Fig. 1. A.pp and BALF of A.pp-infected swine induce NETs, but A.pp grows efficiently in the presence of degraded NETs.

a Primary fresh blood-derived porcine neutrophils were isolated and treated with RPMI as negative control (RPMI, ctr), methyl-β-cyclodextrin (CD) as positive control, A.pp, BALF of uninfected or A.pp-infected pigs. After 3 h incubation at 37 °C and 5% CO₂ the cells were fixed. Afterwards NET staining for immunofluorescence microscopy was conducted (blue = DNA (Hoechst), green = DNA/histone‐1‐complexes (NETs)). Representative pictures are shown. (scale bar = 50 µm). b Statistical analysis of NET induction assays was conducted from three independent experiments. Treated cells were incubated as described in a. Per sample, six pictures were taken on two slides at predefined positions and the number of NET-releasing cells were determined. Compared results of the mean values are presented with ±SD (n = 3, one-way ANOVA P = 0.0083, followed by Tukey’s multiple comparison test). c NET induction was analyzed after treatment of neutrophils with BALF. Compared results of the mean values are presented with ±SD (n = 3, one-way ANOVA P = 0.0031, followed by Tukey’s multiple comparison test). d Neutrophils were incubated with A.pp for 3 h 37 °C, 5% CO₂ and the cells were fixed. Afterwards staining for NETs was conducted (blue = DNA (Hoechst), green = DNA/histone‐1‐complexes (NETs), red = A.pp). Representative pictures are shown (scale bar = 15 µm). e, f A.pp was grown for 1 or 3 h at 37 °C in the absence or presence of neutrophils (PMN) and external added DNase as indicated. The survival factor was calculated based on the CFU at time point 0 h (see Fig. S2) and compared to the end point. Data shown as mean ± SD (n = 4, one-way ANOVA 1 h P = 0.0010, 3 h P < 0.0001, followed by Tukey’s multiple comparison test)