Spatio-temporal dissection of epithelial cell hierarchies in gut inflammation

 描述(由申请人提供)：我们使用最先进的技术和计算分析，在单细胞分辨率下，了解TE末端回肠的选择性扰动或炎性扰动如何改变上皮分化的组织。在持续的动态平衡更新过程中，上皮祖细胞根据沿隐窝-绒毛轴空间排列的一系列信号通路所制定的一套规则逐步分化。几十年的肠道生物学研究已经证实，分化是循序渐进的，分支的决定导致不同功能谱系的分级组织。单个单元格如何 将多个信号通路连贯地整合到一个分级分化程序中，以及当该程序在疾病状态下受到干扰时如何协调改变，这一点还没有定义。在炎症性肠病(IBD)中，有组织的肠道微环境被各种免疫细胞渗透，这些免疫细胞为上皮细胞提供额外的信号输入。我们假设正常的信号组织被这些额外的输入扰乱，反过来，这些额外的输入要么重新安排要么取消上皮分化的分级组织，导致不精确的分化，无法维持成熟状态，以及改变上皮细胞类型的比例。我们将协调两项新兴技术，MultiOmyx显微镜和细胞计数飞行时间(CytoF)，在单细胞分辨率下询问多个信号活动和细胞分化途径，以便使用数据驱动的计算方法来模拟分化层次。在这项提案的两个目标中，我们将应用我们的方法来研究(目标1)特定途径抑制的效果，我们假设它是为了维持层级分化，以及(目标2)慢性炎症的影响，我们假设它是为了将严格的分化层次转换为塑料景观。基质和免疫细胞对分化组织的作用的研究将结合肠样培养、活体成像和组织切片的多重显微镜进行研究。未来的方向包括将我们的实验计算平台直接应用于克罗恩回肠炎的人类患者样本。通过我们的发现，我们的目标是对IBD的理解、诊断和治疗做出重大贡献。这项研究的结果有望成为研究其他复杂疾病的门户，这些疾病的病因不明，许多危险因素在许多成分上的分子分布，以及多个异源系统之间的相互作用。

 DESCRIPTION (provided by applicant): We use state-of-the-art technologies and computational analyses to understand, at single cell resolution, how the organization of epithelial differentiation is altered by selective perturbations or inflammatory perturbations of te terminal ileum. During the continuous homeostatic renewal process, an epithelial progenitor cell progressively differentiates according to a set of rules set forth by a composite of signaling pathways that are spatially arranged along the crypt-villus axis. Decades of intestinal biology research have established that differentiation proceeds stepwise, with branched decisions resulting in a hierarchical organization of the different functional lineages. How individual cells coherently integrate multiple signaling pathways into a hierarchical differentiation program, and how this program is coordinately altered when perturbed in states of disease are undefined. In inflammatory bowel disease (IBD), the organized intestinal microenvironment is infiltrated by a variety of immune cells that provide additional signaling inputs to epithelial cells. We hypothesize that normal signaling organization is perturbed by these additional inputs, which in turn, either rearranges or abolishes the hierarchical organization of epithelial differentiation, leading to imprecise differentiation, failed maintenance of the mature state, and altered proportions of epithelial cell types. We will coordinate two emerging technologies, MultiOmyx microscopy and Cytometry Time-of-Flight (CyTOF), to interrogate multiple signaling activities and cell differentiation pathways at single cell resolution, in order to use data-driven computational approaches to model differentiation hierarchies. In the two aims of this proposal, we will apply our approaches to study (Aim 1) the effects of specific pathway inhibition, which we hypothesize to maintain hierarchical differentiation, and (Aim 2) the effects of chronic inflammation, which we hypothesize to convert the strict differentiation hierarchy into a plastic landscape. The study of stromal and immune cell contributions to the organization of differentiation will be studied using a combination of enteroid cultures, live imaging, and multiplex microscopy of tissue sections. Future directions include the direct application of our experimental-computational platforms to human patient samples of Crohn's ileitis. Through our findings, we aim to make significant contributions to the understanding, diagnosis and treatment of IBD. The outcomes from this research will hopefully be a gateway towards the study of other complex diseases with undefined etiologies, molecular distribution of many risk factors over many components, and interactions between multiple heterologous systems.