PGC-1α activation to enhance macrophage immune function in mycobacterial infections

Joel R. Frandsen; Zhihong Yuan; Brahmchetna Bedi; Zohra Prasla; Seoung-Ryoung Choi; Prabagaran Narayanasamy; Ruxana T. Sadikot

doi:10.1371/journal.pone.0310908

Peer Review History

Original SubmissionOctober 19, 2023
5 Dec 2023 Decision Letter - Frederick Quinn, Editor PONE-D-23-34313PGC-1α Activation to Enhance Macrophage Immune Function in Mycobacterial InfectionsPLOS ONE Dear Dr. Sadikot, 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 Jan 19 2024 11:59PM. If you will need significantly more time 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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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: 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: 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: This manuscript, titled 'PGC-1α Activation to Enhance Macrophage Immune Function in Mycobacterial Infections,' aimed to investigate the impact of NTM infections on host macrophage mitochondria. Importantly, NTM disrupted the ability of macrophages to generate functional mitochondria by attenuating PGC-1α and TFAM, which were subsequently restored by PGC-1α pharmacologic activators. However, the in vivo studies lack solid evidence to conclusively prove that the altered expression of PGC-1α or TFAM, caused by NTM, occurred in macrophages. Furthermore, the fluorescence images are obscure, as evidenced in Fig. 1C, Fig. 3K, Fig. 4K, and Fig. 4L. Reviewer #2: This is a nice manuscript and worthy of publication. However, appreciate author’s comments/responses to the following points: 1. In general, language requires some revision and editing particularly in the “Discussion” section which requires significant revision for better clarity and removal of redundant and repetitive sentences. 2. Line 153: What dose of oral metformin in an average human would be equivalent to the final concentration of 2 mM metformin mentioned in the manuscript. Please add to the manuscript. 3. Line 182: Is the correct number 15,000 RPM and not 150,000? 4. Lines 245, 293, 303: Please specify what the cells are treated with 5. Figure 1C and 1D: a. The MitoSOX staining for CTRL seems to be nothing (at least not visible on the provided image) where as mentioned as about 1 on Figure 1D. Appreciated comment clarification. b. MitoSox-based flow cytometry could be considered as an alternative 6. Figure 2: Whole lung tissue was used for this experiment and thus it is not clear if the results seen are due to changes in macrophages, other cells, or both. This is important as the authors have previously shown that PGC-1a overexpression, resveratrol, and metformin enhance host bronchial epithelial cell mitochondrial function and improve epithelial innate response to P. aeruginosa. Showing the changes specifically in macrophages (by cell sorting or staining) will be important as they are the focus of the manuscript. 7. Figure 3 related: a. There is an issue with discrepancy between the text (line 391 to 403) and figures 3A to 3H where the text states “MAB-mediated” but figures show “MAC”. b. However, Figures 3I and 3J mention MAB. c. Please reconcile. d. Figure 3K states MAB but 3M mentions MAC. Please clarify e. Also, the morphology of the cells in the bottom row of figure 3K appear different from the rest. In our experience, the morphology of cells (roundish) in the 3 top rows is more in line with monocytes while the morphology in the bottom row is more in line with activated macrophages. Appreciate authors’ input. f. Pre-treatment with ZLN005 was used in this experiment, would treatment after MAB or MAC infection produce similar results and be as effective? This is of clinical importance as treatment is usually initiated after the infection. 8. Figure 4 related: a. Would treatment with metformin after the infection produce the similar results? 9. Figure 5: a. SR (I assume stands for SR-18292) is mentioned in the figure but no explanation in the text or figure legend. 10. Line 492: Please clear the track changes (please remove “and macrophages” at the end of the sentence. 11. Lines 521-523: Please revise the sentence for better clarity 12. Lines 532-534: Given that TFAM is downstream from PGC-1a (as also mentioned in the manuscript) why SR-18292 had no measurable effect on TFAM despite significantly reducing PGC-1a expression? 13. Line 596: Please add “(mitochondrial membrane potential)” after ΔΨm. 14. A figure depicting the pathways and findings will be a welcomed addition Thank you ****** 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.0310908.r001
