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，会导致产生胰岛素的 β 细胞遭到破坏 T1D 发生和进展的时间线与诊断发生时间不同。 一些患者在诊断前几年就已出现自身抗体，而其他患者则在诊断前数年就已出现自身抗体。 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），以及 β 细胞的最终损失 在此期间 β 细胞内发生的事件以及由此产生的结果。 对 β 细胞功能的影响仍然未知。该提案的目标是确定 β 细胞的功能。 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 疾病进展和确定可能的治疗靶点是我的总体目标。 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活胰腺功能障碍和损失中的作用。 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 我认为 T1D 进展早期的免疫失调会导致 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 为了验证这一假设，我们使用了活体胰腺。 将使用从人体器官供体组织生成的组织切片，并辅以由以下物质制成的切片： T1D 小鼠模型的胰腺将使用共聚焦显微镜技术来确定胰腺的程度。 组织内的胰岛炎，同时评估胰岛功能。 例如灌注实验，然后进行胰岛素 ELISA 和基因表达来观察 该提案的第一部分侧重于为确定功能而进行的评估。 原位免疫细胞对 β 细胞功能的影响，以更好地控制疾病时间，特别是对疾病时间的控制。 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，将使用活体胰腺组织切片进行监测。 和免疫细胞研究将按照上面讨论的方式进行。 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。