Gastroenterology 2019;156:1951–1968

Genetics, Cell Biology, and Pathophysiology of Pancreatitis

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1Medical Department II, University Hospital, LMU, Munich, Germany; 2Department of Medicine A, University MedicineGreifswald, Greifswald, Germany; 3Institute for Translational Medicine, University of Pécs, Pécs, Hungary; and 4Center forExocrine Disorders, Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of DentalMedicine, Boston, Massachusetts

Julia Mayerle1,2 Matthias Sendler2 Eszter Hegyi3 Georg Beyer1 Markus M. Lerch2 Miklós Sahin-Tóth4

Since the discovery of the first trypsinogen mutation infamilies with hereditary pancreatitis, pancreatic geneticshas made rapid progress. The identification of mutationsin genes involved in the digestive protease–antiproteasepathway has lent additional support to the notion thatpancreatitis is a disease of autodigestion. Clinical andexperimental observations have provided compelling evi-dence that premature intrapancreatic activation of diges-tive proteases is critical in pancreatitis onset. However,disease course and severity are mostly governed by in-flammatory cells that drive local and systemic immuneresponses. In this article, we review the genetics, cellbiology, and immunology of pancreatitis with a focus onprotease activation pathways and other early events.

Keywords: Trypsinogen; Pancreatitis; Genetics; Inflammation;Cell Death.

ancreatitis is the leading cause for gastrointestinal

Abbreviations used in this paper: AP, acute pancreatitis; AP1, activatorprotein 1; CASR, calcium-sensing receptor; CCK, cholecystokinin; CEL,carboxyl ester lipase; CEL-HYB, hybrid carboxyl ester lipase allele; CFTR,cystic fibrosis transmembrane conductance regulator; CLDN2, claudin 2;CP, chronic pancreatitis; CPA1, procarboxypeptidase A1; CPA2, pro-carboxypeptidase A2; CPB1, procarboxypeptidase B1; CTRC, chymo-trypsin C; DAMP, damage-associated molecular pattern; ER, endoplasmicreticulum; EV, endocytic vacuole; GWAS, genomewide association study;HP, hereditary pancreatitis; IKK, inhibitor of nuclear factor kB kinase; IL,interleukin; MODY8, maturity-onset diabetes of the young type 8; MyD88,myeloid differentiation primary response 88; NET, neutrophil extracellulartrap; NFkB, nuclear factor k light-chain enhancer of activated B cells;NLRP3, nucleotide-binding oligomerization domain-like receptor protein3; OR, odds ratio; PRSS1, cationic trypsinogen; PRSS2, anionic trypsin-ogen; RAP, recurrent acute pancreatitis; RIP, receptor-interacting protein;ROS, reactive oxygen species; SPINK1, serine protease inhibitor Kazaltype 1; TLR, Toll-like receptor; TNF, tumor necrosis factor.

Most current article

© 2019 by the AGA Institute0016-5085/$36.00

https://doi.org/10.1053/j.gastro.2018.11.081

Pdisease-related hospital admissions and it is associ-ated with considerable morbidity, mortality, and socioeco-nomic burden.1 Recent years have shed light on thepathophysiology of pancreatitis, opening up new avenuesfor causal treatment. In this review article, we dissect thecomplexity of premature protease activation and its effecton local and systemic inflammation in pancreatitis.

Genetics of PancreatitisAcute pancreatitis (AP), recurrent AP (RAP), and chronic

pancreatitis (CP) form a disease continuum.2 The progres-sion of a sentinel attack of AP to RAP and eventually to CP isoften driven by chronic alcohol consumption or genetic riskfactors. Genetic risk for RAP and CP overlaps, whereas ge-netic studies in AP are difficult to interpret in the absence ofadequate follow-up that can exclude RAP and CP cases.

Most pancreatitis risk genes code for digestive proteases,a trypsin inhibitor, or other proteins highly expressed in thepancreas. Functional studies have classified the variousmutations and other genetic alterations into pathologic

pathways driving pancreatitis onset and progression. Wediscuss the trypsin-dependent, mis-folding–dependent, andductal pathways of pancreatitis risk.

Trypsin-Dependent Pathway of GeneticRisk in CP

Pancreatic acinar cells secrete digestive proteases ininactive precursor forms that are flushed from the ductalsystem in a sodium bicarbonate–rich fluid. Trypsinogen, theprecursor to trypsin, becomes activated by the serine pro-tease enteropeptidase in the duodenum.3 Trypsin activateschymotrypsinogens, proelastases, and procarboxypeptidaseB1 (CPB1), whereas activation of procarboxypeptidases A1(CPA1) and A2 (CPA2) requires the concerted action oftrypsin and chymotrypsin C (CTRC).4 Trypsinogen also canbe activated by trypsin, and this process is called autoacti-vation.3 Premature, intrapancreatic activation of trypsin-ogen can occur by autoactivation or can be catalyzed by thelysosomal cysteine protease cathepsin B. Protective mech-anisms that prevent trypsinogen activation in the pancreasinclude trypsin inhibition by the serine protease inhibitor

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Kazal type 1 (SPINK1) and trypsinogen degradation byCTRC and cathepsin L.5–7 Although the principal action ofCTRC is to promote trypsinogen degradation, it also enhancestrypsinogen activation by processing the trypsinogen activa-tion peptide to a shorter form, which is more sensitive totrypsin-mediated activation7–9 (Figure 1). As discussed below,certain trypsinogen mutations can hijack this mechanism andthereby stimulate trypsinogen activation to a pathologic extent.Human genetic studies strongly support trypsinogen autoac-tivation and CTRC-dependent trypsinogen degradation as keymechanisms determining intrapancreatic trypsin activity,whereas similarly compelling genetic evidence for the role ofcathepsins B and L has been lacking.10

Cationic Trypsinogen MutationsMutations in human cationic trypsinogen (PRSS1) cause

autosomal dominant hereditary pancreatitis (HP) withincomplete penetrance or act as risk factors in sporadicCP.7,11 Approximately 90% of PRSS1-mutation–positive HPfamilies carry the p.N29I, p.R122C, or p.R122H mutation inthe heterozygous state. Mechanistically, the p.R122C andp.R122H mutations prevent CTRC-mediated trypsinogendegradation.9 The p.N29I mutation has multiple distinct ef-fects on trypsinogen biochemistry, the combination of whichmarkedly increases trypsinogen autoactivation. These effectsinclude an increase in N-terminal processing, decreasedCTRC-dependent degradation, and a slightly increased pro-pensity for autoactivation.9 The p.A16V variant sensitizes theactivation peptide of trypsinogen to CTRC-mediated pro-cessing, which in turn enhances autoactivation.8,9 Pathologictrypsin levels generated by mutation p.A16V are lower thanthose seen with the p.R122H variant, which explains thedecreased penetrance of the p.A16V variant. More recently,mutation p.P17T was found to exhibit characteristics thatwere similar to those of p.A16V.12 Rare mutations affectingthe activation peptide of cationic trypsinogen (p.D19A,p.D21A, p.D22G, p.K23R, and p.K23_I24insIDK) robustlystimulate autoactivation independently of CTRC.13–15 Cellculture experiments have indicated that these activationpeptide mutants are secreted poorly because of intracellularactivation and degradation, which can lead to cellular stressand consequent acinar cell death.16 Taken together, PRSS1mutations stimulate activation of cationic trypsinogen bydecreasing CTRC-dependent trypsinogen degradation,increasing CTRC-mediated processing of the activation pep-tide, or directly stimulating autoactivation. Genomewide as-sociation studies (GWASs) have identified a commonlyoccurring haplotype in the PRSS1 and anionic trypsinogen(PRSS2) locus that slightly decreases CP risk (odds ratio [OR]1.5), with a more pronounced effect in alcoholic CP.17–19 Avariant (c.�204C>A) that lies in the promoter region ofPRSS1 and decreases trypsinogen expression appears to beresponsible for this small protective effect.20

