TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by gram-negative bacteria

Abstract

Systemic infections with Gram-negative bacteria are characterized by high mortality rates due to the "sepsis syndrome," a widespread and uncontrolled inflammatory response. Though it is well recognized that the immune response during Gram-negative bacterial infection is initiated after the recognition of endotoxin by Toll-like receptor 4, the molecular mechanisms underlying the detrimental inflammatory response during Gram-negative bacteremia remain poorly defined. Here, we identify a TRIF pathway that licenses NLRP3 inflammasome activation by all Gram-negative bacteria. By engaging TRIF, Gram-negative bacteria activate caspase-11. TRIF activates caspase-11 via type I IFN signaling, an event that is both necessary and sufficient for caspase-11 induction and autoactivation. Caspase-11 subsequently synergizes with the assembled NLRP3 inflammasome to regulate caspase-1 activation and leads to caspase-1-independent cell death. These events occur specifically during infection with Gram-negative, but not Gram-positive, bacteria. The identification of TRIF as a regulator of caspase-11 underscores the importance of TLRs as master regulators of inflammasomes during Gram-negative bacterial infection.

Figure 1. TRIF Is Essential for NLRP3 Inflammasome Activation by EHEC and C. rodentium

(A) IL-1β production by Pam3CSK4-primed BMDMs stimulated with EHEC (MOI of 25), C. rodentium (MOI of 25), or polydAdT for 16 hr or silica for 16 hr. (B–E) ELISA for IL-1β (B) and IL-18 (C), immunoblots for cleaved caspase-1 and IL-1β in the supernatants (D), and immunoblots for proIL-1β, NLRP3, and GAPDH in the lysates (E) of Pam3CSK4-primed BMDMs stimulated with EHEC (MOI of 25 and/or 50), C. rodentium (MOI of 25 and/or 50), or polydAdT for 16 hr or nigericin for 1 hr. (F) Intracellular bacterial numbers at various time points from EHEC-infected BMDMs. (G) Phagosomal pH assessed by ratiometric analysis at 5 and 60 min postinfection in E. coli K12- or EHEC-infected BMDMs. Data are presented as the mean ± SEM of one experiment representative of three (A–E) or two (F and G) experiments. See also Figure S1.

Figure 2. TRIF Signaling Downstream of TLR4-TRAM Mediates Inflammasome Activation by EHEC and C. rodentium

(A) Cleaved caspase-11 in the supernatants of Pam3CSK4-primed BMDMs stimulated for 16 hr with EHEC (MOI of 25 and 50), C. rodentium (MOI of 25 and 50), or polydAdT. (B, D, and F–J) IL-1β or IL-1α production by Pam3CSK4-primed BMDMs from C57BL/6 wild-type and various knockout mice stimulated with EHEC, C. rodentium, polydAdT, or silica for 16 hr or nigericin for 1 hr. (C) Cell death in BMDMs stimulated with EHEC, C. rodentium, polydAdT, or silica for 16 hr. (E) Immunoblots for cleaved caspase-11, caspase-1, and IL-1β in the supernatants of Pam3CSK4-primed BMDMs stimulated with EHEC, C. rodentium, polydAdT, or silica for 16 hr. Data are presented as the mean ± SEM of one experiment representative of three experiments. See also Figure S2.

Figure 3. Type I Interferon Response Triggered by TLR4-TRIF Mediates Caspase-11-Dependent Inflammasome Activation by EHEC and C. rodentium

(A) IFN-β production by BMDMs stimulated with EHEC (MOI of 25 and 50), C. rodentium (MOI of 25 and 50), or polydAdT for 16 hr. (B) ELISA for IL-1β and immunoblots for cleaved caspase-1, caspase-11, and IL-1β in the supernatants of BMDMs stimulated with EHEC, C. rodentium, or polydAdT for 16 hr or nigericin for 1 hr. (C) IL-1α secretion by and cell death in BMDMs stimulated with EHEC for 16 hr or nigericin for 1 hr. (D and E) IL-1β production by Pam3CSK4-primed C57BL/6 wild-type and Stat1^−/− (D) or Irf9^−/− (E) BMDMs stimulated with EHEC, polydAdT, or silica for 16 hr or nigericin for 1 hr. (F and G) Cleaved caspase-1 and caspase-11 in the supernatants and NLRP3 in the lysates (F) and IL-18 in the supernatants (G) of Pam3CSK4-primed BMDMs stimulated with EHEC, C. rodentium, or polydAdT for 16 hr. Cells were subjected to 1,000 U/ml IFN-β treatment at the time of infection as indicated. (H) Secreted IL-1β and cleaved caspase-1 and IL-1β in the supernatants of Pam3CSK4-primed BMDMs treated with indicated doses of IFN-β and stimulated with EHEC or polydAdT for 16 hr. Data are presented as the mean ± SEM of one experiment representative of 2–3 experiments. See also Figures S1A and S3.

