Dear Dr Kabe,

Thank you for submitting your manuscript entitled "Short-chain fatty acids bind to apoptosis-associated speck-like protein to activate inflammasome complex to prevent Salmonella infection" for consideration as a Research Article by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review.

However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Please re-submit your manuscript within two working days, i.e. by Feb 09 2020 11:59PM.

Login to Editorial Manager here: https://www.editorialmanager.com/pbiology

During resubmission, you will be invited to opt-in to posting your pre-review manuscript as a bioRxiv preprint. Visit http://journals.plos.org/plosbiology/s/preprints for full details. If you consent to posting your current manuscript as a preprint, please upload a single Preprint PDF when you re-submit.

Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. Once your manuscript has passed all checks it will be sent out for review.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Lauren A Richardson, Ph.D

Senior Editor

PLOS Biology

Dear Dr Kabe,

Thank you very much for submitting your manuscript "Short-chain fatty acids bind to apoptosis-associated speck-like protein to activate inflammasome complex to prevent Salmonella infection" for consideration as a Research Article at PLOS Biology. Your manuscript has been evaluated by the PLOS Biology editors, an Academic Editor with relevant expertise, and by two independent reviewers.

In light of the reviews (below), we will not be able to accept the current version of the manuscript, but we would welcome re-submission of a much-revised version that addresses all of the reviewers' concerns. We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent for further evaluation by the reviewers.

We expect to receive your revised manuscript within 2 months.

Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension. At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may end consideration of the manuscript at PLOS Biology.

**IMPORTANT - SUBMITTING YOUR REVISION**

Your revisions should address the specific points made by each reviewer. Please submit the following files along with your revised manuscript:

1. A 'Response to Reviewers' file - this should detail your responses to the editorial requests, present a point-by-point response to all of the reviewers' comments, and indicate the changes made to the manuscript.

*NOTE: In your point by point response to the reviewers, please provide the full context of each review. Do not selectively quote paragraphs or sentences to reply to. The entire set of reviewer comments should be present in full and each specific point should be responded to individually, point by point.

You should also cite any additional relevant literature that has been published since the original submission and mention any additional citations in your response.

2. In addition to a clean copy of the manuscript, please also upload a 'track-changes' version of your manuscript that specifies the edits made. This should be uploaded as a "Related" file type.

*Re-submission Checklist*

When you are ready to resubmit your revised manuscript, please refer to this re-submission checklist: https://plos.io/Biology_Checklist

To submit a revised version of your manuscript, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' where you will find your submission record.

Please make sure to read the following important policies and guidelines while preparing your revision:

*Published Peer Review*

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*PLOS Data Policy*

Please note that as a condition of publication PLOS' data policy (http://journals.plos.org/plosbiology/s/data-availability) requires that you make available all data used to draw the conclusions arrived at in your manuscript. If you have not already done so, you must include any data used in your manuscript either in appropriate repositories, within the body of the manuscript, or as supporting information (N.B. this includes any numerical values that were used to generate graphs, histograms etc.). For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5

*Blot and Gel Data Policy*

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare them now, if you have not already uploaded them. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Di Jiang, PhD

Senior Editor

PLOS Biology

*****************************************************

REVIEWS:

Reviewer #1: Short chain fatty acids have been of continued interest due to their various effects on the microbiota and the host. The presented study by Tsugawa et al. sheds light on the mechanism of how SCFAs modulate the immune response and prolong survival after Salmonella infection. The authors determined that SCFAs bind to a lysine-rich domain of ASC, which facilitates binding to NLRPs. Macrophages exposed to SCFAs killed Salmonella more efficiently in vitro. In vivo, SCFA administration to antibiotics-treated mice increased their survival. No increased survival was seen for SCFA administration to ASC-/- mice. Administration of soluble fiber increased survival of Salmonella-infected mice.

This manuscript uncovers an interesting mechanism that should be of interest to a broad audience, as SCFA-mediated enhancement of the immune response will likely occur also with other intestinal pathogens. The experiments were in general performed rigorously and include important controls. The authors used a variety of different methods, including imaging mass spectrometry, to support their hypothesis. However, for some experimental assays, additional information and clarifications need to be provided.

The manuscript has two main weaknesses: 1) co-housing instead of using littermates and the lack of microbiota analysis and 2) no experimentation to exclude that added SCFAs regulate virulence gene expression in Salmonella.

