LPS induces inflammatory chemokines via TLR-4 signalling and enhances the Warburg Effect in THP-1 cells

Philemon Ubanako; Ntombikayise Xelwa; Monde Ntwasa

doi:10.1371/journal.pone.0222614

Peer Review History

Original SubmissionJune 19, 2019
22 Jul 2019 Decision Letter - Partha Mukhopadhyay, Editor PONE-D-19-17316 LPS induces inflammatory chemokines via TLR-4 signalling and enhances the Warburg Effect in THP-1 cells PLOS ONE Dear Professor Ntwasa, 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. WE received positive feed back from reviewers. However, some major issues were raised by one reviewer and require your attention. We would appreciate receiving your revised manuscript by Sep 05 2019 11:59PM. 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. 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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, Partha Mukhopadhyay, Ph.D. Academic Editor PLOS ONE Journal Requirements: 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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: 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: The authors have tried to address the role of infection on induction of metabolic program in THP-1 cells. Addressed that metabolic changes for these cells does not affect its viability and Warburg effect does not play a role. Interestingly, very few cytokines were expressed upon infection in these cells. I would like the authors to address the following concerns. Comments: 1. I assume fig. 1a is the western blot for TLR protein. It does not seem upon induction with LPS 5ng/ml the protein does not increased while mRNA increased. How many repeats were carried out for western blot and if possible quantify after three repeats, if done. 2. No mention of western blot protocol in material method section. Authors has to be careful in this regard and update it. 3. Fig. 3a- I am not sure how many repeats were carried out for ELISA. 4. Fig. 3b-e – Again for western blots- I would like to see how many repeats were carried out and if possible can they quantify the western blots that can be comparable with the RT-PCR result. 5. This is because it is confusing for all the Figures in 3, as an example – the difference between mRNA to protein in Fig. 3b- compared to 5ng/ml+PmB to 10ng/ml LPS the mRNA level is same between these samples while the 10ng/ml LPS the protein level is high. 6. Similarly, for RANTES (Fig. 3C)- as by visualization 10ng/ml LPS has protein while 10ng/ml LPS with PmB no protein while the mRNA profile is different between these samples. 7. Thus, it is important to address this issue upon repeating western for all these genes (Fig. 3c to e) and quantify whether there is any mRNA and protein difference. 8. Since the authors have addressed the impact of cytokines and its linkage with downstream pathways. Why not address few genes that are linked to IL8 or RANTES by knocking down these genes to show its effect on its interactome? 9. I would also like to know NF kappa B expression upon these treats because it will be an important marker to show that even though NF kappa B expression increased in LPS treatment, only few of its target cytokines expression is altered. Reviewer #2: In this manuscript, the authors analyzed cytokine production and Warburg effect induced by LPS in THP-1 AML cell line. While it is already widely known that LPS induces inflammatory cytokine production, the authors report an interesting finding in the glycolytic changes in THP-1 cells. The techniques used by the authors are sound, and the results fully demonstrate their conclusion. ****** 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 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. 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Revision 1
27 Aug 2019 Author Response Reviewer #1: 1. I assume fig. 1a is the western blot for TLR protein. It does not seem upon induction with LPS 5ng/ml the protein does not increased while mRNA increased. How many repeats were carried out for western blot and if possible quantify after three repeats, if done. Fig 1a is an RT-PCR showing TLR4 mRNA expression and not a Western blot. The reviewer must have made a mistake because the legend clearly indicates this. We have highlighted the section in Figure 1 legend that shows that the blots depict RT-PCR. No repeats were performed for TLR4 expression since it is well-known that THP-1 cells express TLR4. This was just to confirm expression and induction by LPS at the low concentrations compared to published ones. The error bars were generated accidentally because the “error bars” item was ticked off on the spreadsheet “Chart Elements” menu. We apologise for this. We generated a new graph and presented it in Fig 1a. 2. No mention of Western blot protocol in material method section. Authors has to be careful in this regard and update it. We did not perform any Western blot experiments. Hence, no Western blot protocol was included. 