SPINK1 MutationsThe association between the most common p.N34S

SPINK1 variant and CP was first described by a candidategene study in 2000.21 A meta-analysis reported a carrier

frequency of 9.7% in patients with CP and 1% in controlswith an average OR of 11, making the p.N34S the clinicallymost significant risk factor for CP.22 When consideringEuropean populations only, p.N34S increased CP risk byapproximately 10-fold.23 Although several studies haveattempted to identify the functional effect of p.N34S and itsassociated haplotype, the molecular mechanism underlyingCP risk remains unclear. Neither p.N34S nor any of the 4linked intronic variants affect trypsin inhibitory function orcellular expression of SPINK1.24–27 Interestingly, in pancre-atic cancer cell lines carrying the heterozygous p.N34Svariant, decreased expression of the mutant allele wasobserved compared with the wild-type allele.28 The in-vestigators suggested that the c.�4141G>T variant or ahitherto unknown variant located in the 50 region of the genemight be responsible for the decreased expression of thep.N34S allele. The second most frequently reported SPINK1haplotype in CP contains the c.�215G>A promoter variantand the c.194þ2T>C variant in intron 3.21,29 This haplotypewas observed more frequently in East Asia than in Europe.7

Functional studies have shown that the c.194þ2T>Cvariant causes skipping of exon 3, which results in dimin-ished SPINK1 expression.27,30,31 However, the c.�215G>Avariant increases promoter activity, which might mitigate theeffect of the c.194þ2T>C mutation and allow for some re-sidual SPINK1 expression even in homozygous carriers.32,33 Alarge number of rare or private alterations in SPINK1 havebeen found in CP, which cause loss of SPINK1 function byvarious mechanisms.7

Protective PRSS2 VariantAlthough PRSS1 and PRSS2 share 90% identity at the

amino acid level and PRSS2 rapidly auto-activates, no path-ogenic PRSS2 variants have been identified in HP or sporadicCP.34,35 The absence of PRSS2 mutations in CP could be dueto the more effective CTRC-mediated degradation of anionictrypsinogen, which would prevent intrapancreatic activationof the enzyme even if it were mutated.36 However, a pro-tective variant p.G191R with a w3- to 6-fold effect andapproximately 5% population frequency was discovered.35,37

The mutation introduces a new trypsin cleavage site intoanionic trypsinogen, which increases autocatalytic proteolysisand inactivation.35

CTRC MutationsDirect DNA sequencing of the CTRC gene in patients with

nonalcoholic CP showed heterozygous mutations in 4% ofpatients that increased CP risk by 5-fold on average.38,39 Themutations cause loss of CTRC function by various mecha-nisms, which include defective secretion from mis-folding,resistance to trypsin-mediated activation, catalytic defi-ciency, or increased degradation by trypsin.40,41 Consideringthe clinically significant variants, p.A73T exhibits a severesecretion defect, p.K247_R254del is inactive and prone todegradation, p.R254W is degraded by trypsin, and p.V235Ihas partly decreased activity.40 Subsequent studies reporteda frequent p.G60¼ variant found in approximately 30% ofpatients with CP.42–45 The heterozygous p.G60¼ increases

Figure 1.Genetic risk factors associated with the trypsin-dependent pathologic pathway. See text for details.

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the risk of CP by 2.5-fold, whereas the homozygous stateincreases the risk by 10-fold.43,45 The variant is associatedwith decreased CTRC mRNA expression (GTEx Portal),possibly because of altered pre-mRNA splicing.

CTRB1–CTRB2 Locus InversionA recent European GWAS identified a large inversion at

the CTRB1–CTRB2 locus that modestly (OR 1.35) modifies therisk for alcoholic and nonalcoholic CP.19 The inversionchanges the expression ratio of the CTRB1 and CTRB2chymotrypsin isoforms in such a manner that protectivetrypsinogen degradation is increased and CP risk is decreased.In China, the reported population frequency of the inverted(major) allele is 99.6%; thus, the allele is virtually fixed anddoes not contribute to CP risk.46 A mouse model with geneticdeletion of the major mouse chymotrypsin CTRB1 exhibitedincreased intra-acinar trypsin activation and more severepancreatitis induced by the secretagogue cerulein.47 Theseobservations provided the first in vivo proof for the protectiverole of chymotrypsin-mediated trypsinogen degradationagainst pancreatitis.

Mis-folding–Dependent Pathway ofGenetic Risk in CP

More recently, an alternative pathomechanism seem-ingly unrelated to premature intrapancreatic trypsinogenactivation has been identified, in which mutation-inducedmis-folding and consequent endoplasmic reticulum (ER)stress lead to acinar cell damage and pancreatitis.48

Mis-folding–Associated PRSS1 MutationsIn 2009, a subset of PRSS1 variants was found to cause

decreased secretion, intracellular retention, and increasedER stress markers, as judged by in vitro cell culture ex-periments.49 These PRSS1 mutations occur rarely and aremostly associated with sporadic disease (eg, p.C139F,

p.C139S, p.G208A), but also have been found in HP familieswith incomplete penetrance (p.L104P, p.R116C).48 Variantp.G208A is prevalent in East Asia (4% of CP cases) and wasdetected in Europe only in a single case thus far.50,51

Mis-folding–Associated CPA1 MutationsA candidate gene study in 2013 reported that mutations

in the CPA1 gene are associated with CP (OR w25), espe-cially with early-onset disease (OR w80).52 The vast ma-jority of pathogenic CPA1 variants occur with low frequencyand are mostly found in sporadic CP. The p.S282P variantwas described in 2 HP families.53 Pathogenic CPA1 variantscause proenzyme mis-folding, resulting in a secretion defect,intracellular retention, and ER stress.52,53 In contrast toCPA1, variants of CPB1 and CPA2 are not associated withCP.54 Interestingly, ER stress-inducing CPA1 and CPB1 var-iants were over-represented in patients with pancreaticcancer without a clinical history of RAP or CP.55 Most ofthese variants caused premature truncation and did notoverlap with those found in CP. A mouse model for the mis-folding–dependent pathway was described recently. Thisstudy showed that CPA1 N256K knock-in mice harboring themost frequent p.N256K human CPA1 mutation developspontaneous and progressive CP and exhibit signs of ERstress in their pancreas.56

