Fig. 4: Signs for NETs found in A.pp-infected swine and correlate with the severity of infection.
From: Degraded neutrophil extracellular traps promote the growth of Actinobacillus pleuropneumoniae
BALF samples (n = 8 for each group) were analyzed for NET markers a–c and DNase1 d. a The amount of free DNA in BALF samples was determined using Pico Green assay. Data of four independent technical replicates are presented as mean. b–d Samples were analyzed by ELISA. The mean of duplicates for each animal was calculated. e, f Stained lung slices of three animals per group were analyzed by ImageJ software to determine the percentage of mean fluorescence of red (NE) and green (DNA/Histone) signal based to the blue signal (nuclei). Per animal 10 immunofluorescence microscopy pictures were analyzed (uninfected n = 29, infected n = 30). High amount of neutrophil elastase and histone signals were detectable in lungs of A.pp-infected swine. Representative pictures of immunofluorescence microscopy are shown (blue = DNA (Hoechst), green = DNA/histone‐1‐complexes (NETs), red = neutrophil elastase). The right picture is a magnification from left (scale bar left pictures = 100 µm, scale bar right picture = 25 µm). e, g A.pp was grown 3 h at 37 °C in the presence of filtered BALF samples. To exclude that PR-39 as antimicrobial peptide influences the survival of bacteria, in the uninfected group three BALF samples without PR-39 detection (0 pg/ml) were used. The mean of DNase I in these samples are 10.82 pg/ml. In the infected group three BALF samples of pigs with the highest amount of PR-39 were used (640–897 pg/ml). The mean of DNase I in these samples are 97.17 pg/ml. The survival factor was calculated based on the CFU at start point compared to 3 h. The experiment was conducted in three independent technical replicates with all six tested samples each run. h–j In addition, the results of free DNA and pig DNase1, as well as NET releasing cells after BALF treatment for 3 h (from Fig. 1c) are grouped in high and low LLS (three pigs each group from the infected group). Information of statistical analysis: Compared results are shown as mean ± SD in all graphs. In g, j data are analyzed with one-tailed paired Student’s t-test. In all other graphs data are analyzed with one-tailed unpaired Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)
