L. pneumophila OMVs alter bacterial replication and increase the vacuole amount per cell.

(A) THP-1 cells were pre-incubated for 20 h with different doses of OMVs (0.1, 1, 10 μg/mL) and were then infected with L. pneumophila (multiplicity of infection (MOI) of 0.5). Pre-stimulation with LPS (200 ng/mL) in combination with IFN-γ (200 ng/mL) served as a positive control for classically activated macrophages (M1). Bacterial replication after OMV pre-stimulation was determined by CFU assay. Cells were lysed after the indicated time points. Results are depicted relative to CFU count of not pre-treated (but infected) cells at every time point. (B) THP-1 cells were pre-treated with OMVs (1 μg/mL) for 20 h or left untreated before infection with L. pneumophila (MOI 0.5) for 48 h. Cells were fixed and stained with an α-LPS antibody (green), phalloidin (red), and DAPI (blue). One representative out of three independent experiments is shown. (C) Quantification of the number of vacuoles per cells of more than 70 cells per condition. Data are shown as mean values of three independent experiments. (D) Quantification of the distribution of vacuoles per cells. Mean values of three independent experiments are shown. Statistics: Mann-Whitney test; *p<0.05 compared to 0 μg/mL OMVs.