Abstract Background Extracellular vesicles (EVs) are key mediators of intercellular communication, and their molecular cargo and surface properties can be profoundly influenced by external stimuli. In the context of inflammation, immune cells increase EV release to regulate immunity and metabolism. We previously demonstrated that BDE-47 alters macrophage immune responses by impairing pro-inflammatory cytokine secretion and modulating both the production and microRNA cargo of macrophage-derived sEVs, which in turn can influence the phenotype of naïve macrophages. These observations suggest that pollutant-induced alterations of EVs may represent an important mechanism regulating cell-to-cell communication and immune signaling pathways. Methods EV enriched fractions were separated from the conditioned media of BDE-47-treated THP-1 M(LPS) macrophages according to their sedimentation coefficient, and density by differential centrifugation. This procedure enables the enrichment of denser EVs at 10,000 × g (10KEVs), whereas less dense vesicles are predominantly recovered at 100,000 × g (100KEVs). The size, concentration, and surface characteristics of the 10KEVs and 100KEVs were assessed by means of NTA, DLS, Western blotting, and lectin-binding assays, while EV-associated miRNAs were profiled by microarray. Pathway enrichment analysis was conducted to identify key biological pathways altered due to BDE-47 treatment. The downstream effects of different enriched fractions of EVs (100KEVs DMSO/BDE−47 and 10KEVs DMSO/BDE−47 ) were evaluated on LNCaP cells by mean of uptake studies, BrdU incorporation and β-galactosidase senescence assays. Furthermore, transcriptional and Western Blot analyses were performed to investigate the expression of genes involved in cell cycle regulation. Results Our results show that BDE-47 does not alter EV size and surface canonical markers but profoundly reshapes their molecular identity. Specifically, we observed changes in glycan surface expression and a selective modulation of miRNA sorting in both the 100KEVs and 10KEVs enriched fractions. Bioinformatic analysis revealed a distinct BDE-47–associated EV-miRNA signature linked to the regulation of cell cycle checkpoint pathways. Functional assays performed in LNCaP cells demonstrated that EV fractions derived from BDE-47–treated macrophages (10KEVs BDE−47/DMSO and 100KEVs BDE−47/DMSO , respectively) were differentially internalized and exerted distinct biological effects on recipient cells. In particular, the two EVs BDE−47/DMSO enriched fractions differed in their capacity to be up taken by LNCaP cells, modulate cell proliferation, induce cellular senescence, and regulate the expression of key cell cycle inhibitors, including the p16 and p21 genes. Conclusions Our findings highlight EVs as central targets and mediators of pollutant-induced cellular effects, unveiling a novel mechanism by which environmental contaminants interfere with EV-mediated communication and influence the behaviour and functions of recipient cells.
Aloi et al. (Wed,) studied this question.