Characterization of beta-cell-specific extracellular vesicle cargo as functional biomarkers for type I DM disease

1型糖尿病(T1D)是一种自身免疫性疾病，在美国有近200万人患有此病。 2在胰腺中产生β细胞会导致对注射胰岛素的绝对需求，从而导致重大风险 3死亡率和发病率。T1D的特征是潜伏期(无症状)，在此期间，自身免疫性或 4炎症性胰岛β细胞损伤被认为会导致胰岛细胞功能/质量下降，并最终导致 5至T1D。T1D的一个关键目标是通过限制免疫介导的损害来阻止自身免疫细胞攻击。这个 6精确的胰腺内信号机制，导致免疫系统激活和早期胰腺 7伤情尚不清楚。确定与这些关键免疫事件密切相关的标记物 8可在外周循环中检测到的T1D的发病机制将允许检测临床前 9疾病和疾病轨迹的跟踪。细胞外小泡(EVS)及其内容物已成为一种新事物 人类代谢性疾病中细胞间信号转导和功能生物标志物的10种介体。数据来自我们的 11个合作小组作为NIH EV生物学努力的一部分(NIH共同基金)建议循环细胞特异性 12电动汽车及其货物作为疾病轨迹或细胞健康的探针，比传统的 13个全血浆循环RNA或蛋白质生物标志物。最近对T1D的研究表明，胰岛β细胞 14在“压力”下产生含有自身抗原的EV和可能介导交流的RNA转录本。 15通过分子货物的转移在胰腺细胞和免疫细胞之间。然而，研究表明， 胰岛β细胞源性EVS在T1D中的16种功能格局及其作为T1D生物标志物的意义 17儿童时期的易感性，是缺乏的。为了回应RFA-DK-21-016，我们假设胰岛细胞- 18个衍生的EV是胰岛细胞生物学的功能报告者，含有与免疫调节相关的RNA小体 19反应和β细胞健康在T1D早期。在目标1中，我们利用成熟的人类细胞系统 20胰腺损伤(人细胞系和捐献的人胰岛，有或无细胞因子介导的损伤) 21以及我们小组建立的提供β-β蛋白组学和转录特征的方法 22个细胞来源的EV，并从患有和患有胰腺疾病的儿童的循环中分离出胰腺β细胞特异性EV 无T1D的23例。在目标2中，我们定义了胰岛β细胞特异性ev在先天免疫中的功能作用。 24种功能(巨噬细胞、中性粒细胞)通过评估推测为胰腺损伤的早期介质 25种免疫代谢表型和体内注射人EVS对易患糖尿病的人的反应 26模型系统。在目标3中，我们将使用rna-seq和生物信息学来鉴定胰岛β细胞特异性。 来自长期NIDDK Teddy研究的儿童的27份与T1D事件相关的文字记录(n=140，1：1 28例T1D病例/对照)，具有较高的免疫学风险。完成后，这项申请将提供广泛的 29个人β细胞来源的EVS的表型、功能和临床特征 30个与早期T1D发育相关的胰腺细胞特异性EV，满足RFA-DK-21-016的目标。

1 Type 1 diabetes (T1D) is an autoimmune disease afflicting nearly 2 million people in the U.S. The loss of insulin- 2 producing β cells in the pancreas results in an absolute requirement for injected insulin, causing significant risks 3 of mortality and morbidity. T1D is characterized by a latent (asymptomatic) phase, during which autoimmune or 4 inflammatory pancreatic beta cell injury is postulated to lead to a decline in beta cell function/mass and ultimately 5 to T1D. A key goal in T1D is halting the autoimmune cellular attack, by limiting immune-mediated damage. The 6 precise intrapancreatic signaling mechanisms that lead to activation of the immune system and early pancreatic 7 injury remain unclear. Identification of markers closely associated with these key immune events in the 8 pathogenesis of T1D that can be detected in peripheral circulation would allow for detection of pre-clinical 9 disease and tracking of disease trajectory. Extracellular vesicles (EVs) and their contents have emerged as novel 10 mediators of intercellular signaling and functional biomarkers in human metabolic diseases. Data from our 11 collaborative group as part of NIH efforts in EV biology (NIH Common Fund) suggest that circulating cell-specific 12 EVs and their cargo as probes for disease trajectory or cellular health provide greater specificity than traditional 13 circulating RNA or protein biomarkers in whole plasma. Recent studies in T1D suggest that pancreatic beta cells 14 under “stress” produce EVs containing auto-antigens and RNA transcripts that may mediate communication 15 between pancreatic and immune cells by transfer of molecular cargo. Nevertheless, studies characterizing the 16 functional landscape of pancreatic beta-cell-derived EVs in T1D, and their implications as biomarkers of T1D 17 susceptibility in childhood, are lacking. In response to RFA-DK-21-016, we hypothesize that pancreatic islet cell- 18 derived EVs are functional reporters of islet cell biology and contain RNA cargo relevant to regulation of immune 19 responses and beta cell health early in T1D. In Aim 1, we utilize well-established human cellular systems of 20 pancreatic injury (human cell-line and donated human islets, with and without cytokine-mediated injury) 21 alongside methods established by our group to provide proteomic and transcriptional characterization of beta- 22 cell-derived EVs, with isolation of pancreatic beta-cell specific EVs from human circulation in children with and 23 without T1D. In Aim 2, we define the functional role for pancreatic beta-cell specific EVs in innate immune 24 function (macrophages, neutrophils) postulated to serve as early mediators of pancreatic injury via assessments 25 of immunometabolic phenotypes and responses to in vivo administration of human EVs to a diabetes-prone 26 model system. In Aim 3, we will use RNA-seq and bioinformatics to identify pancreatic beta cell-specific 27 transcripts associated with incident T1D from children from the long-standing NIDDK TEDDY study (n=140, 1:1 28 T1D case/control) at high immunologic risk for T1D. Upon completion, this application will provide a broad 29 phenotypic, functional and clinical characterization of human beta cell derived EVs toward developing a signature 30 of pancreatic cell-specific EVs relevant to early T1D development, addressing the aims of RFA-DK-21-016.