Dissection of cellular interactions in T1DM with integrated functional genomics

描述（由申请人提供）：认识到系统方法对复杂疾病的作用，在初始资助期间，我们开发了一个高度控制的三色阵列和分析平台，专注于获取高质量数据。我们的目标是利用生物繁殖（BB）大鼠1型糖尿病（T1DM）模型的独特衍生物，将这些工具应用于导致自身免疫的事件。BB DRlyp/lyp大鼠由于Gimap5基因突变而自发性糖尿病，使其缺乏调节性T （TREG）细胞。DR+/+大鼠具有完整的Gimap5基因，不会发生自发性T1DM，但在TREG细胞耗竭后会发生糖尿病。BB大鼠的T1DM也涉及细胞毒性T细胞，因为它们的消耗具有保护作用。我们对糖尿病前期胰淋巴结（PLN）的基因表达研究表明，DRlyp/lyp动物的肥大细胞被最终激活，而DR+/+动物的肥大细胞被负调控；我们已经通过两种不同的肥大细胞抑制药物在大鼠中预防T1DM证实了它们的功能作用。因此，我们假设除了细胞毒性T效应细胞外，肥大细胞在DRlyp/lyp糖尿病发生中起着至关重要的早期作用，尽管它们具有疾病易感性，TREG细胞足以预防DR+/+大鼠的T1DM。我们进一步假设与这些状态相关的是不同的细胞因子环境，因为我们发现，有T1DM风险或新近发病的T1DM的人类个体的血清与健康的外周血单个核细胞（PBMC）孵育可诱导一种不同于正常对照和长期存在的T1DM的特征性基因表达特征。重要的是，我们发现在T1DM发病前5年观察到这种分子特征。可预测的BB大鼠疾病病程可以纵向解剖免疫细胞亚群的活动，并检查这些活动如何在周围表现出来。因此，我们建议1)通过基因表达谱和组织学方法，在确定的发病前时间点对抗原提呈、调节和效应细胞的活动进行时间和空间解剖。2)纵向评价WF、DR+/+和DRlyp/lyp大鼠血清中诱导健康PBMCs基因表达的因素。候选介质和疾病时间点特异性生物标志物的存在将被确认。了解肥大细胞、T效应细胞和TREG细胞在自身免疫中的关系，有可能建立免疫调节的新范式，并为人类疾病创造新的见解。公共卫生相关性：许多炎症性疾病，包括1型糖尿病（T1DM），只有在组织和器官损伤已经进展后才会变得明显。本提案的目的是：1)更好地了解不同类型的免疫细胞如何以及何时进入导致T1DM的免疫级联反应；2)这些事件如何在外周表现为疾病的生物标志物。

DESCRIPTION (provided by applicant): Recognizing the power of a systems approach to complex disease, during the initial funding period we developed a highly-controlled three color array and analysis platform focused on acquisition of quality data. Our goal is apply these tools to the events leading to autoimmunity using a unique derivative of the BioBreeding (BB) rat model of type 1 diabetes mellitus (T1DM). BB DRlyp/lyp rats are spontaneously diabetic due to a mutation in the Gimap5 gene, rendering them deficient in regulatory T (TREG) cells. DR+/+ rats possess an intact Gimap5 gene and do not develop spontaneous T1DM, but become diabetic upon depletion of TREG cells. T1DM in BB rats also involves a cytotoxic T cells, since their depletion is protective. Our gene expression studies of prediabetic pancreatic lymph nodes (PLN) have shown that mast cells are terminally activated in DRlyp/lyp animals versus negatively regulated in DR+/+ animals; we have confirmed their functional role through preventing T1DM in rats with two different mast cell inhibiting drugs. Thus, we hypothesize that in addition to cytotoxic T effector cells, mast cells play a crucial early role in DRlyp/lyp diabetogenesis and in spite of their disease predisposition, TREG cells are sufficient to prevent T1DM in DR+/+ rats. We further hypothesize that associated with these states are distinct cytokine milieus, since we find incubation of sera derived from human individuals at risk for T1DM or recent onset T1DM with healthy peripheral blood mononuclear cells (PBMC) induce a characteristic gene expression signature that is distinct from normal controls and long standing T1DM. Importantly, we find observe this molecular signature as much as 5 years prior to T1DM onset. The predictable disease course of the BB rat enables longitudinal dissection of the activities of immune cell subpopulations and examining how these activities are manifested in the periphery. Thus we propose to 1) temporally and spatially dissect the activities of antigen presenting, regulatory, and effector cells at defined pre-onset timepoints through gene expression profiling and histological approaches. 2) The sera of WF, DR+/+ and DRlyp/lyp rats will be longitudinally evaluated for factors that induce gene expression in healthy PBMCs. Presence of candidate mediators and disease time point-specific biomarkers will be confirmed. Understanding the relationship between mast cells, T effector, and TREG cells in autoimmunity and has the potential to establish a new paradigm for immune regulation and create new insights to human disease. PUBLIC HEALTH RELEVANCE: Many inflammatory diseases, including type 1 diabetes mellitus (T1DM) become evident only after tissue and organ damage have already progressed. The objective of this proposal is to 1) better understand how and when different immune cell types enter into the immunological cascade that results in T1DM; and 2) how these events manifest themselves in the periphery as biomarkers of disease.