Profiles of lipid, protein and microRNA expression in exosomes derived from intestinal epithelial cells after ischemia-reperfusion injury in a cellular hypoxia model

Atsushi Senda; Mitsuaki Kojima; Arisa Watanabe; Tetsuyuki Kobayashi; Koji Morishita; Junichi Aiboshi; Yasuhiro Otomo

doi:10.1371/journal.pone.0283702

Peer Review History

Original SubmissionAugust 20, 2022
24 Nov 2022 Decision Letter - Jon M. Jacobs, Editor PONE-D-22-23365Multi-omics profiles of exosomes derived from intestinal epithelial cells under ischemia–reperfusion injuryPLOS ONE Dear Dr. Kojima, Thank you for submitting your manuscript to PLOS ONE. After review we feel that it has merit but requires addressed various concerns of the reviewers prior to meeting 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. Please address each point made by the reviewers specifically as noted below. Please submit your revised manuscript by Jan 06 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Multi-omics profiles of exosomes derived from intestinal epithelial cells under ischemia–reperfusion injury Title A major focus of the recently updated guidelines of the International Society for Extracellular Vesicles (ISEV) on minimal information for studies of extracellular vesicles (MISEV) is the need for appropriate nomenclature in reporting extracellular vesicle (EV) research. So I advise the authors to use a more precise term Abstract Many unimportant technical details are mentioned in the abstract. I advise the authors get rid of them. Introduction The introduction did not sufficiently shed light on the studies related to hypoxia and its effect on the components of exosomes. Methods: The mentioned method is not well explained. I believe that hypoxia tests should be conducted in a standard atmosphere such as the hypoxia chamber. The authors mentioned that the cell culture was free of serum, what is the reason? ExoQuick-TC Exosome Precipitation Solution Kit is not an accurate method for isolating exosomes. So, the authors should use a more precise phrase. For example, the concentration of extracellular vesicles. The authors did not clarify the amount of media used to obtain a sufficient amount of exosomes. In the Exo-Check Antibody Array, its method is not well explained. As well as the positive sample, which appears to have not worked, shows a clear result. Nanodrop are not a valid method for measuring exosomal RNA concentration and quality. It often shows inaccurate results and residual salts from the precipitate. The best way is by bioanalyzer How the authors ensured that their exosome content was not contaminated with some of the components present in the media. Especially since the isolation was done here by precipitation. Results: Isolation and characterization of exosomes is a major and important experiment. So it is supposed to be in the main body of the manuscript and not in the appendices. Discussion The discussion sheds light on the possible role and impact of exosomes after hypoxia. This discussion was not deep enough. Reviewer #2: Atsushi Senda et al. investigated whether the isolated intestinal epithelial cell-derived exosomes ameliorated intestinal epithelia cells hypoxia reoxygenation injury. I have some concerns. The data without in vivo experiment cannot support any valuable conclusion for clinical translation. The study is too simple and too observational. It needs more mechanism investigation. ****** 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: Faisal A A 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. 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Revision 1
17 Feb 2023 Author Response RESPONSE TO REVIEWERS’ COMMENTS We would like to thank the reviewers for their insightful comments and suggestions. Please see our point-by-point responses to reviewers’ comments below. Our responses are all labelled as “Response.” Reviewer #1: Multi-omics profiles of exosomes derived from intestinal epithelial cells under ischemia–reperfusion injury Title A major focus of the recently updated guidelines of the International Society for Extracellular Vesicles (ISEV) on minimal information for studies of extracellular vesicles (MISEV) is the need for appropriate nomenclature in reporting extracellular vesicle (EV) research. So I advise the authors to use a more precise term Response: We thank the reviewer for their suggestion. We have changed the title to “Profiles of lipid, protein and microRNA expression in exosomes derived from intestinal epithelial cells after ischemia-reperfusion injury in a cellular hypoxia model.” Abstract Many unimportant technical details are mentioned in the abstract. I