THE ROLE OF CITRULLINATION IN THE DEVELOPMENT OF MOUSE AND HUMAN INFLAMMATORY ARTHRITIS by VAN CRANSTON WILLIS B.S., Brigham Young University, 2007 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Molecular Biology Program 2012 This thesis for the Doctor of Philosophy degree by Van Cranston Willis has been approved for the Molecular Biology Program by James DeGregori, Chair V. Michael Holers, Advisor Kevin Deane Kathryn Haskins Linda van Dyk Date 11/20/12 ii Willis, Van, Cranston (Ph.D., Molecular Biology) The Role of Citrullination in the Development of Mouse and Human Inflammatory Arthritis Thesis directed by Professor V. Michael Holers ABSTRACT Rheumatoid arthritis (RA) is associated with the development of autoantibodies to citrullinated protein antigens (ACPAs). Citrullination is the post-translational modification of arginine residues into citrulline by protein arginine deiminases (PADs). ACPAs can be present years before the onset of clinical disease and represent an increased risk for future development of RA. While high ACPA titers are associated with increased disease severity, the fact that ACPAs are present for years prior to the onset of clinically apparent disease questions their contribution, and the contribution of citrullination, to the pathogenesis of RA. In order to understand the contribution of ACPAs and citrullinated epitopes to the pathogenesis of disease, mice with collagen- induced arthritis (CIA) were treated with the PAD inhibitors Cl-amidine, a pan-PAD inhibitor, or GSK283, a PAD4 inhibitor. Mice treated with either PAD inhibitor showed reduced clinical and histological disease severity, eptiope spreading, and in the case of Cl-amidine, total citrulline levels in the joint and serum. Interestingly epitope spreading in mice treated with PAD inhibitors was reduced in a citrulline-independent manner, suggesting that the ameliorative effects of PAD inhibitor treatment are not necessarily derived from the elimination of citrullinated epitopes and/or ACPA. Cl-amidine treatment failed to ameliorate collagen antibody-induced arthritis (CAIA), suggesting that the effector phase of the arthritis response is unaffected by PAD inhibition. As part of a iii CCTSI-funded clinical research project, sputum samples were obtained from subjects at- risk for the future development of RA. These samples were evaluated for the presence of RA-related autoantibodies (ACPAs and rheumatoid factors [RF]) to test the hypothesis that autoimmunity may begin in the lung in RA. RA-related autoantibodies were detected in the sputa of at-risk subjects as well as patients with early RA (≤ 1 year of diagnosis) as compared to healthy controls. Furthermore, the detection of RA-related autoantibodies in the absence of serological detection of these same autoantibodies suggests that in in some subjects, RA may begin in the lung. Together these data suggest that PAD inhibition ameliorates disease by altering the immune response more broadly than eliminating citrullinated epitope availability and that in some cases RA may begin in the lung. The form and content of this abstract are approved. I recommend its publication. Approved: V. Michael Holers iv TABLE OF CONTENTS CHAPTER I. INTRODUCTION............................................................................................................1 General overview of rheumatoid arthritis..........................................................1 General disease background. .................................................................1 Genetic contributions.............................................................................1 Environmental contributions and citrullination.....................................3 Extra articular manifestations of disease...............................................5 Protein Arginine Deiminases (PADs)....................................................7 Antibodies to Citrullinated Protein Antigens (ACPAs). .......................8 Summary of pathogenesis....................................................................10 PAD inhibitors as a tool to investigate the pathogenesis of disease......................11 Animal models of disease......................................................................................13 Dissertation aims ...................................................................................................14 II. MATERIALS AND METHODS..................................................................................15 Mice.......................................................................................................................15 Collagen-induced arthritis .....................................................................................15 Collagen antibody-induced arthritis ......................................................................16 PAD inhibitor treatments.......................................................................................17 Measurement of total citrulline..............................................................................17 Synovial proteome autoantibody microarrays.......................................................18 Autoantigen reactivity by ELISA..........................................................................19 Flow cytometric analysis of immune cell populations ..........................................19 Measurement of antibodies....................................................................................20 Measurement of antibody-producing cells ............................................................21 v mRNA isolation and determination of transcript expression ................................22 Evaluation of antigen presentation ........................................................................23 Determination of macrophage polarization ...........................................................24 Human subjects recruitment and consent ..............................................................25 Sputa collection and processing ............................................................................25 Human sample autoantibody testing......................................................................26 Statistical analysis..................................................................................................26 III. CL-AMIDINE TREATMENT AMELIORATES CIA ...............................................28 Background and rationale......................................................................................28 Results ...................................................................................................................30 Cl-amidine treatment reduces clinical and histological disease severity in CIA. .................................................................................................30 Cl-amidine treatment reduces serum and synovial citrulline content in CIA. .....................................................................................................31 Cl-amidine treatment does not alter immune cell populations............34 Table I. Frequency of immune cells in Cl-amidine-treated and control mice with CIA. ....................................................................................36 Cl-amidine treatment does not ameliorate CAIA................................36 Treatment with Cl-amidine decreases IgG2a and IgG1 anti-mouse CII antibodies.............................................................................................37 Treatment with Cl-amidine decreases antibody responses to both native and citrullinated synovial epitopes. ..........................................39 Summary and conclusions.....................................................................................43 IV. PAD4 INHIBITION IS SUFFICIENT FOR THE AMELIORATION OF CIA.........48 Background and rationale......................................................................................48 Results ...................................................................................................................49 Treatment with GSK283 decreases clinical disease severity in CIA. .49 vi Treatment with GSK283 decreases histological disease severity in CIA. .....................................................................................................50 Treatment with GSK283 does not significantly alter joint total citrulline levels. ...................................................................................52 Treatment with GSK283 does not significantly alter CII autoantibody production............................................................................................56 Treatment with GSK283 reduces epitope spreading in CIA. ..............60 Summary and conclusions.....................................................................................63 V. INVESTIGATION OF THE MECHANISM OF ACTION FOR ARTHRITIS AMELIORATION BY PAD INHIBITORS .....................................................................66 Background and rationale......................................................................................66 Results ...................................................................................................................69 Cl-amidine treatment does not affect autoantibody-producing cell population numbers. ............................................................................69 Cl-amidine treatment does not