Mitochondrial oxidative phosphorylation in peripheral blood mononuclear cells is decreased in chronic HIV and correlates with immune dysregulation

Louie Mar A. Gangcuangco; Brooks I. Mitchell; Chathura Siriwardhana; Lindsay B. Kohorn; Glen M. Chew; Scott Bowler; Kalpana J. Kallianpur; Dominic C. Chow; Lishomwa C. Ndhlovu; Mariana Gerschenson; Cecilia M. Shikuma

doi:10.1371/journal.pone.0231761

Peer Review History

Original SubmissionAugust 28, 2019
14 Nov 2019 Decision Letter - Qigui Yu, Editor PONE-D-19-24311 Mitochondrial oxidative phosphorylation in peripheral blood mononuclear cells is decreased in chronic HIV and correlates with immune dysregulation PLOS ONE Dear Dr. Louie Gangcuangco, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but need a minor revision to fully meet PLOS ONE’s publication criteria. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. ==============================Reviewer #1 Although the work is interesting and relevant to the overall knowledge in this field, I have some specific points which needs to be addressed. 1. What is reason for not incorporating the HIV-seronegative data? Need to incorporate all the HIV-seronegative data (as PLHIV) in the table 1 for better comparison. 2. How specific the method used for measuring Complex I? The authors need to incorporate more data using specific standards/inhibitors of both complex I and IV to measure their data set more stringently. 3. Also, to make the case stronger the authors needs to incorporate immunostaining data (FACs) to measure both complex I & IV. What is the activity level of these enzymes? 4. What is viability of the PBMC used? 5. What is status of mitochondrial ROS in these samples as this is one of the important functional parameters? 6. ‘We found that the presence of mitochondrial oxidative damage, as assessed by mt-specific 8-oxo-dG, was associated with higher CI and CIV protein levels’ – please explain elaborately as you didn’t find any changes on CIV. 7. Needs more explanation in the discussion section about decreased complex I and unchanged complex IV. Reviewer #2This is an innovative study that examines a seldom explored facet of immunometabolism during HIV infection. The paper by Louie Mar A. Gangcuangco et al. explored the relationship between oxidative phosphorylation in peripheral blood mononuclear cells (PBMCs) and the frequency of monocyte subsets and markers of T-cell activation, senescence, and exhaustion. The study was made of 149 samples from people living with HIV (PLHIV) on ART and 44 samples from HIV-negative individuals. Oxidative Phosphorylation was quantified by measuring levels of Complex 1/4 proteins of the electron transport chain as well as an oxidative stress marker. The frequency of monocyte subsets and markers of T-cell activation, senescence, and exhaustion were analyzed using flow cytometry and levels of soluble inflammatory cytokines were determined using a multiplex assay. The PBMC levels of OXPHOS complex I were decreased in PLHIV on ART and the decreased OXPHOS correlated with disease severity and inflammation. This is an innovative study and the significance was well emphasized. Some minor concerns are described as followed; 1) The study compared PLHIV undergoing ART+ and uninfected samples. There should be an additional control of samples from treatment-naive HIV infected individuals. Because ART can effect mitochondria during treatment, there needs to be control without ART to determine what influence ART had on the phenotype and correlation seen in the paper. 2) The paper uses levels of Complex 1/4 proteins as well as an oxidative stress marker as a measurement of mitochondrial dysregulation and OxPhos level. This is not an established method of determining either of the two. The study would be better served measuring the ratio of mitochondrial membrane polarization versus number of mitochondria to represent dysregulation. Moreover, functional analysis of mitochondrial metabolism (OxPhos) is most commonly done via Seahorse Oxygen Consumption Assays. Low levels of complex1/4 proteins may be mitigated by compensatory mechanisms. Directly measuring the levels of oxygen consumption as a marker for OxPhos will, therefore, be a much more accurate method than those used in the study. ============================== We would appreciate receiving your revised manuscript by 02/14/2020. