Figure 7
Effects of silica nanoparticles on bacterial clearance and AM inflammatory responses induced by P.a . Mice primary AMs (105 cells/well) from BALs were treated ex-vivo with FITC-SiO2 (A) or unlabelled SiO2 NPs (B and C) for 5 h at 2.5 μg/cm2 (0.8 μg/well) in culture media (RPMI) (200 μl). In selected experiments, FITC-SiO2 treated cells were fixed and cytoskeleton and nuclei stained with phalloidin–tetramethylrhodamine (red) and DAPI (blue), respectively. Fluorescence images were collected from observation on Apotome microscope (A). After incubation with NPs, supernatants were removed and cells (105 cells/well) challenged with PAK at MOI 0.1 (B left panel) or MOI 0.5 (B right panel and C). After 4 h, supernatants were collected and cells were lysed in 0,1% triton X-100, conditions that allow bacterial growth. Bacterial CFU were counted in cell lysates and supernatants (CFU super + cell lysate) as shown in B. Concentration of cytokines were measured in cells supernatants (200 μl) from untreated cells (RPMI/RPMI), cells treated with SiO2 NPs (SiO2/RPMI), cells challenged with bacteria (RPMI/PAK) and cells exposed to SiO2 NPs and then challenged with bacteria (SiO2/PAK). Means ± SEM of triplicates from three independent pools of alveolar macrophages are represented. Experiments were repeated twice. (n.d.) = not detectable.