Haematological Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom.
Front Immunol. 2018 Nov 9;9:2538. doi: 10.3389/fimmu.2018.02538. eCollection 2018.
Mesenchymal stromal cells (MSCs) are potent regulators of immune responses largely through paracrine signaling. MSC secreted extracellular vesicles (MSC-EVs) are increasingly recognized as the key paracrine factors responsible for the biological and therapeutic function of MSCs. We report the first comprehensive study demonstrating the immunomodulatory effect of MSC-EVs on dendritic cell (DC) maturation and function. MSC-EVs were isolated from MSC conditioned media using differential ultracentrifugation. Human monocyte-derived DCs were generated in the absence or presence of MSC-EVs (20 ug/ml) then subjected to phenotypic and functional analysis . MSC-EV treatment impaired antigen uptake by immature DCs and halted DC maturation resulting in reduced expression of the maturation and activation markers CD83, CD38, and CD80, decreased secretion of pro-inflammatory cytokines IL-6 and IL-12p70 and increased production of anti-inflammatory cytokine TGF-β. MSC-EV treated DCs also demonstrated a diminished CCR 7 expression after LPS stimulation, coupled with a significantly reduced ability to migrate toward the CCR7-ligand CCL21, although they were still able to stimulate allogeneic T cell proliferation . Through microRNA profiling we have identified 49 microRNAs, which were significantly enriched in MSC-EVs compared to their parent MSCs. MicroRNAs with known effect on DC maturation and functions, including miR-21-5p, miR-142-3p, miR-223-3p, and miR-126-3p, were detected within the top 10 most enriched miRNAs in MSC-EVs, with MiR-21-5p as the third highest expressed miRNA in MSC-EVs. analysis revealed that miR-21-5p targets the gene for degradation. To verify these observations, DCs were transfected with miR-21-5p mimics and analyzed for their ability to migrate toward the CCR7-ligand CCL21 . MiR-21-5p mimic transfected DCs showed a clear trend of reduced CCR7 expression and a significantly decreased migratory ability toward the CCL21. Our findings suggest that MSC-EVs are able to recapitulate MSC mediated DC modulation and MSC-EV enclosed microRNAs may represent a novel mechanism through which MSCs modulate DC functions. As MSCs are currently used in clinical trials to treat numerous diseases associated with immune dysregulation, such as graft-versus-host disease and inflammatory bowel disease, our data provide novel evidence to inform potential future application of MSC-EVs as a cell-free therapeutic agent.
间充质基质细胞 (MSCs) 通过旁分泌信号在很大程度上调节免疫反应。MSC 分泌的细胞外囊泡 (MSC-EVs) 越来越被认为是负责 MSC 生物学和治疗功能的关键旁分泌因子。我们报告了第一项全面研究，证明了 MSC-EVs 对树突状细胞 (DC) 成熟和功能的免疫调节作用。使用差速超速离心法从 MSC 条件培养基中分离 MSC-EVs。在不存在或存在 MSC-EV(20ug/ml)的情况下生成人单核细胞衍生的 DC，然后进行表型和功能分析。MSC-EV 处理可损害未成熟 DC 的抗原摄取，并阻止 DC 成熟，导致成熟和激活标志物 CD83、CD38 和 CD80 的表达降低，促炎细胞因子 IL-6 和 IL-12p70 的分泌减少，抗炎细胞因子 TGF-β 的产生增加。经 LPS 刺激后，MSC-EV 处理的 DC 也表现出 CCR7 表达减少，同时向 CCR7 配体 CCL21 迁移的能力显著降低，尽管它们仍能够刺激同种异体 T 细胞增殖。通过 microRNA 谱分析，我们已经确定了 49 个 microRNAs，与亲代 MSC 相比，这些 microRNAs 在 MSC-EVs 中明显富集。microRNAs 对 DC 成熟和功能有已知的影响，包括 miR-21-5p、miR-142-3p、miR-223-3p 和 miR-126-3p，在 MSC-EVs 中最富集的 top10 microRNAs 中检测到，其中 miR-21-5p 是 MSC-EVs 中表达第三高的 miRNA。 分析表明，miR-21-5p 靶向下 基因的降解。为了验证这些观察结果，将 DC 转染 miR-21-5p 模拟物，并分析其向 CCR7 配体 CCL21 迁移的能力。miR-21-5p 模拟物转染的 DC 显示 CCR7 表达明显减少，向 CCL21 迁移的能力显著降低。我们的研究结果表明，MSC-EVs 能够重现 MSC 介导的 DC 调节，并且 MSC-EV 包裹的 microRNAs 可能代表 MSCs 调节 DC 功能的一种新机制。由于 MSCs 目前正在临床试验中用于治疗与免疫失调相关的多种疾病，例如移植物抗宿主病和炎症性肠病，我们的数据为 MSC-EV 作为无细胞治疗剂的潜在未来应用提供了新的证据。
