Systemic alterations in leukocyte subsets and the protective role of NKT cells in the mouse model of diabetic retinopathy.

The involvement of leukocytes in the pathophysiology of DR has mostly examined the role of monocytes and neutrophils with little emphasis on other immune cell types. In this study, we determined the systemic alterations in T cell subsets, myeloid cell types, NK cells, and NKT cells in the streptozotocin (STZ) mouse model of diabetic retinopathy (DR), and the role of NKT cells on retinal leukostasis and permeability changes. C57BL/6 J mice were made diabetic with 60 mg/kg dose of STZ given for 5-days. Flow cytometry assay measured the frequency of leukocyte subsets in the peripheral blood, spleen, and bone marrow of STZ- and vehicle-treated C57BL/6 J mice. Our results showed an increased proportion of memory CD8 T cells and interferon-gamma (IFN-γ) secreting CD8 T cells in the bone marrow of STZ-treated compared to control mice. Subsequently, increased production of inflammatory monocytes in the bone marrow and an enhanced frequency of CD11b + cells in the diabetic retina were seen in STZ-treated compared to control mice. The diabetic mice also exhibited a decrease in total NKT and CD4+NKT cells. A monoclonal antibody-based approach depleted NKT cells from STZ-treated mice, followed by measurements of retinal vascular permeability and leukostasis. The depletion of NKT cells in STZ-treated mice resulted in a significant increase in vascular permeability in the retinal tissue. Together, our results strongly imply the involvement of NKT cells in regulating the pathophysiology of the diabetic retina.

Role of Insulin-Like Growth Factor Binding Protein-3 in the Pathogenesis of Herpes Stromal Keratitis.

Purpose: The goal of this study was to determine the role of insulin-like growth factor-binding 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.

Development of lacrimal gland inflammation in the mouse model of herpes stromal keratitis.

Herpes stromal keratitis (HSK) is a chronic immunoinflammatory condition which develops in response to recurrent herpes simplex virus-1 (HSV-1) infection of the cornea. Patients with HSK often demonstrate the concurrence of corneal desiccation and the loss of blink reflex. However, the relationship between severity of HSK, level of basal tears and inflammation of the lacrimal gland is mostly unexplored. In this study, we compared these variables in extraorbital lacrimal gland (EoLG) after corneal HSV-1 infection in the C57BL/6J mouse model. Our results showed a significant reduction in the volume of tears in infected eyes during the development of HSK. Extensive architectural damage to EoLG, presumably caused by a massive influx of interferon-gamma secreting T cells, was observed during clinical disease period of HSK. A positive correlation between the decrease in tear volume, severity of HSK and the damage to EoLG were evident in infected mice. The presence of infectious virus measured in EoLG during pre-clinical, but not clinical disease period of HSK, suggested that viral cytopathic effects are not the major contributors of extensive damage seen in EoLG. Furthermore, topical administration of lacritin peptide delayed but did not prevent the decrease in tears in HSV-1 infected mice, and had no significant effect in either reducing the severity of HSK or T cell infiltration in EoLG of infected mice. Together, our results showed an interplay between the severity of HSK, inflammation of EoLG, and the reduced level of tears after corneal HSV-1 infection.

Development of Inflammatory Hypoxia and Prevalence of Glycolytic Metabolism in Progressing Herpes Stromal Keratitis Lesions.

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 hypoxia-inducible 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-2α protein was detected in corneal epithelial cells, whereas HIF-1α protein stabilization was observed in infiltrating immune cells. Administration of acriflavine drug to HSV-1-infected mice inhibited nuclear accumulation of HIF-1α and HIF-2α 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.

Loss of Neurokinin-1 Receptor Alters Ocular Surface Homeostasis and Promotes an Early Development of Herpes Stromal Keratitis.

Substance P neuropeptide and its receptor, neurokinin-1 receptor (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 and 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 (DCs), and a reduced volume of basal tears. Interestingly, massive accumulation of CD11c+CD11b+ conventional DCs was noted in the bulbar conjunctiva and near the limbal area of corneas from NK1R-/- mice. After ocular HSV-1 infection, the number of conventional DCs 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 draining lymph nodes of NK1R-/- mice was much higher than in 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 herpes stromal keratitis. Collectively, our results show that the altered corneal biology of uninfected NK1R-/- mice along with an enhanced immunological response after ocular HSV-1 infection causes an early development of herpes stromal keratitis in NK1R-/- mice.

IL-2/anti-IL-2 antibody complex treatment inhibits the development but not the progression of herpetic stromal keratitis.

The IL-2/anti-IL-2 Ab immunocomplex has recently been shown to expand the naturally occurring pool of CD4(+)Foxp3(+) regulatory T cells (Tregs). In this study, we show that administration of the IL-2/anti-IL-2 Ab immunocomplex to C57BL/6 mice, prior to corneal HSV-1 infection, significantly increased the pool of Foxp3(+) Tregs when measured at early time points postinfection. Increased numbers of Foxp3(+) Tregs on days 2 and 4 postinfection resulted in a marked reduction in the development of severe herpetic stromal keratitis (HSK). When compared with 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 postinfection. Reduced viral load was associated with a 2-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 postinfection in the immunocomplex-treated group of infected mice. Immunocomplex treatment given on days 5, 6, and 7 postinfection 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 postinfection. Our findings demonstrate that increasing Foxp3(+) Tregs early but not late postinfection in secondary lymphoid tissues is more efficacious in controlling the severity of HSK.