Transfer Alert

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Dear Dr. Kurosu,

Thank you very much for submitting your manuscript "Dengue virus infection induces selective expansion of Vγ4 and Vγ6TCR γδ T cells in the small intestine and a cytokine storm driving vascular leakage in mice" 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.

The manuscript was evaluated by two experts in the area which appreciated the importance and novelty of the manuscript. The reviewers raised important points regarding to the gating of their population analysis and requested further clarification on the animal models, and protocol and effects of treatments utilized assess cytokine effects on the model, that must be addressed by the authors. The authors also need to mention if and how many times experiments were repeated and attest the reproducibility of results in their hands.

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,

Helton C. Santiago, M.D., Ph.D

Academic Editor

PLOS Neglected Tropical Diseases

Aaron Jex

Section Editor

PLOS Neglected Tropical Diseases

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

The manuscript was evaluated by two experts in the area which appreciated the importance and novelty of the manuscript. The reviewers raised important points regarding to the gating of their population analysis and requested further clarification on the animal models, and protocol and effects of treatments utilized assess cytokine effects on the model, that must be addressed by the authors. The authors also need to mention if and how many times experiments were repeated and attest the reproducibility of results in their hands.

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: Clearly written

Reviewer #2: (No Response)

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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: Clearly written

Reviewer #2: (No Response)

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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: Clearly written

Reviewer #2: (No Response)

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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)

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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: In this study, Kurosu et al showed that a selective expansion of IL-17A-producing γδ T cells in the small intestine is at least partially responsible for the induction of lethal vascular leak caused by dengue virus (DENV) in IFN-α/β/γR KO mice. The critical role of IL-17A in causing severe vascular leak was demonstrated with the 50% protection of mouse survival by the treatment with anti-IL-17A antibodies accompanied with the reduction of cytokine gene expressions, such as TNF-α, in small intestine. In addition, the authors showed that DENV infection causes the infiltration of neutrophils in small intestine, resulting in a massive production of MMP-8, which is also shown to be responsible for the induction of vascular leak. Thus, this study presented novel mechanisms of vascular leak caused by DENV infection in IFN-α/β/γR KO mice; (1) IL-17A produced by γδ T cells and (2) MMP-8 produced by neutrophils. However, I would request more convincing data/explanations to accept the author’s claims.

1. Figure 6 is important data to identify the IL-17A-expressing lymphocytes. The gate was first set on CD45-positive and SSC (A). However, the non-lymphocyte population in infected mice seems totally shifted toward the right side of x-axis and the gate included a lot of non-lymphocyte cells. This arises the possibility that the CD3+γδTCR+ population (B) includes a number of non-specific cells. I understand that the activated lymphocytes may shift their positions to higher SSC intensity, but they should be still distributed within the elongated pattern of lymphocyte population. I urge the authors to re-analyze the data with appropriate gating to obtain more convincing results.

2. The treatment with anti-IL-17A antibodies showed 50% protective efficacy (Fig.5 A). This is unconvincing to me since the treat mice showed similar pattern of weight reduction to untreated mice (Fig.5B) and similar levels of cytokine production to anti-TNF antibody treatment (Fig. 5 E-M), which showed 100% protective efficacy (Fig. 1F) and induced less weight loss (Fig. 1G). How do the authors explain this discrepancy?

Reviewer #2: PNTD-D-23-00821

Takeshi Kurosu and colleagues submitted a manuscript entitled “Dengue virus infection induces selective expansion of V�4 and V�6TCR �� T cells in the small intestine and a cytokine storm driving vascular leakage in mice”. In the manuscript, the authors examined the mechanisms and effects of blockade of TNF-� signaling on DENV infected mice during severe dengue outcome. Using a well-established mouse model of severe dengue which display human symptoms including vascular leakage, the authors performed transcriptomic analysis of the liver and small intestine samples collected chronologically in presence/absence of blockade of TNF-� signaling and evaluated the cytokine and effector level events. They found that (i) blockade of TNF-� signaling has a better effect in pro-inflammatory cytokines in small intestine compared to liver, (ii) a critical role of IL-17A produced by �� T cells in the small intestine during severe dengue, and (iii) detected the presence of neutrophil infiltrate that produced MMP-8 (Matrix Metalloproteinase 8), a collagen cleaving enzyme which is a major player of inflammatory response.

The manuscript addresses a critical question in the field regarding the mechanisms of action following severe dengue. The manuscript uses well-defined approaches to explore the questions and discuss the conclusions. The manuscript is well written, however some concerns detailed below should be addressed by the authors to improve the understanding and the quality of the manuscript.

