Mesenchymal stem cell secretome in lung fibrosis: mitochondria and RNA shuttle

描述（由申请人提供）：肺纤维化是一种致死性疾病，目前尚无成功的治疗方法。实验性矽肺是研究矽肺的理想模型。在美国，每年有超过200万人接触二氧化硅。巨噬细胞在肺纤维化中起重要作用。基于干细胞的纤维化肺病治疗尚未实现。我们以前报道，全身给予骨髓间充质干细胞（MSCs）可改善实验性肺纤维化。在损伤的肺中保留的MSC的数量很少，并且与MSC通过其组织再生能力促进肺内环境稳定的假设不一致。MSC通过影响靶细胞生物学的分泌蛋白质组表现出重要的旁分泌活性，并且我们先前报道了MSC的有益作用部分地通过其限制巨噬细胞炎症的IL 1受体拮抗剂的产生来介导。现在，我们提供的数据表明，除了可溶性介质，MSC转移线粒体和挤出microRNA（miRNA）装载的外泌体作为细胞通讯的机制，以重新编程肺纤维化期间的先天免疫。 我们的中心假设是，MSC采用微泡作为一种手段，提供肽，miRNA和线粒体重新编程先天免疫和改善矽肺。我们提出以下具体目的：1）确定MSC衍生的微泡是否解释MSC对二氧化硅刺激的巨噬细胞的作用。MSC利用微囊泡的产生来建立与巨噬细胞通讯的细胞独立机制，以重新编程它们的免疫活性。我们将确定向暴露于二氧化硅的小鼠中施用MSC衍生的微泡是否与完整的MSC细胞一样有效地防止Ly 6C/Ghi巨噬细胞在肺中的积累，从而保护小鼠免受硅肺。2)目的探讨硅肺患者骨髓间充质干细胞介导的先天免疫重编程中miRNA的表达。MSC衍生的微泡含有miRNA，并且这些在不同的人MSC中高度保守。我们将确定MSC衍生的微泡内的特定miRNA转录物是否重现了微泡介导的巨噬细胞重编程，并确定抑制MSC中的miRNA产生是否消除了巨噬细胞中MSC或微泡介导的反应。将进行功能获得和丧失研究以确定选择的miRNA在调节巨噬细胞功能中的作用。3)确定自噬和外壳蛋白复合物II（COPII）作为MSC线粒体转移决定因素的作用。MSC将其线粒体转移到周围的巨噬细胞，并且在MSC衍生的微泡中存在线粒体样结构。我们将研究自噬和COPII涂层系统是否调节线粒体进入微泡的负载。我们将测试的假设，线粒体转移可以概括的巨噬细胞与MSC衍生的微泡孵育和恢复线粒体复合物I的酶活性在二氧化硅暴露的巨噬细胞。最后，我们将确定是否线粒体转移发生在二氧化硅暴露小鼠的肺。

DESCRIPTION (provided by applicant): Pulmonary fibrosis is a lethal disease for which no successful therapy is available. Experimental silicosis is an excellent model to study this disease. In the US, more than 2 million people are exposed to silica every year. Macrophages play a fundamental role in lung fibrosis. Stem cell-based therapy for fibrotic lung diseases has yet to be realized. We previously reported that systemic administration of bone marrow derived mesenchymal stem cells (MSCs) ameliorates experimental lung fibrosis. The number of MSCs retained in the injured lung is small and inconsistent with the postulate that MSCs promote lung homeostasis by their capacity for tissue regeneration. MSCs demonstrate an important paracrine activity through a secretome that influences the biology of target cells and we previously reported that beneficial effects of MSCs are mediated, in part, by their production of IL1 receptor antagonist that limits macrophage inflammation. Now we present data that in addition to soluble mediators, MSC transfer mitochondria and extrude micro RNA (miRNA) ladened exosomes as a mechanism of cell communication to reprogram the innate immunity during lung fibrosis. Our central Hypothesis is that MSCs employ microvesicles as a mean to deliver peptides, miRNAs, and mitochondria to reprogram the innate immunity and ameliorate silicosis. We propose the following specific Aims: 1) To determine whether MSC-derived microvesicles account for the effects of MSC on silica stimulated macrophages. MSC use micro vesicle production to establish a cell independent mechanism of communication with macrophages to reprogram their immune activity. We will determine whether administration of MSC-derived microvesicles into silica-exposed mice is as effective as intact MSC cells in preventing accumulation of Ly6C/Ghi macrophages in the lung thus protecting mice from silicosis. 2) To determine the role of miRNA transfer in MSC-mediated reprogramming of innate immunity during silicosis. MSC-derived microvesicles contain miRNAs and these are highly conserved among different human MSCs. We will determine whether specific miRNA transcripts inside MSC-derived microvesicles recapitulate the micro vesicle-mediated reprogramming of macrophages and determine if inhibiting miRNA production in MSCs eliminates MSC or micro vesicle-mediated responses in macrophages. Gain and loss-of-function studies will be done to define the role of select miRNAs in regulating macrophage function. 3) To define the role of autophagy and coat protein complex II (COPII) as determinants of MSC mitochondrial transfer. MSCs transfer their mitochondria to surrounding macrophages and that mitochondria like structures are present in MSC-derived microvesicles. We will examine whether autophagy and COPII coat systems regulate the load of mitochondria into microvesicles. We will test the hypothesis that mitochondrial transfer could be recapitulated by incubation of macrophages with MSC-derived microvesicles and restore the enzymatic activity of mitochondrial complex I in silica-exposed macrophages. Finally, we will determine whether transfer of mitochondria occurs in the lung of silica-exposed mice.