3.6 Genetic Models
From human studies there is increasing evidence that genetics plays an important role in the susceptibility to recurrent acute or chronic pancreatitis.Linkage and candidate gene analysis have discovered six major genes that target either acinar cells in a trypsin-dependent pathway（PRSS 1，PRSS2，CTRC，CASR，SPINK1）or ductal cells（CFTR）and their mutations finally lead to loss or gain of function of the proteins（Aghdassi et al.2015；Whitcomb 2012）.In a much broader approach recent studies investigated genetic variants using genomewide association studies（GWAS）that discovered polymorphism in genes，which have not been associated with pancreatitis yet（Derikx et al.2015；Weiss et al.2015；Whitcomb et al.2012）.
3.6.1 Trypsinogen
PRSS1 gain of function mutations，such as p.R122H，are known to increase the risk of recurrent acute and chronic pancreatitis in humans（Whitcomb et al.1996）.This amino acid exchange renders trypsin to be more resistant to degradation.Meanwhile more PRSS1 mutations were identified in association with hereditary pancreatitis.For these reasons a transfer of clinical findings from bedside to bench，i.e.an animal model became attractive:Indeed，a transgenic mouse carrying the PRSS1 mutant R122H，placed under the control of an elastase promoter showed signs of chronic pancreatic disease.Fibrosis and acinar cell degeneration were observed.Changes started at 7 weeks of age and progressed with older age resembling morphology in humans（Archer et al.2006）.This model has two strengths:First it represents an experimental system that shows histologic characteristics similar to those from human disease.Secondly，it is based on pathophysiologic mechanisms that are known from hereditary chronic pancreatitis in humans.It therefore might be a model that most closely mimics the human pathophysiology.Moreover，R122H_mPRSS1 mice displayed an enhanced reaction upon serial caerulein injections:while in wildtype animals the inflammatory reaction is largely resolved during the post-injection phase PRSS 1 transgenic mice showed a chronic inflammatory response with extensive collagen deposition.Unfortunately，the findings have never been replicated in other laboratories and the model and the mouse are no longer available.
3.6.2 CFTR
Cystic fibrosis transmembrane conductance regulator（CFTR）is expressed on epithelial cells such as ductal cells and functions as a low conductance chloride ion selective channel（Wanget al.2014）.Its major function is believed to dilute and alkalinize the protein-rich acinar secretions，thereby preventing the formation of protein plugs（Chen and Ferec 2012）.Various loss-of-function CFTR variants have been reported in patients with chronic pancreatitis，occurring in up to 37%in idiopathic and 15%in alcoholic chronic pancreatitis（Cohn et al.1998；Sharer et al.1998；Weiss et al.2005）.
Snouwaert and collaborators developed a murine model of cystic fibrosis by targeted disruption of the CFTR gene（cftrm1UNC；UNC:University of North Carolina）.Homozygous knockout mice displayed many features common to young human cystic fibrosis patients.Life expectancy of these mice was often no longer than 40 days resulting from intestinal obstruction causing death（Snouwaert et al.1992）.Although overt signs of chronic pancreatitis were absent in young mice pancreata of CFTR-/-mice showed at least mild features of early cystic fibrosis such as a dilation of the apical acinar lumen（Durie et al.2004），impaired acinar endocytosis or acidification of the pancreatic juice（Freedman et al.2001）.In older animals（9-24 months）more severe changes were visible including a higher proportion of connective tissue areas，clogging of the pancreatic duct with mucus and concomitant duct dilation with loss of exocrine tissue，highly resembling human morphology（Durie et al.2004；Dimagno et al.2005）.Untreated CFTR-/-mice showed a reduction of constitutive expression of pancreatic digestive proteins，lipase，pro-elastase and trypsinogen by around 20-25%.Upon caerulein hyperstimulation CFTR-/-mice developed more severe acute pancreatitis but displayed only a blunted increase of pancreatic digestive enzymes，which might suggest mild pancreatic exocrine insufficiency（Dimagno et al.2005）.Cell death was shifted from an apoptotic to a non-apoptotic form.
Limiting factors of this model implythe necessity of longer breeding periods to reach an older stage.In addition，other organs will be involved besides the pancreas because cystic fibrosis is a systemic disease affecting many organs.This means that this experimental model might not be an ideal system for studying pancreatic injury alone and its effects.Moreover life-threatening complications independent of pancreatic insufficiency can occur，such as respiratory airway obstruction due to defective mucociliary clearance or intestinal obstruction that cause early death of mice（Durie et al.2004）.
