SAT0013 The pathogenic role of myeloid CD141+ dendritic cells in inflammatory arthritis
Authors: M Canavan, T McGarry, K Killick, B Moran, D Cluxton, C Orr, H Convery, S Wade, S Wade, R Mullan, J Fletcher, DJ Veale, U Fearon
Journal: Annals of the Rheumatic Diseases (2017)

<h3>Background</h3> Dendritic cell (DC) are a heterogeneous group of antigen presenting cells that can be subdivided into CD1c<sup>+</sup> &amp; CD141<sup>+</sup> DC. CD141<sup>+</sup> DC are a rare population of DC that were first discovered in 2010 in human peripheral blood. Due to their rarity very little is known about the function of these cells in other tissue in or indeed disease. These newly described DC subset have thus never been described in Inflammatory Arthritis (IA) or any of the rheumatic diseases. <h3>Objectives</h3> To identify CD141 DC in IA synovium and functionally assess if these cells play a pathogenic role in IA. <h3>Methods</h3> CD141<sup>+</sup>DC were magnetically purified from synovial fluid mononuclear cells (SFMC) and peripheral blood mononuclear cells (PBMC) stimulated and stained with a panel of fluorochrome conjugated antibodies for multicolour flow cytometry. CD141<sup>+</sup> DC isolated and purified from IA synovial fluid were subsequently cocultured with allogenic CD3<sup>+</sup> T cells for 6d after which intracellular cytokine production was assessed by flow cytometry. Supernatants from this DC-T cell cocultures were used to treat synovial fibroblasts &amp; the expression of adhesion molecules, cytokines &amp; MMPs was measured. Finally using sorted populations of CD141<sup>+</sup> DC from SFMC and PBMC, RNA sequencing was performed and differentially expressed genes and interaction network analysis were identified using the DeSeq2 R package, Ingenuity® Pathway Analysis (IPA) and InnateDB and Cytoscape. <h3>Results</h3> Within IA synovial fluid (SF), CD141<sup>+</sup> DC are significantly enriched compared to WB &amp; express higher levels of the costimulatory activation markers CD80 CD86 and CD40. Following coculture of these SF CD141<sup>+</sup> DC with CD3<sup>+</sup> T cells, CD141<sup>+</sup> DC induce both CD8<sup>+</sup> &amp; CD4<sup>+</sup> T cell proliferation. SF CD141<sup>+</sup> DC induce Granzyme B production from CD8<sup>+</sup> T cells &amp; TNFα, IFNγ &amp; GMCSF from CD4<sup>+</sup> T cells. The IA synovium consists of a complex interplay of multiple cell types. Therefore next we examined the effect of this CD141<sup>+</sup> DC-T cell interaction on the key invasive cells in the synovium – synovial fibroblasts. Supernatants from CD141 activated T cells were cultured with fibroblasts &amp; induced expression of ICAM-1, IL-6, IL-8, MMP1 &amp; MMP3. SF CD141 expressed significantly higher levels of the the hypoxia marker TREM1, activation of which induces further expression of CD80, CD86 and CD40. Coculture of these TREM1 activated CD141 with CD3<sup>+</sup>T cells increases IFNγ and IL-17a production. Finally RNASeq analysis revealed that there are 2089 differentially expressed genes between SF CD141<sup>+</sup>DC &amp; WB CD141<sup>+</sup>DC. These genes are involved in a number of key pathways such as energy metabolism, chemokine &amp; cytokine signalling. Principal Component Analysis (PCA) revealed that CD141<sup>+</sup> DC with the synovium are distinctly different from blood CD141<sup>+</sup> DC <h3>Conclusions</h3> CD141<sup>+</sup> DC are enriched in the IA joint in an active state. RNASeq analysis revealed they are distinct from blood CD141<sup>+</sup>DC and our <i>in vitro</i> data would support the hypothesis that these CD141<sup>+</sup>DC contribute to synovial inflammation and joint destruction. <h3>Disclosure of Interest</h3> None declared