PONE-D-20-19201

Human osteoclastogenesis in Epstein-Barr virus-induced erosive arthritis in humanized NOD/Shi-scid/IL-2Rγnull mice

PLOS ONE

Dear Dr. Takei,

Thank you for submitting your manuscript to PLOS ONE.Your manuscripts are reviewed by two experts in different fields.  I also read your manuscript quickly. We all agree that this is an interesting and potentially important work.  However, they also identified several significant concerns that require further attention. As indicated in the appended comments, a number of specific points have been raised that have to be resolved. Based on the combined assessments of the reviewers, we would be willing to provide you with an opportunity to respond to these issues in a suitably revised version of the manuscript.  After careful consideration, we feel that your manuscript has merit and will be reconsidered for publication in PLoS One after major revision.

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Please submit your revised manuscript by Oct 26 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Luwen Zhang

Academic Editor

PLOS 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: Partly

Reviewer #2: Yes

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

Reviewer #1: No

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: Human osteoclastogenesis in Epstein-Barr virus-induced erosive arthritis in humanized NOD/Shi-scid/IL-2Ryneull mice by Nagasawa et al describes a humanized mouse model in which erosive arthritis during EBV infections can be studied. Their results suggest that human osteoclasts induce erosive arthritis during EBV infection.

This is exciting and significant work but proper controls need to be shown. Without the necessary controls this review can’t properly evaluate the quality of the data. These controls should be added in addition to increasing the number of representative pictures and sample sizes as described below.

Results section. Page 18. Outline the and describe the experiment broadly before jumping into the specific results to make it easier for the reader to follow.

Figure 1. The legend needs to be more descriptive. Are these averages of the 10 infected samples?

Error bars need to be included and statistics should be provided. The data for the uninfected samples needs to be included.

Table 1. Why were different numbers of CD34+ cells used for different sets? What is the rational for different amounts?

Figure 2. Images are only shown for 1 infected and 1 uninfected mouse. More representative pictures should be added. I would like to see pictures representing the different severity of bone erosion described in Table 1. (ie samples from 1, 2, and 3+ in addition to the negative control).

Figure 3. Only 2 mice from EBV infected or uninfected where examined. 10 mice were infected – including more samples would increase the significance. Only one picture is shown for TRAP, Cathepsin and MMP-9 staining. The authors state these antibodies do not react with the mouse proteins but the negative controls are necessary and need to be shown.

Figure 4. An n=2 is low and only single pictures are shown. Negative controls are necessary and should be shown.

Figure 5. n=3 but only one sample is shown.

Supplemental figure doesn’t have a legend.

Reviewer #2: The article “Human osteoclastogenesis in Epstein-Barr virus-induced erosive arthritis in humanized NOD/Shi-scid/IL-2Rgnull mice” is an interesting original study that starts to elucidate the mechanisms of EBV-induced erosive arthritis through a hu-NOG model. The study aims are addressed by its methodology and the three-dimensional computed tomography was a visually appealing tool to demonstrate bone erosive changes. The presentation of results is linear and the data is properly discussed, supporting the authors conclusions. Although this is true, there are some unanswered questions and revisions that will make this study more impactful.

Major Comments

The authors do not show in vitro osteoclast differentiation of bone marrow cells (cultured with human RANKL and M-CSF) from EBV-uninfected NOG (hu-NOG only).

Even though they say that EBV infection did not increase the number of osteoclast progenitors (in a data not shown - sentence line 440-446) they do not provide evidence that EBV-uninfected hu-NOG have human osteoclast progenitor cells in their bone marrow, or that these progenitors are able to differentiate in osteoclast in vitro as for the EBV-infected hu-NOG.

I believe that if the authors provide the data showing in vitro osteoclast differentiation of bone marrow cells from EBV-uninfected hu-NOG, cultured in the presence of human RANKL and human M-CSF it would demonstrate potential for differentiation as well as equal progenitor

Typically in normal bone remodeling there is a balance between osteoblasts and osteoclasts, with the former being responsible for inducing the differentiation of the latter through the production of RANK-L and M-CSF. It’s true that in pathogenic settings the osteoblasts are not the main sources of these cytokines, but they are still important for conferring some osteoprotection. Although the authors bring this to their discussion, there is no information on osteoblasts in their data. It would be informative to understand the ratio of osteoblast to osteoclasts in hu-NOG and after infection with EBV.

