Genetics and Pathophysiology of Pancreatitis

Abstract

Since the discovery of the first trypsinogen mutation in families with hereditary pancreatitis, pancreatic genetics has made rapid progress. The identification of mutations in genes involved in the digestive protease–antiprotease pathway has lent additional support to the notion that pancreatitis is a disease of autodigestion. Clinical and experimental observations have provided compelling evidence that premature intrapancreatic activation of digestive proteases is critical in pancreatitis onset. However, disease course and severity are mostly governed by inflammatory cells that drive local and systemic immune responses. In this article, we review the genetics, cell biology, and immunology of pancreatitis with a focus on protease activation pathways and other early events.

Full text

Genetics, Cell Biology, and Pathophysiology of Pancreatitis
1
Medical Department II, University Hospital, LMU, Munich, Germany;
2
Department of Medicine A, University Medicine
Greifswald, Greifswald, Germany;
3
Institute for Translational Medicine, University of Pécs, Pécs, Hungary; and
4
Center for
Exocrine Disorders, Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental
Medicine, Boston, Massachusetts
Since the discovery of the first trypsinogen mutation in
families with hereditary pancreatitis, pancreatic genetics
has made rapid progress. The identification of mutations
in genes involved in the digestive protease–antiprotease
pathway has lent additional support to the notion that
pancreatitis is a disease of autodigestion. Clinical and
experimental 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 immune
responses. In this article, we review the genetics, cell
biology, and immunology of pancreatitis with a focus on
protease activation pathways and other early events.
Keywords: Trypsinogen; Pancreatitis; Genetics; Inflammation;
Cell Death.
Pancreatitis is the leading cause for gastrointestinal
disease-related hospital admissions and it is associ-
ated with considerable morbidity, mortality, and socioeco-
nomic burden.
1
Recent years have shed light on the
pathophysiology of pancreatitis, opening up new avenues
for causal treatment. In this review article, we dissect the
complexity of premature protease activation and its effect
on local and systemic inflammation in pancreatitis.
Genetics of Pancreatitis
Acute 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 is
often driven by chronic alcohol consumption or genetic risk
factors. Genetic risk for RAP and CP overlaps, whereas ge-
netic studies in AP are difficult to interpret in the absence of
adequate 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 the
pancreas. Functional studies have classified the various
mutations and other genetic alterations into pathologic
pathways driving pancreatitis onset and progression. We
discuss the trypsin-dependent, mis-folding–dependent, and
ductal pathways of pancreatitis risk.
Trypsin-Dependent Pathway of Genetic
Risk in CP
Pancreatic acinar cells secrete digestive proteases in
inactive precursor forms that are flushed from the ductal
system in a sodium bicarbonate–rich fluid. Trypsinogen, the
precursor to trypsin, becomes activated by the serine pro-
tease enteropeptidase in the duodenum.
3
Trypsin activates
chymotrypsinogens, proelastases, and procarboxypeptidase
B1 (CPB1), whereas activation of procarboxypeptidases A1
(CPA1) and A2 (CPA2) requires the concerted action of
trypsin and chymotrypsin C (CTRC).
4
Trypsinogen also can
be 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 the
lysosomal cysteine protease cathepsin B. Protective mech-
anisms that prevent trypsinogen activation in the pancreas
include trypsin inhibition by the serine protease inhibitor
Julia Mayerle
1,2
Matthias Sendler
2
Eszter Hegyi
3
Georg Beyer
1
Markus M. Lerch
2
Miklós Sahin-Tóth
4
Abbreviations used in this paper: AP, acute pancreatitis; AP1, activator
protein 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, endoplasmic
reticulum; 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 extracellular
trap; NFkB, nuclear factor klight-chain enhancer of activated B cells;
NLRP3, nucleotide-binding oligomerization domain-like receptor protein
3; 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 Kazal
type 1; TLR, Toll-like receptor; TNF, tumor necrosis factor.
Most current article
© 2019 by the AGA Institute
0016-5085/$36.00
https://doi.org/10.1053/j.gastro.2018.11.081
Gastroenterology 2019;156:1951–1968
PANCREATITIS

Kazal type 1 (SPINK1) and trypsinogen degradation by
CTRC and cathepsin L.
5–7
Although the principal action of
CTRC is to promote trypsinogen degradation, it also enhances
trypsinogenactivationbyprocessing the trypsinogen activa-
tion peptide to a shorter form, which is more sensitive to
trypsin-mediated activation
7–9
(Figure 1). As discussed below,
certain trypsinogen mutations can hijack this mechanism and
thereby stimulate trypsinogen activation to a pathologic extent.
Human genetic studies strongly support trypsinogen autoac-
tivation and CTRC-dependent trypsinogen degradation as key
mechanisms determining intrapancreatic trypsin activity,
whereas similarly compelling genetic evidence for the role of
cathepsins B and L has been lacking.
10
Cationic Trypsinogen Mutations
Mutations in human cationic trypsinogen (PRSS1)cause
autosomal dominant hereditary pancreatitis (HP) with
incomplete penetrance or act as risk factors in sporadic
CP.
7,11
Approximately 90% of PRSS1-mutation–positive HP
families carry the p.N29I, p.R122C, or p.R122H mutation in
the heterozygous state. Mechanistically, the p.R122C and
p.R122H mutations prevent CTRC-mediated trypsinogen
degradation.
9
The p.N29I mutation has multiple distinct ef-
fects on trypsinogen biochemistry, the combination of which
markedly increases trypsinogen autoactivation. These effects
include an increase in N-terminal processing, decreased
CTRC-dependent degradation, and a slightly increased pro-
pensity for autoactivation.
9
The p.A16V variant sensitizes the
activation peptide of trypsinogen to CTRC-mediated pro-
cessing, which in turn enhances autoactivation.
8,9
Pathologic
trypsin levels generated by mutation p.A16V are lower than
those seen with the p.R122H variant, which explains the
decreased penetrance of the p.A16V variant. More recently,
mutation p.P17T was found to exhibit characteristics that
were similar to those of p.A16V.
12
Rare mutations affecting
the activation peptide of cationic trypsinogen (p.D19A,
p.D21A, p.D22G, p.K23R, and p.K23_I24insIDK) robustly
stimulate autoactivation independently of CTRC.
