Abstract
Purpose :
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 the presence of myofibroblasts, fibrotic markers, and CD45+ cells. To understand the effect of USP10 knockdown on immune cell infiltration we established a corneal alkaline wound mouse model.
Methods :
C57BL/6J mouse corneas were wounded with 0.5 M NaOH for 1 min and either untreated or treated with an in vivo USP10-targeting siRNA (US16). Corneas were imaged daily using fluorescein to visualize a breached epithelium with scarring and fluorescent intensity of images were quantified in FIJI. A single dose of US16 at the time of wounding (t=0) was compared to multi-dosing over the first 48 hours with a range of concentrations. Flow cytometry (Cytek Aurora) was used to quantify live cells, CD45+ cells, macrophages (CD11b+, F4/80+), monocytes (CD11b+, Ly6C+, Ly6G-), as well as neutrophils (CD11b+, Ly6G+) with analysis using FlowJo Software.
Results :
To elucidate the most effective drug concentration and regiment, we analyzed a range of concentration of US16 (0.2 uM – 100 uM) in a single dose at t=0. We found that at day 2 after wounding, 20 uM and 10 uM significantly increased the rate of wound closure and decreased scarring by 87.5% and 68.8%, respectively (p<0.0001). We then compared a single dose at t=0 to multi-dosing (t=0, 24 and 48 hours). We found that multi-dosing did not further improve wound closure or scarring outcomes. Flow cytometry was used to identify immune cell populations after wounding and +/- US16 treatment. The 20 uM treatment resulted in a 90% decrease in CD45+ cells (p>0.05) and 10uM US16 resulted in a 64% decrease (p=0.05). Of the population of CD45+ cells, macrophages were decreased by 66% in both treatment groups (trending towards significance, p=0.07). Monocytes and neutrophils were not significantly different between groups.
Conclusions :
This study builds upon our previous findings in a rabbit cornea model. Our current data in mice suggest that USP10 knockdown improves wound closure, decreases scarring and alters CD45+ populations and that the effect may be immune cell-type specific. Furthermore, a one-time treatment of US16 versus similar concentrations with multiple doses was equally effective in promoting regenerative healing in an alkaline burn mouse model.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.