类风湿性关节炎（RA）是一种以滑膜增生和骨破坏为特征的自身免疫性疾病。巨噬细胞外细胞陷阱（METs）在各种刺激下从巨噬细胞释放，并可能形成稳定的自身抗原-DNA复合物，加重自身抗体产生和自身免疫反应。我们旨在研究METs对RA-FLSs生物学行为的影响。RA患者的滑膜组织和类纤维样滑膜细胞（FLSs）被获取。应用免疫荧光、免疫组化和SYTOX Green染色检测滑膜和滑膜液中的ETs。用CCK-8、伤口愈合实验、Transwell实验和定量实时PCR（qPCR）检测RA-FLSs的细胞存活率、迁移、侵袭和细胞因子表达。进行RNA测序分析以探索其潜在机制，并使用免疫印迹验证其活跃的信号通路。我们发现，RA中ETs的形成丰富，并与抗CCP呈正相关。经纯化的METs刺激后，RA-FLSs表现出明显的促进肿瘤样生物学行为。RA-FLSs中的DNA传感器cGAS在METs刺激后被激活。RNA测序显示差异基因在PI3K/Akt信号通路中显著富集，cGAS抑制剂RU.521可有效逆转METs处理的RA-FLSs的肿瘤样生物学行为促进作用，并下调PI3K/Akt的激活。总之，我们的研究表明，METs通过cGAS介导的PI3K/Akt信号通路的激活，促进了RA-FLSs的病理生物学行为。这些发现为RA的发病机制和巨噬细胞调节RA-FLSs肿瘤样行为的机制提供了新的见解。© 2023年作者。由牛津大学出版社代表白细胞生物学学会出版。版权所有。如需授权，请发送电子邮件至：journals.permissions@oup.com。

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovium hyperplasia and bone destruction. Macrophage extracellular traps (METs) are released from macrophages under various stimulus and may generate stable autoantigen-DNA complexes, aggravate autoantibodies generation and autoimmune responses. We aimed to investigate the role of METs on the biologic behaviors of RA-FLSs. Synovial tissues and fibroblast-like synoviocytes (FLSs) were obtained from RA patients. ETs in synovium and synovial fluids were detected by immunofluorescence, immunohistochemistry and SYTOX Green staining. Cell viability, migration, invasion, and cytokines expression of RA-FLSs were assessed by CCK-8, wound healing assay, Transwell assays and quantitative real-time PCR (qPCR) respectively. RNA-sequencing analysis was performed to explore the underlying mechanism and Western blot was used to validate the active signaling pathways. We found that ETs formation was abundant in RA and positively correlated to anti-CCP. RA-FLSs stimulated with purified METs, demonstrated the obvious promotion in tumor-like biologic behaviors. The DNA sensor cGAS in RA-FLSs was activated after METs stimuli. RNA sequencing revealed that differential genes were significantly enriched in PI3K/Akt signaling pathway and cGAS inhibitor RU.521 effectively reversed the promotion of tumor-like biologic behaviors in METs treated RA-FLSs and downregulated the PI3K/Akt activation. In summary, our study demonstrates that METs promote the pathogenically biological behaviors of RA-FLSs through cGAS-mediated activation of PI3K/Akt signaling pathway. These findings provide a novel insight into the pathogenesis of RA and the mechanisms of macrophages in modulating RA-FLSs tumor-like behaviors.© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.