Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells

Ann-Kathrin Vlacil; Evelyn Vollmeister; Wilhelm Bertrams; Florian Schoesser; Raghav Oberoi; Jutta Schuett; Harald Schuett; Sonja Huehn; Katrin Bedenbender; Bernd T. Schmeck; Bernhard Schieffer; Karsten Grote

doi:10.1371/journal.pone.0228764

Peer Review History

Original SubmissionJanuary 22, 2020
19 Feb 2020 Decision Letter - Bernard Mari, Editor PONE-D-20-01994 Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells PLOS ONE Dear Dr. Grote, Thank you for submitting your manuscript for review to PLoS ONE. I have received to date one completed review (for a total of 6 reviewers contacted) and could not get a second one. I have personally evaluated your article and I totally agree with the comments raised by the reviewer. After reading the reviews and looking at the manuscript, we feel that your study has merit, but is not suitable for publication as it currently stands. Therefore, my decision is "Major Revision”. You must revise accordingly and explain your revisions in a covering letter if you wish for us to consider your paper further for publication. Note that it will have to go through another round of review. We invite you to submit a revised version of the manuscript that addresses the concerns raised by the reviewer. Please pay attention to all the reviewer suggestions and give them due consideration. Specifically: You should clearly respond to the three major concerns raised by the Reviewer. In particular, the first major point about miR-147 and/or miR-298 function(s) appears essential and need additional experiments. Moreover, I have also personally raised one additional point: while the authors claim that all data are available, they did not provide the full dataset of miRNA TLDA assay in a specific supplemental table (or a link to a public database). We would appreciate receiving your revised manuscript by Apr 04 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Bernard Mari, Ph.D Academic Editor PLOS ONE Journal Requirements: 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 1. Thank you for including your ethics statement; "Human Subject Research - local ethic regulations of Philipps University Marburg - AZ 20/16 - written Animal Research - all experiments were approved by the governmental animal ethics committee and are conform to the guidelines from directive 2010/63/EU of the European Parliament. - #50/2015 - for cell isolation, mice at the age of six to ten weeks were sacrificed by cervical dislocation" With regards to your human ethics statement; Please amend your current ethics statement to confirm that your named institutional review board or ethics committee specifically approved this study. Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”). For additional information about PLOS ONE ethical requirements for human subjects research, please refer to http://journals.plos.org/plosone/s/submission-guidelines#loc-human-subjects-research. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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: Please finds enclosed my comments on the manuscript entitled “Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells” by Vlacil et al. This article describes a link between NOD1/2-regulated microRNAs (miRs) and NOD1/NOD2-dependent inflamatory response (IL6/TNF) in pulmonary endothelial cells. The authors found that concomitantly to IL6 and TNF regulation by NODs stimulation miR-147 and miR-298 are transiently downregulated by NODs stimulation. Therefore, using gain of function assay they suggest that these miRs can be involved in the transient induction of IL6 and TNF by NODs stimulation. Finaly the authors try to emphasize the specificity of their mechanisms (NOD-dependent mechanism) The manuscript is well written and is interesting in some regards as it adress for the first time the miRNA regulation by the NOD pathway in pulmonary endothelial cells. The first part of the manuscript (identification of NOD-dependent miRNA regulation) is well designed and executed with several controls, including NODs KO cells. However, the second part of the manuscript that aim to decipher the mechanism by which miR-147 and miR-298 participate to the NOD signaling pathway remains poorly address Therefore several concerns need to be address before is publication. Major: 1) The authors state that the expression level of miR-147 and miR-298 is low (CT=33), therefore, they only use overexpression experiments to demonstrate the role of these miRNA in the regulation of IL6 and TNF. However the authors also claim that the downregulation of miR-147 and miR-298 participate to the upregulation of IL6 and TNF. Therefore the authors should perform loss of function experiments to demonstrate that the downregulation of miR-147 and/or miR-298 participate to the control of TNF/IL6 expression (mRNA and protein). Alternatively the authors could also try to perform experiment in which they overexpressed miR-298 in conbination with target blocking site(s) oligos 2) The authors should cleary state that the mechanism by which miR-147 participates to the control of IL6/TNF remains enigmatic 3) The authors should remove "In silico analysis" from the abstarct as no in silico analysis were performed. At least for the reviewer, looking at putative binding sites in the 3'UTR of two transcripts do not account for in silico analysis. Minor: 1) In Figure 2 downregulated miRNA are in red while up-regulated miRNAs are in blue. It is confusing as in general up-regulated genes/miRNA are represented in red while downregulated genes are represented in blue. 2) In the text the authors should cleary state that miR-147 is mmu-miR-147 which correspond to hsa-miR-147b. It is important, as the existence of hsa-miR-147a (miR-147) remains debate 3) The authors should acknoledge previous work on mmu-miR-147/hsa-miR-147b and the inflamatory responses. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0228764.r001
Revision 1
