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Background <p>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.</p> Methods <p>EV enriched fractions were separated from the conditioned media of BDE-47-treated THP-1&#xa0;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<sup>DMSO/BDE−47</sup> and 10KEVs<sup>DMSO/BDE−47</sup>) 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.</p> Results <p>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<sup>BDE−47/DMSO</sup> and 100KEVs<sup>BDE−47/DMSO</sup>, respectively) were differentially internalized and exerted distinct biological effects on recipient cells. In particular, the two EVs<sup>BDE−47/DMSO</sup> 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.</p> Conclusions <p>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.</p>

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Environmental pollutant BDE-47 alters glycan and microRNA signatures of macrophage-derived extracellular vesicles and modulates senescence signaling

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月6日

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  • Noemi Aloi,
  • Alessia Maria Sampino,
  • Alessia Li Vigni,
  • Ilaria Cosentini,
  • Giuseppa Augello,
  • Fabrizio Chiodo,
  • Gaspare Drago,
  • Silvia Ruggieri,
  • Daniele Romancino,
  • Sabrina Picciotto,
  • Giorgia Adamo,
  • Samuele Raccosta,
  • Mauro Manno,
  • Antonella Bongiovanni,
  • Paolo Colombo,
  • Valeria Longo

摘要

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 (100KEVsDMSO/BDE−47 and 10KEVsDMSO/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 (10KEVsBDE−47/DMSO and 100KEVsBDE−47/DMSO, respectively) were differentially internalized and exerted distinct biological effects on recipient cells. In particular, the two EVsBDE−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.