13–15
Cell
culture experiments have indicated that these activation
peptide mutants are secreted poorly because of intracellular
activation and degradation, which can lead to cellular stress
and consequent acinar cell death.
16
Taken together, PRSS1
mutations stimulate activation of cationic trypsinogen by
decreasing 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 commonly
occurring 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
A
variant (c.204C>A) that lies in the promoter region of
PRSS1 and decreases trypsinogen expression appears to be
responsible for this small protective effect.
20
SPINK1 Mutations
The association between the most common p.N34S
SPINK1 variant and CP was first described by a candidate
gene study in 2000.
21
A meta-analysis reported a carrier
frequency of 9.7% in patients with CP and 1% in controls
with an average OR of 11, making the p.N34S the clinically
most significant risk factor for CP.
22
When considering
European populations only, p.N34S increased CP risk by
approximately 10-fold.
23
Although several studies have
attempted to identify the functional effect of p.N34S and its
associated haplotype, the molecular mechanism underlying
CP risk remains unclear. Neither p.N34S nor any of the 4
linked intronic variants affect trypsin inhibitory function or
cellular expression of SPINK1.
24–27
Interestingly, in pancre-
atic cancer cell lines carrying the heterozygous p.N34S
variant, decreased expression of the mutant allele was
observed compared with the wild-type allele.
28
The in-
vestigators suggested that the c.4141G>T variant or a
hitherto unknown variant located in the 50region of the gene
might be responsible for the decreased expression of the
p.N34S allele. The second most frequently reported SPINK1
haplotype in CP contains the c.215G>A promoter variant
and the c.194þ2T>C variant in intron 3.
21,29
This haplotype
was observed more frequently in East Asia than in Europe.
7
Functional studies have shown that the c.194þ2T>C
variant causes skipping of exon 3, which results in dimin-
ished SPINK1 expression.
27,30,31
However, the c.215G>A
variant increases promoter activity, which might mitigate the
effect of the c.194þ2T>C mutation and allow for some re-
sidual SPINK1 expression even in homozygous carriers.
32,33
A
large number of rare or private alterations in SPINK1 have
been found in CP, which cause loss of SPINK1 function by
various mechanisms.
7
Protective PRSS2 Variant
Although 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 sporadic
CP.
34,35
The absence of PRSS2 mutations in CP could be due
to the more effective CTRC-mediated degradation of anionic
trypsinogen, which would prevent intrapancreatic activation
of the enzyme even if it were mutated.
36
However, a pro-
tective variant p.G191R with a w3- to 6-fold effect and
approximately 5% population frequency was discovered.
35,37
The mutation introduces a new trypsin cleavage site into
anionic trypsinogen, which increases autocatalytic proteolysis
and inactivation.
35
CTRC Mutations
Direct DNA sequencing of the CTRC gene in patients with
nonalcoholic CP showed heterozygous mutations in 4% of
patients that increased CP risk by 5-fold on average.
38,39
The
mutations 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
Considering
the clinically significant variants, p.A73T exhibits a severe
secretion defect, p.K247_R254del is inactive and prone to
degradation, p.R254W is degraded by trypsin, and p.V235I
has partly decreased activity.
40
Subsequent studies reported
a frequent p.G60¼variant found in approximately 30% of
patients with CP.
42–45
The heterozygous p.G60¼increases
1952 Mayerle et al Gastroenterology Vol. 156, No. 7
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the risk of CP by 2.5-fold, whereas the homozygous state
increases the risk by 10-fold.
43,45
The variant is associated
with decreased CTRC mRNA expression (GTEx Portal),
possibly because of altered pre-mRNA splicing.
CTRB1–CTRB2 Locus Inversion
A recent European GWAS identified a large inversion at
the CTRB1–CTRB2 locus that modestly (OR 1.35) modifies the
risk for alcoholic and nonalcoholic CP.
19
The inversion
changes the expression ratio of the CTRB1 and CTRB2
chymotrypsin isoforms in such a manner that protective
trypsinogen 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 and
does not contribute to CP risk.
46
A mouse model with genetic
deletion of the major mouse chymotrypsin CTRB1 exhibited
increased intra-acinar trypsin activation and more severe
pancreatitis induced by the secretagogue cerulein.
47
These
observations provided the first in vivo proof for the protective
role of chymotrypsin-mediated trypsinogen degradation
against pancreatitis.
Mis-folding–Dependent Pathway of
Genetic Risk in CP
More recently, an alternative pathomechanism seem-
ingly unrelated to premature intrapancreatic trypsinogen
activation has been identified, in which mutation-induced
mis-folding and consequent endoplasmic reticulum (ER)
stress lead to acinar cell damage and pancreatitis.
48
Mis-folding–Associated PRSS1 Mutations
In 2009, a subset of PRSS1 variants was found to cause
decreased secretion, intracellular retention, and increased
ER stress markers, as judged by in vitro cell culture ex-
periments.
49
These PRSS1 mutations occur rarely and are
mostly associated with sporadic disease (eg, p.C139F,
p.C139S, p.G208A), but also have been found in HP families
with incomplete penetrance (p.L104P, p.R116C).
48
Variant
p.G208A is prevalent in East Asia (4% of CP cases) and was
detected in Europe only in a single case thus far.
50,51
Mis-folding–Associated CPA1 Mutations
A 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 frequency
and are mostly found in sporadic CP. The p.S282P variant
was described in 2 HP families.
53
Pathogenic CPA1 variants
cause proenzyme mis-folding, resulting in a secretion defect,
intracellular retention, and ER stress.
52,53
In contrast to
CPA1, variants of CPB1 and CPA2 are not associated with
CP.
54
Interestingly, ER stress-inducing CPA1 and CPB1 var-
iants were over-represented in patients with pancreatic
cancer without a clinical history of RAP or CP.
55
Most of
these variants caused premature truncation and did not
overlap with those found in CP. A mouse model for the mis-
folding–dependent pathway was described recently. This
study showed that CPA1 N256K knock-in mice harboring the
most frequent p.N256K human CPA1 mutation develop
spontaneous and progressive CP and exhibit signs of ER
stress in their pancreas.
