Plasmacytoid Dendritic Cells and CXCL4: Key Drivers of Systemic Sclerosis Alsya Jubilly Affandi ISBN: 978-94-92801-30-2 © Alsya Jubilly Affandi, 2018. All rights are reserved. No part of this thesis may be reproduced or transmitted in any form or by any means without the prior permission of the author. The copyright of published articles have been transferred to the respective journals. Cover description: Illustration of plasmacytoid dendritic cell producing CXCL4 and their interaction with other cells during skin inflammation and fibrosis in systemic sclerosis. Color palette depict real skin tones originally designed by frederiksmoeller via Adobe Color CC. Cover design and thesis layout: Alsya Affandi & Karina Binol, in consultation with Guus Gijben. Printed by: Proefschrift AIO Printing of this thesis was financially supported by: Stichting NVLE fonds Peprotech ChipSoft Infection and Immunity Utrecht UMC Utrecht - Department of Rheumatology & Clinical Immunology Plasmacytoid Dendritic Cells and CXCL4: Key Drivers of Systemic Sclerosis Plasmacytoïde dendritische cellen en CXCL4: belangrijke spelers in systemische sclerose (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof.dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op donderdag 26 april 2018 des middags te 2.30 uur door Alsya Jubilly Affandi geboren op 1 juli 1985 te Jakarta, Indonesië Promotor: Prof.dr. T.R.D.J. Radstake Copromotoren: Dr. W. Marut Dr. J.A.G. van Roon This thesis was accomplished with financial support from: Netherlands Organisation for Scientific Research (NWO) Mosaic project number 017.008.014 For my parents Manuscriptcommissie: Prof.dr. B. Burgering (voorzitter) Prof.dr. E. Hack Prof.dr. F. van den Hoogen Prof.dr. B. Prakken Prof.dr. J.M. van Laar Paranymphs: L.L. van den Hoogen, MD E.F.A. Leijten, MD Contents Chapter 1 General introduction 9 PART I: Plasmacytoid dendritic cells dysfunction in SSc Chapter 2 Proteome-wide analysis and CXCL4 as a biomarker in 25 systemic sclerosis Chapter 3 Low RUNX3 expression alters dendritic cell function 55 in patients with systemic sclerosis and contributes to enhanced fibrosis Chapter 4 Association of microRNA-618 expression with altered 77 frequency and activation of plasmacytoid dendritic cells in patients with systemic sclerosis PART II: CXCL4, a key driver of inflammation and fibrosis Chapter 5 CXCL4 exposure potentiates TLR-driven polarization of 101 human monocyte-derived dendritic cells and increases stimulation of T cells Chapter 6 CXCL4 is a novel inducer of human Th17 cells and correlates 121 with IL-17 and IL-22 in psoriatic arthritis Chapter 7 CXCL4 promotes myofibroblast transformation and it is 143 required for fibrosis development in vivo Chapter 8 Summary and discussion 165 Appendix Nederlandse samenvatting 181 English summary Acknowledgements Portfolio - List of publications About the author Chapter 1 General introduction Parts of this chapter have been published in: Dendritic cells in systemic sclerosis: advances from human and mice studies Alsya J. Affandi1,2, Tiago Carvalheiro1,2, Timothy R.D.J. Radstake1,2, and Wioleta Marut1,2 Immunol Lett 2017 DOI: 10.1016/j.imlet.2017.11.003 Update on biomarkers in systemic sclerosis: tools for diagnosis and treatment Alsya J. Affandi1,2, Timothy R.D.J. Radstake1,2, and Wioleta Marut1,2 Semin Immunopathol 2015;37:475–487 DOI: 10.1007/s00281-015-0506-4 Editorial: closing in on the role of thymic stromal lymphopoietin inhibition as a therapeutic entry point for systemic sclerosis Alsya J. Affandi1,2 and Joel A.G. van Roon1,2 Arthritis Rheumatol 2016 Nov;68(11):2571-2574 DOI: 10.1002/art.39815 1Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands. 2Department of Rheumatology and Clinical Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, the Netherlands. Introduction 1 1. Background General introduction Systemic sclerosis (SSc), or scleroderma, is a complex autoimmune disease manifested by fibrosis in the skin and internal organs. It is characterized by a persistent inflammation, vasculopathy, and fibrosis, forming an overlapping tripartite network that drive disease pathogenesis1,2. Patients with SSc can be classified into two major groups according to the extent of skin fibrosis: limited cutaneous SSc (lcSSc), in which skin fibrosis is restricted to the areas distal to the elbows and knees; and diffuse cutaneous SSc (dcSSc), where skin fibrosis is more extensive and internal organs involvement is more severe (Fig. 1). This classification is supported by the association with specific autoantibodies that specifically define the two clinical phenotypes. Anti-centromere antibodies (ACAs) are associated with