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

1 型糖尿病期间人类胰腺细胞组织、表型和功能的共同注册

基本信息

批准号：
10343979
负责人：
MARK A. ATKINSON
金额：
$ 53.3万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-17 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10343979
关键词：
3-Dimensional Acinar Cell Address Age Agonist Alpha Cell Amylases Antibodies Area Arginine Autoantibodies Autoimmunity Autophagocytosis Beta Cell Biological Assay Blood Vessels Carbachol Caring Cell Death Cell physiology Cells Cellular Assay Cellular Stress Cellular Structures Characteristics Cholecystokinin Chromatin Computer Analysis Cytometry Data Defect Detection Deterioration Development Diabetes Mellitus Diabetes autoantibodies Disease Disease Progression Endocrine Enhancers Environment Enzymes Epigenetic Process Epitopes Exocrine pancreas Extravasation Failure Functional disorder GLP-I receptor Glucagon Glucose Histology Hormones Human Image Imatinib Immune Impairment In Situ Individual Infiltration Inflammation Inflammatory Insulin Insulin-Dependent Diabetes Mellitus Intervention Islet Cell Islets of Langerhans Laboratories Link Lipase Location Lymphatic Measures Messenger RNA Metabolic Pathway Methodology Molecular Molecular Profiling Natural History Oligonucleotides Organ Organ Donor Pancreas Pathology Pathway interactions Persons Phenotype Physiological Principal Investigator Production Proinsulin Proteins Proteomics Recovery Residencies Resolution Role Series Serum Signal Pathway Signal Transduction Slice Stimulus Structure Structure of beta Cell of islet System Techniques Testing Therapeutic Intervention Time Tissue-Specific Gene Expression Tissues Transplantation Transposase Trypsin Trypsinogen Tumor-infiltrating immune cells Tyrosine Kinase Inhibitor antibody conjugate base cell type cellular imaging cytokine diabetes pathogenesis diabetogenic endoplasmic reticulum stress epigenomics exenatide experience indexing innovation insight insulitis islet islet cell antibody live cell imaging non-diabetic novel prevent programs reconstruction response senescence single-cell RNA sequencing targeted imaging therapeutic candidate therapeutic target tool trafficking transcriptome transcriptomics

项目摘要

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.

我们小组和其他人提出的最新证据表明 1 型糖尿病 (T1D) 发病机制涉及除了自身免疫和 β 细胞死亡之外，它还具有免疫、胰岛和腺泡胰腺缺陷的组合。明确 T1D 的特点是全器官病理学，包括胰腺尺寸减小、血清中的外分泌酶水平，以及 α 和 β 细胞功能的改变，包括胰岛素加工受损，甚至因此，有必要了解其中每一项。协调一致的各个方面，将细胞表型和功能与人类胰腺组织的研究结合起来在整个 T1D 进展过程中，我们勇敢地面对 β 细胞状态及其变化。周围环境是β细胞功能、外分泌功能和浸润受损的关键决定因素（胰岛炎）。我们建议使用我们的新型胰腺切片培养平台测定胰岛和腺泡组织功能。（目标 1a）测试（原）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确定的内分泌（葡萄糖、精氨酸、KCl）的反应我们将这些功能数据与分子特征相关联。通过 scRNAseq（单细胞 RNA 测序）和基于抗体的 CITEseq（转录组细胞索引）和表位）和 scATACseq（转座酶可访问染色质测序的单细胞测定）；细胞鉴定以及转录组和表观基因组分析的目的（目标 1b）。也将受到这些相同的刺激条件进行 Ca2+ 信号活动的活细胞成像实时在胰岛和腺泡组织区域内进行检测（目标 2a），然后通过成像质谱流式细胞仪进行固定和分析 (IMC)。我们将通过细胞分辨率评估 120 种免疫和胰腺细胞标记物的原位表达（Aim） 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使用我们的 histoCAT（组织学拓扑细胞计数法），表型与组织和细胞功能相关分析工具箱）。这将能够通过连续切片进行 3D 重建进行计算分析（目标 2c）。最后，在目标 3 中，人类供体胰腺切片将受到致糖尿病的刺激（炎症细胞因子、糖毒性）和针对 β 细胞应激的干预措施 [伊马替尼（酪氨酸激酶抑制剂）、MSL-7（自噬增强剂）、艾塞那肽（GLP-1 受体激动剂）]，并通过单细胞和 IMC 分析进行类似的评估。在通过网络采购移植质量的人类胰腺方面拥有超过 14 年的经验对于患有糖尿病 (nPOD) 的胰腺器官捐献者，我们有独特的准备来进行拟议的研究。期望识别连接 β 细胞、全胰岛和腺泡细胞的改变的分子途径和组织特征 AAb+ 和 T1D 胰腺中具有细胞功能的表型我们预计也会出现这些相同的缺陷。受到糖尿病刺激的切片，提供了一个平台来测试已知和新颖的候选药物的目标干预以减少β细胞应激、恢复胰岛和腺泡细胞功能以及预防疾病进展。