The enhancement of CCL2 and CCL5 by human bone marrow-derived mesenchymal stem/stromal cells might contribute to inflammatory suppression and axonal extension after spinal cord injury

Kazumichi Yagura; Hirokazu Ohtaki; Tomomi Tsumuraya; Atsushi Sato; Kazuyuki Miyamoto; Naoto Kawada; Keisuke Suzuki; Motoyasu Nakamura; Koji Kanzaki; Kenji Dohi; Masahiko Izumizaki; Yutaka Hiraizumi; Kazuho Honda

doi:10.1371/journal.pone.0230080

Peer Review History

Original SubmissionOctober 31, 2019
13 Dec 2019 Decision Letter - Xing-Ming Shi, Editor PONE-D-19-28233 The enhancement of CCL2 and CCL5 by human bone marrow-derived mesenchymal stem/stromal cells might contribute to inflammatory suppression and axonal extension after spinal cord injury. PLOS ONE Dear Dr Ohtaki, 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 if you think you can satisfactorily addresses the points raised during the review process. In addition to the concerns from reviewer 1 (see details below), please address following: 1. Provide evidence that hMSCs are present at the injection site 2. What is the survival rate and where are they located if they are not at the injury site 3. Why are there no dendrites on neurons? We would appreciate receiving your revised manuscript by Jan 27 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. 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Your ethics statement must appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please also ensure that your ethics statement is included in your manuscript, as the ethics section of your online submission will not be published alongside your manuscript. 4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [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: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No ****** 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 ****** 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 ****** 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 bone marrow-derived mesenchymal stem/stromal cell (hMSC) transplantation is a promising strategy for improving functional recovery following traumatic spinal cord injury (SCI). However, the mechanism behind the integration of hMSCs and their beneficial effects remains elusive. The authors seek to 1) transcriptionally characterize the role of microglia/macrophages after SCI; 2) identify the cellular localization of CCL2/CCR2 and CCL5/CCR5 chemokine systems; 3) assess the role of hMSCs on CCL2/CCR2 and CCL5/CCR5; and 4) validate the role of CCL5 administration on microglia/macrophages polarization. The authors suggest that hMSCs increase CCL2 and CCL5 expression, increase gene expression of axonal regenerative markers, and increase microglia/macrophage polarization toward the alternatively activated phenotype. Major Comments: • The main objective of the study needs to be clearly indicated and experiments need to coherently follow the main objective. How were the genes selected for the PCR experiment? Why choose CCL2/CCR2 and CCL5/CCR5? • The representation of expressional changes in Figure 1 needs to be revised. Line graphs or heatmaps would be more suitable for representing time-course transcriptional differences. How was the absolute PCR analysis performed? • The functional recovery assessment is singular (relies only on BMS scores) and is terminated at 14 days while the difference between the treatment is continuing to deviate. The authors need to provide a justification for not continuing their analyses to the chronic phase. • The authors need to indicate how their transcriptional work in Figure 1 advances the previous studies performing transcriptional analyses in SCI research. • It’s not clear to the reader why the authors refrained from administration of immunosuppressants or immunodeficient animals, particularly considering the low survival of hMSCs following transplantation. Minor Comments: • The authors need to justify their choice for using a transection model for studying immune cell migration. • The abstract needs to be reorganized and compressed. While the authors are informative and descriptive, they should succinctly describe their major findings and main inferences. • Line 25: “Central” should not be capitalized. ****** 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 [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. https://doi.org/10.1371/journal.pone.0230080.r001
Revision 1
2 Jan 2020 Author Response Thank you very much for the academic editor’s and reviewer’s comments to improve our manuscript of paper (PONE-D-19-28233). We read the comments well, and answered and revised according to your suggestions. We also removed all the term “data not shown” as journal requirements. Comments from editor 1. Provide evidence that hMSCs are present at the injection site 2. What is the survival rate and where are they located if they are not at the injury site Thank you very much for your comments. We thought that the comments 1 and 2 linked each other. So, we answered them together. Injected hMSCs were disappeared quickly from the injective site. However, it is known to rescue several diseases (Prockop DJ. Clin Pharmacol Ther. 2007; https://ascpt.onlinelibrary.wiley.com/doi/full/10.1038/sj.clpt.6100313). Also. in present study, we determined the fate of hMSCs using human GAPDH mRNA (hGAPDH) expression in the mouse spinal