Identification and characterization of gut microbial bioactive molecules that determine predisposition to autoimmune disease and atopy

摘要 我们知道，肠道微生物组作为辅助器官系统发挥作用，在人类 消化;生产必需的维生素，神经递质和其他小分子;以及 调节免疫系统。我们也知道肠道微生物群的变化会导致疾病， 特别是炎性肠病和相关的炎性病症。然而，我们对 将肠道微生物群与这些疾病联系起来的分子和机制。当肠道中的细菌 微生物群用小分子感知并对环境做出反应，很可能其中一些分子 是促炎和抗炎反应的关键调节因子。该项目旨在确定 这些分子及其运作机制。该项目源于丰富的数据集 （糖尿病），揭示了一些强大的相关性之间的出现，或消失， 肠道微生物群成员和婴儿免疫反应失调和疾病的发展 有1型糖尿病的遗传倾向我们将重点关注约40种菌株，这些菌株似乎在其中发挥着巨大作用 疾病进展，以发现它们产生的促炎或抗炎小分子信号。在特定 目的1我们将在各种条件下培养这些细菌的患者来源菌株，捕获小的 产生的分子，并测量它们调节促炎信号（TNF α）和抗炎症信号（TNF α）的能力。 炎症信号（IL-10）。最有效和最有选择性的调节剂将进入特定目标2， 生物测定引导的分级分离将引导活性分子的分离，然后在结构上 用光谱（核磁共振和质谱）和成像（X射线）表征 衍射）技术。有了已知的与已确认的生物活性有关的分子，我们将优先考虑 根据他们在糖尿病粪便样本中的存在，我们已经存档。只有生物活性 将在特定目标3中分析人类样本中存在的分子以确定机制。因为 这些分子发挥其生物学效应的信号通路是复杂的，我们将采用一种 专门设计和新实施的检测，同时测量50多个转录 调节炎症、免疫、代谢和细胞应激的因子。通过分析转录因子的活性 与已知的化合物相比，我们将能够根据它们的活性特征对分子进行分类。我们 将使用多种功能测定进一步表征其作用机制， 髓样细胞以及结肠和回肠粘膜细胞系，其发育成四种不同的细胞类型 包括被分泌的粘液层覆盖的隐窝的三维结构。管道末端 由这三个特定的目标形成，调节免疫反应的小分子沿着注释 分子和机制的鉴定将为我们提供对这两种途径的深入了解。 基础生物学和潜在的治疗干预。

ABSTRACT We know that the gut microbiome functions as an accessory organ system that plays critical roles in human digestion; the production of essential vitamins, neurotransmitters, and other small molecules; and in the regulation of the immune system. We also know that changes in the gut microbiota can lead to disease, especially inflammatory bowel disease and related inflammatory disorders. However, we know very little about the molecules and mechanisms that connect gut microbiota to these diseases. As the bacteria in the gut microbiota sense and respond to their environment with small molecules, it's likely that some of these molecules are key regulators of both pro-inflammatory and anti-inflammatory responses. This project seeks to identify these molecules and the mechanisms by which they operate. The project stems from a rich data set (DIABIMMUNE) that revealed a number of robust correlations between the appearance, or disappearance, of members of the gut microbiota and the development of dysregulated immune responses and disease in infants with genetic pre-disposition to type 1 diabetes. We will focus on ~40 strains that appear to play outsize roles in disease progression to discover what pro- or anti-inflammatory small molecule signals they produce. In Specific Aim 1 we will culture patient-derived strains of these bacteria under a variety of conditions, capture the small molecules produced, and measure their ability to regulate a pro-inflammatory signal (TNFalpha) and an anti- inflammatory signal (IL-10). The most potent and selective regulators will advance to Specific Aim 2 in which bioassay-guided fractionation will guide the isolation of active molecules, which will then be structurally characterized with spectroscopic (both nuclear magnetic resonance and mass spectrometry) and imaging (X-ray diffraction) techniques. With known molecules linked to confirmed biological activity in hand, we will prioritize hits based on their presence in the DIABIMMUNE stool samples, which we have archived. Only bioactive molecules present in human samples will be analyzed in specific Aim 3 to determine mechanism. Because the signaling pathways through which these molecules exert their biological effects are complex, we will employ a specially designed and newly implemented assay that simultaneously measures more than 50 transcription factors that regulate inflammation, immunity, metabolism and cell stress. By profiling transcription factor activity and comparing to known compounds, we will be able to classify molecules based on their activity profiles. We will further characterize their mechanism of action using a variety of functional assays on both patient derived myeloid cells as well as colonic and ileal mucosa cell lines that develop into four distinct cell types that form three-dimensional structures including crypts covered by a secreted mucus layer. At the end of the pipeline formed by these three specific aims, small molecules that regulate immune responses along annotated pathways will be known, and this identification of both molecule and mechanism will provide insights into both basic biology and potential therapeutic interventions.