3. Fig. 3a- I am not sure how many repeats were carried out for ELISA The ELISA assay was performed in a single experiment as a screening tool as stated in the results and discussion section of the manuscript. We validated this expression profile by using RT-PCR in two biological replicates. We are convinced about the validity of the Array result because the RT-PCR expression profile is consistent with the Array. 4. Fig. 3b-e – Again for western blots- I would like to see how many repeats were carried out and if possible can they quantify the western blots that can be comparable with the RT-PCR result. No Western analyses were conducted in this submission. The legend of Figure 3 indicates that there were two biological replicates (highlighted). 5. This is because it is confusing for all the Figures in 3, as an example – the difference between mRNA to protein in Fig. 3b- compared to 5ng/ml+PmB to 10ng/ml LPS the mRNA level is same between these samples while the 10ng/ml LPS the protein level is high. No Western analyses were conducted in this submission. The source of the confusion could be the lack of explanation for the bar graphs. We have now included in the legend that the lower panels represent quantification of the RT-PCR gels bands. They are not protein measurements. 6. Similarly, for RANTES (Fig. 3C)- as by visualization 10ng/ml LPS has protein while 10ng/ml LPS with PmB no protein while the mRNA profile is different between these samples. No Western analyses were conducted in this submission. Please see (5) above. 7. Thus, it is important to address this issue upon repeating western for all these genes (Fig. 3c to e) and quantify whether there is any mRNA and protein difference. No Western analyses were conducted in this submission. Please see (5) above. 8. Since the authors have addressed the impact of cytokines and its linkage with downstream pathways. Why not address few genes that are linked to IL8 or RANTES by knocking down these genes to show its effect on its interactome? We do agree that this study reveals a broad view and complexity of the molecular pathways induced by infection in the tumour microenvironment. However, at this stage the primary questions were about nature of the Warburg Effect in monocytes. The finer studies suggested by the reviewer are being considered for new studies where the tumour microenvironment is simulated in 3D cultures. We do appreciate these comments because they reinforce our views about the future experiments. 9. I would also like to know NF kappa B expression upon these treats because it will be an important marker to show that even though NF kappa B expression increased in LPS treatment, only few of its target cytokines expression is altered. NF Kappa B regulates multiple aspects of innate immunity and has two broad mechanisms: the (i) canonical and (ii) non-canonical pathways. It is not too surprising that not all effectors known to be mediated by it are induced as its activity is contextual (temporal and cell specific). It is, however, notable that in the profile we present, the chemokines (known to be NF-Kappa B targets) are induced but the cytokines (some known known to be NF Kappa B targets) are poorly induced or not at all). We have addressed the NF kappa B influence in the discussion section. Although we appreciate the interest of the reviewer in NF kappa B expression, we do not think that a focussed study on NF kappa be would be useful in understanding the Warburg Effect at this stage. It could probably enlighten us about cytokine expression pathways – which is not the primary interest of the manuscript. Reviewer #2: In this manuscript, the authors analyzed cytokine production and Warburg effect induced by LPS in THP-1 AML cell line. While it is already widely known that LPS induces inflammatory cytokine production, the authors report an interesting finding in the glycolytic changes in THP-1 cells. The techniques used by the authors are sound, and the results fully demonstrate their conclusion. We are grateful for these comments. Attachments Attachment Submitted filename: Response to Reviewers.doc.docx https://doi.org/10.1371/journal.pone.0222614.r002
4 Sep 2019 Decision Letter - Partha Mukhopadhyay, Editor [EXSCINDED] LPS induces inflammatory chemokines via TLR-4 signalling and enhances the Warburg Effect in THP-1 cells PONE-D-19-17316R1 Dear Dr. Ntwasa, 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, Partha Mukhopadhyay, Ph.D. Section 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.0222614.r003
Formally Accepted
12 Sep 2019 Acceptance Letter - Partha Mukhopadhyay, Editor PONE-D-19-17316R1 LPS induces inflammatory chemokines via TLR-4 signalling and enhances the Warburg Effect in THP-1 cells Dear Dr. Ntwasa: 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. Partha Mukhopadhyay Section Editor PLOS ONE https://doi.org/10.1371/journal.pone.0222614.r004

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