PURPOSE: We analyzed the expression and function of Toll-like receptor 5 (TLR5) in human conjunctival epithelial cells (HCjEC).
METHODS: The expression of TLR5 in HCjEC was studied by RT-PCR and flow cytometry. The amount of IL-6 and IL-8 proteins was determined by ELISA. mRNA expression elicited by stimulation with flagellins derived from Pseudomonas aeruginosa, Serratia marcescens, Salmonella typhimurium, and Bacillus subtilis was assayed by quantitative RT-PCR. The localization of TLR5 protein in human conjunctival epithelium was detected immunohistochemically.
RESULTS: HCjEC expressed TLR5-specific mRNA and TLR5 protein. In HCjEC stimulated with flagellins derived from P. aeruginosa and S. marcescens, IL-6 and IL-8 production was increased and IL-6 and IL-8 mRNA was up-regulated. Flagellins from S. typhimurium and B. subtilis did not induce the up-regulation of these genes and proteins. TLR5 protein was detected on the basolateral- but not the apical side of human conjunctival epithelium.
CONCLUSIONS: Human conjunctival epithelium harbors functional TLR5. Considering the spatially selective basolateral localization of TLR5 protein, we postulate that flagellins from ocular-pathogenic bacteria induce inflammatory responses when disruption of the epithelial barrier permits their transmigration to the basolateral side but not under healthy physiological conditions on the ocular surface.
- Ocular surface
- Toll-like receptor 5 (TLR5)
- human conjunctival epithelial cells
- inflammatory cytokines
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Supplemental Figure 1
Production of IL-6 and IL-8 in human conjunctival epithelial cells was dose dependent. Dose analysis was performed by 0.1ng/ml, 1ng/ml, 10ng/ml and 100ng/ml. The strongest response was achieved with 100ng/ml of P. aeruginosa-, and S. marcescense- derived flagellin. Data represents means ± SEM from the experiment with triplicated dishes.
Supplemental Figure 2
The relative expression of IL-6 and IL-8 mRNA in human conjunctival epithelial cells with time-course. Time analysis was performed at 0hr, 1hr, 3hr and 6hr after stimulation with 100ng/ml of P. aeruginosa- derived flagellin. The strongest response was achieved at 1hr after stimulation. Data represents means ± SEM from the experiment with triplicated dishes.
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