Glucocorticoid and dietary effects on mucosal microbiota in canine inflammatory bowel disease

Todd Atherly; Giacomo Rossi; Robin White; Yeon-Jung Seo; Chong Wang; Mark Ackermann; Mary Breuer; Karin Allenspach; Jonathan P. Mochel; Albert E. Jergens

doi:10.1371/journal.pone.0226780

Peer Review History

Original SubmissionAugust 21, 2019
10 Oct 2019 Decision Letter - Pradeep Dudeja, Editor PONE-D-19-23630 Glucocorticoid and dietary effects on mucosal microbiota in canine inflammatory bowel disease PLOS ONE Dear Dr. Jergens, 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 would like you to please address the reviewers concerns with special focus on clarifying several concerns as well as improving the presentation of results, figure legends and discussion We would appreciate receiving your revised manuscript by Nov 24 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. 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, Pradeep Dudeja 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 2. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. 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: No ****** 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: No ****** 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 used two major techniques (IHC and FISH) to determine the effect of glucocorticoid and diet on mucosal microbiota in canine IBD. The manuscript is well written and has very few typographical errors. The authors have provided the raw data that was used for data analysis. Reviewer #2: Glucocorticoids are commonly administered to reduce mucosal inflammation and gastrointestinal signs. The study evaluated the effects of diet and oral prednisone on the spatial distribution of mucosal bacteria in IBD dogs. Whether or not GCs beneficially modulate the mucosal microbiota to reduce intestinal inflammation in dogs with IBD has not been systematically evaluated using fluorescence in situ hybridization. The current study should be interesting as it will provide new information in treating IBD dogs and understand the association of inflammation and microbiome. However, the authors need address the following concerns from the reviewer: 1. The sample size is relatively small (IBD dog 8, health control 15). Please clarify the IBD stages of each dog . The focus of the analysis should be the IBD dogs before and after treatment. 2. Please clarify that E-cadherin is not a tight junction protein. 3. The Results are poorly written. More details are needed. 4. The Figure Legends should be separated from the Results. 5. It is helpful to extend the Discussion and relate the current study to human IBD, which is better established. ****** 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. 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. Attachments Attachment Submitted filename: Reviewer Comments-PONE-D-19-23630.docx https://doi.org/10.1371/journal.pone.0226780.r001
Revision 1
15 Nov 2019 Author Response RESPONSE TO REVIEWERS (PONE-D-19-23630): My specific critiques are below: • Though the authors use the term ‘spatial distribution of microbiota’ in terms of mucosal compartments, however do not provide any analyses of the difference in distribution between ileum and colon, which were used for FISH analysis. The authors should clarify this and provide data obtained from ileal and colonic samples, since these two intestinal regions have been shown to have different composition of microbiota in various experimental animal species and also in humans Intestinal tissues (endoscopic biopsies of ileum and colon) for FISH analysis were combined on glass slides for ease of hybridization and consistency of interpretation by a single operator. In this manner, a single slide would contain both ileal and colonic tissues (3-5 samples of each tissue) in which the operator would perform 10 individual bacterial counts in 4 separate endoscopic biopsies. While care was taken to accurately distinguish between 2 ileal versus 2 colonic mucosal samples for quantitative analysis, a clear distinction between these tissues could not always be determined. Therefore, mucosal bacterial counts for each animal were recorded as a cumulative total of bacteria observed within ileal/colonic intestinal segments rather than separate bacterial counts for the ileum and separate bacterial counts for the colon, respectively. We report a similar study design for FISH analysis in a recent report.1 1White, et al. Randomized, controlled trial evaluating the effect of multi-strain probiotic on the mucosal microbiota in canine idiopathic inflammatory bowel disease. Gut microbes. 2017; 8(5):451-66. We have now modified the methods section to more accurately reflect how mucosal bacteria in endoscopic samples were counted across intestinal segments. • Line 381 in discussion: The authors indicate that there’s a change in the distribution and composition of mucosal microbiota, which is misleading. The authors have only looked at five bacteria and their differential expression in the experimental groups. The authors should change the sentence and not extrapolate the observation to differential microbial composition or intestinal site distribution. Agreed and now modified in discussion. • The authors should speculate and discuss why glucocorticoids may alter the differential expression of the three bacteria (line 383) Speculation is required here since there is an absolute paucity of information on how GCs may modulate gut mucosal bacterial populations in health or disease. We now state: It is clear that dysbiosis of the intestinal bacteria is both a cause and consequence of IBD. As antibiotics and probiotics have