Innate Immune Mechanisms in the Pathogenesis and Management of Acute Pancreatitis

Acute pancreatitis is a common, debilitating condition characterised in its most severe form by organ necrosis as well as systemic inflammation, organ failure and death. The innate immune system, predominantly mediated through neutrophils, has been shown to contribute significantly to both pancreatic injury and respiratory failure: the leading cause of death in severe acute pancreatitis. Neutrophil extracellular traps, anti-microbial nuclear chromatin laced with proteases, are a recent discovery and contribute to the genesis of extracellular free radicals, microvascular thrombosis and abscess formation. Histones and histone fragments are critical active components of neutrophil extracellular traps. They are amongst the most widely conserved biochemical structures, are released as antimicrobial agents by a wide range of organisms and are uniquely toxic to cellular organisms. A central hypothesis for this work, is that the release of neutrophil extracellular traps, and their histone component in particular, is central to the way by which the innate immune response exacerbates acute pancreatitis. By using a 12-injection model of cerulein pancreatitis in mice depleted of specific innate immune cell subsets (Ly6G neutrophils, Ly6C monocytes and CCR2hi inflammatory monocytes), cytokine feedback mechanisms were identified and differential effects on pancreas and lung demonstrated by different cell types. In particular, CCR2hi pro-inflammatory monocytes appeared to contribute greatest to pancreatic injury, whereas Ly6Ghi neutrophils contributed most to lung injury. Using selective simultaneous measurement of portal, systemic venous and arterial blood, the liver was identified as the predominant source of circulating histones in acute pancreatitis, although hepatic immune cells are likely responsible for this signal amplification, similar to that seen with cytokines. Systemic injection of FITC-labelled histones demonstrated concentration within the pancreas only in concomitant pancreatitis. In (acinar) cellular isolates, histones led to dose-dependent necrosis by disrupting the plasmalemma. This in turn led to calcium currents across the membrane, which were not necessary to achieve cytotoxicity. By developing novel methodologies of measuring perfusion on a whole-organ level in mouse pancreas, an overall reduction in pancreatic perfusion is observed in cerulein AP, with patches of ischaemia resulting in focal necrosis on the same time-scale as maximal histone release. Pre-treatment with NETosis inhibitors reduced pancreatic injury, but histone detoxification as treatment (3-hours after induction of acute pancreatitis) was not effective. These results indicate modulation of innate immune mechanisms may allow future therapies to be targeted selectively at different phases of acute pancreatitis, to control extra-pancreatic effects and organ failure independently from pancreatic injury. Significant challenges remain to deliver therapies to a poorly perfused pancreas