Fig. 1

Characterization of bacterial vesicles and response in human macrophages. A Separation of bacterial extracellular vesicles from free proteins via size exclusion chromatography from different bacterial supernatants (Legionella pneumophila (Lp), Klebsiella pneumoniae (Kp), Escherichia coli (Ec), Salmonella enterica serovar Typhimurium (Sal), and Streptococcus pneumoniae (Sp)). Vesicle concentration in each fraction was determined by nano-flow cytometry (nFCM) and proteins were quantified by BCA. B Vesicle size distributions of purified OMVs/MVs were determined by nFCM. C TEM images of purified OMVs/MVs. Scale bar = 50 nm. D–G BDMs were stimulated with OMVs/MVs (1 µg/mL each) from different bacteria or left untreated for control for up to 48 h. D CXCL8 release was determined by ELISA and is depicted in ng/mL. Expression of IL1B (E) and IL12B (F) were determined by qPCR, results are normalized to RPS18 and are depicted relative to untreated control cells. G After 1 h incubation with bacterial vesicles, expression and phosphorylation of IRAK-1, p38 and TBK-1 were determined by Western Blot. Representative results of four biological independent replicates are shown. Bars represent mean values + SEM from three (B) to four (D–F) independent experiments. Statistics: 2-way ANOVA (D-F); *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns = not significant; n = 3–4