Accumulation Of Functionally Mature Cd1c(+) Dendritic Cells Contributes To Synovial Inflammation In Inflammatory Arthritis

Background:Myeloid Dendritic Cells (DC) are potent antigen presenting cells that can be subdivided into CD141 and CD1c+ DC. We have previously reported an unacknowledged role for CD141+DC in the IA synovium. However, the identification and function of CD1c+ DC in the IA synovium has yet to be fully elucidated.Objectives:To investigate if CD1c+DC reside in the IA synovium and ascertain if they represent a unique population, distinct from peripheral CD1c+DC and if they contribute to synovial inflammation.Methods:Synovial tissue (ST) biopsies and synovial fluid mononuclear cells (SFMC) were obtained via arthroscopy and healthy control (HC) ST was obtained during ACL surgery. Synovial tissue single cells suspensions were generated following enzymatic and mechanical digestion. Single cell analysis of synovial tissue cell suspensions, along with PBMC and SFMC was performed by multicolour flow cytometry. CD1c+DC were sorted from IA synovial fluid and peripheral blood and bulk RNA sequencing was performed. CD1c+DC functionality and maturation was assessed using OVA DQ phagocytosis assays, multiplex ELISA and DC: T cell cocultures.Results:Within the circulation the frequency of CD1c+DC are significantly decreased in IA peripheral blood compared to HC (p<0.01) in addition to expressing significantly higher levels of the maturation markers CD80 (p<0.01) and CD40 (p=0.08). IA peripheral blood DC also express significantly higher levels of CXCR3 (p<0.01) and CCR7 (p<0.05) compared to HC - suggestive of DC migration from the periphery to the synovium. Following RNA-seq analysis, IPA and differentially expressed gene (DEG) analysis revealed an enrichment in genes involved in DC maturation, TLR signalling and chemokine signalling in IA peripheral blood compared to HC. In support of the hypothesis that DC migrate and accumulate in the IA synovium, CD1c+ DC were identified in IA ST and were significantly enriched compared to IA peripheral blood (p<0.01). IA ST CD1c+DC express significantly higher levels of the activation marker CD80 compared to IA peripheral blood (p<0.05) or HC ST (p<0.05). Upon examination of IA synovial fluid, we report similar findings to ST, whereby CD1c+DC are enriched in synovial fluid compared to PB (p<0.001). Moreover, RNA sequencing and PCA analysis of synovial versus blood CD1c+DC revealed distinct transcriptional variation between both sites. Functionally, synovial CD1c+DC express higher levels of the maturation markers CD80, CD83, CD40, PD-L1 and BTLA (all p<0.05) and have distinct coexpression of these maturation markers which is unique to the synovium. Synovial CD1c+DC are less phagocytic compared to peripheral blood DC, have decreased production of MMP1 and MMP9 and importantly are still capable of additional activation in-vitro. Finally, synovial CD1c+DC induce the proinflammatory cytokines TNFα, GMCSF, IL-17a and IFNγ from CD4+ T-cells in allogeneic DC: T cells cocultures.Conclusion:Mature circulatory CD1c+DC migrate and accumulate in the IA synovium. Synovial DC are present in the IA synovium in a mature state, have distinct tissue specific characteristics and can induce proinflammatory CD4+T cell responses.Acknowledgements:We would like to thank all the patients who contributed to this studyDisclosure of Interests:Mary Canavan: None declared, Viviana Marzaioli: None declared, Vipul Bhargava Employee of: Janssen Research and Development, Sunil Nagpal Employee of: Janssen Research and Development, Phil Gallagher: None declared, Conor Hurson: None declared, Ronan Mullan: None declared, Douglas Veale Speakers bureau: Abbvie, Janssen, Novartis, Pfizer, MSD, UCB, Consultant of: Abbvie, Janssen, Novartis, Pfizer, MSD, UCB, Grant/research support from: Pfizer, Janssen, AbbVie, UCB, Ursula Fearon Speakers bureau: Abbvie, Grant/research support from: Pfizer, Janssen, Abbvie, UCB