Dear Prof Harris,

Thank you very much for submitting your manuscript "Helminth infection driven gastrointestinal hypermotility is independent of eosinophils and mediated by alterations in smooth muscle instead of enteric neurons" 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.

Your manuscript has been reviewed by three expert reviewers, all of whom appreciated the importance of your study. However, reviewers 1 and 2 have raised concerns regarding the relevance of current study to other helminth models with chronic infections. In light of these comments, I would like to invite you to revise your manuscript to address the points raised by the reviewers. The detailed evaluation reports are attached to this letter for your reference.

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,

Shahid Siddique

Guest Editor

PLOS Pathogens

Margaret Phillips

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

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

Thank you for submitting your manuscript to PLOS Pathogens. It has been reviewed by three expert reviewers, all of whom appreciated the importance of your study. However, reviewers 1 and 2 have raised concerns regarding the relevance of current study to other helminth models with chronic infections. In light of these comments, I would like to invite you to revise your manuscript to address the points raised by the reviewers. The detailed evaluation reports are attached to this letter for your 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: Wang et al present a manuscript investigating the physiological mechanisms by which worm infections stimulate intestinal motility. Previous research involving research with other pathogens has highlighted varying mechanisms of how the gut responds to infection, and the current paper tries to address how this occurs during acute infection with a GI parasite, Nippo. Their hypothesis was that Nippo infection would alter enteric neuron function, perhaps via eosinophils, but neither of these hypothesis were true, instead the infection seems to directly act on smooth muscle.

The authors also use a range of complementary techniques, including some nice imaging and measures of contraction.

Overall it is a well conducted study and the data are well presented, although the advances in knowledge of host-parasite interactions are fairly incremental. The paper is also quite descriptive, and a lot of "negative" data are shown, particularly figures regarding independence of eosinophils for these effects seen. A few comment and questions are listed below:

Reviewer #2: The manuscript by Wang et al. shows that intestinal helminth infection is associated with substantial changes in smooth muscle thickness and tensile strength but surprisingly, little change in the local enteric nervous system (ENS), which is thought to play a key role in regulating intestinal motility. The changes in smooth muscle physiology were not dependent upon the presence of eosinophils. This is an interesting and nicely presented study, unpacking many of the assumptions that we have regarding the regulation of worm expulsion during Nb infection. While many of the data pieces presented are negative, these results are important in identifying the important changes that occur in intestinal physiology during helminth infection. As such, these data represent an important contribution to the literature that will inform future research efforts.

Reviewer #3: The paper by Want et al. is focused on characterizing intestinal motility in Nippostrongylus brasiliensis infection and the role of eosinophils and the enteric nervous system. They investigate several aspects of physiological alterations of the intestine and the effect that eosinophils play. In their observations of neuronal density I appreciated their caveat that the reduction of mysenteric neurons could result from tissue growth rather than loss of neurons. Part of their results suggested that neither Nb infection nor eosinophils had an impact on intestinal ENS structure. I was also interested to see that no eosinophils could be found in the myenteric plexus in naïve or infected mice, nor could they be found in the muscle wall.

Overall the paper is well written, the experimental design and progression is logical and clearly conveyed. This study represents a significant advancement to the field and will be of interest to a broad readership. I had only a few minor suggestions. Well done!

**********

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: 1. It is difficult to know how representative these results are for helminth infections, ie are they specific to acute Nippo infections, or would similar results be seen in any other helminth models? It would be interesting to see if similar mechanisms operate in models such as secondary H. poly infection-

2. The main positive finding of the study is that Nippo infection increases smooth muscle contractillity, but that has been well reported in the literature and the cytokines that drive this response have been well characterised (IL-4/13), which the authors acknowledge. Can the authors elaborate on what information the present study has added in this area? Or, is the main area of novelty the fact that neurons and eosinophils are NOT involved much?

Reviewer #2: Nippostrongylus brasiliensis is expelled quickly from the murine intestine, whereas most endemic parasitic helminths chronically reside in the small intestine in infected humans and other mammals. While Nippo infection is an important model used to study the intestinal physiology associated with worm expulsion, expulsion is not the norm. How does chronic infection with Heligmosomoides polygyrus influence intestinal ENS responses and smooth muscle function? It seems beyond the scope of the current report to investigate the role of eosinophils in these responses in H. polygyrus infection, but investigation of how acute vs. chronic infection affects smooth muscle responses to infection is important in establishing the physiological relevance of the data presented.

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: 3. Lines 123: I would consider adding data from Fig S1 to the main figure set, particularly because the authors have admitted it was "interesting"

Reviewer #2: 1. The coding for the days and genotypes is quite complex, as the reader needs to apply the single “legend” across all 4 panels, and the 4 panels have data from different days post-infection. Is there a way to simplify?

2. The stats indications on the bottom of the panels (genotype vs. infection) are confusing in conjunction with the stats already indicated on the graphs.

Reviewer #3: Much of the paper is about the physiology and physiological changes in the enteric nervous system (ENS) and the myenteric and submucosal plexus. This is introduced in lines 56-68 but it comes back later in the results and discussion. I think a diagram or illustration of these tissues and their relation to lamina propria etc would be informative for the reader and would facilitate understanding of the data. I suggest that the authors add this as a new figure, or perhaps as a panel on existing figure 1.

Minor suggestions:

Line 169: It’s a bit confusing as currently written. I suggest changing to “Ahrends and Aydin et al., reported that…

**********

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

Dear Prof Harris,

We are pleased to inform you that your manuscript 'Helminth infection driven gastrointestinal hypermotility is independent of eosinophils and mediated by alterations in smooth muscle instead of enteric neurons' 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,

Shahid Siddique

Guest Editor

PLOS Pathogens

Margaret Phillips

Section Editor

PLOS Pathogens

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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

Reviewer Comments (if any, and for reference):