Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin.
OBJECTIVE
Flagellin is the major structural protein of the flagella of Gram-negative bacteria and is a potent trigger of innate immune responses in a number of eukaryotic cells and organisms. In this study, we sought to determine whether flagellin induces an inflammation response in cultured human corneal epithelial (HCE) cells and to determine the underlying mechanisms.


METHODS
Flagellin was purified from Pseudomonas aeruginosa (PA) strain PAO1 with ammonium sulfate gradient precipitation and lipopolysaccharide in flagellin preparation was removed by ion exchange chromatography. Purified flagellin was used to challenge HUCL, a telomerase-immortalized HCE cell line, and primarily cultured HCE cells. Inhibitory (I)kappaB-alpha phosphorylation and degradation were detected by Western blot. Interleukin (IL)-6 and -8 expression in mRNA levels and secretion were assessed using RT-PCR and enzyme-linked immunosorbent assay, respectively. TLR5 localization in human cornea was analyzed by immunohistochemistry using anti-TLR5 antibody. Anti-flagellum antiserum and anti-TLR5 antibody were used for functional blocking of flagellin stimulation and TLR5 activation.


RESULTS
Exposure of both HUCL and primary HCE cells to purified PA flagellin (250 ng/mL) resulted in IkappaB-alpha phosphorylation and degradation in a time-dependent manner. Concomitant with NF-kappaB activation, transcriptional expression and subsequent secretion of IL-6 and -8 in these cells were also induced by flagellin. Toll-like receptor (TLR)-5, an innate immunity receptor for flagellin, was expressed in HUCL cells and located at the cell surface of the basal and wing, but not in superficial, cells of human corneal epithelium. Presence of flagellum- or TLR5-antisera in culture medium attenuated flagellin-induced IkappaB-alpha phosphorylation and degradation as well as IL-6 and -8 production.


CONCLUSIONS
Flagellin of Gram-negative pathogens such as PA contributes to the inflammatory responses of corneal epithelium in a TLR5-NF-kappaB signaling pathway-dependent manner.
D019138:Antibodies, Blocking / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000276:immunology; D005408:Flagellin / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D007249:Inflammation / Q000378:metabolism; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D000072000:NF-KappaB Inhibitor alpha; D010766:Phosphorylation; D011550:Pseudomonas aeruginosa / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors
D019138:Antibodies, Blocking; D020672:I-kappa B Proteins; D015850:Interleukin-6; D016209:Interleukin-8; D008562:Membrane Glycoproteins; C000606204:NFKBIA protein, human; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495346:TLR5 protein, human; D051198:Toll-Like Receptor 5; D051193:Toll-Like Receptors; D005408:Flagellin; D000072000:NF-KappaB Inhibitor alpha