Revealing key regulators of neutrophil function during inflammation by re-analysing single-cell RNA-seq

Zhichao Ai

doi:10.1371/journal.pone.0276460

Peer Review History

Original SubmissionMay 11, 2022
3 Aug 2022 Decision Letter - You-Yang Zhao, Editor PONE-D-22-13750Revealing key regulators of neutrophil function during inflammation by re-analysing single-cell RNA-seqPLOS ONE Dear Dr. Ai, 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 a revised version of the manuscript that addresses the points raised during the review process. Especially, what is the new fidnings from this re-analysis, validate gene expression profiles with bulk RNAseq data, and discuss potential clinical significance of targeting IRF7 for treatment of COVID-19 and/or COVID-19 inflammation. Please submit your revised manuscript by Oct 31, 2022. 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. 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Please send your amended statements by return email; we will change the online submission form on your behalf. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The current manuscript (PONE-D-22-13750) re-analyzed scRNA data from GEO and focused on the transcription factors. Some TFs were dig out to involve in neutrophil development, mature, activation under disease conditions such as infection. There were some concerns about the manuscript. 1. For expression assay, usually scRNA-seq is not enough depth. Thus could you find RNA bulk-seq data to support or validate the current finding? 2. In the current analysis, UMAP and t-SNE were used. Can you explain the reasons that different methods were used? 3. In DEG, can you first calculate the mean expression of clusters and then do analysis because FINDMARKER function would pick up genes from major variable features? 4. More details how the data were integrated should be provided in the methods. For example, in method about scRNA-seq analysis, scRNA-seq data from BALF from patients was indicated from four GEO documents. This was wrong. 5. The details how to score in method about biological processes should be indicated. 6. What was the original findings of these scRNA-seq data? What is the difference from re-analysis? Reviewer #2: In this report, the author compared the transcriptional regulatory networks of neutrophils at different developmental stages, activation status and conditions of COVID19 infection. Activation of some TFs has been identified as a results of neutrophil activation, and IRF7 expression was abundant in neutrophils from patients with COVID19 infection. Overall, this report demonstrated few novel interesting findings with very little conceptual advance. The only interesting observation is that IRF7 expression is higher in COVID19 patients, however the author has not explored further how IRF7 signaling might be useful for the treatment of COVID19. ****** 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.0276460.r001
Revision 1
5 Sep 2022 Author Response Comments to the Author Reviewer #1: The current manuscript (PONE-D-22-13750) re-analyzed scRNA data from GEO and focused on the transcription factors. Some TFs were dig out to involve in neutrophil development, mature, activation under disease conditions such as infection. There were some concerns about the manuscript. 1. For expression assay, usually scRNA-seq is not enough depth. Thus, could you find RNA bulk-seq data to support or validate the current finding? We are grateful for the provided suggestion to re-analyze the published bulk-RNA-seq datasets to confirm the change of neutrophil specific transcription factors (TFs) during homeostasis and inflammation, which here we have now done. We utilized two independent published neutrophil RNA-seq datasets (Evrard et al. 2018, Immunity; Tariq. 2022, Nat. Immunol) to examine the expression of the TFs identified and validated in our current study, and other members of their families, during neutrophil maturation at homeostasis. The expression of Runx1 was consistently highest at the earliest stages of neutrophil differentiation, whereas the expression of Irf1, Irf7, Fos, Junb, Jund increased with neutrophil maturation (Rebuttal FigR1A), consistent with what we have observed in our current study. Additionally, enhanced expression of JunD, Fos, Elf2, Irf5, Irf7 were observed during neutrophil transition from the bone marrow, and to the inflammatory sites (air pouch membrane (MEM) and exudate (AP) (Rebuttal FigR1B). The data now are added into the manuscript as Supplementary Figure1. Therefore, we concluded that the list of TFs identified from our current analysis reflected chiefly on the transcriptional change of neutrophil maturation, activation under healthy and disease conditions. Rebuttal Figure R1. Transcriptional change of neutrophil subpopulations. (A) Heatmap showing row-scaled expression of the signature TFs in bulk RNA-seq analysis of neutrophil populations in the bone marrow and blood (Evrard et al. 2018, Immunity). () Heatmap showing the scaled expression of signature genes for each bone marrow neutrophil subpopulation from the bone marrow(BM), blood(BL), air pouch membrane(MEM) and exudate(AP), coloured by the average expression of each gene in each cluster scaled across all clusters (Tariq et al. 2022, Nature Immunology). 