Figure 5
RIPK1, RIPK3 and MLKL are involved on RSV-driven NETosis. Neutrophils were pretreated with: (A) necrostatin-1s (Nec-1s; 50 µM), (B) GW42X (GW; 0.5 µM) or (C) necrosulfonamide (NSA; 5 µM) for 1 h and stimulated with RSV (104 PFU/mL) for 180 min. After this period, NETs were quantified in culture supernatant, using Quant-iT dsDNA HS kit. (D) Supernatants from neutrophils stimulated with RSV (102–106 PFU/mL) for 180 minutes were collected and assayed for LDH release. (E,F) Supernatants from neutrophils pretreated for 1 h with Nec-1s (50 µM) or NSA (5 µM) and stimulated with RSV (104 PFU/mL) for 180 minutes were collected and assayed for LDH release. Cytotoxicity was measured at 490 nm. Data are representative of at least 2 independent experiments performed in triplicates and represent mean ± SEM. Data were analyzed with unpaired Student’s t-test or Mann Whitney test. *p < 0.05, **p < 0.01 when compared to non-infected control (−); +p < 0.05, ++p < 0.01 when compared to RSV-stimulated neutrophils.
