POS1010 MACROPHAGE EXTRACELLULAR TRAPS PROMOTE FIBROBLAST-LIKE SYNOVIOCYTES PROLIFERATION AND PROINFLAMMATORY CYTOKINE IN RHEUMATOID ARTHRITIS

Background Macrophage Extracellular Traps (METs) play an important role in the promotion of tissue injury, inflammation progression and autoimmune diseases. Objectives This study aims to investigate the effects of METs on the proliferation and expression of proinflammatory cytokines in fibroblast-like synoviocytes (FLS) in patients with rheumatoid arthritis (RA). Methods Synovial tissues of RA patients and traumatic controls undergoing joint replacement in our hospital were collected, and FLS of RA patients were cultured in vitro. Peripheral blood mononuclear cells (PBMC) were isolated from RA patients and differentiated into macrophages by M-CSF. The macrophages were co-cultured with serum of RA patients and healthy controls. SYTOX green, which stained the DNA released from cells and was a common method for detecting ETs, was added to observe the formation of METs. The expressions of CD68 and CitH3 in synovium were detected by immunofluorescence. METs were isolated and purified from THP-1 derived macrophages and co-incubated with FLS. The RNA expressions of proinflammatory cytokines TNF-a and IL-1β of RA-FLSs were detected by qPCR. The proliferation ability of RA-FLSs was detected by CCK-8 assay. Results We found the enrichment of macrophages labeled with CD68 and CitH3 in RA synovial tissues but not in traumatic controls. Immunofluorescence co-localization assay displayed that most CitH3 were distributed around CD68, suggesting that macrophages may be the main source of ETs (Figure 1A). PBMC induced macrophages co-incubated with serum from RA patients showed the formation of strip-like METs, while no obvious METs were observed with serum from healthy controls (Figure 1B). Purified METs were isolated and co-incubated with RA-FLSs for 48h. TNF-a and IL-1β were significantly overexpressed, and the proliferation of RA-FLSs was promoted (Figure 1C-D). Conclusion METs were detected in RA synovium but not in traumatic controls. The autoantibodies or inflammatory cytokines presented in the serum or RA patients may be associated with the increased METs formation in RA. In vitro experiments, METs could promote the proliferation and proinflammatory cytokines expression of RA-FLSs, suggesting that clearing or blocking the formation of METs may be a new therapeutic target for RA. Figure 1. A, Immunofluorescence of synovium in RA and traumatic controls. Immunofluorescence showed the areas stained with anti-CD68 (red), anti-CitH3 (green) and DAPI (blue). RA: rheumatoid arthritis, ST: synovial tissue, TC: traumatic controls. B Formation of METs in human monocyte-derived macrophages with serum SYTOX green showed the formation of METs in macrophage with RA-serum. SYTOX green (green) and Hoechst 33342 (blue). C-D The promotion of METs in biologic behaviors of RA-FLSs. METs could promote the proliferation (C) and proinflammatory cytokines expression (D) of RA-FLSs. Acknowledgements This work was supported by the Scientific and Technological Planning Project of Guangzhou City [202102020150], Guangdong Provincial Basic and Applied Basic Research Fund Project [2021A1515111172], National Natural Science Foundation of China Youth Fund [82201998], Third Affiliated Hospital of Sun Yat-Sen University Cultivating Special Fund Project for the National Natural Science Foundation of China [2022GZRPYQN01] and National Natural Science Foundation of China [82271849]. Disclosure of Interests None Declared.