SARS-CoV-2 infection induces mixed M1/M2 phenotype in circulating monocytes and alterations in both dendritic cell and monocyte subsets

Sanja Matic; Suzana Popovic; Predrag Djurdjevic; Danijela Todorovic; Natasa Djordjevic; Zeljko Mijailovic; Predrag Sazdanovic; Dragan Milovanovic; Dejana Ruzic Zecevic; Marina Petrovic; Maja Sazdanovic; Nenad Zornic; Vladimir Vukicevic; Ivana Petrovic; Snezana Matic; Marina Karic Vukicevic; Dejan Baskic

doi:10.1371/journal.pone.0241097

Peer Review History

Original SubmissionOctober 6, 2020
10 Nov 2020 Decision Letter - Aftab A. Ansari, Editor PONE-D-20-31462 SARS-CoV-2 infection induces mixed M1/M2 phenotype in circulating monocytes and alterations in both dendritic cell and monocyte subsets PLOS ONE Dear Dr. Popovic, 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. First of all, I must apologize for the delay in getting a response back to you that was mediated by a failure to receive a response from one of the reviewers. As you may read in the critiques provided by the 2 reviewers, both of them have some concerns hat appear to a large extent technical in nature. Please pay close attention to the suggested changes recommended by the 2 reviewers and address each of these in your revised submission. Please submit your revised manuscript by Dec 25 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Aftab A. Ansari, PhD 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. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary). [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: No ******** 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: No ****** 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: Sanja et al. present an observational study which characterizes changes in circulating immune cells in COVID-19 patients based on severity of disease. This adds to a growing body of literature in this area. The article is well-written, and the methodology appears rigorous where it is sufficiently described. I had several concerns, as follows (in approximately the order they appear in the manuscript): (1) References to preprints should be updated where they have been subsequently published. For example, references 5, 10, 12, etc. (2) Please specify anticoagulant(s) used in blood collection. (3) How were subjects selected for flow cytometry analysis, given that this is only a subset of the total patient sample. (4) Was blood collection standardized by time point or other means (e.g., days from hospitalization or symptom onset)? Observational studies such as these have a limitation in that they may be measuring a kinetic response rather than a severity response, if this is not controlled. (5) It would be useful to have Table 1 information split by severity. For example, were patients with more severe disease older? (6) Please correct D-dimmer to D-dimer. There are several instances of this. (7) Monocyte phenotyping in COVID-19 has yielded vastly different results for different groups, with some showing increased CD16+ subsets, and some (as here) showing decreases. Trafficking to the lungs is one possible explanation, but it would be worthwhile to explore the differences between this study and studies showing divergent results in the discussion. (8) CD23 and CD38 results in monocytes require much more thorough citation and expanded discussion. M1 and M2 phenotypes are not generally used as descriptors for monocytes (rather for macrophages), and these markers are not well-characterized in circulating monocytes. Additionally, since all CD23+ monocytes also expressed CD38, this suggests these markers are not necessarily reflecting the phenotype suggested in the paper. (9) Please clearly label axes in the figures. Some are hard to interpret. Reviewer #2: General Comments: In this manuscript, Sanja et al. described the immunological events in the blood associated with severity of SARS-CoV-2 infection particularly with a focus on innate immunity responses. They indicated that severity of the disease correlated with the decrease in numbers of T and B lymphocytes, DCs, NK cells and HLA-DR expressing cells. Moreover, they determined that the monocyte population was disturbed for expression of M1 and M2 monocyte markers in the intermediate and non-classical subsets. Finally, they concluded that both innate and adoptive immune responses are heavily affected in patients with severe disease but that inmate immunity is preserved in patients with milder symptoms per only slight decreases in the lymphocyte population. A concern, unfortunately, was that the data presentation and flow cytometry analyses should be improved to satisfactorily support the conclusions drawn from this study. Specific Comments: 1. The figure legends should be described and placed outside of the result section. 2. The results section should be described using specific subtitles that indicate the main massage for each section of the study presented. 3. The control data described in S1 Table should be included in Table 2 of the main text. In addition to the % of granulocytes, lymphocytes and monocytes, the absolute numbers of cells/ml would contribute to more informative results. 4. As also suggested for Table 2, the addition of the absolute numbers of cells/ml would be more informative for data presented in Table 3. 5. The entire data presentation and analyses described in Figure 1 should be re-done. None of the figures from panels A-E are informative. For each modified figure, please indicate the take-home message or conclusion for each. To better represent the phenotype changes of NK cells related to severity of disease, the percentage of CD57+ NK cells should be used instead of % of NK cell subsets among leukocytes (F). 6. Figures 2 and 3 should also be modified as recommended for Figure 1. 7. The overlayed figures in Figure 5 and 6 (A-C) are not informative and very difficult to interpret. To better represent the phenotype change of different subsets of monocytes with severity of disease, the percentages of CD38+, CD23+ and CD23/38+ cells should be used for each cell subset. 8. Supplement Figures 1 and 2 should also be modified to clearly indicate the gating strategy of each of the different cell subsets. ****** 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.0241097.r001
