A hierarchy of cell death pathways confers layered resistance to shigellosis in mice
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eLife assessment
This paper reports important findings on the mechanisms by which death pathways are activated by Shigella infection to impact the host response. The methods used provide compelling evidence for the involvement of multiple death cell pathways in the pathogenesis and host response to murine shigellosis. The results presented therein will be of interest to investigators in the field of bacterial pathogenesis, infectious disease and immunology.
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Abstract
Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease driven by bacterial colonization of colonic intestinal epithelial cells. Vertebrates have evolved programmed cell death pathways that sense invasive enteric pathogens and eliminate their intracellular niche. Previously we reported that genetic removal of one such pathway, the NAIP–NLRC4 inflammasome, is sufficient to convert mice from resistant to susceptible to oral Shigella flexneri challenge (Mitchell et al., 2020). Here, we investigate the protective role of additional cell death pathways during oral mouse Shigella infection. We find that the Caspase-11 inflammasome, which senses Shigella LPS, restricts Shigella colonization of the intestinal epithelium in the absence of NAIP–NLRC4. However, this protection is limited when Shigella expresses OspC3, an effector that antagonizes Caspase-11 activity. TNFα, a cytokine that activates Caspase-8-dependent apoptosis, also provides potent protection from Shigella colonization of the intestinal epithelium when mice lack both NAIP–NLRC4 and Caspase-11. The combined genetic removal of Caspases-1, -11, and -8 renders mice hyper-susceptible to oral Shigella infection. Our findings uncover a layered hierarchy of cell death pathways that limit the ability of an invasive gastrointestinal pathogen to cause disease.
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eLife assessment
This paper reports important findings on the mechanisms by which death pathways are activated by Shigella infection to impact the host response. The methods used provide compelling evidence for the involvement of multiple death cell pathways in the pathogenesis and host response to murine shigellosis. The results presented therein will be of interest to investigators in the field of bacterial pathogenesis, infectious disease and immunology.
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Reviewer #1 (Public Review):
Roncaioli et al. build upon their previous findings showing that the NAIP/NLRC4 inflammasome confers host resistance to oral Shigella flexneri infection. They investigate the role of additional programmed cell death pathways in the host response to Shigella infection in mice. They find that in the absence of the NAIP inflammasome, caspase-11 contributes but in a limited capacity due to OspC3 antagonism of caspase-11 activity. Furthermore, in the absence of both NAIP and caspase-11, TNF-mediated caspase-8 activation contributes. Thus, the authors conclude that there is a hierarchy of cell death pathways involving caspase-1, 11, and 8 that all contribute to restrict Shigella infection in mice. Overall, the manuscript is well-written, the studies are logically presented and well-designed, and the data largely …
Reviewer #1 (Public Review):
Roncaioli et al. build upon their previous findings showing that the NAIP/NLRC4 inflammasome confers host resistance to oral Shigella flexneri infection. They investigate the role of additional programmed cell death pathways in the host response to Shigella infection in mice. They find that in the absence of the NAIP inflammasome, caspase-11 contributes but in a limited capacity due to OspC3 antagonism of caspase-11 activity. Furthermore, in the absence of both NAIP and caspase-11, TNF-mediated caspase-8 activation contributes. Thus, the authors conclude that there is a hierarchy of cell death pathways involving caspase-1, 11, and 8 that all contribute to restrict Shigella infection in mice. Overall, the manuscript is well-written, the studies are logically presented and well-designed, and the data largely support the authors' conclusions. The findings will be of great interest to the field. I have a few suggestions for improving the manuscript.
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Reviewer #2 (Public Review):
In this study, the authors follow up on their previous work demonstrating that NLRC4-deficient mice are susceptible to shigellosis and therefore can be used as a model to dissect immune control of infection in a tractable animal host (Mitchell & Roncaioli et al. 2020). This is therefore the first direct, mechanistic study on other immune pathways that contribute to protection of the host from Shigella in vivo. Importantly, the authors report that epithelial cell death is a critical protective mechanism against Shigella infection, and differences in the ability of the bacteria to antagonize host cell death explains in part why humans are susceptible while mice are resistant to infection.
Strengths
The authors use elegant genetic approaches to investigate the roles of distinct cell death pathways in …
Reviewer #2 (Public Review):
In this study, the authors follow up on their previous work demonstrating that NLRC4-deficient mice are susceptible to shigellosis and therefore can be used as a model to dissect immune control of infection in a tractable animal host (Mitchell & Roncaioli et al. 2020). This is therefore the first direct, mechanistic study on other immune pathways that contribute to protection of the host from Shigella in vivo. Importantly, the authors report that epithelial cell death is a critical protective mechanism against Shigella infection, and differences in the ability of the bacteria to antagonize host cell death explains in part why humans are susceptible while mice are resistant to infection.
Strengths
The authors use elegant genetic approaches to investigate the roles of distinct cell death pathways in anti-Shigella defense. The evidence presented convincingly shows that caspase-1, caspase-11, and caspase-8 mediate a hierarchy of protection against Shigella infection. The authors additionally show that the Shigella effector OspC3 drives colonization and infection by blocking caspase-11 function, which is, to this reviewer's knowledge the first demonstration that OspC3 facilitates infection in vivo. Overall, this is an important study and the work will have a large impact on the field by building a foundational understanding of the in vivo immune response against Shigella.
Weaknesses
A major limitation of the current study is absence of direct evidence that cell death within the intestinal epithelium is responsible for the loss of bacterial control. TNF is a pleiotropic cytokine that regulates cell death as well as inflammatory/antimicrobial gene expression. Similarly, caspase-8 has been found to also regulate inflammatory gene expression independent of its cell death functions. The authors propose that caspase-11 and caspase-8 contribute to protection from infection by driving epithelial cell death and extrusion. While this interpretation is supported by studies implicating cell death in eliminating the Shigella replicative niche, a formal demonstration that caspase-11 and TNF⍺/caspase-8 contribute to epithelial cell death in response to Shigella infection in vivo would strengthen the conclusions of the paper.
Furthermore, the claim that TNF⍺/caspase-8 signaling mediates protection against Shigella by driving cell death and not by NF-kB activation may be overstated, based on the evidence presented. While the work demonstrates that there is no difference in inflammatory cytokines in the Casp1/11/8/Ripk3 mice, this interpretation is less straightforward in a setting where there is substantially more CFU. Nonetheless, the study is overall very strong, and this point could be addressed experimentally or by modification to the text that would acknowledge this possibility.
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Reviewer #3 (Public Review):
The authors set out to examine the roles of multiple cell death pathways during a Shigella infection. Shigella oral infection has been classically difficult to perform because wild type C57BL/6 mice naturally resist Shigella infection, likely reflecting the fact that Shigella species are human-specific pathogens. The authors recently developed an oral Shigella infection model that successfully allows mice to be infected orally with Shigella. In this model, NLRC4 inflammasome knockout mice are treated with streptomycin 1 day prior to infection. Streptomycin depletes the microbiota and opens a microbial niche for intestinal infection (this is the same method that is used for Salmonella typhimurium oral infections in C57BL/6 mice). The Nlrc4 deletion removes one innate immune barrier to infection. Now the …
Reviewer #3 (Public Review):
The authors set out to examine the roles of multiple cell death pathways during a Shigella infection. Shigella oral infection has been classically difficult to perform because wild type C57BL/6 mice naturally resist Shigella infection, likely reflecting the fact that Shigella species are human-specific pathogens. The authors recently developed an oral Shigella infection model that successfully allows mice to be infected orally with Shigella. In this model, NLRC4 inflammasome knockout mice are treated with streptomycin 1 day prior to infection. Streptomycin depletes the microbiota and opens a microbial niche for intestinal infection (this is the same method that is used for Salmonella typhimurium oral infections in C57BL/6 mice). The Nlrc4 deletion removes one innate immune barrier to infection. Now the authors examine additional deletions in other regulated cell death genes on this Nlrc4-/- background.
Their results show that three distinct pathways to cell death are important in defending against Shigella infection, but that some pathways are more protective than others. In their previous paper, the authors showed a difference in phenotype between Nlrc4-/- mice on a C57BL/6 (B6) versus a 129S1/SvImJ (129) mouse background. The authors now show that the difference in these phenotypes is primarily driven by Casp11, in which 129 mice are naturally genetically deficient. The authors show that Casp11 is capable of protecting IECs from colonization. This is conceptually at odds with the knowledge that Shigella encodes OspC3, which is a type III secretion effector that inhibits caspase-11. However, it turns out to be that both inhibition by OspC3 and defense by caspase-11 occur in parallel with partial efficiency. The attenuation of the ospC3 Shigella mutant was abolished in mice lacking Casp11.
Further, they show that counter to assumptions in the field, neither myeloid pyroptosis nor IL-1 affected Shigella pathogenesis during this oral infection model.
The authors next examine the role of TNF driven cell death through caspase-8 and RIPK3. They show that TNF does contribute to defense against Shigella infection, but that this protection is secondary to the roles of Nlrc4 and Casp11. Finally, the authors show that quadruple knockout Casp1/11/8-/-Ripk3-/- mice lacking all four of these pathways display far worse disease pathogenesis than any of the other knockout mice studied.
In summary, NLRC4 provides the strongest defense, and caspase-11 and caspase-8/RIP3 provide weaker defense. The authors show that the weakness of the caspase-11 pathway is caused by the OspC3 effector that inhibits caspase-11. We can extrapolate form this to speculate that the weakness of caspase-8 is caused by OspC1 inhibiting it, and the weakness of RIPK3 is caused by OspD3 inhibiting it. This could be proved in future work.
One formal weakness is that Figure 1 is data from just one experiment, however, the key conclusion is verified in Figure 2 by the use of targeted Casp11 knockout.
One omission from the paper is that in Figure 3 and Figure 4, WT mice were not infected with an ospC3 mutant to show the baseline attenuation. It is stated that oral infections have not been studied with this mutant.
One weakness inherent in the use of Casp8-/- mice is that they are not viable unless they carry the Ripk3-/- or equivalent mutation. Therefore, the authors can only assess the simultaneous loss of both pathways. This can be compared to a single Ripk3-/- situation, but, here caspase-8-driven apoptosis could be sufficient. Often RIPK3 serves as a backup defense when a pathogen inhibits caspase-8, thus a hypothetical Casp8-deficient Ripk3-sufficient mouse might remain resistant due to RIPK3 activation. This might be achieved in future work by using recently developed mouse lines that carry specific Casp8 point mutations that cause the loss of apoptosis while retaining mouse viability.
One limitation of the study is that littermate controls arising from heterozygous by knockout breeding were not always used. Co-housing was used for at least 3 weeks, which often, but not always, normalizes the microbiota. This noted, it should be acknowledged that littermate controls would be extremely burdensome to accomplish in the case of some strains where multiple knockouts are used.
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