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

摘要 我们知道，肠道微生物群起着辅助器官系统的作用，在人体内起着至关重要的作用 消化；生产必需的维生素、神经递质和其他小分子；以及在 免疫系统的调节。我们也知道肠道微生物区系的变化会导致疾病， 尤其是炎症性肠病和相关的炎症性疾病。然而，我们对此知之甚少 将肠道微生物区系与这些疾病联系起来的分子和机制。因为肠道中的细菌 微生物群通过小分子感知并响应环境，很可能这些分子中的一些 是促炎和抗炎反应的关键调节者。这个项目试图确定 这些分子和它们的运作机制。该项目源于丰富的数据集 (DIABIMMUNE)揭示了一些强有力的相关性， 肠道微生物区系成员与婴儿失调免疫反应和疾病的发展 有1型糖尿病的遗传倾向。我们将重点关注大约40株似乎在 疾病进展，以发现它们产生的促炎或抗炎小分子信号。具体而言 目的1我们将在各种条件下培养患者来源的这些细菌菌株，捕获小的 产生的分子，并测量它们调节促炎信号(TNFpha)和抗炎信号的能力 炎症信号(IL-10)。最强大和最有选择性的监管者将推进到具体目标2，其中 生物测定引导的分级将指导活性分子的分离，然后从结构上进行 以光谱(核磁共振和质谱学)和成像(X射线)为特征的 衍射)技术。由于已知的分子与已证实的生物活性有关，我们将优先考虑 基于他们在DIABIMMUNE粪便样本中的存在，我们已经存档了。只有生物活性 人体样本中存在的分子将在特定的目标3中进行分析，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 特别设计和新实施的同时测量50多个转录的测试 调节炎症、免疫、新陈代谢和细胞应激的因子。通过分析转录因子的活性 与已知化合物相比，我们将能够根据分子的活性分布对其进行分类。我们 将在两个患者身上使用各种功能分析进一步表征它们的作用机制 髓系细胞以及结肠和回肠粘膜细胞系，它们发育成四种不同的细胞类型，形成 三维结构，包括被分泌粘液层覆盖的隐窝。在管道的尽头 由这三个特定的目标形成的，调节免疫反应的小分子 途径将是已知的，这种分子和机制的识别将提供对两者的洞察 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.