Figure 7. TLR9 participates in OMV-dependent B cell activation.

(A) B cell proliferation was measured by means of [methyl-³H]-thymidine incorporation after 96 h. OMV from the MID-expressing M. catarrhalis wild type (OMV) were combined with different additives as indicated in the figure. OMV isolated from M. catarrhalis growing in presence of DNase (OMV DNase culture) and OMV treated with DNase after isolation (OMV DNase-treated) were also included. The viability of B lymphocytes after each treatment was measured by trypan blue exclusion staining. Error bars indicate SEM from 5 different donors. The presence of DNA associated with OMV from DNase treated bacterial culture (B) or untreated culture (C) were analyzed by TEM using gold labelling anti-DNA pAb. (D) The presence of DNA associated with OMV was confirmed by PCR. DNA extractions from OMV DNase-treated or untreated samples were used as template for amplification of the genomic 16S rRNA gene. M. catarrhalis genomic DNA was used as a positive control and an ultracentrifuge supernatant (free of bacteria and OMV) from DNase treated and untreated cultures were also analyzed to check the presence of extracellular DNA. Representative images from three independent experiments are shown. * p≤0.05, OMV DNase-treated versus OMV and OMV DNase-treated + genomic DNA versus OMV DNase-treated; ** p≤0.01, OMV versus unstimulated cells.