Activation of inflammasome signaling mediates pathology of acute P. aeruginosa pneumonia

Abstract

The respiratory tract is exceptionally well defended against infection from inhaled bacteria, with multiple proinflammatory signaling cascades recruiting phagocytes to clear airway pathogens. However, organisms that efficiently activate damaging innate immune responses, such as those mediated by the inflammasome and caspase-1, may cause pulmonary damage and interfere with bacterial clearance. The extracellular, opportunistic pathogen Pseudomonas aeruginosa expresses not only pathogen-associated molecular patterns that activate NF-κB signaling in epithelial and immune cells, but also flagella that activate the NLRC4 inflammasome. We demonstrate that induction of inflammasome signaling, ascribed primarily to the alveolar macrophage, impaired P. aeruginosa clearance and was associated with increased apoptosis/pyroptosis and mortality in a murine model of acute pneumonia. Strategies that limited inflammasome activation, including infection by fliC mutants, depletion of macrophages, deletion of NLRC4, reduction of IL-1β and IL-18 production, inhibition of caspase-1, and inhibition of downstream signaling in IL-1R- or IL-18R-null mice, all resulted in enhanced bacterial clearance and diminished pathology. These results demonstrate that the inflammasome provides a potential target to limit the pathological consequences of acute P. aeruginosa pulmonary infection.

Figure 1. Role of flagella in P. aeruginosa infection.

(A and B) Numbers of fliC, motAB, and WT PAK recovered from (A) BAL and (B) lung tissue. (C) Mortality 18 hours after infection (10⁸ CFU/mouse; n = 8). (D) H&E (original magnification, ×100) and TUNEL (original magnification, ×400) staining of PAK-infected lungs. (E) Lung injury score. (F) FACS analysis of immune cell populations — neutrophils, AMs, and DCs — in BAL. (G) IL-1β levels in BAL. (H) IL-1β secreted from AMs in vitro. (I) IL-1β secreted from primary murine nasal epithelial cells in vitro. Levels are normalized to WT. (J) Cleavage of pro–caspase-1 in lung tissue of WT, fliC, and motAB PAK–infected mice. Data in A–I are from at least 2 independent experiments.

Figure 2. AMs mediate pathology via IL-1β.

(A) FACS plots of AMs and DCs from BAL of uninfected mice treated with PBS or clodronate liposome (Clod). (B and C) Immune cell populations in (B) BAL and (C) lungs after infection. (D and E) Levels of (D) IL-1β and (E) IL-18 in BAL. (F and G) Numbers of PAK recovered from (F) BAL and (G) lung. (H) Mortality 18 hours after infection (10⁸ CFU/mouse; n = 12 per group). (I) Numbers of PAK recovered from spleen. (J) H&E (original magnification, ×100) and TUNEL (original magnification, ×400) staining of PAK-infected lungs. (K) Lung injury score. Data are representative of at least 2 independent experiments.

Figure 3. Role of the NLRC4 inflammasome in P. aeruginosa pneumonia.

(A and B) Numbers of bacteria in (A) BAL and (B) lungs of WT and Nlrc4^–/– mice. (C) IL-1β concentration and (D) numbers of bacteria in BAL of mice treated with DMSO and caspase-1 inhibitor (CASPinh) after PAK infection. (E) Immune cell populations after infection. PMN, polymorphonuclear neutrophils. (F) H&E (original magnification, ×100) and (G) TUNEL (original magnification, ×400) staining of PAK-infected lungs. (H) Mortality 18 hours after infection (10⁸ CFU/mouse; n = 15). Data are representative of at least 2 independent experiments.

Figure 4. Type I IFN regulation of inflammasome-dependent cytokines.

(A and B) Secretion of (A) IL-1β and (B) IL-18 from AMs pretreated with PBS or type I IFN (IFN-β; 5 μg/ml) in vitro. (C) IL-1β in BAL of uninfected WT and Ifnar^–/– mice. (D) TUNEL staining of uninfected WT and Ifnar^–/– mice (original magnification, ×400). (E and F) Levels of (E) IL-1β and (F) IL-18 in BAL of PAK-infected WT mice pretreated with PBS or poly(I:C). (G) Numbers of bacteria in BAL of PAK-infected WT mice pretreated with PBS or poly(I:C). (H) TUNEL staining of infected mice pretreated with PBS or poly(I:C) (original magnification, ×400). (I–K) Levels of (I) KC, (J) TNF, and (K) IL-1Ra in BAL of infected mice. Data are representative of at least 2 independent experiments.

Figure 5. IL-1 and IL-18 inhibition improves P. aeruginosa clearance.

(A and B) Numbers of bacteria in BAL of (A) Il1r1^–/– and (B) Il18r1^–/– mice compared with WT. (C and D) Immune cell populations in BAL of (C) Il1r1^–/– and (D) Il18r1^–/– mice after infection, compared with WT. Data are representative of at least 2 independent experiments.