ABSTRACT: BACKGROUND:Pathological stimuli cause mitochondrial damage and leakage of mitochondrial DNA (mtDNA) into the cytosol, as demonstrated in many cell types. The cytosolic mtDNA then drives the activation of noninfectious inflammation. Retinal microvascular endothelial cells (RMECs) play an important role in the inner endothelial blood-retinal barrier (BRB). RMEC dysfunction frequently occurs in posterior-segment eye diseases, causing loss of vision. In this study, we investigated the involvement of cytosolic mtDNA in noninfectious immune inflammation in RMECs under pathological stimuli. METHODS:RMECs were stimulated with 100?ng/ml lipopolysaccharide (LPS), 200??M hydrogen peroxide (H2O2), or 25?mM D-glucose. After 24?h, immunofluorescent staining was used to detect the opening of the mitochondrial permeability transition pore (MPTP). Cytosolic mtDNA was detected with immunofluorescent staining and PCR after stimulation. mtDNA was then isolated and used to transfect RMECs in vitro, and the protein levels of cGAS were evaluated with western blotting. Real-time PCR was used to examine cGAS mRNA expression levels at different time points after mtDNA stimulation. The activation of STING was detected with immunofluorescent staining 6?h after mtDNA stimulation. Western blotting was used to determine the expression of STING and IFN?, the phosphorylation status of TBK1, IRF3, and nuclear factor-?B (NF-?B) P65, and the nuclear translocation of IRF3 and NF-?B P65 at 0, 3, 6, 12, and 24?h. The mRNA expression of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were determined with real-time PCR, together with the concentrations of intercellular adhesion molecule 1 (ICAM-1) mRNA. RESULTS:Pathological stimuli caused mtDNA to leak into the cytosol by opening the MPTP in RMECs after 24?h. Cytosolic mtDNA regulated the expression of cGAS and the distribution of STING in RMECs. It promoted ICAM-1, STING and IFN? expression, TBK1, IRF3, and NF-?B phosphorylation and the nuclear translocation in RMECs at 12 and 24?h after its transfection. The mRNAs of proinflammatory cytokines CCL4, CXCL10, and IFNB1, and transcription factor IRF1 were significantly elevated at 12 and 24?h after mtDNA stimulation. CONCLUSIONS:Pathological stimulation induces mtDNA escape into the cytosol of RMECs. This cytoplasmic mtDNA is recognized by the DNA sensor cGAS, increasing the expression of inflammatory cytokines through the STING-TBK1 signaling pathway. Video Abstract. (MP4 37490 kb).
