Dear Aileen Milillo,

Thank you very much for submitting your manuscript "Brucella abortus RNA interferes with the M1 polarization of human macrophages" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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 to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. 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.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

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

Sincerely,

Ruifu Yang

Section Editor

PLOS Neglected Tropical Diseases

Ruifu Yang

Section Editor

PLOS Neglected Tropical Diseases

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

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

Yes

-Is the study design appropriate to address the stated objectives?

Yes

-Is the population clearly described and appropriate for the hypothesis being tested?

Yes

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

Not applicable.

-Were correct statistical analysis used to support conclusions?

Yes.

-Are there concerns about ethical or regulatory requirements being met?

No.

Reviewer #2: The manuscript is methodologically sound.

Reviewer #3: The objectives and the study design are congruent with the objectives, but the hypothesys must be enunciated to the reader. Methods are correct, but please indicate in more detail the flow cytometric analysis: it is not clear if the normalization is performed with percentages or MFI. In addition, please indicate the formula used for normalization.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: -Does the analysis presented match the analysis plan?

Yes.

-Are the results clearly and completely presented?

Yes.

-Are the figures (Tables, Images) of sufficient quality for clarity?

Figure resolution need improvement.

Reviewer #2: The results are properly presented and the figures have sufficient quality.

Reviewer #3: This section is correct, although writing must be improved, since its written in a very informal English. The results are clearly described, but considering the modifications suggested, it must be improved. Regarding the figures, please improve them by indicating, for example in fig. 1, if “normalization to negative” indicates negative control (isotype control) or media condition. It could be useful also to draw a line though the replicate to track the changes as they occur (Media – Ba RNA – IFNg+LPS).

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: -Are the conclusions supported by the data presented?

Yes.

-Are the limitations of analysis clearly described?

No.

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

Not exactly.

-Is public health relevance addressed?

No.

Reviewer #2: A significant part of the manuscript are negative results and, because of this, the main conclusion is that B. abortus RNA activates macrophages and phenotypically inhibits polarization of macrophages under in vitro M1 polarization conditions. Some controls are necessary to properly conclude this (see summary and general comments).

Reviewer #3: Conclusions are supported by the data. Nevertheless, discussion must be improved. i) limitations are not addressed; ii) Public relevance is not highlighted; iii) an in-depth analysis of the observations is not adopted. Please improve this section comprehensively. Discuss the observations regarding “phenotypic changes”: supported by the literature, discuss the implications in cell function in the context of Brucella abortus infection. Please discuss how these data can be helpful to advance our understanding of the topic under study.

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

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Minor Revision

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Brucella abortus (Ba) is an intracellular pathogen which survives in monocytes and macrophages. This manuscript report their findings about the mechanism about Brucella survival in macrophages, which is an interesting field about the interaction between Brucella and host cells. In their previous study, the authors have shown Ba RNA stimulate pro-inflammatory cytokine of monocytes. In this study, they test whether Ba RNA polarize macrophages. Their results showed that Ba RNA diminished t CD64, and MHC-II surface expression on macrophages at 48h, but did not modify CD206, DC-SIGN and CD163 surface expression, indicating that Ba RNA interfere M1 but not M2. Further analysis showed that Ba RNA did not modify M1- glucose consumption or lactate production. but production of Nitrogen Reactive Species diminished in Ba RNA-treated M1 macrophages. All these results show that Ba RNA could alter the proper immune response set to counterattack the bacteria. This work also demonstrates that RNA could also functions as a virulence factor of Brucella.

Specific comments

1, The main findings of this study is that Ba RNA does not activate both M1 and M2 polarization, but interfere M1 polarization of macrophages. This finding should be made more clear both in the title and abstract.

2, Two time points, 24 and 48h, were checked for surface expression of cytokines and biomakers. No explain or discussion about the expression difference between the two time points, particularly from the bacteria host interaction aspects.

3, What effects of the macrophage polarization will be generated on Ba survival or immune response to the bacteria? This should be discussed according to the results.