Mis-folding–Associated CEL Mutations andCEL-HYB Allele

Single-nucleotide deletions in the last exon of the CELgene encoding carboxyl ester lipase cause maturity-onsetdiabetes of the young type 8 (MODY8).57 The deletionsalter the reading frame of the C-terminal variable numbertandem repeat sequence, resulting in CEL proteins withunnatural extensions that are prone to aggregation.58,59 Theexocrine dysfunction in MODY8 is in all likelihood caused bymis-folding–induced ER stress and consequent acinar cellloss. A hybrid CEL allele (CEL-HYB1) formed between CEL

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and its neighboring pseudogene CELP was over-representedin idiopathic CP by approximately 5-fold vs the averagepopulation frequency of 0.5%–1%.60 In cell culture experi-ments, the hybrid protein was secreted poorly because ofintracellular retention, suggesting that the CEL-HYB1 variantcan increase CP risk by the mis-folding–dependent pathway.A second hybrid CEL allele (CEL-HYB2) that does not asso-ciate with CP was described in Asian populations.61 Inter-estingly, the CEL protein carries blood group antigens and aGWAS in 2015 indicated that fucosyl-transferase 2 nonse-cretor status and blood group B are risk factors for CP.62

Although other studies in ethnically mixed cohorts failedto replicate this association,63,64 with the exception ofazathioprine-induced pancreatitis in patients with inflam-matory bowel disease,65 it is still interesting to speculatethat the observed effects might have been due to changes inCEL folding or trafficking.

Ductal Pathway of Genetic Risk in CPCFTR Variants

The cystic fibrosis transmembrane conductance regu-lator (CFTR) is a cyclic adenosine monophosphate–regulated chloride–bicarbonate channel localized to theapical plasma membrane of epithelial cells66 (Figure 2).CFTRmutations disrupt channel activity or affect membranelevels and are associated with various phenotypes, ranging

from asymptomatic state to multiorgan symptoms leadingto the diagnosis of cystic fibrosis in homozygous carriers ofsevere mutations. Observations that heterozygous andcompound heterozygous CFTR mutations are associatedwith CP were reported by 2 studies in 1998.67,68 In the firstanalysis of the entire CFTR coding region, the frequency ofabnormal CFTR alleles in patients with CP was 18.6%compared with 9.2% in controls.69 More recent large cohortanalyses have corroborated the pathogenic role of CFTRvariants in CP, although the effect and frequency of CFTRvariants was less pronounced than reported previ-ously.39,70,71 Heterozygous carrier status of the severep.F508del mutation confers a small risk for CP (OR 2.5),whereas the mild p.R117H mutation increases risk byapproximately 4-fold. Compound heterozygous state for 1severe and 1 mild CFTR allele represents strong risk for CPand can be considered causative.70 The role of commonpolymorphic CFTR alleles (eg, T5, TG12) and the non–cysticfibrosis–causing, so-called bicarbonate-defective CFTR vari-ants in CP remains controversial because the preponderanceof data does not support their association with CP.66 UnlikeCFTR, variants in the solute-linked carrier 26 member 6 aniontransporter (SLC26A6) do not alter the genetic risk in CP.72

CLDN2 VariantsGWASs of CP identified several single-nucleotide pep-

tides in the claudin 2 (CLDN2) and MORC4 locus to be

Figure 2.Genetic risk fac-tors associated with theductal pathologic pathway.See text for details.

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associated with CP risk.17,19 The OR was approximately 2and the effect was more pronounced in alcoholic CP. Withinthis locus, CLDN2 seems to be the clinically relevant risk gene,because it is expressed in pancreatic ducts at low levels as atight junction protein. It was proposed that CLDN2–MORC4variants might cause CLDN2 mis-localization. Additionalwork is required to clarify the mechanism of action of thisrisk locus and to confirm whether assignment to the ductalpathway is appropriate (Figure 2).

CASR VariantsThe calcium-sensing receptor (CASR) regulates calcium

homeostasis through parathyroid hormone secretion andrenal tubular calcium reabsorption. Functional CASR also isexpressed in the pancreas, including ductal cells whereCASR can respond to high calcium concentrations in thejuice by increasing ductal fluid secretion, thereby preventingstone formation and pancreatitis73 (Figure 2). A USpopulation-based study failed to demonstrate the previouslyanticipated association between CASR variants and theSPINK1 p.N34S haplotype, but reported the p.R990G variantincreased CP risk, especially in subjects with moderate orheavy alcohol consumption.74 More recently, a French studyfound over-representation of rare CASR coding variants inidiopathic CP and a significant association of the p.A986Svariant, but only in the homozygous state, with CP.75 How-ever, the previously reported association with the p.R990Gvariant was not observed in this cohort. Taken together,current evidence does not support a clear role for CASRvariants in CP pathogenesis.

In summary, human genetic data indicate that prematureactivation or mis-folding of pancreatic proteases play acentral role in the onset of pancreatitis and progression toCP (Supplementary Table 1).

Role of Proteases in Pathophysiologyand Cell Biology of Pancreatitis

Although genetic evidence for the involvement of theprotease–antiprotease balance in the pathogenesis ofpancreatitis dates back only 2 decades and focuses mainlyon CP, pathophysiologic and biochemical investigations haveimplicated this system for more a century. Because of thelack of adequate animal models and the inability to keepisolated pancreatic acinar cells in culture for long periods,experimental studies have focused primarily on AP. Therelative importance of the pathways discussed below mightchange with respect to etiology. It is our general under-standing that these mechanisms also are relevant to CP,although experimental evidence is mostly lacking.

Autodigestion by Pancreatic ProteasesThe pathophysiologic concept of autodigestion was first

developed by the Austrian pathologist Hans Chiari in Praguemore than 120 years ago. He claimed that pancreatitis wascaused and driven by the glands’ digestive properties.76

Since then, the pathomechanism of premature activationof pancreatic enzymes and its contribution to disease

severity and progression have captured the attention ofmany pancreatologists. Bialek et al77 first reported thatprotease activation during pancreatitis begins in theexocrine pancreas and Hofbauer et al78 reported that itbegins in a membrane-confined vesicular compartment andparallels acinar cell damage. Although the fact that activa-tion of digestive proteases is an early event during AP iswidely accepted, the question of where and through whatmechanism this process is initiated and whether it plays arole in chronicity remain under debate.

