Vitronectin-derived bioactive peptide prevents spondyloarthritis by modulating Th17/Treg imbalance in mice with curdlan-induced spondyloarthritis

Hong Ki Min; JeongWon Choi; Seon-Yeong Lee; A. Ram Lee; Byung-Moo Min; Mi-La Cho; Sung-Hwan Park

doi:10.1371/journal.pone.0262183

Peer Review History

Original SubmissionOctober 12, 2021
15 Nov 2021 Decision Letter - Yeonseok Chung, Editor PONE-D-21-32711Vitronectin-derived bioactive peptide prevents spondyloarthritis by modulating Th17/Treg imbalance in mice with curdlan-induced spondyloarthritisPLOS ONE Dear Dr. Park, 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. Two experts in T cell immunity have reviewed the manuscript and raised a number of concerns that need to be addressed in the revised manuscript. In particular, both reviewers suggested that the authors can improve the flow data in Fig 6 & 7. In addition, the authors are also encouraged to improve the description of their data and to expand discussion on the potential mechanisms. 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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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No 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: No 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: In this manuscript, Min et al. reported that vitronectin-derived peptide, VnP16, ameliorates spondyloarthritis (SpA) in SKG mice treated with curdlan. The authors firstly showed that either VnP16 or VnP16+celecoxb attenuated inflammation at ankle joints and spines of SpA-induced mice compared to that of vehicle-treated mice. Furthermore, VnP16 treatment in SpA-induced mice exhibited potent inhibitory effect on the production of IL-1β, IL-6, TNFα, and IL-17 in nucleus pulposus and cartilaginous. Fluorescence microscopic analyses of SpA-induced mice revealed the reduction of Th17/Treg ratio upon VnP16 treatment. Overall, this manuscript presented novel effects of VnP16 on Th17/Treg balance during SpA. Addressing the following minor points will strengthen the clarity of this study. Minor points 1. In the Legends, the authors should describe which mouse strain/chemical they used to induce SpA along with treatment schedule. 2. Regarding following statement in the Legend of Figure 1: “Data are means ± standard error of the mean (SEM) of three independent experiments.”, it is confusing if those data are pooled from 3 independent experiments or representative of 3 independent experiments. 3. To solidify their conclusion, authors should present more statistical analysis through the overall experimental groups, not just vehicle vs VnP16 or VnP16+celecoxib. 4. In the Legend of Figure 6, the authors appear to analyze splenocytes, not spleen tissue itself. 5. Flow cytometric analyses of Th17 and Treg cells in Figure 7 look ambiguous in terms of staining and gating. Since not all cellular responses in tissues are reconciled systemically, it would be better to exclude Figure 7 if the authors are unable to show more clear data on splenocytes. 6. On page 13, please provide a reference for the following statement: “Therefore, proper management at the acute inflammation stage (bone marrow edema) can prevent fat metaplasia and syndesmophyte formation in patients with SpA.” 7. The manuscript requires editing to conform to correct scientific English. Reviewer #2: In this manuscript, the authors investigated the therapeutic potential of Vitronectin-derived bioactive peptide in combination with anti-inflammatory drug celecoxib for the treatment of spondyloarthritis. The combination between the two drugs was more effective than each monotherapy for ameliorating the arthritis and spondylitis symptoms in an animal model of spondyloarthritis by diverting the balance between Th17 cells and Treg cells toward increased Treg/Th17 ratio. Here are some specific comments. 1. Flow cytometric analysis shown in Figure 6 should be improved or removed. Gated IL-17+ and CD25+Foxp3+ cell populations in the Figure might be cellular debris or noise. 2. Please discuss the potential mechanism for the additive effect between VnP-16 and celecoxib in the regulation of Th17/Treg regulation in SpA. 3. Please describe the data in the Figures in more detail in the Result section. 4. Please indicate the number of experimental repetitions in each figure. ****** 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 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.0262183.r001
Revision 1
9 Dec 2021 Author Response Revised submission of manuscript PONE-D-21-32711 Dear Pf. Yeonseok Chung Academic Editor, PLOS ONE We thank the reviewers for their constructive and helpful comments concerning the manuscript. We have addressed the reviewers' concerns by revising the manuscript or explaining respectfully our rebuttal. The point-by-point replies are given in this letter. We hope that we have addressed satisfactorily all concerns raised by the reviewers, and that this manuscript is now suitable for publication. Thank you again for the comments. Sincerely yours, Mi-La Cho, PhD Conversant Research Consortium in Immunologic disease, College of Medicine, The Catholic University of Korea, Seoul, 222 Banpo-Daero, Seocho-gu, Seoul 06591, Republic of Korea Tel: +82-2-2258-7467, Fax: +82-2-599-4287, E-mail: iammila@catholic.ac.kr Sung-Hwan Park, MD, PhD, Division of Rheumatology, Department of Internal Medicine, College of Medicine, Seoul St. Mary's Hospital, The Catholic University of Korea, 222 Banpo-Daero, Seocho-gu, Seoul 06591, Republic of Korea Tel: +82-2-2258-6011, Fax: +82-2-599-3589, E-mail address: rapark@catholic.ac.kr Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. 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 Answer : We followed the PLOS ONE's style requirement. 2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. Answer : We added the methods for sacrifice / anesthesia / effort to reduce suffering in method section (revised manuscript, line 146-150) “The clinical score was assessed under isoflurane inhalation anesthesia in every mouse, and every effort was made to minimize suffering. After the mice had been euthanized with 100% carbon dioxide within 5 min, in accordance with the use and care of animal guidelines of the Catholic University of Korea, the joint, spleen, and spinal tissues were collected.” 3. 