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，而且也构成了疾病的开始和进展的病因学因素。 肠道然而，微生物影响肠道健康的机制在很大程度上仍然未知。 利用一种强大的遗传模式生物--黑腹果蝇，我们最近发现， 不仅改变肠干细胞（ISC）的增殖，而且还调节分化细胞的相对比例， 上皮中的类型。重要的是，我们发现病原体促进肠内分泌（EE）的命运，而非 致病微生物促进肠上皮细胞（EC）的命运，这表明致病和非致病微生物 以相反的方式影响ISC谱系。根据这些结果，以及我们以前的研究，我们 假设肠道微生物调节ISCs中免疫信号通路以影响其谱系 决策为了验证这一假设，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、毒力/损伤） 果蝇和小鼠肠上皮细胞。同时，我们将确定细胞的相对贡献 丢失、ISC增殖和分化为这一过程。目标2：我们将描述经典免疫系统 NFκB同源物）在果蝇ISCs和小鼠肠样细胞中起作用，影响 对肠道微生物的反应。我们将首先使用免疫荧光技术鉴定ISC特异性Relish靶基因。 细胞类型特异性转录组学和靶向DamID（TaDa）的组合。然后我们将分析 Imd/Relish通路与已知控制ISC分化的其他基因网络相互作用。我们的研究将 因此，证明了Imd/Relish的新作用，它超越了免疫效应物的控制， 这一途径如何改变干细胞谱系和上皮组成的机制的见解。目标3：我们 研究Janus激酶（JAK）-激活物信号转导子（STAT）通路的激活如何触发EE 果蝇ISCs和小鼠肠的命运承诺。我们将确定直接和间接的靶基因， STAT，以表征下游机制并描述JAK-STAT信号传导的影响 ISC分化。最后，我们的目标是阐明JAK-STAT和Imd/Relish之间的相互作用 这两种途径决定了致病性和非致病性微生物在肠道中产生的相反ISC命运。 这项研究的结果将提高我们对微生物群如何改变肠道内稳态的理解， 并证明不同肠道微生物（致病性与非致病性）改变肠道 上皮组成的差异调节ISC分化。我们还将确定途径的新作用， 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是如何对它们的微生物环境做出反应的。因此，我们研究中确定的机械原理将 为更好地了解肠道疾病铺平道路，并加强新疗法的开发。

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.