Protease Activation During Pancreatitis—CluesFrom Mechanistic Studies

Role of Calcium for Intracellular Protease Activa-tion. States of hypercalcemia, such as primary hyperpara-thyroidism, are a risk factor for the development of AP inhumans and rats.79–81 Intracellular calcium concentrationsand compartmental distribution in acinar cells are tightlyregulated because calcium serves as a second messenger forthe physiologic release of digestive enzymes in response tovagal nerve stimulation or humoral activation.82,83 Afterintraperitoneal treatment of rats with supramaximal dosesof cerulein, an analogue of cholecystokinin,84 a time-dependent disruption of the physiologic oscillating intra-cellular calcium signal was observed in rat acini using theCa2þ-sensitive dye fura-2.85 When using for the first timefluorescent trypsin substrates that allow the subcellularimaging, localization, and quantification of protease activa-tion,86 Krüger et al87 found that a specific extended plateaurelease of calcium at the apical pole of acinar cells isnecessary for premature trypsin activation to occur, whichis different from the calcium oscillations required forenzyme secretion. These findings were confirmed by otherswho also found that acetylcholine- or cholecystokinin (CCK)-induced intracellular protease activation was associatedwith the formation of cytoplasmic vacuoles in acinar cellsthat resemble those overserved in experimental pancreatitisin vivo88 and that inhibition of calcium release also inhibitsthe formation of these vesicles.89 Acinar cells can replacecalcium for magnesium in its stores and, when this is done,not only the premature activation of proteases in vitro andin vivo but also the severity of pancreatitis is significantlydecreased.90 Two ongoing clinical trials (1 in AP and 1 inCP) are based on this observation. Orabi et al91 took theconcept of calcium-dependent protease activation further byshowing that AP induced by supramaximal doses of themuscarinic agonist carbachol can be abrogated by inhibitionof the intracellular calcium channel ryanodine receptor,which is located at the basolateral membrane of acinar cells.Therefore, the importance of spatial distribution of calciumto the apical pole might be a specific feature of the ceruleinmodel. Interestingly, carbachol-induced protease activationis more severe if cells are pretreated with ethanol. Calciumsignaling also could play a role in other forms of experi-mental pancreatitis, such as bile acid-induced pancreatitis,92

pressure-induced pancreatitis, and pancreatitis after endo-scopic retrograde cholangiopancreatography.93 Experi-mental pancreatitis can be ameliorated by modulating

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calcium release from intracellular stores or influx throughthe plasma membrane by pharmacologic inhibition ofinositol triphosphate receptor (predominantly types 2 and3) signaling94,95 or calcium release-activated calciummodulator 1.96,97 The calcium-dependent protease activa-tion also heavily depends on calcineurin, a calcium-activatedphosphatase, and its downstream signaling through thetranscription factor nuclear factor of activated T cells. In-hibition of calcineurin by inhibitors or in mice lacking cal-cineurin subunits causes decreased intracellular proteaseactivity in secretagogue or bile acid–induced pancreatitis,without affecting vesicular transport.98,99

Mechanisms of Protease Activation. Three majorconcepts have been investigated over the past decades:autoactivation, spatial redistribution, and fusion of zymogengranules with other organelles and failure of protectivemechanisms.

Autoactivation of Trypsin. There is strong evidencefrom human genetic studies indicating that autoactivation oftrypsinogen100 causes CP in affected humans.9 In contrast,trypsinogen autoactivation is unimportant in experimentalcerulein-induced pancreatitis. Inhibition of active trypsin bythe reversible chemical inhibitor S124 could prevent trypsinactivity in experimental cerulein-induced AP, but had no ef-fect on the generation of the trypsin activation peptide (TAP)or active trypsin after washout of S124, thus indicating thathydrolysis of trypsinogen to trypsin appeared in a trypsin-independent fashion.101

Activation by Lysosomal Proteases. Inhibition of thelysosomal hydrolase cathepsin B by the cysteine proteaseinhibitor E-64d leads to a significant decrease in trypsinactivity and TAP formation, indicating that the trypsinactivation in response to cerulein depends on cathepsinB.101–103 Similarly, in cathepsin B knockout mice, trypsinactivation is significantly inhibited after cerulein adminis-tration.104 In the past, it was thought that cathepsin B105

and trypsinogen under physiologic conditions are notlocated in the same cell organelles (zymogen granules forexocytosis vs lysosomes for degradation of content ofendosomes and autophagosomes). In 1998, investigatorsshowed in subcellular fractions of cerulein-treated acini acolocalization of cathepsin B and digestive enzymes,including trypsin and TAP, in heavy fractions78 containingzymogen granules and lysosomes early in the disease courseand later a shift of trypsin and cathepsin B activity to thecytosol.106 This confirmed earlier findings from in vivostudies that indicated a fusion of zymogen-containing vac-uoles with lysosomes in secretagogue-, duct obstruction-, ordiet-induced AP.107–112 Intracellular activation of proteasesother than trypsin, such as chymotrypsin and carboxypep-tidase B, also depend on non-physiologic colocalization withother cell components, but are independent of cathepsinB.113 Mis-sorting in the exocrine machinery, secretionblockage, and reuptake of previously secreted proteases byendocytosis have been described under experimental con-ditions. The fact that an acidic pH, as found in lysosomes,enhances secretagogue-dependent zymogen activation sup-ports the fusion hypothesis.114 A very recent study reportedthat CCK or ethanol treatment depletes acinar cells of

syntaxin 2, a key regulator of apical exocytosis, thus leadingto increased basolateral exocytosis and formation of auto-lysosomes mediated by syntaxins 3 and 4, in which tryp-sinogen activation takes place.115 Inhibition of syntaxin-4–mediated basolateral exocytosis in experimental pancrea-titis decreases disease severity.116 Another concept in-troduces endocytic vacuoles (EVs) as the site of intracellulartrypsinogen activation. These occur under physiologic andpathophysiologic conditions after compound exocytosis ofzymogen granules.117 EV formation is calcium depen-dent.118 Their content is acidic and calcium rich and, aftersupramaximal CCK or taurocholate stimulation, trypsin ac-tivity within these post-exocytic structures can be visualizedusing fluorescent dyes.119 During pancreatitis, EVs arelarger than normal and tend to fuse with the plasmamembrane or even rupture, discharging active trypsin intothe cytosol or extracellular space. This instability is believedto be caused by disruption of otherwise protective actinfilaments surrounding the EV.120 However, rupture of EVs isindependent of trypsin or cathepsin B activity. Secretoryblockage can contribute to these events. In experimentalpancreatitis, vesicle-associated membrane protein-8–medi-ated secretion is impaired because of a loss of early endo-somal proteins, resulting in retention of trypsinogen andtransformation to active trypsin in a cathepsin-dependentmanner. Knockout of vesicle-associated membraneprotein-8 protects against pancreatitis and restoration ofearly endosomal trafficking decreases severity of pancrea-titis.121,122 Vesicular trafficking is regulated in a calcium-dependent manner.123,124

Loss of Trypsin Inhibitors. Little is known about the roleof failing protective mechanisms for protease activation in theearly phase of pancreatitis. The most potent cellular trypsininhibitor is SPINK1. Although SPINK1 mutations are amongthe most common genetic risk factors for the development ofrecurrent AP and CP, thus far, none of the describedmutations seemed to impair SPINK function and therefore donot explain the increased risk for pancreatitis.24–27 In micecarrying a heterozygous SPINK3 deletion, a significantdecrease of functional SPINK does not lead to the develop-ment of spontaneous pancreatitis or more severe diseaseafter supramaximal cerulein administration compared withwild-type controls.125 The fact that mice with a homozygousSPINK3 deletion develop pancreatic atrophy and that thisphenotype can be rescued by transgenic expression of ratpancreatic secretory trypsin inhibitor 1125 point toward arole of trypsin inhibitors during pancreatic development butdo not explain that role in pancreatitis.

