Abstract
Purpose :
Corneal fibrosis due to ocular trauma is a major cause of visual impairment. Previous studies have shown macrophages secrete soluble pro-fibrotic factors which indirectly promote fibrosis. Here, we investigated if macrophages directly contribute to fibrosis by transitioning into myofibroblasts.
Methods :
Both in vivo and in vitro models were used to test this hypothesis. Male C57BL/6 mice received a 2 mm corneal injury using an AlgerBrush II to remove the epithelium and anterior stroma. Corneas were collected for immunohistochemistry and flow cytometry to identify alpha-smooth muscle actin (aSMA)+F4/80+CD34- macrophages. qRT-PCR and ELISA measured IL-1β, IL-6, IL-10, and TGFβ1 levels. For in vitro studies, human macrophages were derived from monocytic (THP-1) cells and mouse macrophages were collected from bone marrow and peritoneum. Macrophages were pre-stimulated with 100 ng/mL IL-1β then exposed to 100 ng/mL IL-10 + 100 ng/mL TGFβ1 or media alone. Immunohistochemistry and flow cytometry measured aSMA expression. Myofibroblast markers aSMA, desmin (DES), vimentin (VIM), fibronectin (FN1), collagen I (COL I), and COL III were assessed with qPCR. Collagen gel contraction assay tested myofibroblast functionality.
Results :
During corneal wound healing, F4/80+CD34- macrophages have increased aSMA expression from day 3 to day 14. In addition, flow cytometry analysis showed an increased frequency of aSMA+F4/80+CD34- macrophages. Generation of aSMA+ macrophages corresponds to high levels of pro-fibrotic (IL-10 and TGFβ1; p<0.05) and pro-inflammatory (IL-1β and IL-6; p<0.01) factors in an injured cornea. In vitro cultures of human and mouse macrophages showed IL-10 + TGFβ1 exposed macrophages have significantly increased aSMA expression (p<0.0001) compared to untreated macrophages. Furthermore, aSMA+ macrophages had an elongated fibroblastic-like morphology with significantly increased expression of DES (p<0.05), VIM (p<0.01), FN1 (p<0.01), and COL III (p<0.05) compared to untreated macrophages. Only aSMA+ macrophages showed a significant capacity to contract a collagen gel matrix (p<0.001).
Conclusions :
Alpha-smooth muscle actin (aSMA) positive macrophages are generated during a corneal injury. Our mechanistic studies demonstrate that IL-10 and TGFβ1 are essential for the transition of macrophages to aSMA+ cells. These transitioned macrophages are phenotypically and functionally similar to myofibroblasts.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.