Fecal bacteria transplantation replicates aerobic exercise to reshape the gut microbiota in mice to inhibit high-fat diet-induced atherosclerosis

Jie Men; Hao Li; Chenglong Cui; Xuedi Ma; Penghong Liu; Zhengyang Yu; Xueyan Gong; Youhao Yao; Jieying Ren; Chengrui Zhao; Binyu Song; Kaijiang Yin; Jianting Wu; Wei Liu

doi:10.1371/journal.pone.0314698

Peer Review History

Original SubmissionJuly 17, 2024
28 Aug 2024 Decision Letter - Udai P. Singh, Editor PONE-D-24-29677Fecal bacteria transplantation replicates aerobic exercise to reshape the gut microbiota in mice to inhibit high-fat diet-induced atherosclerosisPLOS ONE Dear Dr. Li, 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. Major: 1. Authors are requested to reanalyze the statistical significance test for figure1e, figure 3b, c. Authors are suggested to prepare all the bar graphs with individual data points for better understanding. Authors are also necessary to check that data has been represented either mean ± standard error or mean ± standard deviation as they claimed mean ± standard error. 2. Authors have mentioned in the material and methods section that they used Student's t-test and one-way ANOVA; otherwise, the Kruskal-Wallis. Authors are recommended to mention the tests used in Figure 1 a, e; Figure 2 and Figure 3 b, c with a number of samples/repeats (n =) in figure legends. Minor: 1. Authors are suggested to add the significance test result in Figure 1a as described in result 3.1 section. 2. Authors are recommended to add the lipids’ names and LPS above the graphs of Figure 2 for easy visualization for readers. 3. Authors are suggested to correct the Excel data (supplementary) for the Ace and Shannon index which is written oppositely. 4. Authors are recommended to correct the appropriate reference mentioned as [16] in the Introduction. ============================== Please submit your revised manuscript by Oct 12 2024 11:59PM. 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Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Additional Editor Comments: Major: 1. Authors are requested to reanalyze the statistical significance test for figure 1e, figure 3b, c. Authors are suggested to prepare all the bar graphs with individual data points for better understanding. Authors are also necessary to check that data has been represented either mean ± standard error or mean ± standard deviation as they claimed mean ± standard error. 2. Authors have mentioned in the material and methods section that they used Student's t-test and one-way ANOVA; otherwise, the Kruskal-Wallis. Authors are recommended to mention the tests used in figure 1 a, e; figure 2 and figure 3 b, c with number of samples/repeats (n =) in figure legends. Minor: 1. Authors are suggested to add the significance test result in Figure 1a as described in result 3.1 section. 2. Authors are recommended to add the lipids’ names and LPS above the graphs of Figure 2 to easy visualization for readers. 3. Authors are suggested to correct the Excel data (supplementary) for Ace and Shannon index which is written oppositely. 4. Authors are recommended to correct the appropriate reference mentioned as [16] in the Introduction. [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: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: 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 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 study designed to focus on aerobic exercise influenced fecal microbiota transplantation (FMT) to reestablish the gut microbiota in mice to inhibit high-fat diet-induced atherosclerosis. They claimed that this technique is an innovative therapeutic strategy for clinical treatment of atherosclerosis. The manuscript is well written and easy to understand. However, several points need to be improved. Please, see the comments below. Major: 1. Authors are requested to reanalyze the statistical significance test for figure1e, figure 3b, c. Authors are suggested to prepare all the bar graphs with individual data points for better understanding. Authors are also necessary to check that data has been represented either mean ± standard error or mean ± standard deviation as they claimed mean ± standard error. 2. Authors have mentioned in material and methods section that they used Student's t-test and one-way ANOVA; otherwise, the Kruskal-Wallis. Authors are recommended to mention the tests used in figure 1 a, e; figure 2 and figure 3 b, c with number of samples/repeats (n =) in figure legends. Minor: 1. Authors are suggested to add the significance test result in figure 1a as described result 3.1 section. 2. Authors are recommended to add the lipids’ name and LPS above the graphs of figure 2 to easy visualization for readers. 3. Authors are suggested to correct the excel data (supplementary) for Ace and Shannon index which is written oppositely. 4. Authors are recommended to correct the appropriate reference mentioned as [16] in Introduction. Reviewer #2: The manuscript entitled “Fecal bacteria transplantation replicates aerobic exercise to reshape the gut microbiota in mice to inhibit high-fat diet-induced atherosclerosis” sounds very interesting topic, however reviewer feels it need to modify little bit before published online. # Line 33, 209, 241, 303 and 343: Unknown figure legends with different front sizes. Need do necessary Changes. # Manuscript is pasted twice that need to be corrected and kindly use sample spacing format throughout the manuscript. # Author must include any prebiotics, probiotics, and any standard weight loss medicine to compare the results, without that there is no positive control. Without that it is not rational to evaluate the study ****** 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: Mousumi Mandal Reviewer #2: Yes: Amarinder Singh ******** [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". 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Revision 1
