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

To investigate the anti-inflammatory and wound-healing effects of an M2a macrophage-dominant microenvironment on human corneal endothelial cells (HCECs) in vitro.

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.

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.

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.