ABNORMALITIES IN THE ADHESION AND AGGREGATION PROFILES OF CIRCULATING MONOCYTES IN PSORIASIS By JACKELYN BEZZEG GOLDEN Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Kevin Cooper Department of Pathology CASE WESTERN RESERVE UNIVERSITY January, 2016 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Jackelyn B. Golden _____________________________________________________ Ph.D. candidate for the ______________________ degree *. George Dubyak, Ph.D. (signed)_____________________________________________________ (chair of the committee) Kevin Cooper, M.D. ___________________________________________________________ Alex Huang, M.D./Ph.D. ___________________________________________________________ Eric Pearlman, Ph.D. ____________________________________________________________ Thomas Hamilton, Ph.D. ____________________________________________________________ Clive Hamlin, Ph.D. ____________________________________________________________ August 27, 2015 (date)_______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. 1 For my family: Dad, Mom, Jeff, and Tyler Thank you all for your continuous support. 2 TABLE OF CONTENTS List of Tables 6 List of Figures 7 Acknowledgements 9 List of Abbreviations 10 Abstract 13 Chapter 1: Introduction Psoriasis 16 Clinical pathogenesis and assessment 17 Psoriasis and associated comorbidities 21 Genetics of psoriasis 22 Psoriasis as an immune-mediated inflammatory disease: cellular immune cells mediating psoriasis pathology T-helper subtypes: Th1, Th2, Th17, and Th22 23 Regulatory T cells 26 CD8+ T cells 27 Dendritic cells 27 Neutrophils 28 Monocytes and macrophages 28 Keratinocytes 34 Treatment of psoriasis 34 3 Murine models recapitulate aspects of human psoriasis 36 Murine monocyte populations 38 Objectives and Significance 39 Chapter 2: Chronic psoriatic skin inflammation leads to increased monocyte adhesion and aggregation Introduction 44 Materials and Methods 46 Results 55 Discussion 79 Chapter 3: Chronic, not acute, skin-specific inflammation promotes thrombosis in psoriasis murine models Introduction 86 Materials and Methods 89 Results and Discussion 93 Conclusions 102 Chapter 4: IL-17 in psoriasis: implications for therapy and cardiovascular co-morbidities Introduction 104 Psoriasis pathogenesis and treatment 105 Psoriasis immunology and the role of IL-17 T helper subtypes important in psoriasis pathogenesis 108 IL-17A in human psoriasis 110 IL-17 targeted human therapeutics 112 4 Psoriasis and cardiovascular co-morbidities 113 Th17 cytokines that link psoriasis and CVD 116 Conclusion 119 Chapter 5: Ongoing work and future directions Human Studies: Comparative Effectiveness Research 122 Murine Studies 132 Conclusions and Future Directions 143 Appendix A 151 Appendix B 152 References 154 5 TABLES 2.1: Patient demographics 47 2.2: Antibodies used in Flow Cytometry and Immunofluorescence 50 2.3: Absolute cell count 57 2.4: MFI changes 71 4.1: Common cell types and mediators in psoriasis and atherosclerosis 118 5.1: Patient demographics of human comparative 125 effectiveness research 6 FIGURES 1.1: Histological features of psoriasis 18 1.2: Subsets of circulating human monocytes 33 1.3: Overall hypothesis 42 2.1: Monocyte and doublet gating strategy 49 2.2: CD14++CD16+ (intermediate) cells are increased in psoriasis 58 patients compared to healthy controls 2.3: Psoriasis patients have increased total doublets within PBMCs 60 2.4: Amnis Imagestream visualization of cell surface expression 62 of CD14 (APC) and CD16 (FITC) and monocyte-monocyte doublets 2.5: CD14++CD16+ cells are present and detectable in 64 psoriatic plaques 2.6: CD14, CD16, and CD3 staining with CD31, CD68, or 65 DEC-205 co-localization 2.7: Upregulation of CD16 expression and enhanced adhesion 68 2.8: Increased β2 integrin expression on psoriasis 70 classical monocytes 2.9: Gene networks in doublets and adhered classical monocytes 74 2.10: Gene network of psoriasis doublets 76 2.11: Monocyte doublets are increased in KC-Tie2 mice 78 3.1: Gating strategy to identify the CD11b+Ly6GnegLy6Chigh 92 monocyte population 3.2: Chronic, but not acute, skin inflammation promotes 95 shorter arterial clotting times in the Rose Bengal thrombosis assay 3.3: CD11b+Ly6Chigh monocytosis is increased in acute and 99 chronic inflammatory skin disease mouse models 7 FIGURES 5.1: Correlations of doublet percentage and psoriasis disease 126 severity as measured by PASI 5.2: Time course of changes in PASI and monocyte populations 128 (intermediate and classical) from baseline following systemic therapy at subsequent time points 5.3: Correlations at baseline of circulating blood monocyte 129 populations with MDSC percentage 5.4: KC-Tie2 and