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PONE-D-23-15179Cystine/glutamate antiporter system Xc- deficiency impairs macrophage glutathione metabolism and cytokine production.PLOS ONE

Dear Dr. de Baat,

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Academic Editor

PLOS ONE

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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: Yes

Reviewer #2: Partly

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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: No

Reviewer #3: 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

Reviewer #3: 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 sounds good and well written but sequences should be added to the manuscript

Results should be titled according to method's procedures

Product ID and any modifications or precautions done to procedures should be clarified

Reviewer #2: In this manuscript, the authors investigated the functional implication of glutamate-cystine antiporter (sXc) system in the regulation of macrophage responses. The manuscript is well-written with enough experimental details. Statistical analysis is done carefully and mentioned in the manuscript in a clear manner. However, it is concerning that the manuscript is missing figure 1 and there is duplication of figure 2 instead. Rest of the figures and the respective experimental analysis are quite convincing, and the authors tried to highlight the physiological implication/importance for each experiment. A list of corrections and suggestion regarding the manuscript are mentioned below:

1. In the manuscript, a space is required between effect and or in line 2 (introduction).

2. In line 11, authors mentioned that recent studies have shown how mitochondria modulates immune cell function by regulating metabolism and redox homeostasis but cited only one article. Why?

3. Similarly, citation related correction is needed in lines 23-26. Moreover, in this part of the introduction (and in the discussion), authors tried to draw a connection between their study and another publication that demonstrated the effect of sXc deficiency and inhibited sXc in improved wound healing in diabetic mouse. Additional clarification on this connection would be helpful for the general readers.

4. The end of the introduction is abrupt; hence, a brief (1-2 lines) description of the overall findings would make it complete.

5. A brief description of endotoxemia and how introduction of LPS would mimic the condition would help the readers to follow the study.

6. In line 82, the sentence ends abruptly with a question mark.

7. In line 84, the Cas9 concentration is written as pmol µM, which needs to be corrected.

8. The lack of figure 1 makes it difficult to assess the transcriptional regulation of sXc.

9. Legends are missing for figure 4a and 4b. Also, in figure 4b, the X-axis label WTLPS and KOLPS need to replaced with WT LPS and KO LPS.

10. Figure 5 is also missing the legend.

Overall this manuscript is incomplete without figure 1. The decision can only be made once the data representing figure 1 are submitted (only if it is permitted by the Editor) and thoroughly evaluated.

Reviewer #3: The main idea of the manuscript was investigated earlier and the involvement of the cystine transport system in macrophage activity was reported since 1994 by Piani and Fontana (an author of the current manuscript) and in other publications as well. However, I thoroughly enjoyed reviewing the manuscript and only have some minor requests for revision.

1- The details for both the ex vivo and in vivo experiments should be mentioned clearly in the materials and methods section

2- In line 82, "Product ID and sequences are listed in where?" should be removed

3- In line 92-93, "Journal probably wants article numbers/clones of Ab used here or supplemental table." should be removed

4- Both the ex vivo and in vivo results should be discussed and explained in more details in the discussion section

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Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

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Cystine/glutamate antiporter system x_c^- deficiency impairs macrophage glutathione metabolism and cytokine production.

PONE-D-23-15179R1

Dear Dr. Axel de Baat,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Subhadip Mukhopadhyay, 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 #2: All comments have been addressed

Reviewer #3: (No Response)

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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 #2: Yes

Reviewer #3: (No Response)

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

Reviewer #2: Yes

Reviewer #3: (No Response)

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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 #2: Yes

Reviewer #3: (No Response)

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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 #2: Yes

Reviewer #3: (No Response)

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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 #2: In this revised manuscript, the authors investigated the functional implication of the glutamate-cystine antiporter (sXc) system in the regulation of macrophage responses. The authors did a very careful job of editing and revising the content of the manuscript. The statistical analysis of the data was done thoroughly. Also, figure 1 was added properly, which supports their interpretation and the analysis of the transcriptional regulation of Sxc, Slc7a11. As requested, the authors included a very brief description of their overall findings at the end of the introduction, which might be useful for the general readers to get an overall impression of the study. The authors also added a short definition of the term endotoxemia. Finally, the authors made nearly all the corrections (textual, figure legends, figure labeling, etc.), which were needed for the publication. Overall, this manuscript reads well and would be a nice addition to the field of the cystine transport system in macrophage activity.

Reviewer #3: The authors has done a nice job of revising this manuscript and they have addressed all of my comments.

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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 #2: No

Reviewer #3: No

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