24 Oct 2024 Author Response Response to Reviewers Dear Editorial Board and Reviewers of PLOS ONE, Hello! We sincerely appreciate your time in reviewing our manuscript and providing valuable feedback on our paper titled "Fecal bacteria transplantation replicates aerobic exercise to reshape the gut microbiota in mice to inhibit high-fat diet-induced atherosclerosis" (Manuscript Number: PONE-D-24-29677R1). Your insightful comments have greatly contributed to improving the current version, and we are truly grateful for the opportunity to revise our submission. The authors have carefully considered your suggestions and have provided detailed responses addressing the raised issues. We hope that the revised manuscript, with all modifications highlighted in red, meets your standards. It is our sincere hope that you will consider accepting this revised paper for publication in PLOS ONE. Please find our responses to your comments below: Response to Reviewer 1 Comment #1: Authors are requested to reanalyze the statistical significance test for figure1e, figure 3b, c. Authors are suggested to prepare all the bar graphs with individual data points for better understanding. Authors are also necessary to check that data has been represented either mean ± standard error or mean ± standard deviation as they claimed mean ± standard error. Response from the Authors: Based on your valuable guidance and suggestions, our team has conducted an in-depth and meticulous review of the key data analyses in the manuscript, specifically addressing the statistical significance tests for the data presented in Figures 1e, 3b, and 3c. To ensure the accuracy and scientific rigor of the research results, we not only rechecked all related statistical processes but also conducted multiple verifications for potentially affected data sections, including a thorough review of the information presented in Figure 2. During this process, we utilized GraphPad Prism 9.5 software tool - an industry-leading software - to comprehensively process, analyze, and graphically represent all relevant data. Ultimately, we have decided to express our data using the "mean ± standard deviation" format in Section 2.8 (Statistical Analysis) of the manuscript. This approach not only demonstrates our dedication to scientific rigor but also improves clarity in presenting research findings for peer reviewers and readers' accessibility. We are confident that these enhancements will further elevate both quality and credibility of this manuscript while appreciating your professional feedback that contributed towards refining and improving our work. Comment #2: Authors have mentioned in material and methods section that they used Student's t-test and one-way ANOVA; otherwise, the Kruskal-Wallis. Authors are recommended to mention the tests used in figure 1 a, e; figure 2 and figure 3 b, c with number of samples/repeats (n =) in figure legends. Response from the Authors: We greatly appreciate your suggestion regarding the lack of detailed annotations of the specific statistical analysis methods in the experimental data figures. We sincerely apologize for this oversight in the initial draft and have taken immediate steps to correct and improve this aspect. After thorough research and careful consideration, we confirm the following: Figure 1a was analyzed using Student's t-test; Figure 2 was analyzed using one-way ANOVA; and Figures 1e, 3b, and 3c were analyzed using the Kruskal-Wallis rank sum test, which is more appropriate for non-normally distributed data. Additionally, to enhance transparency and readability, we have precisely listed the sample sizes for each group in their respective figure legends, and we have also clearly indicated the specific statistical analysis methods used under each figure. These adjustments aim to ensure that our research report is more rigorous and standardized while demonstrating our commitment to academic integrity and high-quality scientific outcomes. Once again, we thank you for your insightful suggestions that undoubtedly helped us move towards higher standards. Comment #3: Authors are suggested to add the significance test result in figure 1a as described result 3.1 section. Response from the Authors: We sincerely appreciate and value your insightful suggestions, which have prompted us to carefully optimize Figure 1a in order to enhance its clarity and readability. Specifically, we have now marked the time point at week 11 in the figure, clearly indicating that this marks the official start of fecal microbiota transplantation (FMT) intervention for the F group mice at the end of week 11. To further emphasize the statistical significance of the changing trend, we have added at week 12 based on detailed significance test results and * between weeks 13 and 19 to reflect significant differences in body weight data between AS and F groups over time. These subtle yet meaningful adjustments not only enrich the information presented in the figure but also offer readers a more precise perspective to observe experiment progression and key findings. Once again, we appreciate your thoughtful guidance that has enlightened our path towards continuously refining our research and striving for the highest standards in every outcome. Comment #4: Authors are recommended to add the lipids’ name and LPS above the graphs of figure 2 to easy visualization for readers. Response from the Authors: We have labeled the corresponding lipid names or LPS above each legend in Figure 2 to improve its readability. Comment #5: Authors are suggested to correct the excel data (supplementary) for Ace and Shannon index which is written oppositely. Response from the Authors: We sincerely apologize for misplacing the Ace and Shannon index data in Excel, which has now been corrected in the revised version. We greatly appreciate your scrutiny, and we will be more vigilant to prevent such oversights in the future, ensuring the maintenance of our research quality. Comment #6: Authors are recommended to correct the appropriate reference mentioned as [16] in Introduction. Response from the Authors: We sincerely appreciate the profound insights you have provided. After carefully reviewing the references, we have made necessary changes in the revised version of the manuscript. Specifically, in the Introduction section, we have updated the reference content in [16] to: “Considering the contraindications and risks of exercise, there are obstacles to promoting exercise therapy for AS among the elderly population [1]; Fecal microbiota transplantation (FMT) offers a novel approach for treating this condition.” Once again, we deeply appreciate your keen observation. Your feedback serves as an indispensable source of motivation for our continuous pursuit of excellence. 1. Aengevaeren VL, Mosterd A, Sharma S, Prakken NHJ, Möhlenkamp S, Thompson PD, et al. Exercise and Coronary Atherosclerosis: Observations, Explanations, Relevance, and Clinical Management. Circulation. 2020;141(16):1338-50. Epub 2020/04/21. doi: 10.1161/circulationaha.119.044467. PubMed PMID: 32310695; PubMed Central PMCID: PMCPMC7176353. Response to Reviewer 2 Comment #1: Line 33, 209, 241, 303 and 343: Unknown figure legends with different front sizes. Need do necessary Changes. Response from the Authors: We greatly appreciate your meticulous feedback. In accordance with PLOS ONE guidelines, we have completely removed the blanks in #33, 209, 241, 303, and 343 from the revised manuscript. We sincerely apologize and thank you for bringing this to our attention. Comment #2: Manuscript is pasted twice that need to be corrected and kindly use sample spacing format throughout the manuscript. Response from the Authors: Your insightful suggestions have enabled us to reflect and improve. I am pleased to inform you that we have thoroughly reviewed the revised manuscript in accordance with PLOS ONE's stylistic standards, making necessary adjustments to ensure full compliance with the journal's requirements. We are committed to presenting a more rigorous and professional version of our work. Your attention and guidance serve as the driving force behind our progress, for which we extend our deepest respect and gratitude. Comment #3: Author must include any prebiotics, probiotics, and any standard weight loss medicine to compare the results, without that there is no positive control. Without that it is not rational to evaluate the study Response from the Authors: We deeply appreciate your professional insights and support for our research. Your suggestion to include a positive control group containing prebiotics, probiotics, and standardized weight management drugs has greatly enriched the scope of our study and reflects a forward-thinking approach. However, conducting such supplementary experiments at this stage presents significant technical and methodological challenges due to the following reasons: The primary challenge arises from concerns over sample stability due to the long time span of the project. This research has been ongoing for two years, and although we have employed low-temperature storage (-80°C) to preserve the integrity of the mouse fecal samples, previous studies indicate that long-term freezing may lead to a decline in α-diversity, likely due to microbial DNA degradation[2]. Consequently, restarting the animal model would not only require replicating the initial conditions accurately to ensure consistency for the newly added control group but also addressing uncertainties posed by variations in probiotic product efficacy, individual responses, and other external variables which could complicate comparability of experimental results. Additionally, although an increasing body of evidence emphasizes that dysbiosis of gut microbiota (GM) is a potential contributor to