Targeting peptidylarginine deiminase reduces neutrophil extracellular trap formation and tissue injury in severe acute pancreatitis

Abstract

Recent evidence suggests that neutrophil extracellular traps (NETs) play an important role in the development of acute pancreatitis (AP). Herein, we examined the role of peptidylarginine deiminase (PAD), which has been shown to regulate NET formation, in severe AP. AP was induced by retrograde of taurocholate infusion into pancreatic duct in C57BL/6 mice. PAD was pharmacologically inhibited using Cl‐amidine, a pan‐PAD inhibitor. Pancreata were collected, and histones, citrullinated histone 3, chemokines, myeloperoxidase, and NETs were quantified. Chemokines, matrix metalloproteinase‐9 (MMP‐9), interleukin‐6 (IL‐6), and DNA‐histone complexes were determined in plasma samples. Infusion of taurocholate induced formation of NETs in pancreatic tissues of mice. Pretreatment with Cl‐amidine markedly reduced the NET formation in the inflamed pancreas. Moreover, inhibition of PAD decreased the levels of blood amylase as well as edema, acinar cell necrosis, hemorrhage, and neutrophil infiltration in the pancreas of animals with AP. Administration of Cl‐amidine attenuated the myeloperoxidase levels in the pancreas and lung of mice exposed to taurocholate. In addition, Cl‐amidine decreased pancreatic levels of CXC chemokines, plasma levels of IL‐6, and MMP‐9 in mice with severe AP. This study shows that Cl‐amidine is a potent inhibitor of NET formation in severe AP. Also, our results suggest that PAD regulates pathological inflammation and tissue damage in the inflamed pancreas. Thus, targeting PAD might be a useful strategy to treat patients with severe AP.

Complement Component 3 Is Required for Tissue Damage, Neutrophil Infiltration, and Ensuring NET Formation in Acute Pancreatitis

“…In addition, a recent study has shown that a cold-inducible RNA-binding protein (eCIRP), a novel damage-associated molecular pattern molecule, is present in NETs and that NET-associated eCIRP can cause tissue damage in AP [30]. Furthermore, other studies indicated that NET formation is regulated by the c-Abelson (c-Abl) kinase [31] and the histone-citrullinating enzyme peptidylarginine deiminase 4 (PAD4) in AP [32]. Knowing that the C3 activation product, C3a, exerts proinflammatory functions, we evaluated whether C3a alone can induce histone 3 citrullination and NETs formation in bone marrow-derived neutrophils.…”

The Role of Pancreatic Infiltrating Innate Immune Cells in Acute Pancreatitis

“…During AP, the pancreatic juice is a strong instigator of infiltrating neutrophil chromatin extrusion and thereby lead to the formation of macroscopic NETs aggregates, which may occlude pancreatic duct and drive pancreatic inflammation, inhibition of NETs formation can mitigate AP [57]. For the detection of NETs formation in AP, cell-free DNA (cfDNA) [55], MPO-DNA complex [55], or histone-DNA complex [39,75] in blood samples can be measured by commercial kits. Besides, citrullinated histone H3 (CitH3) [39,75] in pancreatic samples can be measured by western blot.…”

“…For the detection of NETs formation in AP, cell-free DNA (cfDNA) [55], MPO-DNA complex [55], or histone-DNA complex [39,75] in blood samples can be measured by commercial kits. Besides, citrullinated histone H3 (CitH3) [39,75] in pancreatic samples can be measured by western blot. During AP, damaged pancreatic acinar cells release proinflammatory mediators, leading to the recruitment, activation and functional changes of neutrophils and macrophages: (1) Neutrophils release proteases and ROS, which directly damage pancreatic tissue.The released MMP-9 is a potent trypsinogen activator.…”

“…Peptidyl arginine deiminase 4 (PAD4) and c-Abelson (c-Abl) kinase are responsible for regulating the formation of NETs [39,60]. Pharmacologically inhibition of PAD4 by Cl-amidine, a pan-PAD inhibitor, or inhibition of c-Abl by GZD824, a c-Abl kinase inhibitor, can reduce NETs formation and tissue Injury in SAP [39,75]. The autophagy inhibitor chloroquine (CQ) also inhibits NETs formation, thus reduces the severity of AP [55].…”

Machine Learning to Quantitate Neutrophil NETosis

“…These two NETosis pathways utilize different biochemical mediators, for example histone citrullination is a marker for the PAD4-mediated but not the ROS-mediated pathway 22,23 . Targeting PAD4 with pharmacological inhibitors is successful in inhibiting NETosis in vitro 24 and in animal models 25–27 ; however, no targeted therapies for the ROS-pathway exist 27–31 . DNase-1 has also been used to digest the nuclear backbone of NETs and prevent ischemia-reperfusion (I/R) injury 32 , lung injury due to cigarette smoke or mechanical ventilation 33,34 , and deep vein thrombosis 35 .…”