Pitfalls in AR42J-model of cerulein-induced acute pancreatitis

Marcus Hollenbach; Sebastian Sonnenberg; Ines Sommerer; Jana Lorenz; Albrecht Hoffmeister

doi:10.1371/journal.pone.0242706

Peer Review History

Original SubmissionMay 8, 2020
9 Jun 2020 Decision Letter - Zoltán Rakonczay Jr., Editor PONE-D-20-13635 Pitfalls in AR42J-model of Cerulein-Induced Acute PancreatitisPitfalls in AR42J-model of Cerulein-Induced Acute Pancreatitis PLOS ONE Dear Dr. Hollenbach, 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. Your paper has been evaluated by three experts in the field. Overall, the paper is well-written, there are only some minor points that need to be addressed. Novelty is not an issue at PLOS ONE, you do not need to deal with these comments. Please submit your revised manuscript by Jul 19 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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Please note that supplementary tables should be uploaded as separate "supporting information" files. [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: Partly Reviewer #2: Yes Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: 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 Reviewer #2: Yes Reviewer #3: 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 Reviewer #3: 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 manuscript, the authors presented AR42J cells as a cell-culture model of cerulein (CER)-induced acute pancreatitis as well as the conditions for the transfection and induction of proinflammatory markers in CER-treated AR42J cells. Unfortunately, there is no novelty in the study, because it is well known that these amphicrine cells should be treated by dexamethasone that favors their differentiation toward the exocrine phenotype and leads to increased secretion of amylase in response to the secretagogue cholecystokinin. AR42J cells have been also widely used as an “in vitro” model for the transfection to study the exocrine pancreas and it was described in published protocols and reviews (Logsdon CD, Moessner J, Williams JA, Goldfine ID. Glucocorticoids increase amylase mRNA levels, secretory organelles, and secretion in pancreatic acinar AR42J cells. J Cell Biol. 100:1200-1208, 1985; Gonzalez A S-CP, Salido GM (2011) Culture of pancreatic AR42J cell for use as a model for acinar cell function. In: The Pancreapedia: Exocrine Pancreas Knowledge Base 2011; Christophe J. Pancreatic tumoral cell line AR42J: an amphicrine model. Am J Physiol 266: G963-971, 1994; Mareninova, O.A. Orabi, Abrahim I. Husain, Sohail Z. (2015). Experimental acute pancreatitis: In vitro models. In: The Pancreapedia: Exocrine Pancreas Knowledge Base 2015). Reviewer #2: The article written by Hollenbach et al. provides a useful summary about the methodological difficulties that any researcher may face when working with AR42J cell line. The authors also created a guidance of the optimal experimental setup of this cell line that helps to follow good laboratory practice. I have only a few reflections that should be clarified in the manuscript: 1. Abstract/ Results: “CN treatment resulted in increased TNS-alpha production but not secretion and did not influence IL-6.” The second part of this sentence should be corrected. Which characteristics of IL-6 is mentioned? Expression, IL-6 levels, production etc.? 2. Materials and methods / qRT-PCR: The details of cDNA construction should be mentioned here. 3. Materials and methods / Glo-I plasmid generation: Primer orientation should be indicated (5’ – 3’), and heat shock temperature of E.coli transformation should be mentioned. 4. Figure 2C. It is mentioned that LPS was not able to induce a secretion of amylase by AR42J. However, it seems that LPS treatment inhibited the increase of amylase levels after dexamethason pretreatment and CN treatment. According to the Figure2C amylase levels decreased if cells were treated by LPS and CN together compared to CN only treatment. Could you please explain this observation? Is it possible that this figure is inaccurate? Reviewer #3: Hollenbach et al. present a methodological paper on the usability of AR42J cells for investigation of some aspects of the pathophysiology in acute pancreatitis. They show that caerulein can be used in combination with dexamethasone to induce amylase and cytokine secretion. They also investigated the expression of nf-κΒ and transfected cells with a plasmid containing glyoxalase-I or glyoxalase-I siRNA. The authors conclude that AR42J cells are a useful in-vitro model for caerulein induced acute pancreatitis when considering some experimental conditions. The paper covers an important topic when investigating experimental pancreatitis. The methods are clearly presented and the results contain novelty. There are some aspects the authors need to consider: 1.) In the images of figure 1 and figure 4, A1 and A3 the brightness of AR42J cells should be adjusted. 2.) The authors should mention in the introduction why the investigate Glyoxalase-I expression in AR42J cells. 3.) It should be mentioned in the results section (p. 11-12), which concentration of CN leads to the highest amylase secretion. 4.) The figures should be arranged in a numerical order starting with figure 1. Here the authors start with figure 6A (p. 11). ****** 6. 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Revision 1
24 Oct 2020 Author Response Reviewer #1: In this manuscript, the authors presented AR42J cells as a cell-culture model of cerulein (CER)-induced acute pancreatitis as well as the conditions for the transfection and induction of proinflammatory markers in CER-treated AR42J cells. Unfortunately, there is no novelty in the study, because it is well known that these amphicrine cells should be treated by dexamethasone that favors their differentiation toward the exocrine phenotype and leads to increased secretion of amylase in response to the secretagogue cholecystokinin. AR42J cells have been also widely used as an “in vitro” model for the transfection to study the exocrine pancreas and it was described in published protocols and reviews (Logsdon CD, Moessner J, Williams JA, Goldfine ID. Glucocorticoids increase amylase mRNA levels, secretory organelles, and secretion in pancreatic acinar AR42J cells. J Cell Biol. 100:1200-1208, 1985; Gonzalez A S-CP, Salido GM (2011) Culture of pancreatic AR42J cell for use as a model for acinar cell function. In: The Pancreapedia: Exocrine Pancreas Knowledge Base 2011; Christophe J. Pancreatic tumoral cell line AR42J: an amphicrine model. Am J Physiol 266: G963-971, 1994; Mareninova, O.A. Orabi, Abrahim I. Husain, Sohail Z. (2015). Experimental acute pancreatitis: In