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Insights Into the Immune Mechanisms Leading to Lupus-Like Autoimmunity in New Zealand Black Mice

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Insights Into the Immune Mechanisms Leading to Lupus-Like Autoimmunity in New Zealand Black Mice Insights into the immune mechanisms leading to lupus-like autoimmunity in New Zealand Black mice by Evelyn Yin-Wah Pau A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Immunology University of Toronto © Copyright by Evelyn Yin-Wah Pau 2013 Insights into the immune mechanisms leading to lupus-like autoimmunity in New Zealand Black mice Doctor of Philosophy Degree, 2013 Evelyn Yin-Wah Pau Department of Immunology University of Toronto Abstract Systemic lupus erythematosus (SLE) is a chronic, multi-organ autoimmune disease characterized by the production of antibodies against self nuclear antigens. Genetics play a dominant role in disease pathogenesis and functional examination of spontaneously-arising lupus-prone animal models has provided key insights into understanding the genetic complexity of the disease. The overarching goal of the work presented here is to identify the underlying immunologic abnormalities, together with lupus susceptibility loci that produce them, that promote the development of autoimmunity in the lupus-prone New Zealand Black (NZB) background. Chapter 2 identifies the critical role of CD40-CD40L interactions in the pathogenesis of disease in NZB mice. We showed that this interaction is required for the production of class-switched IgG autoantibodies and development of hemolytic anemia and kidney disease. Polyclonal B cell activation, expansion of plasmacytoid dendritic cells (pDC), and elevated gene expression of baff were maintained in CD40L-deficient NZB mice, despite the lack of IgG immune complexes. Chapter 3 utilizes bicongenic mice carrying both NZB chromosomes 1 and 13 to investigate the genetic complexity in disease pathogenesis. In addition to identifying new phenotypes, examination of bicongenic mice showed that chronic stimulation of pDC due to the persistence of nuclear antigens leads to a refractory state with a failure to produce more IFN-α upon subsequent stimulation. Chapter 4 ii identifies a novel lupus susceptibility locus on NZB chromosome 13 associated with impaired clearance of apoptotic debris, a key initiating step in the development of autoimmunity. Using subcongenic mice, this locus was localized and examined its impact on immune function. Work from this thesis will contribute to understanding the complex immunogenetic mechanisms that lead to development of SLE. iii Acknowledgments There are many people who have contributed in different ways to this thesis: My supervisor Dr. Joan Wither, for her wisdom and continuous support. Thank you for being the best supervisor and bringing me into the world of immunology. Dr. Nan-Hua Chang, for shaping me into the researcher I am today and allowing me to coin the term “Nan is always right.” Dr. Christina Loh, for introducing me to the Wither lab, teaching me to do research, and being the best colleague and friend in the lab. I couldn’t have done this without you. Dr. Yui-Ho Cheung, for being a pioneer in the Wither lab and leading me into the world of TAing. Dr. Carolina Landolt-Marticorena, for her humour and wit. We will always celebrate Candlemas. Nafiseh Talaei, for the warmth and kindness she brings to the lab. Yuriy Baglaenko, for supporting me with his sanity. Kimberley Lifeso, for keeping Yuriy in check and being a sassy deskmate. Kieran Manion, for her musical entertainment in the lab when she thinks nobody notices. Gillian Minty, for taking over my c13 project and answering all the remaining questions. Allie Rasiuk, for all her laughter and genotyping help. Babak Noamani, for his constant source of gossip at TWH. Connor Moffatt, our honourary Wither lab member, for the excitement he brings to the lab. Past members of the lab: Julie Kim, Charmaine Ferguson, Gabriel Bonventi, and Yong-Chun Cai. My supervisory committee Dr. Eleanor Fish and Dr. Jennifer Gommerman, for their thoughts, feedback, and expertise. My family, for letting me pursue science, and Timothy Li, for his love and patience. I am grateful for having you all in my life and in my graduate career. Thank you from the bottom of my heart. Research conducted in this thesis was supported by grants from the Canadian Institutes of Health Research and The Arthritis Society. Studentship awards were supported by the Edward Dunlop Foundation Ontario Graduate Scholarships in Science and Technology (2007-2011), and the University of Toronto Doctoral Completion Award (2011-2012). iv Table of Contents Abstract ...................................................................................................................................................... ii Acknowledgments.................................................................................................................................................. iv Table of Contents .................................................................................................................................................... v List of Figures and Tables ..................................................................................................................................... ix List of Publications ................................................................................................................................................ xi List of Abbreviations ........................................................................................................................................... xii Chapter 1 Introduction ................................................................................................................................ 1 1.1 Systemic lupus erythematosus ................................................................................................... 2 1.1.1 Genetic factors in SLE ................................................................................................................. 2 1.1.2 Environmental factors in SLE .................................................................................................... 3 1.2 Mouse models of lupus ............................................................................................................... 3 1.2.1 Spontaneous lupus models ......................................................................................................... 4 1.2.2 Congenic mouse models of lupus .............................................................................................. 6 1.3 Mechanisms of lupus pathogenesis ........................................................................................... 9 1.3.1 Impaired clearance and aberrant response to apoptotic debris ........................................... 10 1.3.2 Aberrant lymphocyte signalling .............................................................................................. 21 1.3.3 Defects that promote survival of autoreactive lymphocytes................................................. 25 1.3.4 Defects that promote end organ damage ................................................................................ 27 1.4 Bridging mouse studies to human SLE .................................................................................. 28 1.5 Thesis objectives ....................................................................................................................... 32 Chapter 2 Abrogation of pathogenic IgG autoantibody production in CD40L gene-deleted lupus-prone New Zealand Black mice .................................................................................... 34 2.1 Abstract ..................................................................................................................................... 35 v 2.2 Introduction ............................................................................................................................. 35 2.3 Materials and Methods ............................................................................................................ 38 2.3.1 Mice ............................................................................................................................................ 38 2.3.2 Flow cytometry staining and analysis ..................................................................................... 39 2.3.3 Measurement of antibody production .................................................................................... 39 2.3.4 Detection of anti-RBC antibodies ........................................................................................... 40 2.3.5 Grading of kidney sections ....................................................................................................... 41 2.3.6 Quantitative real-time PCR analysis ....................................................................................... 41 2.3.7 In vitro cell proliferation and Ig class-switching ................................................................... 42 2.3.8 Statistical analysis ...................................................................................................................... 43 2.4 Results ....................................................................................................................................... 43 2.4.1 Abrogated IgG autoAb and attenuated kidney disease in NZB.CD40L-/- mice. ................. 43 2.4.2 Variable effects of CD40L on the cellular phenotypic abnormalities seen in NZB mice. ........................................................................................................................................... 46 2.4.3
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