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. Answer : We added raw flow cytometry and western blot data of Figure 6 and 7 as supplementary figure 1 and 2 (revised manuscript, line 260, 263). 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. Answer : We added raw blot / gel image as supplementary figures 1 &2 and, and mentioned it in cover letter. 4. 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. Answer : We mentioned the change in reference in rebuttal letter and marked by "track change” in “revised manuscript with track change” file. Reviewers' comments: Reviewer #1: In this manuscript, Min et al. reported that vitronectin-derived peptide, VnP16, ameliorates spondyloarthritis (SpA) in SKG mice treated with curdlan. The authors firstly showed that either VnP16 or VnP16+celecoxb attenuated inflammation at ankle joints and spines of SpA-induced mice compared to that of vehicle-treated mice. Furthermore, VnP16 treatment in SpA-induced mice exhibited potent inhibitory effect on the production of IL-1β, IL-6, TNFα, and IL-17 in nucleus pulposus and cartilaginous. Fluorescence microscopic analyses of SpA-induced mice revealed the reduction of Th17/Treg ratio upon VnP16 treatment. Overall, this manuscript presented novel effects of VnP16 on Th17/Treg balance during SpA. Addressing the following minor points will strengthen the clarity of this study. Minor points 1. In the Legends, the authors should describe which mouse strain/chemical they used to induce SpA along with treatment schedule. Answer: We have added information on the mouse strain and chemicals used to induce SpA, and the treatment schedule, to the figure legends (revised manuscript, lines 559–563). 2. Regarding following statement in the Legend of Figure 1: “Data are means ± standard error of the mean (SEM) of three independent experiments.”, it is confusing if those data are pooled from 3 independent experiments or representative of 3 independent experiments. Answer: We apologize for the typo. The clinical score and histological analysis were assessed by three independent experimenters blinded to the treatments. We calculated the mean values for the three experimenters. We have revised the figure legends (revised manuscript, lines 143–145, 568-569). 3. To solidify their conclusion, authors should present more statistical analysis through the overall experimental groups, not just vehicle vs VnP16 or VnP16+celecoxib. Answer: We agree. We have added a comparison of the groups to all figures and the revised Results section (revised manuscript, lines 228–229, 230–232, 241–242, 243–245, 247–249, and 255–258) 4. In the Legend of Figure 6, the authors appear to analyze splenocytes, not spleen tissue itself. Answer: We apologize for the typos. We have revised the legend of Figure 6 (line 597) 5. Flow cytometric analyses of Th17 and Treg cells in Figure 7 look ambiguous in terms of staining and gating. Since not all cellular responses in tissues are reconciled systemically, it would be better to exclude Figure 7 if the authors are unable to show more clear data on splenocytes. Answer: We have improved the flow cytometry image in Figure 6. 6. On page 13, please provide a reference for the following statement: “Therefore, proper management at the acute inflammation stage (bone marrow edema) can prevent fat metaplasia and syndesmophyte formation in patients with SpA.” Answer: We have cited a reference for this and have added reference number 42 (line 334). 7. The manuscript requires editing to conform to correct scientific English. Answer: We have had the manuscript proofread by English experts. We have added a certificate pertaining to the English editing. The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/pqmY6N Reviewer #2: In this manuscript, the authors investigated the therapeutic potential of Vitronectin-derived bioactive peptide in combination with anti-inflammatory drug celecoxib for the treatment of spondyloarthritis. The combination between the two drugs was more effective than each monotherapy for ameliorating the arthritis and spondylitis symptoms in an animal model of spondyloarthritis by diverting the balance between Th17 cells and Treg cells toward increased Treg/Th17 ratio. Here are some specific comments. 1. Flow cytometric analysis shown in Figure 6 should be improved or removed. Gated IL-17+ and CD25+Foxp3+ cell populations in the Figure might be cellular debris or noise. Answer: We have improved the flow cytometry image in Figure 6. 2. Please discuss the potential mechanism for the additive effect between VnP-16 and celecoxib in the regulation of Th17/Treg regulation in SpA. Answer: We agree. We have added a potential mechanism to the Discussion (revised manuscript, lines 289–296), and have also added references 28 to 31. 3. Please describe the data in the Figures in more detail in the Result section. Answer: We have added more detailed descriptions of the figures to the Results (revised manuscript, lines 228–229, 230–232, 241–242, 243–245, 247–249, and 255–258). 4. Please indicate the number of experimental repetitions in each figure. Answer: We have indicated the number of replicate experiments or total number of samples included in the analysis to each figure legend (revised manuscript, lines 563-564, 572–573, 581, 586, 592, 599, and 604). https://doi.org/10.1371/journal.pone.0262183.r002
19 Dec 2021 Decision Letter - Yeonseok Chung, Editor Vitronectin-derived bioactive peptide prevents spondyloarthritis by modulating Th17/Treg imbalance in mice with curdlan-induced spondyloarthritis PONE-D-21-32711R1 Dear Dr. Park, 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, Yeonseok Chung Academic Editor PLOS ONE Additional Editor Comments (optional): The authors adequately addressed all critiques raised by the reviewers Reviewers' comments: https://doi.org/10.1371/journal.pone.0262183.r003
Formally Accepted
24 Dec 2021 Acceptance Letter - Yeonseok Chung, Editor PONE-D-21-32711R1 Vitronectin-derived bioactive peptide prevents spondyloarthritis by modulating Th17/Treg imbalance in mice with curdlan-induced spondyloarthritis Dear Dr. Park: I'm 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 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 plosone@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 Yeonseok Chung Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0262183.r004

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