Integrated Functional Histopathology of the Diabetic Human Pancreas

摘要 1型糖尿病(T1D)是一种主要的自身免疫性疾病，对患者构成重大问题， 制定有效的治疗干预措施，以及广泛的公共卫生倡议。已启动并 在遗传和环境风险因素的复杂相互作用下，分泌胰岛素的胰岛β- 细胞被异常的免疫反应逐渐破坏，导致血糖水平升高。 作为严重的蛋白质、脂肪和碳水化合物代谢紊乱。目前，没有治愈或有效预防的方法。 尽管接受了胰岛素治疗，但严重的长期并发症仍然经常发生。 在过去的40年里，在T1D研究方面取得的许多进展都来自于动物研究 然而，尽管产生了许多洞察力，但真正认识到人类疾病需要更好的 关于直接在人类胰腺中展开的病理过程的知识。对这个目标的追求， 然而，面临着两大挑战：难以从糖尿病前期/糖尿病捐赠者那里获得合适的组织；以及 相关的病理改变通常是轻微的，并以杂乱的方式分布在整个胰腺。 因此，通过对单个胰腺组织切片的传统分析可以获得的信息是有限的。在 目前的建议，这构成了我们之前由NIH资助的胰腺研究的直接继续 组织病理学，我们将通过利用胰腺器官网络的资源来应对这些挑战 糖尿病供体(NPOD)组织存储库以及最近开发或改编的两种新的， 高度多元化的组织染色技术，并结合专业知识和长期互动的 冯·赫拉斯和霍曼实验室。这一特定的资源、技术和经验星座 使我们能够解决具体的两个关键知识差距：详细的原位表型、功能和 糖尿病前期/糖尿病胰腺中主要免疫和内分泌细胞群的相互作用；以及 在这些细胞中发现的特异性细胞因子信号与组织病变型MHC-I的高表达有关 T1D的特点。 因此，我们制定了一项研究计划，采用高维多路复用策略来 询问准确的表型、激活状态、上下文显微解剖定位和分布 糖尿病前期/糖尿病患者的CD8和CD4T细胞、抗原提呈细胞和B细胞以及内分泌细胞 胰腺(目标1)，这将关联主要细胞因子的表达模式(1型干扰素、干扰素γ、肿瘤坏死因子α)和 功能特征及其细胞来源和靶点是MHC-I高表达(AIM)的潜在原因 2)。此外，通过使用两种不同的多路复用技术对来自相同捐赠者的组织样本进行研究 技术在两个不同的实验室，我们寻求实现稳健性和重复性的实验 读数。总之，我们建议对多达30个表型、功能和拓扑的综合分析 参数将定义时空致病标志，这些标志将允许对高度 动态原位自身免疫过程在T1D病中的应用绘制组织病理学地貌图 糖尿病前期/糖尿病人胰腺的精细细节不仅对我们的疾病发病机制的概念很重要 但最终可能有助于开发新的或改进的预防策略和治疗模式。

ABSTRACT Type 1 diabetes (T1D) is a major autoimmune disease that poses significant problems to afflicted individuals, the development of effective therapeutic interventions, and public health initiatives at large. Initiated and perpetuated by a complex interplay of genetic and environmental risk factors, insulin-secreting pancreatic beta- cells are progressively destroyed by aberrant immune responses leading to elevated blood glucose levels as well as serious disturbances of protein, fat and carbohydrate metabolism. Currently, no cure or effective prevention is available, and despite insulin treatment, serious long-term complications are frequent. Much of the progress in T1D research attained over the past four decades comes from the study of animal models yet despite many insights generated, a genuine appreciation of the human disease requires better knowledge about pathological processes unfolding directly in the human pancreas. The pursuit of this goal, however, faces two major challenges: the difficulty to obtain suitable tissues from pre/diabetic donors; and the fact that pertinent pathological alterations are often mild and distributed in a variegated fashion across the pancreas. Thus, information obtainable by traditional analysis of individual pancreatic tissue sections is limited. In the present proposal, which constitutes a direct continuation of our previously NIH-funded work on pancreatic histopathology, we will address these challenges by leveraging the resources of the Network of Pancreatic Organ Donors with Diabetes (nPOD) tissue repository together with the recent development or adaptation of two novel, highly multiplexed tissue staining technologies, and with the combined expertise and long-standing interactions of the von Herrath and Homann laboratories. This particular constellation of resources, technologies and experience allows us to tackle two critical knowledge gaps in specific: the detailed in situ phenotypes, functionalities, and interactions of major immune and endocrine cell populations in the pre/diabetic pancreas; and the contribution of specific cytokine signatures to as revealed in these cells to the hyperexpression of MHC-I, a histopathognomonic feature of T1D. Accordingly, we have developed a research plan that employs high-dimensional multiplexing strategies to interrogate the precise phenotype, activation status, contextual microanatomical localization and distribution of CD8+ and CD4+T cells, antigen presenting cells and B cells, and endocrine cells across the pre/diabetic human pancreas (Aim 1), and that will correlate major cytokine expression patterns (type 1 interferons, IFNγ, TNFα) and functional signatures with their cellular sources and targets as potential causes for MHC-I hyperexpression (Aim 2). Moreover, by conducting the work with tissue specimens from the same donors using two different multiplexing technologies in two different laboratories, we seek to achieve robustness and reproducibility of experimental readouts. Altogether, we propose that an integrated analysis of up to 30 phenotypic, functional and topological parameters will define spatiotemporal pathogenetic landmarks that will permit a partial reconstruction of the highly dynamic in situ autoimmune processes operative in T1D disease. Mapping the histopathological landscape of the pre/diabetic human pancreas in exquisite detail not only is important for our conception of disease pathogenesis but ultimately may inform the development of novel or improved prevention strategies and treatment modalities.