Radiation exposure of peripheral mononuclear blood cells alters the composition and function of released extracellular vesicles

Normal tissue toxicity is a dose-limiting factor in radiation therapy. One component of normal tissue that is continuously exposed during therapeutic irradiation is the circulating population of blood mononuclear cells (PBMC). PBMCs are highly sensitive to IR, however little is known how ionizing radiation (IR) affects the PBMC response on a systemic level. Therefore it was the aim of this study to investigate whether IR was capable to induce changes in the composition and function of extracellular vesicles secreted from PBMCs after radiation exposure to different doses. Whole blood samples from healthy donors were irradiated with 0 Gy, 0,1 Gy, 2 Gy or 6 Gy X-rays and PBMC-secreted EVs were isolated 72 h later. Proteome analysis of EVs by label-free proteomics identified 606 proteins, of which 283 significantly changed their abundance after irradiation. microRNA expression analysis by small RNA sequencing revealed 379 microRNAs, of which 34 were changed after irradiation. For both, proteome and microRNA data, principal component analysis showed a dose-dependent separation of control and exposed groups. An IPA network analysis of the radiation-regulated EV proteins and microRNAs consistently predicted an association of deregulated components with apoptosis, cell death and survival. Functional studies identified endothelial cells as efficient EV recipient system, whereby irradiation of recipient cells further increased the uptake. Furthermore we detected an apoptosis suppressive effect of EVs from irradiated PBMCs in endothelial recipient cells. In summary, our study demonstrates that IR triggers the communication between PBMCs and endothelial cells. We identified EVs from irradiated PBMC donors as transmitters of protective signals to irradiated endothelial cells. Thus our data may lead to the discovery of biomarker candidates for radiation dosimetry and even more importantly, they suggest EVs a novel systemic communication pathway between irradiated and non-irradiated normal, non-cancer tissues.

正常组织毒性是放射治疗中的剂量限制性因素。治疗性照射过程中持续暴露于辐射的正常组织组分之一为外周血单核细胞（PBMC）。PBMC对电离辐射（IR）具有较高敏感性，但目前对于电离辐射如何在系统层面影响PBMC的应答机制仍知之甚少。因此本研究旨在探究不同剂量电离辐射照射后，是否可诱导PBMC分泌的细胞外囊泡（EVs）的组成与功能发生改变。本研究采集健康供者的全血样本，分别给予0 Gy、0.1 Gy、2 Gy及6 Gy X射线照射，并于照射后72小时分离PBMC分泌的EVs。通过无标记蛋白质组学对EVs进行蛋白质组分析，共鉴定出606种蛋白质，其中283种蛋白质的丰度在照射后发生显著变化。通过小RNA测序开展微小RNA（microRNA）表达分析，共检测到379种微小RNA，其中34种微小RNA的表达在照射后出现改变。针对蛋白质组与微小RNA两组数据，主成分分析（Principal Component Analysis）结果均显示对照组与照射组的样本呈现剂量依赖性分离。对受辐射调控的EV蛋白质及微小RNA进行Ingenuity通路分析（Ingenuity Pathway Analysis, IPA），结果一致预测到失调组分与细胞凋亡、细胞死亡及细胞存活存在关联。功能实验证实内皮细胞可作为高效的EV受体系统，且对受体细胞进行照射可进一步提升其对EVs的摄取能力。此外，本研究还在内皮受体细胞中检测到，受照射PBMC分泌的EVs具有抑制细胞凋亡的效应。综上，本研究证实电离辐射可触发PBMC与内皮细胞之间的信号交流；我们鉴定出受照射供者PBMC分泌的EVs可作为保护信号的载体，传递至受照射的内皮细胞。本研究数据不仅有望筛选出辐射剂量测定的潜在生物标志物，更重要的是，其揭示了EVs作为受照射与未受照射的正常非癌组织之间新型系统通讯通路的潜在价值。