Figure 4.

Infected endothelial exosomes promote a proinflammatory phenotype in monocytes following uptake (A) Expression of proinflammatory markers CD11b and MHCII were analysed on the surface of monocytes by flow cytometry after 24 hours. Data is represented as bar graphs using geometric mean taken from baseline negative control. Significant increases in CD11b and MHCII levels were observed in monocytes that had taken up infected exosomes. (B) Basal levels of IL-6 (35 pg/mL) and IL-10 (4300 pg/mL) were obtained in unstimulated THP-1 monocytes by ELISA. Direct S. aureus infection had the greatest influence on IL-6 and IL-10 production from monocytes. Healthy endothelial exosomes had no effect on cytokine expression compared to unstimulated monocytes. Exosomes from S. aureus infected endothelial cells induced a significant increase in IL-6 and a negatively correlating decrease in IL-10 production in monocytes. (C) Western blot and densitometry analyses of mTOR levels (200 kDa) in monocytes following delivery of healthy or infected exosomes after 24 hours. GAPDH (37 kDa) used as loading control. Positive control (monocytes + S. aureus) showed greatest mTOR protein expression. Significantly elevated mTOR levels were observed in monocytes following uptake of S. aureus infected endothelial exosomes. Data is represented as mean ± SEM (n=3 CD11b; n=4 MHCII; *p<0.05; **p<0.01, ***p<0.001). NS, not significant.