树突状细胞外泌体激活T细胞改善心梗后心功能

Johnny

JMCC:树突状细胞外泌体激活T细胞改善心梗后心功能

CD4+ T细胞的活化在促进心肌梗死（MI）后伤口愈合的过程中起关键作用。树突细胞（dendritic cells，DCs）分泌的外泌体（Exosomes，EXs）在肿瘤模型中激活T细胞；然而，树突细胞来源的外泌体（dendritic cells derived exosomes，DEXs）是否可通过CD4+ T细胞的活化促进心肌梗死（MI）后伤口愈合仍然不清楚。这项研究旨在确定DEXs是否介导的CD4+ T细胞活化，改善小鼠心梗后的心功能。作者使用缺氧的原代或坏死的HL-1心肌细胞的上清液模拟体外梗死后心肌细胞的微环境。用正常（对照组）、缺氧或坏死的HL-1心肌细胞（MI组）的上清液处理小鼠骨髓来源的DCs，以及未刺激（阴性组）。然后从BMDC上清中分离DEXs，孵育CD4+ T细胞或用于小鼠尾静脉注射。在这项研究中，研究者发现，无论是缺氧还是坏死的HL-1心肌细胞的上清均可上调DC成熟的相关基因。注射DEXs后，大量的MI-DEXs被代谢到脾脏，且比对照组和阴性组更快速。共聚焦成像和流式细胞术显示，相比对照组和阴性组-DEXs，MI-DEXs会更容易被脾CD4+ T细胞获取；并且体外与体内结果都显示脾CD4+ T细胞摄取MI-DEXs会表达更多的趋化因子和炎症因子IFN-γ和TNF。此外，注射MI-DEXs改善小鼠MI后的心功能。这些结果表明，DEXs可通过一种内分泌机制介导的CD4+ T细胞的活化，改善MI后心功能。该发现为通过全身递送DEXs治疗MI的新策略提供了基础。

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该研究的设计流程如上图。首先分离培养小鼠骨髓来源的DCs，然后用低氧处理和坏死的HL-1细胞的细胞上清处理DCs，然后分离该DCs培养上清中的DEXs，然后用DEXs分别于体外、体内处理心梗后的小鼠和培养的脾CD4+ T细胞，检测小鼠心功能改善情况，和T细胞对DEXs的摄取情况和基因表达的改变。

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首先对分离得到的外泌体进行了鉴定，分别做了电镜检测和western检测。

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尾静脉注射外泌体后进行活体成像，结果显示三组并无差异。

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接着检测肝脏的外泌体摄取情况，也并无差异。

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再检测脾脏的外泌体摄取情况，发现脾脏对MI-DEXs的摄取较对照组和阴性组快且多。

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通过PKH67标记外泌体检测处理CD4+ T细胞和尾静脉注射24h后的摄取情况。

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接着，通过流式定量检测CD4+ T细胞对三组DEXs摄取的情况，结果显示MI-DEXs被摄取得最多。

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Con-DEXs和MI-DEXs处理CD4+ T细胞后均可促进MIP-1α、MIP-1β、MIP-1、IL-2的分泌，而仅MI-DEXs处理CD4+ T细胞后促进趋化因子和炎症因子IFN-γ和TNF的分泌。

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尾静脉注射外泌体后检测脾CD4+ T细胞的趋化因子和炎症因子表达情况。结果显示MI-DEXs处理促进MIP-1α、MIP-1β、MIP-1、IFN-γ和TNF的表达。

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最后分析，尾静脉注射外泌体后对小鼠心梗后心功能的改善情况。结果显示，注射MI-DEXs组在射血分数、左室短轴缩短率和左室后壁厚度3项心功能指标又明显改善作用。

总结，除了已经报道的胚胎干细胞、血浆、间充质干细胞、心血管前体细胞和成纤维细胞的外泌体对心梗具有保护作用，该研究发现树突状细胞（缺氧或坏死的HL-1心肌细胞培养上清预处理）的外泌体也具有心梗保护作用，补充了外泌体治疗心梗的细胞来源，为全身递送外泌体治疗心梗的新策略提供了新的实验基础。

参考文献：

Haibo, L., et al. (2015). "Exosomes derived fromdendritic cells improve cardiac function via activation of CD4 T lymphocytesafter myocardial infarction." J Mol Cell Cardiol.    IF=4.655

            CD4+ T cell activation plays a key role in facilitatingwound healing after myocardial infarction (MI). Exosomes (EXs) secreted fromdendritic cells (DCs) can activate T cells in tumor models; however, whetherDEXs (DC-EXs) can mediate CD4+ T cell activation and improve wound healingpost-MI remains unknown. This study sought to determine whether DEXs mediateCD4+ T cell activation and improve cardiac function post-MI in mice. We usedsupernatants of hypoxic primary or necrotic HL-1 cardiomyocytes to simulate thepost-MI cardiomyocyte microenvironment in vitro. Cultured bone marrow-derivedDCs (BMDCs) from mice were stimulated with the supernatants of normal (Controlgroup), hypoxic primary or necrotic HL-1 cardiomyocytes (MI group); a subset ofBMDCs remained unstimulated (Negative group). DEXs were then isolated from theBMDC supernatants and either incubated with CD4+ T cells or injected into micevia the tail vein. In this study, we found that the supernatants of bothhypoxic primary and necrotic HL-1 cardiomyocytes upregulate DC maturationmarkers. After the injection of DEXs, a greater number of MI-DEXs are recruitedby the mouse spleen and with greater rapidity than control- or negative-DEXs.Confocal imaging and flow cytometry revealed that MI-DEXs exhibited higheruptake by splenic CD4+ T cells than the control- and negative-DEXs, and thisincrease was correlated with significantly greater increases in the expressionof chemokines and the inflammatory cytokines IFN-gamma and TNF by the CD4+ Tcells in vitro and in vivo. In addition, the injection of MI-DEXs improvedcardiac function in mice post-MI. These results suggest that DEXs could mediatethe activation of CD4+ T cells through an endocrine mechanism and improvecardiac function post-MI. Our findings provide the basis for a novel strategyfor the treatment of MI through the systemic delivery of DEXs.