Revision 1
15 Dec 2020 Author Response Reviewer #1: Sanja et al. present an observational study which characterizes changes in circulating immune cells in COVID-19 patients based on severity of disease. This adds to a growing body of literature in this area. The article is well-written, and the methodology appears rigorous where it is sufficiently described. I had several concerns, as follows (in approximately the order they appear in the manuscript): 1. References to preprints should be updated where they have been subsequently published. For example, references 5, 10, 12, etc. Re: Among reprint references, references 10 is corrected due to recent publishing. Preprint references 5, 11, 12, 14, 15, 18 and 21 are not published yet on 8th December 2020 at 20:00h. 2. Please specify anticoagulant(s) used in blood collection. Re: Anticoagualnt used in blood collection is specified in Patients and method section Flow cytometry (EDTA). 3. How were subjects selected for flow cytometry analysis, given that this is only a subset of the total patient sample. Re: Patients were selected for flow cytometry analysis by creating a randomized list in Microsoft Excel for both groups of patients (SD and MD). The first ten patients from both groups were included in the analysis. The sample size was based on a study of a similar design (Gupta R, Gant VA, Williams B, Enver T. Increased complement receptor-3 levels in monocytes and granulocytes distinguish COVID-19 patients with pneumonia from those with mild symptoms. International Journal of Infectious Diseases. 2020;99:381-5.). 4. Was blood collection standardized by time point or other means (e.g., days from hospitalization or symptom onset)? Observational studies such as these have a limitation in that they may be measuring a kinetic response rather than a severity response, if this is not controlled. Re: We have stated in manuscript, section Patients and methods, subsection Data sampling that blood samples were collected at admission. To be more precisely, we preformulated in present manuscript that blood speciments were collected on the first day of hospitalisation. Thank you for the suggestion. 5. It would be useful to have Table 1 information split by severity. For example, were patients with more severe disease older? Re: Table 1 informations are splited by severity. According to changed concept of Table 1, text of manuscript in section Results, subsection Baseline Characteristics of COVID-19 Patients was adapted. 6. Please correct D-dimmer to D-dimer. There are several instances of this. Re: D-dimmer is corrected to D-dimer throughout the manuscript. 7. Monocyte phenotyping in COVID-19 has yielded vastly different results for different groups, with some showing increased CD16+ subsets, and some (as here) showing decreases. Trafficking to the lungs is one possible explanation, but it would be worthwhile to explore the differences between this study and studies showing divergent results in the discussion. Re: The differences between this study and studies showing divergent results are expored and interpreted in discussion on pages 19 and 20. 8. CD23 and CD38 results in monocytes require much more thorough citation and expanded discussion. M1 and M2 phenotypes are not generally used as descriptors for monocytes (rather for macrophages), and these markers are not well-characterized in circulating monocytes. Additionally, since all CD23+ monocytes also expressed CD38, this suggests these markers are not necessarily reflecting the phenotype suggested in the paper. Re: Short description of CD38 and CD23 molecules and the supporting references are now included in section Discussion (next to the last paragraph). Regarding M1 and M2 monocytes, in the same paragraph we included a statement: “A concept of M1 and M2, pro- and anti-inflammatory monocytes, mirroring M1/M2 macrophage polarization, was introduced by Fukui et al. Ever since, this concept was accepted by other researchers.” with supporting references. In order to introduce more clearly the presence of mixed populations of monocytes Figure 6 is revised in form of more informative bar charts that present the percentages of cells expressing CD38+, CD23+ and CD23+CD38+ in each cell subset in controls, MD and SD patients. Our conclusion about the presence of monocytes with mixed M1/M2 phenotype in COVID-19 patients now can be more clearly observed. 