Co-housing apparently rendered the observed phenotype in Fig. 1 less pronounced. The microbiota analysis would, especially as fiber fermentation is subject of analysis, be a valuable addition to the paper. It could help dispel concerns that phenotypes are due to microbiota difference not resolved during co-housing. A number of studies have shown that SCFAs modulate Salmonella virulence gene expression. This was not addressed experimentally or discussed.

The authors should address the following points:

1) Include analysis of the microbiota to show that WT and ASC-/- mouse microbiota is highly similar before the start of the experiment and during recovery. If there are differences before, differences in the microbiota that recovers after the stop of antibiotic treatment could be partially responsible for the observed phenotype.

2) Related to point 1: The experimental setup in 4B is unclear. Please indicate with horizontal bars the periods when the antibiotic cocktail was administered and when SCFAs were administered. Was administration of the cocktail stopped before Salmonella infection or was a resistant Salmonella strain used?

3) The authors did not indicate for how long they co-housed the mice. Extensive co-housing is accepted by researchers as a way to normalize the microbiota of strains of different origins. However, the authors used male mice for their experiments, where co-housing would presumably result in severe aggression. Please indicate for how long mice were co-housed and how they were housed during experiments (single vs. group).

4) SCFAs like butyrate and propionate are known to down-regulate Salmonella SPI-1 expression at higher concentrations. Experiments with ASC-/- mice and BMDM seem to indicate that this is not a relevant mechanism in this model. No actual numbers, just percentages are given, so it is difficult to interpret. A probably relevant control would be an experiment with a SPI-1 deficient Salmonella strain and WT and ASC-/- BDMDs.

5) Are the pictures in Fig. 5B truly representative? The immune cell recruitment with SCFAs is massive. Fig. 5C shows more moderate recruitment. Please provide pathology scores to assess all and not just representative mice.

6) Were all experiments done exclusively with male mice? The purchased C57BL/6 mice were male, but no indication for other strains is given. Ideally (and most often now requested) experiments should be performed with male and female mice to exclude gender bias.

Minor comments:

Manuscript is well written, but it needs to be thoroughly checked (e.g. "% of survive" or "infection to wild-type mice" and "scarify" in Fig. 4).

Multiple mouse models exist for Salmonella Typhimurium. Please provide the rationale for the choice of the mouse model you use (e.g. lower colonization and cecum inflammation than streptomycin pre-treatment mouse model).

M&M clarifications needed:

* Fiber and composition of the diet is important for the observed phenotype. Please indicate which diet the mice were fed.

* Mice are known to avoid drinking the antibiotic cocktail. How much weight did they lose before the beginning of the experiment? This should be noted to orient the reader to the fact that there are more differences between SPF and antibiotics-treated mice than the presence or absence of bacteria.

* The Salmonella strain used is likely ATCC 14028S and not ATCC 14208S?

* Rationale for using U937 cells, mention also in results.

* Some key information, e.g. how long BMDMs were incubated with Salmonella, is only available in the figure legend and not in the M&M. Please add where appropriate.

Fig. 3: Percent of LDH release is compared to which positive control?

Fig. 7: Imaging mass spectrometry is a very useful tool to illustrate the penetration of SCFAs into the tissue. In uninfected mice, the cecal tissue is easily identified, and also in sections of the infected mice. However, there seems to be absolutely no structure in the remaining parts, rather only huge rips. Are these artefacts from processing? Please explain this a bit more.

Fig. 3E: Why are here CFU/ml and not CFU as percent of control? How did this assay differ from the other similar assays in Fig. 3?

Fig. 3D: Please indicated that levels were below limit of detection and are not missing data

Fig. S4E: I assume you have done multiple tests to confirm the deletion, but this WB does not look convincing.

Fig. S7: Why was the extra SCFA boost necessary? Lines 252-254 seems to indicate that SCFA administration in the drinking water increases SCFA levels to similar levels as in SPF mice. However, the authors seem to measure an undefined boost and not what is available during Salmonella infection.

Fig. S9: Bacterial numbers recovered from mice usually have log-normal distribution and are best displayed on a log scale.

Reviewer #2: Tsugawa et al provide evidence for an interesting model in which short chain fatty acids (SCFAs), which are produced through fermentation of dietary fiber by members of the gut microbiota, facilitate the interaction between the inflammasome pattern recognition receptor Nlrp3 and its adaptor Asc, leading to enhanced inflammasome activation. The authors pinpoint a lysine rich region of the pyrin domain as important for SCFA binding. Furthermore, SCFA treatment of macrophages leads to pyroptosis and restricts Salmonella intracellular survival. In addition, SCFA administration to mice induces inflammation and enhances resistance against Salmonella infection. These results suggest the intriguing possibility that eating a diet rich in fiber may protect against Salmonella infection. However, the manuscript is lacking in sufficient context with respect to previous studies involving Salmonella and SFCAs. A number of references important to this work are omitted and not discussed.

