Inhibition of CCl4-induced liver inflammation and fibrosis by a NEU3 inhibitor

Darrell Pilling; Trevor C. Martinez; Richard H. Gomer

doi:10.1371/journal.pone.0308060

Peer Review History

Original SubmissionAugust 13, 2024
16 Sep 2024 Decision Letter - Matias A Avila, Editor PONE-D-24-29550Inhibition of CCl4-induced liver inflammation and fibrosis by a NEU3 inhibitorPLOS ONE Dear Dr. Gomer, 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 by the reviewers. Please submit your revised manuscript by Oct 31 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, Matias A Avila, Ph.D. Academic Editor PLOS ONE 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 2. We note that you have a patent relating to material pertinent to this article. Please provide an amended statement of Competing Interests to declare this patent (with details including name and number), along with any other relevant declarations relating to employment, consultancy, patents, products in development or modified products etc. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. This information should be included in your cover letter; we will change the online submission form on your behalf. 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. 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. [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: Partly 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: 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: This study was performed to demonstrate the effect of 2-acetyl pyridine (2AP), an inhibitor of neuraminidase 3 (NEU3), on the liver inflammation and fibrosis experimentally induced by CCl4. Results are of interest; however, some issues still need to addressed before further steps in the publication process. The manuscript is too long, it contains repeated information in the introductory paragraph of results section and in the description of discussion section, and authors cited 116 references. So, an original manuscript should be limited to no more than 50 to 60 references. This manuscript can easily be shortened by 30% if authors reorganize its content. Representative pictures of IHC analyses (Fig 3) of anti-S100A8 and anti-Mac2 should be included as evidence of where the quantifications came from. In this document is missing a strong conclusion at the end of the discussion section; instead, authors included a kind of incomplete conclusion at the beginning of discussion section: “In this report, we observed that 2AP attenuated CCl4-induced liver inflammation and fibrosis in both male and female mice. CCl4-induced liver desialylation and increased expression of NEU3 were also reversed by 2AP. The sera from patients with NAFLD and NASH also had increased levels of a desialylated serum protein and patients with NAFLD had increased serum levels of NEU3. These data suggest that increased NEU3 may be associated with liver inflammation and fibrosis and that a NEU3 inhibitor can, in part, reverse these effects”. The clinical relevance of 2AP on liver fibrosis should be clearly stated in the conclusion section of the manuscript. This manuscript contains some semantic and grammar issues that need to be carefully corrected; e.g., in results section, authors exaggerate to use “compare to”, “there was/were”, etc. Reviewer #2: This manuscript is well-written and describes a set of rigorously performed experiments demonstrating that the NEU3 inhibitor, 2AP, attenuates development of CCl4-induced liver inflammation and fibrosis. Experiments were conducted in both male and female mice and data were carefully analyzed in aggregate as well as separately. Appropriate controls were performed, including controls administering vehicle and inhibitor alone. NEU3 activity was also demonstrated in patient samples, increasing the clinical relevance of the paper’s findings. This reviewer has only minor additional comments: 1) Can the authors speculate on the significance of the acute reversible weight loss in mice immediately after each CCl4 injection? In addition, what may be the reason for the lack of acute weight loss in female mice at day 40 treated with 2AP? 2) For Figure 2, please clarify the total number of liver vessels that were evaluated in each sample that generated the reported numbers of vessels with inflammatory cells reported in the quantification graphs I-K. 3) Please explain why inhibitory activity of 2AP on CYP2E1 function was not directly tested. Are CYP2E1 functional assays not available and/or difficult to perform? ****** 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.0308060.r001
Revision 1
25 Sep 2024 Author Response Matias A Avila, Ph.D. Academic Editor PLOS ONE September 23, 2024 Dear Dr. Avila, Thank you for considering our manuscript PONE-D-24-29550 “Inhibition of CCl4-induced liver inflammation and fibrosis by a NEU3 inhibitor” for publication in PLOS ONE. As detailed below, we have carefully responded to the suggestions from the reviewers and have included additional images related to the analysis of MRP8/S100A8 positive cells in the liver (Figure S6) and the raw whole blot images related to the analysis of desialylation of proteins and levels of NEU3 in patient sera (Figure S10). As suggested by reviewer 1, we have also reduced the number of references in the manuscript and added a conclusion section. Following the comments from reviewer 2 we have clarified the methods section regarding assessment of vessel inflammation. We have also uploaded a revised marked-up copy of the manuscript with track changes, an unmarked revised manuscript, and provided an updated competing interests and financial disclosure sections. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. We confirm that the manuscript does conform to PLOS ONE style requirements. 2. We note that you have a patent relating to material pertinent to this article. Please provide an amended statement of Competing Interests to declare this patent (with details including name and number), along with any other relevant declarations relating to employment, consultancy, patents, products in development or modified products etc. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, as detailed online in our guide for authors by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. This information should be included in your cover letter; we will change the online submission form on your behalf. We confirm that all requirements related to financial and competing interest statements have been completed. 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. We confirm that the raw whole blot images related to the analysis of desialylation of proteins and levels of NEU3 in patient sera have been included in the manuscript (Figure S10). 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. 