Failure to process chromatin on apoptotic microparticles in the absence of deoxyribonuclease 1 like 3 drives the development of systemic lupus erythematosus Benjamin Sally Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2017 © 2017 Benjamin Sally All rights reserved ABSTRACT Failure to process chromatin on apoptotic microparticles in the absence of deoxyribonuclease 1 like 3 drives the development of systemic lupus erythematosus Benjamin Sally Systemic lupus erythematosus is an autoinflammatory disorder driven by the development of autoantibodies to self-nucleic acids, in particular to DNA and to chromatin. Loss-of-function mutations of the secreted deoxyribonuclease DNASE1L3 have been implicated in the development of aggressive familial lupus. In addition, recent genome-wide association studies have linked a hypomorphic variant of DNASE1L3 to sporadic lupus. Studies in the lab determined that Dnase1l3-deficient mice develop rapid autoantibody responses against dsDNA and chromatin, and at older ages this leads to a lupus-like inflammatory disease. These disease manifestations were completely independent of the intracellular DNA sensor STING, which has been implicated in other examples of self-DNA driven autoinflammatory diseases. My project focused on developing assays to track the activity of DNASE1L3, as well as identifying the endogenous source of self-DNA normally processed by DNASE1L3. Using mouse models that allow the depletion of specific cell populations, we found that circulating DNASE1L3 is produced by hematopoietic cells, in particular by CD11c+ dendritic cells and by tissue macrophages. Taking into account the unique properties of DNASE1L3, we discovered that this enzyme is uniquely able to digest chromatin contained within and on the surface of apoptotic microparticles. Loss of DNASE1L3 activity in circulation results in elevated levels of DNA in plasma, in particular within microparticles. Microparticles are extensively bound by anti-chromatin autoantibodies isolated from both murine models of lupus as well as prototypical human clones. In addition, Dnase1l3-deficient mice have high levels of circulating IgG that bind to microparticles from young ages, and these titers increased as disease progressed in aged animals. Pretreatment of microparticles with DNASE1L3 largely abrogated this binding, demonstrating that DNASE1L3 directly reduces the immunogenicity of microparticles. We also studied two human patients with null mutations in DNASE1L3, and observed increased DNA circulating in plasma and, in particular, in their microparticles, demonstrating a conserved role for DNASE1L3 in mice and humans. Finally, we obtained plasma samples from a cohort of patients with sporadic SLE, and found that roughly 80% had circulating IgG that avidly bound microparticles. Roughly half of this group failed to bind to microparticles that had been pretreated with DNASE1L3, and this DNASE1L3-sensitive group also presented with lower levels of DNASE1L3 activity. We conclude that extracellular chromatin associated with microparticles acts as a potential self-antigen capable of causing loss of tolerance to self-DNA and inflammatory disease in both mice and humans. The secretion of a DNA- processing enzyme thus represents a novel, conserved tolerogenic mechanism by which dendritic cells restrict autoimmunity. Table of Contents List of figures ii Introduction Systemic lupus erythematosus in humans and mice 1 Processing of self-DNA as a mechanism to prevent autoimmunity 14 Mice lacking Dnase1l3 develop autoimmunity 21 Chapter 1: Development of a functional assay to identify sources of DNASE1L3 Background 26 Results 29 Conclusions 44 Chapter 2: Microparticles as the physiological target of DNASE1L3 Background 46 Results 48 Conclusions 60 Chapter 3: Microparticles as autoantigens Background 62 Results 63 Conclusions 74 Chapter 4: DNASE1L3 and microparticles in human lupus patients Background 76 Results 77 Conclusions 89 Discussion and future directions 91 Materials and methods 107 References 121 i List of figures Figure 1 26 Phylogenetic tree of human and mouse deoxyribonucleases Figure 2 28 Representation of the key features of DNASE1L3 Figure 3 29 Standard curve for detection of DNASE1L3 activity by qPCR Figure 4 31 The C terminal domain of DNASE1L3 allows its activity to be specifically tracked as it confers the ability to digest coated DNA Figure 5 33 The catalytic domain of DNASE1 and DNASE1L3 share extensive homology Figure 6 35 Perturbations of the C terminal domain of DNASE1L3 dramatically affect its function Figure 7 36 Dnase1l3-deficient animals do not process liposome-coated DNA ex vivo or in vivo Figure 8 37 Active DNASE1L3 in circulation is produced by hematopoietic