Mesenchymal stem cell-derived exosome-educated macrophages alleviate systemic lupus erythematosus by promoting efferocytosis and recruitment of IL-17+ regulatory T cell

Extracellular Vesicles

Background Anti-inflammatory polarized macrophages are reported to alleviate systemic lupus erythematosus (SLE). Our previous studies have demonstrated that exosomes from adipose-derived stem cells promote the anti-inflammatory polarization of macrophages. However, the possible therapeutic effect of exosomes from stem cells on SLE remains unexplored. Methods Exosomes were isolated from the conditioned medium of bone marrow-derived mesenchymal stem cells using ultrafiltration and size-exclusion chromatography and were identified by nanoparticle tracking analysis and immunoblotting of exosomal-specific markers. Macrophages were collected from the MRL/lpr mouse kidney. The phenotype of macrophages was identified by immunoblotting for intracellular markers-inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1), and flow cytometry for macrophage markers F4/80, CD86, CD206, B7H4, and CD138. Pristane-induced murine lupus nephritis models were employed for in vivo study. Results When macrophages from the kidney of the MRL/lpr mice were treated with exosomes from bone marrow-derived mesenchymal stem cells (BM-MSCs), the upregulation of CD206, B7H4, CD138, Arg-1, CCL20, and anti-inflammatory cytokines was observed, which suggested that the macrophages were polarized to a specific anti-inflammatory phenotype. These anti-inflammatory macrophages produced low levels of reactive oxygen species (ROS) but had a high efferocytosis activity and promoted regulatory T (Treg) cell recruitment. Moreover, exosome injection stimulated the anti-inflammatory polarization of macrophages and increased the production of IL-17+ Treg cells in a pristane-induced murine lupus nephritis model. We observed that exosomes from BMMSCs depleted of microRNA-16 (miR-16) and microRNA-21 (miR-21) failed to downregulate PDCD4 and PTEN in macrophages, respectively, and attenuated exosome-induced anti-inflammatory polarization. Conclusion Our findings provide evidence that exosomes from BMMSCs promote the anti-inflammatory polarization of macrophages. These macrophages alleviate SLE nephritis in lupus mice by consuming apoptotic debris and inducing the recruitment of Treg cells. We identify that exosomal delivery of miR-16 and miR-21 is a significant contributor to the polarization of macrophages.

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Cigarette smoke (CS) represents one of the most relevant environmental risk factors for several chronic pathologies. Tissue damage caused by CS exposure is mediated, at least in part, by oxidative stress induced by its toxic and pro-oxidant components. Evidence demonstrates that extracellular vesicles (EVs) released by various cell types exposed to CS extract (CSE) are characterized by altered biochemical cargo and gained pathological properties. In the present study, we evaluated the content of oxidized proteins and phospholipid fatty acid profiles of EVs released by human bronchial epithelial BEAS-2B cells treated with CSE. This specific molecular characterization has hitherto not been performed. After confirmation that CSE reduces viability of BEAS-2B cells and elevates intracellular ROS levels, in a dose-dependent manner, we demonstrated that 24 h exposure at 1% CSE, a concentration that only slight modifies cell viability but increases ROS levels, was able to increase carbonylated protein levels in cells and released EVs. The release of oxidatively modified proteins via EVs might represent a mechanism used by cells to remove toxic proteins in order to avoid their intracellular overloading. Moreover, 1% CSE induced only few changes in the fatty acid asset in BEAS-2B cell membrane phospholipids, whereas several rearrangements were observed in EVs released by CSE-treated cells. The impact of changes in acyl chain composition of CSE-EVs accounted for the increased saturation levels of phospholipids, a membrane parameter that might influence EV stability, uptake and, at least in part, EV-mediated biological effects. The present in vitro study adds new information concerning the biochemical composition of CSE-related EVs, useful to predict their biological effects on target cells. Furthermore, the information regarding the presence of oxidized proteins and the specific membrane features of CSE-related EVs can be useful to define the utilization of circulating EVs as marker for diagnosing of CS-induced lung damage and/or CS-related diseases.

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