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

描述(申请人提供)：肺纤维化是一种致命的疾病，目前还没有成功的治疗方法。实验性矽肺是研究本病的良好模型。在美国，每年有200多万人接触二氧化硅。巨噬细胞在肺纤维化中起着基础性作用。以干细胞为基础的治疗纤维化肺疾病的方法尚未实现。我们先前报道，全身应用骨髓间充质干细胞(MSCs)可改善实验性肺纤维化。MSCs滞留在受损肺中的数量很少，与MSCs通过其组织再生能力促进肺内稳态的假设不一致。MSCs通过分泌组表现出重要的旁分泌活性，影响靶细胞的生物学，我们以前报道过MSCs的有益作用部分是通过产生IL1受体拮抗剂来介导的，该拮抗剂可以限制巨噬细胞的炎症。现在我们提出的数据表明，除了可溶性介质外，MSC还可以转移线粒体并挤出携带有miRNA的外切体，作为一种细胞通讯机制，在肺纤维化过程中重新编程先天免疫。我们的中心假设是，MSCs利用微泡作为递送多肽、miRNAs和线粒体的手段来重新编程先天免疫，改善矽肺。我们提出了以下具体目标：1)确定MSC来源的微泡是否在MSC对二氧化硅刺激的巨噬细胞的影响中起作用。MSC利用微泡的产生来建立一种与巨噬细胞沟通的细胞独立机制，以重新编程其免疫活性。我们将确定将MSC来源的微囊注入二氧化硅暴露的小鼠是否与完整的MSC细胞一样有效地防止Ly6C/Ghi巨噬细胞在肺中积累，从而保护小鼠免受矽肺的侵袭。2)探讨miRNA转移在MSC介导的矽肺天然免疫重编程中的作用。MSC来源的微囊含有miRNAs，这些miRNAs在不同的人MSCs中高度保守。我们将确定MSC来源的微泡内特定的miRNA转录是否概括了微泡介导的巨噬细胞的重编程，并确定抑制MSCs中的miRNA产生是否消除了MSC或微泡介导的巨噬细胞反应。将进行功能获得和功能丧失的研究，以确定选定的miRNAs在调节巨噬细胞功能中的作用。3)明确自噬和外壳蛋白复合体II(COPII)在MSC线粒体转移中的作用。MSCs将其线粒体转移到周围的巨噬细胞，在MSC来源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。我们将验证这一假设，即通过将巨噬细胞与MSC来源的微泡孵育，可以重现线粒体转移，并恢复二氧化硅暴露的巨噬细胞中线粒体复合体I的酶活性。最后，我们将确定接触二氧化硅的小鼠肺中是否发生线粒体转移。

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.