Figure 3.

Delivering infected endothelial exosomes to monocytes causes increase in miR-99b and miR-99b levels (A) Endothelial cells were treated with Near-Infrared BF2-azadipyrromethene (NIR-AZA) dye (red) then viewed by fluorescent microscopy at 63X. Nuclei were counterstained with DAPI (blue). (B) Exosomes were isolated from cells and delivered to THP-1 monocytes via 8-channel ibidi chambers and analysed with real-time fluorescent video microscopy. Images were captured every 5 minutes over 16 hours. Images displayed using a time-lapse across 300 minutes (top panel: Cy5 channel, bottom panel: DIC channel). Absence of fluorescence at 0 minutes indicates monocytes have not yet interacted with exosomes (arrowheads). At 300 minutes, fluorescent intensity was greatest, and staining of the plasma membrane and cytosolic components of monocytes suggests exosomes were successfully taken up (arrow). (C) Real-time PCR using standard curves was performed to determine absolute quantification of miR-99a and miR-99b levels in monocytes following delivery of exosomes obtained from healthy and infected endothelial cells. No significant difference in miR-99a/b was noted when monocytes were directly infected with S. aureus. Delivery of healthy endothelial exosomes to monocytes induced a significant increase in miR-99a expression relative to unstimulated monocytes, respectively. Delivery of infected endothelial exosomes to monocytes induced a significantly higher increase in miR-99a and miR-99b levels. The cystolic increase in both miR-99 concentrations in monocytes indicates successful exosomal delivery and uptake. Data was representative of 3 independent experiments as mean ± SEM. Statistics were performed using one-way ANOVA (*p<0.05, **p<0.01). NS, not significant.