PONE-D-24-49430Extracellular vesicle release in an experimental ventilator-induced lung injury porcine modelPLOS 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: No

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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?

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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 authors have conducted an in-vivo study in pigs to evaluate how high tidal volume ventilation after surfactant depletion alters the generation of extracellular vessicles (EVs) in the bronchiolar lavage fluid and in the plasma. They also monitor a marker of lung damage and demonstrate that nucleic acid containing EVs increase in bronchoalveolar lavage fluid but not in the plasma. This is an important finding as it indicates that airspace markers might be more specific for lung injury during mechanical ventilation and that plasma biomarkers may not be indicated for assessing the degreee of lung injury during ventilation. This is an important study that I recommend for publication but I do have several comments that should be addressed prior to publication.

1. My main comment is that the ventilation protocol used, high tidal volume following lung lavage/surfactant inactivation may involve other forms of lung injury besides stretching injury. First, surfactant depletion will cause airway/alveolar atelectasis and even ventilation at the PEEP levels may result in cyclic airway closure and reopening. Recently, this form of injury (atelectrauma) has been implicated as a more damaging force than stretching, see Gabela-Zuniga, Lab on Chip, 2024. Second, the data in Table 2 indicate that there is higher inspiratory and airway pressures in the HTV groups and therefore transmural induced inflammation (barotrauma) may also be occuring. The authors should at minimum note the importance of these other forms of ventilation induced lung injury and note that they may be occuring to some degree in thier animal model.

2. I suggest referencing the novel work by Shaver et al in which protein biomarkers of ARDS were obtained from heat moisture exchange (HME) filter fluid as a way of assessing airway markers. Extension of this technique to obtain nucleic acids from HME fluid might be an important new way to non-invasively assess lung injury markers from the airspace and thus more specifically assess the degree of lung injury in mechanically ventilated patients See Bastarache, Am J Physiol Lung Cell Mol Physiol. 2021 and Rizzo, JCI Insight. 2022.

3. Since the authors use 100% O2 in the HTV group, there could also be hyperoxic lung injury. This should be noted.

4. It should be noted that BAL could contain EVs from both airway as well as alveolar compartments

5. Given that neutrophil counts are going up in the presented model of VILI, it is possible that the increased EV counts seen in BAL are due to these cells. Is there any evidence for which cells these EVs come from? Surface markers?

6. I think table 5 would be better served as a figure (like figure 3-4)

7. Finally, in addition to being a biomarker of disease, it might be worth mentioning that EVs have recently been used as a therapeutic drug delivery vehicle to minize VILI see Salazar‐Puerta, Advanced Materials, 2023.

Reviewer #2: Thank you for asking me to review this paper. Larssen et al use a porcine model of saline lung lavage and high stretch to examine release of extracellular vesicles in relation of VILI. They show increases in bronchoalveolar lavage neutrophils, and EVs containing SYTO-13 and HMGB-1 combined. I have a few comments:

1. I do not think the data add to the knowledge base. It is known that EVs and neutrophils are increased in ARDS/VILI.

2. Further investigation as to where the EVs derived from would be useful, given the use of flow cytometry.

3. Neutrophil EVs are associated with SYTO-13 especially if derived during NETosis induced DNA release.

4. HMGB-1 is also associated with NET release

5. Increased neutrophil numbers in the BAL would also mean higher EVs being detected. Especially given other EVs e.g. platelet derived ones did not increase.

6. What are the downstream effects of the EVs that have been detected?

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Reviewer #1: No

Reviewer #2: No

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Extracellular vesicle release in an experimental ventilator-induced lung injury porcine model

PONE-D-24-49430R1

Dear Dr. Larsson,

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,

Rui Tada, 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

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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

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

Reviewer #1: Yes

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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

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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

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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 #1: (No Response)

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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

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