Flagellin-induced corneal antimicrobial peptide production and wound repair involve a novel NF-kappaB-independent and EGFR-dependent pathway.
BACKGROUND
The bacterial protein flagellin plays a major role in stimulating mucosal surface innate immune response to bacterial infection and uniquely induces profound cytoprotection against pathogens, chemicals, and radiation. This study sought to determine signaling pathways responsible for the flagellin-induced inflammatory and cytoprotective effects on human corneal epithelial cells (HCECs).


RESULTS
Flagellin purified from Pseudomonas aeruginosa (strain PAK) or live bacteria were used to challenge cultured HCECs. The activation of signaling pathways was assessed with Western blot, and the secretion of cytokine/chemokine and production of antimicrobial peptides (AMPs) were measured with ELISA and dot blot, respectively. Effects of flagellin on wound healing were assessed in cultured porcine corneas. L94A (a site mutation in TLR5 binding region) flagellin and PAK expressing L94A flagellin were unable to stimulate NF-kappaB activation, but were potent in eliciting EGFR signaling in a TGF-alpha-related pathway in HCECs. Concomitant with the lack of NF-kappaB activation, L94A flagellin was ineffective in inducing IL-6 and IL-8 production in HCECs. Surprisingly, the secretion of two inducible AMPs, LL-37 and hBD2, was not affected by L94A mutation. Similar to wild-type flagellin, L94A induced epithelial wound closure in cultured porcine cornea through maintaining EGFR-mediated signaling.


CONCLUSIONS
Our data suggest that inflammatory response mediated by NF-kappaB can be uncoupled from epithelial innate defense machinery (i.e., AMP expression) and major epithelial proliferation/repair pathways mediated by EGFR, and that flagellin and its derivatives may have broad therapeutic applications in cytoprotection and in controlling infection in the cornea and other mucosal tissues.
Gao
Nan
N
Department of Ophthalmology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.
Kumar
Ashok
A
Jyot
Jeevan
J
Yu
Fu-Shin
FS
D000818:Animals; D023181:Antimicrobial Cationic Peptides / Q000378:metabolism*; D015153:Blotting, Western; D002460:Cell Line; D003315:Cornea / Q000187:drug effects / Q000378:metabolism; D065306:Corneal Injuries; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D005408:Flagellin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D009924:Organ Culture Techniques; D011485:Protein Binding / Q000187:drug effects; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D015398:Signal Transduction / Q000187:drug effects*; D013552:Swine; D051198:Toll-Like Receptor 5 / Q000378:metabolism; D016211:Transforming Growth Factor alpha / Q000378:metabolism; D014945:Wound Healing / Q000187:drug effects; D023083:beta-Defensins / Q000378:metabolism; D054804:Cathelicidins
D023181:Antimicrobial Cationic Peptides; C106641:DEFB4A protein, human; D015850:Interleukin-6; D016209:Interleukin-8; D016328:NF-kappa B; D051198:Toll-Like Receptor 5; D016211:Transforming Growth Factor alpha; D023083:beta-Defensins; D005408:Flagellin; D066246:ErbB Receptors; D054804:Cathelicidins