Minor comments:

Figure 1: Panel E needs more details as done for panel A. The symbols are confusing. When is the infection and what antibody has been used?

The authors mentioned that the mice are dying at day 4 in this mouse model, but Fig 1F showed day 5. It is not clear if the mice are dying at day 4 or 5.

Typos in line 962: Mice were instead of Mice. Were

Figure S2: The effects of TNF-� blockade are mentioned as limited, why? Even though in panel F, the protection is 100%. This is not clear. Please, clarify.

Typos in line 254-255 and 257: The ��TCR subsets seem mislabeled. i.e CD3+/��TCR- (including CD8 T cells) in Fig6D instead of CD3+/��TCR+ (including CD8 T cells). Please, double check, Figure 6C-G

Figure S5: Add the title of the Y axis.

Figure 8: The gating strategy will be better and easy to follow as one panel instead of 5 panels.

Figure 9: The resolution of the image is not great and the authors should add annotations and labels to indicate what histopathological changes should be seen and compared between treatments.

Moderate comments:

In all figures, the authors did not specify how many times the experiments were done. Moreover, in Figure 1, only 3 to 4 mice have been used. This looks like a one-time experiment and knowing the variability of the mouse experiments, it is not acceptable to do an experiment only once. Please, clarify and/or add more samples to confirm the results.

Figure 4: It is not clear why the authors are not using a better protocol to blockade the IL-6 signaling. The antibody treatment did not effectively suppress IL-6 signaling as measured by serum albumin A levels (Fig 4C). Is it the result of a suboptimal protocol or a bad antibody lot? The use of MR16-1 to suppress IL-6 signaling seems to have better effects in others published manuscripts. The purpose of this figure is not clear if the treatment is not working.

In addition, this mouse model is supposed to succumb to DENV infection at day 4, however this is not the case in panel A. Could the authors clarify why this mouse model is different here?

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Reviewer #1: No

Reviewer #2: 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.

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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. Kurosu,

Thank you very much for submitting your manuscript "Dengue virus infection induces selective expansion of Vγ4 and Vγ6TCR γδ T cells in the small intestine and a cytokine storm driving vascular leakage in mice" 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. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

The reviewers acknowledge the improvement in the manuscript, however there is still a minor point that was still not properly addressed. Reviewer number one points that there may be unspecific IL-17+ population based on the gating strategy of the authors in Figure 6. Please, try to respond this concern properly or describe the limitations that this strategy may bring.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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

[1] A letter containing a detailed list of your responses to all 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.

Thank you again for your submission to our journal. 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,

Helton C. Santiago, M.D., Ph.D

Academic Editor

PLOS Neglected Tropical Diseases

Aaron Jex

Section Editor

PLOS Neglected Tropical Diseases

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

The reviewers acknowledge the improvement in the manuscript, however there is still a minor point that was not properly addressed. Reviewer number one points that there may be unspecific IL-17+ population based on the gating strategy of the authors in Figure 6. Please, try to respond this concern properly or describe the limitations that the current strategy may bring.

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: I would request the Authors for an alternative FCM analysis.

Reviewer #2: (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: Results are described clearly

Reviewer #2: (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: Conclusions are reasonable

Reviewer #2: (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

Reviewer #2: (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: Kurosu et al responded to 2 concerns raised by this Reviewer. Overall, their responses are reasonable. One point they missed to answer regarding the FCM data is the possibility that CD3+γδTCR+ population (Fig. 6B) contains a number of non-specific cells by gating a broad range of SSC intensity (Fig. 6A) and this leads the false positive of IL-17A expression (Fig. 6C). I understand that CD45+/SSClow population is mainly lymphocytes and CD45+/SSChigh population would be mainly neutrophils and monocytes. Gating and analyzing separately these populations would be the best way in order to avoid the concern about false-positive and clearly show that γδ T cells are responsible for IL-17A production in small intestine. It would be very easy to address this point.

Reviewer #2: The authors addressed some of the comments and improved the manuscript. Typos and grammatical mistakes are still in the manuscript, ie L1065, L1150, etc...

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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

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

References

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.

Dear Dr. Kurosu,

We are pleased to inform you that your manuscript 'Dengue virus infection induces selective expansion of Vγ4 and Vγ6TCR γδ T cells in the small intestine and a cytokine storm driving vascular leakage in mice' 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,

Helton C. Santiago, M.D., Ph.D

Academic Editor

PLOS Neglected Tropical Diseases

Aaron Jex

Section Editor

PLOS Neglected Tropical Diseases

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Although the authors did NOT answer the reviewer #1 properly, which is not a good practice, we agree that there is not major reasons of concern with figure 6 and the claims of the manuscript.