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A. Kumar, J. Zhang, L. D. Hazlett, F.-S. X. Yu; Flagellin Pretreatment Sppresses Inflammatory Response and Enhances Bacterial Clearance in Murine Model of Bacterial Keratitis. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3162.
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The aim of this study was to test the hypothesis that pre-exposure of the cornea to flagellin induces protective mechanisms in a murine model of Pseudomonas aeruginosa (PA)-keratitis and the underlying mechanisms can be exploited as novel approaches to treat microbial keratitis and other infectious diseases.
C57BL/6 mice were injected 100 ng PA flagellin subconjunctivally and 125 ng intraperitoneally (i.p.) per mice, with PBS as control. The pretreated mice were inoculated with 1.0 x 106 CFU of PA (ATCC19660) by applying 5-µl aliquot to wounded cornea. The corneal disease response was graded on different days post infection (p.i.) and further examined with slit lamp microscopy and histopathological analysis. Polymorphonuclear leukocyte (PMN) infiltration and expression of the inflammatory cytokines/chemokines in the infected corneas were assessed with MPO and real-time PCR respectively. Bacterial load in the cornea was determined by standard bacterial plate count. To determine whether flagellin also induces protective mechanisms in other tissues, normal and cystic fibrosis (CF) airway epithelial cells were pretreated with 50 ng/ml flagellin for 24 h and production of proinflammatory cytokine/chemokines in culture supernatant upon live PA or flagellin challenge was assessed using ELISA.
Administration of flagellin via subconjunctival and intraperitoneal injection 24 h prior to PA inoculation significantly improved disease outcome, preserved structural integrity and transparence, and thus maintained vision in otherwise perforated corneas of C57BL/6 mice. The flagellin pretreatment resulted in the suppression of PMN infiltration in late, but not in early stage of infection, also decreased the expression of proinflammatory genes (IL-1ß, MIP-2, IL-12, IFN-γ), and greatly enhanced bacterial clearance in the corneas of B6 mice. Strikingly, one time administration of flagellin resulted in total elimination of the invading bacteria in a large portion (60%) of the B6 mouse cornea. Significantly, flagellin exposure substantially reduced proinflammatory cytokine production in normal and CF cells upon subsequent challenge either with live PA or higher dose flagellin.
TLR5 ligand flagellin-induced protective mechanisms not only curb the host inflammatory response but also eliminate the invading pathogen in vivo and thus may be used as a novel therapeutic approach for better management of bacterial keratitis and other infectious diseases caused by PA.
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