Spontaneous liver disease in wild-type C57BL/6JOlaHsd mice fed semisynthetic diet

Onne A. H. O. Ronda; Bert J. M. van de Heijning; Alain de Bruin; Rachel E. Thomas; Ingrid Martini; Martijn Koehorst; Albert Gerding; Mirjam H. Koster; Vincent W. Bloks; Angelika Jurdzinski; Niels L. Mulder; Rick Havinga; Eline M. van der Beek; Dirk-Jan Reijngoud; Folkert Kuipers; Henkjan J. Verkade

doi:10.1371/journal.pone.0232069

Peer Review History

Original SubmissionApril 2, 2020
1 Jun 2020 Decision Letter - Michael W Greene, Editor PONE-D-20-09497 Spontaneous liver disease in wild-type C57BL/6JOlaHsd mice fed semisynthetic diet PLOS ONE Dear Dr. Ronda, 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. 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. Our ability to obtain multiple reviews for your manuscript has been hampered by the COVID-19 pandemic. 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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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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 manuscript is well written and describes extensive, and well done phenotypic investigation of a unique variant in a common mouse model fed a semi-synthetic diet. The authors have done extensive work evaluating a spontaneous phenotype associated with feeding a semisynthetic diet in their C57BL/6JOlaHsd mouse colony. They do an excellent job describing the changes in liver weight and body weight as well as some biochemical and metabolic features of their small liver phenotype and they demonstrate that these changes are independent of dominance behavior. I commend the thorough histologic pathology evaluation. The authors draw fairly broad conclusions from their findings, and they suggest that this phenotype extends to all C57BL/6JOlaHsd mice fed this specific diet. There is discussion on the impact of other unique metabolic phenotypes observed in other stains and the authors suggest that plasma lipids may be used a biomarker for the small liver phenotype. While this paper is overall well written and has extensive analysis, I have a few concerns. The rationale and effects of the fasting time are a bit confusing, and if they are not consistent, would have major metabolic effects. It looks like the PN21 group was not fasted since they were sacrificed at weaning, but this means they were collected following a lipid rich diet. What diet were the Dams fed? It appears the PN42 were fasted for 4 hours during a time where mice would not be expected to eat much (9 am to 1 pm), while the remaining two groups (PN63 and PN70) were fasted for 8 hours during a prime feeding time for the mice (12 am – 9 am). The overall objective of the study is not directly comparing between the time points, but this may affect some of the outcomes in the section that reviews the pathogenesis of the process. IN some areas of the manuscript the authors make direct comparisons between the cohorts where these differences in fasting are more problematic. The choice for the fasting protocol should be clarified or justified, or both. In the discussion, the authors consider portosystemic shunt as a possible cause for the SL phenotype, but I am unclear how that relates to diet. PSS can be intrahepatic or extrahepatic, and I believe are congenital, or acquired secondary to fibrosis or other primary disease processes. Large extra hepatic vessels could be identified grossly, but would be congenital and not diet dependent. Intrahepatic shunts should be able to be identified histologically. The point of this section in the discussion did not seem relevant and distracted from the findings. Given the presence of a small liver, fibrosis, impaired BA processing and alter lipids, and altered hepatic enzymology there should be a more direct statement regarding overt liver failure and early cirrhosis as opposed to simply calling it a small liver phenotype. I have one major concern: It is unclear to me if this small liver phenotype is present in C57BL/6JOlaHsd mice outside of this particular housing unit. At the end of the Introduction, the authors describe some of the aspects of the phenotype as a “spontaneous variation” and I think that is an accurate term. Inbred mouse models have extremely high homozygosity, but they still have variation and are capable of genetic drift and spontaneous polymorphisms that can alter the expected phenotype, particularly within closed breeding groups. The authors discuss their findings in regards to the significance of this phenotype, but it is not clear in my reading of the manuscript that this phenotype is not limited to this specific colony. There is discussion as to unique vendors for the food, but I did not see anything in regards to the mice and it appears all cohorts of mice were bred onsite. If the authors do not have evidence that this phenotype extends beyond their colony, there is very limited impact of all of their hard work in phenotyping the disease/model. The author suggest this is a developmental feature. Is there any information on the lifespan of these mice in this colony? Do they eventually segregate into these two phenotypes and does the diet exacerbate an underlying condition, or is the pathogenesis triggered by this diet alone? This last question is likely beyond the scope of the study, but would make for a more interesting and impactful discussion. This study does such wonderful work with the phenotyping of these mice, but the real importance of the changes stem from the underlying cause, which based on the information provided appears to be developmental and likely genetic. Instead of using lipids post-study it would be more ideal to genotype the mice for this allele prior to putting them on expensive diets. Please note, while this is a major concern, it does not negate the findings or the extensive characterization of the phenotype. It just may limit the value of the work to those who work directly with this specific colony. What would be necessary is extensive clarity in the discussion and to some extent the conclusions to this point. ****** 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 [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
