Leishmanicidal and healing effects of 3β,6β,16β-trihydroxy lup-20 (29)-ene isolated from Combretum leprosum on Leishmania braziliensis infection in vitro and in vivo

Aline Sombra Santos; Naya Lúcia de Castro Rodrigues; Francisco Rafael Marciano Fonseca; Nathalia Braga Fayão Oliveira; Bianca Oliveira Loucard; Fabíola Fernandes Heredia; Teresa Neuma Albuquerque Gomes Nogueira; Ticiana Monteiro Abreu; Hélcio Silva dos Santos; Edson Holanda Teixeira; Luzia Kalyne Almeida Moreira Leal; Regis Bernardo Brandim Gomes; Clarissa Romero Teixeira; Maria Jania Teixeira

doi:10.1371/journal.pone.0287665

Peer Review History

Original SubmissionJanuary 30, 2023
23 Mar 2023 Decision Letter - Manas Ranjan Dikhit, Editor PONE-D-23-02657Leishmanicidal and healing effects of 3β,6β,16β-trihydroxy lup-20 (29)-ene isolated from Combretum leprosum on Leishmania braziliensis infection in vitro and in vivo.PLOS ONE Dear Dr. Teixeira, 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 May 07 2023 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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If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ [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: 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: In this article by Santos et.al, entitled Leishmanicidal and healing……… on Leiahmania braziliensis infection in vitro and in vivo” the authors have demonstrated anti-leishmanial and wound healing effect of Combretum leprosum leaves extract. The article has important information and findings but requires revision with clarification before being accepted for publication. My comments are below. 1. Brief description of isolation and characterization of CLF-1 in page 5 line 106 should be provided. 2. The cream formulation should be elaborated in page 6 line 135. 3. In figure 1, panel A, B, C & D, the only macrophage bar in all the cases- it is not clear against which control was these bars normalized. In panel ‘A’ the macrophage bar is around 120 %, in panel ‘B’ it is around 130%, in ‘C’ it is 110% and in ‘D’ it is less than ‘100’. Please clarify and correct. It is also unclear why 100µg/ml CFL-1 is toxic to macrophage but not to Leishmania in that respect. 4. In figure 2 the authors should explain why there is not much effect of duration of treatment. 5. The data provided in the manuscript shows that higher dose of CFL-1 has no effect on number of amastigotes (Fig 2) but higher dose of CFL-1 do induce the cytokines (Fig 3). How can this be explained in wound healing and clearance of parasites. 6. I would suggest the authors to measure the parasitic load through some molecular method like qPCR. Reviewer #2: Author has shown with different experiments that CLF-1 has in vitro (in J774 macrophage) and in vivo (in Hamster model) anti-leishmanial effect against L. braziliensis. However, author should address the following comments before acceptance. Comments: 1. Author did not study any mechanism of inhibition for CLF-1 in L. braziliensis as previous study already explored the mechanism of inhibition by CLF-1 in L. amazonensis promastigotes and also showed that CLF-1 has anti-leishmanial activity in-vitro. So, in my opinion the only novel part of this study was to explore the effect of CLF-1 cream in Hamster model. Author should study the mechanism of inhibition in amastigote by isolating the amastigotes from infected macrophages. 2. Author used J774 (mouse cancerous cell line) for in-vitro infection study. In my opinion, author may use mouse peritoneal macrophages for this study as this the primary macrophages. 3. For in-vitro study, author used Amphotericin B as a control and concentration used is 16 µg/ml. What is the rationale behind choosing this concentration? Is there any report? 4. Author has developed CLF-1 cream for topical application. However, author did not provide the details about the process of formulation of the cream. 5. Author has shown that promastigotes viability is reduced with increasing concentration of CLF-1 (Figure 1) and in case of intracellular amastigotes, viability is increased with increasing concentration of CLF-1 (Figure 2). What is the explanation of this contradictory results? However, I am quite happy with the work but, author need to address the above issues. ****** 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.0287665.r001
Revision 1
8 May 2023 Author Response ANSWERS TO REVIEWERS: Reviewer #1: In this article by Santos et al, entitled Leishmanicidal and healing……… on Leishmania braziliensis infection in vitro and in vivo” the authors have demonstrated anti-leishmanial and wound healing effect of Combretum leprosum leaves extract. The article has important information and findings but requires revision with clarification before being accepted for publication. My comments are below. 1. Brief description of isolation and characterization of CLF-1 in page 5 line 106 should be provided. More information related to the isolation and characterization of CLF-1 was included in the Material and Methods section as suggested. 2. The cream formulation should be elaborated in page 6 line 135. A more detailed description of CLF-1 cream formulation was included in the Material and Methods section as suggested. 3. In figure 1, panel A, B, C & D, the only macrophage bar in all the cases- it is not clear against which control was these bars normalized. In panel ‘A’ the macrophage bar is around 120 %, in panel ‘B’ it is around 130%, in ‘C’ it is 110% and in ‘D’ it is less than ‘100’. Please clarify and correct. It is also unclear why 100µg/ml CFL-1 is toxic to macrophage but not to Leishmania in that respect. We appreciate that the reviewer’s comment and we have corrected figure 1, the bars were normalized based on the experiment controls. While CLF-1 cytotoxicity to macrophages was only observed with the highest concentration (100µg/ml), promastigotes were more susceptible to the toxic effect, observed also with lower CLF-1 concentrations (10 µg/ml). This is probably resulting from different mechanisms of actions of CLF-1 on such distinct cells (macrophages and Leishmania parasites) and should be further explored on future studies. 