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols 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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Qigui Yu, M.D./Ph.D Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: 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 is an innovative study that examines a seldom explored facet of immunometabolism during HIV infection. The paper by Louie Mar A. Gangcuangco et al. explored the relationship between oxidative phosphorylation in peripheral blood mononuclear cells (PBMCs) and the frequency of monocyte subsets and markers of T-cell activation, senescence, and exhaustion. The study was made of 149 samples from people living with HIV (PLHIV) on ART and 44 samples from HIV-negative individuals. Oxidative Phosphorylation was quantified by measuring levels of Complex 1/4 proteins of the electron transport chain as well as an oxidative stress marker. The frequency of monocyte subsets and markers of T-cell activation, senescence, and exhaustion were analyzed using flow cytometry and levels of soluble inflammatory cytokines were determined using a multiplex assay. The PBMC levels of OXPHOS complex I were decreased in PLHIV on ART and the decreased OXPHOS correlated with disease severity and inflammation. This is an innovative study and the significance was well emphasized. Some minor concerns are described as followed; 1) The study compared PLHIV undergoing ART+ and uninfected samples. There should be an additional control of samples from treatment-naive HIV infected individuals. Because ART can effect mitochondria during treatment, there needs to be control without ART to determine what influence ART had on the phenotype and correlation seen in the paper. 2) The paper uses levels of Complex 1/4 proteins as well as an oxidative stress marker as a measurement of mitochondrial dysregulation and OxPhos level. This is not an established method of determining either of the two. The study would be better served measuring the ratio of mitochondrial membrane polarization versus number of mitochondria to represent dysregulation. Moreover, functional analysis of mitochondrial metabolism (OxPhos) is most commonly done via Seahorse Oxygen Consumption Assays. Low levels of complex1/4 proteins may be mitigated by compensatory mechanisms. Directly measuring the levels of oxygen consumption as a marker for OxPhos will, therefore, be a much more accurate method than those used in the study. Reviewer #2: Although the work is interesting and relevant to the overall knowledge in this field, I have some specific points which needs to be addressed. 1. What is reason for not incorporating the HIV-seronegative data? Need to incorporate all the HIV-seronegative data (as PLHIV) in the table 1 for better comparison. 2. How specific the method used for measuring Complex I? The authors need to incorporate more data using specific standards/inhibitors of both complex I and IV to measure their data set more stringently. 3. Also, to make the case stronger the authors needs to incorporate immunostaining data (FACs) to measure both complex I & IV. What is the activity level of these enzymes? 4. What is viability of the PBMC used? 5. What is status of mitochondrial ROS in these samples as this is one of the important functional parameters? 6. ‘We found that the presence of mitochondrial oxidative damage, as assessed by mt-specific 8-oxo-dG, was associated with higher CI and CIV protein levels’ – please explain elaborately as you didn’t find any changes on CIV. 7. Needs more explanation in the discussion section about decreased complex I and unchanged complex IV. ****** 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: Yes: Fahim Syed 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0231761.r001
Revision 1
