Immune recognition of Pseudomonas aeruginosa mediated by the IPAF/NLRC4 inflammasome

Abstract

Pseudomonas aeruginosa is a Gram-negative bacterium that causes opportunistic infections in immunocompromised individuals. P. aeruginosa employs a type III secretion system to inject effector molecules into the cytoplasm of the host cell. This interaction with the host cell leads to inflammatory responses that eventually result in cell death. We show that infection of macrophages with P. aeruginosa results in activation of caspase-1 in an IPAF-dependent, but flagellin-independent, manner. Macrophages deficient in IPAF or caspase-1 were markedly resistant to P. aeruginosa-induced cell death and release of the proinflammatory cytokine interleukin (IL)-1beta. A subset of P. aeruginosa isolates express the effector molecule exoenzyme U (ExoU), which we demonstrate is capable of inhibiting caspase-1-driven proinflammatory cytokine production. This study shows a key role for IPAF and capase-1 in innate immune responses to the pathogen P. aeruginosa, and also demonstrates that virulent ExoU-expressing strains of P. aeruginosa can circumvent this innate immune response.

Figure 1.

P. aeruginosa–induced macrophage cell death and IL-1β secretion are dependent on IPAF and caspase-1. LPS-primed macrophages from WT, caspase-1–, ASC-, NALP3-, or IPAF-deficient mice were infected with P. aeruginosa strain PAK at a MOI of 20 bacteria per macrophage. Culture supernatants were collected 1 h after infection (A–D) or as indicated (E and F). Cytotoxicity was measured by LDH release and expressed as a percentage of LDH release by Triton X-100 detergent (A, C, and E). IL-1β release into culture supernatants was measured by ELISA (B, D, and F). Determinations were performed in triplicate and expressed as the mean ± the SD. *, P = 0.01. Results are representative of two (E and F) and three (A–D) separate experiments. (G and H) Lysates from LPS-primed WT, IPAF-, and ASC-deficient macrophages infected with P. aeruginosa strain PAK at a MOI of 20 bacteria per macrophage for the indicated times were immunoblotted with antibodies against the p10 subunit of caspase-1. Results are representative of two independent experiments.

Figure 2.

ExoU can mediate macrophage cell death in an IPAF-independent manner. (A) LPS-stimulated macrophages from WT or IPAF-deficient mice were infected with the P. aeruginosa strain PA103, PA103ΔU, PA103ΔT, or PA103ΔUT at a MOI of 20 bacteria per macrophage. 1 h after infection, culture supernatants were collected and cytotoxicity was measured by LDH release and expressed as a percentage of LDH release by Triton X-100 detergent. (B and C) Nonstimulated WT (B) or IPAF^−/− (C) macrophages were infected with the P. aeruginosa strain PA103 or PA103ΔU at a MOI of 20 bacteria per macrophage. At the indicated times after infection, culture supernatants were collected and cytotoxicity was measured by LDH release into the culture supernatant. Determinations were performed in triplicate and expressed as the mean ± the SD. Results are representative of two to three separate experiments.

Figure 3.

P. aeruginosa ExoU inhibits caspase-1 activation. (A–C) Nonstimulated WT macrophages were infected with P. aeruginosa strain PA103 or PA103ΔU at a MOI of 20 bacteria per macrophage. At the indicated times after infection, culture supernatants were collected and IL-1β, TNF-α, and IL-6 release into culture supernatants was measured by ELISA. Results are expressed as the mean ± SEM of four separate experiments, each performed in triplicate. (D and E) Lysates from nonstimulated WT macrophages infected with P. aeruginosa strain PA103 or PA103ΔU at a MOI of 20 bacteria per macrophage for the indicated times were immunoblotted with antibodies against IL-β the p10 subunit of caspase-1, IκBα, phosphorylated-p38, p38, phosphorylated-ERK1/2, or ERK1/2. Results are representative of two independent experiments.

Figure 4.

