A novel system to culture human intestinal organoids under physiological oxygen content to study microbial-host interaction

Tatiana Y. Fofanova; Umesh C. Karandikar; Jennifer M. Auchtung; Reid L. Wilson; Antonio J. Valentin; Robert A. Britton; K. Jane Grande-Allen; Mary K. Estes; Kristi Hoffman; Sashirekha Ramani; Christopher J. Stewart; Joseph F. Petrosino

doi:10.1371/journal.pone.0300666

Peer Review History

Original SubmissionNovember 6, 2023
20 Nov 2023 Decision Letter - Gianpaolo Papaccio, Editor PONE-D-23-36187A novel system to culture human intestinal organoids under physiological oxygen content to study microbial-host interaction.PLOS ONE Dear Dr. Karandikar, 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. This study has been judged to be of interest pending some alterations that the Authors must perform.They must alter the manuscript as the referees have specified in their comments and answer to all the point that have been highlighted.All the amendments in the revised paper must be written in red color.A point to point answer rebuttal letter must be also written in details. Please submit your revised manuscript by Jan 04 2024 11:59PM. 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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 study presents a new protocol do develop intestinal organoids co-culture with anearobic commensal bacteria. The study is well-conducted. However a relevant reference to a similar topic is missing (Hill DR, Huang S, Nagy MS, Yadagiri VK, Fields C, Mukherjee D, Bons B, Dedhia PH, Chin AM, Tsai YH, Thodla S, Schmidt TM, Walk S, Young VB, Spence JR. Bacterial colonization stimulates a complex physiological response in the immature human intestinal epithelium. Elife. 2017 Nov 7;6:e29132. doi: 10.7554/eLife.29132), and an analysis of transcriptional changes in terms of cell subpopulations induced after the contact with bacteria is missing (i.e. a ssGSEA on intestine cell subpopulations) in order to better understand to the question of transcriptional changes induced. Reviewer #2: In this paper Authors generated Intestinal Organoid Physoxic Coculture (IOPC) system, and, using commensal anaerobes with varying degrees of oxygen tolerance, such as nano-aerobe Bacteroides thetaiotaomicron and strict anaerobe Blautia sp., They demonstrated that IOPC can successfully support 24 - 48 hours HIO-microbe coculture. The paper is interesting and the experiments are well conducted. Only little concerns must be addressed. In figure 3c Authors must add a legend in the IF pictures referring to the markers with relative color. Authors must add some WB to confirm the gene expression in figure 4. However relevant references to analysis of transcriptional changes is missing (read and add Journal of Experimental and Clinical Cancer Research, 2023, 42(1), 8 “Proteotranscriptomic analysis of advanced colorectal cancer patient derived organoids for drug sensitivity prediction”; Cell Stem Cell 2020 Jan 2;26(1):17-26.e6. doi: 10.1016/j.stem.2019.10.010. Epub 2019 Nov 21. J Exp Clin Cancer Res 2023 Oct 25;42(1):281. doi: 10.1186/s13046-023-02853-4. ****** 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.0300666.r001
Revision 1
16 Feb 2024 Author Response We thank the reviewers for their positive feedback and thoughtful suggestions. We have revised the manuscript to address the concerns raised by the reviewers. Please see our detailed responses below. 1) As suggested by Reviewer 1, we have added the reference ‘Bacterial colonization stimulates a complex physiological response in the immature human intestinal epithelium. Elife. 2017 Nov 7;6:e29132. doi: 10.7554/eLife.29132’ (line 304). 2) Reviewer 1 suggested, “analysis of transcriptional changes in terms of cell subpopulations induced after the contact with bacteria is missing.” We believe such an analysis will need significant optimization and goes beyond the scope of this ‘proof of principle’ manuscript describing a straightforward bacterial-organoid coculture method using off-the-shelf parts. 3) We have modified the legend to IF images in Figure 3c, as suggested by Reviewer 2. 4) Reviewer 2 suggested, “… add some WB to confirm the gene expression in Figure 4”. Figure 4 shows bacteria-induced differential gene expression in the organoids. Per this suggestion, we conducted western blot analysis for Casp1 and F11R. A widely used Anti-Casp1 showed weak bands at the expected molecular weight for Casp1 and additional bands that haven’t been documented before. On the other hand, a widely used anti-F11R Ab detected multiple bands that have not been reported in previous studies. We believe the discrepancy between our results and previously published studies may reflect differences in the glycosylation pattern between cell lines and intestinal organoids. Based on these results, we conclude that the investigation of gene expression based on western blot analysis needs more significant optimization, including screening of antibodies. Such an analysis is clearly outside of the scope of this manuscript. We strongly believe that the absence of this data does not alter or diminish the coculture method described in this manuscript. 5) Reviewer 2 listed references to be added while discussing the transcriptional changes in organoids. However, we found a reference better suited to discussing transcriptional changes in organoids during coculture with bacteria, so we added that reference (lines 304, 307). The references listed by Reviewer 2 were more appropriate to support the rationale of organoids as a preclinical model (line 350). We appreciate reviewer 2 for pointing out the need to cite relevant references while discussing transcriptional changes during bacterial coculture. In summary, we firmly believe that the manuscript adequately describes a cost-effective and robust method to coculture intestinal organoids with anaerobic bacteria using ‘off-the-shelf parts’ that will be of immense value to the study of many researchers investigating intestinal microbes. Attachments Attachment Submitted filename: Response to reviewer_Fofanova2024.docx https://doi.org/10.1371/journal.pone.0300666.r002
4 Mar 2024 Decision Letter - Gianpaolo Papaccio, Editor A novel system to culture human intestinal organoids under physiological oxygen content to study microbial-host interaction. PONE-D-23-36187R1 Dear Dr. Karandikar, 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, Gianpaolo Papaccio, 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: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: (No Response) ****** 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: (No Response) ****** 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 Response) ****** 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: Authors have addressed all reviewer's comments and concerns. No further comments. 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: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0300666.r003
Formally Accepted
16 Jul 2024 Acceptance Letter - Gianpaolo Papaccio, Editor PONE-D-23-36187R1 PLOS ONE Dear Dr. Karandikar, 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 Prof. Gianpaolo Papaccio Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0300666.r004

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