Intracellular accumulation of free cholesterol in macrophages triggers a PARP1 response to DNA damage and PARP1 impairs lipopolysaccharide-induced inflammatory response

Kenneth K.Y. Ting; Hisham M. Ibrahim; Nitya Gulati; Yufeng Wang; Jonathan V. Rocheleau; Myron I. Cybulsky

doi:10.1371/journal.pone.0318267

Peer Review History

Original SubmissionJuly 9, 2024
9 Jul 2024 Author Response https://doi.org/10.1371/journal.pone.0318267.r001
7 Aug 2024 Decision Letter - Zhiming Li, Editor PONE-D-24-28324Intracellular accumulation of free cholesterol in macrophages triggers a PARP1 response to DNA damage and PARP1 impairs lipopolysaccharide-induced inflammatory responsePLOS ONE Dear Dr. Cybulsky, 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 Sep 21 2024 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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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Ting et al. evidence a pathway stemming from oxLDL/cholesterol-induced metabolic rearrangements yielding reactive species from the mitochondria leading to PARP activation and proinflammatory cytokine production. The study is very relevant from the perspective of foam cell formation and atherosclerosis. The paper needs to be amended as follows: Please do not resolve “PARP”. See PMID: 34323016. The interactions between cholesterol and PARP enzmyes is more complex, please see PMID: 34450194. Please amend the discussion accordingly. Please report (RT)-qPCR reactions according to PMID: 19246619, PMID: 32746458. AG-14361 is not the available best inhibitor. Please repeat key experiments with rucaparib or talazoparib. I am not sure that the inhibition of NAMPT alone is sufficient to block NAD+ salvage, NMNAT blockade would be also necessary. Does PARP inhibition modulate the expression of ABCA1, in other words, is there a difference in cholesterol efflux from cells? The authors introduce HIF1 as a key pathway for the metabolic switch in macrophages, later shown NRF2 activation. HIF1 and NRF2 systems are interconnected. that should be discussed. The PAR blots should be shown as a whole with MW standards. These blots are either cut/cropped or are not showing the usual PARylation pattern. This a key point that needs to be cleared. Please note that pharmacological PARP inhibitors block both PARP1 and PARP2, hence only by pharmacology it is not possible to distinguish between them. PARP2 is also associated with inflammation and cholesterol metabolism (PMID: 38698556). Please either introduce genetic studies or reshape the discussion. Furthermore, PARPs do modulate metabolism, hence the glycolytic switch may be related to PARP activation. This needs to be at least discussed. Typo: line 215 “purines and pyrimidines derivatives” correctly: purine and pyrimidine derivatives. line 335 Please add the missing reference. ****** 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 ******** [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.0318267.r002
Revision 1
7 Jan 2025 Author Response RESPONSE TO THE REVIEW We would like to thank the reviewer for the insightful and helpful comments. Each point raised by the reviewer is in bold font and our response is below. Changes to the text in the revised manuscript are highlighted in yellow. Please do not resolve „PARP”. See PMID: 34323016. We would like to thank the reviewer for pointing out the correct nomenclature for mammalian ADP-ribosyltransferases. We now use the term diphtheria toxin-like ADP-ribosyltransferase (ARTD) family. The interactions between cholesterol and PARP enzymes is more complex, please see PMID: 34450194. Please amend the discussion accordingly. Thank you for highlighting the complex interaction between cholesterol and PARP enzymes. Indeed, as discussed in the review recommended by the reviewer (PMID: 34450194), various lipid-associated molecules are linked to the activation of PARP1. PARP1 inhibition has also been reported to enhance cholesterol efflux in macrophages through ABCA1 in a LXR-dependent manner, thus implicating that PARP1 may play a pivotal role in regulating cholesterol levels and inflammation in foam cells. As part of our revision, we addressed this experimentally (new Supplementary Figure 2A and 2B) and included a discussion of these results in the revised manuscript. Please report (RT)-qPCR reactions according to PMID: 19246619, PMID: 32746458. Thank you for pointing out these publications. Based on section 7 of PMID 19246619, we have provided additional information in the methods section of the revised manuscript, including databased accession numbers of each target and reference gene, the exon locations of each primer, and details about qPCR reactions, such as volumes and primer concentration. AG-14361 is not the available best inhibitor. Please repeat key experiments with rucaparib or talazoparib. We have repeated the experiment shown in Figure 3F with rucaparib. These data are presented in Supplementary Figure 2A and 2B. The data suggest that the enhanced consumption of NAD+ in cholesterol loaded macrophages is PARP1-dependent. This is reproducible with or without LPS stimulation. Other experiments in which AG-14361 was used, such as Figure 3H and 3I, were repeated with siRNA against PARP1, as shown in Figure 6. We consider the siRNA approach more specific