In conclusion, the current cumulative evidence suggestsa cathepsin B–dependent mechanism of protease activationin experimental pancreatitis.

Cell Death Cause or Consequence of ProteaseActivation?

Premature intracellular protease activation in acinarcells leads to cell injury. Type of cell death, be it necrosis,apoptosis, autophagy, necroptosis, or pyroptosis, de-termines disease severity.126 Necrosis is understood as an

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unregulated response to damage. In animal models of AP,approximately 1%–5% of acinar cells undergo apoptosisand severity of pancreatitis is inversely correlated to therate of apoptosis.127 Macroautophagy is a multistep,lysosomal-driven, adaptive process by which cells degradecytoplasmic organelles and long-lived protein.128 Pancrea-titis presents with impaired autophagic flux evident byvacuole accumulation.129 Currently, it is debated whetherimpaired autophagy stimulates cell death through accumu-lation of damaged mitochondria mediating an inflammatoryresponse through a reactive oxygen species (ROS)-dependentmechanism as in lysosome-associated membrane protein2130 or Atg7-knockout animals131 or whether autophagyprevents an inflammatory response as in Atg5-knockoutmice.132,133 The regulated process of necrosis is termednecroptosis and is triggered by tumor necrosis factor (TNF),TNF-related apoptosis-inducing ligand, Fas ligand, interferontype 1, and Toll-like receptors (TLRs), which are released inthe early phase of AP in response to protease activation.134

Receptor-interacting protein 1 (RIP1) and RIP3 form aphosphorylated complex, the necrosome, and phosphorylatemixed-lineage kinase domain-like, resulting in membranerupture. Forty percent of cells undergo necroptosis inpancreatitis and RIP3 deletion or treatment with necrostatinameliorates pancreatitis.135,136 Necroptosis releases damage-associated molecular patterns (DAMPs) and those will acti-vate the nucleotide-binding oligomerization domain-likereceptor protein 3 (NLRP3) pathway, resulting in pyropto-sis.137 Pyroptosis is an innate immune sensing mechanismwith poorly understood upstream signaling. Nevertheless,some interesting inhibitors are known, such as lactate,b-hydroxybutyrate, and aspartate. The term pyroptosis de-scribes activation of the inflammasome through NLRP3. Theinflammasome is a macroscopic cytosolic protein complexthat proteolytically cleaves interleukin (IL) 1b and pro-IL18and releases high mobility group box 1 protein. NLRP3 acti-vation requires lysosomal rupture and cathepsin release,calcium influx, and mitochondria-derived ROS productionand thus is closely linked to pancreatitis.138,139 However,NLRP3 expression is restricted to innate immune cells.140

Cell death pathways in pancreatitis intersect. Caspase 3activation can induce not only apoptosis but also pyroptosis.Necroptosis can shift to pyroptosis by caspase 8 activationand necroptosis activates pyroptosis.141 Currently, we havenot understood why some of our patients deteriorate anddevelop a necrotizing course of pancreatitis. A shift ofregulated cell death from apoptosis to pyroptosis andstimulation of necroptosis might explain this observation.Inhibition of pyroptosis, for example, by using Ringer’slactate for volume resuscitation142 or inhibition of nec-roptosis by necrostatin,143 might well be a way forward inthe treatment of pancreatitis.

An interesting question is whether cell death is a resultof premature protease activation or a consequence ofinflammation. The answer is ambiguous: Talukdar et al106

reported a direct effect on lysosomal stability mediated byactive trypsin and lysosomal rupture leading to the releaseof cathepsins into the cytosol, causing dose-dependentlyapoptosis or necrosis. Our group showed that inhibition of

protease activation, especially trypsin by specific inhibitors,results in a decreased rate of apoptosis but does not affectnecrosis.144 Thus, cell death is a result of intracellular pro-tease activation, but this has been shown only for isolatedacinar cells mimicking the early phase of pancreatitis. Tak-ing into account pyroptosis and necroptosis, inflammation isthe origin and consequence of cell death in pancreatitis(Figure 3).

Protease Activity and Disease SeverityThis raises the question of whether intracellular prote-

ase activation of trypsin as it is linked to cell death also canmediate systemic disease severity. The notion that trypsinactivation is linked to disease severity is supported by thecorrelation of TAP urine levels to severity in patients withAP.145 However, several studies have questioned the role oftrypsin for severity of pancreatitis. Expression of mutanthuman trypsin bearing the HP mutation R122H in miceleads to slightly more severe cerulein pancreatitis,146 but,when compared with mice expressing normal human tryp-sinogen, there is no increase in disease severity. Moreover,the effect seems to be not solely dependent on trypsin,because mice with human trypsin mutations (R122H orN29I) show lower trypsin activity after cerulein hyper-stimulation. This might be explained in part by a higher rateof acinar cell apoptosis even in untreated animals transgenicfor human trypsinogen.147 A similar effect is seen in PACE-tryp(on) mice, which conditionally express an endogenouslyactivated trypsinogen within pancreatic acinar cells. Thosemice will develop AP in a trypsin activity-dependent way,which can lead to organ dysfunction and mortality, but theyalso show pronounced caspase 3 activation with consecu-tive apoptotic loss of acinar cells and replacement by fattytissue.148 Similarly, Archer et al149 described a transgenicmouse, in which the human mutation R122H was inserted inthe murine PRSS1 gene. Those mice developed spontaneouspancreatitis and showed a more pronounced inflammatoryinfiltrate and cellular damage in response to cerulein. Thefact that the predominant way of cell death determines theoverall severity of experimental pancreatitis in mice hasbeen demonstrated in animals with deletion for cathepsin L.Cathepsin L degrades trypsinogen into an inactive elongatedTAP, but cathepsin L deficiency leads to a milder form a ofcerulein-induced pancreatitis, which is linked to a shift fromnecrosis to apoptosis.6

