PPATHOGENS-D-24-02280

Neuregulin-1 inhibits mouse respiratory virus induced necroptosis and death

PLOS Pathogens

Dear Dr. Hussain,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens'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.

Please submit your revised manuscript within 60 days Feb 06 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ 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 editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Makoto Takeda

Academic Editor

PLOS Pathogens

Benhur Lee

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments :

This manuscript has been reviewed by three experts. As a result, some reviewers pointed out that the association between the expression level of NPG1 and its effect on mitigating disease severity has not been sufficiently demonstrated. Additionally, one reviewer has raised questions regarding the novelty of the study. On the other hand, another reviewer acknowledged the novelty of this research and evaluated it as a significant contribution to the field. Please carefully consider all reviewers' comments and provide thorough responses accordingly.

Journal Requirements:

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Mathew S Bochter. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi

GEO accession: GSE281143" . We  noticed that the data is currently private and is scheduled to be released on Nov 04, 2025. We strongly recommend all authors deposit their data before acceptance, as the process can be lengthy and hold up publication timelines. Please note that, though access restrictions are acceptable now, your entire minimal dataset will need to be made freely accessible if your manuscript is accepted for publication. This policy applies to all data except where public deposition would breach compliance with the protocol approved by your research ethics board. If you are unable to adhere to our open data policy, please kindly revise your statement to explain your reasoning and we will seek the editor's input on an exemption.

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: 1. Lack of Novelty: The findings on the protective effect of atopy in viral infections lack novelty, as similar protective mechanisms have been demonstrated in previous studies using influenza virus in mouse models. Studies have already shown that pre-existing atopy can reduce disease severity in influenza infections through mechanisms such as NK cell activation and Type III interferon induction

2. The authors challenged C57BL6 mice intranasally with 10 μg of house dust mite (HDM) extract 3 days before SeV infection, but they have not ruled out the possibility that the HDM extract could induce nonspecific innate immunity, including interferons, which may improve the survival rate of virus-infected mice or suppress SeV replication.

3. Rational: Despite the RNAseq analysis results showing significant differences in the expression levels of various genes, including Adam11, between NA mice and atopic mice, the rationale for focusing on neuregulin-1 is insufficient.

4. Limited Mechanistic Insight: The manuscript does not provide sufficient mechanistic insight into how neuregulin-1 (NRG1) mediates protection against respiratory viral infections. While the authors highlight NRG1’s role in reducing necroptosis, they do not adequately demonstrate how this leads to the observed survival benefit, leaving gaps in the mechanistic understanding.

5. Overemphasis on Correlative Data: Much of the evidence provided in the study appears to be correlational, particularly regarding the association between reduced necroptosis and improved survival. Stronger causal data, such as using knockout models, could strengthen the conclusions.

Reviewer #2: In this manuscript, Hussain SA et al. demonstrated the effect of pre-existing atopy on respiratory virus infection. The authors found that increased NRG1 protects mice from SeV infection via upregulation of repair process. This manuscript presents novel findings, but the results appear somewhat disconnected and lack a cohesive conclusion.

Reviewer #3: Following up on previous work by the authors describing reduced severity of respiratory viral infections in mice made atopic, this study reports upregulation of NRG1 in atopic mice. In a series of in vivo and in vitro experiments, NRG1 is shown to mediate increased survival of animals in a SeV mouse infection model through reduced alveolar leakage and inhibition of virus-induced necroptosis.

These results advance the mechanistic understanding of an unexpected phenotype and identify determinants of disease severity. Data are well presented and overall support the conclusions drawn. Although no direct causality between NRG1 expression levels and greater protection of atopic mice has been established, the authors recognize and appropriately discuss possible limitations of the work. Thorough final editing of the text is recommended to address a few inaccuracies (i.e. reporter gene expression is not equivalent to virus titer (line 256)), but overall this is an important study that will advance the field.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: 1. In line 87: “our high-fidelity model of respiratory viral infection utilizes a natural rodent pathogen, SeV…”, SeV infection cannot be an animal model for RSV infection in humans. It is better to reorganize this section.