lcSSc, whereas anti-topoisomerase I antibodies (ATA) are associated with dcSSc3,4. Both SSc phenotypes can be complicated by severe internal organ dysfunction. Pulmonary fibrosis and pulmonary arterial hypertension (PAH) are the two most feared complications, representing the major causes of mortality in SSc patients, followed by myocardial involvement5. Owing to its complex nature and heterogeneity, SSc remains one of the greatest challenges to both investigators and physicians. Current treatments remains for many complications ineffective, resulting in a high morbidity and mortality6. Fibrosis is attributed to the formation of limited diuse myofibroblast that produces an excessive amount extracellular matrix (ECM), such as collagen. These cells are derived from tissue resident fibroblast or other precursor cells such as endothelial cells and epithelial cells7. Often preceding fibrotic events, immune cell infiltration of affected tissue occurs early in the disease. They release immune mediators that facilitate fibroblast activation and collagen synthesis such as transforming growth factor beta (TGFβ), IL-6, and type 2 cytokines (IL-4, IL-10, IL-13)2,8. Dendritic cells (DCs), in particular, are of main interest due to their capability to regulate cells of the innate and adaptive immune systems, but also cells in the vasculature, as well Figure 1. Classification of systemic sclerosis as fibroblasts. The potential involvement of DCs patients. Schematic representation of 9 affected areas of limited cutaneous systemic in SSc have been reviewed in 2012 , since then sclerosis (left, lcSSc) or diffuse cutaneous multiple studies on DCs and their activation in SSc systemic sclerosis (right, dcSSc) patients. Illustration was adapted from Servier Medical Art. 13 have emerged. In this introduction chapter, I will describe recent work from human studies, mouse models, and other related autoimmune diseases, that have shed light onto the role of DCs in the development of inflammation and fibrosis in SSc. Furthermore, I will discuss the consequence of DC activation on effector cells, in particular CD4 T cell polarization. Finally, the impact of DC-mediated immune activation and myofibroblast transformation will be discussed. 2. Dendritic cell populations and functions Dendritic cells (DCs) are specialized cells in pathogen sensing with high capability of antigen presentation. They express an array of pathogen recognition receptors such as toll-like receptors (TLRs), C-type lectin receptors (CLRs), RIG-I-like receptors (RLRs), and Nod-like receptors (NLRs), to recognize pathogen- or danger-associated molecules in the extracellular and intracellular environment. Upon recognition, DCs upregulate co-stimulatory signal molecules and secrete immune mediators that are required to regulate T cell responses and other cell types. DCs can be broadly categorized into two populations, the conventional DCs (cDCs, also called myeloid DCs) and plasmacytoid DCs (pDCs). The major DC subsets are well conserved between human and mouse although they express different markers10. Human blood cDCs consist of CD1c+/BDCA1+ DCs (cDC2) and CD141+ DCs (cDC1). cDC1 can be distinguished by the expression of CD141+ CLEC9A+ in human and resembles CD8α+ XCR1+ DC in mouse. cDC2 in human are CD1c+ CD11c+ cells that align with mouse CD11b+ CD172+ DCs. pDCs can be identified as CD303+ CD304+ CD123+ cells in human and B220+ Siglec-H+ BST2+ in mouse. DCs can be further subdivided based on their anatomical location, function, and state of inflammation11,12. Furthermore, new data revealed six distinct subtypes of DCs identified in human blood, with a proposed new DC subset of AXL-, SIGLEC1-, and SIGLEC6-expressing cells, named AS DCs13. All three main DC subsets express different sets of TLRs. cDC2s express almost all TLRs, cDC1 mainly express TLR3 and TLR10, while pDCs express TLR7 and TLR914. While cDCs have a high potency in antigen presentation and T cell priming, pDCs specialize in producing type I IFN (IFN-I) that are important in antiviral response, B cell activation and autoantibody production, and NK cells activation, and therefore have been implicated in many autoimmune diseases15,16. 3. Dendritic cells in systemic sclerosis Studies on DC populations in SSc patients have revealed changes in their tissue distribution as well as disturbances of their key functions, in particular their activation and cytokine secretion. Next to clinical research, mouse models of SSc have also been pivotal in unraveling the role of DC in SSc as well as providing means to assess new intervention strategies. DC tissue distribution in SSc cDCs are normally found in