advise the authors get rid of them. Response: We thank the reviewer for their comment. We agree and have reduced the description of technical details in the abstract as suggested. Introduction The introduction did not sufficiently shed light on the studies related to hypoxia and its effect on the components of exosomes. Response: We thank the reviewer for their constructive comments. We have added the following sentence to the third paragraph of the Introduction in line with their suggestion to show the findings between hypoxia and its effects on exosome components: ------------------------------------------------------------------------------------------------------------------ Corresponding to the ischemic state, cultured IECs were first placed under hypoxic conditions. Subsequently, they were returned to an oxygenated environment corresponding to the reperfusion state. Accordingly, this hypoxia reoxygenation model has been used to simulate ischemia-reperfusion injury in an in vitro setting in previous studies [14,15]. 14. Zw D, H L, Ff S, Xz F, Y Z, P L. Cystic fibrosis transmembrane conductance regulator prevents ischemia/reperfusion induced intestinal apoptosis via inhibiting PI3K/AKT/NF-κB pathway. World journal of gastroenterology. 2022;28. doi:10.3748/wjg.v28.i9.918 15. Kip AM, Soons Z, Mohren R, Duivenvoorden AAM, Röth AAJ, Cillero-Pastor B, et al. Proteomics analysis of human intestinal organoids during hypoxia and reoxygenation as a model to study ischemia-reperfusion injury. Cell Death Dis. 2021;12: 95. doi:10.1038/s41419-020-03379-9 Methods: The mentioned method is not well explained. I believe that hypoxia tests should be conducted in a standard atmosphere such as the hypoxia chamber. Response: To generate an environment analogous to that of the reperfusion period in hemorrhagic shock, cells in the hypoxia and normoxia groups were subsequently incubated in the normoxic environment for 24 h. All tests, including biological activity tests, proteome analyses, lipid analyses, and miRNA quantification, were performed in a normoxic environment. We have made appropriate changes to the revised manuscript to clarify this point. The authors mentioned that the cell culture was free of serum, what is the reason? Response: We thank the reviewer for their question. We used serum-free cell culture to avoid the potential contamination of the exosomes that could derive from the serum. We have changed our description in the revised manuscript to ensure that readers understand this point. ExoQuick-TC Exosome Precipitation Solution Kit is not an accurate method for isolating exosomes. So, the authors should use a more precise phrase. For example, the concentration of extracellular vesicles. The authors did not clarify the amount of media used to obtain a sufficient amount of exosomes. Response: We would like to thank the reviewer for raising this point. We used 80 mL of media to obtain sufficient amount of exosomes. We have added this clarification to our revised manuscript. In the Exo-Check Antibody Array, its method is not well explained. As well as the positive sample, which appears to have not worked, shows a clear result. Response: We have included a more detailed explanation in the section kindly pointed out. Our results were positive for ICAM, ALIX, CD81, ANXA5, TSG101, and positive control; intermediate for FLOT1 and EpCAM; and negative for GM130, CD63, and Blank. The difference in concentration between the two positive controls may be caused by a concentration irregularity in the sample or in the blocking agent. As for CD63 expression, the amount and type of surface antigens depends on the cell type from which the exosomes are extracted. In addition, CD63 may not be detected using the kit due to the reactivity between the antibody and cell type. The figure below (manufacturer's manual) also shows that the surface markers on exosomes in human serum and HEK cells are very different. We believe that the relatively low distribution of CD63 on the cell surface of exosomes extracted from the Caco-2 cells used in this study could not be detected, unlike other markers such as CD81. Since this is a simple kit test, if most of the exosome markers are detected, it is considered evidence for the presence of exosomes. Figures from Exo-Check Antibody Array User-manual P.5 http://www.systembio.com/wp/wp-content/uploads/Exo-Check_User-Manual.pdf Nanodrop are not a valid method for measuring exosomal RNA concentration and quality. It often shows inaccurate results and residual salts from the precipitate. The best way is by bioanalyzer Response: We appreciate the reviewer’s highly appropriate