Table_1_E3 ligase RNF5 inhibits type I interferon response in herpes simplex virus keratitis through the STING/IRF3 signaling pathway.xlsx

Herpes simplex keratitis (HSK), caused by the herpes simplex virus 1 (HSV-1), is a major blinding disease in developed countries. HSV-1 can remain latent in the host for life and cannot be eradicated. The infection causes the secretion of various cytokines and aggregation of inflammatory cells. In the early stage of inflammation, mainly neutrophils infiltrate the cornea, and CD4+ T cells mediate the immunopathological changes in herpetic stromal keratitis in the subsequent progression. The STING/IRF3-mediated type I interferon (IFN) response can effectively inhibit viral replication and control infection, but the activity of STING is affected by various ubiquitination modifications. In this study, we found that the expression of RNF5 was elevated in corneal tissues and corneal epithelial cells after infection with HSV-1. Immunofluorescence staining confirmed that RNF5 was mainly expressed in the corneal epithelial layer. We silenced and overexpressed RNF5 expression in corneal epithelial cells and then inoculated them with HSV-1. We found that the expressions of STING, p-IRF3, p-TBK1, and IFN-β mRNA increased after RNF5 silencing. The opposite results were obtained after RNF5 overexpression. We also used siRNA to silence RNF5 in the mouse cornea and then established the HSK model. Compared with the siRNA-control group, the siRNA-RNF5 group showed significantly improved corneal inflammation, reduced clinical scores and tear virus titers, and significantly increased corneal IFN-β expression. In addition, the expressions of the proinflammatory cytokines IL-6 and TNF-α in the corneal tissue were significantly decreased, indicating that RNF5 silencing could effectively promote IFN-I expression, inhibit virus replication, alleviate inflammation, and reduce corneal inflammatory damage. In summary, our results suggest that RNF5 limits the type I IFN antiviral response in HSV corneal epithelitis by inhibiting STING/IRF3 signaling.

单纯疱疹性角膜炎（Herpes simplex keratitis, HSK）由单纯疱疹病毒1型（herpes simplex virus 1, HSV-1）引发，是发达国家境内主要的致盲性眼病。HSV-1可在宿主体内终身潜伏，且无法被彻底清除。该病毒感染会促使多种细胞因子分泌，并引发炎症细胞聚集。在炎症早期，角膜主要以中性粒细胞浸润为特征，而CD4+ T细胞会在疱疹性基质性角膜炎的后续进展过程中，介导免疫病理变化。STING/IRF3介导的I型干扰素（type I interferon, IFN）应答可有效抑制病毒复制、控制感染，但STING的活性会受到多种泛素化修饰的影响。本研究中，我们发现HSV-1感染后，角膜组织与角膜上皮细胞内的RNF5表达水平均升高。免疫荧光染色实验证实，RNF5主要表达于角膜上皮层。我们在角膜上皮细胞中分别沉默与过表达RNF5，随后用HSV-1接种细胞，结果显示：沉默RNF5后，STING、磷酸化IRF3（p-IRF3）、磷酸化TBK1（p-TBK1）以及干扰素β（IFN-β）mRNA的表达量均有所升高；而过表达RNF5则得到相反的实验结果。我们还采用小干扰RNA（siRNA）沉默小鼠角膜内的RNF5表达，随后构建HSK感染模型。与siRNA阴性对照组相比，siRNA-RNF5组小鼠的角膜炎症症状显著减轻，临床评分与泪液病毒滴度均有所降低，角膜内IFN-β的表达水平则显著升高。此外，角膜组织内的白细胞介素6（IL-6）与肿瘤坏死因子α（TNF-α）的表达量均明显下降，这表明沉默RNF5可有效促进I型干扰素的表达、抑制病毒复制、减轻炎症反应并缓解角膜炎性损伤。综上，本研究结果表明，RNF5可通过抑制STING/IRF3信号通路，限制HSV感染引发的角膜上皮炎中的I型干扰素抗病毒应答。