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. We confirm that Figures 1-8 conform to PACE specifications. Review Comments to the Author Reviewer #1: This study was performed to demonstrate the effect of 2-acetyl pyridine (2AP), an inhibitor of neuraminidase 3 (NEU3), on the liver inflammation and fibrosis experimentally induced by CCl4. Results are of interest; however, some issues still need to addressed before further steps in the publication process. The manuscript is too long, it contains repeated information in the introductory paragraph of results section and in the description of discussion section, and authors cited 116 references. So, an original manuscript should be limited to no more than 50 to 60 references. This manuscript can easily be shortened by 30% if authors reorganize its content. We have reduced the number of references by 20 and have shortened the manuscript as much as possible. However, we respectfully disagree that we should reduce our reference list to 50-60. If we reduced the number of references to 60 then all references related to the methods section would have to be removed, and the number of references related to previous work and interpretation of the results would be scant. In addition, the lack of references would make it appear that we do not appreciate previous work in this area of research and it would reduce our ability to interpret our findings with regard to previously published works. This same reasoning is why we did not reduce the manuscript length by 30%. One of the major benefits of publishing in PLOS ONE is that the journal allows space to provide a detailed method section (with relevant references) and allows for a careful explanation of the results and a detailed discussion. Representative pictures of IHC analyses (Fig 3) of anti-S100A8 and anti-Mac2 should be included as evidence of where the quantifications came from. We have now added an additional figure (Figure S6) showing anti-S100A8 staining. For anti-Mac2 staining, we believe that Figure 4 (Fig 4. 2AP injections reduce CCl4 -induced increases in Mac2 positive cells) and Figure S6 (S6 Fig. Mac2 staining in liver parenchyma), (now S7 Fig in the revised manuscript) provide representative images of anti-Mac2 staining. In this document is missing a strong conclusion at the end of the discussion section; instead, authors included a kind of incomplete conclusion at the beginning of discussion section: “In this report, we observed that 2AP attenuated CCl4-induced liver inflammation and fibrosis in both male and female mice. CCl4-induced liver desialylation and increased expression of NEU3 were also reversed by 2AP. The sera from patients with NAFLD and NASH also had increased levels of a desialylated serum protein and patients with NAFLD had increased serum levels of NEU3. These data suggest that increased NEU3 may be associated with liver inflammation and fibrosis and that a NEU3 inhibitor can, in part, reverse these effects”. The clinical relevance of 2AP on liver fibrosis should be clearly stated in the conclusion section of the manuscript. We have now added a conclusion section to the manuscript discussing possible clinical relevance of 2AP on liver fibrosis (lines 562-568 of the revised manuscript with track changes); thank you for this suggestion! This manuscript contains some semantic and grammar issues that need to be carefully corrected; e.g., in results section, authors exaggerate to use “compare to”, “there was/were”, etc. We respectfully disagree with reviewer 1 and believe that our use of the terms indicated above are appropriate. Reviewer #2: This manuscript is well-written and describes a set of rigorously performed experiments demonstrating that the NEU3 inhibitor, 2AP, attenuates development of CCl4-induced liver inflammation and fibrosis. Experiments were conducted in both male and female mice and data were carefully analyzed in aggregate as well as separately. Appropriate controls were performed, including controls administering vehicle and inhibitor alone. NEU3 activity was also demonstrated in patient samples, increasing the clinical relevance of the paper’s findings. This reviewer has only minor additional comments: 1) Can the authors speculate on the significance of the acute reversible weight loss in mice immediately after each CCl4 injection? In addition, what may be the reason for the lack of acute weight loss in female mice at day 40 treated with 2AP? We also find this result interesting. We struggled to find papers where daily weight measurements after CCl4 injections in mice were reported. We did find some papers (Crit Rev Toxicol. 2003;33(2):105-36., Biotechnology & Biotechnological Equipment. 2015;29(6):1164-8., and Toxicology & Applied Pharmacology. 1984;75(1):1-7) which show that in rats there is weight loss at day 1 after CCl4 injections, and that in humans CCl4 exposure leads to cachexia and weight loss. We added to lines 282-283 “This acute reversible weight loss may be due to a general toxicity of CCl4.” The lack of weight loss in the female mice at day 40 may indicate that long-term dosing of 2AP may be hepato-protective, but these data require more extensive experimentation, which is beyond the scope of the current manuscript. Thus, we did not attempt to interpret these data in this manuscript. 2) For Figure 2, please clarify the total number of liver vessels that were evaluated in each sample that generated the reported numbers of vessels with inflammatory cells reported in the quantification graphs I-K. We clarified and expanded the methods section to indicate how the counts were performed and have also included text in the figure legend for Figure 2. We analyzed at least 100 vessels per section for the accumulation of immune cells. This is now in the methods section as lines 241-244 of the revised manuscript with track changes, and in the legend for Figure 2 as lines 348-349 of the revised manuscript with track changes. 3) Please explain why inhibitory activity of 2AP on CYP2E1 function was not directly tested. Are CYP2E1 functional assays not available and/or difficult to perform? Although several companies sell CYP2E1 ELISA kits to measure CYP2E1 protein levels, CYP2E1 functional assays are technically very difficult to perform, and CYP2E1 functional assays are only available as part of drug screening panels from commercial CRO’s. Attachments Attachment Submitted filename: 2024-PONE-D-24-29550 Pilling etal-response to reviewers.docx https://doi.org/10.1371/journal.pone.0308060.r002
27 Sep 2024 Decision Letter - Matias A Avila, Editor Inhibition of CCl4-induced liver inflammation and fibrosis by a NEU3 inhibitor PONE-D-24-29550R1 Dear Dr. Gomer, 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, Matias A Avila, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0308060.r003
Formally Accepted
4 Oct 2024 Acceptance Letter - Matias A Avila, Editor PONE-D-24-29550R1 PLOS ONE Dear Dr. Gomer, 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 Matias A Avila Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0308060.r004

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 .

Inhibition of CCl₄-induced liver inflammation and fibrosis by a NEU3 inhibitor

Inhibition of CCl₄-induced liver inflammation and fibrosis by a NEU3 inhibitor

Peer Review History

Open letter on the publication of peer review reports