β-lactam antibiotics modulate the formation of NETs in
PMA-activated PMNs
To determine the potential effect of β-lactam antibiotics on NET
formation, we preincubated purified peripheral blood neutrophils with
imipenem and ceftriaxone for 2 h. Then, the neutrophils were activated
with PMA and monitored for 3 h. The NETotic index curves are shown in
Fig 1A. Unlike the activated neutrophils, the neutrophils (without PMA)
treated with antibiotics alone did not form NETs (Fig 1B). To confirm
the existence of NETs, both fluorescence and
immunofluorescence
images were obtained to determine the morphology and composition of the
NETs (Fig 1C). All the data suggested that imipenem and ceftriaxone have
opposite effects on the formation of NETs in activated neutrophils.
NADPH oxidase (NOX) is a crucial enzyme in the process of NET formation;
during the classic process of NET formation, which proceeds in a
ROS-dependent manner, NOX may modulate the generation of ROS. β-Lactams
were proven to influence the activity of NOX. Thereafter, we detected
the activity of NOX to explore the mechanism by which β-lactam
antibiotics participate in NET formation. DHR123, an indicator of ROS,
was used to measure the generation of ROS. In both the resting and
activated (activated by PMA) neutrophils, the levels of ROS were
increased in the imipenem group and decreased in the ceftriaxone group
compared with the control group (Fig 1D). Then, we used the NOX
inhibitor DPI to explore the relationship between ROS and NET formation
in response to β-lactam antibiotics (Fig 1E). Consistent with the
results described above, DPI inhibited the formation of NETs and the
generation of ROS in all the groups. Once the exogenous ROS
H2O2 was added, the formation of NETs
increased in all the groups (Fig 1F), which indicated that ROS are
involved in β-lactam antibiotic-induced NET formation.
All these data showed that
β-lactam
antibiotics modulate the formation of NETs in activated neutrophils
through a ROS-dependent manner.