Plasma-derived Extracellular Vesicles Induced STING-mediated Proinflammatory Effects in Dermatomyositis.

Summary: Background/Purpose: Dermatomyositis (DM) is an acquired inflammatory myopathy characterized by chronic skin inflammation. The pathogenesis of DM is still unclear. Extracellular vesicles (EVs) are lipid bilayer membrane vesicles existing in various bodily fluids and implicated in the pathogenesis of autoimmune diseases. As type I interferons, specifically IFN-b, are uniquely elevated in DM, and Stimulator of interferon genes (STING) works as a critical sensor and adaptor in type I IFN signaling, we hypothesized that EVs derived from DM patients’ plasma might trigger STING-mediated proinflammatory effects. Methods: DM patients were recruited in the dermatology clinic at University of Pennsylvania. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient. EVs derived from plasma were isolated via ultracentrifugation. The supernatant was harvested for ELISA and the lysed cells were collected for Western blot after HC-derived PBMCs were stimulated by EVs. Results: Here we found that DM patients’ plasma derived EVs triggered cytokine release (IFNb: (30.24±0.65) vs control (2.683±0.35); TNFα: (1451±98.40) vs control (16.75±1.407); IL6: (945.0±57.40) vs control (0.0±0.0)pg/mL; n=6) with STING phosphorylation. Inhibition of STING significantly attenuated DM patients’ plasma-derived EVs-triggered cytokine production (IFNb: (21.58±2.22) vs (28.34±1.73); TNFα: (434.8±94.50) vs (919.1±133.0); IL6: (611.5±54.22) vs (844.2±73.60)pg/mL; n=6) via suppressing STING and its down-stream signal TBK1, IRF3, and NFκB phosphorylation. Besides, TBK1 inhibitors Amlexanox and MRT67307 also suppressed DM patients’ plasma derived EVs-induced IFNb release by inhibiting TBK1 phosphorylation. To further explore whether STING signaling pathway activation and the proinflammatory effects were caused by EVs-captured dsDNA, EVs were pretreated with Triton X-100 and DNase I to digest DNA. Triton X-100 and DNase pretreatment decreased EVs-triggered cytokine release (IFNb: (4.113±2.08) vs (28.94±5.473); TNFα: (19.00±19.00) vs (1361±293.6); IL6: (210.7±103.6) vs (1020±43.86)pg/mL; n=3-6) and STING activation. To specifically digest dsDNA, EVs were also pretreated with dsDNase. Triton X-100 and dsDNase pretreatment also decreased EVs-triggered cytokine release (IFNb: (1.893±1.893) vs(28.94±5.473); TNFα: (290.3±57.03) vs (1361±293.6); IL6: (617.6±127.4) vs (1020±43.86)pg/mL; n=3-6)and STING activation.. Conclusion: EVs derived from plasma trigger STING-mediated proinflammatory effects in DM. The STING signaling pathway is activated during EVs triggering of proinflammatory effects and was at least partially mediated by dsDNA captured by EVs. Targeting STING pathway might provide insight into a potential therapeutic approach for DM.