ABSTRACT Gene Expression Profiling to Understand the Alterations in the Monocyte Compartment of Pediatric Systemic Lupus Erythematosus Pinakeen Shankarbhai Patel, Ph.D. Mentors: Virginia Pascual, M.D. Jacques Banchereau, Ph.D. Blood monocytes from SLE patients display DC function, as they are able to induce the proliferation of allogeneic T cells. Furthermore, sera from SLE patients induce healthy monocytes to differentiate into DCs. This DC-inducing property is in part due to the presence of type-I IFNs in SLE sera, as well as other, yet uncharacterized factors. To understand these alterations, we performed a thorough phenotypic analysis and gene expression profiling of monocytes from children with active, newly diagnosed and untreated disease. Phenotypic analysis of freshly isolated SLE blood monocytes revealed a modest expansion of CD14highCD16+ cells and an otherwise lack of expression of molecules related to DC function. Further characterization of a fraction of SLE monocytes inducing allogeneic T cell proliferation revealed that upon contact with T cells, SLE monocytes secrete proinflammatory cytokines such as IL-1 and IL-6 and do upregulate expression of innate immunity receptors involved in DC differentiation and molecules responsible for antigen presentation. To recapitulate the initial events leading to monocyte differentiation in this disease, we studied the effects of SLE serum on healthy monocyte at the trasncriptional and protein levels. These studies revealed the upregulation of expression on these cells of chemokine receptor such as CX3CR1 and CCR7, which may lead to the migration of blood monocytes to inflammed tissues and/or secondary lymphoid organs respectively in vivo. There, contact with T cells would lead to the acquisition of antigen presenting function and skewing from tolerogenic to immunogenic responses. Gene Expression Profiling to Understand the Alterations in the Monocyte Compartment of Pediatric Systemic Lupus Erythematosus by Pinakeen Shankarbhai Patel, M.S. A Dissertation Approved by the Department of Biomedical Studies ___________________________________ Rober R. Kane, Ph.D., Chairperson Submitted to the Graduate Faculty of Baylor University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Approved by the Dissertation Committee ___________________________________ Virginia Pascual, M.D., Chairperson ___________________________________ Jacques Banchereau, Ph.D. ___________________________________ Damien Chaussabel, Ph.D. ___________________________________ Christopher J. Kearney, Ph.D ___________________________________ William D. Hillis, M.D. Accepted by the Graduate School August 2008 ___________________________________ J. Larry Lyon, Ph.D., Dean Page bearing signatures is kept on file in the Graduate School. Copyright © 2008 by Pinakeen Shankarbhai Patel All rights reserved TABLE OF CONTENTS TABLE OF CONTENTS iii LIST OF FIGURES v LIST OF TABLES vii LIST OF ABBREVIATIONS viii AKNOWLEDGMENTS xii DEDICATION xiv CHAPTER ONE 1 Introduction 1 Role of Interferon 1 Systemic Lupus Erythematosus 4 Project Objectives 31 CHAPTER TWO 33 Materials and Methods 33 Subjects 33 Blood Collection 34 Isolation/Purificaton of Cell Subsets 34 Cell Staining 37 In Vitro Experiments 39 Microarray Testing and Analysis 41 CHAPTER THREE 44 Results 44 Phenotypic Characterization of SLE Monocytes 44 iii Gene Expression Profiling of SLE Monocytes 48 Characterization of the Effects of SLE Serum on Healthy Monocytes 59 CHAPTER FOUR 67 Discussion 67 APPENDICES 81 APPENDIX A 82 Supplementary figure 82 APPENDIX B 83 Gene lists 83 BIBLIOGRAPHY 360 iv LIST OF FIGURES Figure 1. SLE monocytes display DC function. 14 Figure 2. Induction of DC differentiation is IFN- dependent. 16 Figure 3. Fate of Autoreactive T Cells. 26 Figure 4. Increased bioavailability of IFN-α is fundamental to SLE pathogenesis. 27 Figure 5. Distribution of major blood cells and monocyte subpopulations. 46 Figure 6. Distribution of major monocyte subpopulations in the blood of healthy children. 47 Figure 7. Downregulation of MHC class II expression on ex-vivo SLE monocytes 49 Figure 8. Differentially regulated genes in circulating monocytes of SLE patients. 50 Figure 9. Kinetic study of IFN-inducible genes expressed in circulating blood monocytes from SLE patients. 51 Figure 10. Pathways represented by genes expressed in ex vivo SLE monocytes 53 Figure 11. Flow cytometric representation of purity of blood circulating dendritic cells and monocytes sorted from healthy donors. 54 Figure 12. SLE blood monocytes and healthy blood mDC show little transcriptional overlap. 55 Figure 13. Gene expression profile in SLE monocytes after exposure to naïve CD4 T cells. 57 Figure 14. SLE monocytes that induce CD4 T cell proliferation upregulate MHC class II molecules. 