title: The role of toll-like receptor 4 in corneal epithelial wound healing.
abstract: OBJECTIVE
  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.affiliation: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States.
authors.forename: Medi
authors.initials: M
authors.lastname: Eslani
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States.
authors.forename: Asadolah
authors.initials: A
authors.lastname: Movahedan
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States.
authors.forename: Neda
authors.initials: N
authors.lastname: Afsharkhamseh
authors.affiliation: Oral Medicine and Diagnostic Sciences, College of Dentistry, University of Illinois at Chicago, Chicago, Illinois, United States.
authors.forename: Herve
authors.initials: H
authors.lastname: Sroussi
authors.affiliation: Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, Chicago, Illinois, United States.
authors.forename: Ali R
authors.initials: AR
authors.lastname: Djalilian
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D054443:Cell Migration Assays; D002465:Cell Movement / Q000502:physiology; D049109:Cell Proliferation / Q000502:physiology; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D003646:Debridement; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000293:injuries* / Q000378:metabolism / Q000473:pathology; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000502:physiology; D006801:Humans; D007113:Immunity, Innate; D019394:Ki-67 Antigen / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051197:Toll-Like Receptor 4 / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D014945:Wound Healing / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D016207:Cytokines; D019394:Ki-67 Antigen; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D014157:Transcription Factors; C478359:WDR77 protein, human; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048051:p38 Mitogen-Activated Protein Kinases
keywords: TLR4; Toll-like receptor 4; corneal epithelium; corneal wound healing; innate immunity