Dear Dr Silvestre:

Thank you very much for submitting your manuscript "Glutamine supplementation improves the efficacy of miltefosine treatment for visceral leishmaniasis" (#PNTD-D-19-01729) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

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Associate Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Deputy Editor

PLOS Neglected Tropical Diseases

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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: See "Summary and General Comments"

Reviewer #2: The objectives of the study are clearly articulated with the hypothesis stated, the study is well designed and the ethical requirements are being met. As suggested in the major comments for the authors, the metodology for transcriptome analysis must be better detailed. On this regard, information is missing.

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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: See "Summary and General Comments"

Reviewer #2: Data are well presented, results are clear and match the analysis plan. Figures are also clear.

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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: See "Summary and General Comments"

Reviewer #2: The conclusions are supported by the data presented. However, it is suggested to moderate some of them in order to avoid reductionisms. Limitations of analysis were not described.

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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: See "Summary and General Comments"

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: The study by Ferreira and collaborators provides interesting data indicating that dietary glutamine supplementation may act as a promising adjuvant for the treatment of visceral leishmaniasis. The study is well conducted, but I have some concerns, especially regarding analytical (statistical) procedures. Here are some additional comments.

1) 1) Is there any reason for choosing C57Bl/6J mice? Wouldn't Balb/c mice have been a more susceptible Leishmania donovani?

2) Was the in vitro cytotoxicity assay performed from any previous work? If yes, cite the reference.

3) Line 166: The route of exposure was intraperitoneal rather than intravenous. What is the reason for this? This detail should be clarified in the manuscript.

4) Line 171: The criteria for choosing the dose administered is unclear.

5) What is the sample number of animals per group?

6) Tables 1 and 2 may be reallocated to “supplementary material”.

7) Statistical analyzes are incorrect. Data should be analyzed using a factorial ANOVA. There are different factors which should be considered (gender, time, treatments). Therefore, the authors must redo the analyzes and present the results appropriately (effect of the factors separately and the interaction between them - present F values, degrees of freedom and p values). In addition, ANOVA assumptions must be properly tested.

Reviewer #2: Comment to PNTD-D-19-01729

The manuscript reports on the modulation of glutamine metabolism, in the vertebrate host, by Leishmania donovani and suggests a potential therapeutic use of glutamine in combination with miltefosine for the treatment of visceral leishmaniasis. The modulation of the metabolism of the host cells by these parasites is a relevant issue for the understanding of the immune mechanisms that control the infection. However, this topic is little studied. In this sense, the manuscript addresses an interesting topic and provides important new insights into the crosstalk between glutamine metabolism and T cell-mediated immune response to L. donovani infection. Overall, the manuscript is very well written and the data are well presented. Conclusion could be more cautious as suggested below.

Major comments:

Lines 181-187. Detailed information on the transcriptomic analysis is lacking. Information about whether it was a time course analysis, BM or peritoneal macrophages? Number of experiments? RNA Information, such as it was total or poly-A RNA, if it was treated for depletion of ribosomal DNA, how it was isolated and quantified, which quality control was used. How library was constructed? This information must be included for the correct interpretation of the data by the reader.

In addition, It is not clear whether transcriptomic assays were done at 6, 24 and 48 hours or if they were done just at 6h post-infection and the other time points refer to other assays of gene expression analysis. The design of transcriptomic analysis must be detailed.

Lines 286-287. It would be probably because those cells stopped proliferating due to the lack of glutamine? Please elaborate on that.

Lines 297-298. How do authors explain that there was no difference between the untreated and the group supplemented with glutamine? Shouldn't there be an improvement in animals that received supplementation compared to untreated ones?

Lines 318-320 and figure 3. The supplemented group also decreased the number of CD4 and CD8 T cells. Authors expected that? How to explain this? Shouldn't it be at least the same as the untreated? Or even increase thanks to supplementation? Also, how to explain the reduction of IFN-γ- and TNF produced by CD4 T cells? The authors are encouraged to elaborate on this result.

For this reviewer, the conclusions need to be more moderate because, as it is written, it seems to reduce the complexity the parasite-host relationship. It would be ideal if supplementation with glutamine was sufficient to contribute for controlling the infection, but that would be reductionist, since many other factors contribute to the control of the parasite or, conversely, to the establishment of the infection. In fact, parasites also consume glutamine to maintain their proliferation, and a medium supplemented in this amino acid could also result in parasite survival and establishment within the cells. This aspect deserves to be mentioned by the authors. Could parasites compete with macrophages for glutamine? Please elaborate on that and moderate conclusions.

Minor points:

Figure 3. Captions of this figure should be standardized, i.e., use the same font size.

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Reviewer #1: Yes: Prof. Dr. Guilherme Malafaia

Reviewer #2: No

Dear Silvestre,

We are pleased to inform you that your manuscript 'Glutamine supplementation improves the efficacy of miltefosine treatment for 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 within two working days 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 Neglected Tropical Diseases.

Best regards,

Peter C. Melby, M.D.

Associate Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Deputy Editor

PLOS Neglected Tropical Diseases

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The authors have adequately addressed the concerns of the reviewers. The work is a nice contribution to the field. I have 2 minor editorial comments related to the legend to figure 1: Please identify the method used for determination of gene expression in Figure 1A and Figure 1B. The sentence in line 660-61 ("A two-way ANOVA. . .) is out-of-place.