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

• 批准号: 10673726
• 负责人: MARK A. ATKINSON
• 金额: $ 50.68万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 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/10673726
• 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; Characteristics; Cholecystokinin; Chromatin; Computer Analysis; Cytometry; Data; Defect; Detection; Deterioration; Development; Diabetes Mellitus; Disease; Disease Progression; Dissociation; 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; Maps; 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 of beta Cell of islet; System; Techniques; Testing; Therapeutic Intervention; Time; Tissue-Specific Gene Expression; Tissues; Transplantation; Transposase; Trypsin; Trypsinogen; Tyrosine Kinase Inhibitor; antibody conjugate; cell type; cytokine; diabetes pathogenesis; diabetogenic; endoplasmic reticulum stress; epigenomic profiling; epigenomics; exenatide; experience; immune cell infiltrate; 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

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（T1 D）的发病机制涉及一种免疫抑制剂。 免疫、胰岛和腺泡胰腺缺陷的组合。除了自身免疫和β细胞死亡外， 变得清楚的是，T1 D的特征在于具有胰腺大小减小、胰腺体积减小和胰腺体积减小的全器官病理学。 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 在胰岛自身抗体阳性（AAb+）pre-T1 D条件下。因此，有必要了解其中的每一个 方面的协调一致，连接细胞表型和功能，以及人类胰腺组织的研究 微环境，整个T1 D进展。我们假设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来检测胰岛和腺泡组织功能 (Aim 1a）测试来自以下的（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）分泌： T1 D、AAb+和对照器官供体胰腺对既定内分泌（葡萄糖、精氨酸、KCl）的反应 和外分泌刺激（胆囊收缩素、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scRNAseq（单细胞RNA测序）和基于抗体的CITEseq（转录组的细胞索引） 和表位）和scATACseq（用于转座酶可及染色质测序的单细胞测定）;这对于 细胞鉴定以及转录组学和表观基因组学分析的目的（目的1b）。胰腺切片 也将经受这些相同的刺激条件，用于Ca 2+信号传导活性的活细胞成像 在胰岛和腺泡组织区域内以真实的时间（Aim 2a），然后固定并通过成像质谱细胞术分析 （IMC）。我们将评估120种免疫和胰腺细胞标记物的原位表达，并进行细胞分辨率（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 表型与组织和细胞功能相关，使用我们的histoCAT（组织学拓扑细胞仪 分析工具箱）。这将使计算分析与三维重建从连续切片（目标2c）。 最后，在目标3中，人供体胰腺切片将经受致糖尿病刺激（炎性细胞因子， 葡萄糖毒性）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）、依塞那肽（GLP-1受体激动剂）]，并通过单细胞和IMC谱进行类似评估。与 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， 糖尿病胰腺器官供体（nPOD），我们是唯一准备进行拟议的研究。我们 期望鉴定改变的分子通路和连接β细胞、全胰岛和腺泡细胞的组织特征 表型与AAb+和T1 D胰腺中的细胞功能。我们预计这些同样的缺陷将出现在 切片进行致糖尿病的刺激，提供了一个平台，以测试已知的和新的候选人， 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。