Apoptotic endothelial cells release small extracellular vesicles loaded with immunostimulatory viral-like RNAs

Endothelial cells have multifaceted interactions with the immune system, both as initiators and targets of immune responses. In vivo, apoptotic endothelial cells release two types of extracellular vesicles upon caspase-3 activation: apoptotic bodies and exosome-like nanovesicles (ApoExos). Only ApoExos are immunogenic: their injection causes inflammation and autoimmunity in mice. Based on deep sequencing of total RNA, we report that apoptotic bodies and ApoExos are loaded with divergent RNA cargos that are not released by healthy endothelial cells. Apoptotic bodies, like endothelial cells, contain mainly ribosomal RNA whereas ApoExos essentially contain non-ribosomal non-coding RNAs. Endogenous retroelements, bearing viral-like features, represented half of total ApoExos RNA content. ApoExos also contained several copies of unedited Alu repeats and large amounts of non-coding RNAs with a demonstrated role in autoimmunity such as U1 RNA and Y RNA. Moreover, ApoExos RNAs had a unique nucleotide composition and secondary structure characterized by strong enrichment in U-rich motifs and unstably folded RNAs. Globally, ApoExos were therefore loaded with RNAs that can stimulate a variety of RIG-I-like receptors and endosomal TLRs. Hence, apoptotic endothelial cells selectively sort in ApoExos a diversified repertoire of immunostimulatory "self RNAs" that are tailor-made for initiation of innate immune responses and autoimmunity.