Accumulation of cytosolic dsDNA contributes to fibroblast-like synoviocytes-mediated rheumatoid arthritis synovial inflammation
Accumulation of cytosolic dsDNA contributes to fibroblast-like synoviocytes-mediated rheumatoid arthritis synovial inflammation
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胞浆 dsDNA 的积累导致成纤维细胞样滑膜细胞介导的类风湿性关节炎滑膜炎症
DOI：
10.1016/j.intimp.2019.105791
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发表时间：
2019-11-01
影响因子：
5.6
通讯作者：
Xiao, Youjun
中科院分区：
文献类型：
--
作者：
Wang, Jingnan;Li, Ruiru;Xiao, Youjun
胞质dsDNA的积累在细胞过程的调控中起着重要作用。然而，胞质dsDNA是否参与类风湿关节炎（RA）的发病机制尚不清楚。因此，本研究探讨了胞浆dsDNA在类风湿关节炎患者成纤维细胞样滑膜细胞（FLS）炎症反应调节中的作用。FLS来自活动期RA患者。采用免疫荧光染色和量子比特(R) dsDNA HS法检测细胞质中dsDNA的积累。免疫组化检测滑膜dsDNA和cGMP-AMP合成酶（cGAS）的表达。利用短发夹RNA （shRNA）敲低cGAS和干扰素刺激因子基因（STING）的表达。Western blotting和免疫荧光染色检测蛋白表达。我们观察到在RA患者的FLS和滑膜中细胞质dsDNA和cGAS表达增加。dsDNA和cGAS的表达与类风湿滑膜炎的严重程度相关。将dsDNA转染到RA FLS细胞质中可促进促炎细胞因子的产生。DNaseII过表达下调胞内dsDNA表达，抑制dsDNA诱导的细胞因子分泌。我们还发现，dsDNA和tnf - α增强了cGAS和STING的表达，而ddna诱导的细胞因子分泌被cGAS或STING敲低而减少。此外，我们发现ddna诱导的IRF3和NF-kappa Bp65磷酸化通过DNaseII过表达或cGAS/STING敲低而降低。总体而言，我们的研究结果表明，在RA FLS中，胞质dsDNA水平的增加通过cGAS/STING途径促进了炎症反应，这表明胞质dsDNA积累是FLS介导的类风湿性滑膜炎症的重要因素。
The accumulation of cytosolic dsDNA plays important roles in the regulation of cellular processes. However, whether cytosolic dsDNA is involved in the pathogenesis of rheumatoid arthritis (RA) is not clear. Therefore, the present study investigated the roles of cytosolic dsDNA in the modulation of inflammatory responses of fibroblast-like synoviocytes (FLS) in patients with RA. FLS were obtained from active RA patients. dsDNA accumulation in the cytosol was detected by immunofluorescence staining and the Qubit (R) dsDNA HS Assay. Immunohistochemistry was employed to detect the dsDNA and cGMP-AMP synthase (cGAS) expression in the synovium. Short hairpin RNA (shRNA) was used to knockdown the expression of cGAS and stimulator of interferon genes (STING). Protein expression was detected by Western blotting and immunofluorescence staining. We observed increased cytosolic dsDNA and cGAS expression in FLS and synovium from RA patients. dsDNA and cGAS expression correlated with the severity of rheumatoid synovitis. Transfection of dsDNA into the cytosol of RA FLS promoted pro-inflammatory cytokines production. DNaseII overexpression downregulated cytosolic dsDNA expression and inhibited dsDNA-induced cytokines secretion. We also found that dsDNA and TNF-alpha enhanced cGAS and STING expression, and dsDNA-induced cytokine secretion was reduced by cGAS or STING knockdown. Furthermore, we determined that the dsDNA-induced phosphorylation of IRF3 and NF-kappa Bp65 was decreased by DNaseII overexpression or cGAS/STING knockdown. Overall, our findings show that increased cytosolic dsDNA level promoted inflammatory responses via the cGAS/STING pathway in RA FLS, which suggests that cytosolic dsDNA accumulation is an important contributor to FLS-mediated rheumatoid synovial inflammation.