Survival and potential of insulin-deficient beta cells in type 1 diabetes

摘要/摘要 我们小组和其他人最近提出的证据表明，新发型患者的胰腺 1糖尿病(T1D)包含大量“空的”β细胞，这些细胞已经失去了分泌成熟的能力 胰岛素，但保留了其他特征，如胰岛素原的表达。其背后的机制 这些细胞的形成和它们的命运仍然鲜为人知。我们假设空的贝塔细胞， 对标准的胰岛素免疫染色是不可见的，对免疫系统也可能是不可见的，发挥着重要的作用 在T1D的病理生理学方面，同时拥有逆转糖尿病的治疗潜力。我们深入地提出了 利用MACSima超高含量免疫荧光成像鉴定空β细胞的实验 评估78个免疫和胰腺细胞标记物的原位表达，包括血管和淋巴管 自身抗体中具有炎症、外渗/转运和免疫细胞滞留特征的注释 阳性(AAB+)和T1D献血者与2型糖尿病(T2D)献血者和非糖尿病对照组的比较 可通过糖尿病胰腺器官捐赠者网络(NPOD)储存库获得。我们会比较一下 供者体内和跨供者的含胰岛素胰岛与非胰岛素胰岛的比较，以识别空的β细胞和 确定它们与胰岛、腺泡和免疫细胞表型的相关性。这些数据将用作模板 对于连续切片进行包含胰岛素的胰岛素的激光捕获显微解剖(LCM)，胰岛素阴性 胰岛和腺泡组织区域，这将受到批量RNAseq以及我们的新方法 根据DNA甲基化模式对β细胞进行量化。此外，我们还将使用一种新的小鼠模型(测试版 细胞特异性、三苯氧胺可诱导的ADAR1突变体)和培养的人胰腺切片进行功能询问 空的β细胞形成的分子途径。具体地说，我们建议利用这两个 测试候选治疗药物的模型系统--包括胰岛素模拟物(GLP1)、内质网 (ER)应激抑制剂(ISRIB)，以及针对特定免疫亚群的多种生物制品--因为它们有能力 调节空的β细胞，它们的胰岛素含量和胰岛素分泌。然后，我们将关联这些功能数据 通过MACSima、RNAseq和DNA获得激素阴性胰岛细胞的分子和细胞特征 甲基化。预计这些研究将对一个基本但鲜为人知的过程有深入的了解 放置在人类的T1D中。我们预计空的β细胞可以重新功能化，为治疗铺平道路。 在T1D恢复内源性β细胞功能的开发。归根结底，当与有效的 关于限制自身免疫的干预措施，我们希望这里探讨的代谢模式可以提供 一种在T1D发作后显著减少胰岛素需求甚至实现胰岛素独立的方法 改善这些患者的寿命和生活质量。

SUMMARY / ABSTRACT Recent evidence put forth by our group and others suggests that the pancreas of patients with recent-onset type 1 diabetes (T1D) contains a significant number of “empty” beta cells that have lost the ability to secrete mature insulin but retained other hallmark features such as proinsulin expression. The mechanisms that underlie formation of such cells and their fate remain poorly understood. We hypothesize that empty beta cells, which are invisible to standard insulin immunostaining and potentially also to the immune system, play important roles in the pathophysiology of T1D while holding therapeutic potential for reversion of diabetes. We propose in depth experiments to characterize empty beta cells, using MACSima ultra high-content immunofluorescence imaging to assess in situ expression of 78 immune and pancreas cell markers, including vascular and lymphatic annotation with signatures of inflammation, extravasation/trafficking, and immune cell residency in autoantibody positive (AAb+) and T1D donors as compared to donors with type 2 diabetes (T2D) and non-diabetic controls available through the Network for Pancreatic Organ donors with Diabetes (nPOD) repository. We will compare insulin containing versus insulin negative islets, within and across donors, to identify empty beta cells and determine how they correlate with islet, acinar, and immune cell phenotypes. These data will serve as a template for a serial section to undergo laser capture microdissection (LCM) of insulin containing islets, insulin negative islets, and acinar tissue regions, which will be subjected to bulk RNAseq as well as our novel method for quantifying beta cells based on DNA methylation patterns. In addition, we will use a novel mouse model (beta cell-specific, tamoxifen-inducible Adar1-mutant) and cultured human pancreas slices to functionally interrogate molecular pathways underlying the formation of empty beta cells. Specifically, we propose to leverage these two model systems to test therapeutic candidates— including an incretin mimetic (GLP1), an endoplasmic reticulum (ER) stress inhibitor (ISRIB), and multiple biologics targeting specific immune subsets— for their ability to modulate empty beta cells, their insulin content and insulin secretion. We will then correlate these functional data with molecular and cellular features of hormone negative islet cells via MACSima, RNAseq, and DNA methylation. These studies are expected to yield insights into a fundamental yet little understood process taking place in human T1D. We anticipate that empty beta cells can be re-functionalized, paving the way for therapeutic development to restore endogenous beta cell function in T1D. Ultimately, when combined with effective interventions to constrain autoimmunity, it is our hope that the metabolic modalities explored here could provide a means to reduce insulin requirements or even achieve insulin independence after T1D onset, dramatically improving longevity and quality of life for these patients.