miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasis

Jaqueline Poleto Bragato; Gabriela Torres Rebech; Jéssica Henrique de Freitas; Marilene Oliveira dos Santos; Sidnei Ferro Costa; Flavia de Rezende Eugênio; Paulo Sérgio Patto dos Santos; Valéria Marçal Felix de Lima

doi:10.1371/journal.pone.0265192

Peer Review History

Original SubmissionJuly 5, 2021
3 Sep 2021 Decision Letter - Paulo Lee Ho, Editor PONE-D-21-21939 miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasis PLOS ONE Dear Dr. de Lima, 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. 1) The authors should provide experiments and controls for the transfection efficacy of the micro RNA to be sure that the effect was due to the action or not of the transfected micro RNA; 2) Please, see the comments raised by the both reviewers. Please submit your revised manuscript by Oct 16 2021 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. 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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: Partly ******** 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: 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 paper entitles “miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasis” by de Lima et al., submitted for consideration in PLOS ONE journal (PONE-D-21-21939), shows interesting results in naturally Leishmania infected dogs regarding immune regulation by miRNA 21. However, some critical aspects should be addressed, particularly concerning the limitation of the work. The paper was easy to read but needed some improvement in the discussion section. Please see my comments below. Table S1, please add this table as main table 1. Figure 1: Please indicate in the Y-axis “fold change of miRNA 21”. How the fold change of miRNA 21 expression was calculated is not completely clear. Would you please clarify this information in the figure legend? Figure 2: Please indicate reference description in Figure 2 for blue and red histograms. In the result section, please correct the title of the figure, as “Expression of miR-21 targets gene in splenic leukocytes of CanL and healthy dogs ”. Same comment for figure legend 2 Figure 3: Please indicate in Figure legend 3 title, transfection of what is referring to. In the result section, indicate the reason for using miRNA mimics and inhibitors. Have you been waiting for opposite results? Figure 4: Please also include data of CD4 and CD8 in Figure 4. Discussion section. Even though your results showed no changes in the regulation of FAS, FASL, and CCR7 by miRNA 21, these results could be related to low transfection efficacy, not adequate concentration range of mimic or inhibitor, among others. Did you try different concentrations? Please discuss these aspects very carefully and include them as limitations of the work This sentence is unclear; please revise “FAS and FASL play critical roles in the immune system, particularly in the death of target cells infected by pathogens and autoreactive lymphocytes' death. Same comment as above regarding the effect of miRNA 21 mimic and inhibitor on CD4 and CD8. Did you perform a doses resp Reviewer #2: Dr. Bragato and colleagues have submitted a manuscript entitled: "miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasis." The study explored the role of miR-21 in dogs with CanL comparing the expression of miR-21 targets in samples from infected and non-infected dogs. Additionally, they performed an in vitro experiment with cells transfected with miR-21 mimics and inhibitors. The authors found higher expression of miR-21 in infected dogs and a decrease expression in miR-21 targets in this population. In vitro inhibition of miR-21 decreased CD69 expression in B lymphocytes and IL-10 in supernatants. The authors concluded miR-21 may be a target for CanL. Line 101: The authors stated that “Splenic fragments in control dogs were removed by surgical excision [25]”. I wish to understand if the Animal Ethics Committee approved the collection of splenic fragments from healthy animals. If yes, could you please clarify in which circumstances? Please, provide the Animal Ethics certificate. Also, it is unclear why reference 25 was used. Line 105: Please, explain how the fragments were macerated. Line 110: Please provide the description or the commercial reference for the red blood cell lysis buffer Line 136: Confirm if the DNA was extracted from cells or directly from tissue Line 157: The authors stated “To confirm that miR-21 is upregulated in dogs with CanL obtained by [11],” Please, explain why reference 11 was used. Line 169: A pool of which cDNAs was used for normalization - control, infected, or both? Please explain where the absolute quantification of miR-21 was used. Line 173: Please, insert the reference or complete description for SNORD96A Results Figure 1 legend: Please, insert the number of animals (samples) analyzed. Figure 2 legend: Please, detail which range is referring, min-max, SD? I suggest presenting the control group first in the graph (same order as in figure 1). Figures 4 and 5 legends: Please, detail which range is referring, min-max, SD? I am concerned about the validation of the transfection. The authors did not show a control condition for the experiment (a condition with the transfection reagent only, without the scrambled sequence). Did you perform a pilot experiment with i.e., cells treated with transfection reagent only? Also, it is mandatory to validate the expression of miR-21 after transfection. How do the authors know the transfection efficiency relative to miR-21 inhibition? Is it possible to run an experiment to evaluate the miRNA expression after transfection? Do the authors have frozen samples? Gene expression should be similar in both untransfected cells and cells transfected