Dear Dr. Diaz,

Thank you very much for submitting your manuscript "Host PGD2 acting on DP2 receptor attenuates Schistosoma mansoni infection-driven hepatic granulomatous fibrosis" for consideration at PLOS Pathogens. 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, including performing the additional experiments to target eosinophils and PGD2/DP2.

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,

Meera Goh Nair

Academic Editor

PLOS Pathogens

Dominique Soldati-Favre

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Please address all reviewers' comments including performing the additional experiments to target eosinophils and PGD2/DP2

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: The authors of this study demonstrated that the activation of PGD2 and its receptor may play a protective role in liver fibrosis following S. mansoni infection. Key findings from this research reveal that inhibiting PGD2 synthesis using HQL-79 and blocking the DP2 receptor with an antagonist resulted in elevated levels of TGFb and IL-13 in the liver, which are believed to promote fibrogenesis. An intriguing discovery in this study is that treatment with CAY10471, a known DP2 receptor blocker, led to a decrease in the number of eosinophils in the liver sections of S. mansoni-infected mice. Additionally, CAY10471 treatment also promoted a fibrotic reaction within the hepatic tissue of infected mice. The authors further established a link between LTC4 production and liver fibrosis, identifying eosinophils as the source of LTC4. Both in vitro and in vivo evidence demonstrated reduced levels of LTC4 when mice or eosinophils were treated with CAY10471. The conclusion that PGD2 activates DP2 receptors, stimulating cysLTs production by eosinophils, and subsequently inhibiting liver fibrosis is both interesting and significant. The paper is well-written and deserves publication.

Reviewer #2: (No Response)

Reviewer #3: The liver is the main cause of health problems associated with schistosomiasis. A potential role for PGD2 in promoting liver fibrosis has been demonstrated in vitro. Thus, it was suggested that PGD2 inhibition would benefit people with schistosomiasis. However, there is no direct evidence in a schistosomiasis mouse model.

The Authors describe the effect of inhibiting the production or action of PGD2 in a mouse model of schistosomiasis. They also investigated how this PGD2 effect is linked to eosinophils and to LTC4. Surprisingly the data indicate that PGD2 protects the liver from fibrosis rather than causing it. Thus, its inhibition might aggravate liver failure and not benefit patients with schistosomiasis.

The paper's background is well reasoned and brings the rationale to assess the role of PGD2 in liver fibrosis in Schistosoma mansoni infected mice. The subject is important. The research is exacting, well-executed and well-documented.

However, the manuscript is too complex. I would have suggested that they go more in depth on one of the various directions: which cells besides eosinophils are responsible for the PGD2 pro-fibrotic or anti-fibrotic effects, by which mechanisms in vitro and in vivo is this carried out, etc. For example, the Authors mention the important role of mast cells as main cells producing PGD2 and being present in the liver granulomas but fail to study this.

**********

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: However, there are a few weak points that need to be addressed:

1. It is important to discuss the potential differences in the consequences of DP2 deficiency/blockade on eosinophils in various models. For example, in an allergic inflammatory model of asthma, DP2/CRTH2 knockout mice displayed enhanced eosinophil recruitment into the lung compared to wild-type littermate mice (The Journal of Immunology, 175(4), 2056-2060). Similarly, in cases of renal fibrosis and lung fibrosis, DP2/CRTH2 has been shown to have profibrotic roles (American Journal of Respiratory Cell and Molecular Biology, 67(2), 201-214).

2. It would be beneficial to include in vivo models with DP2/CRTH2 conditional deletion in eosinophils to further elucidate how PGD2/DP2 contributes to liver pathology during S. mansoni infection.

3. If establishing an eosinophil-specific knockout of DP2 is not feasible, the authors should consider supplementing their data to further support the contribution of eosinophils in alleviating liver fibrosis caused by S. mansoni infection, specifically through DP2 and LTC4 production. One possible approach could be to investigate the effects of depleting eosinophils. By depleting eosinophils in an animal model, the authors could observe the impact on liver fibrosis progression and evaluate whether the absence of eosinophils alters the protective effects mediated by the PGD2-DP2-LTC4. This additional data would strengthen the understanding of the role of eosinophils in the context of liver fibrosis and help solidify the conclusions drawn in the study.

Reviewer #2: (No Response)

Reviewer #3: To strengthen the paper, additional experiments could be conducted on:

-- Eosinophil deficient mice in which PGD2 activity is inhibited

-- PGD2 knock-out mice

-- In mice in which pharmacological blockage of LOX5 is achieved

**********

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: The manuscript is too heavy and complex. It is not fluid reading. The itemization -- 1,2,3,4 -- is too much.

It should be re-written and shortened to make it easier to read and understand.

**********

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 on PLOS Biology: 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

Attachments

Attachment

Submitted filename: Reviewer Comments.docx

Dear Dr. Diaz,

Thank you very much for submitting your manuscript "Endogenous PGD2 acting on DP2 receptor counter regulates Schistosoma mansoni infection-driven hepatic granulomatous fibrosis" for consideration at PLOS Pathogens. 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.

There are two outstanding issues raised by reviewer # 4 that shown require your attention  and a mini-revision  prior to acceptance.

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,

Dominique Soldati-Favre

Section Editor

PLOS Pathogens

Dominique Soldati-Favre

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Reviewer Comments (if any, and for reference):

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: The data from additional experiments in the revised manuscript addressed my concerns despite the fact that the eosinophil specific genetic mouse models are not available. The descriptions to put less emphasis on eosinophil specific roles as shown in line 107 to 113, Page 71 were appropriate.

Reviewer #4: The authors demonstrated that PGD2 by activating DP2 receptors stimulates cysLTs production by eosinophils, while endogenously down-regulates the hepatic fibrogenic process of S. mansoni granulomatous reaction – an in vivo protective function which demands caution in the future therapeutic attempts in targeting PGD 2/DP2 in schistosomiasis. The authors inhibited the production or action of PGD2, and antagonized CysLT1 in a mouse model of schistosomiasis. Furthermore, in vitro experiments provided further evidence for the presented conclusions.

The study is well conducted and the data presented represent a valuable contribution to research into schistosomiasis.

**********

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: none

Reviewer #4: Figures 2 A and B: Hepatic Hydroxyproline levels should be checked carefully. There seems to be an error in the presented data. 100-180 µg/g hydroxyproline can be found in the liver of healthy C57BL/6 mice while 300-400µg hydroxyproline per g liver tissue was detected in mice infected with S. mansoni for 9 weeks (PMID: 37990129).

**********

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 #4: 1. Complicated wording makes understanding difficult:

e.g. Lines 298-302: First, distinct from the eosinophilic reaction impairment observed in DP1

receptor-deficient model [19] , HQL-79-unveiled PGD2 role in S. mansoni infection

driven eosinophilia does not correspond to an inevitable consequence of a large

inhibition of S. mansoni parasitism or overall lack of subsequent type 2 immune

response.

2. Perhaps separating the results and discussion into two separate sections would make the manuscript easier to understand.

**********

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 #4: 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. Diaz,

We are pleased to inform you that your manuscript 'Endogenous PGD2 acting on DP2 receptor counter regulates Schistosoma mansoni infection-driven hepatic granulomatous fibrosis' 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.

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 Pathogens.

Best regards,

Dominique Soldati-Favre

Section Editor

PLOS Pathogens

Dominique Soldati-Favre

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Reviewer Comments (if any, and for reference):