Extracellular vesicles (EV) are secretory membranous elements used by cells to transport proteins, lipids, mRNAs, and microRNAs (miRNAs). While their existence has been known for many years, only recently has research begun to identify their function in intercellular communication and gene regulation. Importantly, cells have the ability to selectively sort miRNA into EVs for secretion to nearby or distant targets. These mechanisms broadly include RNA-binding proteins such as hnRNPA2B1 and Argonaute-2, but also membranous proteins involved in EV biogenesis such as Caveolin-1 and Neural Sphingomyelinase 2. Moreover, certain disease states have also identified dysregulated EV-miRNA content, shedding light on the potential role of selective sorting in pathogenesis. These pathologies include chronic lung disease, immune response, neuroinflammation, diabetes mellitus, cancer, and heart disease. In this review, we will overview the mechanisms whereby cells selectively sort miRNA into EVs and also outline disease states where EV-miRNAs become dysregulated.

Exosomes (EXOs) are a type of extracellular nanovesicles released from living cells. Accumulating evidence suggests that EXOs are involved in the pathogenesis of human diseases, including lung conditions. In recent years, the potential of EXO-mediated drug delivery has gained increasing interest. In this report, we investigated whether inhaled EXOs serve as an efficient and practical delivery vehicle to activate or inhibit alveolar macrophages (AMs), subsequently modulating pulmonary immune responses. We first identified the recipient cells of the inhaled EXOs, which were labeled with PKH26. We found that only lung macrophages efficiently take up intratracheally instilled EXOs in vivo. Using modified calcium chloride-mediated transformation, we manipulated small RNA molecules in serum-derived EXOs, including siRNAs, microRNA (miRNA) mimics, and miRNA inhibitors. Via intratracheal instillation, we successfully delivered siRNA and miRNA mimics or inhibitors into lung macrophages using the serum-derived EXOs as vehicles. Furthermore, EXO siRNA or miRNA molecules are functional in modulating LPS-induced lung inflammation in vivo. Beneficially, serum-derived EXOs themselves do not trigger lung immune responses, adding more favorable features to serve as drug delivery agents. Collectively, we developed a novel protocol using serum-derived EXOs to deliver designated small RNA molecules into lung macrophages in vivo, potentially shedding light on future gene therapy of human lung diseases.

BackgroundUncontrolled lung inflammation is one of the prominent features in the pathogenesis of lung infection- associated acute lung injury (ALI). Microvesicles (MVs) are extracellular nanovesicles that are generated via direct membrane budding.MethodsBronchoalveolar lavage fluid (BALF) samples were collected from mice with or without intratracheal lipopolysaccharide (LPS) instillation. BALF MVs were characterised and MV-containing microRNA (miRNA) profiles were assessed and confirmed. Secretion and function of MV-containing miR-223/142 (MV-miR-223/142) were analysed in vivo.ResultsIn BALF, MVs are mainly derived from macrophages in response to LPS. After intratracheal instillation (i.t.) of LPS or Klebsiella pneumoniae, MV-containing miR-223/142 are dramatically induced in both BALF and serum. Mechanistically, miRNA 3′ end uridylation mediates the packing of miR-223/142 into MVs. To investigate the functional role of MV-miR-223/142, we loaded miR-223/142 mimics into unstimulated MVs and delivered them into the murine lungs via i.t. The miR-223/142 mimics-enriched MVs selectively targeted lung macrophages and suppressed the inflammatory lung responses that were triggered by LPS or K. pneumoniae. Mechanistically, miR-223 and miR-142 synergistically suppress Nlrp3 inflammasome activation in macrophages via inhibition of Nlrp3 and Asc, respectively.ConclusionsIn the pathogenesis of lung macrophage-mediated inflammatory responses, MV-miR-223/142 secretion is robustly enhanced and detectable in BALF and serum. Furthermore, restoration of intracellular miR-223/142 via vesicle-mediated delivery suppresses macrophage activation and lung inflammation via inhibition of Nlrp3 inflammasome activation.