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

Abstract

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.

• Authors: Medi Eslani, Asadolah Movahedan, Neda Afsharkhamseh, Herve Sroussi, Ali R Djalilian
• Journal: Investigative ophthalmology & visual science (Invest Ophthalmol Vis Sci) Vol. 55 Issue 9 Pg. 6108-15 (Sep 02 2014) ISSN: 1552-5783 [Electronic] United States
• PMID: 25183764 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Chemical References

• Cytokines
• Ki-67 Antigen
• TLR4 protein, human
• Toll-Like Receptor 4
• Transcription Factors
• WDR77 protein, human
• MAPK1 protein, human
• Mitogen-Activated Protein Kinase 1
• p38 Mitogen-Activated Protein Kinases

Topics

• Animals
• Blotting, Western
• Cell Line
• Cell Migration Assays
• Cell Movement (physiology)
• Cell Proliferation (physiology)
• Cytokines (genetics, metabolism)
• Debridement
• Disease Models, Animal
• Enzyme-Linked Immunosorbent Assay
• Epithelium, Corneal (injuries, metabolism, pathology)
• Fluorescent Antibody Technique, Indirect
• Gene Expression Regulation (physiology)
• Humans
• Immunity, Innate
• Ki-67 Antigen (metabolism)
• Mice
• Mice, Inbred C57BL
• Mitogen-Activated Protein Kinase 1 (metabolism)
• Reverse Transcriptase Polymerase Chain Reaction
• Toll-Like Receptor 4 (physiology)
• Transcription Factors (metabolism)
• Wound Healing (physiology)
• p38 Mitogen-Activated Protein Kinases (metabolism)