56
Mis-folding–Associated CEL Mutations and
CEL-HYB Allele
Single-nucleotide deletions in the last exon of the CEL
gene encoding carboxyl ester lipase cause maturity-onset
diabetes of the young type 8 (MODY8).
57
The deletions
alter the reading frame of the C-terminal variable number
tandem repeat sequence, resulting in CEL proteins with
unnatural extensions that are prone to aggregation.
58,59
The
exocrine dysfunction in MODY8 is in all likelihood caused by
mis-folding–induced ER stress and consequent acinar cell
loss. A hybrid CEL allele (CEL-HYB1) formed between CEL
Figure 1. Genetic risk factors associated with the trypsin-dependent pathologic pathway. See text for details.
May 2019 Genetics and Pathophysiology of Pancreatitis 1953
PANCREATITIS

and its neighboring pseudogene CELP was over-represented
in idiopathic CP by approximately 5-fold vs the average
population frequency of 0.5%–1%.
60
In cell culture experi-
ments, the hybrid protein was secreted poorly because of
intracellular retention, suggesting that the CEL-HYB1 variant
can 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 a
GWAS 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 failed
to replicate this association,
63,64
with the exception of
azathioprine-induced pancreatitis in patients with inflam-
matory bowel disease,
65
it is still interesting to speculate
that the observed effects might have been due to changes in
CEL folding or trafficking.
Ductal Pathway of Genetic Risk in CP
CFTR Variants
The cystic fibrosis transmembrane conductance regu-
lator (CFTR) is a cyclic adenosine monophosphate–
regulated chloride–bicarbonate channel localized to the
apical plasma membrane of epithelial cells
66
(Figure 2).
CFTR mutations disrupt channel activity or affect membrane
levels and are associated with various phenotypes, ranging
from asymptomatic state to multiorgan symptoms leading
to the diagnosis of cystic fibrosis in homozygous carriers of
severe mutations. Observations that heterozygous and
compound heterozygous CFTR mutations are associated
with CP were reported by 2 studies in 1998.
67,68
In the first
analysis of the entire CFTR coding region, the frequency of
abnormal CFTR alleles in patients with CP was 18.6%
compared with 9.2% in controls.
69
More recent large cohort
analyses have corroborated the pathogenic role of CFTR
variants in CP, although the effect and frequency of CFTR
variants was less pronounced than reported previ-
ously.
39,70,71
Heterozygous carrier status of the severe
p.F508del mutation confers a small risk for CP (OR 2.5),
whereas the mild p.R117H mutation increases risk by
approximately 4-fold. Compound heterozygous state for 1
severe and 1 mild CFTR allele represents strong risk for CP
and can be considered causative.
70
The role of common
polymorphic CFTR alleles (eg, T5, TG12) and the non–cystic
fibrosis–causing, so-called bicarbonate-defective CFTR vari-
ants in CP remains controversial because the preponderance
of data does not support their association with CP.
66
Unlike
CFTR, variants in the solute-linked carrier 26 member 6 anion
transporter (SLC26A6) do not alter the genetic risk in CP.
72
CLDN2 Variants
GWASs 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 the
ductal pathologic pathway.
See text for details.
1954 Mayerle et al Gastroenterology Vol. 156, No. 7
PANCREATITIS

associated with CP risk.
17,19
The OR was approximately 2
and the effect was more pronounced in alcoholic CP. Within
this locus, CLDN2 seems to be the clinically relevant risk gene,
because it is expressed in pancreatic ducts at low levels as a
tight junction protein. It was proposed that CLDN2–MORC4
variants might cause CLDN2 mis-localization. Additional
work is required to clarify the mechanism of action of this
risk locus and to confirm whether assignment to the ductal
pathway is appropriate (Figure 2).
CASR Variants
The calcium-sensing receptor (CASR) regulates calcium
homeostasis through parathyroid hormone secretion and
renal tubular calcium reabsorption. Functional CASR also is
expressed in the pancreas, including ductal cells where
CASR can respond to high calcium concentrations in the
juice by increasing ductal fluid secretion, thereby preventing
stone formation and pancreatitis
73
(Figure 2). A US
population-based study failed to demonstrate the previously
anticipated association between CASR variants and the
SPINK1 p.N34S haplotype, but reported the p.R990G variant
increased CP risk, especially in subjects with moderate or
heavy alcohol consumption.
74
More recently, a French study
found over-representation of rare CASR coding variants in
idiopathic CP and a significant association of the p.A986S
variant, but only in the homozygous state, with CP.
75
How-
ever, the previously reported association with the p.R990G
variant was not observed in this cohort. Taken together,
current evidence does not support a clear role for CASR
variants in CP pathogenesis.
In summary, human genetic data indicate that premature
activation or mis-folding of pancreatic proteases play a
central role in the onset of pancreatitis and progression to
CP (Supplementary Table 1).
Role of Proteases in Pathophysiology
and Cell Biology of Pancreatitis
Although genetic evidence for the involvement of the
protease–antiprotease balance in the pathogenesis of
pancreatitis dates back only 2 decades and focuses mainly
on CP, pathophysiologic and biochemical investigations have
implicated this system for more a century. Because of the
lack of adequate animal models and the inability to keep
isolated pancreatic acinar cells in culture for long periods,
experimental studies have focused primarily on AP. The
relative importance of the pathways discussed below might
change 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 Proteases
The pathophysiologic concept of autodigestion was first
developed by the Austrian pathologist Hans Chiari in Prague
more than 120 years ago. He claimed that pancreatitis was
caused and driven by the glands’digestive properties.
76
Since then, the pathomechanism of premature activation
of pancreatic enzymes and its contribution to disease
severity and progression have captured the attention of
many pancreatologists. Bialek et al
77
first reported that
protease activation during pancreatitis begins in the
exocrine pancreas and Hofbauer et al
78
reported that it
begins in a membrane-confined vesicular compartment and
parallels acinar cell damage. Although the fact that activa-
tion of digestive proteases is an early event during AP is
widely accepted, the question of where and through what
mechanism this process is initiated and whether it plays a
role in chronicity remain under debate.
Protease Activation During Pancreatitis—Clues
From 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 in
humans and rats.
