Figure 4.

Tiam1 is required for NETs release and killing of bacteria by neutrophils. (A) NETs. Wild type (grey symbols) and Tiam1^–/– (blue symbols) neutrophils were allowed to adhere to ibidi μ-slides for 30 min before the addition of serum-opsonised S. aureus (ratio 10 bacteria per neutrophil), or mock-treatment. 15 min before a timepoint, Sytox Green and Hoechst 33342 were added, and cells were live-imaged. Representative images from one experiment are shown. White squares denote the magnified areas. White arrows highlight dead cells, red arrows NETs. Quantification by image analysis; data are mean ± SEM of 3 independent experiments. Statistics are two-way ANOVA with Sidak’s multiple comparison corrections on log-transformed raw data. (B) Killing of bacteria. Wild type and Tiam1^–/– neutrophils were primed with 50 ng/ml GM-CSF, 20 ng/ml TNFα for 45 min either in suspension or while adhering to ICAM1-coated coverslips, as indicated. Serum-opsonised S. aureus (ratio 1 bacteria per neutrophil) were added for 30 or 90 min, before samples were permeabilised with saponin, plated on LB-agar, and CFU of surviving bacteria enumerated. Samples with heat-killed neutrophils were processed in parallel to determine the efficacy of killing. Samples containing bacteria but no neutrophils (black symbols) were used as an additional control (right-hand panel). Data are mean ± SEM of 3 independent experiments. Statistics are two-way ANOVA with Sidak’s multiple comparisons corrections.