14 Feb 2020 Author Response February 9, 2020 Qigui Yu, MD, PhD Academic Editor PLOS ONE Re: Mitochondrial oxidative phosphorylation in peripheral blood mononuclear cells is decreased in chronic HIV and correlates with immune dysregulation (PONE-D-19-24311) Dear Dr. Yu: We would like to thank you and the Reviewers for their careful review of our manuscript. The reviewers thought the work was ‘interesting and relevant to the overall knowledge in the field’ and ‘innovative and significant’. We have addressed their minor revisions below and in the revised manuscript. Reviewer 1 Although the work is interesting and relevant to the overall knowledge in this field, I have some specific points which needs to be addressed. 1. What is reason for not incorporating the HIV-seronegative data? Need to incorporate all the HIV-seronegative data (as PLHIV) in the table 1 for better comparison. Unfortunately, mt-specific 8-oxo-dG and other immune parameters (cytokines, monocyte subsets, and NCRs) were not available for the HIV-seronegative controls. We included this in the limitations section in the Discussion. Table 1 would therefore have several missing cells if an HIV-seronegative column is included. To compare and clarify the demographic characteristics of HIV-seronegatives, the following has been added to the first paragraph of the Results section (lines 158-162): “Compared with PLHIV, the HIV-seronegative group (N=44) had similar age (51.0 years in PLHIV vs 54.2 years in HIV-seronegatives, p=0.36), gender (88.6% vs 81.8% male, p=0.25), and ethnicity (57.7% vs 61.4% Caucasian, p=0.67).” 2. How specific the method used for measuring Complex I? The authors need to incorporate more data using specific standards/inhibitors of both complex I and IV to measure their data set more stringently. Complex I was measured as described in reference 9: PMCID: PMC2649940. Complex I and IV protein levels were measured and not activity, thus inhibitors were not used (e.g. rotenone for Complex I activity, etc.). Additional text describing the methodology has been added to lines 112-122. 3. Also, to make the case stronger the authors need to incorporate immunostaining data (FACs) to measure both complex I & IV. What is the activity level of these enzymes? We agree with the reviewer that immunostaining of the PBMCS for Complex I and IV would have been feasible. We did not have enough PBMCs for both FACS and the immunoassays. We measured protein levels, not activity. 4. What is viability of the PBMC used? The viability of the cells was 90-95%. Cell viability was determined using AOPI (acridine orange/propidium iodide). A statement was included in the Methods section. 5. What is status of mitochondrial ROS in these samples as this is one of the important functional parameters? We measured mitochondrial ROS by measuring the amount of oxidized guanine in the mtDNA. We added a reference in the Methods section for PMID 19321503, Gerschenson et al. Mitochondrial 8-oxo-dG damage was assessed using a Gene-Specific Repair Assay. Ten micrograms of PBMC DNA was isolated with a DNeasy Blood and Tissue Kit (Qiagen, Inc.). DNA was then digested with PvuII (New England BioLabs, Inc., Ipswich, MA, USA) overnight to linearize mtDNA. Digested DNA was halved: 5 µg of DNA was treated with human 8-oxoguanine DNA glycosylase (hOGG1) for 1 h at 37°C in a reaction volume of 15 µL and then for 1 h at 65°C for enzyme deactivation, and the remaining 5 µg of DNA was left untreated and stored at 4°C. For analysis, 4 µL of 1× Alkaline Agarose Loading Dye (Boston Bioproducts, MA, USA) was added, and cleaved and non-cleaved products were resolved on a 0.75% alkaline agarose gel. DNA was transferred to nylon (+) membranes using standard Southern blot methodology. Human mitochondrial probes specific for cytochrome b were labelled with digoxigenin-dUTP (Roche) by linear PCR amplification. Primer sequences were: DigFor, GCT ACC TTC ACG CCA A (14 976–15 001); and DigRev, CCG TTT CGT GCA AGA AT (15 357–15 341). Blots were hybridized overnight at 45°C and processed for chemiluminescent detection following Roche protocols. Finally, membranes were developed on a chemilumi imager (Roche) using LumiAnalyst software (Roche). Mitochondrial 8-oxo-dG damage was quantified by calculating break frequencies (BFs) based on the Poisson distribution of DNA treated with the hOGG1 repair enzyme and DNA untreated. 6. ‘We found that the presence of mitochondrial oxidative damage, as assessed by mt-specific 8-oxo-dG, was associated with higher CI and CIV protein levels’ – please explain elaborately as you didn’t find any changes on CIV. We hypothesize that the lack of changes in Complex IV protein levels may be due to the electron transport compensation via Complexes II-IV. Text was added in Lines 272 to 275. 