79–81
Intracellular calcium concentrations
and compartmental distribution in acinar cells are tightly
regulated because calcium serves as a second messenger for
the physiologic release of digestive enzymes in response to
vagal nerve stimulation or humoral activation.
82,83
After
intraperitoneal treatment of rats with supramaximal doses
of cerulein, an analogue of cholecystokinin,
84
a time-
dependent disruption of the physiologic oscillating intra-
cellular calcium signal was observed in rat acini using the
Ca
2þ
-sensitive dye fura-2.
85
When using for the first time
fluorescent trypsin substrates that allow the subcellular
imaging, localization, and quantification of protease activa-
tion,
86
Krüger et al
87
found that a specific extended plateau
release of calcium at the apical pole of acinar cells is
necessary for premature trypsin activation to occur, which
is different from the calcium oscillations required for
enzyme secretion. These findings were confirmed by others
who also found that acetylcholine- or cholecystokinin (CCK)-
induced intracellular protease activation was associated
with the formation of cytoplasmic vacuoles in acinar cells
that resemble those overserved in experimental pancreatitis
in vivo
88
and that inhibition of calcium release also inhibits
the formation of these vesicles.
89
Acinar cells can replace
calcium for magnesium in its stores and, when this is done,
not only the premature activation of proteases in vitro and
in vivo but also the severity of pancreatitis is significantly
decreased.
90
Two ongoing clinical trials (1 in AP and 1 in
CP) are based on this observation. Orabi et al
91
took the
concept of calcium-dependent protease activation further by
showing that AP induced by supramaximal doses of the
muscarinic agonist carbachol can be abrogated by inhibition
of the intracellular calcium channel ryanodine receptor,
which is located at the basolateral membrane of acinar cells.
Therefore, the importance of spatial distribution of calcium
to the apical pole might be a specific feature of the cerulein
model. Interestingly, carbachol-induced protease activation
is more severe if cells are pretreated with ethanol. Calcium
signaling 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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PANCREATITIS

calcium release from intracellular stores or influx through
the plasma membrane by pharmacologic inhibition of
inositol triphosphate receptor (predominantly types 2 and
3) signaling
94,95
or calcium release-activated calcium
modulator 1.
96,97
The calcium-dependent protease activa-
tion also heavily depends on calcineurin, a calcium-activated
phosphatase, and its downstream signaling through the
transcription factor nuclear factor of activated T cells. In-
hibition of calcineurin by inhibitors or in mice lacking cal-
cineurin subunits causes decreased intracellular protease
activity in secretagogue or bile acid–induced pancreatitis,
without affecting vesicular transport.
98,99
Mechanisms of Protease Activation. Three major
concepts have been investigated over the past decades:
autoactivation, spatial redistribution, and fusion of zymogen
granules with other organelles and failure of protective
mechanisms.
Autoactivation of Trypsin. There is strong evidence
from human genetic studies indicating that autoactivation of
trypsinogen
100
causes CP in affected humans.
9
In contrast,
trypsinogen autoactivation is unimportant in experimental
cerulein-induced pancreatitis. Inhibition of active trypsin by
the reversible chemical inhibitor S124 could prevent trypsin
activity 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 that
hydrolysis of trypsinogen to trypsin appeared in a trypsin-
independent fashion.
101
Activation by Lysosomal Proteases. Inhibition of the
lysosomal hydrolase cathepsin B by the cysteine protease
inhibitor E-64d leads to a significant decrease in trypsin
activity and TAP formation, indicating that the trypsin
activation in response to cerulein depends on cathepsin
B.
101–103
Similarly, in cathepsin B knockout mice, trypsin
activation is significantly inhibited after cerulein adminis-
tration.
104
In the past, it was thought that cathepsin B
105
and trypsinogen under physiologic conditions are not
located in the same cell organelles (zymogen granules for
exocytosis vs lysosomes for degradation of content of
endosomes and autophagosomes). In 1998, investigators
showed in subcellular fractions of cerulein-treated acini a
colocalization of cathepsin B and digestive enzymes,
including trypsin and TAP, in heavy fractions
78
containing
zymogen granules and lysosomes early in the disease course
and later a shift of trypsin and cathepsin B activity to the
cytosol.
106
This confirmed earlier findings from in vivo
studies that indicated a fusion of zymogen-containing vac-
uoles with lysosomes in secretagogue-, duct obstruction-, or
diet-induced AP.
107–112
Intracellular activation of proteases
other than trypsin, such as chymotrypsin and carboxypep-
tidase B, also depend on non-physiologic colocalization with
other cell components, but are independent of cathepsin
B.
113
Mis-sorting in the exocrine machinery, secretion
blockage, and reuptake of previously secreted proteases by
endocytosis 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 reported
that CCK or ethanol treatment depletes acinar cells of
syntaxin 2, a key regulator of apical exocytosis, thus leading
to 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 intracellular
trypsinogen activation. These occur under physiologic and
pathophysiologic conditions after compound exocytosis of
zymogen granules.
117
EV formation is calcium depen-
dent.
118
Their content is acidic and calcium rich and, after
supramaximal CCK or taurocholate stimulation, trypsin ac-
tivity within these post-exocytic structures can be visualized
using fluorescent dyes.
119
During pancreatitis, EVs are
larger than normal and tend to fuse with the plasma
membrane or even rupture, discharging active trypsin into
the cytosol or extracellular space. This instability is believed
to be caused by disruption of otherwise protective actin
filaments surrounding the EV.
120
However, rupture of EVs is
independent of trypsin or cathepsin B activity. Secretory
blockage can contribute to these events. In experimental
pancreatitis, vesicle-associated membrane protein-8–medi-
ated secretion is impaired because of a loss of early endo-
somal proteins, resulting in retention of trypsinogen and
transformation to active trypsin in a cathepsin-dependent
manner. Knockout of vesicle-associated membrane
protein-8 protects against pancreatitis and restoration of
early 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 role
of failing protective mechanisms for protease activation in the
early phase of pancreatitis. The most potent cellular trypsin
inhibitor is SPINK1. Although SPINK1 mutations are among
the most common genetic risk factors for the development of
recurrent AP and CP, thus far, none of the described
mutations seemed to impair SPINK function and therefore do
not explain the increased risk for pancreatitis.