The most convincing data questioning the role oftrypsin for disease severity were generated using a mousestrain lacking trypsinogen 7, the murine counterpart ofhuman cationic trypsinogen. Dawra et al150 and Sahet al151 found that a significant decrease in trypsin andchymotrypsin activity after cerulein hyperstimulation inthese mice had no effect on disease severity for cell deathor local or systemic inflammation in AP and CP models,which they claimed was mediated by a mechanismdependent on nuclear factor k light-chain enhancer ofactivated B cells (NFkB). This confirms previous findingsgenerated in the PACE-tryp(on) model, in which NFkB-activation was independent of increased intracellulartrypsin activity in vitro.152

Figure 3. Trypsinogen activa-tion and cell death in pancreaticacinar cells. Intracellular tryp-sinogen activation is an earlyevent at the onset of experi-mental AP. Supramaximalsecretagogue-receptor stimula-tion or activation of bile salt re-ceptors leads to anunphysiologic peak–plateaucalcium signal. This results indisrupted exocytosis ofzymogen granules, secretoryblockage, zymogen retention,and formation of EVs, whichcontain trypsin and trypsin-ogen, taken up from the extra-cellular space. Those EVscolocalize and fuse with lyso-somes containing cathepsin B,which in turn transforms tryp-sinogen into active trypsin.Owing to increasing instability,EVs often rupture, releasingtrypsin and cathepsin B into thecytosol. Active trypsin isbelieved to induce mainlyapoptosis, a silent form of celldeath, which suppressesinflammation. In contrast, if thepathologic calcium releasecannot be contained, then rapidenergy depletion occurs andcells undergo necrosis, duringwhich the plasma membranebecomes leaky and cellularcomponents (eg, DNA or mito-chondria) reach the extracellularspace. Those will be recognizedby leukocytes, which will beactivated by the inflammasomesignaling pathway. IL1b andTNFa release and pyroptosisoccur. If TNFa reaches thebasolateral membrane of previ-ously unaffected or slightlydamaged acinar cells, then itcan induce another form ofprogrammed cell death (ie,necroptosis). PLC, phospholi-pase C.

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Role of Systemic Inflammation inPancreatitisNFkB Activation—Initial Step of Inflammation

The activation of NFkB is an early event duringpancreatitis and occurs within the first minutes after onsetof the disease.153,154 One main function of NFkB is tran-scriptional regulation of the immune response.155 Theprincipal pathway of NFkB signal transduction is depicted inFigure 4. The fact that NFkB is already present in thecytoplasm explains its rapid activation after induction ofpancreatitis.153,154

Intra-acinar protease activation and NFkB activation areearly cellular events during pancreatitis,153,154 which havebeen suggested to occur independent of each other,151 butfollow a similar kinetic. Trypsinogen activation depends onintracellular Ca2þ signaling87 and NFkB activation also canbe induced by protein kinase C and Ca2þ, which could be thereason for parallel kinetics.156 The deletion of trypsinogen 7or cathepsin B, either of which results in greatly decreasedprotease activation,104,144,151 does not influence NFkB acti-vation during cerulein-induced pancreatitis.151 This can beregarded as evidence that protease activation does notdirectly lead to NFkB activation in acinar cells, whereasNFkB activation can still interact with protease activity: thetranscriptional regulation of serine protease inhibitor 2a is

Figure 4. NFkB pathway inpancreatic acinar cells. Theearly activation of NFkBfollows the same timepattern as trypsinogenactivation. They areinduced by cytoplasmicCa2þ influx, but NFkB doesnot depend on trypsinogenactivation. The phosphory-lation of IkBa, followed byproteosomal degradationand nuclear translocationof NFkB (p65–p50) occursin parallel to protease acti-vation. NFkB as a tran-scription factor acts in 2directions; first, transcrip-tions of proinflammatorygenes, such as IL6 orTNFa, initiate the immuneresponse; and second, thetranscription of prosurvivalgenes. Therefore, NFkBcan directly influence pro-tease activity to protectcells by the up-regulationof serine protease inhibitor2a. CTSB, cathepsin B;IkBa, inhibitor of NFkBalpha; IkBb, inhibitor ofNFkB beta; NEMO, NF-kappa-B essential modu-lator; PLC, phospholipaseC; Ub, ubiquitin.

mediated by NFkB and can inhibit trypsin activity in amouse model of acute pancreatitis (Figure 4).157 These re-sults suggest a more complex role of NFkB during diseaseprogression that is not restricted to proinflammation.158

Surprisingly, the pancreas-specific deletion of Rel-A(p65)resulted in increased systemic inflammation and pancre-atic necrosis.159 In contrast, the pancreas-specific deletionof NFkB inhibitor a resulted in nuclear translocation of Rel-A and ameliorated pancreatitis.157 The same phenotype wasobserved in mice deficient in myeloid differentiation pri-mary response 88 (MyD88), which developed a more severedisease phenotype compared with controls.160 MyD88 is acentral adaptor for the TLR–IL1-receptor signaling pathway,which induces NFkB activation. These results ultimatelysuggest a protective role of NFkB activation in pancreaticacinar cells. Other studies attributed a more critical role forNFkB in disease severity. Mice that constitutively over-express the active inhibitor of NFkB kinase subunit b (IKKb)under a pancreas-specific promoter showed chronic infil-tration of immune cells associated with an increased diseaseseverity after cerulein stimulation.161,162 However, theinfiltration of immune cells alone, or the constitutive activityof NFkB in acinar cells, did not result in AP, organ damage,and necrotic or apoptotic cell death162; an additional path-ophysiologic stimulus was still needed. The increasedseverity of pancreatitis in IKKb-overexpressing mice can be

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explained by the presence of leukocytes within thepancreas, which become rapidly activated after disease in-duction and do not need to infiltrate the pancreas. Thepancreas-specific deletion of IKKa, another NFkB inhibitor aphosphorylating kinase, caused spontaneous pancreatitis inmice,163 but this process appeared to be independent ofNFkB. IKKa also regulates autophagic flux, which is essentialfor pancreas homeostasis.164 These data demonstrate thecomplexity of the NFkB network, which often hampers theinterpretation of results. Taken together, the constitutiveactivation of NFkB leads to a chronic infiltration of immunecells, but pancreatitis develops only after induction by anexternal stimulus.161,162 The presence of immune cellswithin the pancreas is required but insufficient for pancre-atitis to develop and these cells need to be activated tocontribute to disease severity. Conversely, data frompancreas-specific RelA deleted mice show that NFkB acti-vation in acinar cells is not essential for the recruitment ofimmune cells to the side of damage.159 Quite to the contrary,the absence of p65 in acinar cells result in greater diseaseseverity. Although all these studies investigated the role ofNFkB in acinar cells, NFkB activation could play a muchlarger role in infiltrating immune cells, which directlyregulate the immune response.

