AAV6-CMV-iCre

Calcineurin is a ubiquitously expressed central Ca2+-responsive signaling molecule that mediates acute pancreatitis, but its role is poorly understood. Here, researchers compared the effects of hematopoietic and pancreatic expression of calcineurin in mouse models of pancreatitis and pancreatitis-associated lung inflammation. Mice with hematopoietic-specific deletion of CNB1 had the same degree of local pancreatic inflammation as control mice after penicillin administration or radiocontrast injection into the pancreatic bile duct. Cnb1^UBC△/△ mice or mice with pancreatic-specific deletion of CNB1 had less severe pancreatitis and reduced pancreatic inflammation after penicillin administration or radiocontrast injection into the pancreatic bile duct compared with control mice. NFAT was activated in pancreas of Swiss Webster mice given caerulein or infusions of radiocontrast into biliopancreatic ducts. Blocking the interaction between calcineurin and NFAT did not reduce pancreatic acinar cell necrosis induced by penicillin or radiocontrast infusion. Mice with hematopoietic-specific but not pancreatic-specific CNB1 deletion had reduced neutrophil infiltration in the lungs. Neutrophil chemotaxis and reactive oxygen species production were reduced after incubation with a calcineurin inhibitor. These findings suggest that the protective effects of blocking or deleting calcineurin against pancreatitis are mediated by its source of expression.

To compare the protective effects of calcineurin expression within the pancreas, the researchers investigated the effects of pancreas-specific calcineurin deficiency on the pancreatitis response. Pancreatic-specific calcineurin deficiency was induced in Cnb1f/f mice by intraductal infusion of adeno-associated virus 6 (AAV6) containing enhanced Cre recombinase (iCre) driven by the ubiquitous cytomegalovirus (CMV) promoter (Figure 1A). Intraductal infusion of AAV6-CMV-iCre was performed in LSL-tdTomato reporter mice and strong pancreas-specific red fluorescence was observed throughout the pancreas, indicating widespread expression of iCre. As expected, CNB1 protein levels were reduced in the pancreas but not in BM cells (Figure 1B). During penicillin hyperstimulatory pancreatitis, mice with pancreas-specific CNB1 deficiency (Cnb1^Panc∆/∆) exhibited significantly reduced pancreatic histological damage (Figure 1C), edema threshold (Figure 1D), and inflammatory infiltrate (Figure 1E). No sex differences in these parameters were observed with or without pancreas-specific Cnb1 deficiency. Neutrophil infiltration, assessed by pancreatic MPO immunostaining, was significantly reduced after pancreas-specific CNB1 deficiency (Figure 1E), whereas macrophage infiltration (by F4/80) was not different. These results suggest that pancreatic calcineurin selectively affects neutrophil infiltration.

Figure 1. During caerulein hyperstimulation pancreatitis (CER), pancreas-specific deletion of CNB1 largely prevents localized pancreatic injury and systemic inflammation, but it does not affect distant organ damage. (Wen L, et al., 2020)