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

描述（由申请人提供）：肺纤维化是一种致命疾病，目前尚无成功的治疗方法。实验矽肺是研究该疾病的良好模型。在美国，每年有超过200万人接触到二氧化硅。巨噬细胞在肺纤维化中起着重要作用。基于干细胞的肺纤维化疾病治疗尚未实现。我们之前报道了骨髓间充质干细胞（MSCs）的全身管理改善实验性肺纤维化。损伤肺中保留的间充质干细胞数量很少，这与间充质干细胞通过其组织再生能力促进肺稳态的假设不一致。MSCs通过影响靶细胞生物学的分泌组表现出重要的旁分泌活性，我们之前报道过MSCs的有益作用部分是通过其产生il - 1受体拮抗剂来调节的，该拮抗剂限制了巨噬细胞的炎症。现在我们提供的数据表明，除了可溶性介质外，间充质干细胞转移线粒体和挤压携带微RNA （miRNA）的外泌体作为细胞通讯机制，在肺纤维化期间重编程先天免疫。我们的中心假设是MSCs利用微泡作为传递肽、mirna和线粒体的手段来重编程先天免疫并改善矽肺。我们提出以下具体目的：1)确定MSC衍生的微泡是否解释了MSC对二氧化硅刺激的巨噬细胞的影响。间充质干细胞利用微泡的产生与巨噬细胞建立细胞独立的通讯机制，以重编程巨噬细胞的免疫活性。我们将确定在暴露于二氧化硅的小鼠中给予MSC来源的微泡是否与完整的MSC细胞一样有效，以防止肺中Ly6C/Ghi巨噬细胞的积累，从而保护小鼠免受矽肺病。2)确定miRNA转移在msc介导的矽肺先天免疫重编程中的作用。骨髓间充质干细胞衍生的微泡含有microrna，这些microrna在不同的人骨髓间充质干细胞中高度保守。我们将确定MSC来源的微泡内的特异性miRNA转录物是否再现了微泡介导的巨噬细胞重编程，并确定抑制MSCs中miRNA的产生是否会消除巨噬细胞中MSC或微泡介导的反应。将进行功能增益和功能丧失研究，以确定选定的mirna在调节巨噬细胞功能中的作用。3)定义自噬和外壳蛋白复合物II （COPII）作为MSC线粒体转移的决定因素的作用。间充质干细胞将其线粒体转移到周围的巨噬细胞，线粒体样结构存在于间充质干细胞衍生的微泡中。我们将研究自噬和COPII外壳系统是否调节线粒体进入微泡的负荷。我们将验证线粒体转移可以通过巨噬细胞与msc衍生的微泡孵育来重现的假设，并恢复二氧化硅暴露的巨噬细胞中线粒体复合物I的酶活性。最后，我们将确定线粒体转移是否发生在二氧化硅暴露小鼠的肺部。

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.