Abstract
Purpose: :
Our previous studies showed that flagellin is an important stimulus of key signaling pathways leading to an inflammatory response in corneal epithelial cells (CECs) challenged with Pseudomonas aeruginosa (PA). In this study, we used PA expressing flagellin mutants to demonstrate whether TLR5 is a sensor for CECs to recognize Gram-negative bacteria and to mediate ocular surface innate immunity.
Methods: :
P. aeruginosa wild type strains PAK, PAO1, and mutants fliC (a non-motile strain with mutation in flagellin encoding gene), L94A (a motile strain with mutation in TLR5 binding region), and Q83A (a motile strain with mutation outside TLR5 binding region) were used to challenge HUCL, a telomerase-immortalized human CEC line in ratio of 100 bacteria/ cell. Phosphorylation and degradation of IkB- and activation of MAP-kinase signaling pathways (p38, JNK-1/2, ERK-1/2) was assessed by Western blotting using phospho-specific antibodies. The accumulation of cytokines in the culture media was determined by ELISA.
Results: :
Exposure of HUCL cells to wild type PAO1 and PAK strains resulted in rapid activation of NF-kB (IkB- phosphorylation and degradation), p38, ERK and JNK within 1h post bacterial challenge. Both fliC and L94A, but not Q83A, failed to activate NFΚB and MAPKs. Concomitant with activation of NF-ΚB and MAPKs signaling, both wild type and Q83A mutant induced the inflammatory response in HUCL cells as indicated by increased accumulation of IL-6 and IL-8 in culture supernatant. In contrast, the fliC and L94A mutant failed to induce the inflammatory response in HUCL cells.
Conclusions: :
Flagellin is an important virulence factor of P. aeruginosa in triggering epithelial inflammatory response and TLR5 is necessary and sufficient for CECs to recognize Gram-negative bacteria at the ocular surface.
Keywords: cornea: epithelium • inflammation • signal transduction