Abstract book (download .pdf file) - Redox and Inflammation ...

Session 1: Cell death Poster 3
In vitro characterization of bovine neutrophil cell death following Escherichia coli
phagocytosis
Sofie Notebaert 1* , Kristel Demeyere 1 *, Dieter Demon 1 , Filip Boyen 2 , Chris Guerin 3 ,
Peter Vandenabeele 4 , Evelyne Meyer 1
1 Laboratory of Biochemistry, Faculty of Veterinary Medicine, Ghent University, Salisburylaan
133, 9820 Merelbeke, Belgium – sofie.notebaert@hotmail.com; kristel.demeyere@ugent.be;
dieter.demon@ugent.be; evelyne.meyer@ugent.be
2 Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine,
Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium – filip.boyen@ugent.be
3 Microscopy Core Facility, Department for Molecular Biomedical Research, VIB, Ghent
University, 9052 Zwijnaarde, Belgium - chris.guerin@dmbr.vib-ugent.be
4 Molecular Signaling and Cell Death Unit, Department for Molecular Biomedical Research,
VIB, Ghent University, 9052 Zwijnaarde, Belgium - peter.vandenabeele@dmbr.vib-ugent.be
* These authors contributed equally to this work.
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-1! 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-1! 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.
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