2. Fig 1B: please provide MLD50 of SeV infection. How much mice are survived from the “regular” dose infection?

3. The authors need to show the severity of asthma in this model. In this model, there is no PAS+ cells as shown in Fig 2E, indicating that the asthma symptoms are very weak. Please examine levels of IgE and granulocytes. Is this a chronic inflammation model?

4. In line 161: “In a dose responsive fashion”, there is no dose response.

5. Fig 3: EBD should be examined in SeV-infected atopic model.

6. Fig 2C: The viral titer does not drop in NRG1 treated mice, even though it does in the atopic model.

7. Fig 5A: The results are very unclear, the authors have to provide WB results with similar lysate amount.

8. It is unclear why necroptosis is inhibited by NRG1 treatment.

9. Is the epithelial leak regulated by necroptosis?

Reviewer #3: (No Response)

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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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. If there are other versions of figure files still present in your submission file inventory at resubmission, please replace them with the PACE-processed versions.

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit 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

PPATHOGENS-D-24-02280R1

Neuregulin-1 inhibits mouse respiratory virus induced necroptosis and death

PLOS Pathogens

Dear Dr. Grayson,

Thank you for submitting your manuscript to PLOS Pathogens. After careful consideration, we feel that it has merit but does not fully meet PLOS Pathogens'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.

Please pay attention to the "Additional Editor Comments" below and respond to reviewer #2 remarks regarding whether the model presented represents necroptosis. These comments are not something that can be addressed by additional experimentation but perhaps additional explication regarding the limitations of the model and/or a change in title is warranted. 

Please submit your revised manuscript within 30 days May 25 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plospathogens@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/ppathogens/ 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 editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Makoto Takeda

Academic Editor

PLOS Pathogens

Benhur Lee

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

Additional Editor Comments:

According to the concerns one of the reviewers raised, it is unclear what pathological condition this model reflects. In other words, it is difficult to determine whether this model has aspects of an atopic model or whether the inflammation-inducing model using HDM is merely a trigger for discovering NRG1. Another particularly important point is whether the observed immune and inflammatory response, and pathological features are completely consistent with necroptosis. This is because the title of this manuscript is based on the claim that NRG1 inhibits necroptosis, so a satisfactory explanation of these points is required. Is it possible to respond appropriately to the reviewer's concern?

Journal Requirements:

1) Please ensure that the CRediT author contributions listed for every co-author are completed accurately and in full.

At this stage, the following Authors/Authors require contributions: Mitchell H Grayson. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

The list of CRediT author contributions may be found here: https://journals.plos.org/plospathogens/s/authorship#loc-author-contributions

2) Please ensure that the funders and grant numbers match between the Financial Disclosure field and the Funding Information tab in your submission form. Note that the funders must be provided in the same order in both places as well.

Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: (No Response)

Reviewer #2: Although HDM was administered, this is not an atopic model but rather an experiment in which mice were infected with artificially induced acute inflammation, not chronic inflammation, and there are discrepancies in the interpretation of the results. Necroptosis, which is an inflammatory cell death, was suppressed by NRG1, but inflammatory responses such as granulocyte recruitment were not suppressed. This suggests that necroptosis is not relevant to this mouse model. The results are not consistent and do not respond to this reviewer's concerns at all. Also, the data could not support the interpretation of the results.

Reviewer #3: My concerns have been addressed. This is a well-executed study that will advance the field.

Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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

[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.]

Figure resubmission:

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. If there are other versions of figure files still present in your submission file inventory at resubmission, please replace them with the PACE-processed versions.

Reproducibility:

To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit 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 Grayson,

We are pleased to inform you that your manuscript 'Neuregulin-1 prevents death from a normally lethal respiratory viral infection' has been provisionally accepted for publication in PLOS Pathogens.

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.

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

Best regards,

Makoto Takeda

Academic Editor

PLOS Pathogens

Benhur Lee

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):