PONE-D-21-02995

Neutrophil stimulation with citrullinated histone H4 slows down calcium influx and reduces NET formation compared with native histone H4

PLOS ONE

Dear Dr. Wagner,

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Michel Simon, Ph. D.

Academic Editor

PLOS ONE

Additional Editor Comments:

the Authors conclusively demonstrated that the citrullinated form of H4 is less effective than the native form to induce NETosis in HL60 cells and human primary neutrophils, because of a lower induced calcium influx. Some of the data concerning mouse neutrophils (Fig 2E and 2G) are less convincing (extra-cellular CitH3 is hardly visible), and better images should be shown. Please, could the Authors also quantify immunodetection data shown on Fig S1A.

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- https://pubmed.ncbi.nlm.nih.gov/32193354/

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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

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

Reviewer #1: Yes

Reviewer #2: No

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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 paper analyzes the ability of “native” vs citrullinated histones to induce NETosis in mouse and human neutrophils and in HL60 cells. The results indicate that histone H4 induces NETosis mediating calcium influx and PADi4 activation, while citrullinated H4 has a lower activity, suggesting that citrullinated histones in NET may act as negative feedback mechanism in NET production.

On the whole, the data presented in the paper are of great interest and relevance; only a minor point can be raised.

Under a quantitative point of view, is NET induction by native histones comparable to what obtained with other “physiological” stimuli (for example lps)?

Reviewer #2: The manuscript submitted by Shi et al. addresses the role of H4 citrullination in NET formation. The topic is interesting and the results suggest that the release of citrullinated H4 may constitute a physiological mechanism to slow down the process of NET formation. The manuscript is well written, but it would need additional controls and changes before considering its publication in PlosOne.

Specific comments:

1. In line 192 the authors state that citrullinated H1 is released into the extracellular space along native H4. The authors would need to demonstrate this statement showing an experiment where native H4 is shown to be released into the extracellular space. Moreover, it would be really helpful to determine the proportion H4 modified by citrullination. These results should be added to Figure 1, or as an additional figure.

2. The statistical analysis shown in the figures must be revised to include all possible pairs. For instance, Figure 2B does not show if the percentage of NET formation in the presence of Cla is significant when compared to the control cells. The same problem can be observed in all the figures in panels with more than two bars.

3. The extent of in vitro citrullination must be determined to ensure that all H4 has been modified in the reaction conditions. These results must be included in Figure S2.

4. Regarding the manuscript format, higher resolution images are needed, especially for immunofluorescence and the supplementary file must be revised because at the moment previous changes are marked in red within the document.

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Reviewer #1: Yes: Paola Migliorini

Reviewer #2: No

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Neutrophil stimulation with citrullinated histone H4 slows down calcium influx and reduces NET formation compared with native histone H4

PONE-D-21-02995R1

Dear Dr. Wagner,

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,

Michel Simon, Ph. D.

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: The point raised has been addressed. No further modifications are required and the paper can be accepted in the present form.

Reviewer #2: Shi et al. have addressed all the comments, although they didn’t determine the extent of H4 citrullination as requested. However, they provided an understandable reason for this issue and highlighted this limitation within the discussion. As a whole, the manuscript is significantly improved in respect of the original version and is now suitable for publication in PLosOne.

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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: Yes: Paola Migliorini

Reviewer #2: No