Bilirubin Modulates Leukocyte Recruitment to Sites of Inflammation

Bilirubin modulates leukocyte recruitment to sites of inflammation

A dissertation presented by

Megan Elizabeth Vogel B.S., Ohio University 2011

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.

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.

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.

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.

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!

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Table of Contents

Title Page…………………………………………………………………………………… i

Abstract …………………………………………………………………………………….. ii

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 production, metabolism and elimination ...... 3

1.1.4 Clinical syndromes causing hyperbilirubinemia ...... 4

1.1.5 Antioxidant properties of bilirubin ...... 5

1.1.5 Anti-inflammatory properties of bilirubin ...... 6

1.2 Role of the endothelium in immune regulation ...... 8

1.2.1 The endothelium ...... 8

1.2.2 Vascular cell adhesion molecule 1 (VCAM-1) ...... 9

1.2.3 Intercellular adhesion molecule 1 (ICAM-1) ...... 9

1.3 Bilirubin as a regulator of leukocyte migration ...... 10

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Chapter II: Bilirubin prevents acute DSS-induced colitis by inhibiting leukocyte infiltration and suppressing up-regulation of inducible nitric oxide synthase 21

2.1 Abstract ...... 23

2.2 Background ...... 23

2.3 Materials and Methods ...... 26

2.3.1 Materials...... 26

2.3.2 Cell isolation and culture ...... 26

2.3.3 Induction of colitis by the administration of DSS ...... 27

2.3.4 Assessment of disease activity ...... 28

2.3.5 Histological analyses of tissue specimens ...... 28

2.3.6 Quantification of leukocytes in blood samples ...... 29

2.3.7 Serum assay for nitric oxide ...... 29

2.3.8 Quantification of cellular interleukin-5 expression ...... 30

2.3.9 Transwell migration assay ...... 30

2.3.10 Statistical analysis ...... 31

2.4 Results ...... 31

2.4.1 Effect of bilirubin on DSS-induced disease activity, colon length, and intestinal histology ...... 31

2.4.2 Influence of bilirubin on eosinophil recruitment in response to DSS treatment ...... 32

2.4.3 Bilirubin modulates the physiological homing of eosinophils to the intestinal tract ...... 34

2.4.4 Effect of bilirubin on the intestinal recruitment of T lymphocytes and monocytes/macrophages ...... 35

2.4.5 Influence of bilirubin on DSS-induced colonic iNOS expression and systemic nitrate production ...... 36

2.5 Discussion ...... 37

Chapter III: Bilirubin prevents atherosclerotic lesion formation in low-density lipoprotein (LDL) receptor-deficient mice by inhibiting endothelial vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) signaling 55

3.1 Abstract ...... 57

3.2 Background ...... 57

3.3 Materials and Methods ...... 60

3.3.1 Materials...... 60

3.3.2 Cell isolation and culture ...... 61

3.3.3 Immunoblot analysis ...... 61

3.3.4 Quantitative RT-PCR analysis ...... 62

3.3.5 Luminex assay for cellular cytokine production ...... 62

3.3.6 Transendothelial migration assay...... 63

3.3.7 Cell adhesion assay ...... 64

3.3.8 Measurement of cellular ROS by confocal microscopy ...... 64

3.3.9 Xanthine oxidase inhibition assay...... 65

3.3.10 Murine model of atherosclerosis ...... 65

3.3.11 Histologic analysis of aortic root lesions ...... 65

3.3.12 Determination of serum bilirubin, lipid, lipid peroxide and chemokine levels ...... 66

3.3.13 Statistical analyses ...... 67

3.4 Results ...... 67

3.4.1 Induction of adhesion molecule expression by HUVEC ...... 67

3.4.2 Bilirubin inhibits monocyte migration across endothelial cell monolayers ... 68

3.4.3 Influence of bilirubin on endothelial cell ROS production in response to activation of VCAM-1 and ICAM-1 ...... 70

3.4.4 Relative contribution of VCAM-1 and ICAM-1 to TNF α-induced monocyte migration ...... 71

3.4.5 Effect of bilirubin on the development of atherosclerotic lesions in Ldlr -/- mice ...... 72

3.5 Discussion ...... 73

Chapter IV: Discussion 94

4.1 Overview of bilirubin ...... 95

4.2 Mechanisms underlying bilirubin’s anti-inflammatory effects...... 95

4.2.1 Bilirubin inhibits the upregulation of inducible nitric oxide synthase

(iNOS) ...... 95

4.2.2 Bilirubin inhibits leukocyte trafficking to sites of inflammation...... 96

4.3 Confirmation of the anti-inflammatory effects of bilirubin ...... 97

4.3.1 Utility of bilirubin in the treatment of inflammatory bowel disease...... 97

4.3.2 Bilirubin for the prevention of atherosclerosis...... 99

4.3.3 Similarities in the pathogenesis of inflammatory bowel disease and atherosclerosis...... 101

4.4 Proposed physiological function of bilirubin ...... 101

4.5 Future studies and directions...... 103

4.5.1 Elucidating the mechanism underlying the inhibitory effect of bilirubin on physiological leukocyte homing to the intestinal tract ...... 103

4.5.2 Validation of bilirubin’s mechanism of action ...... 105

4.5.3 Clinical application of bilirubin in human disease...... 107

4.5.4 Effect of bilirubin on antigen presenting cell (APC) activation and interaction with lymphocytes ...... 109

4.6 Conclusion...... 113

References 119

xii

List of Figures:

1.1 Crystalline bilirubin and its chemical structure ……………………………………. 12

1.2 Overview of bilirubin production, metabolism and elimination …………………… 13

1.3 Species-dependent pathways of heme degradation and elimination ………………. 14

1.4 Bilirubin undergoes intracellular redox cycling……………………………………. 15

1.5 The process of leukocyte adhesion and transmigration ……………………………. 16

1.6 VCAM-1 signal transduction in endothelial cells ………………………………….. 17

1.7 ICAM-1-mediated intracellular signaling in endothelial cells ……………………... 18

1.8 Proposed mechanism of bilirubin modulation of VCAM-1- and ICAM-1- dependent leukocyte migration …………………………………………………….. 19

2.1 Effect of bilirubin on body weight, disease activity, and colonic shortening induced by dextran sodium sulfate (DSS)………………………………………….. 43

2.2 Bilirubin treatment reduces colon histological injury following DSS administration………………………………………………………………………. 44

2.3. Bilirubin inhibits eosinophil infiltration and VCAM-1 expression in the colon of DSS-treated mice ………………………………………………………………... 45

2.4 Effect of bilirubin on peripheral blood leukocytes…………………………………. 46

2.5 Influence of bilirubin on cellular IL-5 production, leukocyte transendothelial migration, and serum nitrate concentration………………………………………… 47

2.6 Bilirubin reduces eosinophil infiltration into the small intestine …………………... 48

2.7 Treatment with bilirubin decreases T lymphocyte infiltration into the colon of DSS-treated mice…………………………………………………………………… 49

2.8 Bilirubin inhibits the recruitment of monocytes/macrophages to the colon in response to DSS ……………………………………………………………………. 50

xiii

2.9 DSS and bilirubin do not alter T lymphocyte levels in the small intestine ………… 51

2.10 Monocyte infiltration into the small intestine is reduced following bilirubin treatment …………………………………………………………………………… 52

2.11 Bilirubin inhibits DSS-induced colonic inducible nitric oxide synthase (iNOS) expression ………………………………………………………………………….. 53

3.1 Time course for TNF-α-induced expression of adhesion molecules by HUVEC….. 78

3.2 Bilirubin inhibits the migration of THP-1 monocytes across activated HUVEC monolayers ………………………………………………………………………… 80

3.3 Bilirubin does not alter cytokine or chemokine expression by activated HUVEC … 81

3.4 Bilirubin does not alter adhesion molecule expression or monocyte binding to HUVEC …………………………………………………………………………….. 82

3.5 Bilirubin suppresses cellular ROS generation following activation of VCAM-1 or ICAM-1 …………………………………………………………………………….. 83

3.6 Nox and XO inhibitors and antibodies against VCAM-1 and ICAM-1 recapitulate the effect of bilirubin on endothelial ROS generation and monocyte transmigration 85

3.7 Effect of bilirubin administration on body weight, serum bilirubin and lipid levels ………………………………………………………………………………. 87

3.8 Bilirubin inhibits the formation of aortic root lesions in Ldlr -/- mice………………. 88

3.9 Bilirubin decreases leukocyte infiltration and oxidative injury in the aortic root of Ldlr-/- mice …………………………………………………………………………. 89

3.10 Bilirubin does not alter VCAM-1 or ICAM-1 expression in Ldlr -/- mice ………….. 90

3.11 Effect of bilirubin on serum parameters in Ldlr -/- mice ……………………………. 91

3.12 Proposed mechanism of bilirubin modulation of VCAM-1- and ICAM-1- dependent monocyte migration …………………………………………………….. 92

4.1 Leukocyte migration in inflammatory bowel disease and atherosclerosis ………… 115

xiv

4.2 The immunological synapse between a dendritic cell and a B cell ...... 117

4.3 Oxidative stress and redox signaling in physiologic and pathophysiologic states…. 118

List of Abbreviations:

ANCA anti-neutrophil cytoplasmic antibodies Ag antigen AP allopurinol; xanthine oxidase inhibitor APC antigen presenting cell apoE apolipoprotein E ATCC American Type Culture Collection B220 pan B-cell marker (CD45R) BAFF B cell activating factor (TNFRSF13C) BCA-1 B cells attracting chemokine 1 (CXCL13) BCR B cell receptor BR bilirubin BVR biliverdin reductase Ca 2+ calcium CBC complete blood count CD3 cluster of differentiation 3; marker of T lymphocytes CD11a integrin alpha L (ITGAL) CD18 integrin beta 2 (ITGB2) CD29 integrin beta 1 (ITGB1) CD45 cluster of differentiation 45; marker of leukocytes CD49d integrin alpha 4 (ITGA4) CD68 cluster of differentiation 68; marker of monocytes/macrophages cDNA complementary deoxyribonucleic acid CMAP 2-chloro-6(methylamino)purine CO carbon monoxide

CO 2 carbon dioxide

xvi

CRP C-reactive protein cSMAC central supramolecular activation complex DAI disease activity index DAPI 4,6-diamidino-2-phenylindole DC dendritic cell DHR dihydrorhodamine DMSO dimethylsulfoxide DSS dextran sodium sulfate F-12K Kaighn’s modification of Ham’s F-12 medium FCS fetal calf serum FDC follicular dendritic cell Fe 2+ free iron GAPDH glyceraldehyde-3-phosphate dehydrogenase H&E hematoxylin and eosin

H2O2 hydrogen peroxide HIF-1α hypoxia-inducible factor 1 alpha HO heme oxygenase HPF high power field HRP horseradish peroxidase HSA human serum albumin HSB-2 human T cell lymphoblastoid line HUVEC human umbilical vein endothelial cell IBD inflammatory bowel disease ICAM-1 intercellular adhesion molecule 1 (CD54) IgG immunoglobulin G

IFN-γ interferon gamma

IL interleukin

xvii iNOS inducible nitric oxide synthase i.p. intraperitoneal IS immunological synapse Jurkat human acute T cell leukemia cell line

K3PO 4 potassium phosphate

LPAM-1 lymphocyte Peyer patch adhesion molecule 1 (α4β7) LDLR low density lipoprotein receptor Ldlr -/- low density lipoprotein receptor-deficient

LFA-1 lymphocyte function-associated antigen 1 (αLβ2, CD11a/CD18) LPS lipopolysaccharide

Mac-1 macrophage 1 antigen (αMβ2, CD11b/CD18) MAdCAM-1 mucosal addressin cell adhesion molecule 1 MCP-1 monocyte chemoattractant protein 1 (CCL2) M-CSF macrophage colony-stimulating factor MDA malondialdehyde; marker of lipid peroxidation MIP-1α macrophage inflammatory protein 1 alpha (CCL3) ML171 2-acetylphenothiazine; NADPH oxidase inhibitor MMP matrix metalloproteinases mRNA messenger ribonucleic acid MRP2 multidrug resistance-associated protein 2 (ABCC2) MW molecular weight NADPH nicotinamide adenine dinucleotide phosphate NCBI National Center for Biotechnology Information NO nitric oxide Nox NADPH oxidase

O2 oxygen

·- O2 superoxide

xviii

OAPT2 organic anion transporting polypeptide 2 (SLC21A6) OVA ovalbumin OVA-IC ovalbumin immune complex PBS phosphate-buffered saline PCR polymerase chain reaction PECAM-1 platelet endothelial cell adhesion molecule 1 (CD31) PMA phorbol myristate acetate P-SCCAI Patient Simple Clinical Colitis Activity Index pSMAC peripheral supramolecular activation complex qRT-PCR quantitative reverse transcription polymerase chain reaction Rac1 Ras-related C3 botulinum toxin substrate 1 RANTES regulated on activation, normal T cell expressed and secreted (CCL5) RhoA Ras homolog family member A RNA ribonucleic acid RNS reactive nitrogen species ROS reactive oxygen species SMAC supramolecular activation complex SMC smooth muscle cells THP-1 human acute monocyte leukemia cell line TNF-α tumor necrosis factor alpha UC ulcerative colitis UCEIS Ulcerative Colitis Endoscopic Index of Severity UGT1A1 UDP glucuronosyltransferase 1 family, polypeptide A1 VCAM-1 vascular cell adhesion molecule 1 (CD106)

VLA-4 very late antigen 4 (α4β1, CD49d/CD29) XO xanthine oxidase

xix

Chapter I:

Introduction

1.1 Introduction to bilirubin

1.1.1 Discovery of bilirubin

Bilirubin is a naturally occurring tetrapyrrole formed during the normal physiological degradation of heme. Formerly termed “Hematoidin”, it was originally discovered in blood extravasates by Virchow in 1849. Stadeler subsequently coined the term bilirubin, derived from the Latin terms, bilis , meaning bile, and ruber , meaning red, as it exhibits an orange-red hue in crystalline form ( Figure 1.1A ). A few years later, Tarchanoff demonstrated that bilirubin is generated as part of the metabolism of the heme moiety of hemoglobin. The physiological isomer, bilirubin IX α, was first synthesized and its structure characterized by Fischer and

Plieninger in 1942.

1.1.2 Structure and properties of bilirubin

Bilirubin IX α adopts a “ridge-tile” conformation as a result of intramolecular hydrogen bonding between the two carboxylic acid groups and opposing dipyrromethenone moieties

(Figure 1.1B ) (1). This distinctive molecular structure results in low aqueous solubility (~70 nM) at physiologic pH (1, 2), while simultaneously causing bilirubin to be poorly soluble in apolar solvents as well (3). These unique solubility properties manifest in unusual membrane binding characteristics. For example, kinetic and thermodynamic analyses of bilirubin interactions with model and native lipid vesicles (4), as well as studies employing parallax analysis of fluorescence quenching (5), all support that bilirubin preferentially localizes to the lipid-water interface of membranes. Bilirubin also has been shown to freely pass through cellular membranes (4), facilitating rapid and quantitative entry into cells. This may, in part, explain why

2 patients with jaundice exhibit yellow discoloration of the skin, sclera (white of the eye), and various organs.

1.1.3 Bilirubin production, metabolism and elimination

Heme is derived primarily from hemoglobin (80%) released during the breakdown of senescent red blood cells and, to a lesser extent, from the turnover of hepatic enzymes (~20%) such as cytochromes, peroxidases, and catalase. Following its dissociation from hemoglobin, heme is oxidized by the membrane-bound heme oxygenase (HO) enzyme to form biliverdin, releasing carbon monoxide (CO) and free iron in the process ( Figure 1.2 ). Biliverdin reductase

(BVR), an NAD(P)-dependent oxidoreductase, catalyzes the conversion of biliverdin to bilirubin

(6). Because of the ubiquitous and constitutive nature of the BVR enzyme (7), HO represents the rate-limiting enzyme in bilirubin synthesis.

Because of its poor aqueous solubility, bilirubin is transported in the plasma principally bound to serum albumin, which possesses one high and two low-affinity binding sites (8, 9). To a minor degree, plasma bilirubin also is associated with high-density lipoproteins through binding to apolipoprotein D (10). The net result of this extensive protein binding is that free serum bilirubin constitutes less than 0.1% of the total plasma concentration (11, 12). Bilirubin is cleared from the circulation exclusively by the liver. Hepatic uptake of bilirubin occurs both by active transport, via organic anion transporting polypeptide 2 (OATP2; SLC21A6) (13, 14), and by passive diffusion through the hepatocyte plasma membrane (4, 15). Within the liver, bilirubin is conjugated with glucuronic acid by the action of the 1A1 isoform of UDP- glucuronosyltransferase (UGT1A1). This conjugation reaction leads to the disruption of the

3 internal hydrogen bonding and the formation of water-soluble mono- and diglucuronides. The canalicular transporter, multidrug resistance protein 2 (MRP2; ABCC2) efficiently secretes bilirubin glucuronides into the bile ( Figure 1.2 ) (16). Once these bilirubin conjugates enter the colon, a proportion are hydrolyzed by bacterial β-glucuronidases back to bilirubin. While most bilirubin precipitates in the colonic lumen and is excreted in the stool, a small amount is reabsorbed in the colon and circulated back to the liver for re-conjugation, a process known as enterohepatic cycling (17, 18).

It is notable that many non-mammalian vertebrates (e.g., birds, reptiles, amphibians) lack the BVR enzyme (19, 20), such that heme degradation terminates at the HO step (Figure 1.3 ).

The resultant biliverdin is efficiently excreted in the bile (21, 22) and urine (23, 24) without adverse consequences. Although bilirubin generally has been regarded as a little more than a metabolic by-product, the evolutionary development in mammals of the energetically costly process of bilirubin production and secretion suggests that this bile pigment may serve an important physiological role. A primary aim of this dissertation is to identify and elucidate key physiologic functions of bilirubin.

1.1.4 Clinical syndromes causing hyperbilirubinemia

Normal concentrations of bilirubin in human serum range from 2 to 24 µM (0.1 – 1.4 mg/dL), with higher levels in men versus women and in Caucasians versus blacks or Hispanics

(25). There are a number of conditions that may cause serum bilirubin levels to exceed the normal range. Increased concentrations of conjugated (direct) bilirubin occur when the hepatic secretion of bilirubin glucuronides is impaired (e.g., Dubin-Johnson, Rotor syndromes) or when

4 bile outflow is impeded (e.g., biliary obstruction). Unconjugated (indirect) hyperbilirubinemia develops when heme breakdown is accelerated (e.g., hemolysis) or hepatic UGT1A1 activity is impaired (e.g., Crigler-Najjar, Gilbert's syndromes). Nearly all neonates exhibit transient unconjugated hyperbilirubinemia, termed “physiologic jaundice”, because of a reduced ability to conjugate bilirubin (26) due to low UGT1A1 expression that gradually rises after birth (16).

Bilirubin levels typically peak (maximum: 170 µM ≈ 10 mg/dL) by one week of age (4) and return to normal within one month (4, 26).

Although markedly elevated serum concentrations of unconjugated bilirubin (> 20 mg/dL

≈ 340 µM) can cause neurologic damage (kernicterus) in newborns, toxicity is negligible in adults. Indeed, bilirubin has been administered intravenously at doses as high as 20 mg/kg

(producing serum levels above 22 mg/dL) without overt sequelae aside from scleral icterus (27).