KC-Tie2 x S100A9-/- mice exhibit 134 sustained skin inflammation + high 5.5: CD11b Ly6C monocytes are increased in KC-Tie2 mice 135 and remain elevated in KC-Tie2 x S100A9-/- animals 5.6: KC-Tie2 and KC-Tie2 x S100A9-/- mice demonstrate 137 increases in IL-6 at the RNA and protein level 5.7: KC-Tie2 mice backcrossed to IL-6 knockout mice have 139 sustained skin inflammation but are not pro-thrombotic 5.8: Immune cell counts in KC-Tie2-IL-6-/- mice 140 5.9: CD11b+Ly6Chigh monocytes decrease in the lymph nodes, 142 but not spleens, of KC-Tie2 x IL-6-/- mice 5.10: Hypothesized mechanism of doublet formation and 147 monocyte differentiation in humans 8 ACKNOWLEDGMENTS Thank you to my committee – I appreciate the helpful feedback, ideas regarding how to think about my experiments and results in a different way, and the suggestions on how to better present my data. The guidance was invaluable in my graduate school career. Thank you to my mentors – Drs. Kevin Cooper and Thomas McCormick – I could not have asked for better examples of successful scientists and outstanding thinkers. I would also like to thank Dr. Nicole Ward for her support and valuable feedback on my projects. Additionally, I’d like to recognize my family – my Mom, Dad, and brother, Jeff – who were supportive of my efforts from the very first day of graduate school until the very last. Finally, my fiancé, Tyler, was a significant contributor to my success - I sincerely appreciate his encouragement and love. 9 ABBREVIATIONS AMP: antimicrobial peptide BMI: body mass index BSA: body surface area CACS: and carotid artery calcium scoring CAD: coronary artery disease CCR: chemokine receptor CD: cluster of differentiation CLA: cutaneous lymphocyte antigen CMML: chronic myelomonocytic leukemia CVD: cardiovascular disease CXCR: C-X-C motif receptor DAMP: damage associated molecular pattern molecule DC: dendritic cell DQLI: dermatology life quality index hBD: human beta-defensin HMVEC-D: human dermal microvascular endothelial cells hsCRP: high-sensitivity C-Reactive Protein EC: endothelial cell ECM: extracellular matrix ERK: extracellular signal-regulated kinases FAK: focal adhesion kinase 10 FDG: fluorodeoxyglucose GWAS: genome-wide association study IFN: interferon IKK: IkappaB kinase IL: interleukin IMIAD: immune-mediated inflammatory autoimmune disease IMQ: imiquimod iNOS: inducible nitric oxide synthase ICAM: intercellular adhesion molecule IPA: ingenuity pathway analysis ITG: integrin JNK: c-Jun N-terminal kinase KC: keratinocyte LC: Langerhans cell LN: lymph node LPS: lipopolysaccharide MAPK: mitogen-activated protein kinase MCP: monocyte chemoattractant protein MDSC: myeloid derived suppressor cell MHC: major histocompatibility complex MI: myocardial infarction MMP: matrix metalloproteinase MP: microparticle MPA: monocyte/platelet aggregate 11 NET: neutrophil extracellular trap oxLDL: oxidized LDL PASI: psoriasis area and severity index PBMC: peripheral blood mononuclear cell PCR: polymerase chain reaction pDC: plasmacytoid DC PPPP: palmoplantar pustular psoriasis PET/MRI: positron emission tomography-magnetic resonance imaging PSORS1: psoriasis susceptibility gene 1 QoL: quality of life RA: rheumatoid arthritis RORγ: RAR-related orphan receptor gamma SD: standard deviation slanDCs: 6-sulfo LacNAc dendritic cells STAT: signal transducer and activator of transcription STEMI: ST segment elevation myocardial infarction Tie-2: tyrosine kinase with immunoglobulin and EGF homology domains TNF: tumor necrosis factor Teff: effector T cell TLR: Toll-like receptor Treg: regulatory T cell VEGF: vascular endothelial growth factor WT: wild type 12 Abnormalities in the Adhesion and Aggregation Profiles of Circulating Monocytes in Psoriasis Abstract By JACKELYN BEZZEG GOLDEN Human psoriasis patients have numerous comorbidities including an increased risk of developing cardiovascular disease (CVD), thus making psoriasis truly more than skin deep. Numerous cellular immune mediators are implicated to initiate, direct, and participate in the chronic inflammation characteristic of psoriasis. A relatively neglected participant in psoriasis pathology is the monocyte. Three main populations of circulating monocytes have been identified in humans based on differential expression of the cell surface markers CD14 and CD16, defining them as “classical” (CD14++CD16-), “intermediate” (CD14++CD16+), and “non-classical” (CD14+CD16++). Intermediate (CD14++CD16+) monocytes are elevated in psoriasis, correlate with psoriasis severity and, interestingly, are predictive of increased CVD risk and outcomes including death, suggesting that monocytes may be an important cellular mediator. A unique feature