Enhanced Neutrophil Extracellular Trap Formation in Acute Pancreatitis Contributes to Disease Severity and Is Reduced by Chloroquine.

Pranav Murthy,Patricia Loughran,Aatur D Singhi,Amer H Zureikat,Michael T Lotze,Mark A Ross,Pedram Paragomi,Brian A Boone,David C Whitcomb,Herbert J Zeh Iii,Georgios I Papachristou

doi:10.3389/fimmu.2019.00028

Abstract

Background: Neutrophil extracellular traps (NETs) are generated when activated neutrophils, driven by PAD4, release their DNA, histones, HMGB1, and other intracellular granule components. NETs play a role in acute pancreatitis, worsening pancreatic inflammation, and promoting pancreatic duct obstruction. The autophagy inhibitor chloroquine (CQ) inhibits NET formation; therefore, we investigated the impact of CQ mediated NET inhibition in murine models of pancreatitis and human correlative studies.Methods: L-arginine and choline deficient ethionine supplemented (CDE) diet models of acute pancreatitis were studied in wild type and PAD4−/− mice, incapable of forming NETs. Isolated neutrophils were stimulated to induce NET formation and visualized with fluorescence microscopy. CQ treatment (0.5 mg/ml PO) was initiated after induction of pancreatitis. Biomarkers of NET formation, including cell-free DNA, citrullinated histone H3 (CitH3), and MPO-DNA conjugates were measured in murine serum and correlative human patient serum samples.Results: We first confirmed the role of NETs in the pathophysiology of acute pancreatitis by demonstrating that PAD4−/− mice had decreased pancreatitis severity and improved survival compared to wild-type controls. Furthermore, patients with severe acute pancreatitis had elevated levels of cell-free DNA and MPO-DNA conjugates, consistent with NET formation. Neutrophils from mice with pancreatitis were more prone to NET formation and CQ decreased this propensity to form NETs. CQ significantly reduced serum cell-free DNA and citrullinated histone H3 in murine models of pancreatitis, increasing survival in both models.Conclusions: Inhibition of NETs with CQ decreases the severity of acute pancreatitis and improves survival. Translating these findings into clinical trials of acute pancreatitis is warranted.

Highlights

Acute pancreatitis (AP) is a common, severe gastrointestinal disease, assessed as the fifth leading cause of in-hospital mortality and an annual incidence that has increased by 30% since 2000 [1, 2]
We first confirmed the role of Neutrophil extracellular traps (NETs) in the pathophysiology of acute pancreatitis by demonstrating that PAD4−/− mice had decreased pancreatitis severity and improved survival compared to wild-type controls
Neutrophils from mice with pancreatitis were more prone to NET formation and CQ decreased this propensity to form NETs

Summary

Introduction

Acute pancreatitis (AP) is a common, severe gastrointestinal disease, assessed as the fifth leading cause of in-hospital mortality and an annual incidence that has increased by 30% since 2000 [1, 2]. AP is characterized by elevated digestive enzyme concentrations in the blood linked to altered acinar cell secretion and/or duct obstruction, which promotes autodigestive injury within the pancreas [2]. These events stimulate tissue injury and activation of the innate immune system, resulting in recruitment and activation of neutrophils with subsequent release of pro-inflammatory cytokines and other substances that lead to systemic inflammation [2, 3]. In addition to release of cytokines, activated neutrophils release their DNA, histone proteins, high mobility group box 1 (HMGB1), and granule components into the extracellular space or circulation to form neutrophil extracellular traps (NETs). The autophagy inhibitor chloroquine (CQ) inhibits NET formation; we investigated the impact of CQ mediated NET inhibition in murine models of pancreatitis and human correlative studies

Methods

Results

Conclusion