Notably, Gilbert’s syndrome is a benign condition principally caused by a polymorphism in the promoter TATA element of the gene encoding UGT1A1 (28) that results in diminished hepatic bilirubin conjugation (16). Individuals with Gilbert’s syndrome commonly manifest mildly elevated serum bilirubin concentrations (29). It has been speculated that the high prevalence of the polymorphism (gene frequency ~40%) is due to potential beneficial effects of modestly elevated bilirubin levels (30), as detailed below (Sections 1.1.5 and 1.1.6 ).

1.1.5 Antioxidant properties of bilirubin

Bilirubin is an efficient chain-breaking scavenger of reactive oxygen (ROS) and nitrogen

(RNS) species, with potency equivalent to that of α-tocopherol (vitamin E) (31). Not only is bilirubin the most abundant endogenous antioxidant in mammalian tissues (7, 32); but it also is

5 unique among other antioxidants in that it undergoes intracellular redox cycling ( Figure 1.4).

This allows for efficient consumption of ROS through oxidation and subsequent regeneration of bilirubin by the action of biliverdin reductase (33) and is believed to be an important mechanism by which bilirubin provides effective cellular protection against oxidative injury (34). Direct evidence that bilirubin protects against oxidative injury is derived from in vitro studies of ventricular muscle cells (35) and neurons (36, 37) and from investigations demonstrating that bilirubin protects against ischemia-reperfusion injury in rodents (38–41).

1.1.6 Anti-inflammatory properties of bilirubin

Indirect evidence that bilirubin exhibits anti-inflammatory effects dates back several millennia. Bezor bovis (dried gallstones from oxen), which was first described between 22 and

250 A.D. in She-nong-ben-cao-jing, is a traditional Chinese medicine (Niu-huang) that is used to this day in Asia to treat inflammatory conditions such as toothache, sore throat, fever, and headache (42). It is notable that ox gallstones are comprised primarily of bilirubin, bile salts

(principally cholate, taurocholate, and glycocholate), and inorganic salts (43–46). In Western countries, it was first described in the early 1930s that patients with rheumatoid arthritis experienced remission after developing jaundice from superimposed liver disease (47, 48). More recent epidemiological studies have correlated increased serum bilirubin levels with a decreased incidence of a number of inflammatory conditions, including asthma (49), multiple sclerosis

(50), Crohn’s disease (51), cardiovascular disease (52–57), and diabetes mellitus-associated metabolic syndrome (58, 59). These observations raise the possibility of clinically relevant anti- inflammatory effects of bilirubin.

At the molecular level, the rate-limiting enzyme in bilirubin synthesis, heme oxygenase

(HO), has an inducible isoform (HO-1) that plays an important role in attenuating inflammation

(60). For example, upregulation of HO-1 protects against LPS-induced hypotension (61), mollifies glomerulonephritis (inflammatory disease of the kidneys) induced by anti-rat nephrotoxic globulin (62), and attenuates chemical-induced colitis (63, 64). Conversely, HO-1 inhibition results in enhanced tissue injury (65). However, despite ample evidence that HO-1 is an important modulator of inflammatory responses, the mechanism(s) underlying its cytoprotective and anti-inflammatory actions remains poorly understood (60). As heme oxygenase is the rate-limiting enzyme in bilirubin production, we have speculated that bilirubin is a key mediator of the anti-inflammatory effects of HO. In support of this proposition, it has been shown that induction of HO-1 suppresses venular leukocyte adhesion and chemotaxis (66), an effect that is abolished by HO inhibitors and reconstituted by the addition of bilirubin (66,

67).

Direct evidence that bilirubin dampens inflammatory responses is derived from experiments showing that bilirubin (but not CO, the other main product of heme catabolism by

HO) blocks ventricular leukocyte adhesion (67) and suppresses endothelial cell cytokine secretion (68) in response to oxidative and inflammatory stimuli. In rodent models, bilirubin prevents autoimmune inflammation of the brain and spinal cord (encephalomyelitis) (69), inhibits liver graft injury (70), improves survival and attenuates liver injury in endotoxemic rats

(71), suppresses carrageenan-induced hindpaw inflammation (71), and ameliorates allergic pneumonitis (72). The bilirubin precursor, biliverdin, also has been shown to protect rodents against ischemic (73) and immune-mediated liver injury (74), prevent chemical-induced colitis

(63), and diminish intestinal transplant injury (75). However, given the ubiquitous nature of biliverdin reductase, the enzyme that catalyzes the conversion of biliverdin to bilirubin, we postulate that bilirubin is the principal effector species in these studies. While the above data provide strong evidence that bilirubin exerts anti-inflammatory effects, the mechanism(s) by which it acts to regulate the immune response remains unclear.

1.2 Role of the endothelium in immune regulation

1.2.1 The endothelium

The endothelium is a highly specialized cell layer that lines the lumen of blood vessels and serves as a barrier between circulating blood constituents and the vascular intima (76) . One key role of the endothelium is to regulate the trafficking of circulating immune cells into target tissues (77). The transendothelial migration of leukocytes is a highly-ordered, multifaceted process (Figure 1.5) that is initiated by the induction of adhesive proteins on the luminal surface of endothelial cells. These proteins interact with specific counter receptors expressed on various leukocyte populations. An initial step in immune cell migration involves leukocyte rolling along the luminal surface of the endothelium, which is mediated by low-affinity endothelial receptors known as selectins (e.g., E-Selectin, P-Selectin) (78). In the presence of chemoattractants, the binding of leukocyte receptors to selectins stimulate “outside-in” signals in the leukocytes that activate specific surface molecules (integrins) that possess high-affinity binding sites for endothelial cell adhesion molecules, principally Vascular Cell Adhesion Molecule 1 (VCAM-1),

Intercellular Adhesion Molecule 1 (ICAM-1), and Platelet Endothelial Cell Adhesion Molecule 1

(PECAM-1) (79). The binding of integrins to VCAM-1 and ICAM-1 causes the arrest of leukocytes on the endothelium (80–82) and activates endothelial cell signaling pathways that

8 lead to the disruption of endothelial tight junctions, thereby facilitating leukocyte transmigration

(83). For reasons outlined below ( Section 1.3 ), we propose that bilirubin suppresses inflammation by impeding this migration process.

1.2.2 Vascular cell adhesion molecule 1 (VCAM-1)

VCAM-1 is a cell surface glycoprotein expressed by endothelial cells in response to inflammatory stimuli (e.g., TNF-α, IL-1β) or shear forces, but generally not under normal circumstances (80). It selectively binds to α4-containing integrins, specifically α4β1 (very late antigen 4, VLA-4) and α4β7 (lymphocyte Peyer patch adhesion molecule 1, LPAM-1), that are expressed primarily by eosinophils, lymphocytes and monocytes (84). The binding of α4β1 or

α4β7 to VCAM-1 triggers a signaling cascade within the endothelial cell that begins with the release of intracellular calcium and the activation of Rac1. This leads to an increase in the activity of isoform 2 of the enzyme NADPH oxidase (Nox) ( Figure 1.6) (85). Nox catalyzes the

·- conversion of molecular oxygen to superoxide (O 2 ), which then rapidly dismutates to form hydrogen peroxide (H 2O2) (86, 87). The Nox-mediated increase in cellular ROS induces actin restructuring and stimulates matrix metalloproteinases (MMP)-2 and -9 activity, which causes disruption of endothelial tight junctions and facilitates leukocyte transmigration (31, 85). The fundamental contribution of VCAM-1 to leukocyte trafficking is highlighted by the finding that inhibition of VCAM-1-mediated signal transduction prevents leukocyte transendothelial migration (88).

1.2.3 Intercellular adhesion molecule 1 (ICAM-1)

ICAM-1 is a cell surface glycoprotein that exhibits low-level constitutive expression on endothelial cells under basal conditions, and is up-regulated by inflammatory stimuli (89). It selectively binds integrin β2, specifically αLβ2 (lymphocyte function-associated antigen 1; LFA-

1) and αMβ2 (macrophage 1 antigen; Mac-1) found on the surface of lymphocytes, monocytes and neutrophils (90, 91). This binding induces several endothelial responses, including stimulation of Ras homolog family member A (RhoA), release of intracellular calcium, and

·- activation of xanthine oxidase (XO) which generates O 2 and H 2O2 within the cell ( Figure 1.7)

(90, 92, 93). Similar to VCAM-1, these reactive oxygen species induce reorganization of the actin cytoskeleton and promote leukocyte transmigration (90).

1.3 Bilirubin as a regulator of leukocyte migration

As detailed in Sections 1.2.2 and 1.2.3 above, activation of VCAM-1 or ICAM-1 triggers signaling cascades within the endothelial cell that result in the generation of ROS through activation of NADPH oxidase or xanthine oxidase, respectively. As bilirubin is a potent, chain- breaking antioxidant ( Section 1.1.5 ), we postulate that it disrupts adhesion molecule-mediated leukocyte migration through the scavenging of these ROS signaling intermediaries ( Figure 1.8), and that this mechanism may underlie bilirubin’s anti-inflammatory effects. In support of this hypothesis, we previously have shown that bilirubin is able to inhibit VCAM-1-mediated lymphocyte migration across murine endothelial cell monolayers in vitro (72, 94). The physiological relevance of these findings is further highlighted by our additional observation that bilirubin administration prevents eosinophil infiltration into the lungs of mice with allergic pneumonitis, a disease model that is known to be VCAM-1-dependent (72). There are a host of clinical disorders in which VCAM-1 and ICAM-1 play an essential role in mediating leukocyte

10 trafficking to sites of inflammation. Two diseases that are believed to be mediated by VCAM-1 and ICAM-1 (95, 96), have well-established animal models (97, 98), and are highly prevalent in the United States (99, 100) are inflammatory bowel disease (101, 102) and cardiovascular disease (103). The next few chapters will validate the important contribution of VCAM-1 and

ICAM-1 to the pathogenesis of these conditions and clarify the molecular mechanisms underlying the ameliorating effect of bilirubin.

Figure 1.1: Crystalline bilirubin and its chemical structure. (A) Bilirubin crystals exhibit an orange-red hue. ( B) Bilirubin adopts a “ridge-tile” conformation due to intramolecular hydrogen bonding between the carboxylic acid and dipyrrinone groups, creating an internal angle of 100° that spontaneously disrupts and reforms in a lepidopterous manner.

Figure 1.2: Overview of bilirubin production, metabolism and elimination. Heme, which is primarily derived from hemoglobin released from senescent red blood cells, is oxidized by heme oxygenase ( HO ) to form biliverdin, carbon monoxide ( CO ) and free iron ( Fe 2+ ). Bilirubin is subsequently generated by the ubiquitously expressed biliverdin reductase ( BVR ) enzyme. In the liver, bilirubin is conjugated by the 1A1 isoform of UDP-glucuronosyltransferase ( UGT1A1 ) to form water-soluble mono- and diglucuronides. Upon conjugation, bilirubin is excreted into bile by the canalicular multidrug resistance protein 2 (MRP2; ABCC2 ) transporter. Once bilirubin glucuronides reach the colon, they are hydrolyzed by bacterial β-glucuronidases back to bilirubin which, when solubilized by intestinal bile salts, diffuses across the intestinal mucosa and circulates back to the liver through the splanchnic system.

Figure 1.3: Species-dependent pathways of heme degradation and elimination. As many birds, reptiles, and amphibians lack biliverdin reductase ( BVR ) activity, following heme oxygenase ( HO )-mediated metabolism of heme, biliverdin is excreted directly into the bile and urine. In contrast, mammals efficiently convert biliverdin to bilirubin, which then requires the additional processes of hepatic uptake, conjugation, and transporter-mediated secretion into bile.

The physiologic rationale for these additional metabolic steps remains elusive.

Figure 1.4: Bilirubin undergoes intracellular redox cycling. Bilirubin is oxidized by reactive oxygen species ( ROS ) to form biliverdin, which is converted back to bilirubin by the ubiquitous biliverdin reductase ( BVR ) enzyme. This continuous recycling of bilirubin facilitates efficient scavenging of intracellular ROS (33).

Figure 1.5: The process of leukocyte adhesion and transmigration. In order to gain access to tissues, circulating leukocytes undergo an orchestrated series of steps that are utilized under both physiologic and pathophysiologic conditions. The initial events involve the capturing and rolling of leukocytes on the luminal surface of endothelial cells, a process that is primarily mediated by low-affinity selectins (e.g., E-Selectin) (78). This leads to an increase in the affinity of leukocyte integrins, a family of receptors that bind to endothelial cell adhesion molecules (e.g., VCAM-1,

ICAM-1) and mediate arrest and firm adhesion (80). Activation of VCAM-1 and ICAM-1 induces signaling cascades within the endothelial cell that promotes leukocyte diapedesis (83).

Adapted from Ley et al., 2007 (104).

Figure 1.6: VCAM-1 signal transduction in endothelial cells. Originally defined by Cook-

Mills et al. (80), the binding of leukocyte α4β1 (VLA-4) or α4β7 (LPAM) to endothelial VCAM-1 triggers intracellular calcium release and Rac1-dependent activation of NAPDH oxidase ( Nox ),

·- which catalyzes the production of superoxide ( O2 ) and hydrogen peroxide ( H2O2). These reactive oxygen species (ROS) induce actin restructuring and activate matrix metalloproteinases

(MMP )-2 and -9, causing disruption of endothelial tight junctions and facilitating leukocyte transmigration from the vascular lumen to the intima (80, 85, 86).

Figure 1.7: ICAM-1-mediated intracellular signaling in endothelial cells. The selective binding of leukocyte integrin αLβ2 (LFA-1) to endothelial ICAM-1 triggers the release of intracellular calcium and the activation of RhoA (90). Additionally, xanthine oxidase ( XO ) is stimulated to convert molecular oxygen to superoxide and hydrogen peroxide. These reactive oxygen species induce cytoskeletal changes and ultimately lead to leukocyte transmigration (90) in a manner analogous to VCAM-1 (Figure 1.6).

Figure 1.8: Proposed mechanism of bilirubin modulation of VCAM-1- and ICAM-1- dependent leukocyte migration. The binding of VCAM-1 and ICAM-1 with their

- corresponding leukocyte integrins leads to the generation of superoxide (O 2 ) and hydrogen peroxide (H 2O2) within the endothelial cell. Bilirubin, a membrane permeant (5) and highly potent chain-breaking antioxidant (31) that undergoes redox cycling (dotted lines) though the action of biliverdin reductase (BVR) (33), scavenges NOX- and XO-derived signaling intermediaries, thereby inhibiting endothelial retraction and preventing leukocyte migration.

Chapter II:

Bilirubin prevents acute DSS-induced colitis by inhibiting leukocyte infiltration and suppressing up-regulation of inducible nitric oxide synthase

Stephen D. Zucker 1, Megan E. Vogel 2, Tammy L. Kindel 2, Darcey L.H. Smith 2, Gila Idelman 2,

Uri Avissar 2, Ganesh Kakarlapudi 2, and Michelle E. Masnovi 2

1Division of Digestive Diseases, University of Cincinnati, Cincinnati, OH [email protected], 2Division of Digestive Diseases, University of Cincinnati, Cincinnati,

Citation: Zucker SD, Vogel ME, Kindel TL, Smith DLH, Idelman G, Avissar U, et al. Bilirubin

prevents acute DSS-induced colitis by inhibiting leukocyte infiltration and suppressing up-

regulation of inducible nitric oxide synthase. Am J Physiol Gastrointest Liver Physiol. 2015;

309(10):G841-G854.

2.1 Abstract

Bilirubin is thought to exert anti-inflammatory effects by inhibiting vascular cell adhesion molecule-1 (VCAM-1)-dependent leukocyte migration and by suppressing the expression of inducible nitric oxide synthase (iNOS). As VCAM-1 and iNOS are important mediators of tissue injury in the dextran sodium sulfate (DSS) murine model of inflammatory colitis, we examined whether bilirubin prevents colonic injury in DSS-treated mice. Male

C57BL/6 mice were administered 2.5% DSS in the drinking water for 7 days, while simultaneously receiving intraperitoneal injections of bilirubin (30 mg/kg) or potassium phosphate vehicle. Disease activity was monitored, peripheral blood counts and serum nitrate levels were determined, and intestinal specimens were analyzed for histological injury, leukocyte infiltration, and iNOS expression. The effect of bilirubin on IL-5 production by HSB-2 cells and on Jurkat cell transendothelial migration also was determined. DSS-treated mice that simultaneously received bilirubin lost less body weight, had lower serum nitrate levels, and exhibited reduced disease severity than vehicle-treated animals. Concordantly, histopathological analyses revealed that bilirubin-treated mice manifested significantly less colonic injury, including reduced infiltration of eosinophils, lymphocytes, and monocytes, and diminished iNOS expression. Bilirubin administration also was associated with decreased eosinophil and monocyte infiltration into the small intestine, with a corresponding increase in peripheral blood eosinophilia. Bilirubin prevented Jurkat migration but did not alter IL-5 production. In conclusion, bilirubin prevents DSS-induced colitis by inhibiting the migration of leukocytes across the vascular endothelium and by suppressing iNOS expression.

2.2 Background

Bilirubin is generated during the physiological breakdown of heme, through the sequential activity of heme oxygenase and biliverdin reductase. Elevated serum levels of bilirubin can occur as a consequence of the accelerated release of heme from hemoglobin (e.g., hemolysis) or diminished hepatic conjugating activity (e.g., Gilbert’s syndrome). It was first postulated over 75 years ago that bilirubin exerts an anti-inflammatory effect when it was noted that patients with rheumatoid arthritis experienced a remission of symptoms after developing jaundice secondary to superimposed liver disease (48, 105). These observations led to small therapeutic trials in which intravenous bilirubin was found to variably induce sustained analgesia and diminished joint swelling in subjects with chronic arthritis (27), although technical hurdles resulted in the abandonment of this line of investigation (106). More recently, bilirubin has been shown to suppress inflammatory responses in animal models of autoimmune encephalomyelitis

(69) and hind paw inflammation (71). With regard to inflammatory conditions of the intestine, indirect evidence supporting a potential protective effect of bilirubin are derived from the finding that individuals possessing the Gilbert’s polymorphism have a decreased risk of Crohn’s disease

(51).

One mechanism whereby bilirubin has been postulated to exert an anti-inflammatory effect is through inhibition of Vascular Cell Adhesion Molecule-1 (VCAM-1)-mediated signaling. VCAM-1 promotes the binding and movement of leukocytes across activated vascular endothelium (87, 107, 108), and is believed to facilitate the immune-mediated tissue injury that occurs in inflammatory bowel disease (101, 102). Specifically, the infiltration of eosinophils into the intestinal mucosa, a VCAM-1-dependent process (109, 110), has been implicated in the pathogenesis of both ulcerative colitis and Crohn’s disease (111–113). In support of this

24 hypothesis, treatment with antibodies (114–116) or antisense oligonucleotides (117) directed against VCAM-1 suppresses intestinal inflammation in rodent colitis models, and blocking antibodies direct against leukocyte integrins that bind VCAM-1 have been shown to ameliorate

Crohn’s disease in humans (118, 119). Bilirubin, a potent chain-breaking antioxidant (31, 33), inhibits VCAM-1-dependent migration of lymphocytes across endothelial monolayers by scavenging NADPH oxidase-generated superoxide (72), an early event in VCAM-1 signaling

(85). Consistent with these in vitro findings, bilirubin has been demonstrated to attenuate allergen-induced pneumonitis in mice by blocking VCAM-1-mediated eosinophil infiltration into the lungs (72).

