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Original research
Preclinical mouse model of a misfolded PNLIP variant develops chronic pancreatitis
  1. Guoying Zhu1,2,
  2. Steven J Wilhelm1,
  3. Leah G George1,
  4. Brett M Cassidy1,
  5. Sammy Zino1,
  6. Cliff J Luke1,3,
  7. Mina Hanna1,
  8. Stephen Stone1,
  9. Nhung Phan1,
  10. Neel Matiwala1,
  11. Samuel J Ballentine4,
  12. Mark E Lowe1,
  13. Xunjun Xiao1
  1. 1Department of Pediatrics, Washington University School of Medicine, St Louis, Missouri, USA
  2. 2Department of Clinical Nutrition, Putuo People’s Hospital, School of Medicine,Tongji University, Shanghai, China
  3. 3Siteman Cancer Center, Washington University School of Medicine, St Louis, Missouri, USA
  4. 4Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri, USA
  1. Correspondence to Dr Xunjun Xiao, Department of Pediatrics, Washington University School of Medicine, St Louis, Missouri, USA; xunjun.xiao{at}wustl.edu

Abstract

Objective Increasing evidence implicates mutation-induced protein misfolding and endoplasm reticulum (ER) stress in the pathophysiology of chronic pancreatitis (CP). The paucity of animal models harbouring genetic risk variants has hampered our understanding of how misfolded proteins trigger CP. We previously showed that pancreatic triglyceride lipase (PNLIP) p.T221M, a variant associated with steatorrhoea and possibly CP in humans, misfolds and elicits ER stress in vitro suggesting proteotoxicity as a potential disease mechanism. Our objective was to create a mouse model to determine if PNLIP p.T221M causes CP and to define the mechanism.

Design We created a mouse model of Pnlip p.T221M and characterised the structural and biochemical changes in the pancreas aged 1–12 months. We used multiple methods including histochemistry, immunostaining, transmission electron microscopy, biochemical assays, immunoblotting and qPCR.

Results We demonstrated the hallmarks of human CP in Pnlip p.T221M homozygous mice including progressive pancreatic atrophy, acinar cell loss, fibrosis, fatty change, immune cell infiltration and reduced exocrine function. Heterozygotes also developed CP although at a slower rate. Immunoblot showed that pancreatic PNLIP T221M misfolded as insoluble aggregates. The level of aggregates in homozygotes declined with age and was much lower in heterozygotes at all ages. The Pnlip p.T221M pancreas had increased ER stress evidenced by dilated ER, increased Hspa5 (BiP) mRNA abundance and a maladaptive unfolded protein response leading to upregulation of Ddit3 (CHOP), nuclear factor-κB and cell death.

Conclusion Expression of PNLIP p.T221M in a preclinical mouse model results in CP caused by ER stress and proteotoxicity of misfolded mutant PNLIP.

  • PANCREATITIS
  • CELL DEATH
  • CHRONIC PANCREATITIS

Data availability statement

All data relevant to the study are included in the article or uploaded as online supplemental information.

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Data availability statement

All data relevant to the study are included in the article or uploaded as online supplemental information.

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Footnotes

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  • Contributors Study conceptual design and supervision (MEL and XX). Study experiments, data analysis and interpretation (GZ, SJW, LGG, BMC, SZ, CJL, MH, SS, NP, NM, SJB, MEL and XX) Manuscript drafting (MEL, XX). Manuscript revision (GZ, SJW, LGG, BMC, SZ, CJL, MH, SS, NP, NM, SJB, MEL and XX). Being fully responsible for the overall work and the conduct of the study as the author guarantor (XX).

  • Funding This work was supported by The Children’s Discovery Institute of Washington University and St. Louis Children’s Hospital MI-II-2018-750-2, NIH/NIDDK R01 DK128188 to XX and by NIH/NIDDK R01 DK080820 to MEL. CJL is supported by NIH/NIDDK R01 DK114047 and The Children’s Discovery Institute. We gratefully acknowledge Dr. Sanja Sviben and Dr. James Fitzpatrick for their assistance in electron microscopy studies conducted at the Washington University Center for Cellular Imaging (WUCCI), which is supported in part by Washington University School of Medicine, The Children’s Discovery Institute (CDI-CORE-2015-505 and CDI-CORE-2019-813), the Foundation for Barnes-Jewish Hospital (3770) and the Washington University Diabetes Research Center (NIH P30 DK020579). We thank the Genome Engineering and iPSC Center at Washington University School of Medicine in St. Louis for the design and validation of CRISPR/Cas9 reagents and genotyping of mice. We thank the Transgenic, Knockout and Micro-Injection Core and the Mouse Genetics Core at Washington University in St. Louis for mutant mouse production and animal husbandry, respectively.

  • Competing interests None declared.

  • Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.