PONE-D-19-27394

In vivo bioluminescent tracking of transplanted macrophages reveals temporal distribution and specific homing in the liver that can be perturbed by clodronate liposomes

PLOS ONE

Dear Dr. Nakayama,

Thank you for submitting your manuscript to PLOS ONE. Your manuscript requires some relevant revisions before it can be sent out for peer review process. Specifically:

- Figure 1A: Authors need to insert a scalebar in all panels describing the bioluminescence in mice.

- Figure 1C: Authors need to insert a scalebar in all panels depicting the bioluminescence in organs.

- Figure 2C: Authors need to resize all panels depicting the bioluminescence in organs, so that the panels will be displayed with identical sizes.

Figure 2D: Authors must insert a scalebar for each depicted panel in this figure.

Figure legends must be revised in accordance to these new modifications.

We would appreciate receiving your revised manuscript by Nov 22 2019 11:59PM. When you are 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Fabrizio Mattei, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

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PONE-D-19-27394R1

In vivo bioluminescent tracking of transplanted macrophages reveals temporal distribution and specific homing in the liver that can be perturbed by clodronate liposomes

PLOS ONE

Dear Dr. Nakayama,

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.In particular, Authors must carefully and thoroughly address all the points raised by the Reviewer 1. Furthermore, the use of liposomal clodronate is not sufficiently motivated in the text. For this reason, Auhtors must extensively clarify in their manuscript why they studied liposomal clodronate raather than other types of liposomal products.

We would appreciate receiving your revised manuscript by May 22 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Fabrizio Mattei, Ph.D.

Academic Editor

PLOS ONE

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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: (No Response)

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: Partly

Reviewer #2: Yes

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

Reviewer #1: No

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 #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: No

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 #1: The authors report the localization of fluorescent labelled macrophages within the lungs, spleen and liver and the impact of liposomal clodronate upon maintenance of fluorescence. They demonstrated that labelled macs accumulated flourescence in the above organs, but not kidney or skin. Clodronate is biphosphonate with a liposome and action is not mentioned within the article.

Issues:

1. thioglycollate recruited macrophages are differentially activated through the recruitment process. The old literature (80s) spoke about the type of thiogycollate used and differential ability to phagocytose bacterial. The authors didn't mention the impact/activation of the type of recruitment upon subsequent homing of cells to the various locations. Some mention of cell surface receptors for endothelial or tissue ligands may be useful. Some of the differences are discussed in old literature, again such as J Immunol November 1, 2003, 171 (9) 4816-4823

2. There should be discussion of liposomal clodronate and the induction of apoptosis. It is not mentioned.

3. there is not discussion of statistical analysis of the findings.

4. the authors keep discussing the possibility of liposomal steroids, but did not study it. Liposomal clodronate has a putative role in autoimmune and transplant related conditions where on may want to remove tissue macrophage damage. It was not mentioned within the context of this manuscript. the fact that inert liposomes did not appear to alter macrophages and clodronate loaded liposomes was associated with loss of macrophage associated fluorescence suggests that this may be a way to gain targeted drug delivery, but it is very round about.

Reviewer #2: Nakayama et al presents a new method to track macrophages in vivo using bioluminescence and probe the method of depletion using clodronate liposomes.

Actually the article checks a macrophage tracking method in vivo. Regarding the methodology used, the necessary controls are considered to validate the technique and the materials and methods are adequate to verify that the method works correctly. Perhaps it would be interesting to test it on immunodeficient mice or another animal species to see the possibilities of the technique. The Editor should assess whether it is of sufficient scientific quality to be published on Plos One.

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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: No

Reviewer #2: Yes: Carlos Alfaro

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-19-27394R2

In vivo bioluminescent tracking of transplanted macrophages reveals temporal distribution and specific homing in the liver that can be perturbed by clodronate liposomes

PLOS ONE

Dear Dr. Nakayama,

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.

As you can see from the comments, the reviewers felt that the scientific soundness of this study should be improved before the acceptance of this work. One reviewer raised the important issue on quantitative analysis, which needs to be addressed. To help me expedite processing, please explicitly address the questions raised by the reviewers in your cover letter and also point out the changes made in the manuscript. I will go back to the reviewers for further input and advice before any final decision on possible publication is made.

Please submit your revised manuscript by Nov 15 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Bing Xu, PhD

Academic Editor

PLOS ONE

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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 #2: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #4: 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

Reviewer #3: No

Reviewer #4: Yes

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

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: 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 #2: Yes

Reviewer #3: Yes

Reviewer #4: 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 #2: Yes

Reviewer #3: Yes

Reviewer #4: 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 made an effort to answer the questions raised by the reviewers and are generally acceptable and correct. The article has improved significantly with the changes introduced.

Reviewer #3: The paper by Nakayama and colleagues aim to image the biodistribution of Raw macrophages and see the effect of Clodronate liposomes on their depletion using optical imaging. Since the authors use the XenolighDIR fluorescent dye they are imaging near infrared fluorescence and not bioluminescence so the title is misleading.

Moreover since the perform direct labelling and the dye bound to cell membranes, there is no proof that cells retain it after 24h so for their analysis at 168h the authors should provide more evidence that they are looking at macrophages in the different organs and not at the free dye. Moreover the paper lacks quantitative analysis. The author should report the fluorescence signal in the organ in relation to weight of the organ so that a comparison of the prevalence of the cells can be claimed and statistical analysis performed.

Finally A. Taylor and colleagues ( doi: 10.1155/2018/2514796) injected bioluminescent RAW macrophages in animals and, although they also see signals in the liver , they were able to see it in the brain area too. So it would be nice to add this organ to the analysis.

I am afraid that in this form the paper is more appropriate as a technical note and not a research article.

Reviewer #4: In this manuscript, the authors studied the biodistribution of implanted macrophages in mice model. The results revealed that the macrophages exhibited organ-specific accumulation. This result provides new knowledge for immunologist. Moreover, the authors developed a method using clodronate liposomes to remove the macrophages accumulated in the liver, spleen and lung. This way could potentially address diseases like Haemophagocytic lymphohistiocytosis (HLH), or macrophage activation syndrome (MAS) which are relevant to the high affinities of macrophages. This work is technically sound. The experiment groups and the controls are well designed. The authors have fully address the concerns raised by previous reviewers. Thus, I recommend the acceptance of this manuscript.

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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 #2: Yes: Carlos Alfaro

Reviewer #3: No

Reviewer #4: 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.

In vivo tracking of transplanted macrophages with near infrared fluorescent dye reveals temporal distribution and specific homing in the liver that can be perturbed by clodronate liposomes

PONE-D-19-27394R3

Dear Dr. Nakayama,

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,

Bing Xu, PhD

Academic Editor

PLOS ONE

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