No abstract

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.

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Active Na(+) transport mediated by epithelial Na(+) channel (ENaC) is vital for fetal lung fluid reabsorption at birth and pulmonary edema resolution. Previously, we demonstrated that αENaC expression and activity are downregulated in alveolar epithelial cells by cycloheximide (Chx) and Pseudomonas aeruginosa. The regulatory mechanisms of αENaC mRNA expression by Chx and lipopolysaccharide (LPS) from P. aeruginosa were further studied in the present work. Both agents decreased αENaC mRNA expression to 50% of control values after 4 h. Chx repressed αENaC expression in a dose-dependent manner independently of protein synthesis. Although extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways were activated by the two treatments, their mechanisms of ENaC mRNA modulation were different. First, activation of the signaling pathways was sustained by Chx but only transiently by LPS. Second, ERK1/2 or p38 MAPK inhibition attenuated the effects of Chx on αENaC mRNA, whereas suppression of both signaling pathways was necessary to alleviate the outcome of LPS on αENaC mRNA. The molecular mechanisms involved in the decrease of αENaC expression were investigated in both conditions. LPS, but not Chx, significantly reduced αENaC promoter activity via the ERK1/2 and p38 MAPK pathways. These results suggest that LPS attenuates αENaC mRNA expression via diminution of transcription, whereas Chx could trigger some posttranscriptional mechanisms. Although LPS and Chx downregulate αENaC mRNA expression similarly and with similar signaling pathways, the mechanisms modulating ENaC expression are different depending on the nature of the cellular stress.