Revision 1
5 Feb 2024 Author Response Reviewer #1: 1) Comment: This manuscript, titled 'PGC-1α Activation to Enhance Macrophage Immune Function in Mycobacterial Infections,' aimed to investigate the impact of NTM infections on host macrophage mitochondria. Importantly, NTM disrupted the ability of macrophages to generate functional mitochondria by attenuating PGC-1α and TFAM, which were subsequently restored by PGC-1α pharmacologic activators. However, the in vivo studies lack solid evidence to conclusively prove that the altered expression of PGC-1α or TFAM, caused by NTM, occurred in macrophages. Response: We appreciate the reviewer comment about the significance of our work, we agree that the in vivo model does not necessarily reflect the expression of the mitochondrial proteins only in the macrophages. They are probably a combination of multiple cell types. The purpose of the in vivo experiment is to demonstrate that the expression of the mitochondrial proteins is altered in the lung. We do agree that more in depth studies are needed to define the in vivo role of macrophages in the lungs. In future studies, we plan to study the in vivo model in depth, however, it is beyond the scope of the current manuscript. 2) Comment: The fluorescence images are obscure, as evidenced in Fig. 1C, Fig. 3K, Fig. 4K, and Fig. 4L. Response: We have included labeling with the colors that are represented by the fluorescent images. This provides the explanation for the images; this is now included in the results and figure legends. Reviewer #2: Comment: This is a nice manuscript and worthy of publication. However, appreciate author’s comments/responses to the following points. We appreciate the reviewer comments and have addressed the concerns raised. 1. Comment: In general, language requires some revision and editing particularly in the “Discussion” section which requires significant revision for better clarity and removal of redundant and repetitive sentences. Response: We appreciate the comment and have now carefully edited the manuscript for language and revised the discussion section for brevity and clarity (Lines 570-705) 2. Comment: Line 153: What dose of oral metformin in an average human would be equivalent to the final concentration of 2 mM metformin mentioned in the manuscript. Please add to the manuscript. Response: This is an important point, thank you for raising this issue. Metformin dosages prescribed for diabetes range up to a maximum of 2.5 g (35 mg/kg) per day, which corresponds to blood sera levels of 10-20 µM. Pharmacokinetic evidence shows metformin’s action in the mitochondria occurs through a gradual build-up in the inner mitochondrial matrix, which can have concentrations orders of magnitude higher than serum levels. In one study, a serum concentration of 10 µM was correlated to mitochondrial concentrations 100 times higher. Thus, the elevated metformin concentrations used in this study and others correlates to intramolecular concentration of metformin in vivo. 3. Comment: Line 182: Is the correct number 15,000 RPM and not 150,000? Response: Correct, the centrifugation speed is “15,000 RPM” (Line 182), this has been corrected 4. Comment: Lines 245, 293, 303: Please specify what the cells are treated with? Response: We have now included specifics about drug treatments and concentrations. Cells were treated with metformin [2 mM], ZLN005 [2 µM], and SR-18292 [10 µM] (Line 245, 293, 305), 5. Comment: Figure 1C and 1D: a. The MitoSOX staining for CTRL seems to be nothing (at least not visible on the provided image) where as mentioned as about 1C on Figure 1D. Response: This figure has now been replaced. b. MitoSox-based flow cytometry could be considered as an alternative Response: We agree that flow cytometry can be alternative, however, the reason for using fluorescent cell staining was to illustrate and visualize the progressive increase in MitoSOX fluorescence in macrophages during the initial stages of bacterial infection. 