Caspase-11 protects against bacteria that escape the vacuole

Abstract

Caspases are either apoptotic or inflammatory. Among inflammatory caspases, caspase-1 and -11 trigger pyroptosis, a form of programmed cell death. Whereas both can be detrimental in inflammatory disease, only caspase-1 has an established protective role during infection. Here, we report that caspase-11 is required for innate immunity to cytosolic, but not vacuolar, bacteria. Although Salmonella typhimurium and Legionella pneumophila normally reside in the vacuole, specific mutants (sifA and sdhA, respectively) aberrantly enter the cytosol. These mutants triggered caspase-11, which enhanced clearance of S. typhimurium sifA in vivo. This response did not require NLRP3, NLRC4, or ASC inflammasome pathways. Burkholderia species that naturally invade the cytosol also triggered caspase-11, which protected mice from lethal challenge with B. thailandensis and B. pseudomallei. Thus, caspase-11 is critical for surviving exposure to ubiquitous environmental pathogens.

Fig. 1. Burkholderia detection and protection conferred by Casp1/11 is independent of all known canonical inflammasomes

Lipopolysaccharide (LPS) primed BMMs were infected with B. pseudomallei (A, G) or B. thailandensis (B, F) for 4h. (A, B) IL-1β secretion was determined by ELISA or (F, G) cytotoxicity was determined by LDH release assay. (C, D, E) Survival curves of wild type C57BL/6 or the indicated knockout mice infected 2×10⁷ cfu i.p. with B. thailandensis. Data are representative of at least 3 (A, B, F, G) or 2 (D, E) experiments. (C) Data are pooled from 3 experiments. For number of mice in each panel see Table S2. Statistically significant differences with respect to controls are indicated (Student's T-test or log rank test for survival; * = p ≤ 0.05, n.s. = p > 0.05).

Fig. 2. Diverse cytosolic bacteria activate pyroptosis independent of NLRC4, NLRP3 and ASC

(A) LPS-primed BMMs were infected for 4h with either B. thailandensis or the indicated mutants and cytotoxicity was determined. BMMs were infected for 8h with (B) S. typhimurium or S. typhimurium ΔsifA, (D) S. typhimurium ΔsifA or S. typhimurium ΔsifA flgB (D) and cytotoxicity was determined. LPS-primed BMM were infected for 8h with S. typhimurium or S. typhimurium ΔsifA (C), S. typhimurium ΔsifA or S. typhimurium ΔsifA flgB (E) and IL-1β secretion was determined. (F, G) Cytotoxicity in wild type or Asc^−/− BMMs infected for 4h with L. pneumophila, L. pneumophila ΔflaA or L. pneumophila ΔsdhA ΔflaA. Cytotoxicity was determined by LDH release and IL-1β secretion by ELISA. Data are representative of at least 3 experiments. Statistically significant differences with respect to controls are indicated (Student's T-test; * = p ≤ 0.05, n.s. = p > 0.05).

Fig. 3. Caspase-11 mediates pyroptosis after infection by cytosolic bacteria. (A–I)

Macrophage cytotoxicity and IL-1β secretion were determined after infection with S. typhimurium ΔsifA (8h), L. pneumophila ΔflaA ΔsdhA (4h), or B. thailandensis (4h). (A) C57BL/6, Casp1^−/−Casp11^−/−, Tlr4^−/−, Trif^−/−, and Myd88^−/− BMM infected with S. typhimurium ΔsifA with or without IFN-γ priming prior to infection. (B–C) Retroviral transduction was used to complement Casp1 or Casp11 in Casp1^−/−Casp11^−/− iBMM. Macrophages were primed with LPS (B) or IFN-γ (C) and responses to B. thailandensis (B) or S. typhimurium ΔsifA (C) infection were examined. (D) Control or complemented Casp1^−/−Casp11^−/− BMM infected with L. pneumophila ΔflaA ΔsdhA. (E–F) Retroviral transduction was used to introduce control or Casp11-targeting shRNAmir into Nlrc4^−/−Asc^−/− iBMM. Macrophages were primed overnight with LPS (E) or IFN-γ (F) and then infected as indicated. (G–I) C57BL/6, Casp1^−/−Casp11^−/−, and Casp11^−/− BMM infected with B. thailandensis (G), S. typhimurium ΔsifA (H), or L. pneumophila ΔflaA ΔsdhA (I). Data are representative of at least 3 (A–C, E, G, H) or 2 (D, F, I) independent experiments. Statistically significant differences with respect to controls are indicated (Student's T-test; * = p ≤ 0.05). nd, none detected.

Fig. 4. Caspase-11 protects against cytosolic bacteria in vivo

(A, B) S. typhimurium or S. typhimurium ΔsifA were injected i.p. into C57BL/6 (1000 cfu) or Casp1^−/−Casp11^−/− mice (250 cfu) and survival was monitored. (C–E) The indicated mice were infected with 5×10⁴ cfu of both wild type S. typhimurium and S. typhimurium ΔsifA marked with ampicillin or kanamycin resistance, respectively. Bacterial loads from 3–4 mice per genotype were determined 48h later and competitive index calculated (CI = log (S. typhimurium ΔsifA/ S. typhimurium)). A CI of −1 corresponds to 10 cfu of S. typhimurium for every 1 cfu of S. typhimurium ΔsifA. (F–G) C57BL/6, Casp1^−/−Casp11^−/−, or Casp11^−/− mice were infected with (F) 2×10⁷ cfu mouse passaged B. thailandensis i.p. or (G) 100 cfu B. pseudomallei i.n. (A, B, F, G) Data are pooled from two independent experiments. (C–E) Representative of 2 experiments. For number of mice in each panel see Table S2. Statistically significant differences with respect to controls are indicated (Student's T-test or log rank test for survival; * = p ≤ 0.05, n.s. = p > 0.05).