17 Mar 2020 Author Response Response to Reviewers We highly appreciate the valued suggestions and criticism of the reviewer and the editor in order to strengthen the impact of our manuscript. Please find below a detailed response to all comments. Please note that we have added some replicates to single experiments in the course of the revision which leads to minor changes in bar graphs and significant values. Reviewer #1: Reviewer: The authors state that the expression level of miR-147 and miR-298 is low (CT=33), therefore, they only use overexpression experiments to demonstrate the role of these miRNA in the regulation of IL6 and TNF. However, the authors also claim that the downregulation of miR-147 and miR-298 participate to the upregulation of IL6 and TNF. Therefore, the authors should perform loss of function experiments to demonstrate that the downregulation of miR-147 and/or miR-298 participate to the control of TNF/IL6 expression (mRNA and protein). Alternatively, the authors could also try to perform experiment in which they overexpressed miR-298 in combination with target blocking site(s) oligos. Authors: We used different methods, i.e. TaqMan low-density array, transfection of mimics and luciferase reporter assays to identify NOD-dependently expressed miRNAs to investigate for Tnf-α and Il-6 mRNA regulation in murine pulmonary endothelial cells. Finally, we could confirm miR-298-5p from a couple of initially identified miRNAs by all methods and a direct physical interaction in case of Il-6. We have repeatedly stated this issue in the manuscript. Following the remarks of the reviewer, we consequently focused on miR-298-5p and performed the suggested loss of function experiments using antagomirs for miR-298-5p and were able to further confirm our findings (result section on page 16/17 and novel Figure 4E and 4F and supplemental Figure S2C). Reviewer: The authors should clearly state that the mechanism by which miR-147 participates to the control of IL6/TNF remains enigmatic Authors: We explicitly stated now that we could not finally elucidate if miR-147-3p is involved in Tnf-α and Il-6 regulation in pulmonary endothelial cells in this context (result section on page 16 and discussion section on page 20). Reviewer: The authors should remove "In silico analysis" from the abstract as no in silico analysis were performed. At least for the reviewer, looking at putative binding sites in the 3'UTR of two transcripts do not account for in silico analysis. Authors: We have deleted the term "In silico analysis" from the abstract, method and result sections. Reviewer: In Figure 2 downregulated miRNA are in red while up-regulated miRNAs are in blue. It is confusing as in general up-regulated genes/miRNA are represented in red while downregulated genes are represented in blue. Authors: We have changed the colors in the heatmap in Figure 2A according to the reviewers´ suggestion. Reviewer: In the text the authors should clearly state that miR-147 is mmu-miR-147 which correspond to hsa-miR-147b. It is important, as the existence of hsa-miR-147a (miR-147) remains debate. Authors: We have added this information to the result section on page 17. Reviewer: The authors should acknowledge previous work on mmu-miR-147/hsa-miR-147b and the inflammatory responses. Authors: Since we investigated both NOD-dependently down-regulated candidates from the initial array – miR-147 and miR-298 – we added additional previous work on inflammatory responses for both miRNAs (ref# 30-32) to the discussion section on page 20. Editor: Editor: You should clearly respond to the three major concerns raised by the Reviewer. In particular, the first major point about miR-147 and/or miR-298 function(s) appears essential and need additional experiments. Authors: We addressed all concerns raised by the reviewer including additional loss of function experiments on miRNA function, please see above. Editor: Moreover, I have also personally raised one additional point: while the authors claim that all data are available, they did not provide the full dataset of miRNA TLDA assay in a specific supplemental table (or a link to a public database). Authors: We now provided the full dataset in the GEO public database (GSE145798) at NCBI and included this information in the method section on page 10 and in the result section on page 13. At the moment, results are deposited as ‘private data’. In case of acceptance, we will set the status to ‘public data’. The reviewer may use: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE145798 and token: atofyckibhmnnwt to view the full dataset. Journal Requirements: Please amend your current ethics statement to confirm that your named institutional review board or ethics committee specifically approved this study. Authors: We have amended our ethics statement on page 6 in the method section as requested. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0228764.r002
11 Apr 2020 Decision Letter - Bernard Mari, Editor PONE-D-20-01994R1 Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells PLOS ONE Dear Dr. Grote, Thank you for resubmitting your manuscript for review to PLoS ONE. While you have adequately addressed the queries in the review and that the revised manuscript is significantly improved from its original submission, we feel that your manuscript still needs a few improvements. Therefore, my decision is "Minor Revision. As requested by the reviewer, the authors should replace the term antagomiR by anti-miRNA and should include the manufacturer of their anti-miRNA. We would appreciate receiving your revised manuscript by May 26 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Bernard Mari, Ph.D Academic Editor PLOS ONE [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: 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 authors did a good job. I only have a minor comment. The authors should replace the term antagomiR by anti-miRNA, as antagomiR refer to a specific class of miR inhibitors (coupled to cholesterol allowing miRNA delivery without adding transfection readgent) In addition, the authors should include the manufacturer of their anti-miRNA ****** 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: Thomas BERTERO [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0228764.r003
Revision 2
14 Apr 2020 Author Response Response to Reviewers Reviewer #1: Reviewer: The authors should replace the term antagomiR by anti-miRNA, as antagomiR refer to a specific class of miR inhibitors (coupled to cholesterol allowing miRNA delivery without adding transfection reagent). In addition, the authors should include the manufacturer of their anti-miRNA. Authors: We have replaced the term antagomir by anti-miRNA throughout the manuscript and included the manufacturer (Dharmacon as for mimics) on page 9 of the material and method section. Attachments Attachment Submitted filename: Response to Reviewers (R2).docx https://doi.org/10.1371/journal.pone.0228764.r004
17 Apr 2020 Decision Letter - Bernard Mari, Editor Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells PONE-D-20-01994R2 Dear Dr. Grote, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Bernard Mari, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0228764.r005
Formally Accepted
21 Apr 2020 Acceptance Letter - Bernard Mari, Editor PONE-D-20-01994R2 Identification of microRNAs involved in NOD-dependent induction of pro-inflammatory genes in pulmonary endothelial cells Dear Dr. Grote: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Bernard Mari Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0228764.r006

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