PONE-D-21-35173Role of sphingosine kinase and sphingosine-1-phosphate receptor on liver pathology in mice infected with Plasmodium berghei ANKAPLOS ONE

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Olga A Sukocheva, PhD

Academic Editor

PLOS ONE

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Additional Editor Comments:

Sound study with interesting findings. However, Introduction and Discussion section should be improved; reference list should be extended and present recent relevant reviews in the field of Sphingosine kinase signaling role during activation of inflammation. All Reviewers' suggestions should be addressed. Immunohistopathology images with higher resolution or magnification should be presented. Suggested/ potential S1P-receptor -targeting agents/or other methods for malaria treatment should be indicated in the Discussion section.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript is interesting; however, there are a few issues that need to be corrected before the manuscript is considered for publication.

1. The main issue is with animal experiment execution. Why were control mice not injected with red blood cells but just saline? This is not a proper control as authors introduce additional variables!

2. The method of erythrocyte isolation, infection, etc., has not been described (and is not included in any of the referenced papers). Moreover, one of the referenced manuscripts on page 96 is a case study of an infected patient and does not include any animal experiment. What is the rationale for referencing it here?

3. Not all abbreviations are adequately explained (e.g., line 110) – please correct.

4. Figure 2 is of low quality, and it is impossible to evaluate the staining results.

5. More appropriate for expression data would be boxplot and not the histogram. Also, Figure 3 is incorrectly referenced in the text as Figure 2.

6. Line 208: Authors contradict themselves. First, they write that there are differences in mRNA level of SphK1, to add later that it did not change… Moreover, S1PR1 and S1PR2 mRNA expression are lower only by 30-40% when compared with appropriate controls, so it is not a 3-fold difference. Not to mention, these results are not statistically significant due to high variability between individual animals.

7. Line 251. Downregulation of mRNA expression was only shown for S1PR3, so writing that it affects all investigated receptors is an overstatement.

8. It is not unusual to not see differences at the mRNA level but notice the differences at the protein level (and vice versa), and authors should discuss this in their discussion. Moreover, it would be beneficial for the readers if possible explanation or more detailed information about receptors processing/degradation could be included in the discussion.

Reviewer #2: The study tested levels of SphK1 mRNA expression in liver tissues of mice infected with malaria pathogens. Authors also found that S1PR1, S1PR2, and S1PR3 expressions are decreased in the liver tissue of Plasmodium berghei-infected mice. Data suggests that the decreased levels of S1PR1, S1PR2, and S1PR3 may be involved in liver injury during malaria infection. The study is very interesting. However, there are several issues to address. The manuscript should be improved/amended.

1. Text requires proper English language editing.

2. Figure 2 resolution is poor. Author should provide images with higher magnification and/or better resolution.

3. Introduction/discussion section sections do not provide enough information; several important citations are missing. Role of SPhK in the infection resolution should be covered in more details ( see these reviews: https://pubmed.ncbi.nlm.nih.gov/31863815/ ; https://pubmed.ncbi.nlm.nih.gov/28075451/

https://pubmed.ncbi.nlm.nih.gov/29385066/

4. Was SphK1 protein level measured/compared? Authors should discuss SPhK1 protein expression and mechanisms of its regulation considering the activation of receptor degradation during inflammation.

5. S1P receptors intracellular processing was not discussed ( see this report https://pubmed.ncbi.nlm.nih.gov/23142484/)

6. The manuscript will benefit if authors include schematic signaling pathway for SPhK/S1P receptor signalling in the infected/normal liver.

7. Study limitations section should be extended.

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Reviewer #1: No

Reviewer #2: No

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Role of sphingosine kinase and sphingosine-1-phosphate receptor in the liver pathology of mice infected with Plasmodium berghei ANKA

PONE-D-21-35173R1

Dear Dr. Punsawad,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Olga A Sukocheva, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The revised version of the manuscript has been improved. Authors addressed all my comments properly.

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7. 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