The NLRP3 Inflammasome Is Required for Protection Against Pseudomonas Keratitis

Abstract

PURPOSE. The current study was designed to examine the role of the NLRP3 inflammasome pathway in the clearance of Pseudomonas aeruginosa (PA) infection in mouse corneas. METHODS. Corneas of wild type and NLRP3-/- mice were infected with PA. The severity of bacterial keratitis was graded on days 1 and 3 post-infection by slit lamp, and then corneas were harvested for: (i) bacterial enumeration, (ii) immune cell analysis by flow cytometry, (iii) immunoblotting analysis of cleaved caspase-1 and IL-1β, and (iv) IL-1β quantification by ELISA. In parallel experiments, severity of keratitis was examined in the wild-type mice receiving a subconjunctival injection of a highly selective NLRP3 inhibitor immediately prior to infection. RESULTS. Compared to wild type mice, NLRP3-/- mice 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. Accordingly, increased severity of infection in the NLRP3-/- mice was associated with reduced levels of cleaved forms of caspase-1 and IL-1β and reduced IL-1β + neutrophil infiltration in infected corneas. Likewise, corneas of mice receiving subconjunctival injections of NLRP3 inhibitor exhibited increased bacterial load, and reduced IL-1β expression. CONCLUSIONS. Activation of NLRP3 pathway is required for the clearance of PA infection in mouse corneas.