Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University School of Medicine, Detroit, Michigan, USA.
J Virol. 2022 Sep 14;96(17):e0101422. doi: 10.1128/jvi.01014-22. Epub 2022 Aug 10.
Reactive oxygen species (ROS) play an important role in tissue inflammation. In this study, we measured the intracellular level of ROS in herpes stromal keratitis (HSK) corneas and determined the outcome of manipulating ROS level on HSK severity. Our results showed the predominance of ROS generation in neutrophils but not CD4 T cells in HSK corneas. NADPH oxidase 2 (NOX2) enzyme is known to generate ROS in myeloid cells. Our results showed baseline expression of different NOX2 subunits in uninfected corneas. After corneal herpes simplex virus-1 (HSV-1) infection, an enhanced expression of NOX2 subunits was detected in infected corneas. Furthermore, flow cytometry results showed a higher level of gp91 (Nox2 subunit) protein in neutrophils from HSK corneas, suggesting the involvement of NOX2 in generating ROS. However, no significant decrease in ROS level was noticed in neutrophils from HSV-1-infected gp91 mice than in C57BL/6J (B6) mice, suggesting NOX2 is not the major contributor in generating ROS in neutrophils. Next, we used diphenyleneiodonium (DPI), a flavoenzyme inhibitor, to pharmacologically manipulate the ROS levels in HSV-1-infected mice. Surprisingly, the neutrophils from peripheral blood and corneas of the DPI-treated group exhibited an increased level of ROS than the vehicle-treated group of infected B6 mice. Excessive ROS is known to cause cell death. Accordingly, DPI treatment resulted in a significant decrease in neutrophil frequency in peripheral blood and corneas of infected mice and was associated with reduced corneal pathology. Together, our results suggest that regulating ROS levels in neutrophils can ameliorate HSK severity. Neutrophils are one of the primary immune cell types involved in causing tissue damage after corneal HSV-1 infection. This study demonstrates that intracellular ROS production in the neutrophils in HSK lesions is not NOX2 dependent. Furthermore, manipulating ROS levels in neutrophils ameliorates the severity of HSK lesions. Our findings suggest that excessive intracellular ROS in neutrophils disrupt redox homeostasis and affect their survival, resulting in a decrease in HSK lesion severity.
活性氧（ROS）在组织炎症中发挥重要作用。在这项研究中，我们测量了单纯疱疹性基质角膜炎（HSK）角膜中的细胞内 ROS 水平，并确定了操纵 ROS 水平对 HSK 严重程度的影响。我们的结果表明，HSK 角膜中的中性粒细胞而不是 CD4 T 细胞中 ROS 的产生占优势。NADPH 氧化酶 2（NOX2）酶已知在髓样细胞中产生 ROS。我们的结果显示，未感染角膜中存在不同 NOX2 亚基的基线表达。单纯疱疹病毒-1（HSV-1）感染后，感染角膜中检测到 NOX2 亚基的表达增强。此外，流式细胞术结果显示，HSK 角膜中的中性粒细胞 gp91（Nox2 亚基）蛋白水平更高，表明 NOX2 参与了 ROS 的产生。然而，与 C57BL/6J（B6）小鼠相比，HSV-1 感染的 gp91 小鼠中性粒细胞中的 ROS 水平并没有明显降低，这表明 NOX2 不是中性粒细胞中产生 ROS 的主要贡献者。接下来，我们使用二苯碘（DPI），一种黄素酶抑制剂，在 HSV-1 感染的小鼠中对 ROS 水平进行药理操纵。令人惊讶的是，与感染 B6 小鼠的载体处理组相比，DPI 处理组外周血和角膜中的中性粒细胞的 ROS 水平升高。过量的 ROS 已知会导致细胞死亡。因此，DPI 处理导致感染小鼠外周血和角膜中的中性粒细胞频率显著降低，并与角膜病变减轻相关。总之，我们的结果表明，调节中性粒细胞中的 ROS 水平可以改善 HSK 的严重程度。中性粒细胞是角膜 HSV-1 感染后导致组织损伤的主要免疫细胞类型之一。这项研究表明，HSK 病变中的中性粒细胞中细胞内 ROS 的产生不依赖于 NOX2。此外，操纵中性粒细胞中的 ROS 水平可改善 HSK 病变的严重程度。我们的研究结果表明，中性粒细胞中过多的细胞内 ROS 破坏了氧化还原平衡并影响其存活，从而导致 HSK 病变严重程度降低。
