NAIP–NLRC4-deficient mice are susceptible to shigellosis
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Abstract
Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease that is a major cause of diarrhea-associated mortality in humans. Mice are highly resistant to Shigella and the lack of a tractable physiological model of shigellosis has impeded our understanding of this important human disease. Here, we propose that the differential susceptibility of mice and humans to Shigella is due to mouse-specific activation of the NAIP–NLRC4 inflammasome. We find that NAIP–NLRC4-deficient mice are highly susceptible to oral Shigella infection and recapitulate the clinical features of human shigellosis. Although inflammasomes are generally thought to promote Shigella pathogenesis, we instead demonstrate that intestinal epithelial cell (IEC)-specific NAIP–NLRC4 activity is sufficient to protect mice from shigellosis. In addition to describing a new mouse model of shigellosis, our results suggest that the lack of an inflammasome response in IECs may help explain the susceptibility of humans to shigellosis.
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##Author Response
###Reviewer #1:
The manuscript by Mitchell et al. finds that the NAIP-NLRC4 inflammasome in mice is a critical host factor that controls intestinal infection with the human specific bacterial pathogen Shigella flexneri. The work suggests that Shigella is actively suppressing the human NAIP-NLRC4 inflammasome possibly using an T3SS effector protein, which does not recognize its substrate in mouse cells. The authors use this information to determine that B6 mice lacking the NAIP or NLRC4 inflammasome components are susceptible to Shigella infection and observe disease symptoms similar to Shigellosis in humans. In addition, 129 mice exhibit additional disease symptoms, and the authors suggest that loss of Caspase-11 in 129 mice is responsible for this phenotype.
The strengths of this manuscript include the introduction …
##Author Response
###Reviewer #1:
The manuscript by Mitchell et al. finds that the NAIP-NLRC4 inflammasome in mice is a critical host factor that controls intestinal infection with the human specific bacterial pathogen Shigella flexneri. The work suggests that Shigella is actively suppressing the human NAIP-NLRC4 inflammasome possibly using an T3SS effector protein, which does not recognize its substrate in mouse cells. The authors use this information to determine that B6 mice lacking the NAIP or NLRC4 inflammasome components are susceptible to Shigella infection and observe disease symptoms similar to Shigellosis in humans. In addition, 129 mice exhibit additional disease symptoms, and the authors suggest that loss of Caspase-11 in 129 mice is responsible for this phenotype.
The strengths of this manuscript include the introduction of a new mouse model that mimics Shigellosis, the demonstration that NAIP/NLRC4 activation is important for epithelial cell defense, and the potential of these findings to clarify aspects of human infectious disease caused by this pathogen. The manuscript is well presented, and the experiments are conducted with a high degree of rigor. Overall, this is an important contribution to the Shigella field and also has significant implications on our understanding of inflammasomes in host defense against pathogens.
Response: We thank the Reviewer for recognizing the impact and rigor of our work.
There are some weaknesses that should be addressed. Experimentally, it has not been directly demonstrated that IECs from NLRC4-/- mice undergo cell death (using biochemical markers). This is a critical aspect of the model.
Response: Prior work in the field (e.g., Sellin et al, 2014; Rauch et al, 2017) has already established that inflammasome activation in IECs results in their death and expulsion from the intestinal epithelium. We are currently working on showing this also occurs with Shigella but we have no reason to doubt that it does; our preliminary data indicate that Shigella-infected propidium iodide (PI)-positive cells are expelled from IEC monolayer cultures in an NLRC4-dependent manner. We intend to provide these data in a revised version of the manuscript.
In addition, it would be useful for the authors to evaluate bacterial burden over the time course in Figure 6. Although this is not absolutely necessary to support the manuscript conclusions, this information would greatly benefit the community that intends to use these mice in the future.
Response: This is indeed an experiment we plan to complete in the future. At present we are constrained by the numbers of available mice. We agree with the reviewer that the timecourse is not essential to establish the main conclusions of the present manuscript, and have thus prioritized other experiments.
There are also some discussion points about the mouse model that would enhance the overall impact of the work. For example, a more in depth discussion about the differences between human Shigella infection and the new model would be helpful. It is important to emphasize that the mouse model requires a much greater inoculum of the pathogen to induce disease and requires microbiota-deficiency to be effective. What are the implications of this finding on our understanding of human disease?
Response: Although it is often (correctly) stated that as few as 10-100 bacteria can infect humans with Shigella, there is actually considerable heterogeneity in the infectious dose. DuPont et al 1989 summarizes several human challenge studies in their Table 1, which shows that while 25-39% of humans exhibit symptoms after low dose infection (<200 CFU), 36-44% of humans are resistant to high doses (10^4-10^8 CFU). Therefore we do not consider the infectious dose in our mouse model to be out of the range of what is ‘normal’ in humans. Indeed, our new model may help us understand some of the factors that confer resistance to certain humans. We used a dose of 5x10^7 in our manuscript to ensure reproducible infection of all mice. However, in limited studies, we have observed disease in oral route infected, antibiotic pre-treated NAIP–NLRC4-deficient mice with 10^6 CFU (4/4 mice) and 10^5 CFU (2/3 mice). We are currently repeating these experiments, which we intend to include in a revised manuscript. We also agree with the reviewer that the infectious dose in humans vs. mice merits more discussion in a revised manuscript.
In lines 274-285 the authors present an either/or scenario in which either macrophage pyroptosis is required for IEC infection or inhibition of NAIP/NRLC4 pyroptosis in IECs is required for IEC infection. However, these scenarios are not mutually exclusive. For example, it is plausible that the extremely low burdens of Shigella required to infect humans (<100 CFUs) is due to the pathogen initially crossing the epithelial barrier (e.g. through M-cells) to infect macrophage, and then re-infection of IECs after macrophage pyroptosis. In this scenario, the NAIP/NLRC4 inflammasome could prevent further expansion of bacterial in IECs by eliminating the cell-to-cell spread that have been described by others. Importantly, the macrophage lifecycle stage may not be necessary in mice in which the microbiota has been removed and Shigella is delivered at a very high inoculum. While, additional ideas could be, and should be, put forth since the mouse model provides new insights or challenges an existing dogma in the field.
Response: We do clearly state in our manuscript (line 277) that our results do not directly address the question of whether Shigella might benefit from inflammasome activation in macrophages. In a revised version of the manuscript we will further expand on the discussion of the role of inflammasomes in macrophages and IECs to acknowledge multiple, non-mutually exclusive scenarios.
###Reviewer #2:
Mitchell et al explore the role of NLRC4 in defending against Shigella infection by demonstrating that NLRC4 contributes to resistance to shigellosis in mice. Using in vitro assays, they first show that mouse but not human macrophages undergo NLRC4-mediated pyroptosis in response to Shigella infection despite an ability for both species to successfully detect Shigella NLRC4 agonists. They then demonstrate that C57BL/6 background mice, which normally resist shigellosis, become susceptible to infection when deficient in NAIPs or NLRC4. In parallel, 129 background mice develop more significant infection including intestinal bleeding. Furthermore, using a mouse line in which NLRC4 expression is restricted to intestinal epithelial cells (IECs), they show that IEC expression of NLRC4 is sufficient to resist shigellosis. Finally, using a known attenuated Shigella mutant, they demonstrate that their shigellosis model can mimic kinetics seen in humans.
Mitchell et al convincingly demonstrate both the importance of NLRC4 in protecting mice against Shigella and the utility of their mouse model for studying Shigella infections, both of which are significant and will push the Shigella field forward. There are mechanistic questions to be addressed in future studies beyond the current manuscript, attesting to the importance of the paper in opening up new areas in the field of research. In some places, the authors draw conclusions that reach beyond what is proven in the data, which should be addressed in text edits to the manuscript. In summary, this article presents an important new model for Shigella infection. The impact of the manuscript is the development of a mouse model with which to study Shigella infection in vivo.
Response: We thank the Reviewer for emphasizing the importance of our new shigellosis model for the field. We have addressed their comments below.
