Immune stimuli shape the small non-coding transcriptome of extracellular vesicles released by dendritic cells. Immune stimuli shape the small non-coding transcriptome of extracellular vesicles released by dendritic cells

PURPOSE: The molecular composition of extracellular vesicles (EV) is regulated by cellular activation and differentiation stimuli, thereby affecting their signaling function to target cells. While activation-associated changes in EV-derived microRNAs (miRNA) have been intensively studied, it is unknown whether cell stimulation affects other classes of small non-coding RNA in EV. METHODS: RNA isolated from primary immune cells that were made tolerogenic with VitD3, activated by LPS, or left untreated (control), and RNA isolated from EV purified from the supernatant of these dendritic cells were analyzed by RNA seq (n = 3 biological replicates) RESULTS: Prominent changes were found in the miRNA, snoRNA and Y-RNA content of EV from differentially stimulated dendritic cells, whereas tRNA and snRNA levels were much less affected. Comparing changes in EV-RNA with changes in the corresponding cells, we found that EV-RNA levels only partly reflected changes in cellular RNA. CONCLUSIONS: Our results implicate that the EV-transcriptome not necessarily predicts RNA levels in EV-producing cells. The acquired knowledge can guide discovery and understanding of non-coding RNA types beyond miRNAs as functional entities in EV and as EV-RNA-based biomarkers. Overall design: SmallRNA isolated from n = 3 biological replicates of cells (3 conditions: LPS, VitD3, control) and corresponding EV (3 conditions: LPS, VitD3, control). Libraries were prepared with NebNext smallRNA kit for Illumina, 150 bp paired end reads on Illumina HiSeq 4000 sequencer