with the negative control. Comment, please. Besides that, the average CD69 expression in the control group was around 40000 (figure 2). When the cells of this group were transfected with the scrambled sequence, the average expression was around 75000 (figure 3). It seems that the scrambled condition is increasing the expression. At the same time, the inhibitor condition is achieving an expression around 40000, similar to the control group expression in figure 2. Results from the negative control should be compared to results from untransfected cells. It seems that adequate controls are missing. Comment, please. The authors found significantly less expression of CD69 in control and canL groups when miR-21 was mimic on the other side the inhibition did not produce the opposite effect. Comment, please. Discussion: Line 334-335: Maybe the low transfection rate of this study does not support the previous sentence “suggesting that, in dogs, these proteins may not be targets of miR-21.” Please, comment. ****** 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.0265192.r001
Revision 1
30 Sep 2021 Author Response Response to Reviewers Manuscript No.: PONE-D-21-21939 Title: miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasis Dear Editor, We appreciate the careful review of our manuscript by the reviewers. Their constructive comments and suggestions are much appreciated, and the new version of the manuscript has benefited considerably from their assistance Please note that all changes made in the text are highlighted in red. Editor Comment: The authors should provide experiments and controls for the transfection efficacy of the micro RNA to be sure that the effect was due to the action or not of the transfected micro RNA; R: The experiment that represents transfection efficacy was added to supporting information;. Reviewer Comments: Author’s answer to Reviewer Comments: We thank the referee for providing constructive comments to improve the article. All criticisms and detailed corrections have been addressed and the following revisions have been made. We have accepted all the suggestions and requests made by the Editor and Reviewers in the manuscript. Sincerely yours, Valéria Marçal Felix de Lima, MSc, PhD São Paulo State University – UNESP Reviewer #1 Author’s answer: We thank the referee for providing constructive comments to improve the article. All criticisms and detailed corrections have been addressed and the following revisions have been made. Reviewer #1’s comments Answers Table S1, please add this table as main table 1 R: Table 1 was put as main table as recommended. Figure 1: Please indicate in the Y-axis “fold change of miRNA 21”. How the fold change of miRNA 21 expression was calculated is not completely clear. Would you please clarify this information in the figure legend? R: The required information was added to the figure legend. Figure 2: Please indicate reference description in Figure 2 for blue and red histograms. In the result section, please correct the title of the figure, as “Expression of miR-21 targets gene in splenic leukocytes of CanL and healthy dogs”. Same comment for figure legend 2 R: The information was modified in the text. Figure 3: Please indicate in Figure legend 3 title, transfection of what is referring to. In the result section, indicate the reason for using miRNA mimics and inhibitors. Have you been waiting for opposite results? R: The legend of the figure was modified. We used both mimics and inhibitor because increased and decreased expression could facilitate the understanding of how miR-21 does the regulation. Figure 4: Please also include data of CD4 and CD8 in Figure 4. R: The figure was added. Discussion section. Even though your results showed no changes in the regulation of FAS, FASL, and CCR7 by miRNA 21, these results could be related to low transfection efficacy, not adequate concentration range of mimic or inhibitor, among others. Did you try different concentrations? Please discuss these aspects very carefully and include them as limitations of the work. R: We tested all the different concentrations recommended by the manufacturer in order to optimize the transfection rate, and used the concentrations that gave the best results. This sentence is unclear; please revise “FAS and FASL play critical roles in the immune system, particularly in the death of target cells infected by pathogens and autoreactive lymphocytes' death. R: This sentence was reformulated. Same comment as above regarding the effect of miRNA 21 mimic and inhibitor on CD4 and CD8. Did you perform a doses resp R: Yes, we tested different doses to optimize the results. Reviewer #2 Author’s answer: We thank the referee for providing constructive comments to improve the article. All criticisms and detailed corrections have been addressed and the following revisions have been made. Reviewer #2’s comments Answers Line 101: The authors stated that “Splenic fragments in control dogs were removed by surgical excision [25]”. I wish to understand if the Animal Ethics Committee approved the collection of splenic fragments from healthy animals. If yes, could you please clarify in which circumstances? Please, provide the Animal Ethics certificate. Also, it is unclear why reference 25 was used. R: Animal Ethics Committee approved the collection of splenic fragments from healthy animals. The owners assigned a term of consentient authorizing the procedure. Reference 25 was used to describe the surgical procedure. Line 105: Please, explain how the fragments were macerated. R: The information was added to the text. Line 110: Please provide the description or the commercial reference for the red blood cell lysis buffer R: The information was added to the text. Line 136: Confirm if the DNA was extracted from