PONE-D-24-13219LFA-1: A potential key player in microglia-mediated neuroprotection against oxygen-glucose deprivation in vitroPLOS ONE

Dear Dr. Jansen,

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PLOS ONE

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"Michael Gliem: Speaking fees from Novartis and Pfizer. 

Mark Pawlitzki  received honoraria for lecturing and travel expenses for attending meetings from Alexion, ArgenX, Bayer Health Care, Biogen, Hexal, Merck Serono, Novartis, Sanofi-Aventis and Teva. His research is funded by the by ArgenX, Biogen, Hexal and Novartis. 

Sven Meuth receives honoraria for lecturing, and travel expenses for attending meetings from Academy 2, Argenx, Alexion, Almirall, Amicus Therapeutics Germany, Bayer Health Care, Biogen, BioNtech, BMS, Celgene, Datamed, Demecan, Desitin, Diamed, Diaplan, DIU Dresden, DPmed, Gen Medicine and Healthcare products, Genzyme, Hexal AG, Impulze GmbH, Janssen Cilag, KW Medipoint, MedDay Pharmaceuticals, Merck Serono, Neuropoint, Novartis, Novo Nordisk, ONO Pharma, Oxford PharmaGenesis, Roche, Sanofi-Aventis, Springer Medizin Verlag, Chugai Pharma, QuintilesIMS ,Teva, Wings for Life international and Xcenda.

His research is funded by the German Ministry for Education and Research (BMBF), Bundesinstitut für Risikobewertung (BfR), Deutsche Forschungsgemeinschaft (DFG), Else Kröner Fresenius Foundation, Gemeinsamer Bundesausschuss (G-BA), German Academic Exchange Service, Hertie Foundation, Interdisciplinary Center for Clinical Studies (IZKF) Muenster, German Foundation Neurology and Alexion,  Almirall, Amicus Therapeutics Germany, Biogen, Diamed, DGM e.v., Fresenius Medical Care, Genzyme, Gesellschaft von Freunden und Förderern der Heinrich-Heine-Universität Düsseldorf e.V., HERZ Burgdorf, Merck Serono, Novartis, ONO Pharma, Roche, and Teva."

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Additional Editor Comments:

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The manuscript was evaluated by two experts. They suggested some important amendments that are needed for acceptance including changes in the images, improvements of the text. At least one further experimental figure showing the mechanism of the observed neuroprotective effect is needed.==============================

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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: Partly

Reviewer #2: Partly

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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?

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: This study shows the critical role of the β2 integrin, LFA-1, in microglia-mediated neuroprotection during oxygen-glucose deprivation (OGD) in hippocampal cell cultures. The research demonstrates that LFA-1 is necessary for neuronal survival under ischemic conditions, as its inhibition with the compound BIRT377 resulted in a dose-dependent increase in neuronal death. ICAM-5, which is strongly expressed in hippocampal neurons, was identified as a potential ligand for LFA-1, indicating an important interaction between microglia and neurons that is essential for neuroprotection. These findings highlight the significance of LFA-1 in neuroprotective signaling and emphasize the need for further investigation into integrin-mediated pathways in the context of ischemic stroke.

Minor concerns:

In the introduction, it is mentioned that IGF-1 is an example of a neurotransmitter, but its canonical function is as a growth factor mainly released from the liver, although also produced in many tissues. IGF-1's main activity is to induce proliferation; however, in the brain, it has been related to survival and neuroprotection.

What happens to the BV-2 microglial cells that are co-incubated with the hippocampal organotypic cultures? Is there any kind of tissue penetration? Do microglial BV-2 cells secrete proinflammatory cytokines? Are they M1 or M2 phenotype? Are they pre-treated to express a specific phenotype?

Did you try higher doses of BIRT377?

Is PI incorporation specific to neuronal cells?

Please include better pictures of the OHCs in Figures 2C and 3C, as they are very small, and without nuclei counterstaining, it is very hard to identify the damage zone and the affected cells.

Authors show that N=7. How was this calculated? What is the effect size? Was it calculated a priori?

Does BIRT377 have a differential effect on endogenous microglia and BV-2?

Major concerns:

The abstract is very general and does not provide important information such as methodology, a summary of results, and a brief conclusion. It also states that there is no single therapy to induce neuroprotection after a stroke in patients, which is hard to believe. I recommend being more cautious with statements like this.

