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.