Canonical Inflammasomes Drive IFN-γ to Prime Caspase-11 in Defense against a Cytosol-Invasive Bacterium

Abstract

The inflammatory caspases 1 and 11 are activated in response to different agonists and act independently to induce pyroptosis. In the context of IL-1β/IL-18 secretion, however, in vitro studies indicate that caspase-11 acts upstream of NLRP3 and caspase-1. By contrast, studying infection in vivo by the cytosol-invasive bacterium Burkholderia thailandensis, we find that caspase-1 activity is required upstream of caspase-11 to control infection. Caspase-1-activated IL-18 induces IFN-γ to prime caspase-11 and rapidly clear B. thailandensis infection. In the absence of IL-18, bacterial burdens persist, eventually triggering other signals that induce IFN-γ. Whereas IFN-γ was essential, endogenous type I interferons were insufficient to prime caspase-11. Although mice transgenic for caspase-4, the human ortholog of caspase-11, cleared B. thailandensis in vivo, they did not strictly require IFN-γ priming. Thus, caspase-1 provides priming signals upstream of caspase-11 but not caspase-4 during murine defense against a cytosol-invasive bacterium.

Figure 1. Both caspase-1 and -11 confer resistance to Burkholderia thailandensis

(A, B) Mice were infected with 2×10⁷ cfu of either unpassaged (u.p.) or mouse-passaged (m.p.) B. thailandensis (Bth) and survival was monitored. Data are representative of two (A) or pooled from at least three experiments (B). (C) Mice were infected with 2 × 10⁷ cfu of m.p. Bth, and bacterial burdens were measured in spleen, liver, or mediastinal lymph nodes 24 hours later. Data are representative of three independent experiments. Dashed line: limit of detection. (D, E) Mice were infected with the indicated dose of m.p. Bth and survival monitored or bacterial burdens assessed at the indicated time. Data are representative of two (D) or pooled from two experiments. * p ≤ 0.05; log rank test (a). For number of mice in each panel see Table S1. See also Figure S1.

Figure 2. Differential contribution of caspase-1 and -11 to defense against B. thailandensis

(A, B, and F) Mice were infected with Bth at the indicated dose and survival was monitored. Data are pooled from at least two (E) or three experiments (A and B). (C, D, and E) Bacterial burdens were determined in spleen, liver, or mediastinal lymph nodes at the indicated time post-infection. Data are representative of two (C) or three (D, and E) independent experiments. *Student’s t test (C, D, and E). For number of mice in each panel see Table S1. See also Figure S2

Figure 3. NLRC4 and NLRP3 both contribute to defense against B. thailandensis

(A) Mice were infected i.p with 2×10⁷ CFU of Bth and survival was monitored. Data are pooled from at least two experiments. (B and C) IFN-γ primed BMMs were stimulated with CTB/LPS for 4h and cytotoxicity was determined by LDH release assay. Histogram represents mean ± SEM of at least two independent experiments. (D and E) Mice were infected i.p. with the indicated doses of Bth and survival was monitored. Data are pooled from 3 experiments. (F) Mice were sacrificed 24 hrs post-infection with 2×10⁷ CFU and the bacterial burdens in organs were assessed. Data are representative of three independent experiments. * Student’s t test (F). For number of mice in each panel see Table S1.

Figure 4. IL-18 is required for defense against B. thailandensis

(A and B) Mice were infected i.p. with the indicated dose of and survival was monitored. Data are pooled from at least three experiments. (C and D) Mice were infected with 2×10⁷ cfu of Bth and bacterial burdens were assessed. Data are representative of three independent experiments. (E–F) Serum IL-18 (E–F) or bacterial burdens (F) were determined. Data are pooled from three (E) or two (F) experiments. * p ≤ 0.05; log rank test (A) and Student’s t test (C, D, and E). For number of mice in each panel see Table S1.

Figure 5. IFN-γ is critical to defend against B. thailandensis

(A, B, and C) Mice were infected i.p. with Bth at the indicated dose, and survival was monitored. Data are pooled from at least experiments. (D and E) Bacterial burdens (D) and serum IFN-γ levels (E) were assessed 24 h after infection at the indicated dose. Data are representative of 3 independent experiments (D), or pooled from 3 experiments (E). (F–J) Mice were infected i.p. with 10⁴ or 2×10⁷ cfu Bth and splenic bacterial burdens and IFN-γ levels were determined at the indicated time. Data are representative of two (F, G, I, J) or three (H) independent experiments. * Student’s t test (D–J). For number of mice in each panel see Table S1. See also Figure S3A.

Figure 6. IFN-γ primes caspase-11 in vivo to protect against B. thailandensis

(A) IFN-γ, IFN-β, or poly(I:C) primed BMMs were infected with L. monocytogenes (Lm) plus LPS for 4hrs. Cytotoxicity was determined by LDH release assay. Line represents mean ± SEM of at least two independent experiments. (B) Mice were infected with Bth at the indicated doses and survival was monitored. Data are pooled from at least two experiments. (C) Western blot for caspase-11 in splenic lysates from WTor Ifng^−/− mice 6h after PBS or poly (I:C) priming. Representative of three experiments. (D) Mice were infected with 10⁴ cfu of Bth. 24 hrs post infection they were injected with PBS or poly (I:C). 48 hrs after PBS or poly (I:C) priming, bacterial burdens in organs homogenates were determined. Data are representative of three independent experiments. (E) Mice were infected with 10⁴ cfu of Bth. 48 hrs post infection they were injected with PBS or poly(I:C). 9 hrs after PBS or poly(I:C) priming, serum HMGB1 levels were assessed by ELISA. Data are pooled from three experiments. (F) Splenic bacterial burdens in WT, Casp11^−/−, and Gbp^chr3−/− 3 days post infection with 10⁴ cfu Bth. Data are representative of two experiments. (G) Survival of WT, Casp11^−/−, and Nos2^−/− mice after infection with 2×10⁷ cfu of Bth. Data are pooled from at least three experiments. * Student’s t test (D and E). For number of mice in each panel see Table S1. See also Figure S3B

Figure 7. Human caspase-4 does not require IFN-γ priming in vivo

(A) LPS primed BMM were infected with Bth for 4hrs and cytotoxicity was determined by LDH release. Histogram represents mean ± SEM of at least two independent experiments. (B and C) Mice were infected with 10⁴ cfu Bth, and survival (B) or bacterial burden in spleen and liver were determined 48 hrs post infection (C). Data are representative of two independent experiments. (D) Casp1^−/−Casp11^−/−CASP4^Tg mice were injected with PBS or 5μg IFN-γ. 6h later spleens were harvested and caspase-4 and actin levels determined by immunoblot. (E) Mice were infected with 10⁴ cfu of Bth. IFN-γ was depleted using two doses of anti-IFN-γ antibodies or isotype controls 24 hrs prior and 24 hrs post infection. Splenic bacterial burdens were determined 72hrs post infection. Data are representative of two independent experiments. * Student’s t test (C and D). For number of mice in each panel see Table S1. See also Figure S4