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Modulation of Corneal Epithelial Innate Immune Response to Pseudomonas Infection by Flagellin Pretreatment

¹From the Kresge Eye Institute, Departments of Ophthalmology and ²Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.

PURPOSE. A prior study showed that Toll-like receptor (TLR)-5 recognizes Pseudomonas aeruginosa flagellin and triggers the production of proinflammatory cytokines in human corneal epithelial cells (HCECs). The present study was conducted to determine how the inflammatory response is modulated after TLR activation in HCECs.

METHODS. HUCL cells, a telomerase-immortalized HCEC line, and primary cultures of HCECs were pretreated with low-dose flagellin and then challenged, with either a high dose of flagellin or with Pseudomonas. NF-B activation was determined by the extent of IB- phosphorylation and degradation and of nuclear p65 DNA binding. The amount of cytokines in the culture media was assessed by ELISA. The activation of p38 and JNK and the cellular expression of TLR5 were determined by Western blot analysis. Cell surface distribution of TLR5 was assessed by flow cytometry. The expression and secretion of antimicrobial peptides were assessed by semiquantitative RT-PCR and slot–blot analysis, respectively.

RESULTS. Pre-exposure (12–24 hours) of HCECs to low-dose flagellin induced a state of tolerance, characterized by impaired activation of the NF-B, p38, and JNK pathways and reduced production of IL-8 and TNF- on subsequent challenge with a high dose of flagellin. Flagellin-induced tolerance did not alter the cellular level and surface distribution of TLR5. Furthermore, flagellin priming of HCECs dampened the inflammatory response of HCECs to live Pseudomonas. Pseudomonas-induced upregulation of antimicrobial genes such as hBD2 and LL-37 was augmented, even in tolerized HCECs.

CONCLUSIONS. Pre-exposure of the ocular surface to TLR agonists may induce protective mechanisms that not only modulate the host inflammatory response but also provide an innate defense against bacterial infection in the cornea.

This article has been cited by other articles:

				A. Kumar, L. D. Hazlett, and F.-S. X. Yu Flagellin Suppresses the Inflammatory Response and Enhances Bacterial Clearance in a Murine Model of Pseudomonas aeruginosa Keratitis Infect. Immun., January 1, 2008; 76(1): 89 - 96. [Abstract] [Full Text] [PDF]