B. A. Boone1, P. Murthy1, X. Liang1, A. D. Singhi1, R. Kang1, D. Tang1, H. J. Zeh1 1University Of Pittsburgh,Surgical Oncology,Pittsburgh, PA, USA
Introduction: Neutrophil extracellular traps (NETs) occur when activated neutrophils release their intracellular contents, including histones, DNA, elastase and other proteins, into the extracellular space, tissues or circulation. Recently, NETs have been implicated in acute pancreatitis, worsening pancreatic inflammation, and promoting pancreatic duct obstruction. We have previously shown that the autophagy inhibitor chloroquine (CQ) inhibits NET formation; therefore we sought to determine if CQ could improve severity and outcome in murine pancreatitis through NET inhibition.
Methods: Acute pancreatitis was induced in C57/Bl6 mice through two hourly injections of L arginine (4g/kg) into the peritoneal cavity. Sham control mice were injected with saline. Mice were sacrificed 48 hours after injection to collect serum. Neutrophils were harvested from bone marrow using density gradient centrifugation and stimulated with platelet activating factor to induce NET formation. Hoechst staining of DNA was then utilized to visualize NETs using fluorescence microscopy. Citrullinated histone H3 (Cit H3), which allows for unwinding and expulsion of neutrophil DNA during NET formation and is a critical marker of NETs, was measured in murine serum using ELISA. Serum DNA was measured using Quant-It picogreen. For survival experiments, mice were injected with L arginine weekly x 3 weeks. Animals were treated with CQ in the drinking water (100 mg/kg/day PO), beginning 1 hour after induction of pancreatitis.
Results: Injection of L arginine resulted in increased serum amylase and trypsin compared with sham controls, consistent with induction of pancreatitis. Neutrophils harvested from mice with pancreatitis were more prone to NET formation than sham injected controls. Induction of pancreatitis resulted in a significant increase in serum DNA and citrullinated histone H3, suggesting upregulated in vivo NET formation. CQ treatment decreased the propensity to form NETs from neutrophils harvested in mice with pancreatitis. Both serum DNA and Cit H3 were significantly decreased with CQ treatment, suggesting a decrease in NETs in response to CQ. CQ lessened the severity of acute pancreatitis, resulting in a reduction in serum amylase and trypsin with CQ treatment. CQ treatment improved survival from pancreatitis (Figure 1, median survival 15 days vs. not reached, p<0.05).
Conclusion: CQ treatment decreases the severity of L arginine induced murine pancreatitis through inhibition of neutrophil extracellular traps, resulting in improved survival. NET inhibition represents a novel treatment strategy in acute pancreatitis. Further study to translate these findings into treatment of patients with severe acute pancreatitis is warranted.
