Background: The pathophysiology of sepsis and its accompanying hyper-inflammatory response are key events that lead to multi-organ failure and death. A growing body of literature now suggests that the vascular endothelium plays a critical role in driving sepsis progression. Upon bacterial infection, the endothelium releases exosomes, which harbour microRNA. A dysregulated exosomal miRNome may be a key driver of the sustained inflammatory response observed in sepsis.

Aims: We aim to identify the role of endothelial exosomes in driving a pro-inflammatory phenotype in leukocytes.

Methods: Exosomes were identified by western blot, flow cytometry and fluorescent imaging. Exosomal miRNA profiling was performed using TaqMan® Microarray Cards. The inflammatory state of monocytes was determined by cytokine analysis, inflammatory marker cytometry, and western blot.

Results: Activation of monocytes was determined by flow cytometry of CD11b. Exosomes, from healthy endothelial cells, were delivered to monocytes and failed to affect CD11b expression. Exosomes isolated from Staphylococcus aureus infected endothelial cells induced a ~2; ~4 fold increase in CD11b expression after t=24; 120 hours respectively (N=5; ±SEM; p< 0.05). These exosomes resulted in heightened production of IL-6 and reduction in IL-10 over 24 hours (N=3; ±SEM; p< 0.05). Our data suggests that exosomes contain RNA capable of influencing monocyte function. Microarray analysis of exosomal RNA identified 17 up-regulated miRNA, including the targets of interest, miR-99a/b. Bioinformatics analysis identified >200 potential miR-99a/b targets that were critically affected following infection including mTOR. Absolute quantification of miR-99a/b in monocytes revealed that these miRNA are poorly expressed (N=3; ±SEM; p=NS). Knockdown of mTOR (N=3; ±SEM; p< 0.05) using miR-99a/b mimetics resulted in an increase in IL-6 production and a decrease in IL-10 production comparable to that of exosome delivery data (N=3; ±SEM; p< 0.05).

Conclusions: Our results suggest that upon infection, endothelial cells release exosomes harbouring miRNAs capable of driving a pro-inflammatory phenotype in monocytes.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/human-vascular-endothelial-cell-derived-exosomes-contribute-to-the-excessive-inflammatory-response-observed-in-sepsis-through-a-dysregulated-microrna-expression-profile/