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Zierke et al. Rare Dis Orphan Drugs J 2023;2:10 https://dx.doi.org/10.20517/rdodj.2022.17 Page 5 of 10

[53]
intracellular inhibitor of trypsin . Besides CTSB, the lysosomal hydrolase cathepsin L and chymotrypsin C
are also capable of cleaving trypsinogen [20,54] . Moreover, the aspartic protease cathepsin D (CTSD) indirectly
[55]
acts on trypsinogen as CTSD proteolytically activates pro-CTSB . In contrast, under pathophysiological
conditions, the activation of trypsinogen and other downstream proteases occurs inside pancreatic acinar
cells, resulting in self-digestion and necrosis of the cells and the release of damage-associated molecular
patterns (DAMPs). In addition, the transcription nuclear factor kappa B (NF-κB) is upregulated in
pancreatic acinar cells, resulting in the overexpression and secretion of pro-inflammatory cytokines and
chemokines . The combination of DAMPs, cytokines, and chemokines augments the activation and
[56]
infiltration of immune cells into the pancreatic tissue. Activation of trypsinogen occurs not only in acini but
also within macrophages that invade the pancreas during acute pancreatitis and phagocytose zymogen-
containing vesicles. This process also depends on pH as well as the lysosomal protease CTSB . Neutrophils
[57]
are a second major immune cell type that invades the pancreas during pancreatitis and can cause local tissue
damage in different ways. In rodents with acute pancreatitis in which neutrophils were depleted by anti-
neutrophil serum or anti-GR-1-antibodies, disease severity was significantly reduced [58,59,59] . Neutrophils are
also able to activate trypsinogen, but the exact underlying mechanism is unknown, although it has been
elucidated for macrophages . One study showed that inhibition of matrix metalloproteinase-9 (MMP9) led
[57]
[58]
to a reduction of trypsinogen activation by neutrophils , Which was NADPH-mediated .
[60]
Moreover, neutrophils release NSPs cathepsin G (CTSG), proteinase 3 (PR3), and neutrophil elastase (NE).
The cysteine protease cathepsin C (CTSC) seems to have a higher-ranking function, as it activates
NSPs [61-63] . Neutrophil serine enzymes are capable of cleaving the tight junction protein E-cadherin, but they
differ in their efficiencies. The strongest cleavage effects have been observed for NE. The breakdown of E-
cadherin results in a loss of cell-cell connections, facilitating the infiltration of immune cells into pancreatic
tissue and the formation of edema . Pharmacological inhibition of NE prevented cleavage of E-cadherin
[43]
[64]
and reduced neutrophil infiltration in the pancreas in vivo .In contrast, CTSG and PR3 only have minimal
E-cadherin cleavage capabilities in acinar cells, and depletion of CTSG neither decreased trypsinogen
activation nor led to an amelioration of pancreatitis severity, although a transient decrease of neutrophil
infiltration in the pancreas and extra-pancreatic organs was observed . Furthermore, neutrophils are
[65]
responsible for the extrusion of neutrophil extracellular traps (NETs), which consist of condensed
chromatin and NSPs and can enhance pancreatic damage, promote platelet aggregation, and produce
further NSP-associated effects . NETs can also promote trypsinogen activation . The main pathogenetic
[66]
[67]
cellular events in the initiation of acute pancreatitis are summarized in Figure 1.
The knowledge of the role of neutrophils in general and NSPs in particular in the pathogenesis and course
of hereditary and autoimmune pancreatitis remains very limited. The pathogenic role of neutrophils in
chronic pancreatitis associated with genetic mutations has been reflected in two murine models. In mice
carrying a heterozygous p.D23A mutation in the mouse cationic trypsinogen isoform 7 (T7D23A knock-in),
strong auto-activation of trypsinogen was observed, resulting in spontaneous acute and subsequent chronic
pancreatitis. Massive infiltration of inflammatory cells including neutrophils and macrophages occurred
during the early disease phase. Signs of disease progression were already visible after 4-5 weeks, while
immune cells became fewer in number . Transgenic mice expressing the PRSS1 mutant R122H
[68]
(R122H_mPRSS1) displayed early-onset acinar cell injury and inflammatory cell infiltration, followed by
pancreatic fibrosis and an enhanced response to the cholecystokinin analog caerulein .
[69]

To the best of our knowledge, no currently available literature has specifically focused on the role of NSPs in
hereditary pancreatitis, although the organ-injuring effects of neutrophils and elastase in acute pancreatitis
are clear in general.