Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1

Abstract

Inflammasomes are cytosolic multiprotein complexes assembled by intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and they initiate innate immune responses to invading pathogens and danger signals by activating caspase-1 (ref. 1). Caspase-1 activation leads to the maturation and release of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18, as well as lytic inflammatory cell death known as pyroptosis. Recently, a new non-canonical inflammasome was described that activates caspase-11, a pro-inflammatory caspase required for lipopolysaccharide-induced lethality. This study also highlighted that previously generated caspase-1 knockout mice lack a functional allele of Casp11 (also known as Casp4), making them functionally Casp1 Casp11 double knockouts. Previous studies have shown that these mice are more susceptible to infections with microbial pathogens, including the bacterial pathogen Salmonella enterica serovar Typhimurium (S. typhimurium), but the individual contributions of caspase-1 and caspase-11 to this phenotype are not known. Here we show that non-canonical caspase-11 activation contributes to macrophage death during S. typhimurium infection. Toll-like receptor 4 (TLR4)-dependent and TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent interferon-β production is crucial for caspase-11 activation in macrophages, but is only partially required for pro-caspase-11 expression, consistent with the existence of an interferon-inducible activator of caspase-11. Furthermore, Casp1(-/-) mice were significantly more susceptible to infection with S. typhimurium than mice lacking both pro-inflammatory caspases (Casp1(-/-) Casp11(-/-)). This phenotype was accompanied by higher bacterial counts, the formation of extracellular bacterial microcolonies in the infected tissue and a defect in neutrophil-mediated clearance. These results indicate that caspase-11-dependent cell death is detrimental to the host in the absence of caspase-1-mediated innate immunity, resulting in extracellular replication of a facultative intracellular bacterial pathogen.

Figure 1. Signaling through TLR4 and Trif is required for activity of the non-canonical inflammasome pathway

a, LDH release from unprimed BMDMs infected with the indicated S. Typhimurium strains for 17 h. b, c, IL-1β secretion, LDH release and immunoblots for processed caspase-1, caspase-11, IL-1β and IL-18 released from unprimed BMDMs infected with Δflag S. Typhimurium for 17 h. d, Induction of pro-caspase-11 and pro-IL-1β expression in unprimed BMDMs infected with Δflag S. Typhimurium. Graphs show the mean ± s.d. of quadruplicate wells and are representative of three (a-c) and two (d) independent experiments.

Figure 2. Type-I-IFN signaling is required for caspase-11 activation, but not for pro-caspase-11 expression

a, c, IL-1β secretion, LDH release and immunoblots for processed caspase-1, caspase-11 and IL-1β released from unprimed BMDMs infected with Δflag S. Typhimurium for 17 h. b, d, Timecourse measuring pro-caspase-11 and pro-IL-1β induction in unprimed BMDMs infected with Δflag S. Typhimurium. e, Cell death in BMDMs treated with recombinant murine IFN-β or vehicle control and infected with Δflag. S. Typhimurium for 17 h. Graphs show the mean ± s.d. of quadruplicate wells and are representative of three (a, c, e), two (b, d) independent experiments.

Figure 3. Casp-1^−/− mice are more susceptible to Salmonella infection than Casp-1^−/−/Casp-11^−/− mice

a, Bacterial burden in mice infected orally with GFP⁺ wild-type S. Typhimurium at day 4. b, Flow-cytometry analysis of neutrophil counts (Ly6G⁺), Salmonella-associated neutrophils and the intracellular distribution of S. Typhimurium (GFP⁺) in macrophages (F4/80) and neutrophils (Ly6G) in the spleen. c, Livers from S. Typhimurium-infected mice showing typhoid nodules, mats of extracellular bacteria (stars) and single Salmonella (arrowheads). Original magnification 10x or 40x (boxed close-ups). Graphs show ± s.e.m of 6–10 mice per genotype and are representative of two to three independent experiments. *, P < 0.05, **, P < 0.01. Dashed line: detection limit, UI, Uninfected controls.