Abstract
Purpose :
In this study, we measured the intracellular level of ROS in neutrophils and CD4 T cells in HSK lesions and determined the outcome of manipulating ROS level on HSK severity.
Methods :
C57BL/6J (B6) mice were infected with HSV-1 after mild scratching of the corneas. Multi-colored flow cytometry (FCM) was carried out to enumerate the ROS producing cells’ frequency in infected mice. qRT-PCR assay measured the transcripts of antioxidant genes and NOX isoforms in uninfected and infected corneas. A slit lamp was used to grade the corneal opacity and angiogenesis in infected corneas of gp91-/-, vehicle and DPI treated groups of B6 mice.
Results :
The flow cytometry data showed that approximately 90% of ROS generating leukocytes in HSK lesions were neutrophils. Antioxidant enzymes play an essential role in regulating intracellular ROS levels. Our results showed significant downregulation in antioxidant genes SOD1, GSS, and NQO1 but an upregulation in Hmox1, SOD2, SOD3, and GSR in infected corneas at 10- and 15-day post-infection (DPOI) as compared to uninfected corneas. ROS generation is regulated by flavoenzymes, including NADPH oxidase 2 (NOX2). qRT-PCR studies showed a two to six-fold increase in the expression of NOX2 subunits in infected corneas at 5- and 12-DPOI than uninfected corneas. FCM experiments showed NOX2 (gp91) protein predominance in neutrophils and monocytes in infected corneas at 15-DPOI. The corneal HSV-1 infection of gp91-/- and B6 mice showed no significant difference in the intracellular level of ROS in HSK lesions, suggesting NOX2 does not regulate ROS formation in HSK lesions. To ascertain the functional significance of ROS in regulating HSK, HSV-1 infected mice received a subcutaneous injection of DPI (1mg/Kg /day), a flavoenzyme inhibitor, from 7- through 15-DPOI. Our results showed significantly reduced corneal opacity and angiogenesis in DPI than vehicle-treated mice. Intriguingly, DPI treatment caused an increased level of ROS in neutrophils. An increased ROS level can cause cell death by apoptosis. However, FCM experiments revealed no significant difference in the frequency of apoptotic neutrophils from treated and vehicle groups, indicating the decreased frequency of neutrophils in the DPI treated group is not due to apoptosis.
Conclusions :
Together, our results suggest that manipulating ROS level in HSK lesions can play an important role of regulating the severity of HSK.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.