Abstract
Abstract: :
Purpose: 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. In this study, we sought to elucidate the role and the underlying mechanisms for flagellin to induce inflammation response in cultured human corneal epithelial cells. Methods: Flagellin was purified from P. aeruginosa strain PAO1 with ammonium sulfate gradient precipitation and used to stimulate HUCL cells (a telomerase-immortalized human corneal epithelial cell line). Phosphorylation and degradation of IΚB-α were detected by Western blot. NF-ΚB translocation was detected with immunocytochemistry. Interleukin-6 and interleukin-8 expression and secretion were assessed using RT-PCR and ELISA, respectively. Flagellum antiserum and TLR5 antibody were used to functionally block flagellin stimulation and TLR5 activation. Results: Exposure of HUCL cells to purified PA flagellin (250ng/ml) resulted in IΚB-α phosphorylation and degradation, NF-ΚB nuclear translocation in time-dependent manner. Concomitant with NF-ΚB activation, transcriptional expression and subsequent secretion of IL-6 and IL-8 in these cells were also induced by flagellin. In cultured HUCL cells, Toll-like receptor-5 (TLR5), an innate immunity receptor for flagellin, was expressed and located at the cell surface of while TLR4, an LPS receptor, is not. In the mouse cornea, TLR5 was observed in both apical and basal membrane while TLR4 staining appeared to be intracellular. Pretreatment of flagellin with antiserum of HUCL cells with TLR5-antibody significantly inhibited flagellin-induced IΚB-α phosphorylation and degradation and IL-6 and IL-8 secretion. Conclusions:Flagellin released by Gram-negative pathogens such as P. aeruginosa contributes to the inflammatory responses of corneal epithelium in a TLR5 signaling pathway dependent manner.
Keywords: inflammation • cornea: epithelium • Pseudomonas