The control in Figures 2C and 3D show a strong discrepancy in propidium iodide incorporation intensity. The timeline indicates that they are exposed to PI for 24 hours; if there is any other difference in the protocol that explains this fluorescence inconsistency, it should be included in the manuscript.

I strongly recommend including a conclusion or summary at the end of the discussion, which includes only technical English, avoiding expressions such as “Like a stone into water.” This can be confusing for some international readers.

Stressed microglia are reported to promote an M1 proinflammatory phenotype, which can be harmful to neural tissue. Why does BIRT377, once blocking OGD in non-stressed BV2, increase survival (Figure 2B)? Also, endogenous microglia and BV2 are likely to have opposing responses since one is stressed and the other is not. Please include a discussion about this.

The number of results seems a little short. It would be interesting to see what the mediators are in the effect exogenous microglia upon survival in hippocampal tissue cultures. Maybe qPCR or ELISA for cytokines and neurotrophins could complement this work.

Reviewer #2: In this manuscript, the authors investigated LFA-1 inhibition with BIRT377 on neuronal death after oxygen-glucose deprivation (OGD) in cultured hippocampal slices. The authors found that BIRT377 treatment enhanced neuronal death after OGD, and abolished the neuroprotective effect of transplanted BV2 cells on OGD-preconditioned slices. Below are some comments on the manuscript.

(1) In figure 1C, the authors show expression of ICAM5 but not ICAM1 in neurons. At which experimental conditions (OGD vs. Normal) was this done?

(2) In figure 3, the authors show that BV2 cell transplantation is neuroprotective on OGD-preconditioned slices, which can be partially abolished by LFA-1 inhibition. The expression of LFA-1 in BV2 cells should be experimentally confirmed.

(3) There is a lack of scale bars on the immunofluorescent images in all figures.

(4) It is difficult to see the morphology of the slices in figure 2C and figure 3C. The authors should either provide bright-light images of the slices together with the fluorescent images or enhance the intensity of the fluorescent images to show the slice morphology.

(5) The manuscript needs careful proof-reading: in several occasions, the authors start the sentence with “previously”, “previous studies” or “previous data” without providing any literature reference; in line 7 of the abstract: “hippocampal cell culture” should be “hippocampal slice culture”; in line 16 of the introduction, IGF-1 is not a neurotransmitter; in the “Organotypic hippocampal slice cultures” section of the method: repeated texts in lines 5-8; for microglia labelling: it is stated in the main text that rhodamine-IB4 was used; however, in the method section it is indicated that Alexa568-IB4 was used.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-24-13219R1LFA-1: A potential key player in microglia-mediated neuroprotection against oxygen-glucose deprivation in vitroPLOS ONE

Dear Dr. Jansen,

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.

==============================

The manuscript was improved, but there are still problems to be solved. The attached clean copy is not the same as the version with tracked changes. The authors need to address ALL comments of both reviewers and these should be incorporated in the revised text as well. I suggest the authors to use color highlight in yellow in revision 2 as compared to the original submission to ensure all changes are visible and clear. 

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Please submit your revised manuscript by Oct 24 2024 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,

Mária A. Deli, M.D., Ph.D.

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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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: (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 #1: Yes

Reviewer #2: (No Response)

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

Reviewer #1: Yes

Reviewer #2: (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 #1: Yes

Reviewer #2: (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 #1: Yes

Reviewer #2: (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 #1: Authors addressed all the concerns, however, I still think the amount of results is a little short for a journal as PLOS ONE

Reviewer #2: The manuscript has not been revised at all in the clean version, except that one or two sentences were inserted in the method section. For the version with tracked changes, some revisions have been made based on reviewers' comments. However, not all comments had been addressed even though the authors claimed that they did so in the responses to reviewers.

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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: No

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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.

LFA-1: A potential key player in microglia-mediated neuroprotection against oxygen-glucose deprivation in vitro

PONE-D-24-13219R2

Dear Dr. Jansen,

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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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,

Mária A. Deli, M.D., 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 #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 #2: Yes

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

Reviewer #2: 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 #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 #2: 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 #2: The authors have addressed the critical concerns sufficiently in the updated version. I have no further comments.

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Reviewer #2: No

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