A potent HNF4α agonist reveals that HNF4α controls genes important in inflammatory bowel disease and Paneth cells

Seung-Hee Lee; Vimal Veeriah; Fred Levine

doi:10.1371/journal.pone.0266066

Peer Review History

Original SubmissionSeptember 20, 2021
27 Oct 2021 Decision Letter - Pradeep Dudeja, Editor PONE-D-21-30275A potent HNF4α agonist reveals that HNF4α controls genes important in inflammatory bowel disease and Paneth cellsPLOS ONE Dear Dr. Levine, 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. Specifically, it was felt that the authors need to perform additional studies to show whether the effects of HNF4 are direct or indirect. The suggested additional studies including western analysis and CHIP assays may further strengthen the impact of the manuscript. Please submit your revised manuscript by Dec 10 2021 11:59PM. 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Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. [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: I Don't Know ****** 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: In this interesting study, the authors have investigated the therapeutic effect of the potent HNF4� agonist, N-trans caffeoyltyramine (NCT) on gene expression in the intestine. This study is based on previous findings that demonstrated the therapeutic potential of the novel HNF4� activator, NCT in the liver by reversing severe NAFLD symptoms (hepatic steatosis) in diet-induced obese mice. For the current study, vehicle (DMSO) or NCT was administered to high fat diet mice for 14 days (2 weeks) followed by RNAseq, RT-PCR, and Immunofluorescence analysis. The results showed that NCT upregulated genes in the small intestine that play important role in IBD and that are shown to be downregulated in IBD. Moreover, NCT also reversed the decrease in lysozyme expression (Paneth cell marker) in the small intestine in high fat diet fed mice. Impaired Paneth cell function and intestinal barrier integrity have shown to be the hallmarks of IBD. Therefore, these data suggest that HNF4� could be a therapeutic target for IBD and that NCT could act as a candidate therapeutic in treating IBD. The manuscript for the most part is easily understandable, straight-forward and the results are clearly presented. The abstract adequately summarizes the data and is concise. The strength of the manuscript includes the therapeutic relevance of the study with respect to the important role of HNF4� agonist, NCT in the upregulation of genes that are decreased in IBD while the weaknesses relate to the lack of utilizing an established mouse model of intestinal disease (colitis) to examine the beneficial effect of NCT. 1. Since most of the IBD associated genes upregulated by NCT are independent of HFD (high fat diet) effects (Fig. 3E), then it must be assumed that NCT will also show a similar phenomenon in mice fed with normal chow diet. The authors should elaborate and discuss more on this aspect. 2. Although, the immunofluorescence (IF) images are convincing (Fig. 1A), it will be important to corroborate IF results with Western blot utilizing small intestinal tissue lysates and specific HNF4� antibody. 3. In addition to the lysozyme expression (Paneth cell marker), the authors should also show the altered expression of certain Paneth cell defensins in HFD mice with or without NCT by qRT-PCR. 4. It is not clear which region of the small intestine (jejunum vs ileum) was used for the studies? Reviewer #2: Study by Seung- Hee Lee et al- Entitled “A potent HNF4α agonist reveals that HNF4α controls genes important in inflammatory bowel disease and Paneth cells” has interesting findings. Although, there are some concerns that need to be addressed to strengthen the manuscript. Major concerns: 1. In this study, the authors have showed that the decreased expression of the IBD associated gene and Paneth cell markers by HFD and their reversal by NCT. However, the data does not suggest that the effects are directly due to HNF4A, so it will be important to examine the activation of HNF4A by NCT. 2. ChIP assays or other studies should be provided to prove that NCT increases the binding of HNF4A to the promoter of its target gene. 3. Authors should also examine or provide detail that NCT is a specific agonist only to HNF4A, and it is not producing effects indirectly. Minor concerns: 1. Authors should rephrase the abstract for better grammar and flow. 2. Detail of RT-PCR primers and sting analysis should be provided in the methods and materials section. 3. Authors should include the previous study done regarding regulation of stem cells in HNF4A KO mice 4. Authors should also include a column in table 2 for associated diseases same as table 1. ****** 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. 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Revision 1
17 Nov 2021 Author Response Reviewer #1: In this interesting study, the authors have investigated the therapeutic effect of the potent HNF4� agonist, N-trans caffeoyltyramine (NCT) on gene expression in the intestine. This study is based on previous findings that demonstrated the therapeutic potential of the novel HNF4� activator, NCT in the liver by reversing severe NAFLD symptoms (hepatic steatosis) in diet-induced obese mice. For the current study, vehicle (DMSO) or NCT was administered to high fat diet mice for 14 days (2 weeks) followed by RNAseq, RT-PCR, and Immunofluorescence analysis. The results showed that NCT upregulated genes in the small intestine that play important role in IBD and that are shown to be downregulated in IBD. Moreover, NCT also reversed the decrease in lysozyme expression (Paneth cell marker) in the small intestine in high fat diet fed mice. Impaired Paneth cell function and intestinal barrier integrity have shown to be the hallmarks of IBD. Therefore, these data suggest that HNF4� could be a therapeutic target for IBD and that NCT could act as a candidate therapeutic in treating IBD. The manuscript for the most part is easily understandable, straight-forward and the results are clearly presented. The abstract adequately summarizes the data and is concise. The strength of the manuscript includes the therapeutic relevance of the study with respect to the important role of HNF4� agonist, NCT in the upregulation of genes that are decreased in IBD while the weaknesses relate to the lack of