Minor comments

In the methods section the authors list CD19+ as one of the monitored percentages of human cells in the blood of hu-NOG. In Fig. 1 the authors do not show CD19+ numbers, although one can assume by subtracting CD4+ and CD8+ from total Human CD45+, it would be easier to observe how the frequency of these cells stay through the experiment course if a CD19+ curve was displayed.

Fig. 5 - it appears that the cells stained for TRAP (in Fig. 5A and B) were also stained for hematoxylin, correct? If that is true, since this was performed in a glass bottom dish, the statement in line 219 of the methods section “After washing, only the plates of serial sections were stained in hematoxylin (cultured cells on the chamber slides, pit formation assay plates, and glass bottom dishes were not stained” is incorrect.

Line 645 - Possible typo/missing verb “be cultured”

Have the authors considered staining for collagen (with picrosirius red staining or by using polarized light microscopy) to assess cathepsin K effects in the joints of EBV-infected hu-NOG.

Aside RANKL and M-CSF there are a number of inflammatory cytokines that are usually present in inflamed joints that impact bone remodeling within that microenvironment. Given that the bone marrow cells of EBV-infected hu-NOG when cultured in vitro (without human RANKL and M-CSF cytokines) resulted in osteoclast differentiation and the authors had previously shown bone marrow edema in EBV-infected mice, one could assume that pro-inflammatory cytokines have an important role in this process. Specially since T cells do not survive for long in culture, even with proper stimuli, cells from the monocytic/macrophage lineage could be the sources of this cytokines in this case. Have the authors investigated IL-1 (alpha and or betta) and TNF-a (that have been previously implicated in osteoclastogenesis), in the bone marrow of EBV-infected hu-NOG?

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Reviewer #1: No

Reviewer #2: Yes: Thiago Alves da Costa

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PONE-D-20-19201R1

Human osteoclastogenesis in Epstein-Barr virus-induced erosive arthritis in humanized NOD/Shi-scid/IL-2Rγnull mice

PLOS ONE

Dear Dr. Takei,

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. Reviewers still have some concerns. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the reviewers.

Please submit your revised manuscript by Apr 01 2021 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,

Luwen Zhang

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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: 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

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: Yes

Reviewer #2: 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: The authors have addressed my initial concern but I still have a few comments and suggestions.

The authors added the statement “In this study, we initially confirmed our previous findings of rapid increase in the number of CD8+ cells and the reversal of CD4/CD8 ratio in the peripheral blood of humanized mice following EBV infection.” to the beginning of the results section. Please provide a reference.

The authors state the information in figure 1 is not an average bu a representation from 1 mouse. I think showing the average with error bars would give a representation of how consistent the results were.

Figure S1 is labeled in this order A,C, B, D. Is this intentional or is A, B, C, D meant.

Figures 3 ,4 and 6 would be easier to read if labels such as A1, A2, B1, etc were replaced with the antibody that was used for staining.

Figure 3 A1, A2. Are these the same picture at different magnifications? When multiple pictures are shown for the the same antibody stain please indicate if it is from the same mouse. The figures would be enhanced with a more descriptive labeling scheme.

Reviewer #2: The introduction of additional images to Figures 2; 3; 4 and 6, were a good improvement of the manuscript, strengthening the authors findings.

However, figure labeling could be improved to ensure better readability. The current panel labeling (“A-1; B-1”) and the amount of panels in each figure, are hard for readers to follow without constantly looking at the figure legend.

The authors could identify the panels with the molecule they are showing on top in the vertical orientation while identifying the panels with the correct experimental group in the horizontal. That would also potentially improve the figure legend itself.

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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: Thiago Alves da Costa

[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.

Human osteoclastogenesis in Epstein-Barr virus-induced erosive arthritis in humanized NOD/Shi-scid/IL-2Rγnull mice

PONE-D-20-19201R2

Dear Dr. Takei,

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,

Luwen Zhang

Academic Editor

PLOS ONE