only modest effects in managing patients with IBD, many clinicians now opt for a top-down therapeutic approach using biologics and/or immunosuppressive drugs, such as glucocorticoids, to induce clinical remission. Only few studies show that GCs (or any other immunosuppressive drug for that matter) can exert favorable effects in the fecal microbiome of IBD affected individuals. However, there are no studies (beyond the present report) which describe how or why GCs selectively alter the mucosal microbiota in IBD. It is also possible that other components of the gut microbiome (i.e., fungi and viruses) are influenced by GC administration and contribute to beneficial modulation of resident bacterial populations as observed in this study. • Just for clarity: Were all the biopsies done at the end of 8 weeks of diet and treatment? Yes, IBD dogs for repeat GI endoscopy were re-examined during treatment within 1-3 days of diet/drug trial completion. Review Comments to the Author Reviewer #1: The authors have used two major techniques (IHC and FISH) to determine the effect of glucocorticoid and diet on mucosal microbiota in canine IBD. The manuscript is well written and has very few typographical errors. The authors have provided the raw data that was used for data analysis. There are no specific comments to address. Reviewer #2: Glucocorticoids are commonly administered to reduce mucosal inflammation and gastrointestinal signs. The study evaluated the effects of diet and oral prednisone on the spatial distribution of mucosal bacteria in IBD dogs. Whether or not GCs beneficially modulate the mucosal microbiota to reduce intestinal inflammation in dogs with IBD has not been systematically evaluated using fluorescence in situ hybridization. The current study should be interesting as it will provide new information in treating IBD dogs and understand the association of inflammation and microbiome. However, the authors need address the following concerns from the reviewer: 1. The sample size is relatively small (IBD dog 8, health control 15). Please clarify the IBD stages of each dog. The focus of the analysis should be the IBD dogs before and after treatment. Agreed that the focus is on the IBD dogs pre- versus post-treatment. Regarding the various clinical stages of IBD dogs at diagnosis (pre-treatment), 3 dogs with IBD had severe clinical disease activity (i.e., CIBDAI scores > 9) and 5 dogs with IBD had moderate disease activity (i.e., CIBDAI scores 6-8). At trial completion, all 8 dogs with IBD were in full clinical remission with a mean post-treatment CIBDAI score of 1. These new data are now included in the revised manuscript. Good suggestion. 2. Please clarify that E-cadherin is not a tight junction protein. Thanks for pointing out that there is a difference between the different classes of apical junction proteins (AJPs), which include the tight junction proteins (occludin, claudin family, and ZO-1) and the adherens junction (AJ) protein, E-cadherin. To avoid mis-characterization of the protein classes we evaluated using IHC, we have now corrected the manuscript to state AJPs and their respective proteins rather than TJPs. Good suggestion and sorry for the confusion. We have now updated the discussion section appropriately. 3. The Results are poorly written. More details are needed. Thank you for this recommendation and we have now expanded the results section regarding patient demographics and FISH analysis to include more detail and clarity as requested. 4. The Figure Legends should be separated from the Results. Now separated as requested. 5. It is helpful to extend the Discussion and relate the current study to human IBD, which is better established. Good suggestion and additional information now provided for the reader. That said, there is no information RESPONSE TO REVIEWERS (PONE-D-19-23630): My specific critiques are below: • Though the authors use the term ‘spatial distribution of microbiota’ in terms of mucosal compartments, however do not provide any analyses of the difference in distribution between ileum and colon, which were used for FISH analysis. The authors should clarify this and provide data obtained from ileal and colonic samples, since these two intestinal regions have been shown to have different composition of microbiota in various experimental animal species and also in humans Intestinal tissues (endoscopic biopsies of ileum and colon) for FISH analysis were combined on glass slides for ease of hybridization and consistency of interpretation by a single operator. In this manner, a single slide would contain both ileal and colonic tissues (3-5 samples of each tissue) in which the operator would perform 10 individual bacterial counts in 4 separate endoscopic biopsies. While care was taken to accurately distinguish between 2 ileal versus 2 colonic mucosal samples for quantitative analysis, a clear distinction between these tissues could not always be determined. Therefore, mucosal bacterial counts for each animal were recorded as a cumulative total of bacteria observed within ileal/colonic intestinal segments rather than separate bacterial counts for the ileum and separate bacterial counts for the colon, respectively. We report a similar study design for FISH analysis in a recent report.1 1White, et al. Randomized, controlled trial evaluating the effect of multi-strain probiotic on the mucosal microbiota in canine idiopathic inflammatory bowel disease. Gut microbes. 