Bilirubin also may attenuate intestinal injury by preventing the up-regulation of inducible nitric oxide synthase (iNOS). Nitric oxide (NO) generated by the iNOS-catalyzed conversion of

L-arginine to L-citrulline is thought to contribute to impaired intestinal mucosal integrity during sepsis (120), and also appears to play a key role in the pathogenesis of intestinal inflammation in animal models of colitis (121–124). In humans, colonic iNOS expression and NO levels correlate with clinical and endoscopic indices of disease activity in patients with ulcerative colitis (125–127). While the influence of bilirubin on intestinal iNOS activity has not been directly examined, bilirubin has been shown to inhibit the up-regulation of iNOS in isolated murine macrophages, and in liver, renal, myocardial, and aortic tissues of endotoxin-treated rats

(71, 128).

The oral administration of dextran sodium sulfate (DSS) to mice induces a form of colitis that mimics many of the clinical and histological features of human ulcerative colitis (129),

25 including eosinophil infiltration. The fundamental role of eosinophils in inducing colonic injury in this murine model is highlighted by the finding that intestinal inflammation is markedly attenuated in mice lacking eosinophil peroxidase (an enzyme that produces oxidizing compounds implicated in inflammatory responses) or eotaxin (the principal eosinophil chemoattractant)

(130). Previous investigations also support a contribution of iNOS activity to DSS-mediated intestinal damage (122, 124). Since bilirubin has been shown to modulate iNOS expression and inhibit VCAM-1-mediated eosinophil migration, we postulated that treatment with this bile pigment would prevent colitis induced by DSS. Hence, the aims of the present studies were to examine the influence of bilirubin on colonic inflammation, eosinophil accumulation, and iNOS activity in DSS-treated mice.

2.3 Materials and Methods

2.3.1 Materials

Unconjugated bilirubin (bilirubin IX α) was obtained from Porphyrin Products (Logan,

UT) and further purified according to the method of McDonagh and Assisi (131) in order to eliminate potential lipid contaminants. Bilirubin stock solutions were freshly prepared in 0.1 M potassium phosphate (pH 12), as previously described by our group (71). The addition of a small aliquot ( ≤ 0.4% v:v) of this stock solution had no effect on the pH of the cultured medium or on cell viability. Phorbol myristate acetate (PMA), ionomycin, collagen, fibronectin, tumor necrosis factor-α (TNF-α), Texas Red dextran 10,000 MW, and CellTrace Far Red were obtained from

Life Technologies (Grand Island, NY).

2.3.2 Cell isolation and culture

Human umbilical vein endothelial cells (HUVEC) were isolated from discarded umbilical cords using collagenase, as previously described (132). Cells were cultured in F-12K Medium supplemented with 10% fetal calf serum (FCS), endothelial cell growth supplement (Corning;

Bedford, MA), 0.1 mg/mL heparin, 100 IU penicillin, and 100 µg/mL streptomycin. All experiments were performed using passages 3 to 7. The human acute T cell leukemia cell line,

Jurkat, was purchased from ATCC and grown in RPMI 1640 supplemented with 10% FCS, 1 mM L-glutamine, 100 IU penicillin, and 100 µg/mL streptomycin. The following reagent was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HSB-2 from

Electro-Nucleonics, Inc (133). HSB-2 cells, a Human T-cell lymphoblastoid line, were cultured in RPMI 1640 medium with 10% FCS.

2.3.3 Induction of colitis by the administration of DSS

Animals were housed in the Laboratory Animal Medicine Services facility at the

University of Cincinnati under controlled conditions (temperature 22 ± 2 o C, relative humidity 50

± 10%, 12 h light-dark cycle) and fed a standard chow diet. Experiments were performed on adult male C57BL6/J mice (Jackson Laboratories, Bar Harbor, ME) because of the previously demonstrated susceptibility of this murine strain to DSS-induced colonic injury (134, 135).

Colitis was induced by administering 2.5% DSS (w/v) in the drinking water for the entire course of the experiment, with control animals receiving filtered water that did not contain DSS.

Twenty-four hours after commencing DSS treatment, animals received intaperitoneal (i.p.) injections of bilirubin (30 mg/kg), or an equivalent volume of the potassium phosphate vehicle, every 8 h for 7 days. All experiments were approved by the University of Cincinnati Institutional

Animal Care and Use Committee.

2.3.4 Assessment of disease activity

Mice were weighed and examined for signs of colitis on a daily basis, using a standard clinical disease activity index (DAI) comprised of the following parameters graded on a 4 point scale (136–138): stool consistency, presence or absence of fecal blood, and weight loss. At the conclusion of the 7 day treatment period, animals were sacrificed by CO 2 inhalation. Blood was immediately obtained by cardiac puncture. The small and large intestines were resected and colon length was promptly measured.

2.3.5 Histological analyses of tissue specimens

Longitudinal sections of the entire colon and of the small intestine were fixed in 4% paraformaldehyde and stained with H&E, employing standard histological techniques. Each colon specimen was independently assessed for injury over serial low-power fields by three blinded observers, employing a well-defined quantitative scoring system (136, 139) that grades the severity of inflammation (on a scale of 0 - 3), the extent of injury (0 - 3), and crypt damage (0

- 4). The value assigned to each parameter is multiplied by a factor reflecting the percentage of tissue involvement and then summed, with a maximum achievable severity score of 40. A minimum of 22 independent fields were examined per specimen per observer. To determine whether treatment effects were site specific, a separate experiment was performed in which 2 cm sections of proximal (between the cecum and descending colon) and distal (between the descending colon and anus) colon were isolated, and contiguous low-power fields graded histologically in a blinded fashion by 3 independent observers.

Eosinophils were identified by staining paraffin-embedded specimens with Sirius Red, according to the method of Meyerholz et al (140). Briefly, slides containing fixed tissue sections were immersed in Harris hematoxylin for 2 min followed by rinses in tap water and 100% ethanol. Slides were then immersed in an alkaline Sirius red solution (pH 8 - 9) for two hours and rinsed in tap water. Immunohistochemical staining for inducible nitric oxide synthase was performed by incubating frozen sections of colonic tissue in the presence of primary anti-mouse iNOS antibody (1:250; Transduction Laboratories) or the isotype control antibody (rat anti- mouse Ig; 1:500) at room temperature for 1 h, followed by an HRP-conjugated secondary antibody (141). Immunohistochemical staining of small intestine and colonic tissue for CD3

(prediluted, Ventana Medical Systems), CD68 (1:25; Abcam), and VCAM-1 (1:500; Biorbyt) was performed by the Cincinnati Children’s Hospital histology core.

2.3.6 Quantification of leukocytes in blood samples

Blood samples were collected in heparinized tubes and total white blood cell counts determined using a hemacytometer. Differential leukocyte counts were performed on blood smears treated with Wright’s stain, with a total of 200 cells counted per sample. Eosinophils were specifically identified by staining blood samples with Discombe’s solution (142, 143).

2.3.7 Serum assay for nitric oxide

Serum samples were filtered (10,000 mol wt) to exclude erythrocytes, and nitrate levels were measured using a Nitrate/Nitrite Colorimetric Assay kit (Cayman Chemical, Ann Arbor,

MI), as previously described by our group (71).

2.3.8 Quantification of cellular interleukin-5 expression

HSB-2 cells were seeded in 12-well plates at a density of 2 x 10 6 per ml and grown for 24 h. Monolayers were induced to express interleukin-5 (IL-5) by the addition of 4 ng/mL PMA and

1 µM ionomycin to the culture medium (144). At the end of the incubation period, total RNA was extracted using a GeneJET PCR Purification Kit (ThermoScientific) and 5 µg of total RNA was reverse transcribed using a Thermo Scientific cDNA kit, according to the manufacturer's instructions. Quantification of IL-5 and GAPDH mRNA (the latter to control for amplification) was achieved using the ABsolute Blue QPCR SYBR Green detection system (ThermoScientific).

Primers pairs were designed using Primer-blast NCBI: IL-5 (sense: 5’-gcttctgcatttgagtttgctagct-

3’, antisense: 5’-tggccgtcaatgtatttctttattaag-3’) and GAPDH (sense: 5’-agaaggctggggctcatttg-3’, antisense: 5’-aggggccatccacagtcttc-3’).

2.3.9 Transwell migration assay

HUVEC were seeded in the upper chamber of 24-transwells with 8-µm pores (Costar,

Cambridge, MA) that were pre-coated with collagen (0.4 mg/cm 2) and fibronectin (2 µg/cm 2) at a density of 4 x 10 5 cells/insert and grown to confluence. Monolayer integrity was validated by overlaying Texas Red dextran 10,000 MW (20 µg/mL) and measuring fluorescence intensity (ex:

595 nm, em: 625 nm) in the lower chamber. To induce adhesion molecule expression, HUVEC monolayers were stimulated with TNF-α (5 ng/mL) for 16 h prior to performing migration studies (145, 146). Jurkat cells were incubated in the presence of 25 µM CellTrace Far Red for

45 min at 37 oC, washed, and migration initiated by overlaying these fluorescently-labeled cells

(1 x 10 5 per insert) onto the HUVEC monolayers in the upper chamber of the transwell. Studies were performed in the presence of bilirubin (0 - 20 µM) or vehicle (0.1 M K 3PO 4), which was

30 added to the assay medium (F-12K plus 0.1% HSA) immediately prior to the start of the experiment. Transendothelial migration was quantified by measuring CellTrace fluorescence intensity (ex: 625 nm, em: 670 nm) in the lower chamber.

2.3.10 Statistical analysis

Data were analyzed using a computer-based statistical program (SSI SigmaStat, San Jose,

CA). Mean values were evaluated by ANOVA with t-test to assess for statistical significance.

For data that were not normally distributed, a Kruskal-Wallis analysis of variance on ranks was performed.

2.4 Results

2.4.1 Effect of bilirubin on DSS-induced disease activity, colon length, and intestinal histology

To determine whether bilirubin is able to suppress DSS-induced colitis, C57BL/6 mice were divided into each of four treatment groups comprised of 9 (vehicle treatments) or 10

(bilirubin treatments) animals: no DSS plus i.p. bilirubin vehicle ( vehicle ); DSS plus vehicle

(DSS + vehicle ); no DSS plus bilirubin ( bilirubin ); and DSS plus bilirubin ( DSS + bilirubin ).

DSS-treated mice that received bilirubin exhibited less weight loss ( Figure 2.1A ), diarrhea, and intestinal bleeding than did animals administered DSS plus vehicle, as reflected in the significantly lower DAI scores ( Figure 2.1B ). Unexpectedly, and despite a normal appearance, mice treated with bilirubin alone failed to gain weight to the same degree as animals receiving vehicle alone. Consistent with the clinical findings, DSS-treated mice that were administered

31 bilirubin demonstrated less colon shortening ( Figure 2.1C ) and reduced mucosal inflammation

(Figure 2.2, upper panels ) compared with animals that received DSS plus vehicle. The colons from DSS-treated mice that were administered vehicle exhibited dense cellular infiltrates, marked architectural destruction, and loss of epithelial lining. Mice receiving DSS plus bilirubin manifest less colonic inflammation and reduced morphologic injury, as evidenced by the significantly lower histologic injury scores ( Figure 2.2A ). Administration of bilirubin alone was found to have no noticeable effect on colon histology as compared with vehicle treatment.

Notably, bilirubin was found to exert a less pronounced effect in the distal ( Figure 2.2B ) as compared with the proximal ( Figure 2.2C ) colon.

2.4.2 Influence of bilirubin on eosinophil recruitment in response to DSS treatment

Since eosinophils are believed to play an important role in the pathogenesis of DSS colitis (130), and since bilirubin has previously been shown to inhibit eosinophil influx in a murine model of airway inflammation (72), we assessed whether bilirubin treatment prevents eosinophil recruitment to the colon of DSS-treated mice. Paraffin-embedded sections of distal colon were stained with the eosinophil marker, Sirius Red ( Figure 2.3, upper panels) , and the average number of eosinophils per high power field was quantified ( Figure 2.3, lower panel ).

Eosinophil infiltration into the colon of animals that received DSS was significantly lower following bilirubin versus vehicle treatment, supporting an inhibitory effect of bilirubin on eosinophil infiltration.

Since VCAM-1 is up-regulated in the colon of mice administered DSS (116) and appears to contribute to tissue injury and eosinophil recruitment (147), we examined the effect of bilirubin on VCAM-1 expression by performing immunohistochemical staining of intestinal specimens. We found that the DSS-induced expression of VCAM-1 in the colon was markedly reduced in mice that were simultaneously administered bilirubin ( Figure 2.3, middle panels ), approaching levels observed in animals that did not receive DSS (data not shown). These findings provide a potential explanation for the ameliorating effect of bilirubin. However, while the number of colonic eosinophils was not statistically different between the non-DSS control groups, there was a strong trend (p = 0.06) toward lower numbers of eosinophils/hpf in mice that were administered bilirubin versus vehicle, despite minimal detectable VCAM-1 in both groups.

These data raise the possibility that bilirubin modulates eosinophil homing via mechanism(s) that are independent of VCAM-1 expression.

To determine whether the diminished number of eosinophils in the colon of bilirubin- treated animals might be due to fewer circulating cells available for recruitment, we measured total leukocyte and differential counts in the peripheral blood of bilirubin- and vehicle-treated mice, both in the presence and absence of DSS. While total peripheral blood leukocyte counts did not differ significantly between the various treatment groups ( Figure 2.4A ), mice that received bilirubin exhibited a selective increase in the percentage of circulating eosinophils

(Figure 2.4B ), regardless of whether the animals were administered DSS. An increase in the absolute number of eosinophils in the peripheral circulation of mice treated with bilirubin was confirmed by staining blood smears with Discombe’s solution in order to specifically label eosinophils ( Figure 2.4C ). These data indicate that the observed reduction in the number of

33 colonic eosinophils in mice treated with bilirubin does not result from fewer circulating eosinophils. On the contrary, the increased peripheral blood eosinophilia noted in bilirubin- treated animals suggests that eosinophil access to peripheral tissues is impeded.

The proliferation, differentiation, and release of eosinophils from the bone marrow into the systemic circulation is mediated primarily by interleukin-5 (IL-5) (148). To investigate whether the observed increase in peripheral blood eosinophils associated with bilirubin administration might be due to augmented production of IL-5, we examined the effect of bilirubin on cellular IL-5 expression by HSB-2 cells, a human T-cell leukemia line that has been shown to produce IL-5 in a manner similar to normal peripheral blood T-cells (144, 149). When stimulated with PMA and ionomycin, HSB-2 cells exhibited a marked (1000-fold) up-regulation in IL-5 mRNA that peaked at 6 h and was unaffected by co-treatment with bilirubin ( Figure

2.5A ). When incubated in the presence of bilirubin alone, HSB-2 cells manifested a non- significant (less than 2-fold) increase in IL-5 mRNA out to 24 h ( Figure 2.5B ). In light of previous studies demonstrating no significant effect of bilirubin on IL-5 levels in isolated T-cells and lung lavage fluid obtained from mice with allergen-induced pulmonary inflammation (72), these data support that bilirubin does not induce peripheral eosinophilia through modulation of

IL-5 production.

2.4.3 Bilirubin modulates the physiological homing of eosinophils to the intestinal tract

We have shown that bilirubin reduces eosinophil infiltration into the colon when inflammation is induced by treatment with DSS ( Figure 2.3). As eosinophils primarily reside in

34 intestinal tissue under non-inflammatory states (148), we assessed whether bilirubin impairs the normal physiological homing of these cells to the gastrointestinal tract by quantifying the number of eosinophils in Sirius Red-stained sections of the mid-jejunum ( Figure 2.6, upper panels ). Notably, mice that received bilirubin had significantly fewer tissue eosinophils than those administered vehicle, regardless of whether DSS was administered ( Figure 2.6, lower panel ). These data, when taken in conjunction with our finding that the number of eosinophils in the peripheral blood increases in bilirubin-treated mice ( Figure 2.4B & C), suggest that bilirubin impedes the physiologic migration of eosinophils from the circulation to the gut.

To further examine this hypothesis, we investigated the effect of bilirubin on the transendothelial migration of leukocytes in vitro . Since VCAM-1 appears to play an essential role in the pathogenesis of DSS colitis (116) and in the recruitment of eosinophils to sites of inflammation (109, 150, 151), we elected to study Jurkat cells, a human acute T cell leukemia cell line that is known to express the VCAM-1-specific integrin VLA-4 (152). HUVEC monolayers were pre-incubated with TNF-α in order to induce the expression of VCAM-1 (145,

146), as validated by measuring both cellular mRNA and protein levels (data not shown).

Paralleling our in vivo findings and the results of our prior studies (72), bilirubin markedly inhibited the migration of Jurkat cells across TNF-α-stimulated HUVEC monolayers ( Figure

2.6C ) without altering VCAM-1 expression, consistent with a direct modulatory effect on

VCAM-1 signaling.

2.4.4 Effect of bilirubin on the intestinal recruitment of T-lymphocytes and monocytes/macrophages

To determine whether the inhibitory effect of bilirubin is specific to eosinophil recruitment, we stained sections of colon and small intestine for the pan-phenotypic T- lymphocyte marker, CD3, and for CD68, a glycoprotein that is expressed exclusively on monocytes and macrophages. Analogous to our findings for eosinophils ( Figure 2.3 ), bilirubin treatment was associated with a significant reduction in the DSS-induced infiltration of CD3

(Figure 2.7) and CD68 ( Figure 2.8) positive cells into the colon. While there appears to be no effect of either DSS or bilirubin on the number of CD3 positive cells in the small intestine

(Figure 2.9), substantially fewer CD68 positive cells were present in small intestinal tissue from bilirubin-treated mice, irrespective of whether they received DSS ( Figure 2.10 ). These latter findings parallel our observation for eosinophils (Figure 2.6), suggesting that bilirubin also impedes the migration of monocytes/macrophages from the peripheral circulation into the small intestine.

2.4.5 Influence of bilirubin on DSS-induced colonic iNOS expression and systemic nitrate production

Nitric oxide generated by iNOS is thought to contribute to the colonic injury induced by

DSS (122, 124, 153). As bilirubin has been shown to inhibit the up-regulation of iNOS in response to inflammatory stimuli (71, 128, 154), we sought to determine whether bilirubin treatment is associated with reduced iNOS expression in the colon of mice treated with DSS.

Frozen sections of colon tissue from untreated and DSS-treated mice that were simultaneously administered bilirubin or vehicle were subjected to immunohistochemical staining for murine iNOS, employing isotype antibody as control. Consistent with prior reports (141, 153), the colonic expression of iNOS was markedly augmented by DSS. Additionally, we found that DSS-

36 induced iNOS expression was substantially abrogated by bilirubin treatment ( Figure 2.11 ).

Consistent with these histologic findings, mice treated with vehicle plus DSS manifested elevated serum nitrate levels (a marker of iNOS activity), which were significantly lower in bilirubin-treated animals ( Figure 2.5D ). Taken together, these data support that bilirubin exerts an inhibitory effect on DSS-induced colonic expression and activity of iNOS.

