Co-registration of Cell Organization, Phenotype and Function in the Human Pancreas During Type 1 Diabetes

我们小组和其他人最近提出的证据表明，1型糖尿病(T1D)的发病机制涉及一种 免疫性、胰岛和腺泡状胰腺缺陷症。除了自身免疫和β细胞死亡外，它还有 很明显，T1D的特点是整个器官的病理，胰腺体积缩小，缩小 血清中的外分泌酶水平，并改变α和β细胞的功能，包括胰岛素处理障碍，甚至 在胰岛自身抗体阳性(AAB)的T1D前状态下。因此，有必要了解其中的每一个 将细胞表型和功能联系在一起的方面，以及对人类胰腺组织的研究 微环境，在整个T1D进程中。我们假设β细胞状态的改变及其 周围环境是β细胞功能受损、外分泌功能受损和渗透的关键决定因素 (绝缘炎)。我们建议使用我们的新的胰腺切片培养平台来分析胰岛和腺泡组织的功能 (目标1a)测试(PRO)激素(胰岛素原、胰岛素、胰高血糖素)和酶(脂肪酶、胰蛋白酶原)的分泌 T1D、AAB和对照器官供体胰腺对已建立的内分泌(葡萄糖、精氨酸、氯化钾)的反应 和外分泌刺激(缩胆囊素、卡巴胆碱)。我们将把这些功能数据与分子特征联系起来 通过单细胞RNA测序和基于抗体的CITEseq(转录本细胞索引) 和scATACseq(用于转座酶可及染色质测序的单细胞测序法)；这对于 细胞鉴定的目的以及转录组和表观基因组分析(目标1b)。胰腺切片 也会受到同样的刺激条件，以进行钙信号活动的活细胞成像 在胰岛和腺泡组织区域内实时定位(目标2a)，然后通过成像质谱仪进行固定和分析 (IMC)。我们将用细胞分辨率(AIM)评估120个免疫和胰腺细胞标记物的原位表达 2b)，然后对IMC数据进行空间和时间分析，以确定胰岛、免疫和腺泡细胞如何 表型与组织和细胞功能相关，使用我们的组织CAT(组织形态结构细胞术 分析工具箱)。这将使从连续切片进行三维重建的计算分析成为可能(目标2c)。 最后，在目标3中，人类供体胰腺切片将受到糖尿病刺激(炎性细胞因子， 糖毒性)和针对β细胞应激的干预[伊马替尼(酪氨酸激酶抑制剂)，msl-7(自噬)] 增强剂)、埃塞那肽(GLP-1受体激动剂)]，并通过单细胞和IMC图谱进行类似的评估。使用 14年以上通过网络采购移植质量的人胰腺的经验 作为糖尿病患者(NPOD)的胰腺器官捐献者，我们准备进行这项拟议的研究。我们 期望识别连接β细胞、整个胰岛和腺泡细胞的改变的分子途径和组织特征 AAB和T1D胰腺的细胞功能表型。我们预计这些同样的缺陷将出现在 受到糖尿病刺激的切片，提供了一个测试已知和新的靶向候选对象的平台 干预以减少β细胞应激，恢复胰岛和腺泡细胞功能，以及防止疾病进展。

Recent evidence put forth by our group and others suggests type 1 diabetes (T1D) pathogenesis involves a combination of immune, islet, and acinar pancreas defects. In addition to autoimmunity and β-cell death, it has become clear that T1D is characterized by a whole-organ pathology with reduced pancreas size, reduced exocrine enzyme levels in serum, and altered α- and β-cell function, including impaired insulin processing, even in the islet autoantibody positive (AAb+) pre-T1D condition. Hence, there is a need to understand each of these facets in concert, linking cellular phenotype and function, together with studies of the human pancreas tissue microenvironment, throughout T1D progression. We hypothesize that alterations to β-cell status and its surrounding environment are key determinants of impaired β-cell function, exocrine function, and infiltration (insulitis). We propose to assay islet and acinar tissue function using our novel pancreas slice culture platform (Aim 1a) to test (pro)hormone (proinsulin, insulin, glucagon) and enzyme (lipase, trypsinogen) secretion from T1D, AAb+, and control organ donor pancreata in response to established endocrine (glucose, arginine, KCl) and exocrine stimuli (cholecystokinin, carbachol). We will correlate these functional data with molecular features via scRNAseq (single cell RNA sequencing) with antibody-based CITEseq (Cellular Indexing of Transcriptomes and Epitopes) and scATACseq (single cell assay for transposase-accessible chromatin sequencing); this, for the purpose of cell identification together with transcriptomic and epigenomic analyses (Aims 1b). Pancreas slices will also be subjected to these same stimulatory conditions for live cellular imaging of Ca2+ signalling activity within islet and acinar tissue areas in real time (Aim 2a), then fixed and analyzed by imaging mass cytometry (IMC). We will assess in situ expression of 120 immune and pancreas cell markers with cellular resolution (Aim 2b), followed by spatial and temporal analysis of IMC data to determine how islet, immune and acinar cell phenotypes correlate with tissue and cellular function, using our histoCAT (histology topography cytometry analysis toolbox). This will enable computational analysis with 3D reconstruction from serial sections (Aim 2c). Finally, in Aim 3, human donor pancreas slices will be subjected to diabetogenic stimuli (inflammatory cytokines, glucotoxicity) and interventions targeting β-cell stress [imatinib (tyrosine kinase inhibitor), MSL-7 (autophagy enhancer), exenatide (GLP-1 receptor agonist)], and similarly evaluated by single cell and IMC profiling. With over 14 years of experience in procurement of transplant-quality human pancreata through the Network for Pancreatic Organ donors with Diabetes (nPOD), we are uniquely poised to perform the proposed studies. We expect to identify altered molecular pathways and tissue features linking β-cell, whole-islet, and acinar cell phenotypes with cellular function in AAb+ and T1D pancreata. We anticipate these same defects will arise in slices subjected to diabetogenic stimuli, providing a platform to test known and novel candidates for targeted intervention to reduce β-cell stress, restore islet and acinar cell function, as well as prevent disease progression.