Mining the Multiple Sclerosis miRNome for Disease Markers

描述（由申请人提供）：多发性硬化症（MS）是最常见的中枢神经系统（CNS）脱髓鞘疾病，导致进行性身体和认知障碍。MS被认为是一种自身免疫性疾病，因为存在活化的髓鞘碱性蛋白（MBP）特异性T细胞。为了研究多发性硬化症的免疫学异常，Racke实验室收集了健康受试者（n=15）和多发性硬化症患者（n=60）的外周血单核细胞（PBMC）。我们建议使用这个集合来定义来自健康和MS患者的T淋巴细胞的miRNA谱。mirna是调节重要的发育、分化和疾病过程的小非编码rna，但它们在MS中的作用仍未被探索。我们假设miRNA失调导致T细胞缺陷是MS发病机制的基础。具体目标多发性硬化初始CD4 T细胞miRNA特征的鉴定。这一目标将检验来自MS患者的初始CD4 T细胞具有miRNA表达基线失调的假设，这可能使它们更容易受到自我反应性免疫反应的影响。在RNA分离之前，将从全血PBMC中分选来自i)健康、ii)初治复发-缓解型多发性硬化症（RRMS）和iii)初治原发性进行性多发性硬化症（PPMS）受试者的未受刺激的初始CD4 T细胞。mirna将通过综合多重实时PCR检测和靶标鉴定。具体目标2。多发性硬化症激活记忆CD4 T细胞miRNA特征的鉴定。这一目的将验证在激活之前和之后，记忆性CD4 T细胞miRNA谱在健康和MS患者之间存在差异的假设。样本将包括静息和肉豆酸酯磷酸酯(PMA)/碘霉素激活的PBMC，分别来自1)健康、2)未治疗的RRMS和3)未治疗的PPMS受试者。如Specific Aim 1所述，在激活、RNA分离和miRNA分析之前，将对这些样本中的记忆性CD4 T细胞进行分类。这些研究将确定MS患者记忆T细胞的miRNA谱和与激活相关的变化。对初始和记忆CD4+ T细胞中miRNA的初步分析将使我们能够确定MS患者中miRNA表达的改变是否会影响介导MS发病机制的脑源性T细胞的发育，并确定潜在的MS生物标志物。初始T细胞、记忆T细胞和活化T细胞的比较将确定T细胞成熟的哪个阶段mirna可能影响ms患者的T细胞功能。尽管这项初步研究将重点关注未接受治疗的患者，但我们从大多数患者的多个时间点获得了PBMC细胞，其中许多人开始接受免疫调节治疗。因此，该数据将为评估特异性免疫调节疗法对可能与治疗反应性相关的miRNA水平正常化的影响提供基础。

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is the most prevalent, demyelinating disease of the central nervous system (CNS), causing progressive physical and cognitive disability. MS is considered an autoimmune disease, as supported by the presence of activated myelin basic protein (MBP)-specific T cells. To study the immunological abnormalities of MS, the Racke laboratory has compiled an extensive collection of peripheral blood mononuclear cells (PBMC) from healthy subjects (n=15) and MS patients (n=60). We propose to use this collection to define the miRNA profile of T lymphocytes from healthy and MS patients. miRNAs are small non-coding RNAs that regulate important developmental, differentiation and disease processes, but their role in MS remains unexplored. We hypothesize that miRNA dysregulation causes the T cell defects that underlie MS pathogenesis. Specific Aim 1. Identification of the miRNA signature of Multiple Sclerosis naive CD4 T cells. This aim will test the hypothesis that naive CD4 T cells from MS patients have a baseline dysregulation of miRNA expression, perhaps making them more susceptible to self-reactive immune responses. Unstimulated naive CD4 T cells from i) healthy, ii) treatment-naive relapsing-remitting multiple sclerosis (RRMS), and iii) treatment-naive primary progressive MS (PPMS)subjects will be sorted from whole blood PBMC prior to RNA isolation. miRNAs will be detected by a comprehensive multiplexed real-time PCR assay and targets will be identified. Specific Aim 2. Identification of the miRNA signature of Multiple Sclerosis activated memory CD4 T cells. This aim will test the hypothesis that, prior to and following activation, the memory CD4 T cell miRNA profile varies between healthy and MS patients. Samples will include resting and phorbol myristate acetate (PMA)/Ionomycin-activated PBMC from i) healthy, ii) treatment-naive RRMS, and iii) treatment-naive PPMS subjects. Memory CD4 T cells from these samples will be sorted prior to activation, RNA isolation and miRNA analysis, as described in Specific Aim 1. These studies will define the miRNA profile of MS patient memory T cells and the changes associated with activation. This initial analysis of miRNA in naive and memory CD4+ T cells will allow us to determine if altered miRNA expression in MS patients influences the development of encephalitogenic T cells that mediate MS pathogenesis and identify potential MS biomarkers. Comparison of naive, memory and activated T cells will determine at which stage of T cell maturation miRNAs may influence T cell function in MS. Although this initial study will focus on treatment-naive patients, we have PBMC cells from multiple time points from most patients, many of whom began an immunomodulatory therapy. Therefore, this data will provide a foundation for evaluating the effects of specific immunomodulatory therapies on normalizing miRNA levels that may be associated with therapy responsiveness. PUBLIC HEALTH RELEVANCE: Multiple Sclerosis (MS) is an immune-mediated disease that destroys the myelin sheath around axons, resulting in impaired nerve conduction and functional loss. Since the cause of MS is unknown, there is no cure, and current therapies are only partially affective, approximately 350,000 Americans live with MS and many will become physically handicapped during their lifetime. The current study uses a new approach to understanding the mechanism by which immune cells which are designed to protect self, actually damage self in MS. This study is designed to identify normally occurring microRNA that may be dysregulating, promoting autoimmunity. The data will enable us to identify new therapeutic targets and disease biomarkers for MS.