remarks. To avoid the complexity of the description, we originally referred to this as Nanodrop, but in the actual measurement, we used both Qubit and Nanodrop. As pointed out, there were samples that showed unexpected values with Nanodrop; thus, we did not perform further measurements on them. In the revised manuscript, we have amended the Methods section regarding this point and added text that we did not use a bioanalyzer as a study limitation. How the authors ensured that their exosome content was not contaminated with some of the components present in the media. Especially since the isolation was done here by precipitation. Response: We are deeply concerned about the points raised by the reviewer. We used serum-free media to avoid contamination. However, as the reviewer pointed out, the existence of contamination cannot be completely ruled out. Therefore, we decided to present a relative evaluation between the hypoxia group and normoxia group in our experimental system: In a preliminary experiment, the protein concentration and exosome particle size distribution were compared between exosome samples subjected to centrifugation at 10,000 × g and those subjected to the ExoQuick-TC followed by 0.2-µm filtration. As pointed out by the reviewer, the amount of 30–150-nm particles was lower in the exosomes obtained using ExoQuick followed by filtration than in those obtained with ultracentrifugation (82% in ultracentrifuge versus 78% ExoQuick, please refer to the figure below). To determine whether the above difference is significant, we compared the protein amount in exosomes obtained by ultracentrifuge with that in the exosomes obtained by ExoQuick. Additionally, as shown in the figure below, the correlation coefficients between the protein amounts between the Ultracentrifuge and ExoQuick groups were over 99.5% (99.3% between ultracentrifuge-isolated exosome compartment and ExoQuick-isolated exosome compartment in the hypoxia group, and 99.6% in the normoxia group).Therefore, we concluded that there was no significant difference between the ExoQuick and ultracentrifugation results and chose to perform the experiment using the Centrifugal Filter Unit for better experimental efficiency. Results: Isolation and characterization of exosomes is a major and important experiment. So it is supposed to be in the main body of the manuscript and not in the appendices. Response: We agree with the comment and have revised our manuscript as suggested. Please see Figure 1. Discussion The discussion sheds light on the possible role and impact of exosomes after hypoxia. This discussion was not deep enough. Response: We thank the reviewer for this useful suggestion. Since this study is only an observation of qualitative relationships, we have added some more to the Discussion section, but being mindful not to exceed the extent to which conclusions can be stated. Reviewer #2: Atsushi Senda et al. investigated whether the isolated intestinal epithelial cell-derived exosomes ameliorated intestinal epithelia cells hypoxia reoxygenation injury. I have some concerns. The data without in vivo experiment cannot support any valuable conclusion for clinical translation. The study is too simple and too observational. It needs more mechanism investigation. Response: We appreciate the valuable comments made by the reviewer. To better understand the significance of this study, please allow us to explain the background of our previous studies. We have focused on in vivo studies using animal models. As described in the background, we focused on in vitro experiments to prove the following assumptions. 1. We first confirmed the presence of exosomes in the lymph fluid of rats in 2017[1]. 2. We have shown that exosomes in mesenteric lymph fluid have biological activity after hemorrhagic shock in an animal model and that these exosomes induce lung injury when administered to the naive animal [2]. 3. Exosomes extracted from rat lymph fluid in a rat intestinal ischemia-reperfusion model have been shown to induce immune cell inflammation, as in the trauma hemorrhagic shock model [3]. 