Major comments:

1) There is no reference to PMID: 29447698 entitled "Genetic ablation of butyrate utilization attenuates gastrointestinal Salmonella disease". Among other things relevant to this manuscript, this paper showed that butyrate decreased Salmonella colonization of Peyer's patches in gnotobiotic mice.

2) If SCFAs induce ROS by neutrophils (pg 13 line 100), why would Salmonella colonization of the cecum decrease (Fig S9), as ROS induction by Salmonella itself leads to generation of alternative TEAs that allows Salmonella to outcompete microbiota (PMID: 27078066)?

3) What is the rationale behind using STm strain A and needing to compare it to ATCC 14208S?

4) An explanation of how Nlrp3 normally gets activated and interacts with Asc is lacking. What's known and how could SCFAs fit in? Would SCFAs affect nucleation of Asc filaments by Nlpr3, as in Lu et al 2014 Cell (PMID: 24630722).

5) Is it possible that the top band in Fig 1B found in the proprionate and butyrate samples but not the acrylate sample is polymerized Asc?

6) Fig 3A: What is shown in the images? If they are wells with infected monolayers, why are the cells growing in clumps? A scale bar would be useful.

7) Why was procasp-1 and casp-1 precipitated out of the infected monolayer supernatant using acid rather than probed from cell lysate (Fig 3B)?

8) Expression of SPI-1 genes in Salmonella were previously shown to modulate SPI-1 gene expression (PMID: 19433544, PMID: 12453229), but this is not mentioned in the manuscript nor are these papers referenced. Since the SPI-1 T3SS induces inflammasome activation, this is important to address.

9) Fig S3A: Why are Salmonella killed even in the absence of SCFAs during intracellular residence even at time points where no pyroptosis is detected?

10) Are the mice in Fig 4C and Fig 6 pre-treated with antibiotics?

11) In Barthel et al 2003 I&I (PMID: 12704158), Salmonella infection in the absence of antibiotic pre-treatment did not lead to cecal inflammation, which is at odds with Fig 5A.

12) Does SCFA treatment of naïve mice alter tissue architecture?

13) Where are the Salmonella replicating in clodronate-treated mice? Inside other cell types? Extracellularly?

Minor comments:

1) Needs to be made clear that bacteria do not make SCFAs only to regulate immune responses (pg. 11 lines 62-3).

2) Grammatical errors are found in the manuscript. For example, pg 11 line 64.

3) Pg 13 line 107, should be SCFA-bound, not SCFA-found.

4) Fig 4B, "scarify" would be "sacrifice".

Dear Dr Kabe,

Thank you for submitting your revised Research Article entitled "Short-chain fatty acids bind to apoptosis-associated speck-like protein to activate inflammasome complex to prevent Salmonella infection" for publication in PLOS Biology. I have now obtained advice from the original reviewers and have discussed their comments with the Academic Editor.

Based on the reviews, we will probably accept this manuscript for publication, assuming that you will modify the manuscript to address the remaining points raised by the reviewers. Please also make sure to address the data and other policy-related requests noted at the end of this email.

We expect to receive your revised manuscript within two weeks. Your revisions should address the specific points made by each reviewer. In addition to the remaining revisions and before we will be able to formally accept your manuscript and consider it "in press", we also need to ensure that your article conforms to our guidelines. A member of our team will be in touch shortly with a set of requests. As we can't proceed until these requirements are met, your swift response will help prevent delays to publication.

*Copyediting*

Upon acceptance of your article, your final files will be copyedited and typeset into the final PDF. While you will have an opportunity to review these files as proofs, PLOS will only permit corrections to spelling or significant scientific errors. Therefore, please take this final revision time to assess and make any remaining major changes to your manuscript.

NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:

https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

*Early Version*

Please note that an uncorrected proof of your manuscript will be published online ahead of the final version, unless you opted out when submitting your manuscript. If, for any reason, you do not want an earlier version of your manuscript published online, uncheck the box. Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

*Submitting Your Revision*

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include a cover letter, a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable), and a track-changes file indicating any changes that you have made to the manuscript.

Please do not hesitate to contact me should you have any questions.

Sincerely,

Di Jiang, PhD

PLOS Biology

------------------------------------------------------------------------

ETHICS STATEMENT:

-- Please create a separate subsection entitled "Ethics Statement" and place it in the beginning of the Methods section, and please include all relevant information described below.

