PONE-D-23-01584A cross-circulatory platform for monitoring innate responses in lung grafts

PLOS ONE

Dear Dr. Schwartz-Cornil,

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.

Please submit your revised manuscript by Apr 22 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Lourdes Chacon Alberty, M.D

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following financial disclosure: 

   "ES received funding from ADETEC-Coeur support (ES), la « Chaire Universitaire de Transplantation Université de Versailles-Saint-Quentin en Yvelines, Hôpital Foch et Fondation Foch, AR received the Association Gregory Lemarchal and the association Vaincre la Mucoviscidose (project number RF20220503016) and ISC received funding from INRAE institutional support." 

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." 

If this statement is not correct you must amend it as needed. 

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

3. Thank you for stating the following in your Competing Interests section:  

    "Authors have no competing interest."

Please complete your Competing Interests on the online submission form to state any Competing Interests. If you have no competing interests, please state "The authors have declared that no competing interests exist.", as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now 

 This information should be included in your cover letter; we will change the online submission form on your behalf.

4. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.

[Note: HTML markup is below. Please do not edit.]

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

**********

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: No

Reviewer #2: Yes

**********

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: This is a very innovative study with great potential to uncover mechanisms of ischemia reperfusion injury and reveal putative targets for intervention. This area of investigation is of great interest to the lung transplant community and has translatable potential to transplantation in general. The manuscript could be enhanced through the following ways

1. The paper would benefit significantly from a native english speaking editor. There are countless awkward statements and grammatical errors throughout the text which can easily be corrected by a native english speaker.

2. The major deficit I see with this manuscript are the lack of different conditions to compare. There is novelty in the technique, but there is a lack of scientific rigor and the lack of varying conditions diminishes confidence that the observed immune phenotypes have a biological underpinning. By showing, for instance, variable injury phenotypes in response to prolonged ischemic times, this would vastly increase the significance of this research.

3. Related to point #2 above, why did the authors chose as the sole condition: a DCD donor model with additional cold ischemia time? Arguably, a cleaner model would use a heart beating donor.

4. The focus of this paper is innate immune responses to ischemia reperfusion injury. I am confused as to why the authors go great lengths to introduce and explain that they specifically chose outbred pigs of differing genders to introduce an "allogeneic" component to the model. If the goal is to understand innate immunity, the model should use syngeneic transplants.

5. Throughout the manuscript, the word "recruited" is used liberally to describe perfusing pig immune cells found in the allograft. This word should be used carefully as "recruited" implies an active process. Passively perfusing the lung with CSFE labeled blood is not an active biologic process...therefore CSFE+ cells in the intravascular space I would not consider as recruitment. Conversely, CSFE+ cells in the alveolar space or interstitium could be considered as "recruited" since there was a process of extravasation. Related to this comment, Fig 3 could be modified to demonstrate this with the IHC showing improved histologic resolution to identify which compartment the CSFE+ cells are in. Currently, that figure is blurry and zoomed out too far so you cannot tell histologic architecture.

6. Fig 2 should include 0, 6, and 10 hr timepoints for lung histology to assess over time. For P/F ratio, is should be better defined where they are drawing samples from.

7. Fig 5, how do you define MHCII positive cells? Need to show the FACS plots.

8. In the discussion I would tone down the discourse on the fallibility of rodent models to study IRI. The mouse is a much higher throughput model of lung IRI and an extremely well established model. Multiple groups throughout time have used both hilar clamp IRI models and even lung transplant models in mice to uncover the immune mechanisms of IRI. Pigs are much more expensive to use. Also, lung transplant techniques were developed in Dogs more so than pigs.

9. A significant weakness in the described cross perfusion platform that needs to be mentioned by the authors is the extremely well known pro-inflammatory stimulus that is the cardiopulmonary bypass circuit (of which all the elements are in this model: pump, membrane oxygenator, reservoir, and LOTS of plastic tubing). Every clinician that uses mechanical circulatory support knows that the blood-plastic interface activates the complement system, coagulation system, and releases a host of pro-inflammatory mediators into circulation. This will certainly affect any study evaluating the innate immune response.

Reviewer #2: Very nice observational molecular work by the French Lung Transplant Group and Prof. Sage. In the current study they used 10 donors and 10 recipients and cross-circulation for 10 hours. The method cross-circulation itself is not novel but the molecular work in regards to cross-ciculation and recrutment of immune cells with a strong clinical translational potential to the donr lung has not been done by the Colombia team or any other group that has implemented the method as far as I know.

I have some additional questions.

Why did the authors set the time to 10 hours of circulation? To make the connection between acute and chronic rejection wouldn’t it be better and more translational to run over 4 days or even more? An unharmed lung would probably last longer on cross-circulation than the injured lungs at Colombia.

Why was a DCD lung donation model used? Would there be any differences using a beating heart donor lung? Brain dead pig models are extremely demanding to achieve but why not just use a beating heart donor?

Was the donor and the recipient matched according to blood type?

Was the lung really put on ice before establishment of cross-circulation? Perhaps it was cold storage? Either way I cannot find the ischemic time in the manuscript please add.

Did the recipient receive any immunosuppressant therapy? If not, why. Please elaborate.

The manuscript lacks many of the hemodynamic, and ventilatory parameters for the 10 hours. Would be interesting to see for example flow through the “donor lung”, oxygen, saturation, end-tidal CO2, compliance, and respiratory rate were continuously monitored. If I understood, it right this have been measured in this model. Perhaps it is in the supplementary file. I was unable to open those. If so, please move them to the main manuscript.

It is a nice manuscript with high quality molecular work. However, if any, this reviewer lacks a interventional angle to increase the novelty of the work.

**********

6. 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

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

A cross-circulatory platform for monitoring innate allo-responses in lung grafts

PONE-D-23-01584R1

Dear Dr. Schwartz,

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,

Lourdes Chacon Alberty, M.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

**********

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

**********

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

Reviewer #1: 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

**********

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: No

**********

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: Thank you for addressing the points. This article is a valuable addition to the literature on lung transplant, especially with the description of the technique of cross circulation

**********

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

**********