Role of dysregulated miRNA in Tregs in Multiple Sclerosis

描述（由申请人提供）：多发性硬化症（MS）是一种免疫介导的中枢神经系统（CNS）脱髓鞘疾病，可导致严重的神经功能缺损。该疾病的原因尚不清楚，对该疾病病理生理学的免疫和神经退行性机制了解甚少，目前的治疗方法在减缓疾病进展方面仅部分有效。我的实验室对未经治疗的MS患者的幼稚和效应/记忆CD 4 T细胞进行了大规模的miRNA分析研究，以确定miRNA是否可能导致疾病易感性或进展。在MS患者中，TGF β信号通路被预测为被鉴定为在CD 4 T细胞中失调的12种miRNA抑制。TGF β在CD 4 + CD 25 hiFoxp 3+调节性T细胞（TCL 3）中起着至关重要的作用，TCL 3在MS患者中有缺陷。TGF β信号转导已被证明是关键的生产iTGF 3，并维持抑制功能和Foxp 3表达在TGF 3。我们假设MS患者的CD 4 T细胞中失调的miRNA表达导致TGF β信号传导受损和CD 4调节性T细胞缺陷，促进MS的发作和进展。目的1：确定MS患者的CD 4 T细胞中鉴定为失调的miRNA是否直接靶向TGF β信号传导途径中的特异性分子。首先，我们将确定MS患者和健康对照的人CD 4 T细胞中特异性miRNA和预测的TGF β靶点之间的基因表达水平是否存在相关性。其次，我们将使用荧光素酶测定来确定特定的miRNA是否抑制TGF β途径中预测的靶基因的3' UTR序列。 目的2：确定靶向TGF β通路的miRNA是否改变iTreg发育和/或TGF β的抑制功能。靶向TGF β的miRNA将在初始CD 4 T细胞中被抑制或过表达，然后在iTreg条件下培养。所得T细胞将针对iTreg特征进行表型分型。此外，将用特异性miRNA抑制剂转染TGFm，评估其对TGF β的敏感性和抑制效应T细胞的能力。 目标3：确定TGF β-相关的miRNA表达水平与体内TGFm数量之间是否存在相关性，或TGF β-相关的miRNA表达与MS患者TGFm抑制能力之间是否存在相关性。MS患者外周血CD 4 T细胞的流式细胞术分析将评估MS患者中TGFP的数量相对于TGF β-相关miRNA的表达。此外，将从MS患者的外周血中分选TGFP，评估其抑制能力，并确定TGFP相关的miRNA与Treg数量和/或功能之间是否存在相关性。 考虑到TcG在维持自身耐受性方面的作用以及观察到MS患者中CD 4 TcG存在缺陷，这项新研究将确定CD 4 T细胞中的miRNA表达失调是否是MS患者中Treg缺陷的基础。尽管一些研究已经鉴定了在Tregs中发挥作用的miRNA 17 -22，但没有研究鉴定出MS患者中与TG通路中的基因特异性相关的差异表达的miRNA及其对自身免疫性疾病中的TG的影响。Treg细胞在保护我们免受环境影响和保护我们免受自身免疫系统影响的微妙平衡中发挥着重要作用，而TGF？具有超越T细胞的多效性作用，对健康和疾病具有巨大的重要性，使其成为一项非常宝贵的研究。 公共卫生相关性：多发性硬化症（MS）是年轻人非创伤性残疾的主要原因，在美国影响超过40万人。该疾病的原因尚不清楚，病理学基础的机制知之甚少，治疗仅部分有效，需要创新策略来了解疾病和易感因素。在拟议的研究中，我们将研究miRNAs在MS患者中已被证明有缺陷的调节性T细胞的发育和功能中的作用，这可能会确定疾病易感性的基本机制。

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) which can result in severe neurological deficits. The cause of the disease is unknown, the immune and neurodegenerative mechanisms underlying the pathophysiology of the disease are poorly understood, and current therapies are only partially effective at slowing disease progression. My laboratory has performed a large miRNA profiling study on naive and effector/memory CD4 T cell in untreated MS patients to determine if miRNA may contribute to disease susceptibility or progression. The TGF¿ signaling pathway was predicted to be suppressed in MS patients by 12 miRNAs identified as being dysregulated in CD4 T cells. TGF¿ has shown to play a vital role in CD4+CD25hiFoxp3+ regulatory T cells (Tregs), and Tregs have been shown to be defective in MS patients. TGF¿ signaling has been shown to be critical for production of iTregs, and the maintenance of suppressor functions and Foxp3 expression in Tregs. We hypothesize that dysregulated miRNA expression in CD4 T cells in MS patients' results in impaired TGF¿ signaling and defective CD4 regulatory T cells, promoting the onset and progression of MS. Aim 1: To determine if miRNAs identified as dysregulated in CD4 T cells in MS patients directly target specific molecules in the TGF ¿ signaling pathway. First, we will determine if there is a correlation in gene expression levels between specific miRNAs and predicted TGF¿ targets in human CD4 T cells from MS patients and healthy controls. Second, we will use luciferase assays to determine if specific miRNAs suppress the 3' UTR sequence of the predicted target genes in the TGF¿ pathway. Aim 2: To determine if the miRNAs that target the TGF¿ pathway alter iTreg development and/or suppressive functions of Tregs. TGF¿ targeting miRNAs will be inhibited or over-expressed in naive CD4 T cells and then cultured under iTreg conditions. The resulting T cells will be phenotyped for iTreg characteristics. In addition, Tregs will be transfected with specific miRNA inhibitors, evaluated for sensitivity to TGF¿, and ability to suppress effector T cells. Aim 3: To determine if there is an association between the level of TGF¿-associated miRNA expression and the number of Tregs in vivo, or an association between TGF¿-associated miRNA expression and suppressive capacity of Tregs in MS patients. Flow cytometric analysis of peripheral blood CD4 T cells from MS patients will evaluate the number of Tregs in MS patients relative to the expression of TGF¿-associated miRNAs. In addition, Tregs will be sorted from peripheral blood of MS patients, evaluated for suppressive capacity, and determine if there is a correlation between TGF¿-associated miRNAs and Treg number and/or function. Given the well-documented role of Tregs in maintaining tolerance to self and the observation that CD4 Tregs are defective in MS patients, this novel study will determine if dysregulated miRNA expression in CD4 T cells underlies this Treg defect in MS patients. Although several studies have identified miRNAs that play a role in Tregs17-22, no studies have identified differentially expressed miRNAs in MS patients that are specifically associated with genes in the TG¿ pathway and their influence on Tregs in an autoimmune disease. Treg cells play an important role in the delicate balance of protection from our environment and protecting ourselves from our own immune system, and TGF¿ has pleiotrophic effects that extend beyond Tregs that have vast importance in health and disease, making this an invaluable study. PUBLIC HEALTH RELEVANCE: Multiple sclerosis (MS) is the leading cause of non-traumatic disability of young adults, affecting over 400,000 people in the USA. The cause of the disease is unknown, the mechanisms that underlie the pathology are poorly understood and treatments are only partially effective, necessitating innovative strategies to understand the disease and susceptibility factors. In the proposed study we will study the role of miRNAs in the development and function of regulatory T cells which have been shown to be defective in MS patients, which may identify a fundamental mechanism underlying disease susceptibility.