vitro models. In: The Pancreapedia: Exocrine Pancreas Knowledge Base 2015). We thank the reviewer for this advice but clearly disagree with the reviewer’s opinion. Indeed, AR42J cells were used for decades as an in-vitro model for cerulein (CN)-induced pancreatitis. However, as demonstrated in detail in Table 1, the used conditions for that model dramatically vary between the published articles. Moreover, important details of the experimental setting often are missing. Our data therefore clearly describe the necessity of dexamethasone-treatment prior to CN-stimulation. In addition, different concentrations of CN and dexamethasone as well as incubation times were analyzed. Furthermore, we present an optimized protocol for successful overexpression and gene knockdown to maximize transfection efficacy. Reviewer #2: The article written by Hollenbach et al. provides a useful summary about the methodological difficulties that any researcher may face when working with AR42J cell line. The authors also created a guidance of the optimal experimental setup of this cell line that helps to follow good laboratory practice. We thank the reviewer for the positive critiques and we hope our article will help researches to optimize their own AR42J experimental setting. I have only a few reflections that should be clarified in the manuscript: 1. Abstract/ Results: “CN treatment resulted in increased TNS-alpha production but not secretion and did not influence IL-6.” The second part of this sentence should be corrected. Which characteristics of IL-6 is mentioned? Expression, IL-6 levels, production etc.? We thank the reviewer for this advice and corrected the corresponding sentence. 2. Materials and methods / qRT-PCR: The details of cDNA construction should be mentioned here. We used the Qiagen QuantiTect one stop qRT-PCR kit. This kit allows the easy and fast qRT-PCR without necessity of checking cDNA. RNA was used as described in the manufacturers’ manual. We updated the information in this section regarding the used PCR protocol. 3. Materials and methods / Glo-I plasmid generation: Primer orientation should be indicated (5’ – 3’), and heat shock temperature of E.coli transformation should be mentioned. This section of material and methods was revised and the requested information provided. 4. Figure 2C. It is mentioned that LPS was not able to induce a secretion of amylase by AR42J. However, it seems that LPS treatment inhibited the increase of amylase levels after dexamethason pretreatment and CN treatment. According to the Figure2C amylase levels decreased if cells were treated by LPS and CN together compared to CN only treatment. Could you please explain this observation? Is it possible that this figure is inaccurate? We are very thankful for this advice. Indeed, fig. 2C was incomplete. Our data showed that LPS treatment alone was not able to induce amylase secretion. In addition, co-treatment of LPS and CN did not further influence the release of amylase compared to CN alone. We corrected fig. 2C and clarified the statement in the manuscript. Reviewer #3: Hollenbach et al. present a methodological paper on the usability of AR42J cells for investigation of some aspects of the pathophysiology in acute pancreatitis. They show that caerulein can be used in combination with dexamethasone to induce amylase and cytokine secretion. They also investigated the expression of nf-κΒ and transfected cells with a plasmid containing glyoxalase-I or glyoxalase-I siRNA. The authors conclude that AR42J cells are a useful in-vitro model for caerulein induced acute pancreatitis when considering some experimental conditions. The paper covers an important topic when investigating experimental pancreatitis. The methods are clearly presented and the results contain novelty. We thank the reviewer for the positive evaluation of our manuscript. Indeed, we hope to provide evidence and guidance for researches that are faced with the AR42J-model of CN-induced pancreatitis. There are some aspects the authors need to consider: 1.) In the images of figure 1 and figure 4, A1 and A3 the brightness of AR42J cells should be adjusted. We thank for this suggestion of optimized brightness of the corresponding images. 2.) The authors should mention in the introduction why the investigate Glyoxalase-I expression in AR42J cells. The Glyoxalase-System demonstrates a main research interest for many years in the authors’ lab. Ongoing studies analyze the role of Glyoxalas-I in acute and chronic pancreatitis as well as pancreatic cancer in vivo and in vitro. This was highlighted in the introduction section. 3.) It should be mentioned in the results section (p. 11-12), which concentration of CN leads to the highest amylase secretion. It was now stated at results section that 100nM CN was used for the following experiments as this concentration induced highest amylase release. 4.) The figures should be arranged in a numerical order starting with figure 1. Here the authors start with figure 6A (p. 11). We agree with the reviewer’s suggestion and referred in the corresponding paragraph to sections below. Now the figures order follows the results presentation in the manuscript. Attachments Attachment Submitted filename: 20_07_21 Rebuttal letter PlosOne MH.docx https://doi.org/10.1371/journal.pone.0242706.r002
9 Nov 2020 Decision Letter - Zoltán Rakonczay Jr., Editor Pitfalls in AR42J-model of Cerulein-Induced Acute Pancreatitis PONE-D-20-13635R1 Dear Dr. Hollenbach, 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. 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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 Reviewer #3: 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 Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: N/A ****** 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 Reviewer #3: 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 Reviewer #3: 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: All of my questions were answered and the revised version of the manuscript was corrected accordingly. Reviewer #3: The authors have clarified the reviewers’ queries and the quality of the manuscript could be improved. ****** 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 Reviewer #3: No https://doi.org/10.1371/journal.pone.0242706.r003
Formally Accepted
26 Nov 2020 Acceptance Letter - Zoltán Rakonczay Jr., Editor PONE-D-20-13635R1 Pitfalls in AR42J-model of Cerulein-Induced Acute Pancreatitis Dear Dr. Hollenbach: 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. Zoltán Rakonczay Jr. Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0242706.r004

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