2.5 Discussion

The present studies demonstrate that colonic inflammation induced by the oral administration of DSS to C57BL6/J mice is suppressed by bilirubin, as evidenced by reduced disease activity scores and by diminished histologic injury. The reason why bilirubin exerts a more pronounced protective effect in the proximal versus the distal colon is uncertain. We speculate that this could be due to the overall less vigorous inflammatory response in the former region, or possibly because levels of bilirubin in the colonic lumen decline caudally as a result of enterohepatic cycling (155). Our findings are consistent with previous reports that the bilirubin precursor, biliverdin, ameliorates DSS colitis in mice (63). Although the mechanism underlying this protective effect was not examined in this prior study, in light of our present findings, and since biliverdin is known to undergo rapid and quantitative conversion to bilirubin via the action of the ubiquitous biliverdin reductase enzyme (1, 156), we speculate that bilirubin is the principal physiologic mediator of the observed cytoprotection. This hypothesis is supported by the results of prior studies in which bilirubin, but not biliverdin, was found to attenuate endothelial cell activation (157).

Our data suggest two potential mechanisms by which bilirubin may exert a modulatory effect on DSS-induced colonic inflammation: 1) inhibition of eosinophil (and other leukocyte) infiltration into intestinal tissues, and 2) suppression of iNOS expression and activity. We previously have demonstrated that bilirubin blocks eosinophil migration into the lungs of mice with allergen-induced pneumonitis through a mechanism that involves the disruption of VCAM-

1 signaling within vascular endothelial cells (72). Our present finding that bilirubin administration reduces eosinophil infiltration into the colon, while simultaneously increasing levels of circulating eosinophils, supports a similar mechanism of bilirubin action in the DSS colitis model. Consistent with this proposition, we have directly shown that bilirubin, at physiological concentrations (20 µM ≈ 1.2 mg/dL), effectively prevents the movement of Jurkat cells across TNF-α activated HUVEC monolayers. Although TNF-α induces HUVEC to express both VCAM-1 and ICAM-1, because Jurkat cells do not bind ICAM-1 (158), our findings imply that bilirubin exerts its effects primarily through disruption of VCAM-1-dependent processes.

When taken in conjunction with our previous finding that bilirubin inhibits the movement of lymphocytes across murine endothelial cell monolayers that constitutively express VCAM-1

(72), these data support that the effects of bilirubin are not eosinophil-specific but rather that it interferes with any VCAM-1-mediated migration process. This hypothesis is supported by our demonstration of reduced numbers of CD3 and CD68 positive cells in the colon of DSS-treated mice that also received bilirubin, as both lymphocytes and monocytes/macrophages are known to express VCAM-1-specific integrins (159, 160).

Since VCAM-1 activation (116) and eosinophil infiltration (130) are important contributing factors to the pathogenesis of DSS colitis, the ability of bilirubin to block VCAM-1-

38 dependent leukocyte migration, and thereby inhibit eosinophil recruitment, likely underlie its protective effects. We show that bilirubin-treated animals manifest markedly attenuated colonic

VCAM-1 expression in response to DSS, which may, at least in part, explain its ameliorating effect on tissue injury. However, since the production of VCAM-1 by endothelial cells is stimulated by pro-inflammatory cytokines, these data cannot differentiate as to whether this reflects an ability of bilirubin to directly inhibit endothelial VCAM-1 expression or, rather, is a consequence of reduced cytokine production. Our demonstration that bilirubin does not alter basal or TNF-α-stimulated VCAM-1 mRNA or protein levels in cultured HUVEC supports the latter theory, with the caveat that other investigators have reported an inhibitory effect of bilirubin on endothelial cell expression of VCAM-1 (68, 157). Our current findings conflict with a prior study by our group employing a murine model of allergic pneumonitis in which we observed that bilirubin did not alter pulmonary VCAM-1 expression. Notably, in this same study, bilirubin also did not influence the production of IL-2, IL-4, IL-6, IL-10, IL-12, TNF-α or the main eosinophil chemoattractants IL-5 and eotaxin (72). While an important limitation of the present experiments is our inability to quantitate IL-5 or eotaxin levels in intestinal tissue, the disparate findings could possibly reflect the fact that allergic pneumonitis is a Th2 cytokine- dependent model (161) while DSS-induced colitis is Th1-mediated (162).

Because eosinophils are tissue resident leukocytes that localize primarily to the gastrointestinal tract (163), our observation that eosinophil levels in the jejunum are significantly reduced in bilirubin-treated animals (irrespective of whether they received DSS) supports that bilirubin also inhibits the normal physiological migration of eosinophils into the gut. This theory is bolstered by the concomitant increase in circulating eosinophils associated with bilirubin

39 administration, a finding that corroborates previous observations by our group (72). Our finding that bilirubin does not alter IL-5 message in unstimulated or activated HSB-2 T-cell leukemia cells, which are a generally accepted in vitro model of IL-5 regulation (144, 149), indicates that the peripheral eosinophilia associated with bilirubin administration is unlikely to be the result of increased eosinophil production and/or release from the bone marrow. Notably, while the number of T-lymphocytes in the small intestine was unaffected by bilirubin treatment, the level of monocytes/macrophages was reduced, paralleling our eosinophil results. These data suggest that eosinophils and monocytes may share a similar trafficking mechanism to the small bowel.

As there was no detectable VCAM-1 expression in the small intestine in any of the treatment groups, it seems unlikely that bilirubin reduces leukocyte infiltration by modulating the expression of VCAM-1 in non-inflamed intestinal tissue. In previous studies, we have shown that physiological concentrations of bilirubin, a potent chain-breaking antioxidant (31), inhibits the transmigration of VLA-4-expressing leukocytes across murine endothelial monolayers by scavenging VCAM-1-dependent reactive oxygen species signaling intermediaries (72). We speculate that this same mechanism may underlie the inhibitory effect of bilirubin on the infiltration of eosinophils and monocytes into the small intestine.

We are perplexed by the observed difference in growth curves between vehicle and bilirubin-treated control animals, as we have not previously observed this phenomenon in mice receiving similar dosing of i.p. (72) or oral (154) bilirubin, which we have shown produces very modest 3- to 4-fold elevations in serum bilirubin levels ( ≤ 0.4 mg/dL) (71). Notably, Gunn rats, which are homozygous (j/j) for a mutation in the UGT1A gene locus leading to persistent, marked hyperbilirubinemia (~5 mg/dL) (154), do not manifest abnormal growth. Since bilirubin

40 is generated as part of the physiologic degradation of heme, it is normally present in the circulation. In newborns, markedly elevated serum bilirubin concentrations (generally over 20 mg/dL ≈ 340 µM) can cause neurologic injury (kernicterus); however, toxicity in adults is negligible. Indeed, bilirubin has been administered intravenously to patients without overt sequelae (27), reaching serum levels as high as 22 mg/dL (normal ≤ 1.2 mg/dL ≈ 20 µM). More modest (< 3-fold) chronic elevations in serum bilirubin are commonly encountered in individuals with Gilbert's syndrome, a benign condition resulting from polymorphisms in the gene encoding

UGT1A1 (28), the principal bilirubin conjugating enzyme. It is notable that our in vitro studies utilizing human cells support that bilirubin exerts substantial inhibitory effects on leukocyte migration at concentrations that are at the upper end of the normal physiologic range (20 µM).

The administration of DSS to mice has been shown to induce iNOS expression in the colon (123, 164) and to increase nitrate concentrations in the serum (123, 153). Although conflicting data exist (165, 166), it is generally held that iNOS-derived NO contributes to the pathogenesis of DSS-induced colitis, as evidenced by the findings that iNOS-deficient mice

(122–124) and animals administered iNOS inhibitors (123, 167) manifest reduced colonic injury.

It is notable that bilirubin has previously been shown to suppress the endotoxin stimulated up- regulation of iNOS (71, 128). Consistent with these prior investigations, our experiments demonstrate that bilirubin treatment reduces colonic iNOS expression and serum nitrate levels in

DSS-treated mice, suggesting that bilirubin may act, in part, by preventing peroxynitrite-induced tissue injury (121). Both iNOS (125, 126) and VCAM-1 (101, 102) are believed to contribute to the pathogenesis of inflammatory bowel disease in humans and, notably, natalizumab (an antibody directed against the α4 integrin subunit that mediates leukocyte binding to VCAM-1)

41 has shown efficacy in inducing and maintaining clinical remission in Crohn’s disease (118, 119).

Hence, our demonstration that bilirubin is able to disrupt both of these processes raises the specter of a potential therapeutic application.

Figure 2.1: Effect of bilirubin on body weight, disease activity, and colonic shortening induced by dextran sodium sulfate (DSS). Mice were administered 2.5% DSS in the drinking water, followed by intraperitoneal injections of bilirubin (shaded squares) or the potassium phosphate vehicle (black squares). Control animals received bilirubin (shaded circles) or vehicle

(black circles) without DSS. A: average daily percent change in weight from baseline ( SE) following the initiation of DSS. B: mean disease activity index for each group. C: average length of resected colon specimens. Compared with the other treatment groups, the colons from mice treated with DSS plus vehicle were significantly shorter and lacked formed stool. *P 0.002 vs. all other groups, **P 0.03 vs. bilirubin and bilirubin DSS, ***P 0.001 vs. vehicle DSS.

Figure 2.2: Bilirubin treatment reduces colon histological injury following DSS administration. Top: representative low-power ( 200) images of hematoxylin and eosin-stained sections of colon from mice treated with vehicle alone, DSS plus vehicle, bilirubin alone, or DSS plus bilirubin. Bottom: histological injury scores based on a blinded analysis of colon specimens using a standard 0–40 scoring system. A: mean histological scores ( SE) assessed over the entire colon. B and C: results of a separate experiment in which histological scoring of sections of distal and proximal colon were performed. *P 0.002 vs. vehicle DSS.

Figure 2.3: Bilirubin inhibits eosinophil infiltration and VCAM-1 expression in the colon of

DSS-treated mice. Top: representative high-power ( 400) images of 2-cm sections of distal colon obtained from DSS-treated mice administered vehicle ( left ) or bilirubin ( right ) and stained with Sirius red to highlight eosinophils (arrows). Middle: results of immunohistochemical staining of proximal colon specimens for VCAM-1 ( 200). Bottom: graph of mean number of eosinophils ( SE) per high-power field (hpf) averaged over 10 separate fields ( n 9-10 per treatment group). *P 0.001 vs. vehicle DSS.

Figure 2.4: Effect of bilirubin on peripheral blood leukocytes. A: average total leukocyte count ( SE). B: mean percentage of neutrophils (PMN), lymphocytes (Lymph), monocytes

(Mono), eosinophils (Eos), and basophils (Baso) in the peripheral blood of mice treated with vehicle alone (shaded bars), DSS plus vehicle (open bars), bilirubin alone (solid bars), or DSS plus bilirubin (hatched bars). C: peripheral blood eosinophil counts determined by Discombe’s staining, plotted as the average number of eosinophils/ml ( 10 ). #P 0.05 vs. vehicle and vehicle DSS; *P 0.01 vs. vehicle and vehicle DSS.

Figure 2.5: Influence of bilirubin on cellular IL-5 production, leukocyte transendothelial migration, and serum nitrate concentration. A: effect of 10 µM bilirubin (shaded symbols) on the time course for the expression of IL-5 mRNA in HSB-2 cells, as determined by quantitative

PCR. Cells were incubated in the presence (squares) or absence (circles) of 4 ng/ml PMA and 1

µM ionomycin with data reflecting IL-5 expression (SE) relative to that in untreated cells at time 0 (n = 4). B: HSB-2 cells were incubated with 10 µM bilirubin for the indicated time intervals and IL-5 mRNA levels are plotted relative to expression at time 0 . C: time course for

Jurkat cell migration across human umbilical vein endothelial cells (HUVEC) monolayers pre- incubated for 16 h in the presence (squares) or absence (circles) of TNF-α (5 ng/ml). CellTrace

Far Red-labeled Jurkat cells were overlaid onto the monolayers in the presence of 20 µM bilirubin (shaded symbols) or vehicle (solid symbols). Displayed is the percentage of overlaid cells that migrated to the lower chamber, as quantified by measuring fluorescence intensity (ex:

625nm, em: 670 nm). D: mean serum nitrate concentration for each treatment group. *P 0.05 vs. all other groups

Figure 2.6: Bilirubin reduces eosinophil infiltration into the small intestine. Top: representative high-power ( 400) images of sections of small intestine stained with Sirius red to identify eosinophils (arrows). Treatment groups are as previously described in Fig. 3.2. Bottom: mean number of eosinophils per hpf ( SE) for each group. An average of 39 separate hpf was examined per specimen ( n 9-10 per group). *P 0.001 vs. all other groups.

Figure 2.7: Treatment with bilirubin decreases T lymphocyte infiltration into the colon of

DSS-treated mice. Shown are representative low-power ( 200) images of colon tissue stained for the T lymphocyte marker CD3. Immunohistochemical staining was performed on sections of colon obtained from mice treated with vehicle alone ( top left ), vehicle plus DSS ( top right ), bilirubin alone ( middle left ), or bilirubin plus DSS ( middle right ). Bottom: mean number of CD3- positive cells per hpf ( SE) for each treatment group. An average of 29 separate hpf was examined per specimen ( n 3-4 per group). *P 0.02 vs. all other groups.

Figure 2.8: Bilirubin inhibits the recruitment of monocytes/macrophages to the colon in response to DSS. Representative colon specimens stained for CD68, a marker for monocytes and macrophages, are displayed ( 200). Conditions are as described under Fig. 3.7. The marked infiltration of CD68-positive cells in animals receiving DSS plus vehicle precluded accurate quantitation.

Figure 2.9: DSS and bilirubin do not alter T lymphocyte levels in the small intestine.

Histological sections of small intestine ( 200) were stained for CD3, as detailed in Fig. 3.7.

Quantification of CD3-positive cells is represented as the number ( SE) per intact villus

(bottom ). An average of 32 villi was examined per specimen ( n 3 per treatment group).

Figure 2.10: Monocyte infiltration into the small intestine is reduced following bilirubin treatment. Representative small intestinal specimens stained for CD68-positive cells (arrows) are shown ( 200) as described under Fig. 3.8. Bottom: mean number of CD68-psoitive cells per hpf ( SE) for each treatment group. An average of 32 separate hpf was examined per specimen

(n 3 per group). *P 0.01 vs. both vehicle treatment groups.

Figure 2.11: Bilirubin inhibits DSS-induced colonic inducible nitric oxide synthase (iNOS) expression. Immunohistochemical staining for iNOS was performed on frozen sections of colon tissue obtained from mice treated with vehicle alone ( top left ), vehicle plus DSS ( top middle ), bilirubin alone ( bottom left ), or bilirubin plus DSS ( lower middle ). Representative low-power

(200) images are shown. Right: specimens stained with an isotype antibody ( iso ) as control.

Chapter III:

Bilirubin prevents atherosclerotic lesion formation in low-density lipoprotein (LDL) receptor-deficient mice by inhibiting endothelial vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1

(ICAM-1) signaling

1 1 2 1 Megan E. Vogel , Gila Idelman , Eddy S. Konaniah , Stephen D. Zucker

1Department of Internal Medicine, Division of Digestive Diseases, University of Cincinnati

College of Medicine, Cincinnati, Ohio USA

2Department of Pathology and Laboratory Medicine, Metabolic Disease Institute, University of

Cincinnati College of Medicine, Cincinnati, Ohio USA

Citation: Vogel ME, Idelman G, Konaniah ES, Zucker SD. Bilirubin prevents atherosclerotic

lesion formation in low-density lipoprotein (LDL) receptor-deficient mice by inhibiting endothelial vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1

(ICAM-1) signaling. J Am Heart Assoc. 2017; in press.

3.1 Abstract

Numerous epidemiologic studies support an inverse association between serum bilirubin levels and the incidence of cardiovascular disease; however, the mechanism(s) by which bilirubin may protect against atherosclerosis is undefined. The goals of the present investigations were to assess the ability of bilirubin to prevent atherosclerotic plaque formation in low density lipoprotein receptor-deficient ( Ldlr -/-) mice and to elucidate the molecular processes underlying this effect. Bilirubin, at physiological concentrations ( ≤ 20 µM), dose-dependently inhibits THP-

1 monocyte migration across tumor necrosis factor α-activated human umbilical vein endothelial cell monolayers without altering leukocyte binding or cytokine production. A potent antioxidant, bilirubin effectively blocks the generation of cellular reactive oxygen species (ROS) induced by the crosslinking of endothelial Vascular Cell Adhesion Molecule 1 (VCAM-1) or Intercellular

Adhesion Molecule 1 (ICAM-1). These findings were validated by treating cells with blocking antibodies or with specific inhibitors of VCAM-1 and ICAM-1 signaling. When administered to

Ldlr -/- mice on a Western diet, bilirubin (30 mg/kg i.p.) prevents atherosclerotic plaque formation, but does not alter circulating cholesterol or chemokine levels. Aortic roots from bilirubin-treated animals exhibit reduced lipid and collagen deposition, decreased infiltration of monocytes and lymphocytes, fewer smooth muscle cells, and diminished levels of chlorotyrosine and nitrotyrosine, without changes in VCAM-1 or ICAM-1 expression. Bilirubin suppresses atherosclerotic plaque formation in Ldlr -/- mice by disrupting endothelial VCAM-1- and ICAM-

1-mediated leukocyte migration through the scavenging of ROS signaling intermediaries. These findings suggest a potential mechanism for the apparent cardioprotective effects of bilirubin.

3.2 Background

Bilirubin is formed during the normal physiological degradation of heme. An inverse association between serum bilirubin concentrations and the incidence of coronary artery disease was first described by Schwertner et al. in 1994 (56). Since this initial report, a number of epidemiological analyses have provided corroborating evidence that individuals with a higher serum bilirubin level exhibit a lower risk of cardiovascular events (52, 55, 168, 169). Notably, heme oxygenase (HO), the rate-limiting enzyme in bilirubin synthesis, has an inducible isoform

(HO-1) that plays an important role in attenuating inflammation (60), including processes that can lead to atherogenesis. HO-1 induction has been shown to suppress venular leukocyte adhesion, an effect that is abolished by HO inhibitors and reconstituted by bilirubin (66, 67), while knockdown of HO-1 accelerates plaque formation in apolipoprotein E (apoE)-deficient mice (170). However, the molecular mechanism(s) by which the products of the HO-1 enzyme act to prevent atherosclerosis have yet to be delineated.

Vascular Cell Adhesion Molecule 1 (VCAM-1) is localized on the surface of activated endothelial cells and has been implicated as a key mediator of atherosclerosis (103). It selectively binds to α4-containing integrins ( α4β1, α4β7) expressed by T lymphocytes and monocytes (84) and facilitates leukocyte migration into the vascular intima, an early and critical event in plaque formation (171, 172). VCAM-1 is detected primarily at atherosclerosis-prone sites (173), and targeted disruption of this adhesion molecule inhibits early plaque formation in low density lipoprotein receptor-deficient ( Ldlr -/-) mice (103). Integrin binding to VCAM-1 triggers a signaling cascade within the endothelial cell that results in the generation of superoxide

·- (O 2 ) through activation of NAPDH oxidase (Nox) (87). The increase in intracellular reactive oxygen species (ROS) induces actin restructuring, leading to disruption of endothelial tight

58 junctions and enabling leukocyte transmigration from the vessel lumen to the intima (85, 87).

