O001 Lymphoid tissue macrophages express peptidylarginine deiminase 2 and 4: new implications in citrullination and anti-citrulline antibodies production in autoimmune arthritis
Authors: MEM El Shikh, R El Sayed, L Fossati-Jimack, C Pitzalis
Journal: Annals of the Rheumatic Diseases (2018)

<h3>Introduction</h3> The role of citrullination in breach of tolerance in rheumatoid arthritis (RA) is well established, but the mechanisms linking protein citrullination and the generation of anti-citrullinated protein/p eptide antibodies (ACPA) are poorly understood. We have recently reported a critical role of peptidylarginine deiminase (PAD) 4 expression and extracellular trap formation by neutrophils in ACPA production in RA.<sup>1</sup> In lymphoid tissues, neutrophils are scarce, whereas macrophages are abundant antigen presenting cells where they regulate T and B cell responses and play fundamental roles in the chronicity of RA. <h3>Objectives</h3> To test our hypothesis that antigen presenting cells (APCs) in lymphoid tissues express PADs and contribute to protein citrullination and ACPA production. <h3>Methods</h3> We tested our hypothesis in collagen induced arthritis (CIA) mice and synovial biopsies from RA patients. PAD4 and 2 expression was assessed in the spleens and lymph nodes of CIA mice and their distribution was revealed by immunohistochemistry (IHC). The impact of macrophage depletion on lymphoid tissues' PAD activity, citrulline content, and serum ACPA levels was analysed; and the effect of macrophage stimulation on extracellular trap formation, PADs expression and release was investigated. Additionally, PAD4 expression in RA synovia was verified by IHC; and the correlation of PADI4 with macrophage-related genes was studied by RNA-Seq analysis. <h3>Results</h3> Our results indicated that serum ACPA were detectable in CIA sera before clinical arthritis; and PAD4 and 2 were expressed in the draining lymph nodes and spleens as shown by qPCR and Western blotting. In addition, PAD4 and 2 labelling specifically co-localised with macrophages but not other APCs. Moreover, PAD4 and −2-expressing tingible body macrophages (TBM) were associated with citrulline-retaining follicular dendritic cells (FDCs) in the germinal centres (GC). <i>In vitro</i> TLR activation of macrophages enhanced PAD4 and 2 expression; and stimulation of TBMs with TNF-α induced Macrophage Extracellular Trap formation (METosis), release of apoptotic bodies, and DNA-associated citrullinated proteins. Whereas retention of citrullinated proteins on FDCs induced ACPA production in <i>in vitro</i> GC reactions, macrophage depletion in CIA mice significantly reduced citrullinated proteins in lymphoid tissues and inhibited serum ACPA levels. In RA synovia, PAD4 colocalized with CD68 macrophages and was expressed in TBMs associated with citrulline-retaining FDCs in ectopic lymphoid structures. RNA-Seq analysis of PADI4 expression in RA synovial biopsies identified significant positive correlation with 279 genes associated with macrophage activation and function. <h3>Conclusions</h3> In conclusion, this work reports new mechanisms by which macrophages contribute to citrulline-induced autoimmunity. Therapeutic targeting of these mechanisms would inhibit citrullination, ACPA production and progression of autoimmune diseases. <h3>Reference</h3> . Corsiero E, Bombardieri M, Carlotti E, Pratesi F, Robinson W, Migliorini P, Pitzalis C. Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs. Ann.Rheum Dis2016;75:1866–1875. <h3>Disclosure of interest</h3> None declared