Reviewer #2: The paper describes that the RNA from B. abortus induces the activation of macrophages but is not capable of polarizing them neither to M1 nor to M2. The authors showed that B. abortus RNA blocks the differentiation to M1 (induced by treatment with LPS and IFN-gamma) but this blockage is not the result of inducing them to express M2 markers. The authors also show that RNA treatment of macrophages induces glucose consumption but not lactate production and a mild reduction (only at 48 hrs post-treatment) of NO production in macrophages although the production of iNOS was not significantly modified.

Major concerns

1- The main conclusion of the manuscript is that RNA from B. abortus is not capable of polarizing macrophages (to M1 or M2) but activates them. Additionally, if the macrophages are polarized to M1 in the presence of RNA, this polarization is phenotypically inhibited. Most of the results presented are negative. This is: no polarization was observed when the macrophages were treated with the RNA (Figures 1 and 3), partial macrophage activation was seen (IL-1 beta only at 24 hrs, TNF-alpha only at 48 hrs but not IL-8, Figure 2), it does not interfere with the secretion of pro-inflammatory cytokines (Figure 6), it does not modulate M2 markers when inhibition of M1 polarization is inhibited (Figure 7) and it does not modulate glucose concentration and lactate production (Figure 8). The main biological conclusion is that the RNA could be inhibiting M1 polarization and, in this way, modulating the immune response during infection but to conclude this a central control should be done (see point below).

2- The authors conclude that B. abortus RNA activates macrophages, but a control should be included to show that the RNA preparations are not contaminated with other molecules that could potentially activate macrophages. Have the authors treated the RNA with RNAse and see if the effect is lost? This control should also be included in the inhibition of M1 polarization, glucose consumption and NO production.

Reviewer #3: The manuscript from Serafino et. al constitutes an interesting and valuable input to the field of Brucella abortus immunology. However, this Reviewer consider that it must be improved to be published by Plos Pathogens Journal.

The Major Revision is requested because this reviewer considers that language must be improved (it is written in a very informal English and the relevant observations are not highlighted), but also additional data analysis must be done to improve the manuscript. Therefore, please calculate the RATIO between TNF-a/IL10 and / or IL1b/IL10 to assess changes in the pro-inflammatory/anti- inflammatory balance induced by Ba RNA. Also, glucose analogue incorporation could be performed to clarify glucose consumption, since de assessment of glucose in culture supernatants presents high variability.

Finally, regarding mouse macrophages: this reviewer considers that this set of experiments does not fit with the rest of the manuscript. Therefore, please take out the figure OR perform the same experiments with HUMAN macrophages.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Maria Florencia Quiroga

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all 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.

Reproducibility:

To enhance the reproducibility of your results, we recommend that 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear M. Aileen Milillo,

Thank you very much for submitting your manuscript "Brucella abortus RNA does not polarize macrophages to a particular profile but interferes with M1 polarization" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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 to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. 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.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

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

Sincerely,

Ruifu Yang

Section Editor

PLOS Neglected Tropical Diseases

Ruifu Yang

Section Editor

PLOS Neglected Tropical Diseases

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

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

Yes

-Is the study design appropriate to address the stated objectives?

Yes

-Is the population clearly described and appropriate for the hypothesis being tested?

Yes

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

Not applicable.

-Were correct statistical analysis used to support conclusions?

Yes.

-Are there concerns about ethical or regulatory requirements being met?

No.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: -Does the analysis presented match the analysis plan?

Yes.

-Are the results clearly and completely presented?

Yes.

-Are the figures (Tables, Images) of sufficient quality for clarity?

Yes.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: -Are the conclusions supported by the data presented?

Yes.

-Are the limitations of analysis clearly described?

No.

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

Yes.

-Is public health relevance addressed?

yes

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The authors have addressed succesfully all the issues raised by this reviewer. As it is now, the manuscript is suitable for publication.

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

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

Reviewer #3: No

Figure 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. 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 us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all 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.

Reproducibility:

To enhance the reproducibility of your results, we recommend that 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Dr. M. Ayelén Milillo,

We are pleased to inform you that your manuscript 'Brucella abortus RNA does not polarize macrophages to a particular profile but interferes with M1 polarization' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

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 now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Ruifu Yang

Section Editor

PLOS Neglected Tropical Diseases

Ruifu Yang

Section Editor

PLOS Neglected Tropical Diseases

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