(LR-041) Regenerative healing immune response after corneal chemical burn

Arpan Banerjee, Bachelor's – Graduate Assistant, Ophthalmology and Vision Sciences, Cell and Development Biology, SUNY Upstate Medical University; Mohammod Anjum Shaik, Master's – Graduate Assistant, Ophthalmology and Vision Sciences, Cell and Development Biology, SUNY Upstate Medical University; Nileyma Castro, MD – Senior Scientist/Lab Manager, Ophthalmology and Vision Sciences, SUNY Upstate Medical University; Tere Williams, Bachelor's – Researcher, Ophthalmology and Vision Sciences, SUNY Upstate Medical University; Audrey Bernstein, PhD – Professor, Ophthalmology and Vision Sciences, Cell and Development Biology, Biochemistry and Molecular Biology, SUNY Upstate Medical University

Introduction: Scarring is driven by the persistence of myofibroblasts and inflammation in healing tissue. After wounding, the gene expression of the deubiquitinase (DUB) USP10 is upregulated. Knockdown of USP10 in rabbit cornea reduced scarring, fibrotic markers, and CD45+ cells. Using an alkaline wound model in mice, we investigated the effects of USP10 knockdown on early immune cell response and corneal scarring.

Methods:

C57BL/6J mouse corneas were wounded with 0.5 M NaOH for 1 min and treated with vehicle (PBS) or with an in vivo USP10-targeting siRNA (US16) in a single dose at the time of wounding. Fluorescein was used to visualize a breached epithelium. Flow cytometry (Cytek Aurora) was used to quantify live cells, CD45+ cells, macrophages (CD11b+, F4/80+), monocytes (CD11b+, Ly6C+), neutrophils (CD11b+, Ly6G+), and apoptotic cells (Annexin V). Analysis by FlowJo Software. OCT imaging was used to visualize corneal scarring and morphology. Cornea depth and scarring intensity were quantified in FIJI.

Results: At day 2 post-wounding, US16 increased the rate of epithelial wound closure by 87.5% (p=0.0159). At 14 days post-wounding, the corneal epithelium reopened demonstrating a persistent corneal epithelial defect. US16 treatment reduced wound reopening by 82% (p=0.003) and corneal thickness by 20% (p=0.0382). The effect of US16 on immune cell populations, at various timepoints port-wounding, was assessed via flow cytometry. While there was an overall trend of reduced CD45+ cells at all timepoints in the US16 treated eyes, day 5 reached significance (p=0.0403). Of CD45+ cells on day 5, macrophages were decreased by 66% in treatment groups whereas monocytes and neutrophils were not significantly different between groups. Importantly, analysis of the total population of CD45+ (dead and live), demonstrated that with US16 treatment, CD45+ cell count dropped 61% (p=0.0310) and US16 treatment increased the ratio of live to dead cells by 30%. Correspondingly, Annexin V staining for apoptosis determined that US16 reduced apoptosis by 41% (p< 0.0001) 12hrs post-wounding and 23% (p=0.0110) 24hrs post-wounding.

Discussion: Our data suggest that USP10 knockdown is a novel method to improve wound closure, decrease scarring, and alter CD45+ populations. Furthermore, that a decline in early apoptotic events post-injury is protective to the cornea and may reduce the influx of immune cells after wounding, ultimately leading to improved healing.