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.

Figure 1.

Activation of NLRP3 pathway is required for clearance of P. aeruginosa infection. (A, B, C) NLRP3^−/− mice develop more severe keratitis: The central corneas of wild-type and NLRP3^−/− mice were scarified with 3 parallel 1-mm incisions using a 26-gauge needle, and a 5-µl drop of bacteria (1 × 10² CFU, PA strain 6077) was applied to the cornea. On day 1 p.i., the severity of bacterial keratitis was graded using a scoring system ranging from 0 to 4 as discribed in Methods, corneal thickness was measured by OCT, and then corneas were harvested for bacterial enumeration. (Ai) Represntative images showing corneal opacity; (Aii) opacity score; B corneal thickness, C bacterial load. (D, E, F) Mice treated with NLRP3 inhibitor develop more severe keratitis. Immediately prioir to infection, C57BL/6 mice received a subconjunctival injection of NLRP3 inhibitor (MCC950, Adipogene) or vehicle (PBS) and on day 1 p.i., severity of infection was graded as decribed above. Di Representative images showing corneal opacity; Dii opacity score; E corneal thickness; F bacterial load. Bacterial load data are reported as log10 CFU/cornea ± SEM. Data show mean values ± SEM. Note that corneal opacity, thickness, and bacterial load are all higher in NLRP3^−/− mice and NLRP3 inhibitor-treated mice compared to the respective control mice. Combined results of 3 separate experiments are shown; 8 to 10 corneas per group were used in each experiment. Statistical levels of significance were analyzed by the student t-test. *P < 0.05, ***P < 0.001.

Figure 2.

NLRP3 deletion impairs IL-1β-Caspase-1 pathway activation in infected corneas. (A, B) Infected corneas of NLRP3^−/− and wild-type mice (6 mice/group) were harvested on day 1 p.i., pooled groupwise (6 corneas/group), and were processed either for Western blot analysis to detect pro- and cleaved caspase-1 and IL-1β, or ELISA to quantify total IL-β. A A representative Western blot showing that expression levels of activated caspase-1 and IL-1β are markedly reduced in corneas of NLRP3^−/− mice, and B quantification of total IL-1β by ELISA. (C, D) Immediately prioir to infection, C57BL/6 mice received a subconjuctival injection of NLRP3 inhibitor (MCC950, Adipogene) or vehicle (PBS), and on day 1 p.i., corneas were harvested (6 eyes/group) and processed for Western blot analysis and ELISA. C A representative Western blot showing that expression levels of activated caspase-1 and IL-1β are markedly reduced in corneas of NLRP3 inhibitor-treated mice. D Quantification of total IL-1β by ELISA. A, C Blots are representative of three independent experiments. B, D Data are plotted as mean ± SEM. Combined results of three independent experiemnts are shown (6 pooled corneas/experiment). Statistical levels of significance were analyzed by the student t-test. **P < 0.01, ***P < 0.001.

Figure 3.

NLRP3 deletion decreases infiltration of activated immune cells. PA-infected corneas of wild-type and NLRP3^−/− mice harvested on day 1 p.i., were processed to prepare single cell suspenssion for FACS anaylsis for quantitifaction of activated IL-1β⁺ CD45⁺ CD11b⁺ Ly6G⁺ neutrophils, and CD45⁺ CD11b⁺ 4/80⁺ macrophages, and quantitifaction of caspase-1 activity. (A) Left: A representative FACS plot showing IL-1β⁺ neutrophils and macrophages; right: quantification of IL-1β⁺ neutrophils and macrophages. (B) Caspase-1 activty in nuetrophils and macrophages was measured by FAM-FLICA caspase-1 assaay kit. Left: FACS histogram showing caspase-1 expression intensity on neutrophils and macorphages; right: quantification of caspase-1 using mean fluorescence intensity. Data show mean ± SEM. Combined results of 3 separate experiments are shown; 10 to 12 pooled corneas per group were used in each experiment. Statistical levels of significance were analyzed by the student t-test. *P < 0.05. Note that infiltration of neutrophils and macrophages expressing IL-1β as well as caspase activity of the infiltrated cells is significantly reduced in the infected corneas of the NLRP3^−/− mice compared to the wild type mice.