PONE-D-21-36309Vertical transmission of attaching and invasive E. coli from the dam to neonatal mice predisposes to more severe colitis following exposure to a colitic insult later in lifePLOS ONE

Dear Dr. Wannemuehler

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Although one reviewer appreciates the importance of the study, the other reviewer has critical concerns about the authors' experimental design and  interpretation of their results. Thus, I would like to ask the authors to respond to the comments raised by the reviewers as far as they can. 

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Please submit your revised manuscript by Feb 6, 2022. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Hiroyasu Nakano, M.D., Ph.D.

Academic Editor

PLOS ONE

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2. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

[The authors thank Mary Jane Long for technical assistance throughout these studies, Ross Darling, Jorrell Fredericks, and Danielle Wagner for assistance with necropsy and initial sample processing. Additionally thank you to Angela Bryan, Jessica Elbert, and Curtis Mosher for  assistance with FISH imaging and counting, Katarina Kohn for assistance with QIIME analyses, and Rachel Phillips for the RNAscope® imaging and Halo® analysis. The authors acknowledge support from the following organizations: Kenneth Rainin Foundation Innovator Award (AEJ, MJW, GJP - https://krfoundation.org/health/grants/innovator-awards/); the NIH NIGMS award R01GM099537-1A1 (GLP, MJW, AEJ, JMH http://www.nigms.nih.gov); NSF DBI 1551363 (IF http://www.nsf.dbi.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript].

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Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

 [Keneth Rainin Foundation Innovator Award (AEJ, MJW, GJP - https://krfoundation.org/health/grants/innovator-awards/);  the NIH NIGMS award R01GM099537-1A1 (GLP, MJW, AEJ, JMH   http://www.nigms.nih.gov); NSF DBI 1551363 (IF http://www.nsf.dbi.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”]

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In this study, Wannemuehler‘s group tried to demonstrate that an early exposure to microbiota beneficially impacts mucosal immunity later in life. To achieve this goal, authors examined whether neonatal colonization with AIEC provides lasting protection from DSS-induced colitis in ASF-colonized gnotobiotic mice. Contrary to the speculation that AIEC-colonized mice as a neonate develop less severe colitis induced by DSS in drinking water as compared with adult-colonized mice, the former group showed slightly increased severity of pathology associated with DSS-induced colitis than the latter group. This study addresses an important biological concept that early microbial exposures regulate the susceptibility against IBD later in life using a well-designed experimental protocol. Most of the statements are supported by experimental evidence and the manuscript is overall coherently written. Therefore, this paper could be accepted for publication to PLOS ONE subject to several amendments as described below.

Comments:

1. In Figure 2, although difference between vertically colonized mice and horizontally colonized ones is statistically significant, severity between two groups of mice is marginal at best. Considered that authors have rigorously performed statistical analysis using large number of mice, which was respectable, this reviewer is afraid whether AIEC-colonization during neonate has little or no impact on the mucosal immunity in terms of the severity of DSS-induced colitis. This reviewer proposes that authors mention about a possibility of pathobionts other than AIEC have larger impact on gut homeostasis in the DISCUSSION section.

2. Authors clearly showed that vertical colonization with AIEC does not change the mucosal pathology in steady-state. Explain more carefully the possible mechanism associated with the enhanced colitis, including enhanced colon IL-17 mRNA level, in mice colonized as a neonate, please. Again, related to comment 1, a possibility of vertical colonization with AIEC do regulate gut homeostasis in some manner but having little impact on the pathology of DSS-induced colitis could not be excluded.

Reviewer #2: The interaction of the microbiome with the mucosal immune system in the gut plays an important role in priming immunity. However, it is not fully understood how the microbiome regulates immune homeostasis. The authors have previously reported that E. coli LF82 colonization increases the susceptibility of adult C3H/HeN mice harboring the altered Schaedler flora (ASF) to dextran sodium sulfate (DSS)-induced colitis. In this study, the authors examined the impact of the pathobiont colonization of neonates on the severity of colitis later in life. The authors colonized gnotobiotic mice harboring the ASF with E. coli LF82 by oral gavage as an adult (LF82A) or by vertical acquisition as a neonate (LF82N) and evaluated the differences in the severity of DSS-induced colitis. The authors concluded that the DSS-induced colitis was exacerbated in the LF82N group compared to the LF82A group. Although the authors’ hypothesis appears to be interesting, the difference between the two groups (LF82N vs. LF82A) appears to be marginal. Moreover, there are several critical concerns about the authors’ experimental conditions.

The followings are specific comments.

Major points;

1. The authors concluded that the DSS-induced colitis was exacerbated in the LF82N group compared to the LF82A group based on the length of the colon. Although the macroscopic scores were correlated with the length of the colon, histological scores of the colitis between the two groups were not different. The authors need to discuss these points or replace them with more representative ones to support the authors’ conclusion.

1. In Figure 2D, the colonic mucosa in DSS-treated mice seemed to be normal. In general, DSS treatment usually induces focal ulceration of the colonic mucosa along with massive infiltration of mononuclear cells, as observed in the colon of LF82N + DSS. Thus, it is unclear why DSS treatment did not induce such changes. The authors need to explain apparent inconsistent results.

1. In Figure 4, it is unclear whether the positive signals actually represent the existence of the indicated bacteria. Why were the positive signals not detected in the lumen of the colon of LF82N mice? Given that LF82N mice harbored E. coli in the colon, the signals of E. coli should be present in the lumen of the colon. The authors should show these results in a more convincing manner.

What do G0 and G1 mean?

1. In Figure 5, there was no significant difference in cytokine production between the control and the DSS-treated grossssssups at least the authors’ experimental conditions. Given that DSS treatment induces severe inflammation and upregulation of many cytokines, the results seem to be surprising. To further substantiate the authors’ findings, the authors need to check the expression of several cytokines of the colon of untreated and DSS-treated mice by qPCR.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-36309R1Vertical transmission of attaching and invasive E. coli from the dam to neonatal mice predisposes to more severe colitis following exposure to a colitic insult later in lifePLOS ONE

Dear Dr. Wannemuehler

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. While both reviewers have been satisfied with the authors' revision, one reviewer still has minor concern. Please respond to the comments raised by the reviewer 2.  

Please submit your revised manuscript by March 31. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Hiroyasu Nakano, M.D., Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: (No Response)

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors have adequately responded to this reviewer's comments. The manuscript is now ready for publication to PLOSONE.

Reviewer #2: The authors have responded to the reviewers’ comments.

However, Figure 4 is still unclear. A low magnification picture showing the lumen and tissue, and a high magnification picture showing infiltration should be presented in the figure. In addition, they need to add the scales in the pictures.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Vertical transmission of attaching and invasive E. coli from the dam to neonatal mice predisposes to more severe colitis following exposure to a colitic insult later in life

PONE-D-21-36309R2

Dear Dr. Wannemuehler

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Hiroyasu Nakano, M.D., Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors have adequately responded to reviewers' requests. The revised version of manuscript satisfies the publication policy of PLOS ONE.

Reviewer #2: (No Response)

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No