Bilirubin is a potent chain-breaking antioxidant (31) that uniquely undergoes intracellular redox cycling (33), facilitating the efficient consumption of ROS. Our group has shown that bilirubin inhibits the transendothelial migration of murine lymphocytes in vitro (72), and also attenuates tissue injury in mouse models of VCAM-1-dependent inflammation (72, 174). Based on these findings, we postulate that bilirubin’s cardioprotective effect is derived from its ability to disrupt

VCAM-1-mediated leukocyte migration by scavenging Nox-derived ROS (72, 94, 174).

Intercellular Adhesion Molecule 1 (ICAM-1) is another endothelial adhesion molecule that is up-regulated at sites of atherosclerosis (175). Studies have shown that levels of soluble

ICAM-1 correlate with the extent of atherosclerosis in humans (176) and that ICAM-1 knockdown is associated with a reduction in the size of vascular lesions in apoE-deficient mice

(177). The selective binding of αLβ2 integrin, which is expressed by lymphocytes, monocytes,

·- and neutrophils (90, 91), to ICAM-1 activates xanthine oxidase (XO) and generates O 2 and hydrogen peroxide (H 2O2) within the endothelial cell (92, 93). While the effect of bilirubin on

ICAM-1 signaling has not previously been studied, we speculate that it scavenges ICAM-1- dependent intracellular ROS in a manner analogous to what we have proposed for VCAM-1, thereby augmenting its ability to inhibit atherosclerosis.

Monocytes represent one of the principal inflammatory cell types in early atherosclerotic plaques and are believed to play an important role in lesion progression (172). T lymphocytes also contribute to atherogenesis by producing cytokines and chemokines that induce adhesion molecule expression and recruit inflammatory cells to sites of vascular injury (178). As both

59 monocytes and T cells express integrins that mediate binding to VCAM-1 and ICAM-1 (175), we postulate that bilirubin impedes atherogenesis by disrupting the trafficking of these leukocytes to the vascular intima. To test this hypothesis, we investigated the modulatory effect of bilirubin on VCAM-1- and ICAM-1-dependent monocyte migration in vitro and validated our findings by assessing the influence of bilirubin on the development of early atherosclerotic lesions in Ldlr -/- mice. Our data indicate that bilirubin impedes the migration of monocytes and lymphocytes to the vascular intima by scavenging ROS that mediate endothelial VCAM-1 and

ICAM-1 signaling, suggesting a potential mechanism for the cardioprotective effects of bilirubin.

3.3 Materials and Methods

3.3.1 Materials

Unconjugated bilirubin (bilirubin IX α) was obtained from Porphyrin Products (Logan,

UT) and further purified according to the method of McDonagh and Assisi (131) to eliminate potential lipid contaminants. Unless otherwise indicated, bilirubin was freshly prepared in 0.1 mol/L potassium phosphate (pH 12), as previously described by our group (72). The addition of a small aliquot ( ≤ 0.4% vol/vol) of this vehicle solution had no effect on the pH of the culture medium or on cell viability (71). Recombinant human tumor necrosis factor α (TNF-α) was purchased from PeproTech (Rocky Hill, NJ) and solubilized in dimethylsulfoxide (DMSO).

Allopurinol (AP) was purchased from MP Biomedicals (Santa Ana, CA). ML171 (2- acetylphenothiazine) and mouse IgG were purchased from Calbiochem (San Diego, CA). Mouse anti-human CD18 ( β2; ab8220) and mouse anti-human CD49d ( α4; clone 2B4) were purchased from Abcam (Paris, France) and R&D Systems (Minneapolis, MN), respectively. Mouse anti- human VCAM-1 (clone P3C4) and mouse anti-human ICAM-1 (clone P2A4) were purchased

60 from Millipore (Temecula, CA). CellTrace Far Red, dihydrorhodamine 123, Texas Red-dextran

10,000 MW, and rhodamine 6G were obtained from Molecular Probes (Eugene, OR). Human serum albumin (HSA) was purchased from Sigma (St. Louis, MO).

3.3.2 Cell isolation and culture

Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cords using type I collagenase (132). Cells were grown in F-12K supplemented with 10% fetal calf serum (FCS), endothelial cell growth supplement (Corning; Bedford, MA), 0.1 mg/mL heparin

(Sigma-Aldrich), 100 IU penicillin, and 100 µg/mL streptomycin (Corning; Manassas, VA). All experiments were performed using cells pooled from multiple donors, passages 3 to 7. The human acute monocyte leukemia cell line THP-1 (ATCC, Manassas, VA) was cultured in RPMI

1640 (Hyclone, Logan, UT) supplemented with 10% FCS, 1 mmol/L L-glutamine, 100 IU penicillin, and 100 µg/mL streptomycin.

3.3.3 Immunoblot analysis

Whole cell lysates were prepared in modified RIPA buffer (50 mM HEPES, 20 mM pyrophosphate, 25 mM β-glycerophosphate, 50 mM NaF, 5 mM Na 2MoO 4, 5 mM EDTA, 150 mM orthophenanthrol, 1% NP-40, 2% deoxycholate, 1% Triton X-100) in the presence of protease and phosphatase inhibitors. Protein concentrations were quantified using Pierce BCA

Protein Assay (Thermo Scientific). Samples (25 µg protein) were denatured, resolved on an 8%

SDS-polyacrylamide gel, and then transferred to an Immuno-blot PVDF membrane (Amersham).

Incubation with primary antibodies (rabbit anti-VCAM-1, mouse anti-ICAM-1, rabbit anti-E-

Selectin, and mouse anti-GAPDH; Santa Cruz Biotechnology) was performed for 2 h, followed

61 by HRP-linked anti-rabbit IgG or anti-mouse IgG (BioRad) secondary antibodies for 40 min at room temperature. Immunoreactive bands were visualized by chemiluminescence using an

Amersham ECL Prime Western Blotting Detection Kit (GE Healthcare, Pittsburgh, PA).

Densitometry was performed using ChemiDoc MP System software (BioRad).

3.3.4 Quantitative RT-PCR analysis

Total cellular RNA was extracted and quantitative RT-PCR (qRT-PCR) was performed with a MX300P system (Stratagene, Cedar Creek, TX) using SYBR Green QPRC Master Mix

(AB-4166, ThermoScientific). Primers for human VCAM-1 (sense: 5’-caggctgtgagtccccatt-3’; antisense: 5’-ttgactgtgatcggcttcc-3’), ICAM-1 (sense: 5’-accatctacagctttccggc-3’; antisense: 5’- tttctggccacgtccagttt-3’), E-Selectin (sense: 5’-ggcagttccgggaaagatca-3’; antisense: 5’- gtgggagcttcacaggtagg-3’), P-Selectin (sense: 5’-cgaggaaacatgacctgcct-3’; antisense: 5’- tagcctcacaggttggcaag-3’) and PECAM-1 (sense: 5’-gctaactgaacccactcccc-3’; antisense: 5’- gacagctgccatgtgactct-3’) were utilized for assessment of adhesion molecule mRNA expression.

Human beta-2-microglobulin (sense: 5’-ggcattcctgaagctgac-3’; antisense: 5’- gaatctttggagtacgctgg-3’) was employed as a control for amplification.

3.3.5 Luminex assay for cellular cytokine production

HUVEC were seeded at a density of 0.8 x 10 5 cells per well and grown to confluence in

24-well plates (Corning; Corning, NY). Cytokine (IL-6, IL-1β, IL-8, M-CSF, MCP-1 [CCL-2], and RANTES [CCL-5]) levels in the cell culture medium were determined 24 h following the addition of the indicated treatments using a human Magnetic Luminex Assay Kit (R&D Systems;

Minneapolis, MN), according to the manufacturer’s instructions, and quantified by Luminex multiplexing suspension array system (Millipore Sigma) (179).

3.3.6 Transendothelial migration assay

HUVEC were seeded at a density of 4 x 10 5 cells per insert and grown to confluence on the upper chamber of 24-well transwells with 8-µm pores (Costar; Cambridge, MA) that were pre-coated with collagen (0.4 mg/cm 2) and fibronectin (2 µg/cm 2). Monolayer integrity was validated by overlaying Texas Red dextran 10 kDa (20 µg/mL) and measuring fluorescence intensity (ex: 595 nm, em: 625 nm) in the lower chamber. HUVEC monolayers were stimulated with 5 ng/mL TNF-α for 24 h prior to performing migration studies in order to induce adhesion molecule expression (180). THP-1 cells were incubated in the presence of 25 µM CellTrace Far

Red for 45 min at 37°C, washed, and migration initiated by the addition of these fluorescently- labeled cells (1 x 10 5 cells per insert) to the upper chamber of the transwell. Studies were performed in the presence of bilirubin (0 - 20 µM) or vehicle, which was added to the medium

(F-12K plus 0.1% HSA) in both the upper and lower chambers. Transendothelial migration was quantified by measuring CellTrace fluorescence intensity (ex: 625 nm, em: 670 nm) in the lower chamber of the transwell at the indicated time intervals using a Biotek Synergy H1 reader (181).

The accuracy of the measurements was validated by direct cell count. Migration studies also were conducted in the presence of maximally effective concentrations of blocking antibodies to

VCAM-1 (10 µg/mL), ICAM-1 (10 µg/mL), α4 (20 µg/mL) and/or β2 (5 µg/mL), which were added to the upper chamber of the transwell. As control, isotype antibody was found to have no effect on THP-1 migration. In experiments employing maximally effective concentrations of the

NAPDH oxidase inhibitor ML171 (10 µM) and/or the xanthine oxidase inhibitor, allopurinol (40

µM), reagents were solubilized in DMSO and added to both the upper and lower chambers (final

DMSO concentration 0.05%).

3.3.7 Cell adhesion assay

HUVEC were seeded on 96-well plates (Greiner Bio-One) at a density of 1 x 10 4 cells per well and grown to confluence. Monolayers were stimulated with 5 ng/mL TNF-α for 24 h in the presence of bilirubin or vehicle, after which THP-1 monocytes (1 x 10 5 cells per well) labeled with 25 µM CellTrace Far Red were added. Following a 30 min incubation at 37°C, non- adherent monocytes were removed by gently vortexing and aspirating the supernatant three sequential times, as previously described (72, 87). Monolayers were then overlaid with 100 µL of PBS and adherence was quantified by measuring CellTrace fluorescence intensity (182).

3.3.8 Measurement of cellular ROS by confocal microscopy

HUVEC were grown on 35-mm glass bottom culture dishes (Ibidi; Munich, Germany) and stimulated with 5 ng/mL TNF-α for 24 h to induce adhesion molecule expression.

Monolayers were subsequently washed and incubated in the presence or absence of 10 µg/mL anti-VCAM-1 or anti-ICAM-1 antibody for 30 min at 37°C. Cells were then washed, loaded with

1 µM dihydrorhodamine, and incubated in the presence of 20 µM bilirubin or vehicle in phenol red-free medium for 15 min. Additional studies were conducted in the presence of ML171 (10

µM) or allopurinol (40 µM). Adhesion molecule activation was triggered by the addition of cross-linking goat anti-mouse (1:100) secondary antibodies (Pierce; Rockford, IL) and the subsequent time-dependent changes in dihydrorhodamine fluorescence intensity (ex: 485 nm,

64 em: 525 nm) were quantified by confocal microscopy (Zeiss 7 LIVE), as previously described

(72).

3.3.9 Xanthine oxidase inhibition assay

Xanthine oxidase (XO) activity was determined by colorimetric assay using an Amplex

Red Xanthine/Xanthine Oxidase Assay Kit (Molecular Probes), according to the manufacturers’ instructions. H 2O2 production was quantified by measuring absorbance at 571 nm. All reactions were conducted at room temperature for 15 min. Bilirubin and the xanthine oxidase inhibitors allopurinol and 2-chloro-6(methylamino)purine (CMAP) were solubilized in DMSO.

3.3.10 Murine model of atherosclerosis

Ldlr -/- mice on a C57BL/6 background were a generous gift from Dr. David Hui

(University of Cincinnati). Animals were maintained in a pathogen-free environment on a 12 h light/dark cycle. Beginning at 12 weeks of age, male mice were fed a Western (Research Diets:

D12108C; 20% fat, 1.25% cholesterol) diet ad libitum while simultaneously receiving daily i.p. injections of vehicle (50 mM K3PO 4 plus 10% serum; n = 4), bilirubin (30 mg/kg body weight in vehicle; n = 7), or sham (n = 7) for 8 weeks. Body weight was monitored weekly. Because the marked lipemia in Western diet-fed Ldlr -/- mice impedes the serologic assay for bilirubin (183,

184), steady-state bilirubin levels were determined in chow fed congenic C57BL/6J (Jackson

Laboratory) mice. All studies were reviewed and approved by the University of Cincinnati

Institutional Animal Care and Use Committee (protocol #14-03-03-01).

3.3.11 Histologic analysis of aortic root lesions

The heart and aorta were removed and prepared as previously described (185, 186).

Briefly, hearts were embedded in OCT compound (Tissue-Tek), frozen, and 7-µm sections obtained with a cryostat (Leica Biosystems), commencing at the valve nubs and appearance of the coronary artery and continuing through the aortic sinus until the valve separates at the base

(186). Serial aortic root sections were stained with Oil Red O to assess for neutral lipid accumulation and with Sirius Red to identify collagen deposition (171). Immunohistochemical staining for α-smooth muscle actin (prediluted; Roche) to detect smooth muscle cells (187) was conducted using an OmniMap detection kit (Roche). Immunofluorescence for VCAM-1 (1:500;

Biorbyt), ICAM-1 (1:75; Abcam), CD68 (macrophages; 1:25; Abcam), CD3 (T lymphocytes; prediluted; Ventana Medical Systems), chlorinated tyrosines (reactive oxygen species; 1:100;

Hycult Biotech), and nitrosylated tyrosines (reactive nitrogen species; 1:100; Life Technologies) was assessed employing anti-rabbit secondary antibodies conjugated to Alexa488 (1:100; Life

Technologies) and 4,6-diamidino-2-phenylindole (DAPI) counter-staining. Images were obtained using an Olympus BX61 microscope. Quantitative analyses of lesion area were performed on digitalized images using ImageJ software (NIH), and expressed as ratios of lesion area to total valve area or as ratios of compositional area to total lesion area, as previously described (185,

188).

3.3.12 Determination of serum bilirubin, lipid, lipid peroxide and cytokine/ chemokine levels

Blood samples were collected at the time of sacrifice. Serum bilirubin levels were determined using the Sigma Diagnostics Total Bilirubin Assay Kit (Sigma Chemical), as

66 previously described (71). Serum cytokines (IL-6, IL-1β, TNF α, IFN-γ, IL-10, IL-12p70, M-

CSF) and chemokines (MCP-1, MIP-1α, RANTES) were detected using an Immunology

Multiplex Assay Kit (EMD Millipore), according to the manufacturer’s instructions, and quantified by Luminex (179). Serum total cholesterol and triglyceride levels were determined using Cholesterol Fluorometric Assay and Triglyceride Colorimetric Assay Kits, respectively

(Cayman Chemical). Serum malondialdehyde (MDA) concentration, a marker of lipid peroxidation, was quantified using a Lipid Peroxidation (MDA) Fluorometric Assay Kit

(Abcam).

3.3.13 Statistical analyses

Data were analyzed using a computer-based statistical package (SSI SigmaStat, San Jose,

CA) with differences between mean values assessed for statistical significance. For normally distributed data, one-way ANOVA with Holm-Sidak post-hoc analysis was used to correct for multiple hypothesis testing. For data that were not normally distributed, a non-parametric

Kruskal-Wallis test was performed. Curve fit analyses were conducted utilizing a mixed model with random effects as implemented in SAS PROC MIXED. Experiment ID was included as a random effect to define non-independent observations, with treatment, time and a treatment*time interaction term included in the model. Identical results were obtained when the “repeated” statement was used.

3.4 Results

3.4.1 Induction of adhesion molecule expression by HUVEC

To facilitate investigation of the effect of bilirubin on VCAM-1- and ICAM-1-mediated leukocyte migration in vitro , we first identified conditions that optimized expression of these two adhesion molecules. HUVEC monolayers were incubated in the presence of TNF-α, and cellular mRNA and protein levels for VCAM-1 and ICAM-1 were quantified by qRT-PCR and Western blotting. We simultaneously assessed for expression of Platelet Endothelial Cell Adhesion

Molecule 1 (PECAM-1), which also facilitates leukocyte transmigration (189), and E-Selectin and P-Selectin, which mediate leukocyte rolling (190). Consistent with prior reports (191), TNF-

α induced a marked increase in mRNA for E-Selectin, VCAM-1, and ICAM-1 at 4 h, while expression of P-Selectin and PECAM-1 was unchanged ( Figure 3.1A,B ). Concordantly, protein levels of E-Selectin, VCAM-1, and ICAM-1 ( Figure 3.1C,D ) were significantly elevated after 4 h of TNF-α treatment, but only VCAM-1 and ICAM-1 exhibited sustained expression out to 24 h

(192). To specifically facilitate an analysis of the effect of bilirubin on these two adhesion molecules, all subsequent experiments employed a 24 h incubation period.

3.4.2 Bilirubin inhibits monocyte migration across endothelial cell monolayers

To determine whether bilirubin modulates human monocyte migration, HUVEC were seeded and grown in the upper chamber of a transwell system. CellTrace Far Red-labeled THP-1 monocytes were overlaid onto confluent HUVEC monolayers and the time course for transmigration was assessed by measuring fluorescence intensity in the lower chamber.

Expression of VCAM-1 and ICAM-1 was stimulated by incubating HUVEC in the presence of

TNF-α, which induced a significant increase in THP-1 cell migration ( Figure 3.2A ). Bilirubin blocked TNF-α-stimulated monocyte migration in a dose-dependent manner ( Figure 3.2B ), with

68 maximal inhibition achieved by bilirubin concentrations within the upper normal physiological range (20 µM ≈ 1.2 mg/dL).

The release of cytokines and chemokines by activated endothelia fosters the recruitment of inflammatory cells that promote atherogenesis (193). To determine whether bilirubin modulates endothelial cell production of cytokines relevant to leukocyte trafficking, we examined its effect on Interleukin (IL)-6, IL-1β, IL-8, Monocyte Chemoattractant Protein 1

(MCP-1), Macrophage Colony-Stimulating Factor (M-CSF), and Regulated on Activation,

Normal T cell Expressed and Secreted (RANTES) release into the culture medium of TNF-α- activated HUVEC. As shown in Figure 3.3 , bilirubin did not alter the cellular generation of any of these cytokines, suggesting that it does not act by regulating inflammatory or chemotactic stimuli. Further support for this conclusion is provided by our finding that addition of MCP-1 to the lower chamber of the transwell did not augment bilirubin-inhibited THP-1 cell transmigration

(data not shown).

Because transendothelial migration is predicated upon the binding of leukocyte integrins to endothelial cell adhesion molecules, we investigated the influence of bilirubin on adhesion molecule expression. As shown in Figure 3.4, incubation of HUVEC in the presence of bilirubin did not alter basal or TNF-α-stimulated mRNA or protein levels of VCAM-1, ICAM-1, PECAM-

1, E-Selectin, or P-Selectin. Consistent with these findings, no effect of bilirubin on monocyte adhesion to untreated or TNF-α-stimulated HUVEC was observed ( Figure 3.4A, lower right panel ), indicating that bilirubin does not prevent monocyte migration by disrupting adhesion molecule expression or binding to leukocyte integrins.

