Microbiomal Mediators of TL1A-induced-Fibrosis

摘要 炎症性肠病（IBD）是由易感性和炎症反应之间的一系列复杂相互作用引起的。 严重性基因、环境和粘膜免疫系统，表现为临床疾病。肠道 微生物组有助于IBD;肠道微生物组成的改变已在肠道 炎症，但很少有研究检查肠道纤维化。大量潜在的遗传、表观遗传 和微生物变量有助于IBD表型的严重性。其中，相对明确的，临床上- 可识别的表型是具有独特的、严重的、纤维化克罗恩病表现的那些。的 肿瘤坏死因子超家族（TNFSF）成员15和肿瘤坏死因子- 类（TL）细胞因子1A（称为：TNFSF 15/TL 1A）影响炎症和纤维化的严重程度， 动物模型和人类疾病。我最近发表的研究结果表明， 肠纤维化，并且TL 1A依赖于其发挥其促纤维化作用。精确的机制和 然而，导致肠纤维化的特定微生物还有待阐明。由于成纤维细胞是一种 负责纤维化的主要细胞类型，TL 1A和微生物群驱动肠道的机制 纤维化可能涉及成纤维细胞活化直接诱导，正如我的初步数据所证明的。我假设 TNFSF 15/TL 1A通过改变微生物组在纤维化的诱导和放大中发挥核心作用， 以增强促进纤维化的微生物种群，并通过直接激活成纤维细胞来表达 纤维化相关因素和/或变得对微生物刺激更敏感。在目标1中，我将确定 无论是小鼠模型中的TL 1A诱导的纤维化还是克罗恩病退伍军人中的TNFSF 15遗传风险， 与微生物组的改变有关。我将通过16 S rRNA评估肠道微生物组的特征 对来自具有和不具有TNFSF 15遗传风险的患者的粪便和组织标本进行测序。确认 这些相关微生物、WT和TL 1A转基因无菌C57/BL 6小鼠的因果效力将被 用候选细菌聚生体定殖以评估体内纤维化的发展。这些微生物群 还将使用代谢组学方法进行表征，以更具体地了解它们在疾病中的作用 发病机制在目标2中，我将描述TL 1A-反应的影响和直接的分子机制， 使用体外原代分离的成纤维细胞在成纤维细胞上的目标1中鉴定的细菌产物和代谢物。在 目的3，我将确定是否特定的细菌代谢产物可以因果介导体内肠纤维化， 在工程小鼠模型中的选择性施用。这项拟议研究的结果将提供深入了解 特定的微生物或其产物，与TL 1A一起驱动肠道纤维化。如果宿主微生物组 相互作用和所产生的促纤维化成纤维细胞表型可以通过操纵TL 1A或TL 1B来减轻。 在微生物组/代谢组中，肠纤维化可以在遗传易感个体中预先发生。这些 因此，研究可以揭示另外的下游途径、分子和潜在的治疗靶点。

ABSTRACT Inflammatory Bowel Disease (IBD) results from a series of complex interactions between susceptibility and severity genes, the environment and the mucosal immune system, presenting as clinical disease. The gut microbiome contributes to IBD; altered gut microbial composition has been demonstrated in intestinal inflammation, but few studies have examined intestinal fibrosis. A vast number of potential genetic, epigenetic and microbiotic variables contribute to the severity of IBD phenotypes. Among these, relatively clear, clinically- identifiable phenotypes are those with a distinct, severe, fibrosing Crohn’s disease presentation. The gene/protein combination of tumor necrosis factor superfamily (TNFSF) member 15 and tumor necrosis factor- like (TL) cytokine 1A (referred to as: TNFSF15/TL1A) influences the severity of inflammation and fibrosis in both animal models and human disease. My recently published findings have implicated the gut microbiome in intestinal fibrosis, and on which TL1A depends to exert its pro-fibrotic effects. The precise mechanisms and specific microbial contributors to intestinal fibrosis have yet to be elucidated, however. As fibroblasts are a predominant cell type responsible for fibrosis, the mechanisms by which TL1A and microbiota drive intestinal fibrosis likely involve direct induction of fibroblast activation, as my preliminary data demonstrates. I hypothesize that TNFSF15/TL1A plays a central role in the induction and amplification of fibrosis by altering the microbiome to enhance fibrosis-promoting microbial populations and through the direct activation of fibroblasts to express fibrosis-related factors and/or to become more susceptible to microbial stimulation. In Aim 1, I will determine whether TL1A-induced fibrosis in mouse models or TNFSF15-genetic risk in Veterans with Crohn’s disease is associated with alterations in the microbiome. I will characterize the gut microbiome as assessed by 16S rRNA sequencing of stool and tissue specimens from patients with and without TNFSF15-genetic risk. To confirm the causal efficacy of these correlated organisms, WT and TL1A-transgenic germ-free C57/BL6 mice will be colonized with the candidate bacterial consortia to evaluate for development of fibrosis in vivo. These microbiota will also be characterized using metabolomic methods to allow more specific insight into their role in disease pathogenesis. In Aim 2, I will characterize the TL1A-responsive effects and molecular mechanisms of direct bacterial products and metabolites identified in Aim 1 on fibroblasts using primary isolated fibroblasts in vitro. In Aim 3, I will determine if specific bacterial metabolites can causally mediate intestinal fibrosis in vivo using selective administration in engineered mouse models. The results of this proposed study will provide insight into the specific microbes, or their products, that drive intestinal fibrosis in concert with TL1A. If host-microbiome interactions and resulting pro-fibrotic fibroblast phenotypes can be mitigated by manipulation of TL1A or the microbiome/metabolome, intestinal fibrosis may be pre-empted in genetically susceptible individuals. These investigations may thus reveal additional downstream pathways, molecules, and potential targets for therapy.