Differences in responses of normal and rheumatoid arthritis peripheral blood t cells to synovial fluid and peripheral blood dendritic cells in allogeneic mixed leukocyte reactions

Background:Rheumatoid arthritis (RA) is an autoimmune disorder in which Th17 cells, B cells and inflammatory cytokines (1-3) contribute to joint tissue damage, however the role of specific myeloid populations to immunopathogenesis of RA remains unclear.Objectives:To address this question, we studied transcriptional, phenotypical and functional characteristics of monocytes (Mo), CD1c+ and CD141+ conventional dendritic cells (cDC) from RA patients.Methods:Frequencies and maturation patterns of Lin-CD14-HLADR+ plasmacytoid (CD11c-), CD1c+ and CD141+ cDC (CD11c+) subsets and CD14+ Mo from n=25 RA patients at baseline were analyzed by multicolor flow cytometry. In addition, longitudinal studies on the evolution of these populations after treatment initiation were conducted on a smaller group of RA patients. Moreover, CD1c+ and CD141+ cDC subsets and total Mo were sorted from the peripheral blood from n=4 untreated RA and healthy individuals and the synovial fluid from n=3 RA and chondrocalcinosis patients. Differential transcriptional patterns within each population were analyzed by RNAseq. Functional validation of targets were performed in vitro with cDC subsets isolated form the synoviual fluid of RA patients. Finally, silencing of expression of NLRC4 and NLRP3 on CD1c+cDCs was performed with specific siRNAs.Results:Both CD1c+ (p=0.0001) and CD141+ (p=0.0008) cDCs were significantly depleted from the blood and enriched in the synovial fluid from untreated RA patients, but proportions of CD1c+ cDCs were more significantly recovered after treatment initiation and associated with improved clinical parameters. In addition, specific increased expression levels of the IgG-Fc receptor CD64 on CD1c+ cDC was associated with higher DAS28 (p=0.0002). Moreover, differential transcriptional patterns of circulating CD1c+cDCs from RA patients were characterized by genes linked to toll-like receptor, Fc-receptor, inflammasome pathways and elevated CCR2 expression (p=0.016), while CD141+cDCs transcribed interferon-related genes. Importantly, CCR2+ CD64Hi CD1c+cDCs from the synovial fluid from RA patients transcribed proinflammatory cytokines such as IL1-β, CCL3 and IL-8, actively expressed the inflammasome mediator caspase 1 and were more effective activating pathogenic IFNγ+IL-17+ CD4+ T cells in vitro than CD141+ cDC (p=0.0019). These functional profiles could be artificially induced stimulating CD1c+ cDCs with dsDNA in the presence of IgGs and was dependent on caspase 1 and the NLRC4 inflammasome.Conclusion:Our data provides novel insights about specific activation and functional patterns on CD1c+cDC contributing to RA pathogenesis and identifies new sensors that could represent novel therapeutic target to treat RA.References:[1]Alvandpur N, Tabatabaei R, Tahamoli-Roudsari A, Basiri Z, Behzad M, Rezaeepoor M, et al. Circulating IFN-gamma producing CD4+ T cells and IL-17A producing CD4+ T cells, HLA-shared epitope and ACPA may characterize the clinical response to therapy in rheumatoid arthritis patients. Human immunology. 2020.[2]Nistala K, Adams S, Cambrook H, Ursu S, Olivito B, de Jager W, et al. Th17 plasticity in human autoimmune arthritis is driven by the inflammatory environment. Proceedings of the National Academy of Sciences of the United States of America. 2010;107(33):14751-6.[3]Chapuy-Regaud S, Nogueira L, Clavel C, Sebbag M, Vincent C, Serre G. IgG subclass distribution of the rheumatoid arthritis-specific autoantibodies to citrullinated fibrin. Clinical and experimental immunology. 2005;139(3):542-50.Disclosure of Interests:None declared

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AB0018 ACCUMULATION OF FUNCTIONALLY MATURE CD1C+ DENDRITIC CELLS CONTRIBUTES TO SYNOVIAL INFLAMMATION IN INFLAMMATORY ARTHRITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.1584 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 1043.2-1043

Author(s):

M. Canavan ◽

V. Marzaioli ◽

V. Bhargava ◽

S. Nagpal ◽

P. Gallagher ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Synovial Fluid ◽

Research And Development ◽

Rna Sequencing ◽

Synovial Tissue ◽

Peripheral Blood ◽

Synovial Inflammation ◽

Research Support

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

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