Interplay between hypoxia and oxidative phosphorylation in herpes stromal keratitis

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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.