Type I Interferons and the Development of Impaired Vascular Function and Repair in Human and Murine Lupus by Seth G Thacker A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Immunology) in The University of Michigan 2011 Doctoral Committee: Associate Professor Mariana J. Kaplan, Chair Professor David A. Fox Professor Alisa E. Koch Professor Matthias Kretzler Professor Nicholas W. Lukacs Associate Professor Daniel T. Eitzman © Seth G Thacker 2011 Sharon, this work is dedicated to you. This achievement is as much yours as it is mine. Your support through all six years of this Ph.D. process has been incredible. You put up with my countless miscalculations on when I would finish experiments, and still managed to make me and our kids feel loved and special. Without you this would have no meaning. Sharon, you are the safe harbor in my life. ii Acknowledgments I have been exceptionally fortunate in my time here at the University of Michigan. I have been able to interact with so many supportive people over the years. I would like to express my thanks and admiration for my mentor. Mariana has taught me so much about writing, experimental design and being a successful scientist in general. I could never have made it here without her help. I would also like to thank Mike Denny. He had a hand in the beginning of all of my projects in one way or another, and was always quick and eager to help in whatever way he could. He really made my first year in the lab successful. Many thanks to Sri, Wenpu, Michelle and Eneida. You have made the years in lab enjoyable. I would also like to thank all members of my dissertation committee. They have always provided me with excellent feedback, suggestions, and collaborations which helped to move my work forward. Finally I wish to thank and give credit to all those who had a direct hand in assisting with my experiments. Wenpu Zhao, Jim Park, and Damon Duquaine all assisted with the endothelial reactivity studies. Celine Berthier assisted with the microarray analysis, and without her help I would still be under a deluge of microarray data and trying to make sense of it all. Wei Luo assisted with the thrombosis and atherosclerosis quantification. Brad Rabquer assisted with the Matrigel assays. I also extend my greatest appreciation and thanks to Maria Pia Rastaldi and Deborah Mattinzoli who performed the IHC on the kidney biopsy sections. Additionally I would like to thank Dr. Anne Davidson for graciously providing the IFN-α expressing adenovirus; Dr. Chaim Jacob for providing breeding pairs of NZM 2328 and NZM 2328 IFNAR deficient mice; and Dr. H.W. Virgin for providing breeding pairs of IFNAR deficient mice. iii Table of Contents Dedication ............................................................................................................. ii Acknowledgments ................................................................................................ iii List of Tables ....................................................................................................... vii List of Figures ..................................................................................................... viii List of Appendices ................................................................................................ x List of Abbreviations ............................................................................................. xi Abstract ............................................................................................................. xiv Chapter 1 Introduction .......................................................................................... 1 Systemic Lupus Erythematosus: ....................................................................... 1 Definition and demographics: ......................................................................... 1 Clinical Manifestations and Treatment: .......................................................... 2 Cardiovascular manifestations in SLE patients: ............................................. 2 Pathogenesis of SLE: ..................................................................................... 3 Type I Interferons and SLE: ............................................................................... 4 Relevant mouse models of lupus ...................................................................... 7 MRL/lpr mice: .................................................................................................. 7 New Zealand Black/New Zealand White F1 hybrid and NZM 2328 mice: ............................................................................................................... 7 Pathogenesis of atherosclerotic vascular disease. ............................................ 8 Vascular Repair ............................................................................................... 11 Atherosclerosis and SLE ................................................................................. 13 Concluding Remarks ....................................................................................... 14 Chapter 2 Material and Methods ........................................................................ 16 Patient selection: ............................................................................................. 16 iv Cell isolation and culture and fluorescent microscopy: .................................... 16 Phosphorylated STAT detection and inhibition of IFN-α signaling pathways: ........................................................................................................ 18 RNA isolation: .................................................................................................. 19 Microarray data processing, analysis and pathway mapping: ......................... 19 Real-time quantitative PCR: ............................................................................ 20 Assessment of serum protein levels, EPC/CAC proliferation and apoptosis: ........................................................................................................ 20 Assessment of myeloid cell phenotype: .......................................................... 20 Human kidney tissue and immunohistochemistry: .......................................... 22 Mice: ................................................................................................................ 24 Genotyping of APOE-/-IFNαβR-/- mice: ............................................................. 25 Adenovirus injections: ...................................................................................... 26 Assessment of vascular function in mice: ........................................................ 27 Quantification of murine endothelial progenitor cells (EPCs): ......................... 28 Assessment of murine EPC differentiation into mature endothelial cells: ....... 29 Induction of murine carotid thrombosis: ........................................................... 30 Quantification of atherosclerosis in mice: ........................................................ 30 In vivo Matrigel plug angiogenesis assay: ....................................................... 31 RNA isolation and real-time PCR of murine cells: ........................................... 31 Statistical analysis: .......................................................................................... 32 Chapter 3 Interferon-α inhibits vasculogenesis in human lupus through modulation of the IL-1 pathway. ........................................................... 34 IFN-α induces an antiangiogenic signature in control and lupus EPCs/CACs: ................................................................................................... 34 IL-1β restores the capacity of lupus EPCs/CACS to differentiate into mature endothelial cells: ................................................................................. 40 IL-1β abrogates the defects in proliferation and viability of lupus EPCs/CACs: ................................................................................................... 43 IL-1β decreases lupus DC differentiation: ....................................................... 45 IFN-α regulates IL-1 pathways in EPCs/CACs through JAK-STAT modulation: ..................................................................................................... 46 v Decreased endothelial density and altered IL-1 pathways are observed in SLE in vivo: ..................................................................................................... 51 Summary ......................................................................................................... 54 Chapter 4 Type I IFNs induce endothelial cell dysfunction, aberrant vascular repair, and acceleration of thrombosis and atherosclerosis. ................ 56 NZB/W mice have impaired endothelium-dependent vasorelaxation: ............. 56 NZB/NZW mice display reduced numbers and increased apoptosis of EPCs: .............................................................................................................. 58 NZB/W EPCs are impaired in their capacity to differentiate into mature endothelial cells: ............................................................................................. 60 Type I IFN signatures are increased in NZB/W EPC compartments: .............. 62 IFN-α induces cytotoxicity of murine EPCs: .................................................... 65 Loss of type I IFN signaling in