Abstract
Purpose :
Ocular infection by herpes simplex virus type-1 (HSV-1) is often manifested as severe inflammation and neovascularization in the stroma of the cornea. Our study demonstrates that viral activation of the enzyme heparanase (HPSE) by Cathepsin-L is a key trigger that initiates the emergence of corneal disease symptoms.
Methods :
Wild-type and HPSE knockout mice as well as cultured cells were used to study HSV-1 infection in the cornea. Flow cytometry, fluorescence microscopy and viral plaque assays were used to study Cathepsin-L in HSV-induced activation of HPSE. ELISA, flow cytometry, confocal fluorescence microscopy, histology, and viral plaque assays were used to compare and contrast the HSV-1 infection-dependent changes in the wild-type and HPSE knockout animals. Murine corneas (with or without fluorescein staining) were examined for tissue damage using slit lamp biomicroscope and scored using a four point scale. Relative expression of proinflammatory cytokines and proangiogenic factors in animal corneas were estimated by qRT-PCR. Quantitative proteomics was used to study the effects of HPSE knockout on HSV-1 infection.
Results :
Here we demonstrate that animals lacking HPSE are less prone to developing stromal keratitis. While the wild-type animals showed a full-blown disease within 2 weeks, the symptoms remained significantly milder in the HPSE knockout animals. The levels of many different cytokines including Interferons alpha and beta, TNF-alpha, IL-6, IL-8 and IL-1b were reported higher in wild-type animals. The upregulation of cytokine expression correlated well with disease symptoms in animals with or without HPSE knockout. Cell culture studies demonstrated that Cathepsin-L was the regulatory factor behind HPSE activation and that HPSE interactome plays an important role in generating pro-inflammatory and pro-angiogenic conditions in the cornea.
Conclusions :
Our findings directly implicate HPSE activation by Cathepsin-L as the cellular trigger for corneal inflammation and neovascularization during herpetic stromal keratitis. We also identify common interventional targets for simultaneously reducing viral load and associated inflammation and additional pathologies.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.