PONE-D-20-06132

Host immunity and the colon microbiota of mice infected with Citrobacter rodentium are beneficially modulated by lipid-soluble compounds from late-cutting alfalfa in the early stages of infection

PLOS ONE

Dear Dr Bobeck,

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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Juan J Loor

Academic Editor

PLOS ONE

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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

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

Reviewer #1: Yes

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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: 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

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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: General comments/questions:

-Introduction is extremely long.

-Add a paragraph justifying the choice of inducing an infection with "Citrobacter rodentium" for this study

- How did you demonstrate that the challenge with Citrobacter rodentium was effective to study a response?

-I did not see assays that specifically measured the lipid-compounds. So please, edit the title of the manuscript accordingly. From "are beneficially modulated by lipid-soluble compounds from late-cutting alfalfa", to "are modulated by late-cutting alfalfa"

1) Please, reduce the introduction to 1.5 pages max.

2) Focus on explaining the most relevant facts about the use of alfalfa, including:

"documented source of bioactive compounds" - which compounds?; which bacterial species in the gut has been affected upon alfalfa diet? increased commensal bacteria?

Material and methods:

3) Include the Animal# for the IACUC

4) Blood samples were not collected? Why?

5) please, explain which procedure (including the anesthesia) was used for the mouse euthanasia

6) how were the colon contents collected (i.e., separated from the tissue)?

7) After collection, were the spleen samples stored in microtubes containing RMPI-FBS or were immediately frozen?

8) why 6-week old mouse, instead of 14 or 21d old mouse? For instance, wouldn't alfalfa benefit the early life gut colonization?

9) Flow cytometry analysis did not include the aqueous extract treatment? Why?

Results:

10) Please, re word the sentences on lines 253-255, that's confusing

11) the examining recovery method was not explained in material and methods, but it was discussed. Please, add a paragraph explaining the recovery examination post-infection.

12) "Production of TNF-� is often associated with macrophage activity", please delete. TNFa is associated with a variety of activities.

13) Please, correct "(PCoA) " to PCA

Discussion:

14) Please, reduce the discussion about BW. It's too long for 2 parameters.

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Reviewer #1: No

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Host immunity and the colon microbiota of mice infected with Citrobacter rodentium are beneficially modulated by lipid-soluble extract from late-cutting alfalfa in the early stages of infection

PONE-D-20-06132R1

Dear Dr. Bobeck,

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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Kind regards,

Juan J Loor

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

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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

**********

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

Reviewer #1: Yes

**********

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

**********

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

**********

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: (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