cells or directly from tissue R: DNA was extracted from cells. Line 157: The authors stated “To confirm that miR-21 is upregulated in dogs with CanL obtained by [11],” Please, explain why reference 11 was used. R: Reference 11 was used to show that the waiting result has already been observed in other paper. Line 169: A pool of which cDNAs was used for normalization - control, infected, or both? Please explain where the absolute quantification of miR-21 was used. R: A pool of both cDNAs. Absolute quantification was used in all samples of both groups. Line 173: Please, insert the reference or complete description for SNORD96A. R: The information was added to the text. Results Figure 1 legend: Please, insert the number of animals (samples) analyzed. Figure 2 legend: Please, detail which range is referring, min-max, SD? I suggest presenting the control group first in the graph (same order as in figure 1). Figures 4 and 5 legends: Please, detail which range is referring, min-max, SD? R: All modifications were done. I am concerned about the validation of the transfection. The authors did not show a control condition for the experiment (a condition with the transfection reagent only, without the scrambled sequence). Did you perform a pilot experiment with i.e., cells treated with transfection reagent only? Also, it is mandatory to validate the expression of miR-21 after transfection. How do the authors know the transfection efficiency relative to miR-21 inhibition? Is it possible to run an experiment to evaluate the miRNA expression after transfection? Do the authors have frozen samples? Gene expression should be similar in both untransfected cells and cells transfected with the negative control. Comment, please. According the manufacturer, “The expression of an endogenous gene, which is known to be a target of the miRNA under study, is measured after mimic/inhibitor transfection. The effect of the mimic/inhibitor is determined by comparing this result with the gene expression in untransfected cells or cells transfected with a negative control. Gene expression is often measured at the protein level, for example, by western blot, as miRNAs often inhibit translation of their target genes and do not cause degradation of the target transcript. This means that the effect of a miRNA mimic or inhibitor can often not be determined using quantitative, real-time PCR.” Besides that, the average CD69 expression in the control group was around 40000 (figure 2). When the cells of this group were transfected with the scrambled sequence, the average expression was around 75000 (figure 3). It seems that the scrambled condition is increasing the expression. At the same time, the inhibitor condition is achieving an expression around 40000, similar to the control group expression in figure 2. Results from the negative control should be compared to results from untransfected cells. It seems that adequate controls are missing. Comment, please. Although the numbers are different, the analysis was performed and there was no statistical difference. According to manufacturer, “A negative control should be transfected in every experiment and will indicate if results are nonspecific. Comparison of results from the negative control with results from the miRNA mimic under study can be used to confirm that the observed results are specific to the miRNA mimic under study.” The graph below represents the control group containing comparative analyzes with non-transfected cells and using only the transfection reagent (HiperFect), demonstrating that there was no statistical difference between the other groups. The authors found significantly less expression of CD69 in control and canL groups when miR-21 was mimic on the other side the inhibition did not produce the opposite effect. Comment, please. The amount of miR-21 present in the samples can be so high that the concentration used in the inhibitor and indicated by the manufacturer was not enough to show an opposite effect. Discussion: Line 334-335: Maybe the low transfection rate of this study does not support the previous sentence “suggesting that, in dogs, these proteins may not be targets of miR-21.” Please, comment. The low transfection rate obtained may not have been sufficient to modulate the expression of these molecules. In Other experimental models, these proteins are targets of miR-21: 1- Liu Y, Ren L, Liu W, Xiao Z. MiR-21 regulates the apoptosis of keloid fibroblasts by caspase-8 and the mitochondria-mediated apoptotic signaling pathway via targeting FasL. Biochem Cell Biol. 2018 Oct;96(5):548-555. doi: 10.1139/bcb-2017-0306. Epub 2018 Mar 10. PMID: 29527928. 2- Shang C, Guo Y, Hong Y, Liu YH, Xue YX. MiR-21 up-regulation mediates glioblastoma cancer stem cells apoptosis and proliferation by targeting FASLG. Mol Biol Rep. 2015 Mar;42(3):721-7. doi: 10.1007/s11033-014-3820-3. Epub 2014 Nov 14. PMID: 25394756. 3- Marega LF, Teocchi MA, Dos Santos Vilela MM. Differential regulation of miR-146a/FAS and miR-21/FASLG axes in autoimmune lymphoproliferative syndrome due to FAS mutation (ALPS-FAS). Clin Exp Immunol. 2016 Aug;185(2):148-53. doi: 10.1111/cei.12800. Epub 2016 May 24. PMID: 27060458; PMCID: PMC4954998. 4- Sayed D, He M, Hong C, Gao S, Rane S, Yang Z, Abdellatif M. MicroRNA-21 is a downstream effector of AKT that mediates its antiapoptotic effects via suppression of Fas ligand. J Biol Chem. 2010 Jun 25;285(26):20281-90. doi: 10.1074/jbc.M110.109207. Epub 2010 Apr 19. PMID: 20404348; PMCID: PMC2888441. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0265192.r002
26 Oct 2021 Decision Letter - Paulo Lee Ho, Editor PONE-D-21-21939R1miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasisPLOS ONE Dear Dr. de Lima, 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: 1) Review the statistic accordingly to reviewer #2 comments;2) Please, answer to the comments raised by both reviewers. Please submit your revised manuscript by Dec 10 2021 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, Paulo Lee Ho, 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: (No Response) Reviewer #2: (No Response) ******** 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 Reviewer #2: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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 Reviewer #2: No ****** 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 Reviewer #2: 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 have made most of the solicited changes, but some additional changes are still needed for a clear presentation of the data. This reviewer considers that the paper can be accepted after performing the following suggested corrections. Figure 1: Include “fold change of miRNA 21” on the Y-axis of the Graph. This will help the reader to quickly understand what fold changes are referring to in that graph. Figure 4: The Y-axis of the 3 Graphs showing data of CD69 expression on CD4, CD8, and CD21 positive cells are incorrectly labeled. Those graphs should state “Mean Fluorescence Intensity of CD69 on CD4+ cells, or CD8+ cells or CD21+ cells” instead of the current labels that are very confusing, Mean CD8+ cells (FL1)/CD69+ cells (FL2). Please correct accordingly. S2 Fig. It is not clear what the authors are measuring as the control of transfection. What do more dead cells (7ADD+) mean in regard to transfection efficacy? please clarify this point in result section. Reviewer #2: The authors answered all points addressed by the reviewer. Unfortunately, the authors were not able to solve the main issue raised by the reviewer. Although the author affirms that there is no difference among scrambled, untransfected, and HiperFect conditions, only in two samples in the scrambled condition the CD69 expression is high. So, my point is that the mimic is not decreasing the expression, but something happens in the scrambled that resulted in a higher expression. I still wish to see the raw data of all of them. I strongly suggest a statistical review as in my checking (with the raw data available) there were no defferences in CD69 expression in control and CanL groups (ANOVA followed by Dunn's multiple comparison test). ****** 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 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. Attachments Attachment Submitted filename: 20211022_Plos_revision.docx https://doi.org/10.1371/journal.pone.0265192.r003
Revision 2
29 Oct 2021 Author Response The response to reviewers is also attached as a Microsoft Word doc. Response to Reviewers Manuscript No.: PONE-D-21-21939 Title: miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasis Dear Editor, We appreciate the careful review of our manuscript by the reviewers. Their constructive comments and suggestions are much appreciated, and the new version of the manuscript has benefited considerably from their assistance Please note that all changes made in the text are highlighted in red. Reviewer Comments: Author’s answer to Reviewer Comments: We thank the referee for providing constructive comments to improve the article. All criticisms and detailed corrections have been addressed and the following revisions have been made. We have accepted all the suggestions and requests made by the Reviewers in the manuscript. Sincerely yours, Valéria Marçal Felix de Lima, MSc, PhD São Paulo State University – UNESP Reviewer #1 Author’s answer: We thank the referee for providing constructive comments to improve the article. All criticisms and detailed corrections have been addressed and the following revisions have been made. Reviewer #1’s comments Answers Figure 1: Include “fold change of miRNA 21” on the Y-axis of the Graph. This will help the reader to quickly understand what fold changes are referring to in that graph. R: The y-axis was renamed as recommended. Figure 4: The Y-axis of the 3 Graphs showing data of CD69 expression on CD4, CD8, and CD21 positive cells are incorrectly labeled. Those graphs should state “Mean Fluorescence Intensity of CD69 on CD4+ cells, or CD8+ cells or CD21+ cells” instead of the current labels that are very confusing, Mean CD8+ cells (FL1)/CD69+ cells (FL2). Please correct accordingly. R: The Y-axis title was modified. S2 Fig. It is not clear what the authors are measuring as the control of transfection. What do more dead cells (7ADD+) mean in regard to transfection efficacy? please clarify this point in result section. R: The information to better explain the transfection rates was added in the methods section. Reviewer #2 Author’s answer: We thank the referee for providing constructive comments to improve the article. All criticisms and detailed corrections have been addressed and the following revisions have been made. Reviewer #2’s comments Answers The authors answered all points addressed by the reviewer. Unfortunately, the authors were not able to solve the main issue raised by the reviewer. Although the author affirms that there is no difference among scrambled, untransfected, and HiperFect conditions, only in two samples in the scrambled condition the CD69 expression is high. So, my point is that the mimic is not decreasing the expression, but something happens in the scrambled that resulted in a higher expression. I still wish to see the raw data of all of them. I strongly suggest a statistical review as in my checking (with the raw data available) there were no defferences in CD69 expression in control and CanL groups (ANOVA followed by Dunn's multiple comparison test). CD69 expression in untransfected cells in CanL and Control group Normality test (don’t pass) Non parametric test to compare the expression in CanL and Control group (Mann Whitney test). Analysis post transfection with mimics and inhibitors of miR-21: CanL group data Normality test (don’t pass) Friedman test (non parametric) Post test (Dunn’s multiple comparisons) Control Group data Normality test Friedman test Dunn’s multiple comparison test Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0265192.r004
24 Nov 2021 Decision Letter - Paulo Lee Ho, Editor PONE-D-21-21939R2miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasisPLOS ONE Dear Dr. de Lima, 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. 1) Provide the statistical analysis as requested by the reviewer #2.2) Please, answer all the comments raised by both the reviewers. Please submit your revised manuscript by Jan 08 2022 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. 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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, Paulo Lee Ho, Ph.D. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 Reviewer #2: (No Response) ******** 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 Reviewer #2: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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 Reviewer #2: 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 Reviewer #2: 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 have included all suggestions and clarified pending questions. I do not have further comments. Reviewer #2: I strongly suggest (again) a revision in the statistics of the results in figure 3. Data of different conditions (mimic, inhibitor, scrambled, hiperfect, and untransfected) must be analyzed by ANOVA followed by multicomparison's posttest for canine and control groups. I guess you used Dunn's multiple comparisons test to compare the mean rank of each condition with the mean rank of a control group. Did you use scrambled as a control? Why didn’t you compare the mean rank of each column with the mean rank of every other column? I checked the statistics with the data provided using Prism version 8.2.1. Please, send me the Prism file. ****** 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 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. 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Revision 3
30 Nov 2021 Author Response We appreciate the reviewers' suggestions. All of them have been answered. The file with the requested statistic data was sent by email. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0265192.r006
4 Jan 2022 Decision Letter - Paulo Lee Ho, Editor PONE-D-21-21939R3miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasisPLOS ONE Dear Dr. de Lima, 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. Please submit your revised manuscript by Feb 18 2022 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, Paulo Lee Ho, 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 #2: (No Response) ******** 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 #2: Partly ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: No ****** 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 #2: No ****** 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 #2: 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 #2: According to the Prism file provided by the authors, the statistical analysis of CD69 expression after transfection must be performed with a parametric test (ANOVA) for the CONTROL group instead of the nonparametric test used. Also, data of HiperFect buffer and untransfected cells must be provided in figure 3. Update methods and results section accordingly. ****** 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 #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.0265192.r007
Revision 4
13 Feb 2022 Author Response Figure 3 was modified and HiperFect and untransfected cells data were added. To claim that our data has a normal distribution, we rigorously pass three different normality tests. Since of the three, only 1 test showed normality in the data, while the other 2 showed that n is too small to present a normal distribution. Given this, we chose to use a non-parametric test, which best represents our data. Similar experiment has already been published by our research group, using this same statistical test, in the work of MELO, et al., 2019. Attached are some links to works with values of n similar to ours, which used non-parametric tests. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4029133/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7023135/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905561/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5962361/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6962187/ Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0265192.r008
28 Feb 2022 Decision Letter - Paulo Lee Ho, Editor miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasis PONE-D-21-21939R4 Dear Dr. de Lima, 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, Paulo Lee Ho, Ph.D. 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 #2: 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: N/A ****** 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 #2: No ****** 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 #2: 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 #2: (No Response) ****** 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 #2: No https://doi.org/10.1371/journal.pone.0265192.r009
Formally Accepted
9 Mar 2022 Acceptance Letter - Paulo Lee Ho, Editor PONE-D-21-21939R4 miRNA-21 regulates CD69 and IL-10 expression in canine leishmaniasis Dear Dr. de Lima: 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. Paulo Lee Ho Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0265192.r010

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