Another master switch in the transcriptional machineryof acinar cells is activator protein 1 (AP1). AP1 is implicatedin multiple transcriptional networks within the acinar cellsregulating pancreatic development, differentiation, celldeath, and inflammation. Mice heterozygous for the orphannuclear receptor NR5A2 develop an acinar cell–autonomousAP1-dependent pre-inflammatory state, which, on a tran-scriptome level, mimics that of early AP.165

However, the extent of NFkB and AP1 activity seems togreatly differ with respect to the cause of pancreatitis. Incerulein models, activation of the 2 transcription factors isdescribed and submaximal CCK stimulation induces acinarcell dedifferentiation and proliferation through the mitogen-activated kinase–c-Jun–AP1 pathway probably as part of apancreatic regeneration program.166,167 In contrast, themetabolites occurring in ethanol-induced experimentalpancreatitis can positively and negatively regulate NFkB andAP1 depending on the predominance of oxidative or non-oxidative alcohol metabolism in the pancreas.168

Role of Infiltrating Immune CellsInfiltration of immune cells starts within minutes after the

onset of disease and plays a crucial role in the severity andprognosis of pancreatitis.169–172 Cells of the innate immunesystem, such as neutrophil granulocytes and monocytes andmacrophages, represent the majority of infiltrating cells.NFkB plays a crucial role in the activation of leukocytes and isa central mediator of the innate and adaptive immune sys-tem.173 Pancreatitis is primarily a sterile inflammation, sopathogen-associated molecular patterns play no role in theactivation of immune cells during the early phase of disease.Thus, in pancreatitis, activation of immune cells is mediatedby cytokines or DAMPs that arise from acinar cell necrosis.Acinar cells release various cytokines and chemokines in

response to CCK stimulation and NFkB activation, such asTNFa,174 IL6, or monocyte chemoattractant protein 1.175

DAMPs and cytokines result in the nuclear translocation ofp65 and p50 within infiltrating immune cells, which enhancesthe cytokine storm through the secretion of proinflammatorymediators175 (Figure 5). DAMPs can act in the same way aspathogen-associated molecular patterns through TLRs176 orspecific receptors such as P2RX7, which uses extracellularadenosine triphosphate as a ligand. Acinar cells, which un-dergo necrotic cell death, release a multitude of differentDAMPs, such as free DNA,177 histones, or free adenosinetriphosphate,138 which can act as immune activators. Acti-vated immune cells increase pancreatic damage andcontribute to systemic inflammation.169,175,178 Several studieshave focused on different populations of immune cells andtheir role during AP.

Neutrophil granulocytes are often used as referencemarkers for pancreatic inflammation by measurements ofmyeloperoxidase activity in tissue and reflect the amount ofinfiltrating neutrophils. One major function of neutrophils isremoving pathogens by the release of proteases, antimi-crobial peptides, and ROS. During pancreatitis, neutrophilsare the major source of ROS production; they can induceoxidative damage on acinar cells and enhance trypsinogenactivation.178 The release of proteases such as PMN-elastasecontributes to tissue destruction and acinar cell dissocia-tion.179 These data indicate that neutrophils have a directeffect on disease severity. This was confirmed by thedepletion of neutrophils using antineutrophil serum, whichresulted in decreased pancreatic damage and proteaseactivation.169,178 Recent studies have investigated the role ofneutrophil extracellular trap (NET) formation in the contextof pancreatitis. NETs are extracellular networks consistingof neutrophil DNA and are used to bind pathogens.180 Thissuicide mechanism of neutrophils is a last line of defenseagainst bacterial infections. NET formation is induced byTLR4 activation and the activation of nicotinamide adeninedinucleotide phosphate oxidase, which lead to the oxidationof peptidyl-arginine-deiminase 4.181,182 TLR4 is responsiblefor the detection of pathogens, although DAMPs can activatethe TLR-signaling pathway.176 Merza et al183 reported thatNET formation enhances the immune response during se-vere AP and is accountable for trypsinogen activation.Treatment with DNAse prevents NET formation and de-creases disease severity.183 In addition to bacterial in-fections, crystals can induce NET formation.184 Anothergroup showed that NET formation is a critical step in bilestone development and plays an important role in ductalobstruction contributing to onset and severity of pancrea-titis.185,186 Neutrophil infiltration during AP is an unspecificreaction of the immune system, which enhances localdamage by the formation of NETs and the release of ROS oractivates digestive enzymes.

Monocytes and macrophages belong to cells of the innateimmune system. In contrast to neutrophil granulocytes,macrophages are characterized by high plasticity. Classicactivated macrophages (M1) act in a proinflammatorymanner and secrete large amounts of IL6, TNFa, IL12, andIL1b, and increased expression of inducible nitric oxide

Figure 5. NFkB activation in inflammatory cells. There are multiple pathways of how NFkB could be activated within leuko-cytes; the major pathways that play a role during AP are depicted. Cytokines such as IL1b or TNFa and DAMP signals actingby TLRs can induce the translocation of p65 and p50 into the nucleus. In leukocytes, most inflammatory mediators are underthe control of NFkB: cytokines, chemokines, adhesion molecules, and components of the inflammasome pathway. Leukocyte-mediated NFkB activation enhances the immune response in a very prominent manner and therefore has a different role inpancreatitis compared with NFkB activation within the acinar cell. ASC, Apoptosis-associated speck-like protein containing aCARD; Casp-1, caspase 1; ICAM-1, intercellular adhesion molecule 1; IL1-R, IL1 receptor; IRAK, interleukin-1 receptor-associated kinase; IkBa, inhibitor of NFkB alpha; MCP-1, monocyte chemoattractant protein 1; MIP-2, macrophage inflam-matory protein 2; MMPs, matrix metalloproteinases; NEMO, NF-kappa-B essential modulator; RANTES, regulated on acti-vation, normal T-cell expressed, and secreted; TAK1, transforming growth factor beta-activated kinase 1; TNFR, TNF receptor;TRADD, tumor necrosis factor receptor type 1-associated DEATH domain protein; TRAF2, TNF receptor-associated factor 2;Ub, ubiquitin; VCAM-1, vascular adhesion molecule 1.

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synthase results in the release of nitric oxide.187 Alterna-tively, activated macrophages (M2) are associated withwound healing, tissue regeneration, and fibrogenesis and actin an anti-inflammatory manner through the release of IL10or transforming growth factor b. They are characterized by

decreased inducible nitric oxide synthase and increased argi-nase 1 expression.187 Macrophages are phagocytosing cells thatremove tissue debris and necrotic and apoptotic cells. DuringAP, macrophage infiltration correlates to a greater extent withpancreatic damage and necrosis than with the number of