6. Comment: Figure 2: Whole lung tissue was used for this experiment and thus it is not clear if the results seen are due to changes in macrophages, other cells, or both. This is important as the authors have previously shown that PGC-1a overexpression, resveratrol, and metformin enhance host bronchial epithelial cell mitochondrial function and improve epithelial innate response to P. aeruginosa. Showing the changes specifically in macrophages (by cell sorting or staining) will be important as they are the focus of the manuscript. Response: We agree with the reviewer the in vivo experiments do not discriminate between the cell types are demonstrating the upregulation. We agree that the in vivo model does not necessarily reflect the expression of the mitochondrial proteins only in the macrophages. They are probably a combination of multiple cell types. The purpose of the in vivo experiment is to demonstrate that the expression of the mitochondrial proteins is altered in the whole lung. We do agree that more in depth studies are needed to define the in vivo role of macrophages in the lungs. In future studies, we plan to study the in vivo model in depth, however, it is beyond the scope of the current manuscript since the focus of the current manuscript is to investigate the effects on macrophages in vitro. While we cannot be certain the effect seen was due to cells other than alveolar macrophages, the in vivo experiments demonstrate changes in the whole lung tissue. We agree that in future experiments, it would be beneficial to isolate macrophages from the lung tissue to determine their specific influence on PGC-1α expression during NTM infection. 7. Comment: Figure 3 related: a. There is an issue with discrepancy between the text (line 391 to 403) and figures 3A to 3H where the text states “MAB-mediated” but figures show “MAC”. b. However, Figures 3I and 3J, 3K mention MAB. Please reconcile. 7a-d. Response: We regret the oversight in labeling, figures and captions, are all revised. e. Also, the morphology of the cells in the bottom row of figure 3K appear different from the rest. In our experience, the morphology of cells (roundish) in the 3 top rows is more in line with monocytes while the morphology in the bottom row is more in line with activated macrophages. Appreciate authors’ input. Response: We appreciate the comment, the immunocytochemistry images are that of macrophages, which exhibit both circular and irregular/stellate shaped morphology. Comment: Pre-treatment with ZLN005 was used in this experiment, would treatment after MAB or MAC infection produce similar results and be as effective? This is of clinical importance as treatment is usually initiated after the infection. Response: We agree that treatment post infection would have translational implications, these experiments need careful dose response and time course experiments, are ongoing and will be tested in the future. 8. Comment: Figure 4 related: Would treatment with metformin after the infection produce the similar results? Response: We anticipate similar results with metformin post treatment, since it is a potent PGC-1α activator. 9. Comment: Figure 5: . SR (I assume stands for SR-18292) is mentioned in the figure but no explanation in the text or figure legend. Response: Correct, we have included this in the figure caption SR-18292 and in the abbreviation SR. (Line 520) 10. Comment: Line 492: Please clear the track changes (please remove “and macrophages” at the end of the sentence. Response: Thank you, we have corrected this. “and macrophages” was deleted from sentence. (Line 492). 11. Comment: Lines 521-523: Please revise the sentence for better clarity Response: We have revised lines 521-523 and made corrections for better clarity. 12. Comment: Lines 532-534: Given that TFAM is downstream from PGC-1a (as also mentioned in the manuscript) why SR-18292 had no measurable effect on TFAM despite significantly reducing PGC-1a expression? Response: There are multiple signaling pathways and transcription factors involved with the activation and regulation of PGC-1α and TFAM, and both can be activated through PPARγ signaling. Thus there may be other potential pathways for activation of TFAM, additionally, post transcriptional modifications may be involved in differential gene expression between PGC-1α and TFAM. 13. Comment: Line 596: Please add “(mitochondrial membrane potential)” after ΔΨm. Response: Thank you, this has been corrected. 14. Comment: A figure depicting the pathways and findings will be a welcomed addition Response: We appreciate the suggestion and have added a figure (Figure 8). Attachments Attachment Submitted filename: Response to reviewers Jan 30 2024.docx https://doi.org/10.1371/journal.pone.0310908.r002
