Investigating type 1 diabetes pathogenesis using the live pancreas tissue slice platform

使用活体胰腺组织切片平台研究 1 型糖尿病发病机制

• 批准号: 10620116
• 负责人: Mollie K Huber
• 金额: $ 4.19万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-16 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10620116
• 关键词: Academic Medical Centers; Adoptive Transfer; Age Years; Antigens; Autoantibodies; Autoimmune Diseases; Autoimmune Responses; Autoimmunity; Beta Cell; C-Peptide; Cell Communication; Cell Death; Cell Survival; Cell physiology; Cells; Cessation of life; Complement; Confocal Microscopy; Coupled; Data; Development; Diabetes Mellitus; Diagnosis; Disease; Disease Progression; Environment; Enzyme-Linked Immunosorbent Assay; Event; Functional disorder; Future; Gene Expression; Generations; Glucose; Goals; Human; Immune; Immune mediated destruction; Immune system; In Situ; Inbred NOD Mice; Incidence; Individual; Infiltration; Insulin; Insulin-Dependent Diabetes Mellitus; Investigation; Kinetics; MHC Class I Genes; Mediating; Mitochondria; Modeling; Monitor; Mus; Onset of illness; Organ Donor; Pancreas; Pathogenesis; Pathology; Pathway interactions; Patients; Pharmaceutical Preparations; Play; Population; Principal Investigator; Process; Production; Research; Research Personnel; Role; Sampling; Scientist; Slice; Splenocyte; Stress; Structure of beta Cell of islet; T-Lymphocyte; Techniques; Testing; Time; Tissue Donors; Tissues; Training; United States; Work; autoreactive T cell; clinical diagnosis; diabetes pathogenesis; experimental study; glucose metabolism; glucose tolerance; human tissue; immune cell infiltrate; inflammatory milieu; insulin dependent diabetes mellitus onset; insulitis; islet; mouse model; non-diabetic; preservation; prevent; protein expression; response; success; therapeutic target; therapy design; timeline

Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of the insulin-producing β cells of the pancreas. The timeline of T1D development and progression is heterogenous with diagnosis occuring at a few years of age in some patients versus other patients having autoantibodies for years before diagnosis. In T1D, immune dysregulation occurs leading to autoantibody production, immune cell infiltration into the islets (insulitis), and eventual loss of β cells. The events that occur within the β cell during this time and the resulting impacts on β cell function remain unknown. The goal of this proposal is to determine how β cells function during the course of T1D development, particularly during the critical early, asymptomatic stages. Understanding these functional changes within β cells or islets is critical for a complete understanding of disease progression and for the identification of possible therapeutic targets. The overall objective of my proposal is to use live pancreas tissue slices to explore how β cells function during T1D development, specifically I will identify the role immune cell infiltration plays in β cell dysfunction and loss. Live pancreas tissue slices are ideal for these studies because these samples keep the islet in its native microenvironment and preserve the extant tissue pathologies, allowing for studies of both β cell function and immune cell populations. I hypothesize that immune dysregulation early in T1D progression results in dysfunction of the β cell glucose metabolism pathway primarily mediated by mitochondrial stress, resulting in MHC class I hyperexpression and increased β cell visibility to the immune system. To test this hypothesis, live pancreas tissue slices generated from human organ donor tissue will be used and complemented by slices made from pancreata of mouse models of T1D. Confocal microscopy techniques will be used to determine the degree of insulitis within the tissue while islet function is simultaneously assessed. Additional functional assessments such as perifusion experiments followed by insulin ELISAs and gene expression will be employed to look at function in greater detail. The first part of this proposal focuses on assessments conducted to determine the impacts of in situ immune cells on β cell function. To gain more control over the disease timeline, particularly to investigate the earliest stages of disease, HLA-matched T cell avatars will be introduced to control human pancreas tissue slices, creating an insulitic environment. Disease initiation and progression will be studied further through adoptive transfer experiments with splenocytes from NOD mouse models. Progression to T1D will be monitored with live pancreas tissue slices being made as disease develops. Functionality assessments and immune cell studies will be conducted as discussed above. I expect the contribution of the proposed research will be the identification of the role that immune dysregulation plays in β cell dysfunction as well as the mechanistic origination of β cell dysfunction. This will provide critical information about how T1D develops and will indicate possible therapeutic targets to halt disease progression.

1型糖尿病（T1D）是一种自身免疫性疾病，导致产生胰岛素的β细胞破坏 胰腺。 T1D发展和进展的时间表是异质的，诊断发生在 与其他患者相比，一些患者在诊断前几年都有几岁的患者。在 T1D，免疫失调会导致自身抗体产生，免疫细胞浸润到胰岛 （胰岛炎），最终丧失β细胞。在此期间发生的β细胞内发生的事件以及结果 对β细胞功能的影响仍然未知。该提案的目的是确定β细胞的功能 在T1D开发过程中，特别是在关键的早期无症状阶段。 了解β细胞或胰岛内的这些功能变化对于完全理解 疾病进展和鉴定可能的治疗靶标。我的总体目标 建议是使用活胰腺组织切片来探索β细胞在T1D发育过程中的作用， 具体而言，我将确定免疫细胞浸润在β细胞功能障碍和损失中的作用。现场胰腺 组织切片是这些研究的理想选择，因为这些样品将胰岛保存在其天然微环境中 并保留额外的组织病理，从而可以研究β细胞功能和免疫细胞 人群。我假设在T1D进展的早期免疫失调导致β功能障碍 细胞葡萄糖代谢途径主要由线粒体应力介导的主要介导，导致MHC I类 过度X级和β细胞对免疫系统的可见性提高。为了检验这一假设，现场胰腺 由人体器官捐赠组织产生的组织切片将由由由 T1D小鼠模型的胰腺。共聚焦显微镜技术将用于确定程度 简单地评估组织中的胰岛炎，同时胰岛功能。其他功能评估 例如，将胰岛素ELISA和基因表达进行促进实验，以查看 更详细地发挥作用。该提案的第一部分着重于确定的评估 原位免疫细胞对β细胞功能的影响。为了获得对疾病时间表的更多控制，尤其是 研究疾病的最早阶段，HLA匹配的T细胞化身将被引入以控制人 胰组织切片，创造了胰岛环境。疾病倡议和进展将研究 进一步通过来自NOD小鼠模型的脾细胞的自适应转移实验。发展为T1D 将通过将活的胰腺组织切片作为疾病发展来监测。功能评估 并将进行免疫细胞研究，如上所述。我希望该提议的贡献 研究将是免疫失调在β细胞功能障碍以及 β细胞功能障碍的机械起源。这将提供有关T1D如何发展的关键信息 并将表明可能阻止疾病进展的治疗靶点。