Respiratory syncytial virus fusion protein promotes TLR-4-dependent neutrophil extracellular trap formation by human neutrophils

Abstract

Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV) is the most common respiratory illness in children in the first year of life. RSV bronchiolitis generates large numbers of hospitalizations and an important burden to health systems. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease. Neutrophil Extracellular Traps (NETs) are formed by the release of granular and nuclear contents of neutrophils in the extracellular space in response to different stimuli and recent studies have proposed a role for NETs in viral infections. In this study, we show that RSV particles and RSV Fusion protein were both capable of inducing NET formation by human neutrophils. Moreover, we analyzed the mechanisms involved in RSV Fusion protein-induced NET formation. RSV F protein was able to induce NET release in a concentration-dependent fashion with both neutrophil elastase and myeloperoxidase expressed on DNA fibers and F protein-induced NETs was dismantled by DNase treatment, confirming that their backbone is chromatin. This viral protein caused the release of extracellular DNA dependent on TLR-4 activation, NADPH Oxidase-derived ROS production and ERK and p38 MAPK phosphorylation. Together, these results demonstrate a coordinated signaling pathway activated by F protein that led to NET production. The massive production of NETs in RSV infection could aggravate the inflammatory symptoms of the infection in young children and babies. We propose that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis.

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.