P. aeruginosa ExoU phospholipase activity is required for inhibition of caspase-1 activation. (A and B) LPS-stimulated WT macrophages were either left untreated or pretreated with 100 μM AACOCF₃ for 30 min, and then infected with the P. aeruginosa strain PA103 or PA103ΔU at a MOI of 20 bacteria per macrophage. 1 h after infection, culture supernatants were collected. Parallel wells of untreated or AACOCF₃-pretreated LPS-stimulated WT macrophages were stimulated with 5 mM ATP for 30 min, and then culture supernatants were collected. Cytotoxicity was measured by LDH release into culture supernatants, and IL-1β release was measured by ELISA. Determinations were performed in triplicate and expressed as the mean ± the SD. *, P = 0.03; **, P = 0.0001. Results are representative of two separate experiments. (C) Nonstimulated WT macrophages were either left untreated or pretreated with 100 μM AACOCF₃ for 30 min, and then infected with the P. aeruginosa strain PA103 or PA103ΔU at a MOI of 20 bacteria per macrophage. 90 min after infection, the combined lysate of cells and supernatant were immunoblotted with antibodies against the p10 subunit of caspase-1. (D) Nonstimulated WT macrophages were infected with the P. aeruginosa strain PA103, PA103ΔUT, PA103ΔU, PA103ΔT, PA103ΔUT/LS608, or PA103ΔUT/S142A at a MOI of 20 bacteria per macrophage. 3 h after infection, culture supernatants were collected and IL-1β release was measured by ELISA. Determinations were performed in triplicate and expressed as the mean ± the SD. Results are representative of three separate experiments.

Figure 5.

In vivo cytokine production induced by P. aeruginosa peritonitis. (A–C) WT mice were injected intraperitoneally with 1 × 10⁷ CFU of either PA103 (n = 15 mice) or PA103ΔU (n = 15 mice); 4 h after infection, mice were killed and blood was collected by cardiac puncture. Serum IL-1β, IL-18, and TNFα concentrations were measured by ELISA. Data represent the mean ± the SEM. *, P = 0.036; **, P < 0.0001. (D–F) WT or IPAF^−/− mice were injected intraperitoneally with 1 × 10⁷ CFU of PA103ΔU; 4 h after infection, mice were killed and blood was collected by cardiac puncture. Serum IL-1β (n = 10 mice per group), IL-18 (n = 10 mice per group), and TNFα (n = 10 WT mice, n = 9 IPAF^−/− mice) concentrations were measured by ELISA. Data represent the mean ± the SEM, ***, P = 0.023; ****, P = 0.014. (G) WT (n = 8), IPAF^+/− (n = 6), or IPAF^−/− (n = 10) mice were injected intraperitoneally with 5 × 10⁶ CFU of PA103ΔU; 12 h after infection, mice were killed and peritoneal lavage with 10 ml of PBS was collected, spleens were removed and homogenized, and dilutions were plated on Vogel-Bonner minimal media for enumeration of CFUs. Bacterial counts from peritoneal lavage and spleen of IPAF^−/− mice were significantly higher compared with WT mice (P < 0.0001 and P = 0.0009, respectively) and with IPAF^+/− mice (P = 0.0005 and P = 0.0017, respectively).

Figure 6.

An intact TTSS is required for P. aeruginosa-mediated caspase-1 activation. LPS-stimulated WT macrophages were infected with the P. aeruginosa strain PA103ΔUT, PA103MutN, PA103Mut1, PA103ΔpilA, PA103ΔpopB, or PA103ΔpopD at a MOI of 20 bacteria per macrophage. At the indicated time (A and B) or 3 h (D and E) after infection, culture supernatants were collected and cytotoxicity was measured by LDH release and expressed as a percentage of LDH release by Triton X-100 detergent. IL-1β release into the culture supernatant was measured by ELISA. Determinations were performed in triplicate and expressed as the mean ± the SD. Results are representative of two (A and B) or three (D and E) separate experiments. (C) Lysates from LPS-stimulated WT macrophages that were infected with the P. aeruginosa strain PA103ΔUT, PA103MutN, PA103Mut1, or PA103ΔpilA for 3 h were immunoblotted with antibodies against the p10 subunit of caspase-1.

Figure 7.

IPAF-dependent activation of caspase-1 by P. aeruginosa does not require flagellin. (A) LPS-stimulated macrophages from WT or IPAF-deficient mice were infected with P. aeruginosa strain PAK or the flagellin-deficient PAKΔfliC at a MOI of 20 bacteria per macrophage. 1 h after infection, culture supernatants were collected, and IL-1β release into culture supernatants was measured by ELISA. Determinations were performed in triplicate and expressed as the mean ± the SD. Results are representative of three separate experiments. (B and C) LPS-stimulated macrophages from WT or IPAF-deficient mice were infected with P. aeruginosa strain PAKΔfliC at a MOI of 20 bacteria per macrophage. (B) At the indicated times after infection, culture supernatants were collected and IL-1β release into culture supernatants was measured by ELISA. (C) Lysates were immunoblotted with antibodies against the p10 subunit of caspase-1. Determinations were performed in triplicate and expressed as the mean ± the SD. Results are representative of two separate experiments.