7. Needs more explanation in the discussion section about decreased complex I and unchanged complex IV. See lines 271 and 272. For instance, decreased Complex I activity in skeletal muscle has been associated with normalized Complex IV in human peripheral arterial disease (Pubmed: 11158957). Reviewer #2 This is an innovative study that examines a seldom explored facet of immunometabolism during HIV infection. The paper by Louie Mar A. Gangcuangco et al. explored the relationship between oxidative phosphorylation in peripheral blood mononuclear cells (PBMCs) and the frequency of monocyte subsets and markers of T-cell activation, senescence, and exhaustion. The study was made of 149 samples from people living with HIV (PLHIV) on ART and 44 samples from HIV-negative individuals. Oxidative Phosphorylation was quantified by measuring levels of Complex 1/4 proteins of the electron transport chain as well as an oxidative stress marker. The frequency of monocyte subsets and markers of T-cell activation, senescence, and exhaustion were analyzed using flow cytometry and levels of soluble inflammatory cytokines were determined using a multiplex assay. The PBMC levels of OXPHOS complex I were decreased in PLHIV on ART and the decreased OXPHOS correlated with disease severity and inflammation. This is an innovative study and the significance was well emphasized. Some minor concerns are described as followed; 1) The study compared PLHIV undergoing ART+ and uninfected samples. There should be an additional control of samples from treatment-naive HIV infected individuals. Because ART can effect mitochondria during treatment, there needs to be control without ART to determine what influence ART had on the phenotype and correlation seen in the paper. We concur with the reviewer that it would be useful to have a treatment-naive HIV infected individuals assayed. These PBMCs are unavailable in our current banked specimens, since most PLHIV are treated as soon as they are diagnosed per CDC recommendations (https://www.helpstoptheviruspro.com/start-hiv-treatment/treat-hiv-asap?sitelink=treating+hiv+asap&utm_medium=cpc&utm_campaign=2019_Treatment+-+Broad&utm_content=Strategy&utm_term=%2Bguideline+%2Bhiv&moc=HSTV01&utm_source=bing&gclid=CK_F48GtxecCFTuJxQIdNHcCVg&gclsrc=ds). 2) The paper uses levels of Complex 1/4 proteins as well as an oxidative stress marker as a measurement of mitochondrial dysregulation and OxPhos level. This is not an established method of determining either of the two. The study would be better served measuring the ratio of mitochondrial membrane polarization versus number of mitochondria to represent dysregulation. Moreover, functional analysis of mitochondrial metabolism (OxPhos) is most commonly done via Seahorse Oxygen Consumption Assays. Low levels of complex1/4 proteins may be mitigated by compensatory mechanisms. Directly measuring the levels of oxygen consumption as a marker for OxPhos will, therefore, be a much more accurate method than those used in the study. We agree with the Reviewer that that the Seahorse assays and membrane polarization would have given mechanistic information. We developed the OXPHOS immunoassays (reference 9, 26762648, 26002840) and have used the mitochondrial 8-oxo-guanine assays (PMID 19321503, 2257461, 25574857) as an initial assay to evaluate mitochondrial function. We do not have viable PBMCs to conduct Seahorse experiments and will do so in future studies. Once again, we thank the reviewers for their valuable feedback. We hope that we have addressed the queries adequately. Sincerely, Louie Mar Gangcuangco, MD Attachments Attachment Submitted filename: Response to Reviewers PlosOne Oxphos and NCRs 12Feb2020.docx https://doi.org/10.1371/journal.pone.0231761.r002
1 Apr 2020 Decision Letter - Qigui Yu, Editor Mitochondrial oxidative phosphorylation in peripheral blood mononuclear cells is decreased in chronic HIV and correlates with immune dysregulation PONE-D-19-24311R1 Dear Dr. Gangcuangco, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Qigui Yu, M.D./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 Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes: Fahim Syed Reviewer #2: No https://doi.org/10.1371/journal.pone.0231761.r003
Formally Accepted
20 Apr 2020 Acceptance Letter - Qigui Yu, Editor PONE-D-19-24311R1 Mitochondrial oxidative phosphorylation in peripheral blood mononuclear cells is decreased in chronic HIV and correlates with immune dysregulation Dear Dr. Gangcuangco: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Qigui Yu Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0231761.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .