M2a Macrophage-Dominant Microenvironment in Inflammation Attenuation and Wound Healing of Human Corneal Endothelial Cells

PURPOSE: To investigate the anti-inflammatory and wound-healing effects of an M2a macrophage-dominant microenvironment on human corneal endothelial cells (HCECs) in vitro. METHODS: Two in vitro corneal endothelial inflammation models were developed: a lipopolysaccharide (LPS)-induced inflammation-only model and a dual inflammation-and-wound model. HCECs were co-cultured with M2a macrophages or treated with M1 macrophage-derived exosomes (M1-exo), M2a macrophage-derived exosomes (M2a-exo), or epidermal growth factor (EGF)-preconditioned M2a-derived exosomes (EGF-M2a-exo). Gene and protein expression of inflammatory markers was assessed, and HCEC proliferation was evaluated using cell growth curves. RESULTS: Scratch wounding alone did not induce significant inflammation in HCECs, whereas LPS stimulation combined with scratch wounding markedly increased inflammatory responses. In the inflammation-only model, co-culture with M2a macrophages or M2a-exo treatment significantly suppressed LPS-induced upregulation of IL6, IL1B, and ICAM1 at the mRNA level in HCECs and reduced IL-6 protein secretion. In the dual inflammation-and-wound model, M2a-exo showed limited efficacy, but EGF-M2a-exo significantly reduced inflammatory marker expression. Cell growth analysis revealed that M2a-exo treatment promoted faster HCEC proliferation compared to M1-exo treatment under non-inflammatory conditions, despite no significant differences in cell cycle-related genes. In LPS-stimulated HCECs, EGF-M2a-exo treatment restored proliferation to levels comparable with non-inflammatory controls by day 5. CONCLUSIONS: An M2a macrophage-dominant microenvironment demonstrates anti-inflammatory and regenerative effects on inflamed HCECs. EGF preconditioning enhances these properties, suggesting a potential therapeutic approach for managing corneal endothelial inflammation and injury.