19 Mar 2024 Decision Letter - Frederick Quinn, Editor PONE-D-23-34313R1PGC-1α Activation to Enhance Macrophage Immune Function in Mycobacterial InfectionsPLOS ONE Dear Dr. Sadikot, 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, particularly major concern from Reviewer 1. Please submit your revised manuscript by May 03 2024 11:59PM. If you will need significantly more time 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, Frederick Quinn 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: Partly Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes 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 #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: No ****** 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 revised manuscript has been greatly improved. However, the in vivo studies can not prove that the altered expression of PGC-1α or TFAM, caused by NTM, occurred in macrophages. Reviewer #2: I thank the authors for their responses. 1. Line 152 (revised version): What dose of oral metformin in an average human would be equivalent to the final concentration of 2 mM metformin mentioned in the manuscript. Please add to the manuscript. Appreciate authors’ comments. It would be a plus for the manuscript if their explanation could be incorporated into the manuscript. 2. Line 305 (revised version): Please add what the cells are treated with. 3. Figure 1C and 1D: I think there should be a mistake as the picture appears to be the same and not replaced (please see below). a. The MitoSOX staining for CTRL seems to be nothing (at least not visible on the provided image) where as mentioned as about 1 on Figure 1D. Appreciated comment clarification. b. MitoSox-based flow cytometry could be considered as an alternative. 4. Figure 2: I appreciated author’s comments. As mentioned in the original comments showing the changes specifically in macrophages (by cell sorting or staining) is very important given the focus of this manuscript and the author’s prior work. These experiments should be very straightforward and will significantly improve the manuscript. However, if further experiments are not feasible or can’t be done then this section could be omitted particularly given that the per authors the in vivo studies are beyond the scope of this manuscript or at least the limitation clearly mentioned. 5. Lines 434-436: Language needs revision for better clarity. 6. Figure 4 related: a. Text mentions PGC-1a while figure A and B mention “ppargc1a” (e.g., line 440 could change to “decreases in PGC1-a mRNA (ppargc1a)”. b. Figure 4 (K, L, M, N): While figures K and L mention “ZLN + MAC”, figures M and N and the text mention MET + MAC. Which one is it? The correct image should be MET + MAC to confirm to the text and the rest of images. Please rectify. 7. Figure 5: Again: a. SR (I assume stands for SR-18292) is mentioned in the figure but no mention or explanation of SR in either the text or the figure legend. b. Although SR is mentioned on line 510 (and not 520) and under next topic and in relation to Figure 6, it should be mentioned at the first appearance for clarity. 8. Figure 6: a. A and B. Moving rows 7 to just above current row 4 (MAC) will help the clarity in relative to C and D. b. Also, the legend incorporated in the figures C and D needs to be adjusted to incorporate explanation for the “infection” part of the images. 9. Line 541-542: Please consider changing “and enhances” to “and enhancement of”. 10. Figure 7: a. Are graphs B and C based on Western Blot (not shown) or immunofluorescent images (A). If based on Western Blot then please add blot images. If based on immunofluorescent then the almost doubling of the expression noted in “B” (MET vs CTRL) does not appear to be supported by the image in “A”. 11. Lines 575 ot 577: a. “Bactericidal activity” (used later in the text) could be considered instead of “bacterial killing”. 12. Line 579: Please consider “involved” instead of “functioning”. 13. Line 612: Please consider removing “our”. 14. Line 614: Please consider changing “resulting” to “results” and the “ΔΨm” to “MMP” as mentioned earlier in the manuscript. 15. Line 618: Please either consider removing “and immune cells” or adding “perhaps other” before it. 16. Lines 631-632: Please consider minor revision for clarity. 17. Lines 650-651: Please consider revision/breaking the sentence. 18. Lines 672-674: Please consider revision for better clarity. 19. Line 691: Please consider adding “and” after “bioenergetics,”. ****** 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. https://doi.org/10.1371/journal.pone.0310908.r003
Revision 2