than pharmacological inhibition. I am not sure that the inhibition of NAMPT alone is sufficient to block NAD+ salvage, NMNAT blockade would be also necessary. The reviewer raises an interesting point. Our data show that NAMPT blockade is sufficient to block NAD+ salvage in LPS-activated, but not in unstimulated, macrophages. For instance, in unstimulated macrophages without cholesterol loading (white bars in the left panel of Figure 3E), the abundance of NAD+ did not change during NAMPT inhibition. However, when these cells were stimulated with LPS (white bars in the right panel of Figure 3E), NAD+ was reduced by NAMPT inhibition. In fact, this phenomenon was reported by Edward Pearce’s group (Cameron et al., Nat Immunol 2019). These authors found that NAD+ was reduced in LPS-stimulated macrophages and NAD+ levels were heavily dependent on the activity of NAMPT. Based on our data and published data from the Pearce lab, we feel that NAMPT blockade is sufficient to block NAD+ salvage in LPS-activated macrophages and NMNAT blockade is not necessary in this context. Does PARP inhibition modulate the expression of ABCA1, in other words, is there a difference in cholesterol efflux from cells? The reviewer raises an interesting point by inquiring if PARP inhibition may modulate the expression of ABCA1 and cholesterol efflux. Shrestha et al (JBC, 2016) found that inhibition of PARP1 in RAW264.7 macrophages and bone-marrow derived macrophages is linked to the augmentation of ABCA1 expression in a LXR-dependent manner. To determine if these findings can be reproduced in peritoneal macrophages, we inhibited PARP activity with rucaparib 1h prior to cholesterol loading and perform immunoblotting to measure ABCA1 expression. As shown in Supplementary Figure 2C, cholesterol loading of peritoneal macrophages significantly induced the expression of ABCA1. However, PARP1 inhibition did not modulate ABCA1 protein expression, which suggests that PARP1 inhibition in peritoneal macrophages may not play a significant role in modulating ABCA1 expression and cholesterol efflux. The authors introduce HIF1 as a key pathway for the metabolic switch in macrophages, later shown NRF2 activation. HIF1 and NRF2 systems are interconnected. that should be discussed. As suggested, we have modified the discussion. We discuss the involvement of NRF2 and HIF-1 in our model, as well as their interconnection. The PAR blots should be shown as a whole with MW standards. These blots are either cut/cropped or are not showing the usual PARylation pattern. This a key point that needs to be cleared. Thank you for pointing out the lack of MW standards in our PAR blots. We have now included these in all immunoblots in the revised manuscript. The PAR blots that we have presented are indeed cropped, specifically we have cropped all the bands below 63 kDa. Based on the titrated concentration of PARP1 inhibitor (AG-14361) shown in the uncropped original blot of Figure 2C from our initial submission (see below), we believed that all the bands below the 63 kDa marker are nonspecific as their expression persists at higher concentrations of AG-14361, whereas signals at higher molecular weight decreased. As per PLOS ONE policy, authors are required to provide all original uncropped blots with MW standards as supplementary files. We have now provided the uncropped immunoblots with MW standards as supplementary files. Please note that pharmacological PARP inhibitors block both PARP1 and PARP2, hence only by pharmacology it is not possible to distinguish between them. PARP2 is also associated with inflammation and cholesterol metabolism (PMID: 38698556). Please either introduce genetic studies or reshape the discussion. Furthermore, PARPs do modulate metabolism, hence the glycolytic switch may be related to PARP activation. This needs to be at least discussed. Although the reviewer raises a valid point in pointing out pharmacological PARP inhibitors can block both PARP1 and PARP2, the lack of PARP inhibitor specificity was addressed in Figure 6 by siRNA knockdown of PARP1 in RAW 264.7 macrophages. We would like to thank the reviewer for pointing out that PARPs can modulate metabolism and hence the glycolytic switch. As suggested, we have modified the discussion. Typo: line 215 „purines and pyrimidines derivatives” correctly: purine and pyrimidine derivatives. We have fixed these typos. line 335 Please add the missing reference. We have added the missing reference. Attachments Attachment Submitted filename: 1_Response_to_review.docx https://doi.org/10.1371/journal.pone.0318267.r003
14 Jan 2025 Decision Letter - Zhiming Li, Editor Intracellular accumulation of free cholesterol in macrophages triggers a PARP1 response to DNA damage and PARP1 impairs lipopolysaccharide-induced inflammatory response PONE-D-24-28324R1 Dear Dr. Cybulsky, 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, Zhiming Li, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #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.0318267.r004
Formally Accepted
Acceptance Letter - Zhiming Li, Editor PONE-D-24-28324R1 PLOS ONE Dear Dr. Cybulsky, 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. Zhiming Li Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0318267.r005

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