24–27
In mice
carryingaheterozygousSPINK3deletion,asignificant
decrease of functional SPINK does not lead to the develop-
ment of spontaneous pancreatitis or more severe disease
after supramaximal cerulein administration compared with
wild-type controls.
125
The fact that mice with a homozygous
SPINK3 deletion develop pancreatic atrophy and that this
phenotype can be rescued by transgenic expression of rat
pancreatic secretory trypsin inhibitor 1
125
point toward a
role of trypsin inhibitors during pancreatic development but
do not explain that role in pancreatitis.
In conclusion, the current cumulative evidence suggests
a cathepsin B–dependent mechanism of protease activation
in experimental pancreatitis.
Cell Death Cause or Consequence of Protease
Activation?
Premature intracellular protease activation in acinar
cells 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
1956 Mayerle et al Gastroenterology Vol. 156, No. 7
PANCREATITIS

unregulated response to damage. In animal models of AP,
approximately 1%–5% of acinar cells undergo apoptosis
and severity of pancreatitis is inversely correlated to the
rate of apoptosis.
127
Macroautophagy is a multistep,
lysosomal-driven, adaptive process by which cells degrade
cytoplasmic organelles and long-lived protein.
128
Pancrea-
titis presents with impaired autophagic flux evident by
vacuole accumulation.
129
Currently, it is debated whether
impaired autophagy stimulates cell death through accumu-
lation of damaged mitochondria mediating an inflammatory
response through a reactive oxygen species (ROS)-dependent
mechanism as in lysosome-associated membrane protein
2
130
or Atg7-knockout animals
131
or whether autophagy
prevents an inflammatory response as in Atg5-knockout
mice.
132,133
The regulated process of necrosis is termed
necroptosis and is triggered by tumor necrosis factor (TNF),
TNF-related apoptosis-inducing ligand, Fas ligand, interferon
type 1, and Toll-like receptors (TLRs), which are released in
theearlyphaseofAPinresponsetoproteaseactivation.
134
Receptor-interacting protein 1 (RIP1) and RIP3 form a
phosphorylated complex, the necrosome, and phosphorylate
mixed-lineage kinase domain-like, resulting in membrane
rupture. Forty percent of cells undergo necroptosis in
pancreatitis and RIP3 deletion or treatment with necrostatin
ameliorates pancreatitis.
135,136
Necroptosis releases damage-
associated molecular patterns (DAMPs) and those will acti-
vate the nucleotide-binding oligomerization domain-like
receptor protein 3 (NLRP3) pathway, resulting in pyropto-
sis.
137
Pyroptosis is an innate immune sensing mechanism
with 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. The
inflammasome is a macroscopic cytosolic protein complex
that proteolytically cleaves interleukin (IL) 1band pro-IL18
and releases high mobility group box 1 protein. NLRP3 acti-
vation requires lysosomal rupture and cathepsin release,
calcium influx, and mitochondria-derived ROS production
and 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 3
activation can induce not only apoptosis but also pyroptosis.
Necroptosis can shift to pyroptosis by caspase 8 activation
and necroptosis activates pyroptosis.
141
Currently, we have
not understood why some of our patients deteriorate and
develop a necrotizing course of pancreatitis. A shift of
regulated cell death from apoptosis to pyroptosis and
stimulation of necroptosis might explain this observation.
Inhibition of pyroptosis, for example, by using Ringer’s
lactate for volume resuscitation
142
or inhibition of nec-
roptosis by necrostatin,
143
might well be a way forward in
the treatment of pancreatitis.
An interesting question is whether cell death is a result
of premature protease activation or a consequence of
inflammation. The answer is ambiguous: Talukdar et al
106
reported a direct effect on lysosomal stability mediated by
active trypsin and lysosomal rupture leading to the release
of cathepsins into the cytosol, causing dose-dependently
apoptosis 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 affect
necrosis.
144
Thus, cell death is a result of intracellular pro-
tease activation, but this has been shown only for isolated
acinar cells mimicking the early phase of pancreatitis. Tak-
ing into account pyroptosis and necroptosis, inflammation is
the origin and consequence of cell death in pancreatitis
(Figure 3).
Protease Activity and Disease Severity
This raises the question of whether intracellular prote-
ase activation of trypsin as it is linked to cell death also can
mediate systemic disease severity. The notion that trypsin
activation is linked to disease severity is supported by the
correlation of TAP urine levels to severity in patients with
AP.
145
However, several studies have questioned the role of
trypsin for severity of pancreatitis. Expression of mutant
human trypsin bearing the HP mutation R122H in mice
leads 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 or
N29I) show lower trypsin activity after cerulein hyper-
stimulation. This might be explained in part by a higher rate
of acinar cell apoptosis even in untreated animals transgenic
for human trypsinogen.
147
A similar effect is seen in PACE-
tryp(on) mice, which conditionally express an endogenously
activated trypsinogen within pancreatic acinar cells. Those
mice will develop AP in a trypsin activity-dependent way,
which can lead to organ dysfunction and mortality, but they
also show pronounced caspase 3 activation with consecu-
tive apoptotic loss of acinar cells and replacement by fatty
tissue.
148
Similarly, Archer et al
149
described a transgenic
mouse, in which the human mutation R122H was inserted in
the murine PRSS1 gene. Those mice developed spontaneous
pancreatitis and showed a more pronounced inflammatory
infiltrate and cellular damage in response to cerulein. The
fact that the predominant way of cell death determines the
overall severity of experimental pancreatitis in mice has
been demonstrated in animals with deletion for cathepsin L.
Cathepsin L degrades trypsinogen into an inactive elongated
TAP, but cathepsin L deficiency leads to a milder form a of
cerulein-induced pancreatitis, which is linked to a shift from
necrosis to apoptosis.
6
Themostconvincingdataquestioningtheroleof
trypsin for disease severity were generated using a mouse
strain lacking trypsinogen 7, the murine counterpart of
human cationic trypsinogen. Dawra et al
150
and Sah
et al
151
found that a significant decrease in trypsin and
chymotrypsin activity after cerulein hyperstimulation in
these mice had no effect on disease severity for cell death
or local or systemic inflammation in AP and CP models,
which they claimed was mediated by a mechanism
dependent on nuclear factor klight-chain enhancer of
activated B cells (NFkB). This confirms previous findings
generatedinthePACE-tryp(on)model,inwhichNFkB
-activation was independent of increased intracellular
trypsin activity in vitro.
