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Mihaela G. Gadjeva; Macrophage Migration Inhibitory Factor Regulates The Activation Of Nadph oxydase To Produce Reactive Oxygen Species In Response To P.aeruginosa During Ocular Keratitis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5821.
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Ulcerative keratitis due to Pseudomonas aeruginosa is a sight-threatening disease leading to loss of vision. Here, we investigated the role of reactive oxygen species (ROS) release in regulating susceptibility to P.aeruginosa-induced keratitis.
gp91 knock-outs, MIF knock-outs, and C57Bl6 control mice were anesthetized with a ketamine and xylazine injection, a scratch was made on the cornea, and an inoculum of P. aeruginosa was applied. For evaluation of corneal pathology, daily scores were recorded based on a scoring system from 0 to 4. To determine the levels of bacteria in the cornea, mice were sacrificed and corneas harvested at 24, 48, and 72 h after infection. To determine the bacterial levels of P. aeruginosa, corneas were excised, the tissue was vortexted, and bacteria plated on P. aeruginosa selective cetrimide plates. To analyze the ability of murine PMN to respond to P. aeruginosa by ROS production, bone marrow derived PMNs were purified, stimulated with P. aerginosa, and the amount of released ROS species was quantified by luminol based-assay.
gp91 knock-out mice were significantly more sensitive to P.aeruginosa-induced keratitis than control mice. This sensitivity was associated with dramatic increase in the bacterial burden in the eyes, and faster disease progression. P. aeruginosa strains 6294 and PAO1 induced robust production of ROS from PMNs derived from the C57Bl6 mice, but not from the gp91 knock-out mice, which were incapable of the assembly of the NADPH oxydase. The activation of the NADPH oxydase depended on the presence of macrophage migration inhibitory factor (MIF), a cytokine/enzyme with complex biological functions. We found that high concentrations of MIF inhibited NADPH-dependent ROS production via the MAPK p42/44 pathway, whereas low concentrations activated NADPH-dependent ROS release, suggesting that different concentrations of MIF in the environment initiated distinct PMN responsnes.
Initially, MIF was described as an immunosuppressive molecule that contributes to the immune privilege of the eye. However, MIF is known to have pro-inflammatory functions. Here, we demonstrate that these activities of MIF are not mutually exclusive, and are dose, and cell-type dependent. This is especially important with regard to the ocular innate responses to infection, where local increase in MIF levels during infection, may have immunosuppressive effect on the bactericidal properties of PMNs.
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