Implications of Changes in Islet Exosomal Cargo in Type 1 Diabetes

项目摘要 细胞外囊泡（EV）是膜结合的纳米颗粒，可以与其他细胞相互作用 as a means手段of cell细胞：cell细胞communication通信.新出现的数据表明，β细胞来源的外泌体， EV亚型由多泡体的胞吐作用释放，可能在 健康岛考虑到外泌体在β细胞通讯中发挥作用的潜力， 胰岛微环境中的细胞周围，迫切需要进行深入研究 生理和病理生理条件下β细胞外泌体生物学的定义机制 情节跨膜免疫检查点蛋白死亡配体的β细胞表达 1（PD-L1）在支持1型糖尿病（T1 D）中β细胞存活方面起着关键作用。初步数据 提示β细胞外泌体膜携带PD-L1，且外泌体PD-L1被上调 通过细胞因子处理亲本β细胞。我们的中心假设是T1 D中的应激β细胞 微环境改变其外泌体内容物，包括保护机制，旨在 避免与自身免疫相关的β细胞破坏。目标1将阐明 改变的β细胞外泌体PD-L1货物的病因学。我们假设促炎干扰素 信号传导激活细胞内β细胞PD-L1的分子调节因子， 外泌体生物发生的介质，增加总外泌体PD-L1。化学和遗传 操作将用于测试这些途径对β细胞外泌体PD-L1的影响。目的2 将检验以下假设：通过转移到周围的β细胞和T细胞或与周围的β细胞和T细胞相互作用， 细胞外泌体PD-L1负载是保护β细胞免受自身免疫的保护机制 杀伤性从人诱导多能干细胞系分化的辟田胞的用途 过表达GFP标记的PD-L1将允许直接追踪PD-L1转移和结合至 其他β细胞和CD 8 + T细胞。Aim 3将采用一种新的微流体装置来测试 假设PD-L1+外泌体释放将在患有T1 D或有T1 D风险的人中增加。 我们将量化人血浆或胰腺中总EV PD-L1和胰岛衍生EV PD-L1的差异 将培养基切片并与非糖尿病对照比较。还将在来自 具有或不具有残留可检测C肽的长期T1 D的个体，以确定是否 血浆胰岛来源的EV PD-L1与功能性β细胞存活相关。这项工作将导致 该领域对β细胞与周围细胞通讯的理解发生了范式转变， 确定胰岛来源的PD-L1外泌体货物作为治疗靶向的临床潜力 β细胞存活率或生物标志物来剖析T1 D疾病相关异质性。

PROJECT SUMMARY Extracellular vesicles (EVs) are membrane bound nanoparticles that can interact with other cells as a means of cell:cell communication. Emerging data suggest that β cell-derived exosomes, an EV subtype released by exocytosis of multivesicular bodies, may act as paracrine effectors in islet health. Given the potential for exosomes to play a role in β cell communication with surrounding cells in the islet microenvironment, a critical need exists for deliberate research defining mechanisms of β cell exosome biology under physiologic and pathophysiologic circumstances. β cell expression of the transmembrane immune checkpoint protein death-ligand 1 (PD-L1) plays a key role to support β cell survival in type 1 diabetes (T1D). Preliminary data suggest that β cell exosome membranes carry PD-L1, and that exosomal PD-L1 is upregulated by cytokine treatment of parent β cells. Our central hypothesis is that stressed β cells in the T1D microenvironment alter their exosomal contents to include protective mechanisms aimed at evading β cell destruction associated with autoimmunity. Aim 1 will elucidate the mechanistic etiology of altered β cell exosome PD-L1 cargo. We hypothesize that proinflammatory interferon signaling activates molecular regulators of intracellular β cell PD-L1 that, in concert with mediators of exosome biogenesis, increase total exosomal PD-L1. Chemical and genetic manipulation will be used to test impacts of these pathways on β cell exosomal PD-L1. Aim 2 will test the hypothesis that via transfer to or interaction with surrounding β cells and T cells, β cell exosomal PD-L1 loading is a protective mechanism shielding β cells from autoimmune destruction. Use of β cells differentiated from a human induced pluripotent stem cell line overexpressing GFP-tagged PD-L1 will allow for direct tracking of PD-L1 transfer and binding to other β cells and CD8+ T cells. Aim 3 will employ a novel microfluidic device to test the hypothesis that PD-L1+ exosome release will be increased in humans with or at risk for T1D. We will quantify differences in total and islet-derived EV PD-L1 in human plasma or pancreas slice media and compare to nondiabetic controls. Testing will also be performed in plasma from individuals with longstanding T1D with or without residual detectable C-peptide to determine if plasma islet-derived EV PD-L1 is linked to functional β cell survival. This work will lead to a paradigm shift in the field’s understanding of β cell communication with surrounding cells, and determine the clinical potential of islet-derived PD-L1 exosome cargo as a therapeutic targeting β cell survival or a biomarker to dissect T1D disease-related heterogeneity.