Interplay between hypoxia and oxidative phosphorylation in herpes stromal keratitis

疱疹间质角膜炎(HSK)，一种因复发而发展的角膜慢性炎症性疾病 角膜单纯疱疹病毒1型感染，可造成永久性疤痕和视力丧失。慢性炎症 由于缺乏血管，血液供应减少，新陈代谢高，常使组织缺氧 免疫细胞渗入的需求。缺氧的发展导致缺氧诱导因子的稳定 转录因子(HIF)，增强糖酵解调节基因的表达，从而促进 糖酵解代谢。然而，阻断HIF信号可能会促进氧化磷酸化以维持 炎症组织中炎性细胞的能量需求，提示缺氧和氧化之间的相互作用 磷酸化以防止持续炎症的消退。我们的初步结果显示， 单纯疱疹病毒性角膜炎角膜低氧的发生。此外，低氧的发展与 单纯疱疹病毒1型感染角膜中性粒细胞的分布。当低氧相关信号通路PCR阵列和 对进展期HSK皮损进行RT-qPCR，我们检测到编码基因的表达上调 关键的糖酵解酶，包括感染的角膜中的PFKFB3。PFKFB3是一种糖酵解激活剂，据报道 以促进血管生成。我们还发现乳酸转运蛋白MCT4和MCT4的表达水平增加 单纯疱疹病毒性角膜炎(HSK)角膜中的MCT1。RT-qPCR结果经共聚焦显微镜和流式细胞仪证实。我们的 结果还显示，HIF-2在上皮细胞中有核定位，HIF-1在浸润性中性粒细胞和 HSK角膜中的CD4T细胞。有趣的是，在感染HSV-1的患者中，在使用吖黄素的同时，阻断HIF二聚化 小鼠角膜混浊程度加重，但血管生成减少。我们的初步结果和 支持已发表的证据一起使我们假设HSK病变中的中性粒细胞塑造了 缺氧导致HIF调节的糖酵解代谢促进HSK的流行 HIF的发病机制和阻滞剂，当与线粒体呼吸或 活性氧(ROS)可减轻HSK的严重程度。提出了三个目标来检验这一点 假设。目的1验证HSK病变中中性粒细胞形成缺氧的假说，以及 低氧可加重HSK的严重程度。目标2将检验以下假设：抑制糖酵解激活剂 PFKFB3和乳酸转运蛋白MCT4和MCT1蛋白可减轻HSK的严重程度。目标3将测试 假设HIF阻滞剂与线粒体呼吸或ROS抑制剂联合使用将减少 HSK的严重程度。根据这些研究的结果，治疗HSK病变的新治疗方法 可能被开发出来。从该应用程序中获得的知识将提供创新信息，这些信息可能 适用于其他眼部感染性疾病模型。

Herpes stromal keratitis (HSK), a corneal chronic inflammatory condition that develops in response to recurrent corneal herpes simplex virus-1 infection, can cause permanent scarring and vision loss. Chronic inflammation often makes tissue hypoxic due to the lack of the blood vessel, reduced supply of blood, and a high metabolic demand of infiltrating immune cells. Development of hypoxia leads to the stabilization of hypoxia inducible factor (HIF), a transcription factor, which enhances the expression of glycolysis regulating genes and thereby promotes glycolytic metabolism. However, blocking of HIF signaling may promote oxidative phosphorylation to sustain the energy demand of inflammatory cells in inflamed tissue, suggesting an interplay between hypoxia and oxidative phosphorylation to prevent the resolution of an ongoing inflammation. Our preliminary results showed the development of hypoxia in corneas with HSK. Furthermore, the development of hypoxia was linked with the extent of neutrophils in HSV-1 infected corneas. When hypoxia associated signaling pathway PCR array and RT-qPCR were carried out on progressing HSK lesions, we detected an elevated expression of genes encoding key glycolytic enzymes, including PFKFB3 in infected corneas. PFKFB3 is a glycolytic activator, which is reported to enhance hemangiogenesis. We also found an increased level of expression of lactate transporters, MCT4 and MCT1, in corneas with HSK. RT-qPCR results were confirmed with confocal microscopy and flow cytometry. Our results also showed the nuclear localization of HIF-2 in epithelial cells, and HIF-1 in infiltrating neutrophils and CD4 T cells in HSK corneas. Interestingly, blocking of HIF dimerization, while using acriflavine, in HSV-1 infected mice exacerbated the corneal opacity, but decreased the hemangiogenesis. Our preliminary results and supportive published evidence together led us to hypothesize that neutrophils in HSK lesion shape the development of hypoxia resulting in the prevalence of HIF-regulated glycolytic metabolism that promotes HSK pathogenesis, and blockers of HIF, when given in association with inhibitors of mitochondrial respiration or reactive oxygen species (ROS) should reduce the severity of HSK. Three aims are proposed to test this hypothesis. Aim 1 will test the hypothesis that neutrophils in HSK lesion shape the development of hypoxia, and hypoxia enhances the severity of HSK. Aim 2 will test the hypothesis that inhibition of glycolytic activator PFKFB3, and the lactate transporter MCT4 and MCT1 protein reduces the severity of HSK. Aim 3 will test the hypothesis that blockers of HIF in association with an inhibitor of mitochondrial respiration or ROS will reduce the severity of HSK. Given the results from these studies, novel therapeutic approaches for treating HSK lesions may be developed. The knowledge gained from this application will provide innovative information that could be applicable to other ocular infection disease models.