Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, 200031, China.
Eye Institute, Eye and ENT Hospital of Fudan University, Shanghai, 200031, China.
Cell Commun Signal. 2020 Oct 28;18(1):172. doi: 10.1186/s12964-020-00637-3.
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.
RMECs were stimulated with 100 ng/ml lipopolysaccharide (LPS), 200 μM hydrogen peroxide (HO), 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 IFNβ, the phosphorylation status of TBK1, IRF3, and nuclear factor-κB (NF-κB) P65, and the nuclear translocation of IRF3 and NF-κB 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.
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 IFNβ expression, TBK1, IRF3, and NF-κB 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.
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).
在许多细胞类型中，病理刺激会导致线粒体损伤和线粒体 DNA（mtDNA）漏出线粒体进入细胞质，这一点已经得到证实。然后，细胞质中的 mtDNA 会驱动非传染性炎症的激活。视网膜微血管内皮细胞（RMECs）在血管内内皮血视网膜屏障（BRB）中发挥重要作用。RMEC 功能障碍经常发生在后部眼疾中，导致视力丧失。在这项研究中，我们研究了在病理刺激下细胞质 mtDNA 在 RMECs 中的非传染性免疫炎症中的作用。
用 100ng/ml 脂多糖（LPS）、200μM 过氧化氢（HO）或 25mM D-葡萄糖刺激 RMECs。刺激 24 小时后，用免疫荧光染色检测线粒体通透性转换孔（MPTP）的开放情况。刺激后用免疫荧光染色和 PCR 检测细胞质 mtDNA。然后提取 mtDNA 并在体外转染 RMECs，用 Western blot 检测 cGAS 蛋白水平。用实时 PCR 检测 mtDNA 刺激不同时间点 cGAS mRNA 表达水平。mtDNA 刺激 6 小时后用免疫荧光染色检测 STING 的激活。用 Western blot 检测 STING 和 IFNβ的表达、TBK1、IRF3 和核因子-κB（NF-κB）P65 的磷酸化状态以及 IRF3 和 NF-κB P65 的核转位情况，0、3、6、12 和 24 小时。用实时 PCR 检测促炎细胞因子 CCL4、CXCL10 和 IFNB1 的 mRNA 表达以及转录因子 IRF1 的表达，同时检测细胞间黏附分子 1（ICAM-1）mRNA 的浓度。
病理刺激后 24 小时，RMECs 中 MPTP 的打开导致 mtDNA 漏出线粒体进入细胞质。细胞质 mtDNA 调节 RMECs 中 cGAS 和 STING 的分布。它在转染后 12 和 24 小时促进 RMECs 中 ICAM-1、STING 和 IFNβ 的表达、TBK1、IRF3 和 NF-κB 的磷酸化和核转位。mtDNA 刺激后 12 和 24 小时，促炎细胞因子 CCL4、CXCL10 和 IFNB1 的 mRNA 和转录因子 IRF1 的表达明显升高。
病理刺激诱导 RMECs 中线粒体 DNA 逃逸到细胞质中。这种细胞质 mtDNA 被 DNA 传感器 cGAS 识别，通过 STING-TBK1 信号通路增加炎症细胞因子的表达。
