Toll or interleukin-1 receptor (TIR) domain-containing adaptor inducing interferon-β (TRIF)-mediated caspase-11 protease production integrates Toll-like receptor 4 (TLR4) protein- and Nlrp3 inflammasome-mediated host defense against enteropathogens

Abstract

Enteric pathogens represent a major cause of morbidity and mortality worldwide. Toll-like receptor (TLR) and inflammasome signaling are critical for host responses against these pathogens, but how these pathways are integrated remains unclear. Here, we show that TLR4 and the TLR adaptor TRIF are required for inflammasome activation in macrophages infected with the enteric pathogens Escherichia coli and Citrobacter rodentium. In contrast, TLR4 and TRIF were dispensable for Salmonella typhimurium-induced caspase-1 activation. TRIF regulated expression of caspase-11, a caspase-1-related protease that is critical for E. coli- and C. rodentium-induced inflammasome activation, but dispensable for inflammasome activation by S. typhimurium. Thus, TLR4- and TRIF-induced caspase-11 synthesis is critical for noncanonical Nlrp3 inflammasome activation in macrophages infected with enteric pathogens.

FIGURE 1.

Caspase-11 is required for inflammasome signaling in macrophages infected with C. rodentium and E. coli, but dispensable during S. typhimurium infection. Bone marrow-derived macrophages from WT and caspase-11^−/− mice were infected with C. rodentium (m.o.i. 20), E. coli (m.o.i. 20), and S. typhimurium (m.o.i. 5) as described under “Experimental Procedures.” A–C, caspase-1 activation was determined by Western blotting of cell lysates. D–I, secreted IL-1β and IL-18 in supernatants of infected macrophages were quantified by ELISA. Data represent means ± S.E. (error bars) and are representative of three independent experiments.

FIGURE 2.

Role of NLRs and adaptor molecules in caspase-1 activation and IL-1β secretion from macrophages infected with Gram-negative bacteria. Bone marrow-derived macrophages from WT, Asc^−/−, Aim2^−/−, Nlrp3^−/−, Nlrc4^−/−, Nod1^−/−, Nod2^−/−, Nlrp6^−/−, and Nlrp12^−/− mice were infected with C. rodentium (m.o.i. 20), E. coli (m.o.i. 20), or S. typhimurium (m.o.i. 5) for 24 h as described under “Experimental Procedures.” Caspase-1 activation was determined by Western blotting of cell lysates (A, C, and E), and secreted IL-1β in supernatants of infected macrophages was quantified by ELISA (B, D, and F). Data represent means ± S.E. (error bars) and are representative of at least three independent experiments.

FIGURE 3.

Role of TLR4 in inflammasome activation and secretion of IL-1β and IL-18 by C. rodentium-, E. coli-, and S. typhimurium-infected macrophages. WT, Tlr2^−/−, Tlr4^−/−, and Tlr7^−/− bone marrow-derived macrophages were infected with C. rodentium (m.o.i. 20), E. coli (m.o.i. 20), and S. typhimurium (m.o.i. 5) for 24 h as described under “Experimental Procedures.” Secreted IL-1β and IL-18 in supernatants of infected macrophages were quantified by ELISA (A–F), and caspase-1 activation was determined by Western blotting of cell lysates (G–I). Data represent means ± S.E. (error bars) and are representative of at least three independent experiments.

FIGURE 4.

TRIF is critical for C. rodentium- and E. coli-induced inflammasome activation. WT, Tlr2^−/−, Tlr4^−/−, Myd88^−/−, Trif^−/−, and Myd88^−/−Trif^−/− macrophages were infected with C. rodentium (m.o.i. 20), E. coli (m.o.i. 20), or S. typhimurium (m.o.i. 5) as described under “Experimental Procedures.” Caspase-1 activation was determined by Western blotting of cell lysates (A–C), and secreted IL-1β in supernatants of infected macrophages was quantified by ELISA (D–F). Data represent means ± S.E.(error bars) and are representative of at least three independent experiments.

FIGURE 5.

The TLR4-TRIF axis regulates caspase-11 expression. A, B, E, and F, WT, Tlr2^−/−, Tlr4^−/−, Myd88^−/−, Trif^−/−, and Myd88^−/−Trif^−/− macrophages were infected with C. rodentium and E. coli. RNA was extracted 6 h after infection, and mRNA expression of caspase-11 and Nlrp3 was determined as described under “Experimental Procedures.” RNA expression were normalized to the expression of GAPDH and then depicted as -fold increase. C, D, G, and H, cell lysates of WT, Tlr4^−/−, and Trif^−/− macrophages infected with C. rodentium or E. coli for 24 h were analyzed for caspase-11 and Nlrp3 expression by Western blotting. Data represent means ± S.E. (error bars) and are representative of at least three independent experiments.

FIGURE 6.

Roles of TLR4-TRIF and caspase-11 during C. rodentium infection in vivo. A, survival of WT, Tlr4^−/−, Myd88^−/−, Trif^−/−, and Myd88^−/−Trif^−/− mice infected with 1 × 10¹⁰ C. rodentium by oral gavage. B and C, bacterial burden in fecal pellets of infected mice determined at day 7 after infection. D, working model for TLR4-/TRIF-mediated regulation of caspase-11 expression and noncanonical inflammasome activation in C. rodentium- and E. coli-infected macrophages.