Gut microbes modulate immune pathways in intestinal stem cells to influence their lineage

越来越多的流行病学研究表明，肠道微生物不仅是 维持宿主健康，也是疾病发生和发展的致病因素 肠道。然而，微生物影响肠道健康的机制在很大程度上仍不清楚。 利用一种强大的遗传模式生物--黑腹果蝇，我们最近发现，微生物不能 不仅改变肠道干细胞(ISC)的增殖，而且还调节分化细胞的相对比例 上皮细胞的类型。重要的是，我们发现病原体促进了肠道内分泌(EE)的命运，而非 致病微生物促进肠上皮细胞(EC)的命运，表明致病微生物和非致病微生物 以相反的方式影响ISC血统。基于这些结果，以及我们之前的研究，我们 假设肠道微生物调节ISCs中的免疫信号通路以影响其谱系 决定。为了验证这一假设，我们提出了以下具体目标：目标1：我们将确定 调节肠道细胞组成的微生物特征(免疫原性、毒性/损伤) 果蝇和小鼠的肠样上皮细胞。同时，我们将确定细胞的相对贡献 在此过程中，ISC的丢失、增殖和分化。目标2：我们将描述一个经典的免疫系统如何 作用于果蝇间充质干细胞和小鼠肠道的IMD/AREISH通路(核因子κB同源物) 对肠道微生物作出反应的分化。我们将首先使用一种新的 结合细胞类型特异性转录和靶向DamID(TADA)。然后我们将分析如何 IMD/AREISH途径与其他已知的控制ISC分化的基因网络相互作用。我们的研究将 因此展示了IMD/RAISH的新角色，它超越了免疫效应器的控制，并提供了 对这一途径如何改变干细胞谱系和上皮成分的机械性洞察。目标3：我们将 研究Janus Kinase(JAK)-信号转导的激活剂(STAT)通路如何触发EE 果蝇间充质干细胞和小鼠肠道中的命运承诺。我们将确定直接和间接的靶基因 STAT，以便表征下行机制并描述JAK-STAT信令具有的影响 论ISC的差异化。最后，我们的目标是澄清JAK-STAT和IMD/AREISH之间的相互作用 途径决定了致病微生物和非致病微生物在肠道中产生的相反的ISC命运。 这项研究的结果将提高我们对微生物区系如何改变肠道内环境平衡的理解。 健康和疾病，并证明不同的肠道微生物(致病和非致病)改变肠道 通过差异调节ISC分化的上皮成分。我们还将确定路径的新角色 经典定义为影响ISC分化的免疫途径，提供了新的机制见解 ISCs如何对其微生物环境做出反应。因此，我们研究中确定的机械原理将 为更好地了解肠源性疾病铺平道路，并促进新疗法的开发。

An increasing number of epidemiological studies suggest that intestinal microbes are not only central to maintaining host health, but also constitute etiological factors for the initiation and progression of diseases of the intestinal tract. However, the mechanisms by which microbes affect intestinal health remain largely unknown. Using a powerful genetic model organism, Drosophila melanogaster, we have recently shown that microbes not only alter intestinal stem cell (ISC) proliferation, but also modulate the relative proportions of differentiated cell types in the epithelium. Importantly, we found that pathogens promote an enteroendocrine (EE) fate while non- pathogenic microbes promote an enterocyte (EC) fate, suggesting that pathogenic and non-pathogenic microbes influence ISC lineage in an opposing manner. Based on these results, and from our previous studies, we hypothesize that gut microbes modulate immune signaling pathways in ISCs to influence their lineage decisions. To test this hypothesis, we propose the following specific aims: Aim 1: We will determine the microbial characteristics (immunogenicity, virulence/damage) that modulate the cellular composition of the gut epithelium in both Drosophila and murine enteroids. In parallel, we will determine the relative contributions of cell loss, ISC proliferation and differentiation to this process. Aim 2: We will characterize how a classical immune pathway, the Imd/Relish pathway (NFκB homologue), acts in Drosophila ISCs and murine enteroids to influence differentiation in response to gut microbes. We will first identify ISC-specific Relish target genes using a combination of cell type-specific transcriptomics and targeted DamID (TaDa). We will then analyze how the Imd/Relish pathway interacts with other gene networks known to control ISC differentiation. Our studies will therefore demonstrate a new role for Imd/Relish that goes beyond the control of immune effectors and provide mechanistic insight into how this pathway alters stem cell lineage and epithelial composition. Aim 3: We will investigate how activation of the Janus kinase (JAK)-signal transducer of activator (STAT) pathway triggers EE fate commitment in Drosophila ISCs and murine enteroids. We will identify direct and indirect target genes of STAT in order to characterize downstream mechanisms and delineate the impact that JAK-STAT signaling has on ISC differentiation. Finally, we aim to clarify how the interaction between the JAK-STAT and Imd/Relish pathways determines the opposite ISC fates produced in the gut by pathogenic and non-pathogenic microbes. Outcomes of this research will improve our understanding of how the microbiota alters intestinal homeostasis in health and disease and demonstrate that different gut microbes (pathogenic vs non-pathogenic) alter gut epithelial composition by differentially modulating ISC differentiation. We will also identify a new role for pathways classically defined as immune pathways in affecting ISC differentiation, providing new mechanistic insight into how ISCs respond to their microbial environment. The mechanistic principles identified in our study will therefore pave the way to a better understanding of diseases of gut origin and potentiate the development of new therapies.