Figure 4.

Rv0888NS sphingomyelinase induced formation of neutrophil extracellular traps (NETs) in vitro. (A) Human neutrophils were treated with 0.4 mM Rv0888NS or 0.8 mM Rv0888NS, 0.4 mM H481N or 0.8 mM H481N, and 0.4 mM D438A or 0.8 mM D438A. Alternatively, neutrophils were treated with 100 nM PMA (positive control) to induce the formation of NETs. Extracellular DNA was confirmed to be NETs by immunofluorescence microscopy by staining for DNA/H1 (green), myeloperoxidase (MPO; red), and DNA (DAPI; blue). Images are representative of one of three independent experiments with three wells per group. Scale bars = 50 µm. (B) Quantification of NETs release with the extracellular DNA stain Sytox green. The error bars show the SEM of three independent experiments with three wells per group. (B) One-way ANOVA followed by Bonferroni’s multiple comparison post hoc test, ***P < 0.001.