Chronic inflammation in tissues often causes the development of hypoxia. Herpes stromal keratitis (HSK) is a corneal chronic inflammatory condition that develops in response to recurrent HSV-1 infection. In this study, we investigated the development of hypoxia, the expression of hypoxia-associated glycolytic genes in HSV-1 infected corneas, and the outcome of blocking hypoxiainducible factor (HIF) dimerization on the severity of HSK. Our results showed the development of hypoxia, an elevated expression of hypoxia-associated glycolytic genes, and an increased level of lactate in corneas with progressing HSK lesions. The magnitude of hypoxia correlated with the extent of neutrophils infiltrating the infected corneas, and the depletion of neutrophils reduced the development of hypoxia in infected corneas. Additionally, in progressing HSK lesions, nuclear localization of HIF-2a protein was detected in corneal epithelial cells, whereas HIF-1a protein stabilization was observed in infiltrating immune cells. Administration of acriflavine drug to HSV-1-infected mice inhibited nuclear accumulation of HIF-1a and HIF-2a protein in immune cell types and epithelial cells, respectively, in infected corneas. As a result, a decreased influx of CD4 T cells and nongranulocytic myeloid cells, but an increased influx of neutrophils, was noted in developing HSK lesions. Interestingly, acriflavine treatment given during the clinical disease period decreased neovascularization but increased the opacity in HSV-1-infected corneas. Taken together, the results of our study lay the foundation to dissect the role of inflammatory hypoxia and hypoxia-associated genes in the pathogenesis of HSK.

Interleukin-2 (IL-2) and anti-IL-2 antibody immune complex has recently been shown to expand the naturally occurring pool of CD4+Foxp3+ regulatory T cells (Foxp3+ Tregs). In this report, we showed that administration of IL-2/anti-IL-2 antibody immunocomplex to C57BL/6 mice, prior to corneal herpes simplex virus-1 (HSV-1) infection, significantly increased the pool of Foxp3+ Tregs when measured at early time-points post-infection. Increased numbers of Foxp3+ Tregs on day 2 and day 4 post-infection resulted in a marked reduction in the development of severe HSK. When compared to corneas from the control group, corneas from the immunocomplex-treated group showed a significant reduction in the amount of infectious virus on day 2 but not on day 4 post-infection. Reduced viral load was associated with two-fold increase in NK cell numbers in corneas from the immunocomplex-treated group of mice. Moreover, a dramatic reduction in the influx of CD4 T cells in inflamed corneas was determined on days 7 and 16 post-infection in the immunocomplex-treated group of infected mice. Immunocomplex treatment given on days 5, 6 and 7 post-infection significantly increased Foxp3+ Tregs in draining lymph nodes and in the spleen but failed to reduce the severity of HSK. In terms of the influx of CD4 T cells and granulocytes into inflamed corneas, no significant differences were noted between both groups of mice on day 16 post-infection. Our findings demonstrate that increasing Foxp3+ Tregs early but not late after infection in secondary lymphoid tissues is more efficacious in controlling the severity of HSK.

Substance P neuropeptide and its receptor neurokinin-1 (NK1R) are reported to present on the ocular surface. In this study, mice lacking functional NK1R exhibited an excessive desquamation of apical corneal epithelial cells in association with an increased epithelial cell proliferation, increased epithelial cell density, but decreased epithelial cell size. The lack of NK1R also resulted in decreased density of corneal nerves, corneal epithelial dendritic cells, and a reduced volume of basal tears. Interestingly, massive accumulation of CD11c+CD11b+ conventional dendritic cells (cDCs) was noted in the bulbar conjunctiva and near the limbal area of corneas from NK1R−/− mice. After ocular HSV-1 infection, the number of cDCs and neutrophils infiltrating the infected corneas was significantly higher in NK1R−/− than C57BL/6J mice. This was associated with an increased viral load in infected corneas of NK1R−/− mice. As a result, the number of IFN-γ secreting virus specific CD4 T cells in the DLNs of NK1R−/− mice was much higher than infected C57BL/6J mice. An increased number of CD4 T cells and mature neutrophils (CD11b+Ly6ghigh) in the inflamed corneas of NK1R−/− mice was associated with an early development of severe HSK. Collectively, our results show that the altered corneal biology of uninfected NK1R−/− mice along with an enhanced immunological response after ocular HSV-1 infection cause an early development of HSK in NK1R−/− mice.

The goal of this study was to determine the role of insulin-like growth factorbinding protein-3 (IGFBP-3) in the pathogenesis of herpes stromal keratitis (HSK). METHODS. In an unbiased approach, a membrane-based protein array was carried out to determine the level of expression of pro-and anti-angiogenic molecules in uninfected and HSV-1 infected corneas. Quantitative RT-PCR and ELISA assays were performed to measure the amounts of IGFBP-3 at mRNA and protein levels. Confocal microscopy documented the localization of IGFBP-3 in uninfected and infected corneal tissue. Flow cytometry assay showed the frequency of immune cell types in infected corneas from C57BL/6J (B6) and IGFBP-3 knockout (IGFBP-3 −/−) mice. Slit-lamp microscopy was used to quantitate the development of opacity and neovascularization in infected corneas from both groups of mice. RESULTS. Quantitation of protein array dot blot showed an increased level of IGFBP-3 protein in HSV-1 infected than uninfected corneas and was confirmed with ELISA and quantitative RT-PCR assays. Cytosolic and nuclear localization of IGFBP-3 were detected in the cells of corneal epithelium, whereas scattered IGFBP-3 staining was evident in the stroma of HSK developing corneas. Increased opacity and hemangiogenesis were noted in the corneas of IGFBP-3 −/− than B6 mice during the clinical period of HSK. Furthermore, an increased number of leukocytes comprising of neutrophils and CD4 T cells were found in HSK developing corneas of IGFBP-3 −/− than B6 mice. CONCLUSIONS. Our data showed that lack of IGFBP-3 exacerbates HSK, suggesting the protective effect of IGFBP-3 protein in regulating the severity of HSK.