cord by real-time PCR (Fig. 1A). The estimate number of hMSCs was calculated by standard curve of the gene expression. Standard curves were generated by adding serial dilutions of hMSCs (1 × 102 to 1 × 106 cells) to spinal cord samples (T7 to T12 vertebrae) of uninjured mice prior to homogenization (see page 13, line 95~). We have also reported previously the retention of hMSCs by hAlu genomic DNA expression and the localization of PKH26-labeled hMSCs by immunohistochemistry in same animal model (Fig. 2 in Tsumuraya et al. J Neuroinflammation 2015; https://jneuroinflammation.biomedcentral.com/articles/10.1186/s12974-015-0252-5). We showed in the study that the injected hMSCs are seen to have migrated along the spinal cord toward the injury site at 7 days after SCI. The fate of hMSCs in the present study was shown in Fig. 1A. Immediately after hMSC injection (pod 1), approximately 260,000 hMSCs were detected (representing 50% of the injected cells) including the site of injection. The numbers of hMSCs decreased to approximately 40,000 cells on pod 3(15% of pod1), and have few-to-no hMSCs were detected on pod 14 (see page 21, line 10~). The reason, we choose hGAPDH, but not hAlu in present study is to eliminate a possibility of genomic DNA from hMSCs debris although the result was almost similar with previously. We have reported the fate of hMSCs in our other studies of brain ischemia (Ohtaki et al. Proc Natl Acad Sci U S A. 2008; https://www.pnas.org/content/105/38/14638.long) and STZ-induced diabetes (Murai et al. PLoS One 2017; https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186637). These studies also support in the present results. The question of “where are they located if they are not at the injury site” is very difficult to answer. In the present study, the cells as the hGAPDH level almost disappeared from the spinal cord on pod 14. The cells might eliminate by phagocytes and/or move to somewhere. We have examined the distribution of hMSCs in systemic organs by an in vivo fluorescence imager (Murai et al. PLoS One 2017). However, the detection of small amount of cells was extremely difficult. So, I am sorry we do not have accurate answer of this. 3. Why are there no dendrites on neurons? In present study, we used anti-Neu N antibody as a neuronal marker. Neu N is known to be stained neuronal nuclei and the peri-nucleic region (nerve cell body), but not or few in fibers. So the staining of neurons seems to round and triangle sometimes. Comments to reviewer Thank you very much for your supportive comments. We revised our manuscript (indicated yellow highlight in the text) and commented to you below. Major Comments: 1. The main objective of the study needs to be clearly indicated and experiments need to coherently follow the main objective. How were the genes selected for the PCR experiment? Why choose CCL2/CCR2 and CCL5/CCR5? In present study was designed according to our previous studies. We have focused on the communication between transplanted hMSCs and microglia/macrophages (MG/MΦ) on SCI. Therefore, we firstly selected mainly the MG/MΦ including monocyte-related chemokines and the receptors. Then, from expressing pattern we further focused on the CCL2/CCR2 and CCL5/CCR5. CCL5/CCR5 is a chemokine and receptors increased at chronic periods. Although many chemokines (receptors) increased at acute periods, we selected the most popular MG/MΦ-related chemokine CCL2/CCR2 axis. We think that we should examine the other chemokines in the future study. Although this is unpublished data, we are recognizing in another project that CCL2/CCR2 axis is an important signal to communicate hMSCs and recipient cells. Would you wait for wait a little more to reveal our next publication? 2. The representation of expressional changes in Figure 1 needs to be revised. Line graphs or heatmaps would be more suitable for representing time-course transcriptional differences. How was the absolute PCR analysis performed? Thank you very much. We revised bar to line graphs in Fig.1 (now, this is Fig.2). Moreover, we have not described the unit in the text including the legend and graphs clearly. They are relative level (fold) to compare with day 0. This mentioned in the methods section (see page 14 line 125~) and figure legend. As described below, we reconsidered the order of the figures from your comment #3. Fig.1 in original manuscript changed to Fig.2. 3. The functional recovery assessment is singular (relies only on BMS scores) and is terminated at 14 days while the difference between the treatment is continuing to deviate. The authors need to provide a justification for not continuing their analyses to the chronic phase. We have reported injection of hMSCs suppress neural injury through the macrophage or microglial modulation in the acute phase (within a week) after ischemia (Ohtaki et al. Proc Natl Acad Sci U S A. 2008). Moreover, the phenomenon has been reproduced after SCI as well (Tsumuraya et al. J Neuroinflammation 2015). Therefore, we decided the experiment periods based on the previous our studies and checked the reproducibility by BMS and gene expression of neuron specific enolase (Eno2) in the present study. However, we thought your comments also very reasonable if we showed figures in present order. We reconsidered again the order of the figures. We inserted Fig. 4 prior to Fig. 1 to be logically. Thank you for your supportive comments. 