Figure 4. EHEC-Induced Caspase-11 Transcriptional Induction Is TRIF and IFNAR Dependent

(A) Caspase-11 transcript levels in BMDMs infected with EHEC for 12 hr or stimulated with IFN-β (500 units/ml) or IFN-γ (40 ng/ml). (B) Procaspase-11 and HMGB-1 (as a loading control) in the lysates of BMDMs stimulated with indicated treatments for 16 hr. (C) Caspase-11 transcript and protein levels and pro-IL-1β protein levels in C57BL/6 and MyD88^−/− BMDMs stimulated with EHEC, IFN-β, LPS (200 ng/ml), or Pam3CSK4 (400 ng/ml) for 12 hr (transcript) or 16 hr (protein). (D) Pro- and processed caspase-11 in the lysates of BMDMs treated with EHEC, IFN-β, IFN-γ, or LPS (200 ng/ml) for 16 hr. (E) Cleaved caspase-1 and IL-1β in the supernatants of Pam3CSK4-primed BMDMs treated with indicated doses of IFN-γ and stimulated with EHEC or polydAdT for 16 hr. (F) Cell death in immortalized C57BL/6 and 129S6 BMDMs stimulated with IFN-β (250 U/ml and 500 U/ml) or IFN-γ (40 ng/ml) for 40 hr or etoposide (50 µM) for 24 hr. (G) Oligomerization of ASC and caspase-1 in the inflammasome-enriched and cross-linked lysates of immortalized C57BL/6 and 129S6 BMDMs treated with EHEC or C. rodentium for 6 hr or polydAdT for 3 hr or nigericin for 30 min. Monomers, dimers, and oligomers of ASC and caspase-1 are indicated accordingly. Data are presented as the mean ± SEM of one experiment representative of three (A–D) or two (E–G) experiments. See also Figure S4.

Figure 5. NLRP3 Inflammasome Activation by All Gram-Negative Bacteria Requires TRIF-IFNβ-Caspase-11 Axis

(A–C) IL-1β production by Pam3CSK4-primed C57BL/6 wild-type and Trif^−/− (A) or Ifnar1^−/− (B) or caspase-11^−/− (C) BMDMs stimulated with indicated bacteria for 16 hr. (D) Cleaved caspase-1 and IL-1β in the supernatants of Pam3CSK4-primed BMDMs stimulated with indicated bacteria for 16 hr. (E) IL-1β secretion by Pam3CSK4-primed C57BL/6 wild-type and Trif^−/− BMDMs stimulated with P. aeruginosa PAK wild-type strain or pscC mutant strain (that lacks the type III secretion system) for 16 hr. (F) Secreted and cleaved IL-1β in the supernatants of BMDMs primed as indicated and stimulated with cholera toxin B for 16 hr. Data are presented as the mean ± SEM of one experiment representative of 2–3 experiments. See also Figure S5 and Table S1.

Figure 6. Type I Interferon Signaling Is Essential for Inflammasome Activation in an E. coli-Induced Acute Peritonitis Model

(A) Cytokines in the serum and peritoneal lavages of C57BL/6 and Ifnar1^−/− mice infected with E. coli BL21 for 6 hr (n = 4–5). Data are presented as the mean ± SEM of one experiment representative of two experiments. (B) Integrative model of TLR4-TRIF-IFN and NLRP3 signaling to activate caspase-11-dependent caspase-1, IL-1β, as well as IL-18 processing. See also Figure S6.