Major comments:
Many questions remain concerning why NLRC4-deficient THP1 cells still undergo pyroptosis. The authors provide evidence that Shigella activates PYRIN and/or AIM2 inflammasomes in humans, and that somehow mouse macrophages would fail to have this same detection. At face value, the data would suggest that humans are able to detect Shigella by Pyrin and AIM2, but for some reason these two inflammasomes are insufficient, and instead NLRC4 is required for in vivo defense. Then in mice, it would imply that everything is flipped - for some reason detection by Pyrin and AIM2 is not important, but now the bacteria can be detected by NLRC4 and this is important. The NLRC4 focused conclusions are consistent with the in vivo data, that NLRC4 in humans fails to detect, but NLRC4 in mice succeeds in detecting Shigella. However, the data that Pyrin and AIM2 in human cells successfully detect Shigella are inconsistent with the overall conclusions of the paper. I suspect that this is an artifact of THP1 cells, and that the in vivo situation in humans is that these two inflammasomes will fail to detect Shigella. There is published precedent from other infections where in vitro detection belies in vivo lack of detection (e.g. Listeria is detected by AIM2 in vitro, but probably not in vivo). It may be difficult to make direct comparisons between how inflammasomes act in THP1 cells as compared to BMMs, due to artifacts arising from the different origins and passage levels of the two cell types. It may be that the inflammasomes response is most important in IECs, as proposed by the authors, and that IECs may not express Pyrin or AIM2. There is evidence from publicly available IEC transcriptional profiles that IECs do not express Pyrin (Mefv) (Reikvam, doi: 10.1371/journal.pone.0017996), although this profile does show Aim2 expression in IEC. It is my understanding that BMMs do not express Pyrin unless they are strongly stimulated with some TLR agonist. As it stands, the in vitro data appear to contradict one of the main conclusions of the paper, because it would seem that human Pyrin and AIM2 inflammasomes can detect Shigella, and so these should compensate for NLRC4. The explanation as to why Pyrin and AIM2 are insufficient to compensate for NLRC4 evasion in human infection should be addressed at least in discussions of the data to explain the apparent discrepancy.
Response: The reviewer states that our claim that human PYRIN and AIM2 inflammasomes can detect Shigella in THP1 cells is “inconsistent” with the overall conclusion of our paper, which is that the NLRC4 inflammasome provides necessary defense of mouse intestinal epithelial cells. We do not agree that there is an inconsistency and indeed many of the points the reviewer makes in their comments fit with our view, so perhaps there is less disagreement than it might seem.
As the reviewer discusses, differences in inflammsome expression in humans vs. mice, and in IECs vs. macrophages vs. THP1 cells, and the kinetics of inflammasome responses, as well as several other factors, can easily account for the results we obtain. It appears that PYRIN is not well expressed in mouse IECs (Price et al. 2016), at least not uniformly at levels in all cells that are sufficient to confer protection. AIM2 is expressed in colonic IECs (Price et al. 2016), but it is not clear that it would be engaged in every infected IEC. For example, AIM2 detects bacterial DNA, which might only be released if the Shigella bacteria lysed in the cytosol. As noted by the reviewer, this may be a relatively rare event, as previously documented for AIM2 activation by Listeria-infected macrophages (Sauer JD et al, 2010). AIM2 activation may also be kinetically delayed in IECs. It appears instead that NLRC4 is the main inflammasome that can respond to Shigella in mouse IECs; thus loss of NLRC4 is sufficient to lead to susceptibility of mice. It remains possible that there is some functional AIM2 or PYRIN (or CASP11 or NLRP1B) in mouse IECs; thus, the further removal of these inflammasomes might lead to even greater susceptibility. Alternatively, a low level of activation mediated by these additional inflammasomes (perhaps in macrophages instead of in IECs) might even be necessary to produce the inflammation that causes disease symptoms.
