May 2006
Volume 47, Issue 13
ARVO Annual Meeting Abstract  |   May 2006
A Novel Protective Role of TLR5 Agonist Flagellin in Inducing Tolerance of Human Corneal Epithelial Cells Against Pseudomonas Aeruginosa Infection in vitro
Author Affiliations & Notes
  • A. Kumar
    Ophthalmology, Wayne, Detroit, MI
  • J. Zhang
    Ophthalmology, Wayne, Detroit, MI
  • F.–S.X. Yu
    Ophthalmology, Wayne, Detroit, MI
  • Footnotes
    Commercial Relationships  A. Kumar, None; J. Zhang, None; F.X. Yu, None.
  • Footnotes
    Support  RO1–EY14080, RO1–EY10869 , RPB
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 5870. doi:
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      A. Kumar, J. Zhang, F.–S.X. Yu; A Novel Protective Role of TLR5 Agonist Flagellin in Inducing Tolerance of Human Corneal Epithelial Cells Against Pseudomonas Aeruginosa Infection in vitro . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5870.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : We previously showed that TLR5 recognized Pseudomonas aeruginosa flagellin and triggers the expression and/or release of proinflammatory cytokines in human corneal epithelial cells (HCECs). In the present study we sought to determine whether activation of HCECs by flagellin–TLR5 results in tolerance as manifested by decreased cytokine production and hyporesponsiveness to subsequent P. aeruginosa infection.

Methods: : HUCL, a telomerase–immortalized HCEC line, and primary culture of HCECs were pretreated with low dosages of P. aeruginosa (PA01) flagellin for a period of 12 to 24 h to induce tolerance. The flagellin–tolerized HCECs were then challenged either with higher dose of flagellin or live P. aeruginosa at MOI of 50–100. The activation of NF–ΚB and MAPKs (p38, JNK, ERK) was assessed by Western blotting using phosphor–specific antibodies. RT–PCR was used to detect the changes in mRNA levels of negative–regulators of TLR–signaling pathways such as Tollip, SIGIRR, IRAK–M, IRAK1, and IRAK2. Secretion of proinflammatory cytokine/chemokines IL–6, IL–8, and TNF–α in the culture media was assessed using ELISA.

Results: : Prolonged incubation (12 to 24h) of HUCL cells and primary HCECs with flagellin (as low as 10ng/ml) induced a state of hyporesponsiveness (tolerance) characterized by impaired activation of TLR–down streaming signaling pathways such as NF–kB and MAPKs (p38, JNK, ERK) and reduced cytokine (IL–6 and IL–8) production upon subsequent second challenge with higher (250ng/ml) doses of flagellin. Furthermore, priming of HCEs with flagellin greatly reduced the inflammatory response of HCEs, with diminished accumulation of cytokine/ chemokines (IL–6, –8 and TNF–α) in their culture media upon live P. aeruginosa infection. Pre–treatment with flagellin does not alter the total protein levels of TLR5 and adaptor molecule MyD88. Similarly no significant change in mRNA levels of Tollip, SIGIRR, IRAK–M, IRAK1, and IRAK2, was detected in flagellin tolerized HCECs.

Conclusions: : Exposure of HCECs to flagellin resulted in HCEC "tolerance", as manifest by greatly decreased cytokine production and hypo–responsiveness to PA challenge. Further study to elucidate the underlying mechanism of flagellin induced tolerance in HCECs is warranted.

Keywords: cornea: epithelium • Pseudomonas • inflammation 

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