4. The lipid concentration in exosomes was altered after ischemia-reperfusion injury, and some lipids, such as polyunsaturated fatty acids and lysophosphatidylcholines, revealed significant changes in exosomes [3]. The above in vivo studies suggest that inflammatory exosomes extracted from the intestinal tract travel through the lymphatic system to cause inflammation in the lungs and other organs. However, lymphatic fluid contains not only exosomes derived from intestinal cells, but also exosomes secreted from vascular endothelial cells and blood cell components, and not only exosomes derived from intestinal epithelial cells. Therefore, in this study we decided to focus only on intestinal epithelial cells to clarify how their composition is altered by hypoxic stimuli and how they are involved in inflammation. We fully agree with the reviewer's point. However, we have now presented an in vitro focused study with the aim of selecting targets for future animal studies by analyzing lipids, proteins, and miRNAs in detail. We appreciate the valuable comments made by the reviewer. To better contextualize the significance of this study, we would like to provide additional information about our previous research. Our previous work has mainly focused on in vivo studies using animal models. As described in the background, we conducted in vitro experiments to confirm the following hypotheses: 1. We confirmed the presence of exosomes in the lymph fluid of rats in 2017 [1]. 2. We demonstrated that exosomes in mesenteric lymph fluid have biological activity after hemorrhagic shock in an animal model, and that these exosomes induce lung injury when administered to naive animals [2]. 3. Exosomes extracted from rat lymph fluid in a rat intestinal ischemia-reperfusion model induced immune cell inflammation, similar to the trauma hemorrhagic shock model [3]. 4. Lipid concentration in exosomes was found to be altered after ischemia-reperfusion injury, with significant changes in polyunsaturated fatty acids and lysophosphatidylcholines [3]. The above in vivo studies suggest that inflammatory exosomes extracted from the intestinal tract may cause inflammation in the lungs and other organs. However, since lymphatic fluid contains exosomes from various sources, including vascular endothelial cells and blood cell components, we decided to focus on intestinal epithelial cell-derived exosomes in this study to investigate how their composition is altered by hypoxic stimuli and their potential role in inflammation. We agree with the reviewer's point that our study is limited in scope and lacks in vivo experiments. However, we present an in vitro study to provide insights into the mechanisms underlying the protective effects of intestinal epithelial cell-derived exosomes against hypoxia reoxygenation injury, which could serve as a basis for future animal studies. We analyzed the lipids, proteins, and miRNAs in detail to identify potential targets for future studies. References 1. Kojima M, Gimenes-Junior JA, Langness S, Morishita K, Lavoie-Gagne O, Eliceiri B, et al. Exosomes, not protein or lipids, in mesenteric lymph activate inflammation: Unlocking the mystery of post-shock multiple organ failure. J Trauma Acute Care Surg. 2017;82: 42–50. doi:10.1097/TA.0000000000001296 2. Kojima M, Gimenes-Junior JA, Chan TW, Eliceiri BP, Baird A, Costantini TW, et al. Exosomes in postshock mesenteric lymph are key mediators of acute lung injury triggering the macrophage activation via Toll-like receptor 4. FASEB J. 2018;32: 97–110. doi:10.1096/fj.201700488R 3. Senda A, Morishita K, Kojima M, Doki S, Taylor B, Yagi M, et al. The role of mesenteric lymph exosomal lipid mediators following intestinal ischemia-reperfusion injury on activation of inflammation. J Trauma Acute Care Surg. 2020;89: 1099–1106. doi:10.1097/TA.0000000000002897 Attachments Attachment Submitted filename: Reviewer_0217.docx https://doi.org/10.1371/journal.pone.0283702.r002
14 Mar 2023 Decision Letter - Jon M. Jacobs, Editor Profiles of lipid, protein and microRNA expression in exosomes derived from intestinal epithelial cells after ischemia-reperfusion injury in a cellular hypoxia model PONE-D-22-23365R1 Dear Dr. Kojima, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Jon M. Jacobs, 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 #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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? 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 #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: I have no more comment. My concerns have been addressed. I suggest language editing before fully acceptance. ****** 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.0283702.r003
Formally Accepted
20 Mar 2023 Acceptance Letter - Jon M. Jacobs, Editor PONE-D-22-23365R1 Profiles of lipid, protein and microRNA expression in exosomes derived from intestinal epithelial cells after ischemia-reperfusion injury in a cellular hypoxia model Dear Dr. Kojima: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr Jon M. Jacobs Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0283702.r004

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