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

Inflammasomes are cytosolic multi-protein complexes assembled by intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) and initiate innate immune responses to invading pathogens and danger signals by activating caspase-1¹. Caspase-1 activation leads to maturation and release of the proinflammatory cytokines interleukin (IL)-1β and IL-18 as well as a lytic inflammatory cell death termed pyroptosis². Recently, a novel non-canonical inflammasome was described that activates caspase-11, a proinflammatory caspase required for LPS-induced lethality³. This study also highlighted that previously generated caspase-1 knockout mice lack a functional allele of Casp-11, making them functionally Casp-1/Casp-11 double-knockouts^3–6. Previous studies have shown that these mice are more susceptible to infections with microbial pathogens¹, including the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium)^7,8, however 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. TLR4/Trif-dependent IFN-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. Finally, Casp-1^−/− mice were significantly more susceptible to infection with S. Typhimurium than mice lacking both proinflammatory caspases (Casp-1^−/−/Casp-11^−/−). This phenotype was accompanied by higher bacterial counts, the formation of extracellular, bacterial micro-colonies in the infected tissue and a defect in neutrophil-mediated clearance. These surprising 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.

Previous studies have shown that logarithmic phase S. Typhimurium induce a rapid NLRC4-dependent cell death in cultured macrophages that requires the SPI-1 Type 3 Secretion System (T3SS)⁹. We previously reported that Salmonella grown to stationary phase (decreased SPI-1 expression) do not induce rapid activation of NLRC4, but establish themselves in an intracellular niche¹⁰. Intracellular Salmonella are detected by inflammasome receptors NLRP3 and NLRC4 and mature IL-1β/IL-18 are released 12–17 hours post-infection (Supplementary Fig. 1 and 2a, b)¹⁰. In addition, intracellular Salmonella induce an uncharacterized form of lytic cell death that is independent of the SPI-1 T3SS¹¹. To investigate host factors involved in this type of Salmonella-induced macrophage death, we infected macrophages deficient for specific inflammasome components with stationary phase wild-type Salmonella. Macrophage death, which required infection with live bacteria, did not require NLRP3 or the adaptor protein ASC, but was partially dependent on NLRC4 (Supplementary Fig. 2a and 3). Since macrophages from Casp-1^−/−/Casp-11^−/− mice did not release LDH, we investigated if caspase-1 and caspase-11 were activated and processed in response to intracellular Salmonella (Supplementary Fig. 2a). Processed caspase-1 p20 and caspase-11 p30 subunits were detected during Salmonella infection, indicating that caspase-11 activation correlated with cell death (Supplementary Fig. 2a). Consistently, Casp-11^−/− macrophages were significantly more resistant to death than WT macrophages (Fig. 1a), demonstrating an important role for caspase-11 in cell death caused by intracellular Salmonella. In contrast to L. pneumophila infectons¹², intracellular growth of Salmonella in WT and Casp-11^−/− macrophages was not significantly different (data not shown).

Macrophage death was not totally abrogated in Casp-11^−/− macrophages infected with wild-type Salmonella, indicative of an additional cell death pathway (Fig. 1a and Supplementary Fig. 2c). Since NLRC4 contributed to macrophage death (Fig. 1a and Supplementary Fig. 2a), we determined if NLRC4 activation accounted for the remaining cell death seen in Casp-11^−/− macrophages. The second T3SS (SPI-2), which is used by intracellular Salmonella to inject effector proteins into the host cell, can also inject flagellin¹⁰, a ligand for NLRC4^13–16. Comparable levels of cell death were observed in Casp-1^−/−/Casp-11^−/− and Casp-11^−/− macrophages infected with a ΔSPI-2 or a Δflagellin strain of Salmonella (Fig. 1a and Supplementary Fig. 2c), suggesting that wild-type Salmonella induce cell death through two separate pathways, one controlled by NLRC4 and the other requiring caspase-11.

Finally we compared the levels of cell death in macrophages derived from WT, Casp-1^{−/ −} Casp-11^{−/ −}, Casp-11^{−/ −} or Casp-1^{−/ −} (Casp-1^{−/ −}/Casp-11^tg) mice³ infected with Salmonella. Cell death in Casp-1^−/− macrophages infected with a SPI-2-deficient strain was similar to levels seen in WT macrophages (Supplementary Fig. 2d), indicating that Salmonella strains that cannot inject flagellin exclusively engage caspase-11-dependent cell death. Consistently, Casp-11^−/− macrophages infected with the ΔSPI-2 strain did not die (Supplementary Fig. 2d). In contrast, wild-type Salmonella induced cell death via canonical (NLRC4/caspase-1) and non-canonical signaling pathways (caspase-11) (Supplementary Fig. 1 and 2d).

NLRP3-mediated cytokine production triggered by non-canonical inflammasome stimuli depends on both caspase-1 and caspase-11³. We therefore investigated if the NLRP3 pathway induced by intracellular Salmonella required both caspases. Since IL-1β and IL-18 release in response to intracellular Salmonella is exclusively mediated by NLRC4 and NLRP3 (Supplementary Fig. 2a, b), we studied the response to SPI-2- or flagellin-deficient Salmonella¹⁰. Cytokine maturation in response to these strains required both caspase-1 and caspase-11 (Supplementary Fig. 4). In contrast, cytokine maturation induced by wild-type Salmonella, which activates both NLRP3 and NLRC4, was only partially dependent on caspase-11, but absolutely required caspase-1 (Supplementary Fig. 4). These results indicate that intracellular Salmonella activate a non-canonical inflammasome similar to LPS/CTB and the enteric bacteria C. rodentium, V. cholerae and E. coli³. Formation of ASC foci (specks), a measure of NLRP3/ASC complex formation, required caspase-11 but not caspase-1 (Supplementary Fig. 5a, b), indicating that caspase-11 acts upstream of the NLRP3/ASC complex.

Stimulation of resting macrophages with LPS or IFN-g induces pro-caspase-11 expression^3,4,17. To determine whether Salmonella-dependent activation of the non-canonical inflammasome was dependent on TLR-mediated recognition, we infected Tlr4^−/− macrophages with flagellin-deficient Salmonella (which activate the caspase-11-dependent, non-canonical inflammasome pathway exclusively; Supplementary Fig. 2 and 4). Caspase-11 activation, IL-1β secretion, and cell death depended on TLR4 (Fig. 1b). Caspase-11 processing and cell death required the TLR4-dependent signaling adaptor Trif, but not the TLR4-dependent signaling adaptor MyD88 (Fig. 1c). In contrast, IL-1β maturation was reduced in both MyD88^−/− and Trif^−/−, suggesting that cytokine maturation requires both adaptors. Expression of pro-IL-1β required MyD88-signaling (Supplementary Fig 6a, b), explaining the lack of mature IL-1β release in MyD88^−/− macrophages (Fig. 1c). Since cytokine production and cell death required Trif, we measured pro-caspase-11 expression in MyD88^−/−, Trif^−/−, and MyD88^−/−/Trif^−/− macrophages infected with Salmonella. Although induction of pro-caspase-11 expression was delayed in MyD88^−/− and Trif^−/− macrophages, the levels of pro-caspase-11 protein in MyD88^−/−/Trif^−/− macrophages were significant (Fig. 1d and Supplementary Fig. 6a). Thus, pro-caspase-11 protein expression is partially dependent on TLR-signaling. However, other pathways likely contribute. Intriguingly, non-canonical inflammasomes were not activated in Trif^−/− macrophages (Fig. 1c) even though significant amounts of pro-caspase-11 were present. These results indicate that caspase-11 activity requires a Trif-dependent signal.

The adaptor Trif induces NFkB activation and signals through IRF3 to induce the expression of type-I-interferons (type-I-IFNs)¹⁸. To investigate if type-I-IFNs could be the Trif-dependent signal required for caspase-11 activation, we compared the levels of IL-1β release, cell death and pro-caspase-11 expression in WT, Casp-1^−/−/Casp-11^−/− and IRF3^−/− macrophages (Fig. 2a,b and Supplementary Fig. 6c). IRF3^{−/ −} macrophages were significantly impaired in their ability to initiate caspase-11-dependent IL-1β release and cell death even though significant levels of pro-caspase-11 were present, albeit at slightly reduced levels when compared to WT macrophages. To confirm the requirement of type-I-IFN signaling for caspase-11 activation, we measured non-canonical inflammasome activation in macrophages lacking components of type-I and type-II interferon signaling cascade. IFNαR^−/− or STAT-1^−/− macrophages infected with Salmonella did not process caspase-11 or induce non-canonical cell death (Fig. 2c), and this was not due to a lack of pro-caspase-11 expression (Fig. 2d). Macrophages lacking IFNγR were indistinguishable from WT thereby excluding an involvement of IFN-γ. To confirm a dependency on type-I-IFN signaling for caspase-11 activation, macrophages infected with Δflag Salmonella were treated with recombinant murine IFN-β (Fig. 2e). Consistent with an important role for type-I-IFN in caspase-11 activation, exogenous IFN-β restored cell death and caspase-11 processing in infected MyD88^−/−/Trif^−/− but not Casp-1^−/−/Casp-11^−/− macrophages (Fig. 2e and Supplementary Fig. 7). Importantly, uninfected macrophages treated with IFN-β did not induce LDH release, confirming that IFN-β alone cannot induce non-canonical cell death in the absence of an infection. Our results reveal a previously unreported requirement for type-I-IFN signaling in caspase-11 activation that is consistent with a model in which an interferon inducible activator mediates caspase-11 activation in response to intracellular Salmonella (Supplementary Fig. 1).

Finally, to extend our findings to an in vivo setting, we infected WT, Casp-1^−/−/Casp⁻11^−/−, Casp-1^−/− (Casp-1^−/−/Casp-11^tg) and Casp-11^−/− mice orally with wild-type Salmonella. As reported previously, Casp-1^−/−/Casp-11^−/− mice had significantly higher bacterial loads in tissues compared to WT mice (Fig. 3a)^7,8. Surprisingly, the levels of bacteria in Casp-1^−/− mice were significantly higher compared to Casp-1^−/−/Casp-11^−/− mice (Fig. 3a). Interestingly, bacterial loads in Casp-11^−/− mice were comparable to WT mice in all organs examined. Unexpectedly, these results imply that activation of proinflammatory caspase-11 is detrimental to the host in the absence of caspase-1. Consistent with these findings, the bacterial loads in Nlrp3^−/−/Nlrc4^−/− animals, which activate caspase-11, but not caspase-1 in response to Salmonella (Supplementary Fig. 2), were significantly higher than in Casp-1^−/−/Casp-11^−/− mice (Supplementary Fig. 8), essentially phenocopying Casp-1^−/−.

Although previous studies have shown that Casp-1^−/−/Casp-11^−/− mice are more susceptible to many pathogens, the exact mechanism underlying these findings is not well understood^1,19. Host defense against systemic Salmonella infection requires neutrophils, since Salmonella replication in the liver and spleen is exacerbated in neutropenic mice^20,21. In addition, Salmonella becomes vulnerable to neutrophil-mediated clearance when it transits between host cells²². We therefore investigated if caspase-1 or caspase-11-deficiency resulted in reduced neutrophil influx into the spleen. Splenic neutrophil counts (Ly6G⁺ cells) were reduced in mice lacking either caspase-1 or caspase-11 compared to WT mice, with Casp-1^−/−/Casp-11^−/− mice having the most significant reduction of neutrophil counts compared to WT animals (Fig. 3b). Since the levels of neutrophils in the mice deficient for caspase-1 or -11 were very similar to each other, and did not correlate with the bacterial loads, we concluded that differences in neutrophil levels alone could not account for the higher bacterial levels in Casp-1^−/− animals (Fig. 3a). We next examined if caspase-1 deficiency resulted in any functional defects in splenic neutrophils. Neutrophils have been previously implicated in phagocytosing and killing extracellular Salmonella released by pyroptotic macrophages²². Interestingly, the percentage of neutrophils (Ly6G⁺) among all Salmonella-associated cells (Salmo⁺) was significantly reduced in Casp-1^−/− and Casp-1^−/−/Casp-11^−/− mice compared to WT and Casp-11^−/− mice (Fig. 3b). These data suggested that the lack of caspase-1 resulted in reduced bacterial uptake by neutrophils or reduced association with Salmonella during infection. However, since this reduction was observed in both Casp-1^−/− and Casp-1^−/−/Casp-11^−/− mice, it did not explain the significantly higher CFUs in Casp-1^−/− mice (Fig. 3a).

NLRC4/caspase-1 induced lysis releases intracellular Salmonella, thus making them accessible to neutrophil-mediated uptake and killing²². Since both caspase-1 and caspase-11 can induce lysis of infected cells, we speculated that the lack of caspase-11 in Casp-1^−/−/Casp-11^−/− mice might delay the egress of Salmonella from infected macrophages. Consistent with this model, we found that Salmonella were present to a higher degree in macrophages in Casp-1^−/−/Casp-11^−/− mice compared to WT, Casp-11^−/− and Casp-1^−/− mice (Fig. 3b). Gram stain and specific anti-Salmonella antibody stainings of tissues revealed that WT and Casp-11^−/− liver sections contained low levels of Salmonella, consistent with the CFU data (Supplementary Fig. 9). Casp-1^−/−/Casp-11^−/− liver sections contained high levels of bacteria within cells in the sinusoids (Supplementary Fig. 9), which is consistent with our FACS data indicating that a larger proportion of Salmonella are associated with macrophages in these mice (Fig. 3b). Casp-1^−/− liver sections contained mats of extracellular bacteria within typhoid nodules and expanded sinusoids (Fig. 3c and Supplementary Fig. 9, 10a). Finally, mice infected with Salmonella were injected with the membrane-impermeable antibiotic gentamicin to distinguish intracellular bacteria from extracellular bacteria. Gentamicin treatment significantly reduced bacterial counts in Casp-1^−/− mice, consistent with our histological finding that Salmonella is largely extracellular in these mice (Supplementary Fig. 10b).

We conclude that caspase-1-deficiency results in reduced neutrophil-mediated clearance of Salmonella released from infected macrophages, thus supporting extracellular growth of this facultative intracellular pathogen. In keeping with this observation, it has been reported that Salmonella rapidly replicates extracellularly in the liver of neutropenic mice²³. This phenotype is alleviated in Casp-1^−/−/Casp-11^−/− animals, since bacterial egress from the infected macrophages is delayed. Thus, caspase-11-mediated cell death results in detrimental effects to the host in the absence of caspase-1. Our results indicate that the ability of neutrophils to phagocytose bacteria is dependent on a caspase-1-mediated function. Since Casp-1^−/−/Casp-11^−/−, IL-1R^−/− or IL-1β^−/−/IL-18^−/− mice have similar levels of Salmonella⁸, the mechanism is not likely dependent on IL-1β/IL-18 maturation. Future studies are required to identify and characterize this function. Finally, we provide evidence that caspase-11-dependent cell death is exploited by Salmonella in the absence of caspase-1 to cause disease in the host, highlighting the need to determine if caspase-11 activation has similar detrimental effects for the host in other infectious disease models.