30 Jul 2020 Author Response 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 manuscript is well written and describes extensive, and well done phenotypic investigation of a unique variant in a common mouse model fed a semi-synthetic diet. The authors have done extensive work evaluating a spontaneous phenotype associated with feeding a semisynthetic diet in their C57BL/6JOlaHsd mouse colony. They do an excellent job describing the changes in liver weight and body weight as well as some biochemical and metabolic features of their small liver phenotype and they demonstrate that these changes are independent of dominance behavior. I commend the thorough histologic pathology evaluation. The authors draw fairly broad conclusions from their findings, and they suggest that this phenotype extends to all C57BL/6JOlaHsd mice fed this specific diet. There is discussion on the impact of other unique metabolic phenotypes observed in other stains and the authors suggest that plasma lipids may be used a biomarker for the small liver phenotype. While this paper is overall well written and has extensive analysis, I have a few concerns. 1. The rationale and effects of the fasting time are a bit confusing, and if they are not consistent, would have major metabolic effects. It looks like the PN21 group was not fasted since they were sacrificed at weaning, but this means they were collected following a lipid rich diet. What diet were the Dams fed? It appears the PN42 were fasted for 4 hours during a time where mice would not be expected to eat much (9 am to 1 pm), while the remaining two groups (PN63 and PN70) were fasted for 8 hours during a prime feeding time for the mice (12 am – 9 am). The overall objective of the study is not directly comparing between the time points, but this may affect some of the outcomes in the section that reviews the pathogenesis of the process. IN some areas of the manuscript the authors make direct comparisons between the cohorts where these differences in fasting are more problematic. The choice for the fasting protocol should be clarified or justified, or both. ---------------- AUTHOR RESPONSE: We agree that the differences in the fasting times merit additional clarification and have added this to the new version of the manuscript. The dams were fed AIN-93G throughout breeding, pregnancy and lactation. We do agree and have now stated that we cannot exclude that differences in fasting may have affected some of the biochemical parameters, such as plasma triglyceride levels. Indeed, these levels show variability and tend to be elevated at PN21 compared to our other cohorts. However, other parameters, such as liver weight or the relative composition of essential fatty acids (dependent on slow multi-step enzymatic conversions) in the liver are not expected to differ much between unfasted and 9 h fasted mice. The occurrence of a spontaneous divergence in liver weight under the used experimental conditions is not affected by the duration of fasting immediately prior to dissection. 2. In the discussion, the authors consider portosystemic shunt as a possible cause for the SL phenotype, but I am unclear how that relates to diet. PSS can be intrahepatic or extrahepatic, and I believe are congenital, or acquired secondary to fibrosis or other primary disease processes. Large extra hepatic vessels could be identified grossly, but would be congenital and not diet dependent. Intrahepatic shunts should be able to be identified histologically. The point of this section in the discussion did not seem relevant and distracted from the findings. ---------------- AUTHOR RESPONSE: We agree with the reviewer that we have no data which prove or disprove the occurrence of congenital portosystemic shunting and its possible relationship to the small liver phenotype. Considering this uncertainty, we agree with the reviewer’s opinion that it distracts from the findings. Therefore, we have omitted this section in the new version of the discussion. 3. Given the presence of a small liver, fibrosis, impaired BA processing and alter lipids, and altered hepatic enzymology there should be a more direct statement regarding overt liver failure and early cirrhosis as opposed to simply calling it a small liver phenotype. ---------------- AUTHOR RESPONSE: Agreed. The biochemical differences between SL and NL mice resemble, to some extent, human liver cirrhosis and the early stages of liver failure. Our histological analyses did not (yet) find clear evidence of cirrhosis at the indicated time points. Indeed, extended longitudinal studies could indicate that the SL phenotype evolves to liver cirrhosis and ultimately to end-stage liver failure and early mortality. To honor this valuable remark of the reviewer, we have included this statement in the new version of the manuscript. 4. I have one major concern: It is unclear to me if this small liver phenotype is present in C57BL/6JOlaHsd mice outside of this particular housing unit. At the end of the Introduction, the authors describe some of the aspects of the phenotype as a “spontaneous variation” and I think that is an accurate term. Inbred mouse models have extremely high homozygosity, but they still have variation and are capable of genetic drift and spontaneous polymorphisms that can alter the expected phenotype, particularly within closed breeding groups. The authors discuss their findings in regards to the significance of this phenotype, but it is not clear in my reading of the manuscript that this phenotype is not limited to this specific colony. There is discussion as to unique vendors for the food, but I did not see anything in regards to the mice and it appears all cohorts of mice were bred onsite. If the authors do not have evidence that this phenotype extends beyond their colony, there is very limited impact of all of their hard work in phenotyping the disease/model. The author suggest this is a developmental feature. Is there any information on the lifespan of these mice in this colony? Do they eventually segregate into these two phenotypes and does the diet exacerbate an underlying condition, or is the pathogenesis triggered by this diet alone? This last