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Human conjunctival epithelial cells express functional Toll-like receptor 5
  1. Kentaro Kojima (kkojima{at}ophth.kpu-m.ac.jp),
  2. Mayumi Ueta (mueta{at}ophth.kpu-m.ac.jp),
  3. Junji Hamuro (jhamuro{at}a4.rimnet.ne.jp),
  4. Yukako Hozono (yukako.nannan{at}nifty.com),
  5. Satoshi Kawasaki (skawasak{at}eye.ophth.kpu-m.ac.jp),
  6. Norihiko Yokoi (nyokoi{at}ophth.kpu-m.ac.jp),
  7. Shigeru Kinoshita (shigeruk{at}ophth.kpu-m.ac.jp)
  1. Kyoto Prefectural University of Medicine, Japan
  2. Kyoto Prefectural University of Medicine, Japan
  3. Kyoto Prefectural University of Medicine, Japan
  4. Kyoto Prefectural University of Medicine, Japan
  5. Kyoto Prefectural University of Medicine, Japan
  6. Kyoto Prefectural University of Medicine, Japan
  7. Kyoto Prefectural University of Medicine, Japan

    Abstract

    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
    • Conjunctiva
    • Inflammation
    • Toll-like receptor 5 (TLR5)
    • flagellin
    • 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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