2. In the current analysis, UMAP and t-SNE were used. Can you explain the reasons that different methods were used? Many thanks for the provided comment, which leads to the better interpretation of our results. For UMAP, as the Figure1 demonstrated, UMAP-based trajectory analysis suggests the developmental relationship: (1) from Hematopoietic stem and progenitor cells ->, and (2) from retinal progenitor cells -> Early neutrophil precursors -> Immature and mature neutrophils cells. This branching closely captured the temporal order of cell types that arose along neutrophil generation and overall a more accurate presentation of neutrophil developmental trajectory. For t-SNE, it moves the high dimensional graph to a lower dimensional space points by points at a higher resolution. Both UMAP and t-SNE are non-linear, graph-based methods based on PCA analysis and used for dimensionality reduction. However, for the regulon network analysis executed by the SCENIC package (illustrated in Figure2&3), the tSNE-based visualization is the only available option to use in the package. 3. In DEG, can you first calculate the mean expression of clusters and then do analysis because FINDMARKER function would pick up genes from major variable features? We thank the review for providing the comment that help us improve our analysis. We are well aware that the function FINDMARKERS from the Seurat package for examining the differential expression between groups of cells with a focus on major variable features, which could result in biased results. Therefore, for the differential expression analysis (specifically, Figure4 B&C), we extracted the average counts with the function AverageExpression(from Seurat) for each cell cluster, before deriving the differentially expressed genes for gene ontology enrichment/other downstream analysis, in agreement with what has been suggested by the reviewer. 4. More details how the data were integrated should be provided in the methods. For example, in method about scRNA-seq analysis, scRNA-seq data from BALF from patients was indicated from four GEO documents. This is wrong. Very grateful for the reviewer for pointing out our mistake, which we have corrected in the method section and provided information that is more detailed on. 5. The details how to score in method about biological processes should be indicated. Many thanks for pointing it out, and it is now indicated in the method section. 6. What was the original findings of these scRNA-seq data? What is the difference from re-analysis? It is very good question for us especially when our current analysis stands on the shoulder of numerous giants (including but not limited to Evrad et al, Immunity 2018; Grassi, Cell Reports 2018; Adrover et al, Immunity 2019; Kwok et al, Immunity 2020; Xie et al, 2020; Ballesteros et al, Cell 2020), by whom several reference datasets have been generated. These datasets are of great importance to explore some potential regulators for neutrophil targeting therapy. The main discoveries of our current analysis are: 1, Integrated analysis of scRNA-seq of neutrophils under during homeostasis and inflammatory conditions supported distinct sets of putative transcription factors associated with control of neutrophil maturation an.d inflammatory responses. 2, Re-analysis of COVID-19 scRNA-seq datasets with a focus on the immunological feature of neutrophils found the correlation of neutrophil-specific IRF7 expression to the patient’s disease severity. 3, The current study highlights IRF7 as a key regulator for modulating neutrophil function without causing excessive tissue damage. Currently, some IRF5 inhibitors has been in the preclinical stage for ensuring appropriate inflammation during infection. Reviewer #2: In this report, the author compared the transcriptional regulatory networks of neutrophils at different developmental stages, activation status and conditions of COVID19 infection. Activation of some TFs has been identified as a result of neutrophil activation, and IRF7 expression was abundant in neutrophils from patients with COVID19 infection. Overall, this report demonstrated few novel interesting findings with very little conceptual advance. The only interesting observation is that IRF7 expression is higher in COVID19 patients; however, the author has not explored further how IRF7 signaling might be useful for the treatment of COVID19. Many thanks for your comments, and our current study utilizing a list of publicly available single cell RNA-seq datasets to be further explored in identifying novel therapeutic targets for treating neutrophil-related diseases. Our findings offer a response to the classical but unresolved question of “good or bad” neutrophil function regulators and identify IRF7 as a key regulator for modulating neutrophil inflammatory response. We propose IRF7 inhibition as a therapeutic strategy to protect against excessive tissue damage and bacterial infection, as evidenced by the company Pharma SelectImmune Pharma, which is developing a siRNA therapy to inhibit interferon regulatory factor 7 (IRF7) and thus inhibit inflammation in infected tissue, reducing the risk of sepsis and renal infections(detailed below). Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0276460.r002
7 Oct 2022 Decision Letter - You-Yang Zhao, Editor Revealing key regulators of neutrophil function during inflammation by re-analysing single-cell RNA-seq PONE-D-22-13750R1 Dear Dr. Ai, 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, You-Yang Zhao Academic Editor PLOS ONE Additional Editor Comments (optional): 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: All concerns were addressed properly. I think that the current MS is enough quality to publish on Plos One. Thus I suggested the MS should be acceptable. ****** 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: No ******** https://doi.org/10.1371/journal.pone.0276460.r003
Formally Accepted
13 Oct 2022 Acceptance Letter - You-Yang Zhao, Editor PONE-D-22-13750R1 Revealing key regulators of neutrophil function during inflammation by re-analysing single-cell RNA-seq Dear Dr. Ai: 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. You-Yang Zhao Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0276460.r004

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