Macrophage-P2X4 receptors pathway is essential to persistent inflammatory muscle hyperalgesia onset, and is prevented by physical exercise

Graciana de Azambuja; Fernando Moreira Simabuco; Maria Cláudia Gonçalves de Oliveira

doi:10.1371/journal.pone.0318107

Peer Review History

Original SubmissionAugust 25, 2024
25 Aug 2024 Author Response https://doi.org/10.1371/journal.pone.0318107.r001
15 Nov 2024 Decision Letter - Santosh Kumar Mishra, Editor PONE-D-24-35584Macrophage-P2X4 receptors pathway is essential to persistent inflammatory muscle hyperalgesia onset, and is prevented by physical exercisePLOS ONE Dear Dr. Oliveira, 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 Dec 30 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, Santosh Kumar Mishra Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. 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: São Paulo Research Foundation (FAPESP) – process number: 2018/13599-1; 2020/10585-0; 2021/02921-2 Coordination of Superior Level Staff Improvement (CAPES) – 001 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. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. 4. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files. 5. 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. 6. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 7. 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. [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: Yes 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: Yes 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: "The macrophage-P2X4 receptor pathway plays a crucial role in the onset of persistent inflammatory muscle hyperalgesia, which can be mitigated through physical exercise" - upon reviewing this manuscript, I offer the following observations: 1.The paper is composed with clarity and precision. 2.The proposed hypothesis demonstrates considerable strength. Kindly address the following points in the revised version: 1. - Elucidate the process for preparing the carrageenan solution and specify its concentration. 2. - Provide justification for the exclusive use of male mice in this research. 3. - Describe the nature of the mechanical stimulus applied at the injection site and its duration. 4. - Consider restructuring and enhancing the discussion section for improved clarity and impact. Reviewer #2: Minor comments: 1. Please provide a reference for the “previously standardized time” mentioned in the following statement: “For protein analysis of p38 MAPK…stimulated with carrageenan (100 mg/mL) for 30 minutes (previously standardized time)” 2. Please provide catalog numbers for antibodies. 3. List out Primer sets for all genes tested with SYBR green. These can be in a supplementary file/table. 4. Which outlier calculation tool was used? GraphPad has several options. 5. Does exogenous ATP (P2X4’s ligand) on Day 6 or 17 cause a significant change in behavior on top of the reported hyperalgesia result? 6. It is difficult to identify what the arrows in figure 3 are pointing at. Major comments: 1. There are no figure legends available. 2. The authors demonstrate that P2X4 causes an increase in p-p38 MAPK and that exercised mice have a decrease in p-P38 MAPK due to increased PPARγ activation. Is phosphorylation of p38 MAPK affected by 5-BDBD administration in the exercised mice, since P2X4 is proposed to also alter p-p38 MAPK levels? What about if ATP is administered in these mice? The link between the PPARγ and the rest of the paper about P2X4 is not clear. Are these independent pathways that affect p-p39 MAPK levels? Are they parts of the same cell signaling chain that produces musculoskeletal hyperalgesia? Does activation of P2X4 affect PPARγ expression or function? ****** 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: Yes: Debjeet Sur 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. https://doi.org/10.1371/journal.pone.0318107.r002
Revision 1
30 Dec 2024 Author Response # Response to academic editor’s comments: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. Manuscript was adjusted to meet PLOS ONE’s style requirements. 2. Thank you for stating the following financial disclosure: São Paulo Research Foundation (FAPESP) – process number: 2018/13599-1; 2020/10585-0; 2021/02921-2; Coordination of Superior Level Staff Improvement (CAPES) – 001. We state that "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." 3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. When trying to link the ORCID iD for the corresponding author we received a message stating that the ORCID iD already existed. Please, let us know whether this is a bug that we can fix it. 4. Please include your tables as part of your main manuscript and remove the individual files. Please note that supplementary tables (should remain/ be uploaded) as separate "supporting information" files. Tables were included in the main manuscript. 5. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Captions for main and supporting information files were included at the end of the manuscript. In-text citations were revised and corrected. 6. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. Uncropped and unadjusted original blot results for all assays are included as supporting information files with captions. 