cells Figure 9 39 The production of autoantibodies in Dnase1l3-deficient mice is driven by hematopoietic cells Figure 10 40 Restoration of circulating DNASE1L3 into Dnase1l3-deficient animals delays the development of autoimmunity Figure 11 42 DNASE1L3 is expressed primarily by dendritic cells and macrophages Figure 12 44 DNASE1L3 in circulation is produced primarily by dendritic cells and macrophages ii Figure 13 49 Treatment with staurosporine induces rapid cell death and microparticle release Figure 14 52 DNASE1L3 is uniquely able to process microparticle DNA Figure 15 54 A membrane permeable DNA dye extensively stains microparticles only in the absence of DNASE1L3 Figure 16 55 DNASE1L3 treatment of microparticles alters their surface composition Figure 17 57 Dnase1l3-deficient mice have higher levels of DNA circulating in plasma Figure 18 58 Microparticles from Dnase1l3-deficient mice carry increased amounts of DNA compared to those of WT mice Figure 19 59 Dnase1l3-deficient mice fail to process exogenous microparticles in vivo Figure 20 65 DNASE1L3-sensitive chromatin on the surface of microparticles is antigenic in Dnase1l3-deficient mice Figure 21 66 Microparticles from Dnase1l3-deficient mice at young ages are more coated by IgG and have higher levels of exposed self-antigens Figure 22 68 Immunization with microparticles drives anti-chromatin antibody responses in inflammatory contexts Figure 23 69 Treatment of microparticles with DNASE1L3 but not DNASE1 prevents binding by DNA-specific autoantibodies Figure 24 71 The C terminal domain of DNASE1L3 is required for the processing of microparticle chromatin iii Figure 25 73 A subset of human 9G4+ antibodies bind to microparticles only in the absence of DNASE1L3 Figure 26 78 Analysis of human microparticles Figure 27 80 DNASE1L3 null mutations in human patients result in defective DNA processing that parallels Dnase1l3-deficient mice Figure 28 82 DNASE1L3 null mutations in humans result in defective processing of endogenous microparticles in circulation Figure 29 83 DNASE1L3-sensitive binding of patient IgG to exogenous microparticles Figure 30 85 Extensive binding of microparticles by the plasma of patients with sporadic SLE Figure 31 86 Classification of sporadic SLE patients based on DNASE1L3 sensitivity of IgG binding Figure 32 88 The activity of DNASE1L3 in the circulation of sporadic SLE patients differs according to the observed pattern of staining Figure 33 89 Changes in DNASE1L3 activity in sporadic patients impacts the DNA cargo of microparticles Figure 34 97 Proposed mechanism by which DNASE1L3 prevents the development of autoimmunity iv Acknowledgements I would be remiss to not begin by acknowledging my PI, Dr. Boris Reizis. He is not only a brilliant scientist and educator, but a phenomenal mentor as well. Special mention must also go to Dr. Vanja Sisirak, a postdoctoral researcher in the lab with whom I worked very closely on this project. Indeed, Vanja was instrumental in my training after I joined the lab, and his input and guidance have proved vital many times over. I have been fortunate enough to have worked with two wonderful departments; the Department of Microbiology and Immunology of Columbia and the Department of Pathology of NYU. In addition, I would like to thank all of my fellow lab members. The environment is truly wonderful; everyone is always willing to assist with troublesome experiments or give advice. Furthermore, I would like to acknowledge all of our collaborators, who have provided invaluable reagents, materials, and discussions. In particular, I’d like to acknowledge Dr. Jill Buyon and Dr. Robert Clancy, who have graciously allowed us access to all of the human samples analyzed. In addition, they have been tremendously generous with their time and expertise. I would also like to thank all of the patients for providing material for this study; without such contributions our work would not be possible. Finally, my profound thanks to my advisory committee at Columbia: Dr. Donna Farber, Dr. Uttiya Basu, Dr. Kang Liu, and the non-affiliated evaluator Dr. Robert Clancy. Your advice and support has been paramount during my time as a PhD student, and I appreciate your willingness to be available for this whole process. v To my family, friends, and Mariana: Your love and support has been the cornerstone for anything and everything I’ve accomplished vi Introduction Systemic lupus erythematosus: prevalence and therapeutic options Systemic lupus erythematosus (SLE or lupus) is an autoinflammatory disease wherein pathogenesis is driven by the production of antibodies (Abs) to self-nuclear antigens. Targets may include ribonucleoproteins and/or nucleic acids. Following the initial