Fig 7. A B. parapertussis secreted Bvg-regulated virulence factor is involved in PMA-induced NETs inhibition.

CM was prepared by incubating 2x10⁸ CFU/ml of virulent B. parapertussis (Bvg⁺ CM) or avirulent B. parapertussis (Bvg^- CM) in medium for 2 h. In some samples CM was treated with proteinase K (PK) (100 μg/mL) for 60 min at 37°C prior use. Neutrophils were incubated with each CM 30 min before treatment with PMA. Neutrophils incubated with PMA alone were used as positive control. Four hours after PMA treatment samples were fixed and permeabilized prior to labeling the NE in green and neutrophil DNA in red. Samples were analyzed by fluorescence microscopy and the number of NETting cells was determined by the ImageJ softaware. At least 500 cells were counted per sample. (A) Representative microscopy images of each condition are shown. Scale bar: 20 μm. (B) The bars represent the percentage of neutrophils that underwent NETosis. The data represent the mean ± SD of three experiments with neutrophils from different donors. The percentage of NETting cells in neutrophils incubated with Bvg⁺ CM prior to PMA treatment was significantly different from the percentage of NETting cells observed in the other samples (*P <0.05).