In humans, consistent with our data in Fig. 1, we propose that the NLRC4 inflammasome is antagonized or otherwise evaded by Shigella. The reviewer wonders why PYRIN or AIM2 cannot compensate for NLRC4, and is suspicious that the activation of PYRIN/AIM2 we observe in THP1 cells is not representative of what would occur in vivo. Certainly we agree that THP1 cells are non-physiological and we do not attempt to make claims in the manuscript that our observation of AIM2/PYRIN activity in these cells means anything for human shigellosis.
The reviewer states: “the in vitro data [in THP1 cells] appear to contradict one of the main conclusions of the paper, because it would seem that human Pyrin and AIM2 inflammasomes can detect Shigella, and so these should compensate for NLRC4.” For all the reasons discussed above, we do not agree there is a contradiction. There are many reasons why PYRIN and AIM2 might function in THP1 cells (and possibly even human macrophages) but would not compensate for NLRC4 in IECs.
In sum, we agree that there is more to learn about which inflammasomes, if any, are activated by Shigella in human IECs, but given the many uncertainties, we do not feel it is fair to say that our results are internally contradictory. We will endeavor to discuss some of these points in a revised manuscript.
###Reviewer #3:
Mitchell et al describe the development of a mouse model for shigella gastroenteritis, the lack of which has been a serious impediment to Shigella research. They identified a difference in recognition of shigella between human and mouse Naip/NLRC4 which contributes to the resistance of mice to Shigella gastroenteritis. They suggest that Shigella specifically inhibits human Naip/NLRC4 activation and that the difference between mice and human susceptibility to infection is due to differential inhibition. This was confirmed by the ability of NLRC4-/- mice can recapitulate human infection. Furthermore they show that it is inhibition of NAIP-NLRC4 in IEC that is required for infection to occur. This manuscript therefore describes a number of important findings and uses these to develop a very useful animal model of shigellosis.
We are grateful for the Reviewer’s comments and suggestions, and provide point-by-point responses below:
I have three suggestions that I believe would improve the manuscript:
- Determine the inflammasome that causes cell death in Shigella-infected THP1's. WT Shigella infection did not induce pyroptosis of colchicine-treated (PYRIN inhibitor) AIM2-/- THP1 cells, indicating one or both of these inflammasomes is responsible for the cell death observed in shigella infected THP1 cells. Why not test these separately to determine which?
Response: We have now made *AIM2/MEFV–/– *THP-1 cells. Our preliminary finding is that cell death and IL-1B levels in these cells are impaired in response to Shigella infection. We intend to include these data in a revised manuscript.
- Markers of inflammation during disease. Clinical features of the disease (diarrhoea, weight, CFU/organ, fecal blood) are described well. But since Shigellosis is an inflammatory disease, it would have been nice to have seen some inflammatory molecules/cytokine levels measured, in addition to clinical features. The authors did measure levels of MPO, but that was as a marker for neutrophil recruitment.
Response: We agree that additional readouts of inflammatory disease are warranted. We are planning to repeat our experiments and measure cytokines in the blood. We intend to provide these data in a revised manuscript.
- Further refinement of the mouse model. The authors present the inhibition of human NAIP/NLRC4 as the main factor that affects the difference in infection between humans and mice but a high innolcum (5 x 10(7) cfu/mouse compared to approx. 100 cfu for humans) is still required in addition to streptomycin treatment. It is not discussed whether any refinement of these procedures was attempted or why such a high inoculum and streptomycin treatment is still required. Presumably microbiota differences in addition to naip-/nlrc4 is an important species specific determinant of infection, hence the streptomycin treatment. Why is such a high innoculum required?
