Figure 1
RSV induces the classical ROS-dependent NETosis in human neutrophils. (A) Human neutrophils (2 × 106/mL) were stimulated with RSV (102 – 106 PFU/mL), PMA (100 nM) or left unstimulated for either 10 or 180 min. (B) Neutrophils (2 × 106/mL) were stimulated with Staphylococcus aureus at bacteria to neutrophil ratios of 10:1 or left unstimulated for 10 min. NETs were quantified in culture supernatants using Quant-iT dsDNA HS kit (Invitrogen). (C) Neutrophils (2 × 106 cells/microtube) were stimulated with PMA (50 nM) or left unstimulated for 60 min and incubated with CM-H2DCFDA (0.5 µM) for 30 min at 37 °C with 5% CO2. (D) Neutrophils (2 × 106 cells/microtube) were pretreated with DPI (10 µM), stimulated with RSV (104 PFU /mL) or left unstimulated for 60 min and incubated with CM-H2DCFDA (0.5 µM) for 30 min at 37 °C with 5% CO2. Cytosolic ROS production was measured by flow cytometry, using FACSCanto II flow cytometer. Neutrophils were pre-treated for 1 h with: (E) diphenyleneiodonium (DPI) or (F) apocynin (APO). Afterwards, cells were stimulated with RSV (104 PFU/mL) for 180 min. NETs were quantified in culture supernatants using Quant-iT dsDNA HS kit. Data are representative of 3 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with Mann Whitney test. *p < 0.05; **p < 0.01 when compared to negative control (−); +p = 0.05; ++p < 0.05 when compared to RSV-stimulated neutrophils.