152
May 2019 Genetics and Pathophysiology of Pancreatitis 1957
PANCREATITIS

Figure 3. Trypsinogen activa-
tion and cell death in pancreatic
acinar cells. Intracellular tryp-
sinogen activation is an early
eventattheonsetofexperi-
mental AP. Supramaximal
secretagogue-receptor stimula-
tion or activation of bile salt re-
ceptors leads to an
unphysiologic peak–plateau
calcium signal. This results in
disrupted exocytosis of
zymogen granules, secretory
blockage, zymogen retention,
and formation of EVs, which
contain trypsin and trypsin-
ogen, taken up from the extra-
cellular space. Those EVs
colocalize and fuse with lyso-
somes containing cathepsin B,
which in turn transforms tryp-
sinogen into active trypsin.
Owing to increasing instability,
EVs often rupture, releasing
trypsin and cathepsin B into the
cytosol. Active trypsin is
believed to induce mainly
apoptosis, a silent form of cell
death, which suppresses
inflammation. In contrast, if the
pathologic calcium release
cannot be contained, then rapid
energy depletion occurs and
cells undergo necrosis, during
which the plasma membrane
becomes leaky and cellular
components (eg, DNA or mito-
chondria) reach the extracellular
space. Those will be recognized
by leukocytes, which will be
activated by the inflammasome
signaling pathway. IL1band
TNFarelease and pyroptosis
occur. If TNFareaches the
basolateral membrane of previ-
ously unaffected or slightly
damaged acinar cells, then it
can induce another form of
programmed cell death (ie,
necroptosis). PLC, phospholi-
pase C.
1958 Mayerle et al Gastroenterology Vol. 156, No. 7
PANCREATITIS

Role of Systemic Inflammation in
Pancreatitis
NFkB Activation—Initial Step of Inflammation
The activation of NFkB is an early event during
pancreatitis and occurs within the first minutes after onset
of the disease.
153,154
One main function of NFkB is tran-
scriptional regulation of the immune response.
155
The
principal pathway of NFkB signal transduction is depicted in
Figure 4. The fact that NFkB is already present in the
cytoplasm explains its rapid activation after induction of
pancreatitis.
153,154
Intra-acinar protease activation and NFkB activation are
early cellular events during pancreatitis,
153,154
which have
been suggested to occur independent of each other,
151
but
follow a similar kinetic. Trypsinogen activation depends on
intracellular Ca
2þ
signaling
87
and NFkB activation also can
be induced by protein kinase C and Ca
2þ
, which could be the
reason for parallel kinetics.
156
The deletion of trypsinogen 7
or cathepsin B, either of which results in greatly decreased
protease activation,
104,144,151
does not influence NFkB acti-
vation during cerulein-induced pancreatitis.
151
This can be
regarded as evidence that protease activation does not
directly lead to NFkB activation in acinar cells, whereas
NFkB activation can still interact with protease activity: the
transcriptional regulation of serine protease inhibitor 2a is
mediated by NFkB and can inhibit trypsin activity in a
mouse model of acute pancreatitis (Figure 4).
157
These re-
sults suggest a more complex role of NFkB during disease
progression 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 deletion
of NFkB inhibitor aresulted in nuclear translocation of Rel-
A and ameliorated pancreatitis.
157
The same phenotype was
observed in mice deficient in myeloid differentiation pri-
mary response 88 (MyD88), which developed a more severe
disease phenotype compared with controls.
160
MyD88 is a
central adaptor for the TLR–IL1-receptor signaling pathway,
which induces NFkB activation. These results ultimately
suggest a protective role of NFkB activation in pancreatic
acinar cells. Other studies attributed a more critical role for
NFkB 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 disease
severity after cerulein stimulation.
161,162
However, the
infiltration of immune cells alone, or the constitutive activity
of NFkB in acinar cells, did not result in AP, organ damage,
and necrotic or apoptotic cell death
162
; an additional path-
ophysiologic stimulus was still needed. The increased
severity of pancreatitis in IKKb-overexpressing mice can be
Figure 4. NFkBpathwayin
pancreatic acinar cells. The
early activation of NFkB
follows the same time
pattern as trypsinogen
activation. They are
induced by cytoplasmic
Ca
2þ
influx, but NFkBdoes
not depend on trypsinogen
activation. The phosphory-
lation of IkBa,followedby
proteosomal degradation
and nuclear translocation
of NFkB(p65–p50) occurs
in parallel to protease acti-
vation. NFkB as a tran-
scription factor acts in 2
directions; first, transcrip-
tions of proinflammatory
genes, such as IL6 or
TNFa, initiate the immune
response; and second, the
transcription of prosurvival
genes. Therefore, NFkB
can directly influence pro-
tease activity to protect
cells by the up-regulation
of serine protease inhibitor
2a. CTSB, cathepsin B;
IkBa, inhibitor of NFkB
alpha; IkBb, inhibitor of
NFkB beta; NEMO, NF-
kappa-B essential modu-
lator; PLC, phospholipase
C; Ub, ubiquitin.
May 2019 Genetics and Pathophysiology of Pancreatitis 1959
PANCREATITIS

explained by the presence of leukocytes within the
pancreas, which become rapidly activated after disease in-
duction and do not need to infiltrate the pancreas. The
pancreas-specific deletion of IKKa, another NFkB inhibitor a
phosphorylating kinase, caused spontaneous pancreatitis in
mice,
163
but this process appeared to be independent of
NFkB. IKKaalso regulates autophagic flux, which is essential
for pancreas homeostasis.
164
These data demonstrate the
complexity of the NFkB network, which often hampers the
interpretation of results. Taken together, the constitutive
activation of NFkB leads to a chronic infiltration of immune
cells, but pancreatitis develops only after induction by an
external stimulus.
161,162
The presence of immune cells
within the pancreas is required but insufficient for pancre-
atitis to develop and these cells need to be activated to
contribute to disease severity. Conversely, data from
pancreas-specific RelA deleted mice show that NFkB acti-
vation in acinar cells is not essential for the recruitment of
immune cells to the side of damage.