3.4.3 Influence of bilirubin on endothelial cell ROS production in response to activation of VCAM-1 and ICAM-1

Because bilirubin is a potent antioxidant (31), we assessed whether it is able to inhibit the

·- generation of ROS (i.e., O 2 , H 2O2) that mediate endothelial cell signaling through VCAM-1 (via activation of Nox) and/or ICAM-1 (by activation of XO) (87, 92, 93). To directly examine the influence of bilirubin on endothelial ROS signaling, TNF-α-activated HUVEC were incubated with anti-VCAM-1 or anti-ICAM-1 antibodies and then loaded with the redox-sensitive fluorophore, dihydrorhodamine. Adhesion molecule activation was triggered by the addition of cross-linking antibodies (72, 87) and fluorescence intensity was quantified by confocal microscopy. As expected, activation of VCAM-1 ( Figure 3.5, left panels ) or ICAM-1 ( Figure

3.5, right panels ) produced a robust time-dependent increase in cellular fluorescence (indicative of ROS generation) that was markedly attenuated by bilirubin (Figure 3.5, lower panels ). To validate these findings, we utilized ML171, which specifically blocks Nox activity by interfering with the catalytic subunit (but without affecting other cellular ROS-producing enzymes) (194), and allopurinol, a purine analog that competitively inhibits XO (195). As anticipated, treatment with ML171 completely suppressed cellular ROS production in response to activation of

VCAM-1 ( Figure 3.6A ) while exerting no effect on ICAM-1-generated ROS ( Figure 3.6B ), while allopurinol significantly abrogated ICAM-1-dependent ROS generation ( Figure 3.6B ) without altering VCAM-1-generated ROS ( Figure 3.6A ).

3.4.4 Relative contribution of VCAM-1 and ICAM-1 to TNF-α-induced monocyte migration

To confirm that inhibition of VCAM-1 or ICAM-1 signaling disrupts monocyte transendothelial migration, and to delineate the relative contributions of VCAM-1 and ICAM-1 to this process, the effect of ML171 and/or allopurinol on the movement of THP-1 cells across

HUVEC monolayers was assessed using a transwell system. As shown in Figure 3.6C , treatment of HUVEC with maximal inhibitory concentrations of the Nox inhibitor ML171 was associated with a substantial reduction in TNF-α-stimulated monocyte migration, while the XO inhibitor allopurinol had minimal effect, supporting a primary role for VCAM-1 in monocyte transmigration. Consistent with these findings, incubation of HUVEC monolayers with anti-

VCAM-1, but not anti-ICAM-1, caused significant inhibition of THP-1 cell migration, with modest synergy when both antibodies were combined (Figure 3.6D ). These data were further validated using antibodies that specifically block leukocyte integrin binding to VCAM-1 (anti-

α4) or ICAM-1 (anti-β2) ( Figure 3.6E ). While bilirubin has been shown to suppress Nox- mediated ROS production in vitro (94), data regarding its effect on xanthine oxidase are lacking.

Using an Amplex Red assay system, we studied the XO-catalyzed generation of hydrogen peroxide. Reciprocal plots ( Figure 3.6F ) demonstrate that bilirubin inhibits XO-catalyzed H 2O2 production in a competitive manner, with potency nearly twice that of allopurinol. Taken together, our results indicate that VCAM-1 constitutes the principal adhesion molecule mediating

TNF-α-induced monocyte migration, and support the hypothesis that bilirubin inhibits monocyte transmigration by disrupting adhesion molecule-dependent ROS signaling.

3.4.5 Effect of bilirubin on the development of atherosclerotic lesions in Ldlr -/- mice

To determine whether bilirubin prevents atherogenesis in vivo , we employed a murine model in which Ldlr -/- mice were fed a Western (high-fat) diet. We administered a once daily dose of bilirubin (30 mg/kg), which produced a 3-fold increase (1.7 mg/dL ≈ 28.9 µM) in steady- state serum levels ( Figure 3.7A ). Mice received i.p. bilirubin ( n = 7), vehicle ( n = 4), or sham ( n

= 7) once daily for a total of 8 weeks. All animals appeared outwardly healthy throughout the course of the study. There were no significant differences in body weight ( Figure 3.7B ), serum triglycerides ( Figure 3.7C ) or serum cholesterol ( Figure 3.7D ) between the treatment groups, although there was a strong trend toward reduced triglyceride levels in the mice that received bilirubin. At the end of treatment, cross-sectional analysis of Oil Red O-stained aortic root specimens from sham and vehicle-treated mice demonstrated large, well-established atherosclerotic plaques ( Figure 3.8A ), while bilirubin-treated animals had significantly smaller lesions ( Figure 3.8A ) with reduced deposition of extracellular matrix and diminished numbers of smooth muscle cells ( Figure 3.8B,C ). Mice that received bilirubin also had fewer CD68 ( Figure

3.9A ) and CD3 ( Figure 3.9B ) positive leukocytes in aortic root lesions, despite unchanged expression of VCAM-1 and ICAM-1 ( Figure 3.10 ) and no differences in serum levels of the key cytokines (193, 196), IL-6, IL-1β, TNF α, IFN-γ, IL-10, or IL-12 (Figure 3.11A-F) or, the principal leukocyte chemoattractants (197), M-CSF, MCP-1, Macrophage Inflammatory Protein

1α (MIP-1α), or RANTES ( Figure 3.11G-J). These data suggest that bilirubin impedes the infiltration of monocytes and lymphocytes into the aortic root in spite of adequate adhesion molecule expression and inflammatory and chemotactic stimuli. Bilirubin-treated animals also manifested significantly (albeit modestly) reduced serum indicators of lipid peroxidation ( Figure

3.11K), and aortic root lesions from these mice exhibited substantially reduced immunoreactivity to chlorotyrosine and nitrotyrosine ( Figure 3.9C,D ), markers of tissue oxidation (185). These observations support a mitigating effect of bilirubin on oxidative and nitrosative stress and, when taken in conjunction with our finding that bilirubin suppresses VCAM-1- and ICAM-1- stimulated endothelial ROS production ( Figure 3.5), are consistent with the hypothesis that bilirubin prevents atherosclerotic plaque formation by inhibiting leukocyte migration through the scavenging of ROS signaling intermediaries.

3.5 Discussion

While numerous epidemiological studies have identified an inverse association between serum bilirubin levels and the incidence of cardiovascular disease (52, 55, 56, 168, 169), it has not previously been shown that bilirubin is able to prevent atherosclerosis. In the present studies, we demonstrate that administration of bilirubin to Ldlr -/- mice impedes plaque formation and significantly reduces the infiltration of monocytes and lymphocytes into aortic root lesions, processes that are VCAM-1 and ICAM-1-dependent (87, 171, 198, 199). We further show that bilirubin effectively blocks the VCAM-1- and ICAM-1-mediated migration of monocytes across activated endothelial monolayers at bilirubin concentrations ( ≤ 20 µM) that are within the normal physiologic range (women: 3 - 20 µM; men: 5 - 29 µM) (25). These findings correlate well with epidemiological data demonstrating that patients in the highest quartile of serum bilirubin (> 12 -

17 µM) exhibit a decreased incidence of coronary (56, 168, 169) and carotid (200) artery disease as compared with those in the lowest quartile (< 7 - 10 µM). Concordantly, individuals possessing the prevalent Gilbert’s polymorphism, which is associated with mildly elevated serum bilirubin levels as a consequence of reduced expression of the bilirubin-specific 1A1 isoform of

UDP-glucuronosyltransferase (28), have been found to develop ischemic heart disease at significantly lower rates than the general population (55, 201). As the recruitment and subendothelial accumulation of leukocytes are key initiating events in the formation of atherosclerotic plaques (172, 178), our findings suggest a potential mechanism as to how bilirubin exerts a cardioprotective effect.

The binding of leukocyte integrins to VCAM-1 or ICAM-1 has been shown to trigger signaling cascades that lead to the production of superoxide and hydrogen peroxide within the endothelial cell (80, 93). These ROS induce downstream alterations in the endothelial junctional structure that facilitate the transmigration of leukocytes (174). As bilirubin is a potent, chain- breaking antioxidant (31), we postulated that it would disrupt the endothelial response to adhesion molecule activation by scavenging superoxide and hydrogen peroxide signaling intermediaries ( Figure 3.12). Our demonstration that bilirubin attenuates ROS generation by activated HUVEC in response to VCAM-1 or ICAM-1 cross-linking and inhibits H 2O2 production by isolated XO enzyme supports this hypothesis. These findings are in line with previous studies in which bilirubin has been shown to suppress VCAM-1-dependent ROS

·- generation by murine high endothelial cells (72) and to scavenge Nox-derived O 2 and H 2O2 in murine macrophages (94). That these mechanisms are relevant primarily at the tissue level in vivo are supported by the more pronounced inhibition by bilirubin of chlorinated and nitrosylated tyrosine formation in the aortic root as compared with its modest effect on serum markers of lipid peroxidation.

Our data demonstrating that bilirubin does not alter IL-6, IL-1β, or IL-8 production by

HUVEC or, IL-6, IL-1β, TNF α, IFN-γ, IL-10, and IL-12 in Ldlr -/- mice, and does not affect

VCAM-1 or ICAM-1 expression in vitro or in vivo , suggest that bilirubin does not modulate endothelial activation. These findings are concordant with our previous work showing no impact of bilirubin on VCAM-1 expression by isolated murine (72) or human endothelial cells (174), or in the pulmonary vasculature of mice with allergen-induced asthma (72). While we are unable to explain conflicting reports that bilirubin inhibits endothelial VCAM-1 and ICAM-1 expression in vitro (68, 157, 202), our demonstration that bilirubin has no influence on monocyte adhesion to

TNF-α-activated HUVEC monolayers under conditions where transmigration is completely inhibited supports an effect exerted beyond the step of integrin-adhesion molecule interaction.

Our observations that bilirubin also does not modulate the production of the chemoattractants

MCP-1, M-CSF, and RANTES by activated HUVEC, or circulating levels of M-CSF, MCP-1,

MIP-1α, and RANTES in Ldlr -/- mice, are consistent with previous reports (72) and suggest that bilirubin does not inhibit leukocyte recruitment by regulating chemotactic stimuli (203).

As atherosclerotic lesions mature, smooth muscle cells infiltrate the intima where they proliferate and produce extracellular matrix proteins (e.g., collagen) (204). We found that bilirubin treatment was associated with a marked reduction in collagen deposition and in the number of smooth muscle cells in aortic root lesions, suggesting that bilirubin impedes plaque progression. While our data do not elucidate whether the decrease in plaque-associated smooth muscle cells results from impaired migration, induced apoptosis, or reduced proliferation, our findings are consistent with previous reports that bilirubin directly inhibits smooth muscle cell proliferation in vitro and in response to balloon injury in vivo (205). Since it also has been shown

75 that chlorotyrosine promotes aortic smooth muscle cell migration (206), our demonstration that bilirubin reduces the formation of this oxidative byproduct suggests an additional mechanism by which bilirubin may prevent plaque maturation. We note that Ldlr -/- mice treated with bilirubin manifested a strong trend towards reduced serum triglyceride concentrations. This finding is consistent with prior reports describing lower total triglyceride levels in congenitally hyperbilirubinemic rats, as well as in humans with elevated bilirubin levels (207). While a number of explanations as to how bilirubin may modulate lipid metabolism have been proposed

(207), data in support of these hypotheses remain scant.

In summary, we show that bilirubin inhibits monocyte migration across activated human endothelial cells by disrupting VCAM-1 and ICAM-1 signaling through the scavenging of Nox- and XO-generated reactive oxygen species, findings that were recapitulated using specific enzyme inhibitors and blocking antibodies. We propose that this effect of bilirubin underlies the inverse association between serum bilirubin levels and cardiovascular disease, and have validated this hypothesis in a murine model of atherosclerosis, in which we show that treatment with bilirubin diminishes the number of monocytes, lymphocytes, and smooth muscle cells, decreases collagen deposition, and reduces oxidative stress in aortic root lesions, without altering adhesion molecule expression or circulating cytokine/ chemokine levels. While other antioxidants, such as tocopherols (208), have the potential to exert similar effects, bilirubin is unique in its ability to readily diffuse into cells (4) and to be continuously regenerated by intracellular redox cycling through the action of the ubiquitous biliverdin reductase enzyme (33).

It is notable that endothelial adhesion molecules have been implicated in the pathogenesis of a number of inflammatory disorders (50, 95, 96, 103, 115, 174, 209), and bilirubin has been shown

76 to ameliorate animal models of VCAM-1- and ICAM-1-mediated inflammation, including colitis

(174), allergic pneumonitis (72), and encephalomyelitis (69). As the inducible isoform of heme oxygenase (HO-1), which catalyzes the rate-limiting step in bilirubin synthesis, appears to play a key role in attenuating inflammation (65), we speculate that HO-1-generated bilirubin may serve a fundamental physiological function with regard to the regulation of inflammatory responses.

Figure 3.1: Time course for TNF-α-induced expression of adhesion molecules by HUVEC.

HUVEC monolayers were incubated in the presence of 5 ng/mL TNF-α (squares ) or the TNF vehicle ( Veh; circles ) and the expression of VCAM-1, ICAM-1, E-Selectin, P-Selectin, and

PECAM-1 was determined at the indicated time points by qRT-PCR and Western blotting. Panel

A shows the time-dependent changes in mRNA for E-Selectin ( black symbols ) and P-Selectin

(white symbols ), while panel B displays the results obtained for VCAM-1 ( grey symbols),

ICAM-1 ( black symbols ), and PECAM-1 ( white symbols ). Data reflect mRNA levels (± S.E.M.) relative to untreated cells ( n = 4 separate sets of experiments). Panel C depicts representative immunoblots for E-Selectin, ICAM-1 and VCAM-1, with graphs ( Panel D) quantifying expression at the indicated time points relative to unstimulated cells at time 0 ( Con ) and corrected for GAPDH ( n = 3 sets of experiments). *p<0.05 vs. Veh at that time point

Figure 3.2: Bilirubin inhibits the migration of THP-1 monocytes across activated HUVEC monolayers. Panel A depicts the time course for the migration of CellTrace Far Red-labeled

THP-1 cells across confluent HUVEC monolayers that were pre-incubated with ( squares ) or without ( circles ) 5 ng/mL TNF-α for 24 h. Studies were conducted in the presence of 20 µM bilirubin ( BR ; white symbols ) or the bilirubin vehicle ( Veh ; black symbols ). Displayed is the percentage of THP-1 cells in the lower chamber of the transwell (expressed relative to the total number added to the upper chamber) at the indicated time points. Panel B shows the dose- dependent effect of 10 and 20 µM bilirubin on THP-1 migration at 120 min expressed as the percentage of TNF-stimulated migration in the presence of the bilirubin vehicle . Bars reflect the mean (± S.E.M.) of 4 sets of experiments. *p<0.05 vs. No TNF, TNF + BR; **p<0.05

Figure 3.3: Bilirubin does not alter cytokine or chemokine expression by activated

HUVEC. HUVEC monolayers were incubated with TNF-α (5 ng/mL) in the presence of bilirubin ( BR ; 20 µM) or the bilirubin vehicle ( Veh ) for 24 h, and protein levels of IL-6, IL-8,

IL-1β, MCP-1, M-CSF, and RANTES in the cell culture medium were determined by Luminex.

Bars reflect mean (± S.E.M.) expression relative to unstimulated HUVEC ( Con ; n = 4 sets of experiments). *p<0.05 vs. Con

Figure 3.4: Bilirubin does not alter adhesion molecule expression or monocyte binding to

HUVEC. HUVEC monolayers were incubated with or without TNF-α (5 ng/mL) in the presence of bilirubin ( BR ; 20 µM) or the bilirubin vehicle ( Veh ). Panel A displays mRNA levels for

ICAM-1, VCAM-1, PECAM-1, E-Selectin, and P-Selectin at baseline and at 4 h ( n = 4 sets of experiments). The right lower panel depicts the results of an adhesion assay measuring the binding of CellTrace Far Red-labeled THP-1 cells to HUVEC monolayers incubated with or without TNF-α for 24 h. Bars reflect mean fluorescence intensity (± S.E.M.) relative to non-

TNF-α activated HUVEC ( n = 4 sets of experiments). Panel B shows representative immunoblots for E-Selectin, ICAM-1, and VCAM-1, which are quantified ( Panel C ) as described in Figure 3.1 (n = 4 sets of experiments). *p<0.05 vs. Veh, BR at that time point;

**p<0.001 vs. Veh, BR

Figure 3.5: Bilirubin suppresses cellular ROS generation following activation of VCAM-1 or ICAM-1. TNF-α-activated HUVEC monolayers were incubated with anti-VCAM-1 ( left panels , αVCAM-1; 10 µg/mL) or anti-ICAM-1 ( right panels , αICAM-1; 10 µg/mL) for 30 min, and then loaded with dihydrorhodamine. Adhesion molecule activation was triggered by the addition of a cross-linking antibody, and ROS generation quantified by confocal microscopy.

The upper panels display representative time-lapse images of non-stimulated and antibody- activated cells treated with 20 µM bilirubin ( BR ) or the bilirubin vehicle ( Veh ). Scale bars represent 100 µm. The lower panels plot the time-dependent changes in fluorescence intensity following VCAM-1 ( left panel ) or ICAM-1 ( right panel ) activation ( squares ), in the presence

(white symbols ) or absence ( black symbols ) of bilirubin. Cells that were not treated with cross- linking antibodies ( Nonstimulated ; circles ) serve as control, with curves reflecting mean

83 fluorescence intensity (± S.E.M.) expressed relative to maximal activation at 60 min ( n = 3 sets of experiments). *p<0.001 vs. Nonstimulated, αVCAM-1 + BR and p<0.001 vs. Nonstimulated,

αICAM-1 + BR

Figure 3.6: Nox and XO inhibitors and antibodies against VCAM-1 and ICAM-1 recapitulate the effect of bilirubin on endothelial ROS generation and monocyte transmigration. ROS production by TNF-α-stimulated HUVEC monolayers was assessed by

85 monitoring dihydrorhodamine fluorescence following activation of VCAM-1 or ICAM-1, as described in Figure 3.5. Panels A and B display the time-dependent changes in fluorescence intensity following VCAM-1 ( panel A ) or ICAM-1 ( panel B ) activation ( squares ), in the absence ( black symbols ) or presence of 10 µM ML171 (white symbols ), or 40 µM allopurinol

(AP; grey symbols ). Curves reflect mean fluorescence intensity (± S.E.M.) expressed relative to maximal activation at 60 min ( n = 3 sets of experiments). Panel C compares the effect of the

DMSO vehicle ( Veh ), 40 µM allopurinol ( AP ), and/or 10 µM ML171 on THP-1 cell migration across HUVEC monolayers, as described in Figure 3.2 (n = 4 sets of experiments). Panels D and E depict the results of analogous studies examining THP-1 migration in the presence or absence of antibodies against ICAM-1 ( ICAM ; 10 µg/mL), VCAM-1 ( VCAM ; 10 µg/mL), β2 (5

µg/mL), and/or α4 (20 µg/ mL). Panel F displays a Lineweaver-Burk plot of H 2O2 produced by isolated xanthine oxidase in the presence of 50 µM bilirubin ( diamonds ; Ki = 3.4 µM), 30 µM of the competitive inhibitor, allopurinol ( AP ; triangles ; Ki = 6.7 µM), 30 µM of the non- competitive inhibitor, 2-chloro-6(methylamino) purine ( CMAP ; squares ; K i = 4.7 µM), or the

DMSO vehicle ( circles ). Data reflect the mean (± S.E.M.) of 3 sets of experiments. *p<0.001 vs.

