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来以单细胞分辨率了解回肠末端的选择性扰动或炎症性扰动如何改变上皮分化的组织。在连续的稳态更新过程中，上皮祖细胞根据由信号传导途径的复合物提出的一组规则进行分化，所述信号传导途径在空间上沿沿着隐窝-绒毛轴排列。几十年的肠道生物学研究已经确定，分化是逐步进行的，分支决定导致不同功能谱系的分层组织。单个细胞如何 将多个信号通路连贯地整合到分级分化程序中，以及当疾病状态受到干扰时，该程序如何协调地改变是不确定的。在炎症性肠病（IBD）中，有组织的肠道微环境被各种免疫细胞浸润，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受到这些额外输入的干扰，这反过来又会重新排列或废除上皮分化的分层组织，导致不精确的分化，未能维持成熟状态，以及上皮细胞类型的比例改变。我们将协调两种新兴技术，MultiOmyx显微镜和Cytometry飞行时间（CyTOF），以单细胞分辨率询问多种信号传导活动和细胞分化途径，以便使用数据驱动的计算方法来建模分化层次。在这一建议的两个目标，我们将应用我们的方法来研究（目标1）特定的通路抑制的影响，我们假设保持分层分化，和（目标2）慢性炎症的影响，我们假设转换成一个塑料景观严格的分化层次。将使用肠样培养物、活成像和组织切片的多重显微镜检查的组合来研究基质和免疫细胞对分化组织的贡献。未来的发展方向包括将我们的实验-计算平台直接应用于克罗恩氏回肠炎的人类患者样本。通过我们的研究结果，我们的目标是为IBD的理解，诊断和治疗做出重大贡献。这项研究的结果将有望成为研究其他复杂疾病的门户，这些疾病的病因不明，许多风险因素在许多组分中的分子分布，以及多种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.