Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This research is support by the European Research Council (ERC) consolidator grant EVICARE (ERC-2016-COG-725229). Background Repair of damaged heart tissue upon myocardial infarction remains a major challenge. Transplantation of cardiac progenitor cells (CPCs) has been studied as a potential regenerative therapy, but recent studies have shown that the cardioprotective effect of CPCs is mediated by the release of extracellular vesicles (EVs). The benefits of CPC-derived EVs have mostly been associated with stimulation of angiogenesis and inhibition of cell death. Although macrophages have been suggested to be key for cardiac repair, the effect of CPC-EVs on macrophage polarization is poorly explored. Purpose Here, we hypothesized that CPC-derived EVs can modulate the inflammatory response after myocardial infarction by interacting with macrophages. Methods EVs were isolated from serum-starved CPCs by ultrafiltration followed by size exclusion chromatography. Human monocytes were isolated from the blood of healthy donors and differentiated into macrophages with M-CSF. Macrophages were subsequently stimulated with LPS + IFNy or with IL4 in order to induce an inflammatory M1 and reparative M2 phenotype, respectively. A third group of macrophages were cultured in media without LPS, IFNy or IL4 to resemble naive M0 macrophages. The obtained macrophages were exposed to CPC-EVs and analyzed by flow cytometry, bulk RNA sequencing and confocal microscopy to assess macrophage phenotype changes. Gene candidates were selected based on the p-value and fold-change, and validated by RT-PCR. Small molecule inhibitors were used to further investigate the mechanism by which CPC-EVs are taken up by macrophages and the pathways involved in macrophage response. Results Stimulation of macrophages with CPC-EVs enhanced the expression of the pro-inflammatory marker CD80, while slightly decreasing the anti-inflammatory marker CD206, in M0 and M2 macrophages. CPC-EV-stimulated macrophages also adopted a morphology that reflects the inflammatory macrophage. In line with these findings, bulk RNA sequencing on M0 and M2 polarized macrophages revealed upregulation of genes involved in inflammatory response, including cytokine and both type I and II interferon signaling. EV exposure did not significantly affect gene expression in M1 polarized macrophages. Ongoing investigations will provide in-depth insight into the mechanism by which CPC-EVs induce this response in macrophages. Conclusion Our data suggests that CPC-EVs are able to induce macrophage polarization towards an inflammatory phenotype, which might have implications for CPC-EV treatment after myocardial infarction. This underlines an urgent need to understand the molecular mechanisms underlying the immunomodulatory effect of CPC-derived EVs before moving into a clinical setting.

中文翻译：

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心脏祖细胞衍生的细胞外囊泡使人类巨噬细胞倾向于促炎表型

资金致谢 资金来源类型：公共拨款——欧盟资金。主要资金来源：这项研究得到了欧洲研究委员会 (ERC) 整合资助 EVICARE (ERC-2016-COG-725229) 的支持。背景心肌梗塞时受损心脏组织的修复仍然是一项重大挑战。心脏祖细胞 (CPCs) 的移植已被研究为一种潜在的再生疗法，但最近的研究表明，CPCs 的心脏保护作用是由细胞外囊泡 (EVs) 的释放介导的。CPC 衍生的 EV 的好处主要与刺激血管生成和抑制细胞死亡有关。尽管巨噬细胞被认为是心脏修复的关键，但 CPC-EV 对巨噬细胞极化的影响却鲜有研究。目的在这里，我们假设 CPC 衍生的 EV 可以通过与巨噬细胞相互作用来调节心肌梗死后的炎症反应。方法通过超滤和尺寸排阻色谱法从血清饥饿的 CPC 中分离 EV。从健康供体的血液中分离出人单核细胞，并用 M-CSF 分化成巨噬细胞。随后用 LPS + IFNγ 或用 IL4 刺激巨噬细胞，以分别诱导炎症性 M1 和修复性 M2 表型。第三组巨噬细胞在不含 LPS、IFNγ 或 IL4 的培养基中培养，类似于幼稚 M0 巨噬细胞。将获得的巨噬细胞暴露于 CPC-EV，并通过流式细胞术、大量 RNA 测序和共聚焦显微镜进行分析，以评估巨噬细胞表型的变化。根据 p 值和倍数变化选择基因候选者，并通过 RT-PCR 验证。小分子抑制剂用于进一步研究 CPC-EVs 被巨噬细胞摄取的机制以及参与巨噬细胞反应的途径。结果 CPC-EVs 刺激巨噬细胞增强了 M0 和 M2 巨噬细胞中促炎标志物 CD80 的表达，同时略微降低了抗炎标志物 CD206。CPC-EV 刺激的巨噬细胞也采用了反映炎性巨噬细胞的形态。与这些发现一致，对 M0 和 M2 极化巨噬细胞的大量 RNA 测序揭示了参与炎症反应的基因的上调，包括细胞因子以及 I 型和 II 型干扰素信号传导。EV 暴露没有显着影响 M1 极化巨噬细胞中的基因表达。正在进行的研究将深入了解 CPC-EV 在巨噬细胞中诱导这种反应的机制。结论我们的数据表明，CPC-EVs 能够诱导巨噬细胞极化向炎症表型，这可能对心肌梗死后的 CPC-EV 治疗有影响。这强调了在进入临床环境之前迫切需要了解 CPC 衍生 EV 的免疫调节作用的分子机制。