4. The authors need to indicate how their transcriptional work in Figure 1 advances the previous studies performing transcriptional analyses in SCI research. Yes. Some of SCI studies have reported to the gene expression of chemokines. However, we have not determined the expressions in this model so far. Therefore, we did them. 5. It’s not clear to the reader why the authors refrained from administration of immunosuppressants or immunodeficient animals, particularly considering the low survival of hMSCs following transplantation. Thank you for your comments. As your suggestion, many studies administer administration of immunosuppressants or use immunodeficient animals. However, to observe inflammation or immune systems, we were afraid that they might interfere to the systems. We have previously examined injection of hMSCs into brain parenchyma and compared the fate of hMSCs between immunodeficient and immunocompetent animals. Resulting, no significant differences were recognized (Ohtaki et al. Proc Natl Acad Sci U S A. 2008). Moreover, repetitive injections of hMSCs into immunocompetent animals did not show any anaphylactic reactions or die (Murai et al. PLoS One 2017). The following manuscript explain the reason to inject hMSCs into immunocompetent animals. We added this in our references. Prockop DJ, Oh JY, Lee RH. Data against a Common Assumption: Xenogeneic Mouse Models Can Be Used to Assay Suppression of Immunity by Human MSCs. Mol Ther. 2017 2;25(8):1748-1756. doi: 10.1016/j.ymthe.2017.06.004. Minor Comments: 6. The authors need to justify their choice for using a transection model for studying immune cell migration. To compare with contusion model, the transection model is localized the injury to a small region. Therefore, we thought to be clear cell-cell communications. Moreover, as the other reason, we continuously studied SCI using this model and there are some evidences for the model. 7. The abstract needs to be reorganized and compressed. While the authors are informative and descriptive, they should succinctly describe their major findings and main inferences. Thank you. We revised. 8. Line 25: “Central” should not be capitalized. Thank you. We revised. Attachments Attachment Submitted filename: Response to Reviewers (PONE-D-19-28233R1).docx https://doi.org/10.1371/journal.pone.0230080.r002
28 Jan 2020 Decision Letter - Xing-Ming Shi, Editor PONE-D-19-28233R1 The enhancement of CCL2 and CCL5 by human bone marrow-derived mesenchymal stem/stromal cells might contribute to inflammatory suppression and axonal extension after spinal cord injury. PLOS ONE Dear Dr Ohtaki, 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 (details below). We would appreciate receiving your revised manuscript by Mar 13 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, Xing-Ming Shi, Ph.D Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] 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) ******** 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: 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 manuscript is much improved. However, grammar mistakes and the writing style at the end of the introduction should be addressed. Additionally, the authors may want to mention the justification for the transection model and not administering immunosuppressants in the discussion. ****** 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 [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. https://doi.org/10.1371/journal.pone.0230080.r003
Revision 2
7 Feb 2020 Author Response Thank you very much for the academic editor’s and reviewer’s comments to improve our manuscript of paper (PONE-D-19-28233). We read the comments well, and revised according to your suggestions. Comments to reviewer 1. The manuscript is much improved. However, grammar mistakes and the writing style at the end of the introduction should be addressed. Additionally, the authors may want to mention the justification for the transection model and not administering immunosuppressants in the discussion. Thank you very much for your supportive comments. We revised our manuscript (indicated yellow highlight in the text) according to your comments. Moreover, we checked entire of manuscript again and slightly changed the order of references. Attachments Attachment Submitted filename: Response to Reviewers (PONE-D-19-28233R2).docx https://doi.org/10.1371/journal.pone.0230080.r004
21 Feb 2020 Decision Letter - Xing-Ming Shi, Editor The enhancement of CCL2 and CCL5 by human bone marrow-derived mesenchymal stem/stromal cells might contribute to inflammatory suppression and axonal extension after spinal cord injury. PONE-D-19-28233R2 Dear Dr. Ohtaki, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Xing-Ming Shi, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0230080.r005
Formally Accepted
25 Feb 2020 Acceptance Letter - Xing-Ming Shi, Editor PONE-D-19-28233R2 The enhancement of CCL2 and CCL5 by human bone marrow-derived mesenchymal stem/stromal cells might contribute to inflammatory suppression and axonal extension after spinal cord injury. Dear Dr. Ohtaki: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr Xing-Ming Shi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0230080.r006

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