-->PONE-D-25-61554-->-->Temporal Transcriptomic Profiling of Pulmonary Thromboembolism Reveals a Persistent NETosis-Ferroptosis Axis and a Therapeutic Window for DNase I-Enhanced Thrombolysis-->-->PLOS One

Dear Dr. Yan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Academic Editor

PLOS One

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Additional Editor Comments:

Dear Authors,

Thank you for submitting your manuscript and for the substantial effort invested in this comprehensive and technically demanding study. The reviewers recognize the strengths of the longitudinal design and the breadth of experimental approaches.

After careful evaluation, the reviewers and I agree that the manuscript requires **major revision** before it can be considered further. The main concerns relate to the interpretation of whole-blood transcriptomic data, the level of mechanistic support for the proposed NETosis-ferroptosis axis, and the need for additional validation and clarification of the therapeutic and statistical analyses. In particular, the current version tends to overinterpret associative findings, and the conclusions should be more cautiously framed.

We encourage you to address the reviewers’ comments carefully, strengthen validation where feasible, and moderate the mechanistic and translational claims. We believe that, with substantial revision, the work could make a valuable contribution.

We look forward to receiving your revised manuscript.

Best regards,

Tomasz W Kaminski

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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: No

Reviewer #3: I Don't Know

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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: Comments to the Authors

General Assessment

The study addresses an important clinical problem and combines multiple experimental layers to explore temporal changes after PTE. The manuscript is very well written and connect the main ideas properly. The experimental design and methodology -including techniques- are in general extensive and robust. However, the manuscript require certain major revisions. Attached, I provide detailed comments of specific subjects and comments by section.

Reviewer #2: Dear Authors,

This is a large and carefully executed preclinical study that combines animal modeling, longitudinal transcriptomics, histology, circulating biomarkers, functional cardiopulmonary readouts, and a therapeutic intervention. The authors clearly invested significant effort, and the longitudinal design (days 1, 3, 7, and 14) is a real strength. The dataset is internally consistent, technically solid, and the timing of the intervention is biologically motivated rather than arbitrary. Overall, this is a serious piece of work with clear relevance to thrombo-inflammatory disease biology.

That said, the manuscript currently overstates its mechanistic and translational conclusions, and several key claims are not fully supported by the data. The central concepts - NETs involvement in thrombus persistence, impaired fibrinolysis, and improved thrombolysis with DNase I are already well described in the literature. The main added value here is the temporal transcriptomic profiling, but the study does not convincingly demonstrate a true mechanistic “NETosis-ferroptosis axis.” What the data show is prolonged activation of neutrophil-related and iron-associated pathways in whole blood. This supports coexistence or parallel activation, but not causal interaction, which is frequently implied in the text.

The strength of the data is uneven. Histology, serology, and functional measurements are thorough and well presented. However, all transcriptomic analyses are based on whole blood, which limits biological specificity. Many highlighted genes (e.g., S100A8/9, LCN2, LTF) are expected markers of neutrophil activation and inflammation and do not uniquely demonstrate NET formation or ferroptosis. Importantly, ferroptosis is inferred from gene enrichment alone; there are no direct measurements of lipid peroxidation, iron-dependent cell death, or GPX4 activity at the tissue or cellular level. Similarly, NETs are inferred mainly from MPO-NA and gene signatures, without direct visualization or quantification within thrombi or lung tissue.

From a statistical and analytical standpoint, the methods are generally appropriate, but interpretation goes too far. Many biomarkers and outcomes are tested, yet correction for multiple comparisons outside RNA-seq is not clearly addressed. The “improvement rate” calculation inflates effect sizes and should be complemented with absolute post-treatment values. The intervention study (n=6 or so per group) is small for the number of endpoints assessed, and the conclusions drawn from it are too broad for the sample size.