16 May 2024 Author Response Response to Reviewers: Reviewer #1: Comment: The revised manuscript has been greatly improved. However, the in vivo studies can not prove that the altered expression of PGC-1α or TFAM, caused by NTM, occurred in macrophages. Response: We agree with the reviewer and have now removed the in vivo data and are working on more in details studies. This manuscript will focus on the in vitro mechanistic studies. Reviewer #2: I thank the authors for their responses. Comment: Line 152 (revised version): What dose of oral metformin in an average human would be equivalent to the final concentration of 2 mM metformin mentioned in the manuscript. Please add to the manuscript. Appreciate authors’ comments. It would be a plus for the manuscript if their explanation could be incorporated into the manuscript. Response: Appreciate the reviewer bringing up the issue of dose of Metformin. The maximum dose of oral metformin in clinical practice is 2.5–3 g per day, which is approximately 35–42 mg/kg. In humans, the plasma concentration range of metformin after passing through the liver is usually 10–40 μM. For an invitro dose, 2 mM is would be comparable to the human doses on the higher side. This explanation has been added to the discussion of the manuscript. Comment: Line 305 (revised version): Please add what the cells are treated with. Response: We have now added the cells. “THP-1 cells were cultured, treated with metformin [2 mM], ZLN005 [2 µM], and SR [10 µM], and infected in 96-well plates (Grenier Bio-One) as previously described.” Comment: Figure 1C and 1D: I think there should be a mistake as the picture appears to be the same and not replaced (please see below). a. The MitoSOX staining for CTRL seems to be nothing (at least not visible on the provided image) where as mentioned as about 1 on Figure 1D. Appreciated comment clarification. b. MitoSox-based flow cytometry could be considered as an alternative. Response: We appreciate the comment and regret the oversight. Figure 1C was updated to include a new image panel for the Control group, and 1D was revised with updated statistical analysis. The Control group exhibited low expression of MitoSOX staining in all replicates used for analysis. This was seen in multiple experiments performed. Comment: Figure 2: I appreciated author’s comments. As mentioned in the original comments showing the changes specifically in macrophages (by cell sorting or staining) is very important given the focus of this manuscript and the author’s prior work. These experiments should be very straightforward and will significantly improve the manuscript. However, if further experiments are not feasible or can’t be done then this section could be omitted particularly given that the per authors the in vivo studies are beyond the scope of this manuscript or at least the limitation clearly mentioned. Response: We appreciate the reviewer comments and have now opted to remove the in vivo data. Comment: Lines 434-436: Language needs revision for better clarity. Response: This was related to the in vivo data, which has been removed, the paragraph has been revised accordingly. Comment: Figure 4 related: a. Text mentions PGC-1a while figure A and B mention “ppargc1a” (e.g., line 440 could change to “decreases in PGC1-a mRNA (ppargc1a)”. b. Figure 4 (K, L, M, N): While figures K and L mention “ZLN + MAC”, figures M and N and the text mention MET + MAC. Which one is it? The correct image should be MET + MAC to confirm to the text and the rest of images. Please rectify. Response: a. The text was edited to specify the protein and RNA forms of PGC-1α and TFAM; PGC-1α mRNA as “ppargc1a” and TFAM mRNA as “tfam” b. The figures are edited to add the proper treatment conditions. Comment: Figure 5: Again: a. SR (I assume stands for SR-18292) is mentioned in the figure but no mention or explanation of SR in either the text or the figure legend. b. Although SR is mentioned on line 510 (and not 520) and under next topic and in relation to Figure 6, it should be mentioned at the first appearance for clarity. Response: Appreciate the comment, regret the oversight. This has been corrected. SR-18292 is referred to as “SR” first in the materials and methods (line 154), and in the main text body (line 540) Comment: Figure 6: a. A and B. Moving rows 7 to just above current row 4 (MAC) will help the clarity in relative to C and D. b. Also, the legend incorporated in the figures C and D needs to be adjusted to incorporate explanation for the “infection” part of the images. Response: The image panels were adjusted for better clarity and organization. The figure legend was updated to include “Uninfected” and “MAC” on the X axis. Comment: Line 541-542: Please consider changing “and enhances” to “and enhancement of”. Response: This has been corrected, “and enhances” was revised to “and enhancement of” Comment: Figure 7: a. Are graphs B and C based on Western Blot (not shown) or immunofluorescent images (A). If based on Western Blot then please add blot images. If based on immunofluorescent then the almost doubling of the expression noted in “B” (MET vs CTRL) does not appear to be supported by the image in “A”. Response: The graphs for Figure 7 are quantified from the captured immunofluorescent images. The data from the immunofluorescent images was re-quantified and reanalyzed to provide a revised graphical representation. Comment: Lines 575 ot 577: a. “Bactericidal activity” (used later in the text) could be considered instead of “bacterial killing”. Response: This has been revised, we show that activation of PGC-1α enhances phagocytosis and bactericidal activity in NTM infected macrophages. Comment: Line 579: Please consider “involved” instead of “functioning”. Response: This is edited to, “the immune response generated against NTM infections encompasses multiple cell types, however macrophages are the primary cells involved in initial defense against inhaled mycobacteria, making their activity indispensable for an efficient immune response” Comment: Line 612: Please consider removing “our”. Response: “our” has been removed Comment: Line 614: Please consider changing “resulting” to “results” and the “ΔΨm” to “MMP” as mentioned earlier in the manuscript. Response: revised to “In accordance with other literature, MAB and MAC induce mitochondrial damage in macrophages that result in induced deficits in integrity of both mtDNA and MMP.” Comment: Line 618: Please either consider removing “and immune cells” or adding “perhaps other” before it. Response: Deficits in energy production and mitochondrial activity negatively impact the ability of macrophages and perhaps other immune cells to effectively combat bacterial infections. Comment: Lines 631-632: Please consider minor revision for clarity. Response: this has been revised, “ In addition to control over mitochondrial biogenesis, PGC-1α exerts multiple beneficial effects in immune cells, including expression of cytoprotective and antioxidant proteins. Comment: Lines 650-651: Please consider revision/breaking the sentence. Response: this has been revised, Our data show that NTM infections in vitro downregulate both PGC-1α and TFAM, two critical determinants of mitochondrial function that can impact the immune function of macrophages. Comment: Lines 672-674: Please consider revision for better clarity. Response:Sentences were revised to: “Metformin has been shown to reduce the severity of bacterial infections in preclinical and clinical retrospective studies, although the exact mechanisms remain elusive.The potential mechanisms by which Metformin exerts beneficial effects and rescues mitochondrial bioenergetics are related to its activation of AMPK and downstream activation of PGC-1α.” Comment: Line 691: Please consider adding “and” after “bioenergetics,”. Response: The wording was modified to read “In conclusion, our study shows that NTM infected macrophages exhibit mitochondrial damage with impaired bioenergetics and increased ROS production.” Attachments Attachment Submitted filename: Response to Reviewers PlosOne April 1 2024.pdf https://doi.org/10.1371/journal.pone.0310908.r004
19 Jun 2024 Decision Letter - Frederick Quinn, Editor PONE-D-23-34313R2PGC-1α Activation to Enhance Macrophage Immune Function in Mycobacterial InfectionsPLOS ONE Dear Dr. Sadikot, 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 Aug 03 2024 11:59PM. If you will need significantly more time 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, Frederick Quinn 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 #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: 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: 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 #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: Few issues: 1. Ethics section, lines 320-322 of the first revised version has been omitted from the second (current) revised version. Please clarify. 2. Line 355 of the current version (ppargc1a) should be in italic 3. Line 357 of the current version (tfam) should be in italic 4. Figure 2 (2A to 2L) corresponding to data related to ZLN005 is missing. ****** 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.0310908.r005
Revision 3
