Abstract
Purpose :
Recently, we showed the presence of hypoxic pockets in developing HSK lesions (J Immunology 2019). We also showed NOX2-independent reactive oxygen species (ROS) production in the neutrophils of HSK lesions (J Virol 2022). Since hypoxia and increased ROS can instigate oxidative stress, we hypothesized that HSK corneas also endure such stress.
Methods :
C57BL/6J mice corneas were infected with HSV-1 after mild scratching. RT-qPCR and flow cytometry (FCM) were used for studying gene expression. Ebselen was administered (100 mg/kg) orally, and a hand-held slit lamp was used to grade HSK severity. Viral load was studied by conventional plaque assay for wild type HSV-1 and by FCM using HSV-1-gC-GFP mutant.
Results :
Differential expression of antioxidant and oxidative stress-related transcripts were observed at 5-, 10-, and 15- day post-ocular infection (DPOI), using PCR-array and RT-qPCR assays. A significantly increased level of malondialdehyde, a product of lipid peroxidation, in corneal lysates at 15DPOI than 5DPOI suggested the development of oxidative stress. NRF2 stabilization is reported to occur in the cells experiencing hypoxia-induced oxidative stress. Our FCM data revealed intracellular NRF2 expression in major immune-cell subsets at 10 and 15DPOI, indicating prevailing oxidative stress. A significant surge in GPX activity, a countermeasure to oxidative stress, was noticed in HSK corneas at 15-DPOI. Ebselen treatment given during HSK development (9-14DPOI) to mitigate oxidative stress showed a significant decrease in opacity but no change in pathological hemangiogenesis. We also observed a significant decrease in transcripts of proinflammatory cytokines (TNF-a and IL1-b) and Th1 effector cytokine (IFN-g) with no change in anti-inflammatory cytokine (IL-10) after treatment. Immune cell infiltration studies by FCM revealed no difference in corneal stroma and epithelium amongst treated and control cohorts. We also investigated the antiviral property of Ebselen on HSV-1 and observed a significant decrease in viral load at 2DPOI.
Conclusions :
Our results indicated an impaired redox homeostasis in HSK corneas. Ebselen, a GPX mimetic organo-selenium compound known for exerting antioxidant effect, reduces HSK opacity. It attenuates the effector functions of the infiltrating cells without modulating their infiltration.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.