Novel characterization of CXCR4 expressing cells in uninfected and herpes simplex virus-1 infected corneas.

PURPOSE: To characterize CXCR4-expressing cells in uninfected and herpes simplex virus-1 (HSV-1) infected corneas. METHODS: The corneas of C57BL/6J mice were infected with HSV-1 McKrae. The RT-qPCR assay detected CXCR4 and CXCL12 transcripts in uninfected and HSV-1-infected corneas. Immunofluorescence staining for CXCR4 and CXCL12 protein was performed in the frozen sections of herpes stromal keratitis (HSK) corneas. Flow cytometry assay characterized the CXCR4-expressing cells in uninfected and HSV-1-infected corneas. RESULTS: Flow cytometry data showed CXCR4 expressing cells in the separated epithelium and stroma of uninfected corneas. In the uninfected stroma, CD11b + F4/80+ macrophages are the predominant CXCR4-expressing cells. In contrast, most CXCR4 expressing cells in the uninfected epithelium were CD207 (langerin)+, CD11c+, and expressed MHC class II molecule, documenting the Langerhans cells (LCs) phenotype. After corneal HSV-1 infection, CXCR4 and CXCL12 mRNA levels increased significantly in HSK corneas than in uninfected corneas. Immunofluorescence staining showed CXCR4 and CXCL12 protein localization in the newly formed blood vessels in the HSK cornea. Furthermore, the infection resulted in LCs proliferation, causing an increase in their numbers in the epithelium at 4 days post-infection (p.i.). However, by 9-day p.i., the LCs numbers declined to the counts observed in naïve corneal epithelium. Our results also showed neutrophils and vascular endothelial cells as the prominent CXCR4-expressing cell types in the stroma of HSK corneas. CONCLUSIONS: Together, our data demonstrate the expression of CXCR4 on resident antigen presenting cells in the uninfected cornea and on infiltrating neutrophils and newly formed blood vessels in the HSK cornea.

Flow cytometry protocol to quantify immune cells in the separated epithelium and stroma of herpes simplex virus-1-infected mouse cornea.

Existing flow cytometry approaches identify immune cells using the whole infected/inflamed cornea, which limits its ability to distinguish the immune cells infiltrating the corneal epithelium from the corneal stroma. Here, we present a protocol to analyze immune cells in the separated epithelium and stroma from naïve and herpes simplex virus-1 (HSV-1)-infected mouse corneas. We describe steps for viral infection, separation of corneal epithelium from stroma, preparation of a single-cell suspension of the individual epithelium and stroma, and flow cytometry assay.

Diphenyleneiodonium Treatment Inhibits the Development of Severe Herpes Stromal Keratitis Lesions.

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. IMPORTANCE 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.