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Noorjahan A Panjwani, Zhiyi Cao, Vijay Rathinam, Mihaela G Gadjeva, Gerald Pier, Tanweer Zaidi, Abdulraouf Ramadan; Activation of NLRP3 pathway is required for clearance P. aeruginosa infection in C57BL/6 mouse corneas. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1546.
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© ARVO (1962-2015); The Authors (2016-present)
The role of inflammasome pathway in the regulation of P. aeruginosa (PA) keratitis is unclear. The current study was designed to examine the role of the NLRP3 inflammasome pathway in the clearance of PA infection in mouse corneas.
Two sets of experiments were performed. In the first set, corneas of C57BL/6 mice were infected with PA (strain 6077 or 6294). Immediately prior to infection, each animal received a subconjunctival injection of the NLRP3 inhibitor (MCC950, AdipoGen, and San Diego, CA 300µM in 10 µl PBS) or PBS. The severity of bacterial keratitis was graded on day 1 and day 3 post-infection (pi) by slit lamp using a scoring system ranging from 0 to 4, and then corneas were harvested for: (i) bacterial enumeration, (ii) immune cell analysis by flow cytometry, (iii) analysis of cleaved caspase-1 and IL1-β by Western blot analysis, and (iv) IL1β quantification by ELISA. In the second set, the severity of PA keratitis and infiltration of immune cells were compared between the PA-infected corneas of NLRP3 knockout (KO) mice and wild-type (WT, C57BL/6) mice
Mice receiving subconjunctival injections of NLRP3 inhibitor exhibited more severe infection as indicated by an increase in opacity score and an increase in bacterial load. The hallmark of inflammasome assembly is the activation of proinflammatory caspase-1 and IL-1β by cleavage of their precursors, pro-caspase-1 and pro-IL-1β, respectively; cleaved/activated IL-1β ultimately results in neutrophil recruitment that, in turn, leads to bacterial cell death to clear the infection. Accordingly, increased severity of infection in the inhibitor-treated mice was associated with reduced levels of cleaved forms of caspase-1 and IL-1β and reduced neutrophil infiltration in infected corneas. Additional experiments revealed that just like NLRP3 inhibitor-treated mice, the NLRP3 KO mice exhibited increased bacterial load, reduced IL-1b expression and reduced infiltration of IL-1β+ neutrophils. Similar results were obtained regardless of whether the infection was induced with the cytotoxic strain PA 6077 or an invasive strain PA 6294.
Activation of NLRP3 pathway is required for clearance of PA infection in mouse corneas.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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