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: 10388460
• 负责人: Mollie K Huber
• 金额: $ 4.02万
• 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 2025-05-15
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10388460
• 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; TimeLine; Tissue Donors; Tissues; Training; United States; Work; autoreactive T cell; clinical diagnosis; diabetes pathogenesis; education pathway; experimental study; glucose metabolism; glucose tolerance; human tissue; inflammatory milieu; insulin dependent diabetes mellitus onset; insulitis; islet; mouse model; non-diabetic; preservation; prevent; protein expression; response; success; therapeutic target; therapy design

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，会导致产生胰岛素的 β 细胞遭到破坏 胰腺的。 T1D 发生和进展的时间线是异质的，诊断发生在 一些患者只有几年的年龄，而其他患者在诊断前已有多年的自身抗体。在 T1D，发生免疫失调，导致自身抗体产生，免疫细胞浸润到胰岛 （胰岛炎），并最终导致 β 细胞丧失。这段时间内β细胞内发生的事件以及由此产生的结果 对 β 细胞功能的影响仍不清楚。该提案的目标是确定 β 细胞如何发挥作用 在 T1D 发展过程中，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全面了解 疾病进展并确定可能的治疗靶点。我的总体目标 建议使用活体胰腺组织切片来探索 β 细胞在 T1D 发展过程中的功能， 具体来说，我将确定免疫细胞浸润在 β 细胞功能障碍和丧失中所起的作用。活胰腺 组织切片是这些研究的理想选择，因为这些样本使胰岛保持在其天然微环境中 并保留现有的组织病理学，以便研究 β 细胞功能和免疫细胞 人口。我假设 T1D 进展早期的免疫失调导致 β 功能障碍 细胞葡萄糖代谢途径主要由线粒体应激介导，导致 MHC I 类 过度表达并增加 β 细胞对免疫系统的可见性。为了验证这一假设，活体胰腺 将使用由人体器官捐赠组织生成的组织切片，并辅以由以下材料制成的切片： T1D 小鼠模型的胰腺。共聚焦显微镜技术将用于确定 组织内的胰岛炎，同时评估胰岛功能。额外的功能评估 例如灌注实验，然后进行胰岛素 ELISA 和基因表达来观察 功能更详细。该提案的第一部分重点关注为确定 原位免疫细胞对β细胞功能的影响。为了更好地控制疾病的时间表，特别是 研究疾病的最早阶段，将引入 HLA 匹配的 T 细胞化身来控制人类 胰腺组织切片，创造一个胰岛素环境。将研究疾病的发生和进展 进一步通过 NOD 小鼠模型脾细胞的过继转移实验。进展为 T1D 随着疾病的发展，将通过制作活体胰腺组织切片进行监测。功能评估 免疫细胞研究将如上所述进行。我期望拟议的贡献 研究将确定免疫失调在 β 细胞功能障碍中所起的作用以及 β细胞功能障碍的机制起源。这将提供有关 T1D 如何发展的重要信息 并将指出阻止疾病进展的可能治疗目标。