3.6.3 Cytokines/Chemokines
Many other genes are involved in acute and chronic inflammation and immune response in humans.Whether they affect human chronic pancreatitis as well is still a matter of debate and they will be good candidates for prospective genetic linkage analysis.During inflammation pro-inflammatory cytokines such as interleukin-6（IL-6），interleukin-1β（IL-1 β）or TNFα and chemokines，e.g.CXCL1 and CXCL2 are released causing an influx of immune cells（Steele et al.2015）.Invading inflammatory cells themselves augment local damage，perpetuate protease activation and further release of cytokines（Sendler et al.2013）.
Transgenic mice with overexpression of human IL-1β（sshlL-1β）were followed up for 2 years.The pancreas showed typical histologic features of chronic pancreatitis，i.e.organ atrophy，dilatation of the pancreatic and biliary tract，secondary to proximal fibrotic stenosis（Marrache et al.2008）.Overt sign of exocrine or endocrine insufficiency were not observed in elastase sshlL-1p mice.
This model also addressed the question of malignant transformation in the setting of chronic inflammation.Pancreatic adenocarcinoma can arise on the basis of chronic pancreatitis and p53 mutations are frequently seen in pancreatic cancer（Hingorani et al.2005；Tuveson and Hingorani 2005）.When crossed with heterozygous p53R172H/+mice to create a double transgenic mouse an increased frequency of tubular complexes including some evidence of acinar-ductal metaplasia，that are considered to be preneoplastic lesions，were detected.However pancreatic ductal adenocarcinoma was hardly seen.
CXCR-/-mice were protected from pancreatic damage when chronic pancreatitis was induced by serial caerulein injections（Steele et al.2015）.In particular pancreata showed less organ atrophy and a reduced leukocyte infiltration.Although neutrophil infiltration significantly increases during acute and chronic pancreatitis selective neutrophil depletion（by anti-Ly6G antibody）was less effective suggesting that CXCR2 on non-neutrophils contributes to the development of chronic pancreatitis.Extension of fibrosis was comparable to wildtypes indicating that activation of stellate cells is still maintained despite CXCR2 knockout.
Monocyte chemoattractant protein 1（MCP-1）is classified as a CC-chemokine.Rats were treated with dibutyltin dichloride（DBTC）and received intramuscular injections of mutant MCP-1（mMCP-1）plasmids for several days.Pancreatic fibrosis induced by DBTC was attenuated by transgenic expression of mutant MCP-1.Concomitantly a decrease of serum MCP-1 concentrations and less inflammation were seen.Rats carrying mMCP-1 were heavier than controls suggesting that exocrine insufficiency is abrogated in these animals（Zhao et al.2005）.(https://www.daowen.com)
3.6.4 Autoimmune Mediated
MRL/MpJ mice bearing a mutated lymphoproliferative gene，lymphoproliferation（lpr），（MRL/MpJ-lpr/lpr）spontenously develop autoimmune disorders such as glomerulonephritis，arthritis and sialadenitis.Mice lacking the lpr-gene also develop autoimmune diseases，but at later stage of life（Andrews et al.1978；Kanno et al.1992）.Inflammatory lesions with acinar destruction and fatty tissue replacement were found in up to 74%of female mice at 34-38 weeks.Endocrine function was preserved，as pancreatic islets remained unaffected.Interestingly male mice later developed pancreatitis that was less intense and only present in less than 40%of 46-50 week old mice.Sex-related factors are discussed to cause the attenuated form，since administration of androgens retarded autoimmune disease in female mice as well（Steinberg et al.1980）.Histology of MRL/Mp mice，in particular their inflammatory infiltrate and fibrosis pattern was comparable with autoimmune pancreatitis type I as seen in humans.Characteristic signs including periductal lymphoplasmocytic infiltration，storiform fibrosis or elevated antibody levels（lactoferrin，carboanhydrase）were present（Schwaiger et al.2014）.Application of polyinosinic:polycytidylic acid（poly I:C）accelerated and enhanced disease progression.Blockage of cytotoxic T-lymphocyte associated protein 4（CTLA-4），one of the most potent modulators of T-cell response，increased the severity of autoimmune pancreatitis as well（Schwaiger et al.2014）.
Lymphotoxin receptors are membrane proteins of the TNF superfamily and are involved in intracellular signaling.Pleiotropic functions including control of an adequate immune response are maintained lymphotoxins（Wolf et al.2010）.Mice with transgenic expression of lymphotoxin α andβ（Elal-LTαβ）resembled features of autoimmune pancreatitis.When lymphocytes were depleted（Elal-LTab/Ragl（-/-））autoimmunity was lost whereas deletion of monocytes（Elal-LTab/Ccr2（-/-））preserved autoimmune disease but prevented early pancreatic tissue damage（Seleznik et al.2012）.