Nonstimulated, αVCAM-1 + ML171 and p<0.001 vs. Nonstimulated, αICAM-1 + AP; **p<0.05

Figure 3.7: Effect of bilirubin administration on body weight, serum bilirubin and lipid levels. Panel A shows serum bilirubin levels in chow fed C57BL/6J mice that were administered i.p. injections of bilirubin (30 mg/kg), vehicle, or sham once daily for five days. Bars reflect the mean (± S.E.M.) for each treatment group ( n = 5 - 6). Panel B displays body weights of Western diet-fed Ldlr -/- mice receiving i.p. bilirubin ( light grey symbols ), vehicle (dark grey symbols ) or sham ( black symbols ). Data points reflect the mean (± S.E.M.) for each treatment group ( n = 4 -

7). Panels C and D depict serum triglyceride and cholesterol levels, respectively, in Ldlr -/- mice after 8 weeks on a Western diet, with bars reflecting the mean (± S.E.M.) for each treatment group ( n = 4 - 7). While not statistically significant, bilirubin-treated mice exhibited a strong trend toward reduced serum triglyceride concentrations when compared with the sham (p=0.066) and vehicle (p=0.053) groups. *p<0.05

Figure 3.8: Bilirubin inhibits the formation of aortic root lesions in Ldlr -/- mice. Animals received i.p. bilirubin (30 mg/kg), vehicle, or sham for 8 weeks while on a Western diet. Panels

A-C display representative photomicrographs of aortic root sections stained for lipid (Oil Red O;

A), extracellular matrix (Sirius Red; B) or smooth muscle cells ( α-smooth muscle actin; C). Scale bars represent 100 µm. Panels on the left indicate the results of quantitative morphometric analysis, with Panel A expressed as the ratio of lesion area to total valve area and Panels B and

C expressed as the ratio of compositional area to total lesion area. The geometric mean is indicated with a line for each treatment group ( n = 4 - 7). *p<0.01; **p<0.001; †p<0.05;

†† p<0.005

Figure 3.9: Bilirubin decreases leukocyte infiltration and oxidative injury in the aortic root of Ldlr -/- mice. Panels A-D display representative photomicrographs of aortic root sections stained with immunofluorescent antibodies against CD68 (macrophages; green; A), CD3

(lymphocytes; yellow; B), nitrotyrosine (red; C), or chlorotyrosine (pink; D) and counterstained with DAPI (blue). Arrows mark the luminal margin of atherosclerotic lesions. Scale bars represent 100 µm. Panels on the left indicate the pooled results of morphometric analysis of the aortic root images, with the horizontal line indicating the geometric mean and data expressed as the ratio of positively-stained areas to total lesion area. Treatment groups are as described in

Figure 3.8. *p<0.001; †p<0.05; †† p<0.005

Figure 3.10: Bilirubin does not alter VCAM-1 or ICAM-1 expression in Ldlr -/- mice. Panels

A and B show representative photomicrographs of sections of aortic root stained with immunofluorescent antibodies against VCAM-1 (green; A) or ICAM-1 (cyan; B). Data are presented and analyzed as described in Figure 3.9.

Figure 3.11: Effect of bilirubin on serum parameters in Ldlr -/- mice. Blood was collected from Ldlr -/- mice after 8 weeks on a Western diet, with panels A-J showing serum levels of IL-6,

IL-1β, TNF α, IFN γ, IL-10, IL-12, M-CSF, MCP-1, MIP-1α, and RANTES as determined by

Luminex. Panel K is serum concentrations of malondialdehyde (MDA) as quantified by fluorometric assay, with bars reflecting the mean (± S.E.M.) for each treatment group ( n = 4 - 7).

Treatments are as described in Figure 3.8. *p<0.05; **p<0.001

Figure 3.12: Proposed mechanism of bilirubin modulation of VCAM-1- and ICAM-1- dependent monocyte migration. The ligation of VCAM-1 and ICAM-1 with their corresponding integrins, α4β1 / α4β7 and αLβ2, leads to the Rac-1 and calcium-dependent activation of NADPH oxidase (Nox) and xanthine oxidase (XO). These enzymes generate the

·- reactive oxygen species (ROS) superoxide (O 2 ) and hydrogen peroxide (H 2O2) that comprise a signaling cascade which leads to activation of matrix metalloproteinases (MMP)-2 and -9 and the disruption of endothelial tight junctions. Bilirubin, a potent antioxidant that undergoes intracellular redox cycling (dashed lines) through the action of biliverdin reductase (BVR), scavenges Nox- and XO-derived ROS, thereby inhibiting leukocyte migration.

Chapter IV: Discussion

4.1 Overview of bilirubin

Bilirubin is a principal product of heme catabolism that is produced by the sequential activity of heme oxygenase (which converts heme to biliverdin) and biliverdin reductase (which reduces biliverdin to bilirubin). Bilirubin is cleared from the circulation by the liver, where it is conjugated to water-soluble glucuronides that are actively secreted into bile. While conventionally referred to as a “bile pigment”, the unconjugated form of bilirubin is primarily found in the systemic circulation, with only trace quantities present in bile. Notably, the pathways of heme breakdown and elimination vary between species, with mammals requiring the additional energetically costly processes of bilirubin production and elimination ( Figure 1.3 ).

This suggests that bilirubin may serve an important physiological function. Based on previous epidemiological and animal studies (Section 1.1.6 ), and on the results of the experiments detailed in the preceding chapters, we propose that a principal role of bilirubin is to modulate inflammatory responses.

4.2 Mechanisms underlying bilirubin’s anti-inflammatory effects

Our lab has identified several potential molecular mechanisms by which bilirubin suppresses inflammatory responses:

4.2.1. Bilirubin inhibits the upregulation of inducible nitric oxide synthase

(iNOS)

The enzyme nitric oxide synthase (NOS) generates nitric oxide (NO) from the catalyzed conversion of L-arginine to L-citrulline. The low levels of NO derived from NOS that is

95 constitutively expressed by endothelial cells (eNOS) are thought to facilitate tissue homeostasis and repair (210). In contrast, the isoform of NOS that is induced by a variety of pro- inflammatory cytokines (iNOS) generates high levels of NO that augment tissue injury.

Although the contribution of iNOS to the pathogenesis of human disease is controversial (211), in rodent models of sepsis involving the intravenous administration of lipopolysaccharide (LPS), selective iNOS inhibitors prevent cardiovascular collapse and abrogate liver, lung, renal, and gastrointestinal injury (212), supporting that NO mediates many of the harmful consequences of endotoxemia (213). Our group has demonstrated that the administration of bilirubin to LPS- treated rats ameliorates tissue injury, reduces serum nitrate concentrations (an indicator of NO production), and attenuates the expression of iNOS mRNA in the liver (71), without altering levels of eNOS. We have further shown that physiological concentrations of bilirubin suppresses

LPS-stimulated iNOS up-regulation and nitrate production by murine macrophages, specifically by preventing activation of HIF-1α through scavenging of Nox-derived reactive oxygen species

(71, 94). As summarized below ( Section 4.3.1 ), experiments detailed in the present dissertation validate that bilirubin administration inhibits iNOS up-regulation and protects against tissue injury in a murine model of colitis.

4.2.2. Bilirubin inhibits leukocyte trafficking to sites of inflammation

It previously has been shown in rodent models that the induction of HO-1 results in decreased venular leukocyte adhesion and chemotaxis (66), an effect that is abolished by HO inhibitors and reconstituted by the addition of bilirubin (66, 67). Our group was the first to elucidate a potential mechanism underlying this effect, demonstrating that bilirubin blocks

VCAM-1-mediated signaling and leukocyte migration across murine endothelial cells by

96 scavenging Nox-derived ROS (72). Evidence that these findings translate into relevant in vivo effects was provided by studies employing a murine model of allergic pneumonitis, in which ovalbumin-innoculated mice develop robust eosinophil-predominant pulmonary infiltrates in response to inhaled ovalbumin. Our lab demonstrated that i.p. administration of bilirubin caused specific inhibition of eosinophil and lymphocyte infiltration into the lungs of ovalbumin-primed mice (72), a process that has been shown to be mediated primarily by VCAM-1 (109, 214). As detailed below ( Sections 4.3.1 and 4.3.2 ), the experiments described in the present dissertation not only confirm that bilirubin disrupts endothelial VCAM-1 signaling, but also demonstrate that bilirubin suppresses ICAM-1-mediated ROS production in vitro. Additionally, the ability of bilirubin to protect against inflammatory responses in vivo was validated in murine models of colitis and atherosclerosis.

4.3 Confirmation of the anti-inflammatory effects of bilirubin

The experiments detailed in this dissertation have corroborated and expanded upon the prior work from our laboratory detailed above ( Section 4.2 ) by: 1) providing a broader understanding of the mechanisms of bilirubin action, 2) confirming the effectiveness of bilirubin as an anti-inflammatory agent in vivo , and 3) identifying a potential therapeutic role for bilirubin in the treatment of specific pathophysiological conditions, including inflammatory bowel disease and atherosclerosis. An added benefit of bilirubin is that it is a naturally occurring compound that is generally nontoxic in adults, even at concentrations exceeding ten times normal (27), making it ideally suitable for clinical use.

4.3.1 Utility of bilirubin in the treatment of inflammatory bowel disease

The studies detailed in Chapter II examine the influence of bilirubin on colonic inflammation and iNOS activity in DSS-treated mice, an animal model of ulcerative colitis

(174). We found that treatment with bilirubin diminishes disease severity and histologic injury, and is specifically associated with decreased iNOS expression and reduced infiltration of eosinophils, monocytes, and T lymphocytes into the colon. Notably, these classes of leukocytes are known to express the α4β1 integrin that selectively binds to VCAM-1. As VCAM-1 has been shown to be an important mediator of tissue injury in the DSS model (130), our findings are consistent with the hypothesis that bilirubin inhibits VCAM-1-mediated leukocyte trafficking.

This conclusion is bolstered by our in vitro observation that bilirubin effectively prevents the migration of the Jurkat T-lymphocyte cell line across TNF-α activated HUVEC monolayers.

Although we show that TNF-α induces HUVEC to express both VCAM-1 and ICAM-1, because

Jurkat cells do not bind to ICAM-1 (158), our findings imply that bilirubin exerts its effects primarily through disruption of VCAM-1-dependent processes.

Corroborating support for a potential therapeutic role for bilirubin in human inflammatory bowel disease is derived from studies of natalizumab, a monoclonal antibody directed against the α4 integrin subunit that mediates leukocyte binding to VCAM-1. Clinical trials have demonstrated the utility of this drug in inducing and maintaining clinical remission in

Crohn’s disease (118, 119) and ulcerative colitis (215). While natalizumab is not widely utilized because of the rare, but potentially life-threatening, occurrence of progressive multifocal leukoencephalopathy [demyelinating injury to the central nervous system caused by reactivation of the JC virus (216)], its efficacy in treating inflammatory bowel disease provides proof of concept that agents capable of disrupting VCAM-1 signaling, such as bilirubin, can exert

98 positive clinically relevant anti-inflammatory effects. Further epidemiological support for this hypothesis is derived from studies showing that individuals possessing the Gilbert’s polymorphism (which is associated with hyperbilirubinemia) have a significantly reduced incidence of Crohn’s disease (51).

4.3.2 Bilirubin for the prevention of atherosclerosis

Evidence that bilirubin may exert cardioprotective effects is derived from a host of epidemiological studies showing an inverse association between serum bilirubin levels and the risk of cardiovascular disorders (52, 56, 168, 169), and from findings that individuals possessing the Gilbert’s polymorphism exhibit a significantly reduced incidence of coronary artery disease

(55, 201). The studies presented in Chapter III provide insight into the mechanisms underlying bilirubin’s anti-inflammatory properties, and how these may lead to the prevention of atherosclerosis, which is thought to primarily be an inflammatory condition (217, 218). We not only demonstrate that bilirubin administration causes a marked reduction in atherosclerotic plaque formation in low density lipoprotein receptor-deficient ( Ldlr -/-) mice, a well-established murine model of atherosclerosis (97), but further show that the principal leukocytes that mediate atherogenesis, specifically monocytes/macrophages and T lymphocytes (172, 178), are reduced in aortic root lesions of bilirubin-treated mice. As the infiltration of these leukocytes into the aortic root is VCAM-1- and ICAM-1-dependent (175), our data are consistent with the notion that bilirubin impedes adhesion molecule-mediated cellular migration.

This proposition is further supported by our finding that bilirubin, at physiological concentrations, blocks THP-1 monocyte migration across HUVEC monolayers induced to

99 express VCAM-1 and ICAM-1. We further show that bilirubin effectively abolishes the production of cellular reactive oxygen species (ROS) intermediaries that are generated in response to the cross-linking of endothelial VCAM-1 or ICAM-1, which induce the activation of

Nox and XO, respectively. We not only demonstrate that bilirubin is a potent competitive inhibitor of hydrogen peroxide production by the isolated XO enzyme, but also validate our findings in cell systems using specific Nox and XO inhibitors and blocking antibodies to

VCAM-1 and ICAM-1 (as well as their respective binding integrins). That these mechanisms are operational in vivo is supported by our observation that bilirubin-treated Ldlr -/- mice have significantly reduced oxidative injury in the aortic root, as evidenced by decreased nitrotyrosine and chlorotyrosine immunoreactivity. Taken together, these data support the hypothesis that bilirubin prevents atherosclerotic plaque formation by inhibiting leukocyte migration through the scavenging of ROS signaling intermediaries.

The oxidative modification hypothesis of atherosclerosis holds that oxidized LDL is a key inciting trigger of atherogenesis (219). This has led to the proposed use of antioxidants for the prevention of cardiovascular disease (220), and the utility of α-tocopherol (vitamin E) has been assessed in both animal models of atherosclerosis (221–223) and in large-scale clinical trials (224–227). In overall, these studies have failed to show a beneficial effect. We believe this is, in part, because investigations principally have focused on the role of antioxidants in the prevention of systemic LDL oxidation. On the other hand, we propose that antioxidants exert their effects on atherosclerosis primarily at the site of plaque formation through the scavenging of local ROS involved in adhesion molecule signaling. In support of this hypothesis, we show a marked reduction in leukocyte infiltration into and oxidation of proteins within aortic root

100 plaques from bilirubin-treated Ldlr -/- mice, despite only very modest reductions in serum lipid peroxidation products and no effect on circulating cholesterol or triglyceride levels. While bilirubin is an antioxidant of equivalent potency to α-tocopherol (31), bilirubin is unique in its ability to rapidly traverses cellular membranes (4, 5) and to regenerate intracellularly through continuous redox cycling (33). We believe that these distinctive properties of bilirubin greatly augment its potency in inhibiting adhesion molecule signaling, a contention supported by our data demonstrating a much more robust effect of bilirubin in the prevention of atherosclerosis in

Ldlr -/- mice as compared with what previously has been shown for vitamin E (221, 223).

4.3.3 Similarities in the pathogenesis of inflammatory bowel disease and atherosclerosis

While inflammatory bowel disease (IBD) and atherosclerosis may appear, at first glance, to be quite distinct disease entities, the mechanism by which leukocytes infiltrate into the respective tissues are highly analogous ( Figure 4.1). In both disorders, integrin binding to

VCAM-1 and ICAM-1 generates ROS signaling intermediaries within the vascular endothelium which promotes leukocyte transmigration that leads to tissue injury. Since inflammatory responses in IBD and atherosclerosis are contingent upon adhesion molecule-mediated ROS signaling, it becomes apparent how bilirubin, a potent ROS scavenger that regenerates intracellularly, is able to exert a protective effect in both conditions.

4.4 Proposed physiological function of bilirubin

Individuals with Gilbert’s syndrome express reduced levels of the bilirubin conjugating enzyme, UDP-glucuronosyltransferase 1A1, leading to modestly elevated serum levels of 101 unconjugated bilirubin (28). We speculate that the high prevalence of the Gilbert’s polymorphism (gene frequency ~40%) is due to beneficial anti-inflammatory effects of mild hyperbilirubinemia. Notably, higher serum bilirubin levels have been correlated with a decreased incidence of asthma (49), multiple sclerosis (50), Crohn’s disease (51), and cardiovascular disease (52–57). Further evidence that bilirubin functions to counter overly exuberant inflammatory responses is provided by observations that the body has developed physiological mechanisms that drive an increase in bilirubin levels, both locally and systemically, under pro- inflammatory circumstances. A prime example is “jaundice of sepsis”, in which circulating bilirubin levels rise, sometimes as high as 60 mg/dL ( ≈1.0 mM), in the setting of systemic bacterial infection (228, 229). While the pathogenesis of hyperbilirubinemia in gram positive infections is not well understood, one mechanism underlying this effect in gram negative sepsis appears to be the specific down-regulation by lipopolysaccharide (LPS) of transporters involved in the hepatic uptake (OATP2, or SLC21A6) and canalicular secretion (MRP2, or ABCC2 ) of bilirubin (230). Additionally, IL-10 has been demonstrated to induce HO-1 expression in response to LPS (231), which may further contribute to hyperbilirubinemia. Correspondingly, our lab and others have shown that treatment of animals with bilirubin reduces tissue injury induced by endotoxin (71, 232). Conversely, HO-1 knock-down or deficiency, which results in reduced bilirubin production, is associated with an exaggerated inflammatory response, including leukocytosis and increased tissue injury (233–236). Notably, individuals vary in their ability to generate a heme oxygenase response based upon a GT length polymorphism in the promoter of

HO-1 (237). Those with short GT repeats have augmented expression of HO-1 and a lower susceptibility to inflammatory injury, such as restenosis following angioplasty (238, 239), chronic allograft rejection (240), and emphysema (241). These findings support the physiological

102 relevance of HO-1 in modulating inflammation, and we hypothesize that bilirubin is the key mediator of these anti-inflammatory effects.

4.5 Future studies and directions

Because of the potential for bilirubin to be a safe and effective anti-inflammatory therapy, future investigations of bilirubin’s mechanism(s) of action may provide additional insight into its physiological function, potential utility, and possible drawbacks in specific clinical circumstances.

4.5.1 Elucidating the mechanism underlying the inhibitory effect of bilirubin on physiological leukocyte homing to the intestinal tract

In Chapter II , we observed that bilirubin-treated mice have significantly fewer eosinophils in the jejunum and a corresponding increase in the number of eosinophils circulating in the blood. As eosinophils are primarily tissue resident leukocytes (163), these findings suggest that bilirubin disrupts physiological leukocyte homing to the intestinal tract . Mucosal addressin cell adhesion molecule 1 (MAdCAM-1), which binds α4β7 integrins and is constitutively expressed in intestinal microvascular endothelia (242–244), is believed to be the primary mediator of leukocyte trafficking to the intestinal mucosa (245). While data in humans are scarce, Artis et al.

