Fig 1.
RSV particles and RSV Fusion protein induce NET formation.
(A) Human neutrophils (2 x 106/mL) were stimulated with RSV (102–104 PFU/mL) or left unstimulated for 3 h at 37°C with 5% CO2. (B) Neutrophils (2 x 106/mL) were stimulated with RSV F protein (0.1–5 μg/mL), PMA (100 nM) or medium alone for 3 h at 37°C with 5% CO2. NETs were quantified in culture supernatants using Quant-iT dsDNA HS kit. Data are representative of at least 3 independent experiments performed in triplicates and represent mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 when compared to negative control (NCtrl). (C-F) Neutrophils (2 x 105/300 μL) were stimulated with (C) medium, (D) LPS (100 ng/mL), (E) PMA (100 nM) or (F) F protein (1 μg/mL) for 3 h at 37°C with 5% CO2. Cells were then fixed with 4% PFA and stained with Hoechst 33342 (1:2000). Images are representative of at least 4 independent experiments. (G-L) Neutrophils (2 x 105/300 μL) were stimulated with F protein (1 μg/mL) for 3 h at 37°C with 5% CO2. Cells were fixed with 4% PFA and stained with: (G-I) Hoechst 33342 (1:2000), anti-elastase (1:1000), followed by anti-rabbit Cy3 (1:500) antibodies; (J-L) Hoechst 33342 (1:2000), anti-myeloperoxidase PE (1:1000) antibody. Overlay of the fluorescence images are shown in the last panels (I,L). Images are representative of 2 independent experiments. Images were taken in a Zeiss LSM 5 Exciter microscope. Scale bars = 50 μm.
Fig 2.
Effect of different treatments on F protein-induced NETs generation.
Human neutrophils (2 x 106/mL) were stimulated with: (A) F protein (1 μg/mL) or LPS (100 ng/mL) in the presence or absence of DNase-1 (100U/mL); (B) F protein (1 μg/mL) or LPS (100 ng/mL) in the presence or absence of polymyxin B (Pmx B, 1 μg/mL); (C) F protein (1 μg/mL), boiled F protein (1 μg/mL, 10 min at 100°C) or F protein (1 μg/mL) treated with proteinase K (1 mg/mL for 90 min) for 3 h at 37°C with 5% CO2. (D) F protein solution was treated with monoclonal anti-F protein (10 μg/mL) or isotype-matched (10 μg/mL) antibody and neutrophils (2 x 106/mL) were stimulated with these preparations for 3 h at 37°C with 5% CO2. NETs were quantified in culture supernatants using Quant-iT dsDNA HS kit. Data are representative of at least 2 independent experiments performed in triplicates and represent mean ± SEM. *p<0.05; ***p<0.001 when compared to negative control (NCtrl); #p<0.05 when compared to LPS- or F protein-treated cells.
Fig 3.
F protein-induced NET formation is dependent on TLR-4 activation.
(A) Human neutrophils (2 x 106/mL) were pretreated with monoclonal anti-TLR4 (10 μg/mL) or isotype-matched (10 μg/mL) antibody for 1 h and stimulated with F protein (1 μg/mL) or medium for 3 h at 37°C with 5% CO2. NETs were quantified in culture supernatants using Quant-iT dsDNA HS kit. Data are representative of at least 3 separate experiments performed in triplicates and represent mean ± SEM. *p<0.001 when compared to negative control (NCtrl); #p<0.05 when compared to F protein-treated cells. (B) Neutrophils (2 x 105/300 μL) were pretreated with anti-TLR4 (10 μg/mL) for 1 h at 37°C with 5% CO2 and stimulated with F protein (1 μg/mL) or medium for 3 h at 37°C with 5% CO2. Cells were fixed with 4% PFA and stained with Hoechst 33342 (1:2000). Confocal images were taken in a Zeiss LSM 5 Exciter microscope. Image is representative of 2 independent experiments. Scale bars = 50 μm.
Fig 4.
Essential role for NADPH Oxidase-derived ROS on F protein-induced NET generation.
(A,C) Neutrophils (2 x 106/mL) were pretreated with NAC (1 mM) or DPI (10 μM) for 1 h and stimulated with F protein (1 μg/mL) for 3 h at 37°C with 5% CO2. NETs were quantified in culture supernatants using Quant-iT dsDNA HS kit. Data are representative of 3 separate experiments performed in triplicates and represent mean ± SEM. ***p<0.001 when compared to negative control (NCtrl); #p<0.001 when compared to F protein-treated cells. (B,D) Neutrophils (2 x 106/microtube) were pretreated with NAC (1 mM) or DPI (10 μM) for 1 h, stimulated with F protein (1 μg/mL) for 1 h at 37°C with 5% CO2 and incubated with 0.5 μM CM-H2DCFDA for 30 min. ROS generation was analyzed by flow cytometry using FACSCanto II flow cytometer. Neutrophils gate was based on FSC x SSC distribution. Data are representative of 2 independent experiments performed in triplicates with similar results.
Fig 5.
F protein activates ERK and p38 MAPK to induce NET formation.
(A,B) Neutrophils (2 x 106/mL) were pretreated with PD98059 (30 μM) or SB203580 (10 μM) for 1 h and stimulated with F protein (1 μg/mL) for 3 h at 37°C with 5% CO2. NETs were quantified in culture supernatants using Quant-iT dsDNA HS kit. Data are representative of 3 separate experiments performed in triplicates and represent mean ± SEM. ***p<0.001 when compared to negative control (NCtrl); #p<0.001 when compared to F protein-treated cells. (C,D) Neutrophils (1 x 106/mL) were stimulated with F protein (1 μg/mL) for 5 min at 37°C with 5% CO2 and stained for phosphorylated proteins (ERK 1/2 and p38 MAPK), according to Materials and Methods. Proteins phosphorylation was analyzed by flow cytometry using FACSCanto II flow cytometer. Neutrophils gate was based on FSC x SSC distribution. Phosphorylation of protein pathways are presented as fold increase relative to unstimulated neutrophils (NCtrl). Data are representative of 2 separate experiments with similar results.
Fig 6.
Mechanisms involved in RSV Fusion protein-induced NET formation in human neutrophils.
(I) RSV F protein binds to and activates TLR-4, expressed by neutrophils, stimulating ROS production via NADPH Oxidase, which is essential for NET formation. (II) F protein is also able to activate ERK and p38 MAPK to induce NET release. RSV F protein stimulates the production of NETs decorated with the granular proteins NE and MPO.