9. Please clearly label axes in the figures. Some are hard to interpret. Re: In revised Figures all axes are clearly labeled. Reviewer #2: General Comments: In this manuscript, Sanja et al. described the immunological events in the blood associated with severity of SARS-CoV-2 infection particularly with a focus on innate immunity responses. They indicated that severity of the disease correlated with the decrease in numbers of T and B lymphocytes, DCs, NK cells and HLA-DR expressing cells. Moreover, they determined that the monocyte population was disturbed for expression of M1 and M2 monocyte markers in the intermediate and non-classical subsets. Finally, they concluded that both innate and adoptive immune responses are heavily affected in patients with severe disease but that inmate immunity is preserved in patients with milder symptoms per only slight decreases in the lymphocyte population. A concern, unfortunately, was that the data presentation and flow cytometry analyses should be improved to satisfactorily support the conclusions drawn from this study. Specific Comments: 1. The figure legends should be described and placed outside of the result section. Re: Figure legends are removed from the main text and placed at the end of the manuscript. 2. The results section should be described using specific subtitles that indicate the main massage for each section of the study presented. Re: Section Results is divided into subsections by subtitles that describe the main findings of presented set of data. 3. The control data described in S1 Table should be included in Table 2 of the main text. In addition to the % of granulocytes, lymphocytes and monocytes, the absolute numbers of cells/ml would contribute to more informative results. Re: Control data are included in Table 2. Absolute numbers (cells/ml) of granulocytes, lymphocytes and monocytes are included in Table 2 and S1 Table. 4. As also suggested for Table 2, the addition of the absolute numbers of cells/ml would be more informative for data presented in Table 3. Re: Absolute numbers (cells/ml) of peripheral blood leukocytes are included in Table 3. Due to the large format of the revised Table 3, layout of page 11 in manuscript is changed from portrait to landscape. 5.The entire data presentation and analyses described in Figure 1 should be re-done. None of the figures from panels A-E are informative. For each modified figure, please indicate the take-home message or conclusion for each. To better represent the phenotype changes of NK cells related to severity of disease, the percentage of CD57+ NK cells should be used instead of % of NK cell subsets among leukocytes (F). Re: Take-home message is now indicated in Figure’s captions. Figure 1 is revised. Overlaid histograms and contour plots are replaced with representative pseudocolor plots presenting gating strategy and analysis of overall and activated NK cells in controls, MD and SD patients separately. To better present the phenotype changes of NK cells related to severity of disease, on bar chart CD57+ and CD57- cells are shown inside the bars presenting percent of overall NK cell population, therefore percent of these cells can be observed both within leukocyte and NK cell populations. 6. Figures 2 and 3 should also be modified as recommended for Figure 1. Re: Figures 2 and 3 have been revised following the example of Figure 1. Overlaid histograms and contour plots are replaced with representative pseudocolor plots presenting gating strategy and analysis of overall HLADR+ population in Figure 2, as well as overall DCs, pDCs, mDCs and activated DCs in Figure 3, in controls, MD and SD patients separately. On bar charts in Figure 2 percent of HLADR+ and HLADR- populations are shown inside the bars presenting percent of overall CD14+ and CD19+ population. In Figure 3, separate pDCs, mDCs and activated DCs are presented in perspestive of overall DCs on bar charts. 7. The overlayed figures in Figure 5 and 6 (A-C) are not informative and very difficult to interpret. To better represent the phenotype change of different subsets of monocytes with severity of disease, the percentages of CD38+, CD23+ and CD23/38+ cells should be used for each cell subset. Re: In Figure 5 overlaid contour plots are replaced with representative pseudocolor plots separately for controls, MD and SD patients. The percentages of CD38+, CD23+ and CD23+CD38+ cells in each cell subset are presented by bar charts in Figure 6. 8. Supplement Figures 1 and 2 should also be modified to clearly indicate the gating strategy of each of the different cell subsets. Re: In Suppl. Figure 1 gating strategy (A) and CD4+ and CD8+ T lymphocytes (B and C) are presented by representative pseudocolor plots separately for controls, MD and SD patients. Gating strategy for monocytes and monocyte subsets is presented step-by-step in Suppl. Figure 2 and described in figure legend. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0241097.r002
17 Dec 2020 Decision Letter - Aftab A. Ansari, Editor SARS-CoV-2 infection induces mixed M1/M2 phenotype in circulating monocytes and alterations in both dendritic cell and monocyte subsets PONE-D-20-31462R1 Dear Dr. Popovic, 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, Aftab A. Ansari, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0241097.r003
Formally Accepted
21 Dec 2020 Acceptance Letter - Aftab A. Ansari, Editor PONE-D-20-31462R1 SARS-CoV-2 infection induces mixed M1/M2 phenotype in circulating monocytes and alterations in both dendritic cell and monocyte subsets Dear Dr. Popovic: 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. Aftab A. Ansari Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0241097.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 .