Response: this comment is similar to one of the comments of Reviewer 1. As we state above, it is actually not entirely clear that the infectious dose for humans is consistently ~100 CFU. Indeed, there appears to be great variation, with some humans exhibiting resistance to doses more than 10^5 CFU. Although we used high inoculums in our experiments, this was just to ensure consistent infection of all mice. Preliminary experiments in which we reduce the dose suggests that, like some humans, some mice are also susceptible to lower doses (e.g., 10^5 CFU). Thus our model exhibits an infectious dose within the range of what is observed in humans and we do not feel there is a large discrepancy here, though it appears that we do not recapitulate the extreme susceptibility seen in some humans. We don’t find this particularly surprising as Shigella is a human-specific pathogen and it is likely that at least some of its virulence factors may not work well in mice. Instead, we think what is most surprising is that loss of one host defense component (NLRC4) is sufficient to produce disease symptoms that are strikingly similar to what is seen in humans. We acknowledge that one difference is the need for streptomycin in our model. Clearly this suggests, as the reviewer states, that the microbiota can influence susceptibility. This is a well-described phenomenon with many enteric pathogens and it will be of interest in future studies to determine what components of the microbiota afford protection in our model.
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###Reviewer #3:
Mitchell et al describe the development of a mouse model for shigella gastroenteritis, the lack of which has been a serious impediment to Shigella research. They identified a difference in recognition of shigella between human and mouse Naip/NLRC4 which contributes to the resistance of mice to Shigella gastroenteritis. They suggest that Shigella specifically inhibits human Naip/NLRC4 activation and that the difference between mice and human susceptibility to infection is due to differential inhibition. This was confirmed by the ability of NLRC4-/- mice can recapitulate human infection. Furthermore they show that it is inhibition of NAIP-NLRC4 in IEC that is required for infection to occur. This manuscript therefore describes a number of important findings and uses these to develop a very useful animal model of shigellosis.
###Reviewer #3:
Mitchell et al describe the development of a mouse model for shigella gastroenteritis, the lack of which has been a serious impediment to Shigella research. They identified a difference in recognition of shigella between human and mouse Naip/NLRC4 which contributes to the resistance of mice to Shigella gastroenteritis. They suggest that Shigella specifically inhibits human Naip/NLRC4 activation and that the difference between mice and human susceptibility to infection is due to differential inhibition. This was confirmed by the ability of NLRC4-/- mice can recapitulate human infection. Furthermore they show that it is inhibition of NAIP-NLRC4 in IEC that is required for infection to occur. This manuscript therefore describes a number of important findings and uses these to develop a very useful animal model of shigellosis.
I have three suggestions that I believe would improve the manuscript:
Determine the inflammasome that causes cell death in Shigella-infected THP1's. WT Shigella infection did not induce pyroptosis of colchicine-treated (PYRIN inhibitor) AIM2-/- THP1 cells, indicating one or both of these inflammasomes is responsible for the cell death observed in shigella infected THP1 cells. Why not test these separately to determine which?
Markers of inflammation during disease. Clinical features of the disease (diarrhoea, weight, CFU/organ, fecal blood) are described well. But since Shigellosis is an inflammatory disease, it would have been nice to have seen some inflammatory molecules/cytokine levels measured, in addition to clinical features. The authors did measure levels of MPO, but that was as a marker for neutrophil recruitment.
Further refinement of the mouse model. The authors present the inhibition of human NAIP/NLRC4 as the main factor that affects the difference in infection between humans and mice but a high innolcum (5 x 10(7) cfu/mouse compared to approx. 100 cfu for humans) is still required in addition to streptomycin treatment. It is not discussed whether any refinement of these procedures was attempted or why such a high inoculum and streptomycin treatment is still required. Presumably microbiota differences in addition to naip-/nlrc4 is an important species specific determinant of infection, hence the streptomycin treatment. Why is such a high innoculum required?
