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: 10490416
• 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/10490416
• 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; 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 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

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）发病机理涉及 免疫，胰岛和腺泡胰腺缺陷的组合。除了自身免疫性和β细胞死亡之外，它还具有 清楚地表明，T1D的特征是胰腺大小降低的全器官病理学的特征 血清中的外分泌酶水平以及改变α-和β细胞功能，包括胰岛素加工受损，甚至 在胰岛自身抗体（AAB+）前T1D条件中。因此，有必要理解这些 一致的方面，将细胞表型和功能联系起来，以及人类胰腺组织的研究 微环境，整个T1D进展。我们假设对β细胞状态及其的改变 周围环境是β细胞功能受损，外分泌功能和浸润的关键决定者 （胰岛炎）。我们建议使用我们的新胰岛切片培养平台测定胰岛和腺泡组织功能 （AIM 1A）从 T1D，AAB+和对照器官供体胰腺响应已建立的内分泌（葡萄糖，精氨酸，KCL） 和外分泌刺激（胆囊动蛋白，卡尔巴乔尔）。我们将将这些功能数据与分子特征相关联 通过基于抗体的CITESEQ（转录组的细胞索引）通过SCRNASEQ（单细胞RNA测序） 和表位）和scatacseq（用于转座酶可访问的染色质测序的单细胞测定）；这是 细胞识别的目的以及转录组和表观基因组分析（AIMS 1B）。胰腺切片 还将遵循这些相同的刺激条件，以进行CA2+信号活性的活细胞成像 实时内部和腺泡组织区域内（AIM 2A），然后通过成像质量细胞术进行固定并分析 （IMC）。我们将评估具有细胞分辨率的120个免疫和胰腺细胞标记物的原位表达（AIM 2B），然后对IMC数据进行空间和临时分析，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，使用我们的组织学（组织学地形术） 分析工具箱）。这将通过串行部分的3D重建（AIM 2C）进行计算分析。 最后，在AIM 3中，人类供体胰腺切片将受到糖尿病刺激（炎性细胞因子，，，， 葡萄毒性）和靶向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），艾烯肽（GLP-1受体激动剂）]，并通过单细胞和IMC分析进行了类似的评估。和 超过14年的通过网络采购移植质量的人类胰腺的经验 胰腺器官供体（NPOD），我们被毒死了，可以进行拟议的研究。我们 期望识别有联系的分子途径和组织特征连接β细胞，全islet和acinar细胞的特征 AAB+和T1D胰腺中具有细胞功能的表型。我们预计这些相同的缺陷将会出现 经受糖尿病性刺激的切片，为有针对性的候选者提供了一个测试已知和新颖候选者的平台 干预措施以减少β细胞应力，恢复胰岛和腺泡细胞功能，并防止疾病进展。