7. Please review your reference list to ensure that it is complete and correct. Reference list was revised and no changes were made. Citation style was adapted to Plos One format. # Response to reviewer’s comments: Reviewer #1: "The macrophage-P2X4 receptor pathway plays a crucial role in the onset of persistent inflammatory muscle hyperalgesia, which can be mitigated through physical exercise" - upon reviewing this manuscript, I offer the following observations: 1.The paper is composed with clarity and precision; 2.The proposed hypothesis demonstrates considerable strength. Thank you for your positive feedback regarding the clarity and precision of our manuscript and the strength of the proposed hypothesis. We deeply appreciate your encouraging remarks, which affirm the efforts we put into developing and presenting our work. Kindly address the following points in the revised version: 1. Elucidate the process for preparing the carrageenan solution and specify its concentration. The revised information has been updated and included in the revised manuscript under [Procedures for intramuscular injections, drugs, and doses, page 6]: Lambda Carrageenan was freshly prepared at a concentration of 1 mg/mL and subsequently diluted to a working solution of 5 µg/µL in 0.9% isotonic saline. 2. Provide justification for the exclusive use of male mice in this research. Thank you for raising this important point. The exclusive use of male mice in our research was based on the fact that both the exercise model and the hyperalgesia model were initially validated in male mice as part of our initial exploration. However, we recognize the importance of understanding sex-based differences. Our group is in the process of submitting a new paper that includes the validation of these approaches in female mice and explores differences in molecular mechanisms involved in exercise and muscle hyperalgesia. 3. Describe the nature of the mechanical stimulus applied at the injection site and its duration. The nature of the mechanical stimulus is pressure. Mechanical stimulation was conducted using an analgesimeter, which applies gradually increasing pressure to the injection site. The device's concave tip delivers mechanical stimuli that target deep tissues, including skeletal muscle and fascia. This is clarified in the revised manuscript under [Mechanical testing of muscle nociceptive threshold to assess the muscle hyperalgesia, page 6]. 4. Consider restructuring and enhancing the discussion section for improved clarity and impact. We have incorporated the comments from Reviewer #1 and Reviewer #2 and have made improvements to the manuscript's structure, particularly in the Discussion section. We believe these revisions enhance the clarity of the results interpretation, strengthen the presentation of new hypotheses and open questions, and ultimately improve the impact of the study. Reviewer #2: Minor comments: 1. Please provide a reference for the “previously standardized time” mentioned in the following statement: “For protein analysis of p38 MAPK…stimulated with carrageenan (100 mg/mL) for 30 minutes (previously standardized time)” An initial experiment was conducted in cells to determine the phosphorylation timing of the p38 MAPK protein upon stimulation with Carrageenan at a concentration of 100 mg/mL. This information has been included in the revised manuscript under and an additional figure has been provided as a supplementary information file ([Sup Fig. 3]). 2. Please provide catalog numbers for antibodies. This information is updated in the revised manuscript under [Procedure with primary culture of peritoneal macrophages, page 8-9; and Procedures with RAW 264.7 macrophage culture, page 11]. 3. List out Primer sets for all genes tested with SYBR green. These can be in a supplementary file/table. List of primer sets were added in a table in the manuscript under [Procedures with RAW 264.7 macrophage culture, page 10]. 4. Which outlier calculation tool was used? GraphPad has several options. Indeed, outliers were calculated using GraphPad Quick calc tool which performs the Grubbs' test. Significance level was set to 0.05. This information is updated in the manuscript under [Statistical analysis, page 13]. 5. Does exogenous ATP (P2X4’s ligand) on Day 6 or 17 cause a significant change in behavior on top of the reported hyperalgesia result? These experiments have not been performed in the current study. While outside the scope of this research, our group has previously tested the administration of a non-hydrolyzable form of ATP (α,β-meATP) in muscle and observed that it induces muscle hyperalgesia by activating P2X3 receptors, while P2X4 receptors were not evaluated in this study (Schiavuzzo et al., Neuroscience, 2015 Jan 29:285:24-33). Furthermore, prior studies have demonstrated that exogenous ATP can trigger muscle hyperalgesia and is involved in the molecular processes that lead to muscle hyperalgesia in rodent models. Previous research has shown that α,β-meATP or a metabolite cocktail (combining protons, lactate, and ATP) can induce skeletal muscle hyperalgesia in rodent models (Gregory et al., 2015, PLoS One. 2015 Sep 17;10(9):e0138576; Pollak et al., 2014, Exp Physiol. 99(2):368-80). Additionally, it we provide evidence that carrageenan-induced muscle hyperalgesia is mediated in part by P2X4 receptors, suggesting that there is an increase on ATP concentration. Based on this background, we anticipate that in the present model, administration of exogenous ATP would likely exacerbate the reported muscle hyperalgesia. 