: Bilirubin, at physiological concentrations ( ≤ 20 µM), was found to dose-dependently block THP-1 (monocyte) and Jurkat (lymphocyte) migration across tumor necrosis factor α-activated HUVEC monolayers, without altering leukocyte binding or cytokine/ chemokine production. Bilirubin also effectively abolished endothelial ROS generation induced by the crosslinking of VCAM-1 or ICAM-1, as validated by treatment with blocking antibodies and with specific inhibitors of VCAM-1 and ICAM-1 signaling. Bilirubin, when administered to mice with DSS-induced colitis, abrogated disease activity, specifically reducing eosinophil, lymphocyte and monocyte infiltration into the colon. Ldlr -/- mice maintained on a high-fat diet showed a marked reduction in atherosclerotic plaque formation when treated with bilirubin, without changes in circulating cholesterol, triglyceride, or cytokine/chemokine levels. Aortic roots from bilirubin-treated animals manifested reduced lipid and collagen deposition, diminished numbers of macrophages, lymphocytes and smooth muscle cells, and decreased protein oxidation, without altered VCAM-1 or ICAM-1 expression. These data support that bilirubin ameliorates inflammatory responses by inhibiting VCAM-1- and ICAM-1-mediated iii leukocyte migration through the scavenging of ROS signaling intermediaries, suggesting a potential mechanism for the anti-inflammatory effects of bilirubin. iv Copyright Notice Chapters II and III of this dissertation are adaptations of manuscripts published in the open access journals, American Journal of Physiology - Gastrointestintal and Liver Physiology and, the Journal of the American Heart Association. The American Journal of Physiology permits whole published articles to be reproduced in dissertations with the requirement of full citation. The Journal of the American Heart Association distributes under the terms of the Creative Commons Attribution Non-Commercial License, which permits use, distribution, and reproduction in any medium, provided the original work and authors are credited. The citation for each article can be found on the title page of the corresponding chapter. v Acknowledgements This dissertation would not be possible without the tremendous support and guidance from many individuals. I would like to express sincere gratitude to: My mentor, Dr. Stephen Zucker, who has been tremendously supportive since day one. Your enthusiasm for science, more specifically bilirubin and its many characteristics is how I strive to be with all projects I may endeavor in the future. Thank you for all your inspiration and guidance throughout my graduate career, whether it was in the laboratory or day to day life challenges. Without your confidence not only in my work but also in me, I would not be where I am today. I am forever grateful for these past five years I got to spend in your laboratory. To my thesis committee members, Drs. George Deepe, David Hui, Alex Lentsch and Florence Rothenberg, whom I significantly appreciate your willingness to serve on my committee. Your feedback and support have been fundamental in guiding me through my dissertation work and in my growth as a confident researcher. My lab mate, Dr. Gila Idelman, who has taught me a plethora of lab techniques, a variety of phrases in Hebrew and Russian, and how to be a team player. Without your guidance, knowledge and expertise in bench work, I still would be miscalculating dilutions. Thank you for always dedicating the time to help me throughout the course of my graduate studies, whether it was enduring long hours of excising aortas or the many failed experiments, you were always there for me and I am beyond grateful. Also, I would like to thank Sue Rouster (“Lab Mom”) for her continuous encouragement and support throughout these last couple of years of my graduate work. Without your words of wisdom and the distraction of upcoming concerts, I would not have survived. Thank you for always reminding me that there is light at the end of the tunnel. vi The Patholobiology and Molecular Medicine program faculty and students, who have listened to my many seminars about bilirubin and provided a diverse and incredible learning environment. I would especially like to thank Heather Anderson for always responding rapidly to my vast array of emails and being an incredible friend and listener. You have made this experience worth the five (plus) years. I would also like to acknowledge my extraordinary and entertaining classmates, Drs. Dusten Unruh and Eleanor Powell, and Jamie Tweedle. Without our laughter during those long hours of studying our first year, I would not have survived. I will forever be grateful for your friendships and always remember, “Hepatocytes!” My family – where do I even begin. My parents, Jeff and Karen, for your support, love, and encouragement I will always be entirely thankful for. Who would have thought that that overly stubborn child of yours who always talked during class would one day go on to graduate with a Ph.D.? You instilled in me, at a young age, to always do what I love, even if it meant being completely different from everyone else. I would not be who (or where) I am today without you both and I love you very much. To my siblings, Jeffrey and Brittany, you have taught me to be persistent and never give up. I am forever appreciative of your love and support, and you both continuously make me proud every day. I love you and thank you for always encouraging my “strange” science endeavors. To the rest of my family and friends, thank you for your constant love and support. The Gauerke’s and Hunt’s, you have shown me nothing but love and encouragement these past few years, and for that I am beyond grateful. Ayrton, thank you for your endless motivation and for being the epitome of following your dreams. Your continuous love and support for me and my life endeavors, means more to me than you’ll ever know Bee. Thank you for laughter and for your music that gets me through the toughest of times! vii Table of Contents Title Page…………………………………………………………………………………… i Abstract …………………………………………………………………………………….. ii Copyright Notice …………………………………………………………………………... v Acknowledgements ………………………………………………………………………... vi Table of Contents ………………………………………………………………..…………. viii List of Figures…………………………………………………………………….………… xiii List of Abbreviations ………………………………………………………………………. xvi Chapter I: Introduction 1 1.1 Introduction to bilirubin .......................................................................................... 2 1.1.1 Discovery of bilirubin .................................................................................... 2 1.1.2 Structure and properties of bilirubin .............................................................. 2 1.1.3 Bilirubin