159
Quite to the contrary,
the absence of p65 in acinar cells result in greater disease
severity. Although all these studies investigated the role of
NFkB in acinar cells, NFkB activation could play a much
larger role in infiltrating immune cells, which directly
regulate the immune response.
Another master switch in the transcriptional machinery
of acinar cells is activator protein 1 (AP1). AP1 is implicated
in multiple transcriptional networks within the acinar cells
regulating pancreatic development, differentiation, cell
death, and inflammation. Mice heterozygous for the orphan
nuclear receptor NR5A2 develop an acinar cell–autonomous
AP1-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 to
greatly differ with respect to the cause of pancreatitis. In
cerulein models, activation of the 2 transcription factors is
described and submaximal CCK stimulation induces acinar
cell dedifferentiation and proliferation through the mitogen-
activated kinase–c-Jun–AP1 pathway probably as part of a
pancreatic regeneration program.
166,167
In contrast, the
metabolites occurring in ethanol-induced experimental
pancreatitis can positively and negatively regulate NFkB and
AP1 depending on the predominance of oxidative or non-
oxidative alcohol metabolism in the pancreas.
168
Role of Infiltrating Immune Cells
Infiltration of immune cells starts within minutes after the
onset of disease and plays a crucial role in the severity and
prognosis of pancreatitis.
169–172
Cells of the innate immune
system, such as neutrophil granulocytes and monocytes and
macrophages, represent the majority of infiltrating cells.
NFkB plays a crucial role in the activation of leukocytes and is
a central mediator of the innate and adaptive immune sys-
tem.
173
Pancreatitis is primarily a sterile inflammation, so
pathogen-associated molecular patterns play no role in the
activation of immune cells during the early phase of disease.
Thus, in pancreatitis, activation of immune cells is mediated
by 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 as
TNFa,
174
IL6, or monocyte chemoattractant protein 1.
175
DAMPs and cytokines result in the nuclear translocation of
p65 and p50 within infiltrating immune cells, which enhances
the cytokine storm through the secretion of proinflammatory
mediators
175
(Figure 5). DAMPs can act in the same way as
pathogen-associated molecular patterns through TLRs
176
or
specific receptors such as P2RX7, which uses extracellular
adenosine triphosphate as a ligand. Acinar cells, which un-
dergo necrotic cell death, release a multitude of different
DAMPs, such as free DNA,
177
histones, or free adenosine
triphosphate,
138
which can act as immune activators. Acti-
vated immune cells increase pancreatic damage and
contribute to systemic inflammation.
169,175,178
Several studies
have focused on different populations of immune cells and
their role during AP.
Neutrophil granulocytes are often used as reference
markers for pancreatic inflammation by measurements of
myeloperoxidase activity in tissue and reflect the amount of
infiltrating neutrophils. One major function of neutrophils is
removing pathogens by the release of proteases, antimi-
crobial peptides, and ROS. During pancreatitis, neutrophils
are the major source of ROS production; they can induce
oxidative damage on acinar cells and enhance trypsinogen
activation.
178
The release of proteases such as PMN-elastase
contributes to tissue destruction and acinar cell dissocia-
tion.
179
These data indicate that neutrophils have a direct
effect on disease severity. This was confirmed by the
depletion of neutrophils using antineutrophil serum, which
resulted in decreased pancreatic damage and protease
activation.
169,178
Recent studies have investigated the role of
neutrophil extracellular trap (NET) formation in the context
of pancreatitis. NETs are extracellular networks consisting
of neutrophil DNA and are used to bind pathogens.
180
This
suicide mechanism of neutrophils is a last line of defense
against bacterial infections. NET formation is induced by
TLR4 activation and the activation of nicotinamide adenine
dinucleotide phosphate oxidase, which lead to the oxidation
of peptidyl-arginine-deiminase 4.
181,182
TLR4 is responsible
for the detection of pathogens, although DAMPs can activate
the TLR-signaling pathway.
176
Merza et al
183
reported that
NET 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
Another
group showed that NET formation is a critical step in bile
stone development and plays an important role in ductal
obstruction contributing to onset and severity of pancrea-
titis.
185,186
Neutrophil infiltration during AP is an unspecific
reaction of the immune system, which enhances local
damage by the formation of NETs and the release of ROS or
activates digestive enzymes.
Monocytes and macrophages belong to cells of the innate
immune system. In contrast to neutrophil granulocytes,
macrophages are characterized by high plasticity. Classic
activated macrophages (M1) act in a proinflammatory
manner and secrete large amounts of IL6, TNFa, IL12, and
IL1b, and increased expression of inducible nitric oxide
1960 Mayerle et al Gastroenterology Vol. 156, No. 7
PANCREATITIS

synthase results in the release of nitric oxide.
187
Alterna-
tively, activated macrophages (M2) are associated with
wound healing, tissue regeneration, and fibrogenesis and act
in an anti-inflammatory manner through the release of IL10
or transforming growth factor b. They are characterized by
decreased inducible nitric oxide synthase and increased argi-
nase 1 expression.
187
Macrophages are phagocytosing cells that
remove tissue debris and necrotic and apoptotic cells. During
AP, macrophage infiltration correlates to a greater extent with
pancreatic damage and necrosis than with the number of
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 IL1bor TNFaand DAMP signals acting
by TLRs can induce the translocation of p65 and p50 into the nucleus. In leukocytes, most inflammatory mediators are under
the 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 in
pancreatitis compared with NFkB activation within the acinar cell. ASC, Apoptosis-associated speck-like protein containing a
CARD; 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.
May 2019 Genetics and Pathophysiology of Pancreatitis 1961
PANCREATITIS

infiltrating neutrophils.
175
The reason for that is that macro-
phages are required for the removal of necrosis and thus miti-
gate pancreatic damage. Phagocytosing macrophages have been
observed in different models of AP and CP.
130,164,169,175
In
contrast to apoptosis, necrosis is a proinflammatory cell death
because it entails the release of multiple DAMPs that induce an
M1 polarization of macrophages.
188
Therefore, acinar cell
apoptosis is suggested to be protective against hyper-
inflammation and decrease disease severity.