21 Jul 2024 Author Response We appreciate the reviewers detailed review and comments for correction. We have incorporated all the changes. Reviewer Two Comment 1. Ethics section, lines 320-322 of the first revised version has been omitted from the second (current) revised version. Please clarify. Response The studies do not have any human specimens therefore the ethics section is omitted Comment 2. Line 355 of the current version (ppargc1a) should be in italic Response This has been reformatted Comment 3. Line 357 of the current version (tfam) should be in italic Response: This has been formatted Comment: 4. Figure 2 (2A to 2L) corresponding to data related to ZLN005 is missing. Response: ZLN005 has been added Attachments Attachment Submitted filename: Response to reviewers PlosOne June 20 2024.docx https://doi.org/10.1371/journal.pone.0310908.r006
20 Aug 2024 Decision Letter - Frederick Quinn, Editor PONE-D-23-34313R3PGC-1α Activation to Enhance Macrophage Immune Function in Mycobacterial InfectionsPLOS ONE Dear Dr. Sadikot, 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 Oct 04 2024 11:59PM. If you will need significantly more time 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, Frederick Quinn 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 #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: 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: 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 #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: Although this manuscript is interesting and worthy of publication, it appears that the manuscript is not carefully prepared evident by multiple errors over several revisions which has unfortunately continued even in the most recent version. Figure 2, is not incorporated into the figures and is separately added out of place at the end of the figures. The quality of the figure 2 is not great and apparently does not have a corresponding TIFF file (or at least this reviewer could not download it). Therefore, as is, figure 2 is not suitable for publication. Please make sure that a good quality figure is added and properly incorporated in its right place among figures Line 363: It seems that “and TFAM (FIG 2F)” should be “TFAM (FIG 2G)” Line 367: It seems that “PGC-1a (FIG 2G)” should be “PGC-1a (FIG 2F)” Please make the above correction and make sure that next version is thoroughly reviewed and does not have any more errors. Thank you ****** 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.0310908.r007
Revision 4
22 Aug 2024 Author Response All requested changes have been incorporated into the manuscript and figure files. Reviewer Two Comment: Reviewer #2: Although this manuscript is interesting and worthy of publication, it appears that the manuscript is not carefully prepared evident by multiple errors over several revisions which has unfortunately continued even in the most recent version. Figure 2, is not incorporated into the figures and is separately added out of place at the end of the figures. The quality of the figure 2 is not great and apparently does not have a corresponding TIFF file (or at least this reviewer could not download it). Therefore, as is, figure 2 is not suitable for publication. Please make sure that a good quality figure is added and properly incorporated in its right place among figures Response: Figure 2 was reformatted to a higher quality TIFF file and uploaded in the proper sequence. Comment: Line 363: It seems that “and TFAM (FIG 2F)” should be “TFAM (FIG 2G)” Response: The proper figure title was added Comment: Line 367: It seems that “PGC-1a (FIG 2G)” should be “PGC-1a (FIG 2F)” Response: The proper figure title was added Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0310908.r008
9 Sep 2024 Decision Letter - Frederick Quinn, Editor PGC-1α Activation to Enhance Macrophage Immune Function in Mycobacterial Infections PONE-D-23-34313R4 Dear Dr. A little , 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Frederick Quinn 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: (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: 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: 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 #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: Acceptable for publication with following corrections: - Line 406: "FIG 3F" to "FIG 3G" - Line 409: "FIG 3G" to "FIG 3F" ****** 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.0310908.r009
Formally Accepted
28 Nov 2024 Acceptance Letter - Frederick Quinn, Editor PONE-D-23-34313R4 PLOS ONE Dear Dr. Sadikot, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Frederick Quinn Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0310908.r010

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