In vitro characterization of bovine neutrophil cell death following Escherichia coli phagocytosis

Abstract

Neutrophils are known to play a key role in the early host defense towards coliform mastitis. The molecular events during phagocytosis-induced cell death (PICD) are rarely described, especially for bovine neutrophils. Therefore, our objective was to elucidate the type of cell death of bovine neutrophils after in vitro phagocytosis of Escherichia coli. Peripheral blood samples were collected from clinically healthy heifers. Neutrophils were isolated by density gradient centrifugation, resuspended in RPMI with 10 % fcs and incubated with E. coli strain P4:O32 (moi 5:1) at 37°C. PS exposure and cell membrane integrity loss was determined flow cytometrically by Annexin-V-FITC/PI. ROS were measured as luminol-amplified chemiluminescence. Equal amounts of total cell lysates were analyzed by western blotting using anti caspase-1 (C-1), cleaved C-3 and C-7 and X-IAP. C-3/-7 activity was determined using the caspase-Glo®assay and bovine IL-1β was quantified with ELISA. The higher rate of PS exposure in activated than in control neutrophils undergoing spontaneous apoptosis, indicates that bovine neutrophil cell death is accelerated following phagocytosis of E. coli, as recently described. In contrast to control cells, PICD occurred with significant ROS production but independent from C-3/-7 activation. The latter finding is in line with a recent report describing caspase-independent cell death of human neutrophils incubated with E. coli, although no ROS production was seen. In both human and mouse neutrophils, NADPH oxidase prevents caspase activation following phagocytosis of S. aureus. Nevertheless, all these data do not corroborate a study with human neutrophils incubated with E. coli where C-3 activation was demonstrated. This discrepancy could be either species and/or micro-organism related. Interestingly, the well-described X-IAP inhibitor for C-3 and C-7, was only present in control samples, suggesting that X-IAP may control C-3/-7 activity in spontaneous bovine neutrophil apoptosis but is superfluous in E. coli PICD in vitro. Life cell imaging using GFP-labeled E. coli and PI staining showed no clear morphological features of either netosis or autophagy, but late PI positivity and membrane blebbing without apoptotic bodies. Complementing these features with C-1 activation and intracellular IL-1β suggests that the PICD in our setup is executed by pyroptosis. Taken together, our in vitro data indicate that bovine neutrophils undergo C-3/-7-assisted spontaneous apoptosis and that E. coli stimulated PICD most likely can be characterized as C-3/-7-independent, but C-1- and ROS-dependent pyroptosis