(245) reported that β7-deficient mice exhibit significantly fewer duodenal eosinophils than wild- type animals. Since eosinophils express α4β7 (246), our findings raise the possibility that bilirubin may prevent normal eosinophil homing to the intestine by inhibiting MAdCAM-1 signaling, in a manner analogous to what we have shown for VCAM-1 and ICAM-1. MAdCAM-

1 also appears to be involved in the intestine-specific homing of eosinophils in response to

103 inflammation, as supported by studies demonstrating a marked reduction in eosinophil recruitment to the jejunum, but not the lungs, of β7-deficient mice following an allergen challenge (247). Mice lacking β7 also exhibit an impaired eosinophil response to infection with the small intestinal helminth, Trichinella spiralis (245). As there is scant understanding of

MAdCAM-1 signaling within endothelial cells, we propose to utilize techniques similar to those described in Chapters II and III , to investigate whether intracellular signaling via MAdCAM-1 involves the induction of intracellular ROS (in a manner analogous to what occurs with VCAM-

1 and ICAM-1 activation) and if the scavenging of these ROS by bilirubin may explain its inhibitory effect on eosinophil trafficking to the intestine.

Because the underlying molecular pathways involved in MAdCAM-1 signaling are unknown, we propose to employ a similar experimental approach to that described in Chapter III to: 1) investigate whether MAdCAM-1 activation stimulates the production of intracellular ROS in murine endothelial cells; 2) determine the contribution of MAdCAM-1 to the binding and migration of α4β7–expressing leukocytes across endothelial cell monolayers; and 3) identify the molecular mechanisms whereby bilirubin inhibits MAdCAM-1-mediated transendothelial migration in vitro . To accomplish these aims, we propose to utilize Boyden chambers to study the migration of isolated murine splenic lymphocytes expressing α4β7 across monolayers of murine high endothelial venule cells (mHEV) that we have shown in preliminary studies to express MAdCAM-1 in response to TNF α stimulation. We hypothesize that activation of

MAdCAM-1 facilitates leukocyte migration across vascular endothelia via a signaling mechanism that involves an increase in intracellular ROS. We further propose that bilirubin inhibits the process MAdCAM-1-mediated transmigration by scavenging ROS signaling

104 intermediaries, in a manner analogous to its effects on VCAM-1 and ICAM-1. Assuming that our experiments confirm the above hypotheses, we plan to pursue additional studies to confirm that our findings using murine cells translate to human microvascular endothelia. For these investigations, we propose to examine the effect of bilirubin on the migration of an α4β7- expressing human lymphoblastoid T cell line (RPMI 8866) (248) across monolayers of isolated human intestinal microvascular endothelial cells (HIMEC), which constitutively expresses

MAdCAM-1 (248).

4.5.2 Validation of bilirubin’s mechanism of action

The studies outlined in this thesis demonstrate that bilirubin blocks in vitro leukocyte transendothelial migration by inhibiting intracellular signaling through the adhesion molecules

VCAM-1 and ICAM-1. We further show that bilirubin markedly reduces the number infiltrating leukocytes in animal models of atherogenesis and toxin-induced colitis, the pathogenesis of which are known to involve VCAM-1 and ICAM-1. However, while these in vivo findings are consistent with the hypothesis that bilirubin inhibits leukocyte migration, the evidence is circumstantial. To directly examine whether bilirubin is able to inhibit transendothelial leukocyte migration in vivo , we plan to utilize two-photon intravital microscopy (2P-IVM), which permits real-time visualization of immune cell adherence to and migration across vascular endothelia in living animals (250). For the proposed studies, mice will receive an i.p. injection of bilirubin or vehicle to elevate serum bilirubin concentrations (71). Mice will then receive a mixture of TNF α and IL-1β, in order to induce acute vascular inflammation (251), followed by in vivo labeling of leukocytes by infusion with the fluorophore Rhodamine G (252). Mice are then anesthetized and the right carotid artery is exposed and visualized using a Zeiss 7 LIVE confocal microscope.

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Interactions between leukocytes and endothelium are defined by duration: tethering (< 1 second), rolling ( ≥ 1 sec and < 30 sec), and adherence ( ≥ 30 sec) (249, 250). Leukocyte migration from the circulation into tissue is quantified by counting the number of cells in a 100 x 50 µm 2 area on either side of a defined 100 µm vessel segment (249). The effect of bilirubin on leukocyte interaction with and migration across the vascular endothelium will be determined and compared with that of anti-VCAM-1 and/or anti-ICAM-1 antibodies.

One substantial challenge in studying the effects of bilirubin in vivo is that long-term administration is challenging. Because bilirubin is poorly soluble in aqueous solution, oral administration results in minimal absorption because most remains precipitated in the intestinal lumen unless a solubilizing agent (e.g., bile salts) are added, which confounds interpretation of results. However, the trauma from repeated intraperitoneal injection, irrespective of what is administered, causes harm to the animal over time. Hence, we propose the generation of a hyperbilirubinemic mouse through conditional knockdown of the bilirubin-specific 1A1 isoform of UDP-glucuronsyltransferase (251). We propose the use of a conditional knockdown in order to circumvent several potential limitations, including a high rate of death from kernicterus encountered in neonatal mice by keeping the gene deletion silent until the animals have matured, and avoiding the development of compensatory responses to chronic hyperbilirubinemia. This approach would allow us to assess the effect of elevated bilirubin levels at various stages of inflammation, and the use of a murine model allows for cross-breeding with other transgenic mice.

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Our plan is to develop the conditional knockdown on a C57BL/6J background because many of the murine models to be employed in future studies (e.g., Ldlr -/-) utilize this strain. We will use PCR amplification of the substrate specific exon 1 for UGT1A1 from a mouse genomic library, followed by cloning into an appropriate base targeting vector, which include a neomycin resistance marker surrounded by FLP recombinase target (FRT) sites, and Cre recognition sites

(loxP) bounding exon 1 of UGT1A1. Subsequent coding and flanking of the intronic regions of the final construct will be sequenced to confirm cloning and PCR fidelity. Although, a potential limitation of this hyperbilirubinemic mouse is that serum bilirubin levels are not easily regulated, it uniquely solves the problem of long-term bilirubin administration. We propose to cross these animals with murine inflammatory models, such as the Ldlr -/- mouse, which will facilitate studies of the effect of bilirubin on the initiation, progression, and/or stabilization of atherosclerotic plaques.

4.5.3 Clinical application of bilirubin in human disease

We have shown that bilirubin is a potent inhibitor of inflammation in animal models of asthma, colitis, and cardiovascular disease. Since bilirubin normally is present in humans and modest elevations of serum bilirubin levels have no apparent clinical sequelae (27), and since we have observed no overt toxicity of bilirubin in rodents ( Chapters II and III ), we speculate that it may be an ideal candidate therapeutic agent for the treatment of inflammatory disorders. As the process of atherosclerosis begins years prior to the first clinical manifestations of disease, clinical trials would be logistically and financially challenging; therefore, we propose an initial focus on the treatment of ulcerative colitis (UC), which is the most common form of inflammatory bowel disease in the United States, affecting approximately 238 per 100,000 (99). It is a mucosal

107 disorder of the colon that consistently involves the rectum, thereby facilitating a topical approach to treatment. Indeed, some current therapies for UC (e.g., 5-aminosalicylates, cortisone) involve rectal administration by means of suppository or enema (255). We propose a trial of topical bilirubin treatment for UC using a similar approach. As solubilized bilirubin freely diffuses into cells (4, 5), it should readily access the colonic mucosa from the lumen.

We propose to compare the efficacy of rectally administered bilirubin emulsion with vehicle (carbomer 934P, edetate disodium, potassium acetate, potassium metabisulfite, purified water, xanthan gum) or tap water in patients with mild, active ulcerative colitis. Following informed consent, patients would undergo initial assessment by: 1) completing a standard clinical survey to establish symptom severity, 2) receiving a physical examination, 3) having serologic testing to assess overall severity of inflammation and disease activity, and 4) undergoing flexible sigmoidoscopy with biopsies of the rectum and sigmoid for both visual and histological scoring of disease severity. Subjects will be randomized to receive bilirubin or placebo via suppository or enema twice daily for twelve weeks. Patients will repeat the symptom survey and physical examination periodically during the study, and will undergo lab testing and sigmoidoscopy at the completion of the study in order to assess for changes in disease activity.

The specific survey tool that will be used is the Patient Simple Clinical Colitis Activity

Index (P-SCCAI) that includes six domains of questions, such as bowel frequency (during the day and night), urgency of defecation, blood in stool, general well-being, and manifestations of several extracolonic features of UC (e.g., arthritis, erythema nodosum, uveitis) (253, 254).

Serological variables including levels of C-reactive protein (CRP), complete blood counts

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(CBC), and bilirubin, will be assessed (255, 256), as well as fecal calprotectin (257), as markers of inflammation severity and disease activity. The Ulcerative Colitis Endoscopic Index of

Severity (UCEIS), which surveys vascular pattern, bleeding and ulceration, will be used to assess endoscopic severity of disease (258). Histological assessment will utilize the Geboes scoring system (259). Endpoints will be symptom relief, improvement in markers of inflammation, and decreases in endoscopic and histologic activity (i.e., mucosal healing).

4.5.4 Effect of bilirubin on antigen presenting cell (APC) activation and interaction with lymphocytes

We have shown that bilirubin is a potent endogenous anti-inflammatory agent and provide support that its effects are mediated, at least in part, by disrupting endothelial cell responses to VCAM-1 and ICAM-1 activation. However, these adhesion molecules also are expressed by antigen-presenting cells (APCs) and have been shown to play an important role in

T- and B-lymphocyte activation by providing adhesive connections between APCs and lymphocytes and, termed the immunological synapse (IS) (263, 264). The IS consists of a complex collection of molecules organized at the point of contact between a lymphocyte and

APC, with cellular interactions occurring in sequential steps that involve alterations in cell polarity and redistribution of cell membrane receptors (265). The IS establishes a close connection between the two immune cells, allowing for the bidirectional exchange of signals

(266). Key molecules that are expressed by lymphocytes at this interface, known as the supramolecular activation complex (SMAC), are the T or B cell receptor and the integrins LFA-1

(which binds ICAM-1) and VLA-4 (which binds VCAM-1) ( Figure 4.2 ) (267, 268). Initial cell- cell contact is mediated by the interaction of lymphocyte LFA-1 and VLA-4 with ICAM-1,

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ICAM-2, and VCAM-1 on the APC. This contact initiates an intracellular signaling cascade that leads to lymphocyte activation and antigen capture (269–274).

While a plethora of data exists regarding the intracellular events that are triggered in lymphocytes following interaction with APCs, there is scant information regarding any coincidental signaling that occurs within the APC and the role this may play in lymphocyte activation. Therefore, we propose to elucidate the intracellular events that are triggered within the APC following the engagement of ICAM-1 and VCAM-1 with their corresponding integrins, and how these signals may facilitate lymphocyte activation. We postulate that VCAM-1- and

ICAM-1-mediated signaling in APC involve intracellular production of superoxide and hydrogen peroxide (in a manner analogous to what is observed in endothelial cells), and that inhibition or scavenging of these ROS (as with bilirubin) will suppress lymphocyte activation. In support of this hypothesis, investigators have shown that in an adoptive transfer murine model of experimental autoimmune encephalomyelitis (EAE), bilirubin treatment reduces T cell responsiveness thereby ameliorating neurologic injury (69, 275). Hence, we propose to study the modulatory effect of bilirubin on APC-specific signaling and how this may alter lymphocyte activation and function.

Because it previously has been shown that the binding of VLA-4 expressed on B lymphocytes to VCAM-1 on follicular dendritic cells represents a key initial step in B cell activation (273, 274), experiments examining the effect of this interaction on dendritic cell responses are proposed. As follicular dendritic cells (FDCs) are known to express VCAM-1

(276), we propose to isolate FDCs from mouse lymph nodes, using reported methodologies (277,

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278). Appropriate culture conditions required for FDC expression of VCAM-1 will be determined using qRT-PCR and Western blot analysis, as we have previously describe for endothelial cells ( Sections 3.3.3 and 3.3.4 ). To validate surface expression of VCAM-1, FACS analysis using co-staining with anti-VCAM-1 and the follicular dendritic cell marker, FDC-M1 also will be performed (279). If follicular dendritic cells in culture are found not to constitutively express VCAM-1, induction of this adhesion molecule will be attempted by activating the cells in the presence of ovalbumin immune complexes (OVA-ICs) comprised of OVA/rabbit anti-

OVA (ratio of 1:6), as previously reported (277, 280). Once appropriate conditions for achieving

VCAM-1 expression on FDC are identified, we will utilize crosslinking antibodies to stimulate

VCAM-1 activation, as described in Section 3.3.8 . As dendritic cells have been shown to express

NADPH oxidase (281), we presume that VCAM-1-mediated intracellular signaling in these cells will be analogous to what previously has been described in endothelial cells (85, 86, 282). Thus, we will assess for cellular responses by examining for ROS production in response to VCAM-1 cross-linking, which will be quantified using previously described fluorescence microscopy techniques (see Section 3.3.8 ). We also will examine for intracellular calcium release using the calcium-sensitive fluorophore, indo-1 acetoxymethyl ester (Indo 1-AM), and for Rac1 activation by Western blotting for total and active Rac1 (Rac1-GTP) (85). As it previously has been shown that VCAM-1 activation modulates actin structure (87), we also plan to assess for morphological alterations in dendritic cells in response to VCAM-1 activation by confocal microscopy using

TRITC-phalloidin-labeling (87). Another marker of FDC activation is the production of

CXCL13 (BCA-1; B cell attracting chemokine-1) (279) and BAFF (283), which we will quantify by ELISA (see Section 3.3.5 ). If difficulties are encountered with VCAM-1 activation using standard crosslinking, we will attempt to generate a glycosylphosphatidylinositol-anchored

111 version of VLA-4 and assess for activation of the FDC VCAM-1 following interaction with the planar lipid bilayers, as previously reported (273).

Once the mechanism(s) underlying VCAM-1-mediated signaling in dendritic cells has been characterized, the next step will be to determine the contribution of these events to B cell activation. Naïve B cells will be isolated from murine lymph nodes and purified using a B cell isolation kit, as previously reported (277). The B cells will be subjected to FACS analysis using co-staining with anti-VLA-4 and the B cell marker anti-B220 (284) to insure an appropriate cell population to study. B cell activation is initiated by co-culture of naïve B cells with OVA-IC- loaded FDCs (as described above). Because FDCs are known to stimulate B cell secretion of Igs

(285), we will assess for activation by quantifying B cell secretion of total and OVA-specific

IgM in the culture supernatant by ELISA (277). We also will assess for enhanced B cell proliferation by measuring bromodeoxyuridine (BrdU) incorporation into cellular DNA by

FACS analysis (286). Alternatively, intracellular calcium release is another key feature in B cell activation (287), which can be quantified using Indo 1-AM, as described above.

Once we have established a reliable B cell activation assay, we will assess the contribution of VCAM-1-mediated DC signaling to B cell activation. We plan to determine the efficacy of blocking antibodies for VCAM-1, VLA-4 and/or α4 (see Section 3.3.6), NADPH oxidase inhibitors (e.g., ML171), and ROS scavengers (e.g., bilirubin) in preventing B cell activation when co-cultured with these pretreated DCs. Additionally, we plan to assess the role of ICAM-1 (using similar approaches as mentioned above) and possibly whether VCAM-1 and

ICAM-1 act synergistically within the dendritic cell to activate B cells. If we determine that

112 bilirubin modulates B cell activation, we plan to investigate whether similar mechanisms are involved in T cell activation and function. The findings of these studies hopefully will provide insight into the inverse association between serum bilirubin levels and the incidence of autoimmune disorders such as rheumatoid arthritis (288), multiple sclerosis (50), and Crohn’s disease (51).

4.6 Conclusion

The studies described in this thesis demonstrate that bilirubin is a potent inhibitor of the inflammatory processes that drive colitis and cardiovascular disease in vivo , and that these effects are mediated in large part by the disruption of VCAM-1 and ICAM-1 signaling through the scavenging of ROS intermediaries. The application of antioxidants to the treatment of human disease has generally focused on the ability of these compounds to prevent oxidative tissue injury

(e.g., lipid peroxidation, DNA damage, apoptosis). However, these types of injury occur as a consequence of exposure to high concentrations (> 100 µM) of reactive oxygen species (260–

264), which would be expected to overwhelm the scavenging capacity of most physiological or exogenously administered antioxidants ( Figure 4.3A). However, it has become increasingly evident that much lower levels of ROS, in the range of 1 – 10 µM, mediate a variety of normal cellular functions (e.g., proliferation, immune response) (86, 90, 265–267). Not surprisingly, the process of redox signaling is exquisitely regulated by a delicate balance between pro- and antioxidants (268, 269) (Figure 4.3B). In many disease states, this balance is tipped by a variety of factors to favor an inflammatory phenotype. We propose that higher bilirubin levels, within normal physiologic range, drive the redox balance back toward equilibrium, thereby opposing these pro-inflammatory forces. While it is conceivable that, under normal circumstances,

113 increased concentrations of bilirubin could over tip the redox balance toward an antioxidant state, there is scant clinical or epidemiological evidence that individuals with Gilbert’s syndrome have an increased susceptibility to infection. Therefore, we speculate that modest elevations in bilirubin to within the upper normal physiological range (20 µM ≈ 1.2 mg/dL), have positive anti-inflammatory effects with little to no adverse consequences.

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Figure 4.1: Leukocyte migration in inflammatory bowel disease and atherosclerosis.

Though the leukocytes involved in these two inflammatory states are different, the mechanisms by which the leukocytes transmigrate are analogous. For inflammatory bowel disease ( upper panel ), circulating leukocytes bind to VCAM-1 stimulating an intracellular signaling cascade leading to ROS generation. Monocytes and T lymphocytes are the two main immune cells in atherogenesis ( lower panel ). They bind to VCAM-1 and/or ICAM-1 that are expressed on activated endothelial cells, which induce signal transduction pathways that ultimately lead to production of ROS. The overlap in intracellular signaling cascades illustrates how bilirubin exerts a protective effect against these two inflammatory diseases by inhibiting leukocyte migration through the scavenging of ROS signaling intermediaries.

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Figure 4.2: The immunological synapse between a dendritic cell and a B cell. The initiation of B cell activation involves the interaction between several receptors and ligands found on the surface of the B cell and the dendritic cell (DC). This interaction, known as the immunological synapse, has two distinct spatial structures in which these receptors and ligands are located. The

B cell receptor ( BCR ) on the B cell, as well as the complement receptor ( CR ) and the Fc receptor ( FcR ) on the DC, are located in the central supramolecular activation cluster ( cSMAC ).

In the surrounding peripheral SMAC ( pSMAC ) are found the adhesion molecules VCAM-1 and

ICAM-1 and the integrins VLA-4 and LFA-1. It is thought that the interaction between adhesion molecules and their integrin partners draw the interface between DC and B cell closer, allowing for improved presentation of antigen ( Ag ) and, ultimately, enhanced B cell activation. 117

Figure 4.3: Oxidative stress and redox signaling in physiologic and pathophysiologic states.

Panel A depicts the general concept of how antioxidants may be utilized for the treatment of human disease. Under conditions of high oxidative stress, such as with a neutrophil burst, levels of reactive oxygen species (ROS) can exceed 100 µM. Due to the high concentrations of ROS present in these circumstances, the scavenging capacity of endogenous or exogenously administered antioxidants is likely to be overwhelmed. In contrast, panel B illustrates physiological cellular signaling mediated by low levels of ROS (redox signaling), in which the balance between pro- and antioxidants are tightly regulated within the cell. In certain disease states this balance becomes weighted towards an inflammatory phenotype. We hypothesize that antioxidants modulate cellular redox signaling by scavenging ROS and thereby driving the cell back towards an anti-inflammatory state and re-establishing equilibrium.

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