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infiltrating neutrophils.175 The reason for that is that macro-phages are required for the removal of necrosis and thus miti-gate pancreatic damage. Phagocytosingmacrophages have beenobserved in different models of AP and CP.130,164,169,175 Incontrast to apoptosis, necrosis is a proinflammatory cell deathbecause it entails the release of multiple DAMPs that induce anM1 polarization of macrophages.188 Therefore, acinar cellapoptosis is suggested to be protective against hyper-inflammation and decrease disease severity.189 M1 macro-phages release large amounts of TNFa, which have a directeffect on pancreatic acinar cells.174 Two independent groupsreported that TNFa secreted from infiltrating monocytes isresponsible for pancreatic damage and digestive proteaseactivation.169,190 Depletion of macrophages by clodronate-containing liposomes decreased disease severity and pro-tected mice from cerulein-induced pancreatitis.169,191 TNFaacts on cells by cell death receptors and is necessary to inducecell death by necroptosis through the RIP1–RIP3 pathway,135

which has been suggested to be the major cell death pathwayduring AP.136 Therefore, infiltrating macrophages are respon-sible for the induction of necroptosis and for the clearance ofnecrotic areas within the damaged pancreas.175

In addition to TNFa, macrophages produce large amountsof IL1b, a proinflammatory cytokine associated with theacute disease phase. In contrast to other cytokines, IL1bneeds to be processed. During maturation, pro-IL1b and pro-IL18 undergo activation by the caspase 1–inflammasomecomplex and are released by the gasdermin D–pore complexfrom the cytosol to the extracellular space.192 In consequenceof this process, the cell undergoes pyroptotic cell death.192,193

During pancreatitis, the activation of the inflammasomecomplex and the release of IL1b contribute critically to dis-ease severity.138,139,175,177 Inflammasome complex formationand pyroptotic cell death are mainly known from macro-phages and are not present in acinar cells.175 Inflammasomeactivation is a complex mechanism requiring 2 signals foractivation: the first signal induces transcriptional up-regulation of inflammasome components by NFkB and thesecond signal induces oligomerization of the inflammasomecomplex and the activation of procaspase 1. The majorinducer of the inflammasome pathway is the TLR–MyD88cascade. The second signal, which is necessary for inflam-masome complex formation, can be a high potassium influx,TNFa, or the release of cathepsins from phagosomes into thecytosol.194 Phagocytosis of zymogens by macrophages resultsin a colocalization of trypsinogen and cathepsin B in non–acinar cell phagolysosomes and results in activation of tryp-sinogen175 and macrophage activation. The rupture of thesetrypsin- and cathepsin B–containing vesicles leads to acytosolic redistribution of cathepsins that acts as a secondsignal of inflammasome activation and indirectly links tryp-sinogen activation to the NFkB pathway through IL1b release.IL1b acts as an activator for other immune cells, with the IL1receptor being directly linked to the MyD88–NFkB pathway.The importance of IL1b during pancreatitis was nicely shownby a transgenic mouse model of pancreas-specific IL1b-overexpressing mice that developed CP, including completeloss of pancreatic function within weeks after birth.195

Therefore, M1 macrophages contribute prominently to the

systemic immune response syndrome, which is associatedwith multiorgan dysfunction syndrome and increased mor-tality.170 In contrast to M1 macrophages, the M2 phenotype isassociated with organ regeneration and fibrosis devel-opment.196–198

In conclusion, early protease activation and NFkB acti-vation are essential characteristics of pancreatitis; these 2events occur in parallel during disease manifestation andstrongly influence each other. Recent studies have provedthat not only the activation of proteases and NFkB but alsothe type of cell play a critical role, in which where theiractivation takes place is of importance. Pancreatitis is not adisease of acinar cells alone.

Supplementary MaterialNote: To access the supplementary material accompanyingthis article, visit the online version of Gastroenterology atwww.gastrojournal.org, and at https://doi.org/10.1053/j.gastro.2018.11.081.

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Received September 7, 2018. Accepted November 16, 2018.

Reprint requestsAddress requests for reprints to: Markus M. Lerch, MD, FRCP, Department ofMedicine A, University Medicine Greifswald, Ernst-Moritz-Arndt-UniversityGreifswald, Ferdinand-Sauerbruch-Strasse 1, 17475 Greifswald, Germany.e-mail: lerch@uni-greifswald.de.

AcknowledgmentsThis article is dedicated to the memory of Walter Halangk, PhD, to honor hiswork as a pioneer unraveling the pathophysiology of pancreatitis.

Conflicts of interestThe authors disclose no conflicts.

FundingThis study was funded by the PePPP Center of Excellence (MV ESF/14-BM-A55-0045/16; ESF MV V-630-S-150-2012/132/133; DFG-CRC 1321.-P14;and DFG SE 2702/2-1) and the National Institutes of Health (grants R01DK058088 and R01 DK117809 to Miklós Sahin-Tóth).

Supplementary Table 1.Genetic Risk Factors in Chronic Pancreatitis

Gene/protein Mutation Functional effect Phenotype

PRSS1/cationictrypsinogen

p.R122C, p.R122H Increased trypsinogen activation owing toprevention of CTRC-mediateddegradation

Autosomal dominant HP,familial pancreatitis, orsporadic CP

pA16V, p.P17T, p.N29I Increased CTRC-dependent trypsinogenautoactivation

p.D19A, p.D21A, p.D22G,p.K23R, p.K23_I24insIDK

Increased CTRC-independent trypsinogenautoactivation

c.�204C>A Decreased trypsinogen expression Protection against alcoholicCP

p.C139F, p.C139S,p.L104P,p.R116C, p.G208A

Decreased secretion, intracellular retention,and increased ER stress owing to mis-folding

Sporadic or familial CP

PRSS2/anionictrypsinogen

p.G191R Introduction of new trypsin cleavage site,increased autocatalytic inactivation

Protection against CP

SPINK1/trypsin inhibitor p.N34S unknown Increased susceptibility for CPc.194þ2T>C Skipping of exon 3 resulting in lower SPINK1

levelsCTRC/chymotrypsin C p.A73T Secretion defect Increased susceptibility for CP

p.K247_R254del Inactive CTRCp.R254W CTRC degradation by trypsinp.V235I Decreased CTRC activityp.G60¼ Decreased CTRC mRNA

CTRB1–2/chymotrypsin B1and B2

Inversion at CTRB1–CTRB2locus

Changes expression ratio of CTRB1 andCTRB2

Protection against CP

CPA1/carboxypeptidaseA1

p.S282P, p.N256K Proenzyme mis-folding, secretion defect,intracellular retention, and ER stress

Sporadic and familial CP

CEL/carboxyl ester lipase Single-nucleotide deletions Protein retention and ER stress Exocrine pancreaticinsufficiency associatedwith MODY8

CEL-HYB1 Protein retention and ER stress? Increased susceptibility for CPCFTR/cystic fibrosis

transmembraneconductance regulator

p.F508del, p.R117H Loss of CFTR function due to mis-foldingand degradation or disrupted channelactivity

Increased susceptibility for CP

CLDN2/claudin 2 CLDN2–MORC4 variants Tight junction defect? Increased susceptibility for CPCASR/calcium-sensing

receptorp.R990G Altered bicarbonate and fluid secretion from

disrupted ductal calcium sensing?Increased susceptibility for

CP?p.A986S

May 2019 Genetics and Pathophysiology of Pancreatitis 1968.e1