Lung function adjusted for body length and gestational age in male (black) and female (gray) premature infants.

<p>Data are represented as the mean (+SD). Sixty-six (35 female) were exposed to HCA (combined Grade 1 and Grade 2) and 29 not exposed to HCA (17 female). There was a significant sex by HCA interaction for FEF₅₀ (F = 8.76; p = 0.004), FEF_25–75 (F = 8.11; p = 0.005) and FEV_0.5 (F = 4.81; p = 0.031). Post hoc analyses revealed a significant reduction in lung function in exposed female preterm infants when compared to females not exposed to HCA. The effect of exposure to HCA was not significant in males. *<i>p</i><0.05, **<i>p</i><0.01 (Post hoc Holm-Sidak test).</p

Lung function values, stratified by sex and chorioamnionitis.

<p>Lung function, Weight/age, and Length/age expressed in Z scores. Data are mean±SD.</p>*<p>p<0.05; **p<0.01; for Mann-Whitney Test for continuous variables between male versus female preterm infants and between None versus HCA Grade 1 and HCA Grade 2 combined.</p>#<p>p<0.05 for Jonckheere–Terpstra trend test for continuous variables between None, HCA Grade 1 and HCA Grade 2.</p

Lung function variables, expressed as mean z-score by HCA level in premature infants.

<p>*<i>p</i><0.05 for Jonckheere–Terpstra trend test.</p

Essential role for NADPH Oxidase-derived ROS on F protein-induced NET generation.

<p>(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.</p

Mechanisms involved in RSV Fusion protein-induced NET formation in human neutrophils.

<p>(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.</p

F protein-induced NET formation is dependent on TLR-4 activation.

<p>(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.</p

RSV particles and RSV Fusion protein induce NET formation.

<p>(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.</p

Effect of different treatments on F protein-induced NETs generation.

<p>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.</p

F protein activates ERK and p38 MAPK to induce NET formation.

<p>(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.</p