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F. X. Yu, A. Kumar, G. Yoon; Topical Application of Flagellin Prevents the Development of Pseudomonas Keratitis in Mice via Induced Expression of Antimicrobial Peptide Cramp. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5539.
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We previously showed that subconjuctival administration of flagellin prevents the development of Pseudomonas aeruginosa (PA) keratitis in C57BL6 mice. In the present study, we tested whether a topical application of flagellin exhibit similar protective effects and determined the underlying mechanisms.
Flagellin (500 ng/eye) was topically applied on intact or needle wounded B6 mice cornea. At various time points, the corneas were either harvested and assayed for expression of cytokines and antimicrobial peptide CRAMP (mouse homologue of human LL-37) or subjected to PA infection by inoculation of injured corneas with 106 CFU/eye (ATCC 19660 strain). The corneal disease response was graded on different days post infection (p.i.) and slit lamp microscopy was performed. The corneas were also subjected to histopathological analysis and determination of cytokines and CRAMP expression using ELISA and Western blot, respectively.
Wounding induced rapid up-regulation of MIP-2 and IL-1β in the epithelium of B6 mouse cornea, peaking at 6 h, followed by a rapid decline at 12 and 24 h. While the induced expression of the cytokines was not affected by topical application of flagellin, the up-regulation of CRAMP was markedly increased. The CRAMP expression was induced by flagellin as early as 3h and remained elevated up to 24 h. Topical application of flagellin (500ng/eye) 6-24 h prior to PA inoculation significantly improved disease outcome (an average clinical score of 0.4 flagellin treated group vs. 3.2 in PBS group at 3 dpi), preserved structural integrity and transparency, and thus maintained vision in otherwise perforated corneas of the C57BL/6 mice.
The topical application of flagellin on wounded cornea is very effective to prevent the development of keratitis and the induced expression of antimicrobial peptide CRAMP plays an important role in innate defense of the cornea.
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