Stage-specific Beta Cell Response and Biomarker Profile During Virus-induced T1D

 描述(申请人提供)：T1D启动和进展的潜在机制(S)仍然难以捉摸，因为个体没有症状，在疾病的早期阶段很难识别和研究。即使生物标记物可以在疾病过程的早期识别人类，胰腺样本也不容易用来直接研究目标器官中发生的事件。为了解决这个问题，我们建议使用病毒诱导的Bbdr大鼠T1D模型。Bbdr大鼠是唯一一个在病毒感染后出现T1D的自然动物模型。它与人类T1D病理学的密切相似性，研究β细胞应激的新方法的可用性，以及纯化的β细胞的RNAseq分析和识别血浆中microRNA(MiRNA)特征的新技术，现在使系统地识别T1D早期阶段的事件成为可能。Bbdr大鼠的糖尿病发生在四个阶段：1)β细胞应激的启动和2)胰岛素炎症前期的死亡。胰腺炎(自身免疫期，3期)和T1D(4期)发生在β细胞完全破坏后。我们假设β细胞应激和死亡发生在胰岛炎症开始之前，并且反映β细胞应激的血浆生物标记物将在这一“前胰岛素炎期”疾病中被检测到。AIM 1将在T1D发育的不同阶段鉴定β细胞中的mRNA和microRNA特征。我们假设，mRNA和microRNAβ细胞表达谱反映了在T1D进展的每个阶段起作用的分子机制。我们将使用RNAseq来生成mRNA和microRNA图谱，以确定在T1D的特定阶段支持β细胞反应的分子途径。AIM 2将使用单细胞测序技术询问胰岛细胞的异质性，并识别具有不同MHC I类或胰岛素表达的β细胞的mRNA谱。我们假设，在T1D的发育过程中，β细胞对压力的反应是不同的，这可能导致在大鼠(和人类)T1D中观察到的“斑点状”岛炎和β细胞丢失。我们将使用分离的胰岛细胞的单细胞RNAseq分析来询问在糖尿病进展的特定阶段导致β细胞异质性的基因表达变化，并将mRNA签名与MHC I类或胰岛素表达相关联。AIM 3将识别特定阶段的血浆microRNA信号作为T1D生物标记物。我们假设，血浆中的miRNA图谱将提供阶段特异性信号，作为T1D进展的生物标志物。我们将使用新技术从循环血浆中回收小RNA，并在病毒诱导的Bbdr大鼠和我们的新模型柯萨奇B4感染的免疫缺陷小鼠中执行RNAseq，该模型将使我们能够识别人类miRNA签名。这些研究将使我们能够详细分析胰岛炎症和糖尿病发展之前的β细胞衰退的机制，并将它们与β细胞中正在发生的事件联系起来，从而确定疾病最早事件背后的基本因素。这将为未来的研究寻找可能易患T1D的人类miRNA生物标记物信号奠定基础。

 DESCRIPTION (provided by applicant): The mechanism(s) underlying T1D initiation and progression remain elusive as individuals are asymptomatic and difficult to identify and study during the early stages of disease. Even if biomarkers could identify humans early in the disease process, pancreas specimens are not readily available to permit direct study of the events that occur in the target organ. To address this, we propose to use the virus-inducible BBDR rat model of T1D. The BBDR rat is the only natural animal model that develops T1D following viral infection. Its close similarity with human T1D pathology, availability of new approaches for studying beta cell stress, and novel technologies for RNAseq analyses of purified beta cells and for identifying microRNA (miRNA) signatures in plasma now permit systematic identification of events underlying early stages of T1D. Diabetes in BBDR rats occurs in four stages: 1) initiation of beta cell stress and 2) death during the pre-insulitis period. Inslitis (autoimmunity, stage 3), and T1D (stage 4) occurs after complete beta cell destruction. We hypothesize that beta cell stress and death occurs prior to the initiation of insulitis and that plasma biomarkers reflecting beta cell stress will be detectable during this "pre-insulitis" stage f disease. Aim 1 will identify mRNA and microRNA signatures in beta cells at discrete stages of T1D development. We hypothesize that mRNA and microRNA beta cell expression profiles reflect the molecular mechanisms that are operational during each stage of progression of T1D. We will use RNAseq to generate mRNA and microRNA profiles to identify the molecular pathways that underlie beta cell responses at defined stages of T1D. Aim 2 will interrogate islet cell heterogeneity using single cell sequencing technology and identify mRNA profiles of beta cells with disparate MHC class I or insulin expression. We hypothesize that beta cell responses to stress during the development of T1D are heterogeneous, and that this may lead to the "spotty" insulitis and beta cell loss observed in rat (and human) T1D. We will use single cell RNAseq analyses of isolated islet cells to interrogate gene expression changes that underlie beta cell heterogeneity at defined stages of diabetes progression and correlate mRNA signatures with MHC class I or insulin expression. Aim 3 will identify stage-specific plasma microRNA signatures as T1D biomarkers. We hypothesize that the miRNA profile in the plasma will provide stage-specific signatures as biomarkers of T1D progression. We will use novel technology to recover small RNA from circulating plasma and perform RNAseq in the virus-induced BBDR rat and in our new model of Coxsackie B4-infected immunodeficient mice engrafted with human islets that will permit us to identify human miRNA signatures. These studies will permit detailed analyses of the mechanisms underlying beta cell decay prior to the development of insulitis and diabetes and associate them with ongoing events in the beta cells, leading to the identification of fundamental factors that underlie the earliest events of the disease. This will lay the foundation for future studies to look for miRNA biomarker signatures in humans that may be predisposed to T1D.