58 Figure 15. Expression level of HLA-DR molecules on SLE monocyte samples after contact with CD4 T cell for 48 hours. 58 Figure 16. Levels of IL-1 and IL-6 in supernatant of SLE monocyte induce MLR. 59 v Figure 17. SLE serum induces significant changes in transcriptional program of healthy monocytes. 61 Figure 18. Pathways represented by genes induced by SLE serum in healthy Monocytes. 62 Figure 19. Comparison between SLE serum or IFN on healthy monocytes and ex vivo SLE monocytes. 63 Figure 20. SLE serum induces functional chemokine receptor CCR7 at both the RNA and protein level. 64 Figure 21. SLE serum induces expression of the fractalkine receptor, CX3CR1, at both the RNA and protein level. 65 Figure 22. How lupus monocytes encounter T cells? 79 Figure 23. Potential therapeutic targets to treat SLE. 80 vi LIST OF TABLES Table 1. Clinical Symptoms of Systemic Lupus Erythematosus. 5 Table 2. Revised ACR Criteria for Classification of SLE. 6 Table 3. Clinical and Treatment Information for SLE Patients 355 Table 4. Antibodies used for immunofluorescence staining. 38 Table 5. Subsets of monocytes 48 vii LIST OF ABBREVIATIONS IFNs Interferons IL Interleukin ACR American college of Rheumatology ANAs Antinuclear antibodies BAFF B cell-activating factor BCR B cell receptor CCL19 Chemokine ligand 19 CCL21 Chemokine ligand 21 CCR7 Chemokine receptor 7 CFSE Carboxy fluoroscein succinimidyl ester CNS Central nervous system CTLA-4 Cytotoxic T lymphocyte-associated 4 CX3CR1 Chemokine, CX3C motif, receptor 1 CXCL10 Chemokine, CXC motif, ligand 10 CXCL11 Chemokine, CXC motif, ligand 11 CXCL9 Chemokine, CXC motif, ligand 9 CXCR3 Chemokine, CX3C motif, receptor 3 DCs Dendritic cells DILE Drug-induced lupus erythematosus dsDNA Double stranded DNA viii EBNA-1 Epstein-Barr nuclear antigen 1 EBV Epstein-Barr virus EMG Electromyogram FCS Fetal calf serum GSMA Genome scale meta analysis HLA Human leukocyte antigen IBM Inclusion body myositis ICs Immune complexes IDDM Insulin dependent diabetes mellitus IFNAR1 Interferon-apha receptor 1 IFNAR2 Interferon-apha receptor 2 IIMs Idipathic inflammatory myopathies IRF Interferon regulatory factor IRF5 Interferon regulatory factor 95 IRF9 Interferon regulatory factor 9 ISGF3 Interferon stimulated gene factor 3 ISREs Interferon stimulated response elements ITIM Immunoreceptor tyrosine-based inhibitory motif IV Intravenous JAK1 Janus activated kinase 1 JAKs Janus activated kinases LPS Lipopolysachharide MAC Membrane attack complex ix MDA5 Melanoma differntiation associated gene 5 MHC Major hystocompatibility complex MLR Mixed lymphocyte reaction MxA Myxovirus resistance 1 NKT cell Natural killer T cells NOD Nucleotide binding oligomerization domain NSAIDs Non-steroidal anti-inflammatory drugs NZB New Zealand black NZW New Zealand white PAMPs Pathogen associated membrane proteins PBMCs Peripheral blood mononuclear cells PBS Phosphate buffered saline pDC Plasmacytoid dendritic cell PDCD1 Programmed cell death 1 PGE2 Prostaglandin E2 PRR Pattern recognition receptors PTPN22 Protein tyrosine phosphatase, non receptor-type, 22 RAG Recombination-activating gene RANK Receptor activator of NF-Kappa-B RANKL Receptor activator of NF-Kappa-B ligand RIN RNA integration number RUNX1 Runt-related transcription factor 1 SLAM Signaling lymphocyte activation molecule x SLC Secondary lymphoid-tissue chemokine SLE Systemic lupus erythematosus SLEDAI Systemic lupus erythematosus disease activity index snRNP Small nuclear ribonucleoprotein SS Sjogren’s syndrome STAT1 Signal transducer and activator of transcription 1 STAT2 Signal transducer and activator of transcription 2 Tfh Follicular helper T cells Th1 Helper T cells 1 Th2 Helper T cells 2 TLR Toll-like receptor TNF Tumor necrosis factor TYK2 Tyrosine kinase 2 WBC White blood count Yaa Y-linked autoimmune accelerator xi AKNOWLEDGMENTS It was a great journey for me to reach this stage in life to finish my Ph.D.. Without helps of following people, it would be impossible for me to stand up here. I would like to thank my mentors, Dr. Virginia Pascual and Dr. Jacques Banchereau, for giving me this opportunity to work with them, advising me during this process, and nurturing me intellectually. I would like to thank Dr. Edsel Arce, who has taught me basic techniques in the beginning of my studies. I would like to thank Jeanine for helping me in scientific writing; Vicky and Minning for RNA extraction and labeling of samples; Linda and Benedicte for helping me in various in vitro experiments; Dorothee Stitchweh, Gabriela, and Natalie for patients information; Quynh and Jin from Microarray core for hybridizing samples on microarray chips; Elizabeth and Sebastien for cell sorting and flow cytometry; Lynnette for providing frozen