Type I interferon signaling is required for activation of the inflammasome during Francisella infection

Abstract

Francisella tularensis is a pathogenic bacterium whose virulence is linked to its ability to replicate within the host cell cytosol. Entry into the macrophage cytosol activates a host-protective multimolecular complex called the inflammasome to release the proinflammatory cytokines interleukin (IL)-1beta and -18 and trigger caspase-1-dependent cell death. In this study, we show that cytosolic F. tularensis subspecies novicida (F. novicida) induces a type I interferon (IFN) response that is essential for caspase-1 activation, inflammasome-mediated cell death, and release of IL-1beta and -18. Extensive type I IFN-dependent cell death resulting in macrophage depletion occurs in vivo during F. novicida infection. Type I IFN is also necessary for inflammasome activation in response to cytosolic Listeria monocytogenes but not vacuole-localized Salmonella enterica serovar Typhimurium or extracellular adenosine triphosphate. These results show the specific connection between type I IFN signaling and inflammasome activation, which are two sequential events triggered by the recognition of cytosolic bacteria. To our knowledge, this is the first example of the positive regulation of inflammasome activation. This connection underscores the importance of the cytosolic recognition of pathogens and highlights how multiple innate immunity pathways interact before commitment to critical host responses.

Figure 1.

WT cytosolic F. novicida induces a type I IFN transcriptional response in BMMs, whereas the vacuole-restricted mglA mutant does not. At 8 h postinfection (PI), 68 genes were statistically differentially regulated between mglA and WT F. novicida–infected BMMs. The arrays corresponding to the biological replicates and the 21 genes with the higher changes between mglA and WT infected macrophages are shown.

Figure 2.

F. novicida in the host cytosol induces IFN-β secretion in a TLR-independent IRF-3–dependent manner. IFN-β mRNA levels were determined by quantitative RT-PCR in uninfected BMMs (Un) or at various times PI (A) and 7 (C), 9 (D), or 8 (E) h PI with either mglA or WT F. novicida. IFN-β levels were determined by ELISA in the supernatant of BMMs infected at the indicated MOI for 9 h (ND, nondetectable; B). Various vacuole-restricted mutants do not induce IFN-β mRNA, whereas their complemented counterparts (c.) do. Cytosolic localization is shown (C). BMMs from WT or from IRF-3^−/−, ASC^−/−, MyD88/TRIF^DKO, Ipaf^−/−, RIP2^−/− (D), MAVS^−/− mice, or WT littermates (E) were analyzed for their IFN-β mRNA levels. Error bars represent SEM.

Figure 3.

Type I IFN induction and signaling is required for F. novicida–mediated but not for S. typhimurium–mediated cell death. Cell death of WT, IFNR^−/−, ASC^−/−, and caspase-1 (casp1)^−/− BMMs was assayed by lactate dehydrogenase (LDH) release. BMMs either unactivated (B) or preactivated with heat-killed F. novicida (A, C, and D [left]) or pretreated with recombinant IFN-β (D, right) were infected for 8 (A), 12.5 (B), 3 (C), or 6 h (D) with F. novicida (A, B, and D) or S. typhimurium (C) strains at the indicated MOI. In agreement with previous data (30), cell death required the S. typhimurium gene sipB. Error bars represent SEM.

Figure 4.

Type I IFN signaling is necessary for activation of the inflammasome during F. novicida and L. monocytogenes infections but not upon ATP treatment. (A) WT, IFNR^−/−, and ASC^−/− BMMs uninfected (Un) or infected with mglA (m) or WT F. novicida (W) were lysed at 9 h PI. Caspase-1 processing was visualized by detection of the p20 subunit only in WT macrophages infected with WT F. novicida. (B and C) IL-1β (B) and -18 (C) were quantified by ELISA in the supernatant of preactivated BMMs. Similar levels were detected in WT and IFNR^−/− BMMs treated for 3 h with 5 mM ATP (left), whereas high levels were only detected in WT macrophages upon infection with F. novicida for 5 h (right). (D) Cell death (left) was strongly reduced in IFNR^−/− compared with WT BMMs at 6 h PI with L. monocytogenes. Similarly, IL-1β (middle) and -18 (right) levels were lower in activated BMMs infected for 2.5 h with L. monocytogenes at the indicated MOI.

Figure 5.

IFN-β is up-regulated in vivo upon infection with F. novicida, leading to IFNR-dependent cell death and depletion of BMMs in the spleen at 48 h PI. Each symbol represents the value of one individual mouse. Horizontal bars correspond to the geometric mean. (A) IFN-β mRNA levels in the skin and spleen of WT uninfected (open symbols) or infected (closed symbols) mice were determined by quantitative RT-PCR. (B) TUNEL staining (green) performed on a spleen section of uninfected (left), infected (middle), WT, or infected IFNR^−/− mice (right) nuclei (blue), and F. novicida (red) stainings are shown. Representative images are shown. Bar, 100 μm. (C–E) Quantification of the number of TUNEL-positive cells (C), splenic macrophages (D), and F. novicida CFU in various organs from WT (closed symbols) and IFNR^−/− (open symbols) mice (E) are presented. (F) Model for the inflammasome activation in response to F. novicida (see Conclusion and model: cytosolic sensing of F. novicida … for details). Error bars represent SEM.