Characterisation of Inflammasome-Induced Extracellular Vesicles, Their Uptake by and Effect on Bystander Cells

Abstract

Inflammasomes are multimeric complexes whose activation triggers caspase 1 cleavage resulting in processing of the cytokines interleukin (IL) 1β and IL-18, and the pore-forming protein gasdermin D. This, in turn, initiates programmed inflammatory cell death (pyroptosis). However, even in the absence of IL 1β and IL 18 signalling, inflammation develops, pointing towards the importance of other factors released upon inflammasome activation, such as activated inflammasome itself or extracellular vesicles (EVs). We therefore hypothesised that EVs enhance the paracrine inflammatory effects of inflammasome-activated cells. <br /> To investigate this, we isolated EVs from inflammasome-activated cells using differential centrifugation and size-exclusion chromatography. We next analysed the ribonucleic acid (RNA) and protein content of EVs and investigated their uptake by and their effect on different bystander cells. <br /> Our results show that EV secretion is increased in macrophages stimulated with inflammasome activators relative to controls. Inflammasome-elicited EVs can be identified by the presence of N-terminal gasdermin D, as well as by their distinct RNA signatures. Thus, they have the potential to be used as biomarkers in clinical settings. <br /> EVs can exert their effects over long distances and may therefore contribute to the propagation of inflammatory signals from one part of the body to cells at distant sites. We could show that inflammasome-elicited EVs are taken up by diverse recipient cells, including macrophages, endothelial cells, epithelial cells, fibroblasts, and T cells (primarily activated T cells). We then used transcriptomics to determine the effect of inflammasome-elicited EVs on these recipient cells. They for example induced inflammatory gene sets in endothelial cells and led to the upregulation of marker genes of activated endothelium, including adhesion molecules, which are known to facilitate the attachment and tissue invasion of immune cells. Upon interaction with fibroblasts EVs have the potential to induce inflammatory signalling, further propagating inflammation. Gene sets such as interferon (IFN)-α/ γ, and tumour necrosis factor (TNF)-α signalling were enriched in these cells. <br /> Taken together, these findings indicate that EVs may not only serve as diagnostic markers for inflammatory disease, but also play an important role in the systemic response towards inflammasome activation.