atherosclerosis (AS), our understanding of its exact pathophysiological mechanisms remains limited [3]. While current research is gradually uncovering the functional interactions between GM and the host, the specific bacterial strains that should be introduced and the minimum effective dose required for intervention have not yet been clearly established[4]. A review of past studies[5, 6] shows that although changes in the microbiome and metabolites following FMT in AS model mice have demonstrated potential benefits, further systematic investigation is needed to comprehensively understand how these microbial components regulate the progression of AS as well as their distinct biological activities. Without a clear understanding of these underlying mechanisms, expanding the scope of FMT treatment could lead to unpredictable and difficult-to-interpret results. Once again, we express our deep gratitude for your valuable feedback, which has significantly enhanced our study. We are committed to continuing our focus on investigating the molecular mechanisms through which exercise-mediated improvements in GM contribute to inhibiting AS. Our aim is to identify key bacterial strains and incorporate your suggestions into a more rigorous and refined experimental design. We firmly believe that by adhering to a rigorous, evidence-based scientific approach, we can unravel the complexities of biological phenomena and ultimately uncover the underlying causes of diseases, thereby contributing to human health and well-being. 2. Carroll IM, Ringel-Kulka T, Siddle JP, Klaenhammer TR, Ringel Y. Characterization of the fecal microbiota using high-throughput sequencing reveals a stable microbial community during storage. PLoS One. 2012;7(10):e46953. Epub 2012/10/17. doi: 10.1371/journal.pone.0046953. PubMed PMID: 23071673; PubMed Central PMCID: PMCPMC3465312. 3. Kim ES, Yoon BH, Lee SM, Choi M, Kim EH, Lee BW, et al. Fecal microbiota transplantation ameliorates atherosclerosis in mice with C1q/TNF-related protein 9 genetic deficiency. Exp Mol Med. 2022;54(2):103-14. Epub 2022/02/05. doi: 10.1038/s12276-022-00728-w. PubMed PMID: 35115674; PubMed Central PMCID: PMCPMC8894390. 4. de Groot PF, Frissen MN, de Clercq NC, Nieuwdorp M. Fecal microbiota transplantation in metabolic syndrome: History, present and future. Gut Microbes. 2017;8(3):253-67. Epub 2017/06/14. doi: 10.1080/19490976.2017.1293224. PubMed PMID: 28609252; PubMed Central PMCID: PMCPMC5479392. 5. Weingarden AR, Vaughn BP. Intestinal microbiota, fecal microbiota transplantation, and inflammatory bowel disease. Gut Microbes. 2017;8(3):238-52. Epub 2017/06/14. doi: 10.1080/19490976.2017.1290757. PubMed PMID: 28609251; PubMed Central PMCID: PMCPMC5479396. 6. Parker A, Romano S, Ansorge R, Aboelnour A, Le Gall G, Savva GM, et al. Fecal microbiota transfer between young and aged mice reverses hallmarks of the aging gut, eye, and brain. Microbiome. 2022;10(1):68. Epub 2022/05/04. doi: 10.1186/s40168-022-01243-w. PubMed PMID: 35501923; PubMed Central PMCID: PMCPMC9063061. We sincerely appreciate the valuable feedback provided by the esteemed editorial board and reviewers on our manuscript. We eagerly anticipate your response Thank you and best regards! Yours sincerely, Jie Men Corresponding author: Name: Jie Men E-mail: menjie2020@126.com Attachments Attachment Submitted filename: Response to Reviewers.pdf https://doi.org/10.1371/journal.pone.0314698.r002
15 Nov 2024 Decision Letter - Udai P. Singh, Editor Fecal bacteria transplantation replicates aerobic exercise to reshape the gut microbiota in mice to inhibit high-fat diet-induced atherosclerosis PONE-D-24-29677R1 Dear Dr. LI, 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. 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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: All comments have been addressed ******** 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 entitled “Fecal bacteria transplantation replicates aerobic exercise to reshape the gut microbiota in mice to inhibit high-fat diet-induced atherosclerosis” focused on aerobic exercise influenced fecal microbiota transplantation (FMT) to reestablish the gut microbiota in mice to inhibit high-fat diet-induced atherosclerosis. The MS has been written well and easy to understand for the readers. The authors critically evaluated the FMT from aerobic exercise group of mice ameliorates high-fat diet-induced atherosclerosis. Authors have addressed carefully all the comments and suggestions of the reviewers in the revised version of MS. They also address the technical limitations for adding positive control in current stage. The work is promising for future therapeutics for atherosclerosis. According to my opinion, this manuscript is now suitable to accept for the publication. ****** 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: Yes: Mousumi Mandal ******** https://doi.org/10.1371/journal.pone.0314698.r003
Formally Accepted
29 Nov 2024 Acceptance Letter - Udai P. Singh, Editor PONE-D-24-29677R1 PLOS ONE Dear Dr. Li, 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. Udai P. Singh Section Editor PLOS ONE https://doi.org/10.1371/journal.pone.0314698.r004

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