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###Reviewer #2:
Mitchell et al explore the role of NLRC4 in defending against Shigella infection by demonstrating that NLRC4 contributes to resistance to shigellosis in mice. Using in vitro assays, they first show that mouse but not human macrophages undergo NLRC4-mediated pyroptosis in response to Shigella infection despite an ability for both species to successfully detect Shigella NLRC4 agonists. They then demonstrate that C57BL/6 background mice, which normally resist shigellosis, become susceptible to infection when deficient in NAIPs or NLRC4. In parallel, 129 background mice develop more significant infection including intestinal bleeding. Furthermore, using a mouse line in which NLRC4 expression is restricted to intestinal epithelial cells (IECs), they show that IEC expression of NLRC4 is sufficient to resist shigellosis. …
###Reviewer #2:
Mitchell et al explore the role of NLRC4 in defending against Shigella infection by demonstrating that NLRC4 contributes to resistance to shigellosis in mice. Using in vitro assays, they first show that mouse but not human macrophages undergo NLRC4-mediated pyroptosis in response to Shigella infection despite an ability for both species to successfully detect Shigella NLRC4 agonists. They then demonstrate that C57BL/6 background mice, which normally resist shigellosis, become susceptible to infection when deficient in NAIPs or NLRC4. In parallel, 129 background mice develop more significant infection including intestinal bleeding. Furthermore, using a mouse line in which NLRC4 expression is restricted to intestinal epithelial cells (IECs), they show that IEC expression of NLRC4 is sufficient to resist shigellosis. Finally, using a known attenuated Shigella mutant, they demonstrate that their shigellosis model can mimic kinetics seen in humans.
Mitchell et al convincingly demonstrate both the importance of NLRC4 in protecting mice against Shigella and the utility of their mouse model for studying Shigella infections, both of which are significant and will push the Shigella field forward. There are mechanistic questions to be addressed in future studies beyond the current manuscript, attesting to the importance of the paper in opening up new areas in the field of research. In some places, the authors draw conclusions that reach beyond what is proven in the data, which should be addressed in text edits to the manuscript. In summary, this article presents an important new model for Shigella infection. The impact of the manuscript is the development of a mouse model with which to study Shigella infection in vivo.
Major comments:
Many questions remain concerning why NLRC4-deficient THP1 cells still undergo pyroptosis. The authors provide evidence that Shigella activates PYRIN and/or AIM2 inflammasomes in humans, and that somehow mouse macrophages would fail to have this same detection. At face value, the data would suggest that humans are able to detect Shigella by Pyrin and AIM2, but for some reason these two inflammasomes are insufficient, and instead NLRC4 is required for in vivo defense. Then in mice, it would imply that everything is flipped - for some reason detection by Pyrin and AIM2 is not important, but now the bacteria can be detected by NLRC4 and this is important. The NLRC4 focused conclusions are consistent with the in vivo data, that NLRC4 in humans fails to detect, but NLRC4 in mice succeeds in detecting Shigella. However, the data that Pyrin and AIM2 in human cells successfully detect Shigella are inconsistent with the overall conclusions of the paper. I suspect that this is an artifact of THP1 cells, and that the in vivo situation in humans is that these two inflammasomes will fail to detect Shigella. There is published precedent from other infections where in vitro detection belies in vivo lack of detection (e.g. Listeria is detected by AIM2 in vitro, but probably not in vivo). It may be difficult to make direct comparisons between how inflammasomes act in THP1 cells as compared to BMMs, due to artifacts arising from the different origins and passage levels of the two cell types. It may be that the inflammasomes response is most important in IECs, as proposed by the authors, and that IECs may not express Pyrin or AIM2. There is evidence from publicly available IEC transcriptional profiles that IECs do not express Pyrin (Mefv) (Reikvam, doi: 10.1371/journal.pone.0017996), although this profile does show Aim2 expression in IEC. It is my understanding that BMMs do not express Pyrin unless they are strongly stimulated with some TLR agonist. As it stands, the in vitro data appear to contradict one of the main conclusions of the paper, because it would seem that human Pyrin and AIM2 inflammasomes can detect Shigella, and so these should compensate for NLRC4. The explanation as to why Pyrin and AIM2 are insufficient to compensate for NLRC4 evasion in human infection should be addressed at least in discussions of the data to explain the apparent discrepancy.
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###Reviewer #1:
The manuscript by Mitchell et al. finds that the NAIP-NLRC4 inflammasome in mice is a critical host factor that controls intestinal infection with the human specific bacterial pathogen Shigella flexneri. The work suggests that Shigella is actively suppressing the human NAIP-NLRC4 inflammasome possibly using an T3SS effector protein, which does not recognize its substrate in mouse cells. The authors use this information to determine that B6 mice lacking the NAIP or NLRC4 inflammasome components are susceptible to Shigella infection and observe disease symptoms similar to Shigellosis in humans. In addition, 129 mice exhibit additional disease symptoms, and the authors suggest that loss of Caspase-11 in 129 mice is responsible for this phenotype.
The strengths of this manuscript include the introduction of a new mouse model …
###Reviewer #1:
The manuscript by Mitchell et al. finds that the NAIP-NLRC4 inflammasome in mice is a critical host factor that controls intestinal infection with the human specific bacterial pathogen Shigella flexneri. The work suggests that Shigella is actively suppressing the human NAIP-NLRC4 inflammasome possibly using an T3SS effector protein, which does not recognize its substrate in mouse cells. The authors use this information to determine that B6 mice lacking the NAIP or NLRC4 inflammasome components are susceptible to Shigella infection and observe disease symptoms similar to Shigellosis in humans. In addition, 129 mice exhibit additional disease symptoms, and the authors suggest that loss of Caspase-11 in 129 mice is responsible for this phenotype.
The strengths of this manuscript include the introduction of a new mouse model that mimics Shigellosis, the demonstration that NAIP/NLRC4 activation is important for epithelial cell defense, and the potential of these findings to clarify aspects of human infectious disease caused by this pathogen. The manuscript is well presented, and the experiments are conducted with a high degree of rigor. Overall, this is an important contribution to the Shigella field and also has significant implications on our understanding of inflammasomes in host defense against pathogens.
There are some weaknesses that should be addressed. Experimentally, it has not been directly demonstrated that IECs from NLRC4-/- mice undergo cell death (using biochemical markers). This is a critical aspect of the model. In addition, it would be useful for the authors to evaluate bacterial burden over the time course in Figure 6. Although this is not absolutely necessary to support the manuscript conclusions, this information would greatly benefit the community that intends to use these mice in the future.
There are also some discussion points about the mouse model that would enhance the overall impact of the work. For example, a more in depth discussion about the differences between human Shigella infection and the new model would be helpful. It is important to emphasize that the mouse model requires a much greater inoculum of the pathogen to induce disease and requires microbiota-deficiency to be effective. What are the implications of this finding on our understanding of human disease? In lines 274-285 the authors present an either/or scenario in which either macrophage pyroptosis is required for IEC infection or inhibition of NAIP/NRLC4 pyroptosis in IECs is required for IEC infection. However, these scenarios are not mutually exclusive. For example, it is plausible that the extremely low burdens of Shigella required to infect humans (<100 CFUs) is due to the pathogen initially crossing the epithelial barrier (e.g. through M-cells) to infect macrophage, and then re-infection of IECs after macrophage pyroptosis. In this scenario, the NAIP/NLRC4 inflammasome could prevent further expansion of bacterial in IECs by eliminating the cell-to-cell spread that have been described by others. Importantly, the macrophage lifecycle stage may not be necessary in mice in which the microbiota has been removed and Shigella is delivered at a very high inoculum. While, additional ideas could be, and should be, put forth since the mouse model provides new insights or challenges an existing dogma in the field.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary
In this manuscript, the authors introduce a new mouse model of Shigellosis, provide evidence for NAIP/NLRC4 activation as being important for epithelial cell defense, and apply these findings to observations made in humans infected by this pathogen. These are important findings and provide an opportunity to further advance the field in ways not previously possible. However, there are areas where the in vitro and in vivo data presented contradict each other, and there are inconsistencies with previously …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.
###Summary
In this manuscript, the authors introduce a new mouse model of Shigellosis, provide evidence for NAIP/NLRC4 activation as being important for epithelial cell defense, and apply these findings to observations made in humans infected by this pathogen. These are important findings and provide an opportunity to further advance the field in ways not previously possible. However, there are areas where the in vitro and in vivo data presented contradict each other, and there are inconsistencies with previously published work by the authors. In addition, with the development of the new mouse model being a major highlight of this manuscript, significantly more detail and discussion must be added to explain this mouse model.
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