Abstract
Purpose :
Corneal fibrosis due to ocular trauma is a major cause of visual impairment worldwide. Studies in non-corneal tissue show macrophages have a role in fibrosis during the early stages of wound healing. Here, we investigated whether infiltrating macrophages contribute to fibrosis by differentiating into αSMA-positive cells
Methods :
C57BL/6 mice received a 2mm corneal injury using an AlgerBrush II to remove the epithelium and anterior stroma. Corneas were collected at day 3 for immunohistochemistry and stained with F4/80 (macrophage marker) and αSMA (fibrosis marker). Human monocytic (THP-1) cell-derived macrophages were stimulated with or without10ng/mL IL-1β for 3 days. Afterwards macrophages were divided into four treatment groups: 1) control, 2) 20ng/mL IL-10, 3) 20ng/mL TGFβ, and 4) 20ng/mL IL-10 and 20ng/mL TGFβ. αSMA expression was assessed with qRT-PCR and immunocytochemistry at day 3 and day 6.
Results :
Injured corneas showed increased infiltration of macrophages compared to control. Furthermore, macrophages were predominantly located at the anterior portion of the stroma and were αSMA positive. qRT-PCR analysis of in vitro cultured macrophages stimulated in inflammatory conditions (IL-1β) did not significantly increase αSMA expression following IL-10 only or TGFβ only treatment at tested timepoints compared to controls. However, IL-1β stimulated macrophages incubated with a combination of IL-10 and TGFβ had significantly increased αSMA expression compared to controls at day 3 (p<0.001) and day 6 (p<0.0001). Remarkably, resting macrophages co-treated with IL-10 and TGFβ had a two-fold increase in αSMA expression at both timepoints compared to controls; however, IL-1β stimulated macrophages had a greater increase compared to resting macrophages. Immunocytochemistry confirmed resting and IL-1β simulated macrophages treated with IL-10 and TGFβ expressed αSMA at all tested timepoints.
Conclusions :
Macrophages at the injured cornea express αSMA, which our data suggest depends on co-stimulation with IL-10 and TGFβ. Together these findings suggest that regulatory cytokines-induced αSMA-positive macrophages may play a critical role in corneal fibrosis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.