6. It is difficult to identify what the arrows in figure 3 are pointing at. Figure 3 was updated and we expect to be improved. We included an inset for each treatment condition, to highlight the expression of the cell markers. Updated version is found as figure 3. Major comments: 1. There are no figure legends available. We acknowledge that the figure legends were not positioned correctly in the original manuscript. In the revised version, the figure legends have been relocated to the end of the manuscript, following the reference list, in accordance with PLOS ONE’s manuscript formatting guidelines. 2. The authors demonstrate that P2X4 causes an increase in p-p38 MAPK and that exercised mice have a decrease in p-P38 MAPK due to increased PPARγ activation. Is phosphorylation of p38 MAPK affected by 5-BDBD administration in the exercised mice, since P2X4 is proposed to also alter p-p38 MAPK levels? What about if ATP is administered in these mice? The link between the PPARγ and the rest of the paper about P2X4 is not clear. Are these independent pathways that affect p-p39 MAPK levels? Are they parts of the same cell signaling chain that produces musculoskeletal hyperalgesia? Does activation of P2X4 affect PPARγ expression or function? Thank you for these insightful questions. We present a point-by-point response for discussion with the reviewer and when appropriate, we have addressed them in the revised manuscript at the discussion section, as follows: 1. Effect of 5-BDBD on p-p38 MAPK in exercised mice: Administration of 5-BDBD in exercised mice was not specifically analyzed for its effect on p-p38 MAPK levels in the current study. Based on the proposed pathway, we hypothesize that exercise pathways are inhibiting P2X4 and would prevent the phosphorylation of p38 MAPK. Injecting 5-BDBD in exercised mice could potentiate exercise effects on the P2X4 pathways. However, we did not proposed to test a synergistic effect of the antagonist and the exercise, thus this subject could become a topic for future experiments. 2. Effect of ATP administration on p-p38 MAPK in exercised mice: The effect of ATP administration on p-p38 MAPK levels in exercised mice was not directly assessed in our study. While we did not explore ATP levels in skeletal muscle tissue, either in sedentary or exercised mice, we hypothesize that ATP administration could enhance p-p38 MAPK phosphorylation, increase inflammatory responses, and exacerbate persistent skeletal muscle hyperalgesia in this model. Importantly, we speculate that exercised mice may have improved metabolic function and potentially greater resilience to inflammatory processes or the effects of inflammatory products like ATP. However, further studies are needed to confirm this hypothesis. 3. Clarifying the link between PPARγ and P2X4 in the context of p-p38 MAPK: We agree that the connection between PPARγ and P2X4 requires further clarification, and our study does not fully address this relationship. In the revised manuscript discussion section, we emphasize that PPARγ activation by exercise appears to mitigate p38 MAPK phosphorylation, potentially counteracting the effects of P2X4 activation. While both exercise-activated PPARγ and carrageenan-induced P2X4 converge on p-p38 MAPK levels, it remains unclear whether they function independently, repressing each other, or are part of a shared signaling cascade. We propose that skeletal muscle from exercised mice exhibits increased PPARγ activation, which may contribute to greater resilience to inflammatory insults. The underlying mechanisms require further investigation. Specifically, we hypothesize that exercise-induced PPARγ activation could either enhance the anti-inflammatory response in muscle tissue, reducing P2X4-p38 MAPK signaling in inflammatory macrophages, or directly/indirectly repress P2X4 activation in macrophages cells, thus preventing inflammation exacerbation. 4. Impact of P2X4 activation on PPARγ expression or function: To date, there is no direct evidence in our study, or others, that P2X4 activation affects PPARγ expression or function. This remains an open question and we may include this in future research to better understand the interplay between these pathways in musculoskeletal hyperalgesia. Attachments Attachment Submitted filename: PlosONE_Azambuja_response to reviewers.docx https://doi.org/10.1371/journal.pone.0318107.r003
10 Jan 2025 Decision Letter - Santosh Kumar Mishra, Editor Macrophage-P2X4 receptors pathway is essential to persistent inflammatory muscle hyperalgesia onset, and is prevented by physical exercise PONE-D-24-35584R1 Dear Dr. Gonçalves de Oliveira, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Santosh Kumar Mishra Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0318107.r004
Formally Accepted
Acceptance Letter - Santosh Mishra, Editor PONE-D-24-35584R1 PLOS ONE Dear Dr. Gonçalves de Oliveira, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, 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. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Santosh Kumar Mishra Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0318107.r005

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .