Encephalitozoon cuniculi takes advantage of efferocytosis to evade the immune response

Luciane Costa Dalboni; Anuska Marcelino Alvares Saraiva; Fabiana Toshie de Camargo Konno; Elizabeth Cristina Perez; Jéssica Feliciana Codeceira; Diva Denelle Spadacci-Morena; Maria Anete Lallo

doi:10.1371/journal.pone.0247658

Peer Review History

Original SubmissionNovember 12, 2020
4 Dec 2020 Decision Letter - Selvakumar Subbian, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-20-35590 Encephalitozoon cuniculi takes advantage of efferocytosis to evade the immune response PLOS ONE Dear Dr. Lallo, 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. As mentioned by Reviewer# 1, please make changes on the manuscript to improve the visibility and clarity. Also, make sure to include scale bars in all images. Please submit your revised manuscript by Jan 18 2021 11:59PM. 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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 [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: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Overall, an interesting series of experiments and the data is intriguing. The manuscript needs editing for style and clarity. Comments 1. In Figure 2 B it is shown that ingestion by macrophages of ACs (either infected or uninfected) leads to those cells having more spores at 24 hours following infection by E. cuniculi, and also that if the macrophage ingested an infected AC (IAC) then the amount of spores at 24 hours following infection by E cuniculi is less than that seen in macrophages that ingest an uninfected AC. This is an interesting observation, but after reading the paper there is no clear mechanism identified that leads to this effect. What is different about an IAC that causes the macrophage to have this lower growth rate for E cuniculi following infection. 2. While an 1 or 2 images of this process at the EM level would be useful the extensive EM presented does not add significant data to the paper. I would suggest that most of these EM images could become supplemental figures and that EM within the actual paper be limited to 1 or 2 images demonstrating the present of IACs and that replication is not occurring following ingestion by the macrophages. 3. The resolution of figures 10 and 11 needs to be improved (line drawings should be 600dpi at this size to be able to clearly read these labels) 4. The data on M2 polarization is interesting, but as noted by the authors (line 551-553) the results were ambiguous with respect to the M1 and M2 profiles. The authors to to refine the text to provide a clear explanation of the results and the implications of this work, especially as it relates to Figure 2B. 5. While increased phagocytosis might explain increased infection, this could also be due to other changes in these macrophages rendering them permissive to infection. Was a control done with heat killed spores? This could provide a way to look at phagocytosis without infection (via the polar tube) as the spores that were heat killed would not germinate and otherwise infect the cells. In addition, phagocytosis rates could also be measured by the uptake of latex beads by the macrophages. It would be useful in understanding figure 2B if some measures of phagocytosis efficiency were provided for macrophages treated with AC and IAC compared to controls untreated macrophages. Reviewer #2: The authors present clear and elegant evidence of how efferocytosis of infected apoptotic cells shows a suppressive effect on the activity of macrophages with an M2 profile and the production of large amounts of IL-10 and IL-6, suggesting that E. cuniculi can take advantage of efferocytosis to enter the cell, multiply and spread throughout the body and modulate an anti-inflammatory environment, thus constituting a mechanism to avoid the immune response. The manuscript shows originality and is novel in approaching the subject since few reports of how efferocytosis promotes an anti-inflammatory environment in infections with E. cuniculi; the microscopy images add much weight to the research. I also consider that it provides novel information regarding the immune response in this disease; the results adequately and understandably are presented; they are justified and are related to the objectives clearly and objectively. improve the quality (sharpness) of the figures, mainly figures 7, 10, and 11. When trying to download figure 10, and figure 9 of the document opens, the link or hyperlink of the image is damaged. Line 70 class instead of "clade" ****** 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: Yes:** Uziel Castillo Velazquez [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.0247658.r001
Revision 1
11 Jan 2021 Author Response Reviewer's Responses to Questions We are pleased to resubmit the Manuscript " Encephalitozoon cuniculi takes advantage of efferocytosis to evade the immune response" for revision. We thank the reviewers for their critiques, which have enabled us to sharpen the manuscript considerably. We believe that we have addressed all the points raised by the reviewers and trust that the manuscript is now suitable for publication in PlosOne. A new version was attached as the main file and the corrected version is attached as a supplementary file. Changes are bold and highlighted in red color in the text manuscript, as detailed below. 4. Is the manuscript presented in an intelligible fashion and written in standard English? Reviewer #1: No A. We have provided a new correction to make the text clearer. Reviewer #2: Yes 5. Review Comments to the Author Reviewer #1: Overall, an interesting series of experiments and the data is intriguing. The manuscript needs editing for style and clarity. Comments 1. In Figure 2 B it is shown that ingestion by macrophages of ACs (either infected or uninfected) leads to those cells having more spores at 24 hours following infection by E. cuniculi, and also that if the macrophage ingested an infected AC (IAC) then the amount of spores at 24 hours following infection by E cuniculi is less than that seen in macrophages that ingest an uninfected AC. This is an interesting observation, but after reading the paper there is no clear mechanism identified that leads to this effect. What is different about an IAC that causes the macrophage to have this lower growth rate for E cuniculi following infection. 4. The data on M2 polarization is interesting, but as noted by the authors (line 551-553) the results were ambiguous with respect to the M1 and M2 profiles. The authors to to refine the text to provide a clear explanation of the results and the implications of this work, especially as it relates to Figure 2B. A. Efferocytosis may eliminate the pathogen or may allow the pathogen to infect the efferocyte in a Trojan-horse type maneuver. The efferocyte will initiate anti-inflammatory or proinflammatory signaling depending upon the combined presence of immune-silencing signals and pro-inflammatory pathogen-associated molecular patterns and damage-associated molecular patterns. To answer these questions, we went back to evaluate the groups. When observing the control macrophages pre-incubated with uninfected (unchallenged) apoptotic cells, there was a low expression of CD40, CD206, CD80/86 and MHC II compared to macrophages pre-incubated with IACs. Thus, the greater expression of the activation molecules (CD80/86, MHCII) could indicate potential microbicidal activity in this population, which may explain the finding of a smaller number of spores in these macrophages. Costimulatory molecules are one class of receptors which have been implicated as fulfilling this role in the innate immune response. CD80 and CD86 represent one class of costimulatory receptors. We added a paragraph to explain these interactions and possible explanation for the observed phenomenon. 2. While an 1 or 2 images of this process at the EM level would be useful the extensive EM presented does not add significant data to the paper. I would suggest that most of these EM images could become supplemental figures and that EM within the actual paper be limited to 1 or 2 images demonstrating the present of IACs and that replication is not occurring following ingestion by the macrophages. 3. The resolution of figures 10 and 11 needs to be improved (line drawings should be 600dpi at this size to be able to clearly read these labels) A. We have reduced the number of figures and improved the quality. 5. While increased phagocytosis might explain increased infection, this could also be due to other changes in these macrophages rendering them permissive to infection. Was a control done with heat killed spores? This could provide a way to look at phagocytosis without infection (via the polar tube) as the spores that were heat killed would not germinate and otherwise infect the cells. In addition, phagocytosis rates could also be measured by the uptake of latex beads by the macrophages. It would be useful in understanding figure 2B if some measures of phagocytosis efficiency were provided for macrophages treated with AC and IAC compared to controls untreated macrophages. A. In these experiments, we maintained as controls of macrophage activity to observe possible factors independent of efferocytosis, the exclusive challenge with E. cuniculi spores in all phases. Tests with dead spores or latex spheres could also be used to control macrophage activity, however, we consider that the effects related to the pathogen reflect natural events more realistically. Unfortunately in this pandemic moment, we could not repeat the experiments. Our group has been conducting in vitro experiments with E. cuniculi and macrophages of various strains, immortalized as the RAW, and primaries, marrow or peritoneum macrophages. In none of the previous experiments have we observed, so evident, the multiplication of spores inside macrophages with efferocytosis. We would like to reinforce that efferocytosis affects phagocytosis and spore load inside macrophages, but the most striking phenomenon is the multiplication of the pathogen inside macrophages with efferocytosis and M2 profile, events described for the first time in the literature for microsporids, identifying possible evasion mechanism of the immune response Reviewer #2: The authors present clear and elegant evidence of how efferocytosis of infected apoptotic cells shows a suppressive effect on the activity of macrophages with an M2 profile and the production of large amounts of IL-10 and IL-6, suggesting that E. cuniculi can take advantage of efferocytosis to enter the cell, multiply and spread throughout the body and modulate an anti-inflammatory environment, thus constituting a mechanism to avoid the immune response. The manuscript shows originality and is novel in approaching the subject since few reports of how efferocytosis promotes an anti-inflammatory environment in infections with E. cuniculi; the microscopy images add much weight to the research. I also consider that it provides novel information regarding the immune response in this disease; the results adequately and understandably are presented; they are justified and are related to the objectives clearly and objectively. Improve the quality (sharpness) of the figures, mainly figures 7, 10, and 11. When trying to download figure 10, and figure 9 of the document opens, the link or hyperlink of the image is damaged. Line 70 class instead of "clade" A. We answered the request and changed to class. We have reduced the number of figures and improved the quality. https://doi.org/10.1371/journal.pone.0247658.r002
11 Feb 2021 Decision Letter - Selvakumar Subbian, Editor Encephalitozoon cuniculi takes advantage of efferocytosis to evade the immune response PONE-D-20-35590R1 Dear Dr. Lallo, 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, Selvakumar Subbian, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0247658.r003
Formally Accepted
26 Feb 2021 Acceptance Letter - Selvakumar Subbian, Editor PONE-D-20-35590R1 Encephalitozoon cuniculi takes advantage of efferocytosis to evade the immune response Dear Dr. Lallo: 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. Selvakumar Subbian Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0247658.r004

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