4. In figure 2 the authors should explain why there is not much effect of duration of treatment. We believe that we did not observe a significant effect on amastigotes at 24h, but not at 48h, probably due to the kinetics of CLF-1 effect on the intracellular parasites. Unfortunately, we were not able to test in vitro for a longer period. However, the CLF-1 anti-leishmanial effect was evident in vivo on infected hamsters where we observe a clear reduction of parasite numbers and lesions were not ulcerated with a necrotic center compared to the group treated with Glucantime. 5. The data provided in the manuscript shows that higher dose of CFL-1 has no effect on number of amastigotes (Fig 2) but higher dose of CFL-1 do induce the cytokines (Fig 3). How can this be explained in wound healing and clearance of parasites. The mechanism of CLF-1 treatment on amastigotes (intracellular) and macrophages and the kinetics of the effect observed is probably different and it remains to be elucidated. CLF-1 has been previously described as having an antimicrobial, pro-healing and antiparasitic activities. The wound healing and clearance of parasites is probably resulting, at least in part, by direct modulation of cytokine production that results in parasite killing and promote lesion healing. 6. I would suggest the authors to measure the parasitic load through some molecular method like qPCR. Although qPCR is a sensitive and specific method, limiting dilution is more accessible, widely used, and a reliable method to quantify live parasites. Thus, we decided to measure the parasite burden using the limiting dilution method to determine the parasite load. This method has been used by our group in other studies (Dutra, et al., 2020 and Freire et al., 2022). Reviewer #2: Author has shown with different experiments that CLF-1 has in vitro (in J774 macrophage) and in vivo (in Hamster model) anti-leishmanial effect against L. braziliensis. However, author should address the following comments before acceptance. Comments: 1. Author did not study any mechanism of inhibition for CLF-1 in L. braziliensis as previous study already explored the mechanism of inhibition by CLF-1 in L. amazonensis promastigotes and also showed that CLF-1 has anti-leishmanial activity in-vitro. So, in my opinion the only novel part of this study was to explore the effect of CLF-1 cream in Hamster model. Author should study the mechanism of inhibition in amastigote by isolating the amastigotes from infected macrophages. The reviewer made an important point about previous work demonstrating the potential protective and healing effect of CLF-1. Infection caused by Leishmania braziliensis and Leishmania amazonensis induce skin lesions, but disease progression and immune evasion mechanisms are distinct reinforcing the importance of testing CLF-1 against each individual Leishmania species. Investigation of the mechanism of action of CLF-1 treatment on amastigotes and macrophages are the subject of a future study. 2. Author used J774 (mouse cancerous cell line) for in-vitro infection study. In my opinion, author may use mouse peritoneal macrophages for this study as this the primary macrophages. Although we have not used a primary culture of macrophages due to a limitation on the number of experimental hamsters/mice available at the time, we believe that J774 cells, that are widely used for in vitro tests, also provide reliable and representative results. 3. For in-vitro study, author used Amphotericin B as a control and concentration used is 16 µg/ml. What is the rationale behind choosing this concentration? Is there any report? We have used this concentration of Amphotericin B as a control for parasite killing. It was based on previous work published by the group (Pinheiro et al., 2021). This reference was included in the Material and Methods. 4. Author has developed CLF-1 cream for topical application. However, author did not provide the details about the process of formulation of the cream. Details concerning the formulation of CLF-1 cream were included in the Material and Methods section as suggested. 5. Author has shown that promastigotes viability is reduced with increasing concentration of CLF-1 (Figure 1) and in case of intracellular amastigotes, viability is increased with increasing concentration of CLF-1 (Figure 2). What is the explanation of this contradictory results? However, I am quite happy with the work but, author need to address the above issues. We observed a different effect on promastigotes and amastigotes probably as a result of the mechanism of action and kinetics of CLF-1 on the different stages (intracellular x extracellular). Unfortunately, we were not able to test in vitro for a longer period as we moved forward for in vivo experiments. The CLF-1 anti-leishmanial effect was evident in vivo on infected hamsters where we observe a clear reduction of parasite and lesions were not ulcerated with a necrotic center compared to the group treated with Glucantime. Attachments Attachment Submitted filename: ResponsetoReviewers.docx https://doi.org/10.1371/journal.pone.0287665.r002
12 Jun 2023 Decision Letter - Manas Ranjan Dikhit, Editor Leishmanicidal and healing effects of 3β,6β,16β-trihydroxy lup-20 (29)-ene isolated from Combretum leprosum on Leishmania braziliensis infection in vitro and in vivo. PONE-D-23-02657R1 Dear Dr. Maria, 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, Manas Ranjan Dikhit Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (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 #1: No ******** https://doi.org/10.1371/journal.pone.0287665.r003
Formally Accepted
10 Jul 2023 Acceptance Letter - Manas Ranjan Dikhit, Editor PONE-D-23-02657R1 Leishmanicidal and healing effects of 3β,6β,16β-trihydroxy lup-20 (29)-ene isolated from Combretum leprosum on Leishmania braziliensis infection in vitro and in vivo Dear Dr. Teixeira: 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. Manas Ranjan Dikhit Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0287665.r004

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