There is also interpretive overreach in several places. Claims that NETosis and ferroptosis form a self-reinforcing cycle are not experimentally tested. The designation of “stage-specific biomarkers” is overstated, as many of the identified genes reflect general stress or remodeling rather than PTE-specific stage markers. Finally, the idea of a defined “therapeutic window” is premature, as only one intervention time point is examined.

Suggested experiments to strengthen the paper

Add a causal intervention targeting ferroptosis or NETs independently

For example, inhibiting ferroptosis (e.g., with a ferroptosis inhibitor) or NET formation (independent of DNase I) would help determine whether these pathways actively drive pathology or are downstream bystanders.

Provide tissue-level validation of key mechanisms

Direct visualization of NETs within pulmonary thrombi (immunofluorescence for citrullinated histones/MPO) and direct assessment of ferroptosis markers in lung tissue would substantially strengthen the biological claims.

In summary, this is a strong descriptive study with a valuable longitudinal dataset, but the current manuscript overinterprets associative data as mechanism and overstates translational relevance. With clearer framing, toned-down conclusions, stronger mechanistic validation, and more cautious interpretation, the work could make a solid contribution. At present, major revision is required.

Reviewer #3: Manuscript Number: PONE-D-25-61554

Full Title: Temporal Transcriptomic Profiling of Pulmonary Thromboembolism Reveals a

Persistent NETosis-Ferroptosis Axis and a Therapeutic Window for DNase I-Enhanced

Thrombolysis

Overall comments: In this study, Chen J. et al. aims to demonstrate the dynamic pathophysiology of pulmonary thromboembolism in rabbit autologous thrombus PTE model and focusing on the roles of NETosis and ferroptosis, and to evaluate combination therapy of intravenous DNase-I and recombinant tissue plasminogen activator. First of all, this manuscript is well-written. However, there are still many things that need to be resolved before consideration for publication.

1. The readability of the entire manuscript is good. However, in order to improve, the language editing by a native speaker or a professional translation office should be required.

2. Please ensure that all microscopy images contain visible scale bars and that these scale bars should have legible text alongside them in the figure panel itself and its legends.

3. Did authors use both gender of rats or any specific gender for this model.

4. As mentioned by the authors, the main limitation of the study is using the whole blood for Transcriptomic study.

5. It is always better to use immunofluorescence methods to identify the immune cells infiltration at the injury site in the fixed lung tissue instead of H&E staining. Even, looks for immune cells infiltration in tissue lysate using specific immune cell markers by western blot.

6. Did the authors check the neutrophil elastase and citrullinated histone-3 levels (either ELISA or western blot) in the plasma/serum. MPO is not a specific marker for NETosis. Even HO-1 is good marker for ferroptosis.

7. Addition of pro/anti-inflammatory marker detection will strengthen the manuscript.

8. Disclose all data points in all the experimental data and mention the number of experiments done for each analysis in the figure legend.

9. Did authors use iron chelating agents as therapy in this model?

10. The materials used with the proper details (company name and catalog number) should be in separate section, either in main manuscript or supplementary file.

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Reviewer #1: Yes: Jose R Pittaluga-Villarreal

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: Comments to the Authors.pdf

Temporal Transcriptomic Profiling of Pulmonary Thromboembolism Reveals Persistent NETosis- and Ferroptosis-Associated Gene Signatures and Enhanced Thrombolysis with Adjunctive DNase I

PONE-D-25-61554R1

Dear Dr. Yan,

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.

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Kind regards,

Tomasz W. Kaminski

Academic Editor

PLOS One

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

Reviewer #3: 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

Reviewer #3: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

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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 #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: 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: Dear Authors,

The manuscript has considerably improved, taking into consideration the current limitations.

All previous review comments have been properly addressed by authors.

Reviewer #2: (No Response)

Reviewer #3: The authors have sufficiently addressed all of my concerns. The paper can be accepted in the current version.

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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.

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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: Yes: Marta Wolosowicz

Reviewer #3: No

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