Response to ReviewersRevised Manuscript with Track ChangesManuscript

We look forward to receiving your revised manuscript.

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

Additional Editor Comments :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: Haruka Mizobuchi, Junya Yamagishi, Chizu Sanjoba, and Yasuyuki Goto. 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/plosntds/s/authorship#loc-author-contributions

2) We do not publish any copyright or trademark symbols that usually accompany proprietary names, eg ©,  ®, or TM  (e.g. next to drug or reagent names). Therefore please remove all instances of trademark/copyright symbols throughout the text, including:

- ® on page: 10

- TM on pages: 8, and 9.

3) Tables should not be uploaded as individual files. Please remove these files and include the Tables in your manuscript file as editable, cell-based objects. For more information about how to format tables, see our guidelines:

https://journals.plos.org/plosntds/s/tables 

4) Please amend your detailed Financial Disclosure statement. This is published with the article. It must therefore be completed in full sentences and contain the exact wording you wish to be published.

1) State the initials, alongside each funding source, of each author to receive each grant. For example: "This work was supported by the National Institutes of Health (####### to AM; ###### to CJ) and the National Science Foundation (###### to AM)."

2) State what role the funders took in the study. If the funders had no role in your study, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.".

Reviewers' comments:

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: Fig. 4B. It is not clear how may mice/organs/sections were evaluated to conclude that KMP11+ cells are detectable in fetal liver, but ‘only a few KMP11+ cells were observed in the placentas’, especially when Fig. 4B only shows a single positive cell in the image. Quantification of microscopy would strengthen the observation of this novel finding.

Line 307: Quantification is needed to support ‘the density of the LZ was lower than that of naïve controls’.

Was IFNa/b or IFNg increased in RNASeq data? IFNg is decreased with pregnancy in infected mice, but it is still elevated compared to naïve/pregnancy or infected/non pregnant. Similarly, were type I IFNs elevated in the serum like IFNg in Fig. 2B?

Fig. 5C, lines 324-325. The data does not support more uNK cells, so the emphasis should be decreased on their infiltration in line 325, 467, and 473.

Describe how estimate Ld number in fetus was calculated in M&M (Fig. 4A).

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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: Discussion: What causes the vessel diameter? Are vascular mediators like VEGF-A elevated in the RNASeq dataset that couple help explain the dilation?

Discussion: Is elevated ICAM and VCAM due to elevated IFNg?

Discussion: What restricts the CD3 T cells from moving throughout the tissue or why or the cells cluster?

Line 335-336: no control group is mentioned. Placentas of Ld infected compared to...

Line 448: Is BALA/c a typo?

Line 451 need a citation

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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: Many genes mentioned in Table 3 are induced by either type I IFNs or IFNg, using STAT1 as a prime example. Therefore, the conclusion that type I IFNs are solely responsible is not obvious or supported without pathway analysis or uncoupling the roles of each pathway through blockade strategies.

To make the conclusion that barrier integrity is affected, the authors would need to assess barrier integrity using a common assay like evans blue which measures leakiness, or a comparable approach.

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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: Arrows denoting LZ, trophoblast atrophy, and vasodilation would help the reader for Fig. 4C. For instance, it should be made clear if the nuclei are of villi or trophoblasts?

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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: This study shows that chronic Ld infection induces decreased pregnancy rate, vertical transmission, placental degeneration with T cell infiltration. The manuscript is well-written and addresses an important unanswered question in NTDs. Plus, the new experimental model developed will be informative for future studies dissecting the immunopathology associated with pregnancy in VL. However, the conclusion that type I IFN signaling in the placentas is responsible for the phenotype is less convincing as many of the targets identified by RNASeq can be induced by type I IFNs and IFNg. Therefore, the conclusion that type IFNs are important should be dampened (in the results, discussion, etc) as the current study does not uncouple IFNa/b from IFNg.

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Reviewer #1: No

Figure resubmission: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 Ms Mizobuchi,

We are pleased to inform you that your manuscript 'Vertical transmission of Leishmania donovani with placental degeneration in the pregnant mouse model of visceral leishmaniasis' 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,

Peter E Kima

Guest Editor

PLOS Neglected Tropical Diseases

Laura-Isobel McCall

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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