2017; 8(5):451-66. We have now modified the methods section to more accurately reflect how mucosal bacteria in endoscopic samples were counted across intestinal segments. • Line 381 in discussion: The authors indicate that there’s a change in the distribution and composition of mucosal microbiota, which is misleading. The authors have only looked at five bacteria and their differential expression in the experimental groups. The authors should change the sentence and not extrapolate the observation to differential microbial composition or intestinal site distribution. Agreed and now modified in discussion. • The authors should speculate and discuss why glucocorticoids may alter the differential expression of the three bacteria (line 383) Speculation is required here since there is an absolute paucity of information on how GCs may modulate gut mucosal bacterial populations in health or disease. We now state: It is clear that dysbiosis of the intestinal bacteria is both a cause and consequence of IBD. As antibiotics and probiotics have only modest effects in managing patients with IBD, many clinicians now opt for a top-down therapeutic approach using biologics and/or immunosuppressive drugs, such as glucocorticoids, to induce clinical remission. Only few studies show that GCs (or any other immunosuppressive drug for that matter) can exert favorable effects in the fecal microbiome of IBD affected individuals. However, there are no studies (beyond the present report) which describe how or why GCs selectively alter the mucosal microbiota in IBD. It is also possible that other components of the gut microbiome (i.e., fungi and viruses) are influenced by GC administration and contribute to beneficial modulation of resident bacterial populations as observed in this study. • Just for clarity: Were all the biopsies done at the end of 8 weeks of diet and treatment? Yes, IBD dogs for repeat GI endoscopy were re-examined during treatment within 1-3 days of diet/drug trial completion. Review Comments to the Author Reviewer #1: The authors have used two major techniques (IHC and FISH) to determine the effect of glucocorticoid and diet on mucosal microbiota in canine IBD. The manuscript is well written and has very few typographical errors. The authors have provided the raw data that was used for data analysis. There are no specific comments to address. Reviewer #2: Glucocorticoids are commonly administered to reduce mucosal inflammation and gastrointestinal signs. The study evaluated the effects of diet and oral prednisone on the spatial distribution of mucosal bacteria in IBD dogs. Whether or not GCs beneficially modulate the mucosal microbiota to reduce intestinal inflammation in dogs with IBD has not been systematically evaluated using fluorescence in situ hybridization. The current study should be interesting as it will provide new information in treating IBD dogs and understand the association of inflammation and microbiome. However, the authors need address the following concerns from the reviewer: 1. The sample size is relatively small (IBD dog 8, health control 15). Please clarify the IBD stages of each dog. The focus of the analysis should be the IBD dogs before and after treatment. Agreed that the focus is on the IBD dogs pre- versus post-treatment. Regarding the various clinical stages of IBD dogs at diagnosis (pre-treatment), 3 dogs with IBD had severe clinical disease activity (i.e., CIBDAI scores > 9) and 5 dogs with IBD had moderate disease activity (i.e., CIBDAI scores 6-8). At trial completion, all 8 dogs with IBD were in full clinical remission with a mean post-treatment CIBDAI score of 1. These new data are now included in the revised manuscript. Good suggestion. 2. Please clarify that E-cadherin is not a tight junction protein. Thanks for pointing out that there is a difference between the different classes of apical junction proteins (AJPs), which include the tight junction proteins (occludin, claudin family, and ZO-1) and the adherens junction (AJ) protein, E-cadherin. To avoid mis-characterization of the protein classes we evaluated using IHC, we have now corrected the manuscript to state AJPs and their respective proteins rather than TJPs. Good suggestion and sorry for the confusion. We have now updated the discussion section appropriately. 3. The Results are poorly written. More details are needed. Thank you for this recommendation and we have now expanded the results section regarding patient demographics and FISH analysis to include more detail and clarity as requested. 4. The Figure Legends should be separated from the Results. Now separated as requested. 5. It is helpful to extend the Discussion and relate the current study to human IBD, which is better established. Good suggestion and additional information now provided for the reader. That said, there is no information Attachments Attachment Submitted filename: Response to reviewers_Oct_2019.docx https://doi.org/10.1371/journal.pone.0226780.r002
6 Dec 2019 Decision Letter - Pradeep Dudeja, Editor Glucocorticoid and dietary effects on mucosal microbiota in canine inflammatory bowel disease PONE-D-19-23630R1 Dear Dr. Jergens, 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, Pradeep Dudeja 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: In the revision, the authors addressed my concerns. There is no further question for this manuscript. ****** 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.0226780.r003
Formally Accepted
18 Dec 2019 Acceptance Letter - Pradeep Dudeja, Editor PONE-D-19-23630R1 Glucocorticoid and dietary effects on mucosal microbiota in canine inflammatory bowel disease Dear Dr. Jergens: 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. Pradeep Dudeja Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0226780.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 .