189
M1 macro-
phages release large amounts of TNFa, which have a direct
effect on pancreatic acinar cells.
174
Two independent groups
reported that TNFasecreted from infiltrating monocytes is
responsible for pancreatic damage and digestive protease
activation.
169,190
Depletion of macrophages by clodronate-
containing liposomes decreased disease severity and pro-
tected mice from cerulein-induced pancreatitis.
169,191
TNFa
acts on cells by cell death receptors and is necessary to induce
cell death by necroptosis through the RIP1–RIP3 pathway,
135
which has been suggested to be the major cell death pathway
during AP.
136
Therefore, infiltrating macrophages are respon-
sible for the induction of necroptosis and for the clearance of
necrotic areas within the damaged pancreas.
175
In addition to TNFa, macrophages produce large amounts
of IL1b, a proinflammatory cytokine associated with the
acute disease phase. In contrast to other cytokines, IL1b
needs to be processed. During maturation, pro-IL1band pro-
IL18 undergo activation by the caspase 1–inflammasome
complex and are released by the gasdermin D–pore complex
from the cytosol to the extracellular space.
192
In consequence
of this process, the cell undergoes pyroptotic cell death.
192,193
During pancreatitis, the activation of the inflammasome
complex and the release of IL1bcontribute critically to dis-
ease severity.
138,139,175,177
Inflammasome complex formation
and pyroptotic cell death are mainly known from macro-
phages and are not present in acinar cells.
175
Inflammasome
activation is a complex mechanism requiring 2 signals for
activation: the first signal induces transcriptional up-
regulation of inflammasome components by NFkB and the
second signal induces oligomerization of the inflammasome
complex and the activation of procaspase 1. The major
inducer of the inflammasome pathway is the TLR–MyD88
cascade. 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 the
cytosol.
194
Phagocytosis of zymogens by macrophages results
in a colocalization of trypsinogen and cathepsin B in non–
acinar cell phagolysosomes and results in activation of tryp-
sinogen
175
and macrophage activation. The rupture of these
trypsin- and cathepsin B–containing vesicles leads to a
cytosolic redistribution of cathepsins that acts as a second
signal of inflammasome activation and indirectly links tryp-
sinogen activation to the NFkB pathway through IL1brelease.
IL1bacts as an activator for other immune cells, with the IL1
receptor being directly linked to the MyD88–NFkB pathway.
The importance of IL1bduring pancreatitis was nicely shown
by a transgenic mouse model of pancreas-specificIL1b-
overexpressing mice that developed CP, including complete
loss of pancreatic function within weeks after birth.
195
Therefore, M1 macrophages contribute prominently to the
systemic immune response syndrome, which is associated
with multiorgan dysfunction syndrome and increased mor-
tality.
170
In contrast to M1 macrophages, the M2 phenotype is
associated with organ regeneration and fibrosis devel-
opment.
196–198
In conclusion, early protease activation and NFkB acti-
vation are essential characteristics of pancreatitis; these 2
events occur in parallel during disease manifestation and
strongly influence each other. Recent studies have proved
that not only the activation of proteases and NFkB but also
the type of cell play a critical role, in which where their
activation takes place is of importance. Pancreatitis is not a
disease of acinar cells alone.
Supplementary Material
Note: To access the supplementary material accompanying
this article, visit the online version of Gastroenterology at
www.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 requests
Address requests for reprints to: Markus M. Lerch, MD, FRCP, Department of
Medicine A, University Medicine Greifswald, Ernst-Moritz-Arndt-University
Greifswald, Ferdinand-Sauerbruch-Strasse 1, 17475 Greifswald, Germany.
e-mail: lerch@uni-greifswald.de.
Acknowledgments
This article is dedicated to the memory of Walter Halangk, PhD, to honor his
work as a pioneer unraveling the pathophysiology of pancreatitis.
Conflicts of interest
The authors disclose no conflicts.
Funding
This 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 R01
DK058088 and R01 DK117809 to Miklós Sahin-Tóth).
1968 Mayerle et al Gastroenterology Vol. 156, No. 7
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Supplementary Table 1.Genetic Risk Factors in Chronic Pancreatitis
Gene/protein Mutation Functional effect Phenotype
PRSS1/cationic
trypsinogen
p.R122C, p.R122H Increased trypsinogen activation owing to
prevention of CTRC-mediated
degradation
Autosomal dominant HP,
familial pancreatitis, or
sporadic CP
pA16V, p.P17T, p.N29I Increased CTRC-dependent trypsinogen
autoactivation
p.D19A, p.D21A, p.D22G,
p.K23R, p.K23_I24insIDK
Increased CTRC-independent trypsinogen
autoactivation
c.204C>A Decreased trypsinogen expression Protection against alcoholic
CP
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/anionic
trypsinogen
p.G191R Introduction of new trypsin cleavage site,
increased autocatalytic inactivation
Protection against CP
SPINK1/trypsin inhibitor p.N34S unknown Increased susceptibility for CP
c.194þ2T>C Skipping of exon 3 resulting in lower SPINK1
levels
CTRC/chymotrypsin C p.A73T Secretion defect Increased susceptibility for CP
p.K247_R254del Inactive CTRC
p.R254W CTRC degradation by trypsin
p.V235I Decreased CTRC activity
p.G60¼Decreased CTRC mRNA
CTRB1–2/chymotrypsin B1
and B2
Inversion at CTRB1–CTRB2
locus
Changes expression ratio of CTRB1 and
CTRB2
Protection against CP
CPA1/carboxypeptidase
A1
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 pancreatic
insufficiency associated
with MODY8
CEL-HYB1 Protein retention and ER stress? Increased susceptibility for CP
CFTR/cystic fibrosis
transmembrane
conductance regulator
p.F508del, p.R117H Loss of CFTR function due to mis-folding
and degradation or disrupted channel
activity
Increased susceptibility for CP
CLDN2/claudin 2 CLDN2–MORC4 variants Tight junction defect? Increased susceptibility for CP
CASR/calcium-sensing
receptor
p.R990G Altered bicarbonate and fluid secretion from
disrupted ductal calcium sensing?
Increased susceptibility for
CP?
p.A986S
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