question is likely beyond the scope of the study, but would make for a more interesting and impactful discussion. This study does such wonderful work with the phenotyping of these mice, but the real importance of the changes stem from the underlying cause, which based on the information provided appears to be developmental and likely genetic. Instead of using lipids post-study it would be more ideal to genotype the mice for this allele prior to putting them on expensive diets. Please note, while this is a major concern, it does not negate the findings or the extensive characterization of the phenotype. It just may limit the value of the work to those who work directly with this specific colony. What would be necessary is extensive clarity in the discussion and to some extent the conclusions to this point. ---------------- AUTHOR RESPONSE: We thank the reviewer for (also) this indeed important aspect on the generalizability of the findings with respect to the mice and the environment. The cohorts described in this manuscript were bred from commercially obtained C57BL/6JOlaHsd breeders. These breeders were specifically purchased for this study and did not originate from our local mouse colony. The breeders were used to breed the mice used throughout this manuscript. Only the F1 offspring were used. Separate batches of breeders were ordered from a commercial supplier in the years 2016, 2017 and 2018. Each batch was used separately to breed pups to be used in this study. The cohorts PN63 and PN70 were highly similar in the assessed parameters, although these cohorts originated from separate breeders, and had been studied during different years. This suggests that, by keeping experimental conditions similar, the occurrence of the SL phenotype and its metabolic consequences have been highly reproducible. The mice were obtained from a commercial breeder which had the resources to carefully monitor and control health, reproduction, and genetic drift. The usage of a large number of breeders, obtained in separate batches across separate years gives, in our view, a fair representative of what one may expect from a commercial animal supplier. This approach, however, does not completely exclude the possibility of a genetic cause. It could be possible that the ‘JOlaHsd’ sub-strain experienced genetic drift away from the ‘J’ sub-strain. To investigate whether a similar phenomenon occurs in the ‘J’ sub-strain, we briefly analysed a ‘J’ cohort at our own facility. This cohort was not obtained from the commercial animal supplier used previously. In S6 Fig, we showed that a similar phenomenon does appear to occur in C57BL/6J mice. In the ApoE3L.CETP model, which is used both in our facility but also elsewhere, a comparable phenotypic heterogeneity has been extensively described, although not characterized as well as we now have reported for the ‘JOlaHsd’ mice. Recently, this phenotypic heterogeneity has been attributed to the presence of two distinct sub-groups in cohorts, rather than to heterogeneity in the classic sense (Tarasco et al., 2018 Am J Physiol Gastrointest Liver Physiol.). The ‘non-responders’ (compared to the ‘responders’, as the subgroups have been named) in ApoE3L.CETP mice appear to have very similar characteristics as our SL phenotype. These data strongly suggest that the SL phenotype is possibly not a ‘JOlaHsd’ idiosyncrasy or a breeder-specific phenomenon. We highly value these comments of the reviewer and have summarized this discussion in the new version of the manuscript. We do not have information regarding the lifespan of these mice. It is unclear to us whether the diet plays a major (if any) role in the occurrence of the SL phenotype. We agree with the reviewer that understanding whether the diet exacerbates an underlying condition or whether the diet triggers the phenotype per se, would make for a more impactful discussion. We were not able to pinpoint any one cause for the SL phenotype, what would have further strengthened our manuscript. We have added these aspects, and the points above, to the new version of the discussion. Theoretically, the cause of the SL phenotype could still be due to specific environmental conditions present at our facility, which could then affect each new imported cohort. Single-center experiments are more vulnerable to biases and methodological pitfalls compared to multi-center experiments. The ApoE3L.CETP mice results obtained in another facility suggest, however, but do not prove, that the current observations are not unique for our facility. In addition, why such an environmental factor would only affect some mice, but not others, would then still need to be resolved. It is therefore not (yet) possible to generalize our observations and characterisations to all C57BL/6JOlaHsd mice or indeed to the ubiquitously used C57BL/6J sub-strain. We have also added these points to the new version of the discussion. Attachments Attachment Submitted filename: Response to Reviewers.docx* https://doi.org/10.1371/journal.pone.0232069.r002
18 Aug 2020 Decision Letter - Michael W Greene, Editor Spontaneous liver disease in wild-type C57BL/6JOlaHsd mice fed semisynthetic diet PONE-D-20-09497R1 Dear Dr. Ronda, 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, Michael W Greene, Ph.D. Academic Editor PLOS ONE Additional Editor Comments: I appreciate your effort and attention to detail in the revision. I also appreciate your willingness to proceed with just one reviewer for your manuscript. The COVID-19 pandemic has resulted in real stress in the review process. 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: All of the original comments have been sufficiently addressed by the authors in the Comments and Discussion. Thank you. ****** 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.0232069.r003
Formally Accepted
11 Sep 2020 Acceptance Letter - Michael W Greene, Editor PONE-D-20-09497R1 Spontaneous liver disease in wild-type C57BL/6JOlaHsd mice fed semisynthetic diet Dear Dr. Ronda: 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. Michael W Greene Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0232069.r004

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