utilizing an established mouse model of intestinal disease (colitis) to examine the beneficial effect of NCT. CRITIQUE 1. Since most of the IBD associated genes upregulated by NCT are independent of HFD (high fat diet) effects (Fig. 3E), then it must be assumed that NCT will also show a similar phenomenon in mice fed with normal chow diet. The authors should elaborate and discuss more on this aspect. RESPONSE: The reviewer is correct that most of the IBD-associated genes were not affected by HFD. We agree that this point is worthy of greater emphasis and so have modified the manuscript to make this point more clearly (Lines 210-212, 255-256). CRITIQUE 2. Although, the immunofluorescence (IF) images are convincing (Fig. 1A), it will be important to corroborate IF results with Western blot utilizing small intestinal tissue lysates and specific HNF4� antibody. RESPONSE: To address the reviewer’s request for additional data on HNF4a expression, we performed RT-PCR analysis for HNF4a mRNA, finding that there was a significant increase induced by NCT. That has been added as Figure 1C. CRITIQUE 3. In addition to the lysozyme expression (Paneth cell marker), the authors should also show the altered expression of certain Paneth cell defensins in HFD mice with or without NCT by qRT-PCR. RESPONSE. To address the concern of the reviewer, we have added additional data (Figure 4C, D). CRITIQUE 4. It is not clear which region of the small intestine (jejunum vs ileum) was used for the studies? RESPONSE. Ten cm of intestine proximal to the duodenum, and so consisting primarily of jejunum, was removed. This information has been added to the Materials and Methods- section on Mice. Reviewer #2: Study by Seung- Hee Lee et al- Entitled “A potent HNF4α agonist reveals that HNF4α controls genes important in inflammatory bowel disease and Paneth cells” has interesting findings. Although, there are some concerns that need to be addressed to strengthen the manuscript. Major concerns: CRITIQUE 1. In this study, the authors have showed that the decreased expression of the IBD associated gene and Paneth cell markers by HFD and their reversal by NCT. However, the data does not suggest that the effects are directly due to HNF4A, so it will be important to examine the activation of HNF4A by NCT. RESPONSE. The issue of whether NCT interacts directly with HNF4a is an important question. Our previous work with NCT examined the issue of direct interaction and we have modified the manuscript to make this important point requested by the reviewer (Lines (79-81). CRITIQUE 2. ChIP assays or other studies should be provided to prove that NCT increases the binding of HNF4A to the promoter of its target gene. RESPONSE. The reviewer requests ChIP assays to determine whether the effects of NCT on downstream genes are direct or indirect. Fortunately, a comprehensive ChIP-Seq analysis of the role of HNF4 in the intestine was published recently: Chen L, et al. The nuclear receptor HNF4 drives a brush border gene program conserved across murine intestine, kidney, and embryonic yolk sac. Nat Commun. 2021 May 17;12(1):2886. doi: 10.1038/s41467-021-22761-5. PMID: 34001900; PMCID: PMC8129143. They did not find that the Paneth cell genes that we studied to be direct targets of HNF4a, but a few of the genes associated with IBD were identified as direct targets of HNF4a. The manuscript has been modified to discuss more extensively the results from this paper along with additional discussion of the important issue identified by the reviewer about direct versus indirect effects of HNF4a (Lines 266-270). CRITIQUE 3. Authors should also examine or provide detail that NCT is a specific agonist only to HNF4A, and it is not producing effects indirectly. RESPONSE. As pointed out by the reviewer, the specificity of small molecule compounds is always a concern. We have examined this issue in our previous paper on NCT using siRNA to HNF4a, finding that it ablated the effect of NCT in cultured cells (Lines (79-81). Minor concerns: 1. Authors should rephrase the abstract for better grammar and flow. RESPONSE. The abstract has been edited as requested. 2. Detail of RT-PCR primers and sting analysis should be provided in the methods and materials section. RESPONSE. This information has been added as Table 1. 3. Authors should include the previous study done regarding regulation of stem cells in HNF4A KO mice RESPONSE. We have added a citation to a recent paper on the role of HNF4a in intestinal stem cell renewal (Chen L, et al. HNF4 Regulates Fatty Acid Oxidation and Is Required for Renewal of Intestinal Stem Cells in Mice. Gastroenterology. 2020 Mar;158(4):985-999.e9. doi: 10.1053/j.gastro.2019.11.031. Epub 2019 Nov 22. PMID: 31759926; PMCID: PMC7062567) and hope that this is the citation requested by the reviewer. 4. Authors should also include a column in table 2 for associated diseases same as table 1. RESPONSE. We did not include a column for associated diseases in table 2, since that table included only genes expressed in Paneth cells and the point of the table is to emphasize that many genes expressed in Paneth cells were affected by NCT. To the best of our knowledge, the only disease that has been well studied with regard to Paneth cells is IBD, an association that is discussed at length in the manuscript. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0266066.r002
14 Mar 2022 Decision Letter - Pradeep Dudeja, Editor A potent HNF4α agonist reveals that HNF4α controls genes important in inflammatory bowel disease and Paneth cells PONE-D-21-30275R1 Dear Dr. Levine, 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, 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: The authors have duly addressed all the concerns raised by the reviewer. The revised version of the manuscript is now ready to be accepted 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:** Seema Saksena Reviewer #2: No https://doi.org/10.1371/journal.pone.0266066.r003
Formally Accepted
17 Mar 2022 Acceptance Letter - Pradeep Dudeja, Editor PONE-D-21-30275R1 A potent HNFα agonist reveals that HNF4α controls genes important in inflammatory bowel disease and Paneth cells Dear Dr. Levine: 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. Pradeep Dudeja Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0266066.r004

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