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.
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.