The role of toll-like receptor 4 in corneal epithelial wound healing.

PURPOSE: We evaluated the role of Toll-like receptor 4 (TLR4) in corneal epithelial wound healing.

METHODS: The expression of TLR4 during in vivo corneal epithelial wound healing was examined by immunostaining in mice. The expression and activation of TLR4 was studied in primary or telomerase-immortalized human corneal epithelial cells (HCEC). Scratch assay was performed to evaluate in vitro wound closure using live time-lapse microscopy. Transwell migration assay and Ki67 immunostaining were done to evaluate migration and proliferation, respectively. Lipopolysaccharide (LPS) was used to activate TLR4, whereas CLI-095 was used for its inhibition. The expression of inflammatory cytokines was determined by RT-PCR and ELISA. The activation of p42/44 and p38 was determined by immunoblotting.

RESULTS: In the murine model, TLR4 immunostaining was noted prominently in the epithelium 8 hours after wounding. There was a 4-fold increase in the expression of TLR4 6 hours after in vitro scratch wounding (P < 0.001). Confocal microscopy confirmed the membrane localization of TLR4/MD2 complex. There was a significant increase in migration, proliferation, and wound closure in HCEC treated with LPS (P < 0.05), while there was significant decrease with TLR4 inhibition (P < 0.05). Addition of LPS to wounded HCEC resulted in a significant increase in the expression of IL-6, TNF-α, CXCL8/IL8, and CCL5/RANTES at the mRNA and protein levels. Likewise, LPS increased the activation of p42/44 and p38 in wounded HCEC.

CONCLUSIONS: These results suggest that epithelial wounding induces the expression of functional TLR4. Toll-like receptor 4 signaling appears to contribute to early corneal epithelial wound repair by enhancing migration and proliferation.