Figure 7.

Neutrophils kill S. aureus first by phagocytosis, and then through NETs. Neutrophils were prestimulated with 10 nM PMA (a) or 100 mU/ml GO (b) for the indicated time or left unstimulated (a and b, 0 min). Before infection with S. aureus (MOI 1), the cells were treated with DNase or not (untreated). In the absence of DNase (open bars, untreated), neutrophils kill bacteria by both phagocytosis and NETs. In the presence of DNase, the NETs are dismantled and bacteria are killed only by phagocytosis. The differences in bacterial killing in cultures with and without DNase reflect the antimicrobial activity of NETs. Neutrophils that were not prestimulated killed S. aureus efficiently exclusively by phagocytosis because treatment with DNase did not affect the killing. However, cells prestimulated for 60 min showed decreased bacterial killing. Under both conditions, DNase treatment had no effect on killing the bacteria, as NETs had not yet emerged. After 120 min stimulation, when NETs are formed, DNase reduced killing of S. aureus. 3–4 h after stimulation, neutrophils killed S. aureus as efficiently as unstimulated cells. This killing was completely caused by NETs, as it was affected by the DNase treatment. (c) Unstimulated and PMA-activated (240 min) neutrophils of patients with CGD showed impaired killing of S. aureus. Their defective bacterial killing was circumvented when NETs were induced with GO (for 240 min). The experiment was repeated at least five times with neutrophils from independent donors with similar results. The data shown is a representative triplicate experiment and presented as a mean value ± the SD.