-- Please include the full name of the IACUC/ethics committee that reviewed and approved the animal care and use protocol/permit/project license. Please also include an approval number.

-- Please include the specific national or international regulations/guidelines to which your animal care and use protocol adhered. Please note that institutional or accreditation organization guidelines (such as AAALAC) do not meet this requirement.

-- Please include information about the form of consent (written/oral) given for research involving human participants. All research involving human participants must have been approved by the authors' Institutional Review Board (IRB) or an equivalent committee, and all clinical investigation must have been conducted according to the principles expressed in the Declaration of Helsinki.

------------------------------------------------------------------------

DATA POLICY:

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication.

Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it: Figures 2CD, 3ACDEF, 4ACD, 5DE, 6ABD, 7A-D, S1, S3A-C, S5AB, S6A-D, S7A, S8, S9, S10, S11, S12AB, S15. NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.

Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

------------------------------------------------------------------------

BLOT AND GEL REPORTING REQUIREMENTS:

For manuscripts submitted on or after 1st July 2019, we require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

------------------------------------------------------------------------

Reviewer remarks:

Reviewer #1: The authors very carefully and thoughtfully revised their manuscript. They added a large amount of additional experimental data to support their hypothesis and address the reviewer's concerns. The additional clarifications helped to make the manuscript more accessible to a larger readership. I have only two minor comments.

Comments:

The authors added a microbiome analysis as requested. Unfortunately, the graphs that were chosen for the manuscript do not actually show that there is no difference in the microbiome composition. The graphs show that there is no difference in alpha diversity, which measures the number and abundance of taxa in a given sample. Alpha diversity can be exactly the same in individuals of two groups while the community composition (which taxa are present) between the groups can be completely different. In order to show that there are no significant differences in community structure between the experimental groups, beta diversity needs to be shown. The microbiota analysis is clearly not the focus of the paper or the expertise of the authors. However, the authors went through the trouble of adding this important piece of data for their mouse experiments. It should therefore be actually significant data that supports their hypothesis. The microbiome data are already available, so adding beta diversity PCA plots should be a minor modification.

The description of the "boost" is misleading. In the results section, the concentrations of the SCFAs in water are not given, only the concentrations for the "boost". This gives the impression that the concentration given by oral gavage is higher than what the mice are drinking throughout the experiment. Through the clarification in M&M I learned that this is not the fact, that the concentration in the boost and in the water are the same. As you explained in the reply to my comment, this was more a "forced drinking" to prevent differences in measurements resulting from that once mouse had recently drank and another an hour ago. You might want to clarify this in the results section.

Reviewer #2: 1) Why is intracellular growth data presented as % CFU in Fig 3 but as CFU/mL in Fig S6B? Also, line 257 should read "non-significant effect" rather than "weak effect".

2) The SPI-1 knockdown mutant induced ASC-dependent IL-1� in BMDMs synergistically with SCFAs, but SCFAs did not potentiate LDH release nor diminish intracellular growth of the SPI-1 mutant. This is in contrast to WT bacterial infection during SCFA treatment, where SCFAs led to significantly less intracellular growth of WT Salmonella and more LDH and IL-1� release. This result is not sufficiently explained.

1) "Butyrate" is misspelled in line 143.

2) Grammatical errors persist.

Dear Dr Kabe,

On behalf of my colleagues and the Academic Editor, Matthew K. Waldor, I am pleased to inform you that we will be delighted to publish your Research Article in PLOS Biology.

The files will now enter our production system. You will receive a copyedited version of the manuscript, along with your figures for a final review. You will be given two business days to review and approve the copyedit. Then, within a week, you will receive a PDF proof of your typeset article. You will have two days to review the PDF and make any final corrections. If there is a chance that you'll be unavailable during the copy editing/proof review period, please provide us with contact details of one of the other authors whom you nominate to handle these stages on your behalf. This will ensure that any requested corrections reach the production department in time for publication.

Early Version

The version of your manuscript submitted at the copyedit stage will be posted online ahead of the final proof version, unless you have already opted out of the process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have not yet opted out of the early version process, we ask that you notify us immediately of any press plans so that we may do so on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Thank you again for submitting your manuscript to PLOS Biology and for your support of Open Access publishing. Please do not hesitate to contact us if we can provide any assistance during the production process.

Kind regards,

Pamela Berkman

Publishing Editor,

PLOS Biology

on behalf of

Gabriel Gasque

Senior Editor

PLOS Biology