C5 is a factor of the complement system and complement activation drives many inflammatory responses.Cleavage of the C5 molecule generates C5a and b.C5a exerts a predominant pro-inflammatory activity mediating leukocyte chemotaxis and release of proinflammatory cytokines.An increased vascular permeability facilitates neutrophil transmigration（Kohl 2001）.C5b holds cytolytic functions through the formation of the membrane attack complex（MAC）but also possess a multitude of non-cytolytic immune functions as well（Woodruff et al.2011）.Trypsin is known to act as a complement activator，and is able to cleave both C3 and C5（Acioli et al.1997）.
C5 is functionally linked to liver fibrogenesis，as its receptor（C5R1）is expressed on endothelial and Kupffer cells and activates myofibroblasts.Deletion of C5，either genetically or pharmacologically resulted in reduced liver fibrosis upon CCL4 treatment（Hillebrandt et al.2005）.C5 exerts pro-fibrogenic effects in chronic pancreatitis as well.Sendler and coworkers subjected C5 deficient mice to either pancreatic duct ligation for up to 3 weeks or serial caerulein injections for 10 weeks.In both models pancreatic fibrosis was reduced in C5-/-animals and most predominant at later time points.Pharmacological anti-C5 treatment using a C5-receptor antagonist or a peptide inhibitor achieved comparable results to the knockout mouse model.Isolated pancreatic stellate cells were activated by C5a and synthesized massive amounts of extracellular proteins（Sendler et al.2015）.
3.6.5 WBN/Kob Rats
An example of a rat model mimicking chronic pancreatitis is given by the WBN/Kob（Wistar-Bonn/Kobori）strain.This strain is derived from Wistar rats and was originally generated as a model susceptible to gastric tumors.Starting with an age of 3-6 months male WBN/Kob rats show progressive fibrosis around the pancreatic ducts and vessels.A degradation of Langerhans islets leads to a reduction of number and size of the islets（Mori et al.2009；Ohashi et al.1990）.At 9-12 months of age full manifestation of diabetes mellitus occurs with impaired glucose tolerance，hyperglycemia and glycosuria.Exocrine function measured by BT-PABA（n-benzoyl-l-tyrosyl-paminobenzoic acid）urinary excretion was diminished as well.Chromosomal mapping revealed two potentially responsible loci at chromosome 7 and X，Pdwkl and 2（pancreatitis and diabetes mellitus in WBN/Kob locus 1 and 2）.Candidate genes were found in the Pdwkl locus that makes a genetic origin of this phenotype likely（Mori et al.2009）.
More genetically engineered animals models exist describing chronic fibrotic disease in the pancreas.Good examples are a pancreas specific Kif3a knockout model that resulted in cyst formation and pancreatic fibrosis in aged mice.The Kif3a gene encodes a subunit of the kinesin-2 complex that is essential in cilia formation and its defect is associated with several human genetic diseases，including polycystic kidney disease（PCKD）Bardet-Biedl syndrome and primary ciliary dyskinesia（Cano et al.2006）.Perk-/-mice experienced a rapid loss of their endocrine and exocrine function accompanied with increasing cell death（Harding et al.2001）.Absence of PERK（Protein kinase R-line endoplasmic reticulum kinase），a transmembrane protein of the endoplasmic reticulum and usually highly expressed in the pancreas，renders cells to be more susceptible to ER stress and protein misfolding.For further information on genetic animal models for chronic pancreatitis please see Table 5.3.
Apparently the majority of studies using mouse models employ the C57BL/6 strain for their experiments.This strain is most frequently used in biomedical research.Moreover genetically modified mouse models are often derived from a C57BL/6 background.Meanwhile due to existence of various mouse breeding facilities and separate inbred colonies a high number of C57BL/6 substrains exist that have important genetic and phenotypic differences（Bourdi et al.2011；Ulmasov et al.2013；Watkins-Chow and Pavan 2008）.In a recent study it could be shown that substrains of C57BL/6 mice show different disease severities of chronic pancreatitis upon repetitive caerulein injections.Fibrosis，acinar atrophy and inflammatory infiltrate were markedly more in B6J than in B6N substrains（Ulmasov et al.2013）.Knowledge of the exact background of genetically engineered animals and their control is of high importance，as an erroneous choice of the wrong genetic substrain most probably produces misleading results.
Table 5.3 Overview of genetic animal models for chronic pancreatitis（adapted from Lerch and Gorelick，2013）
ko knockout，tg transgenic，+yes，-no，n.a.no information available