The role of HNF4a in maintaining intestinal epithelial cell homeostasis in the presence of microbes

摘要 失去与微生物区系的动态平衡关系会导致炎症性疾病，如炎症性疾病 肠道疾病(IBD)。维持微生物区系的动态平衡取决于宿主细胞的调节能力 它们的转录程序响应来自微生物区系的信号。肠上皮细胞(IECS)服务 作为微生物区系屏障的关键作用。IEC通过将外部信号集成到各种 产生适当生理反应的转录程序。肝细胞核因子4α HNF4a是一种核受体转录因子，在脊椎动物的消化道中高表达。 在IECS中，HNF4a主要作为转录激活剂调节参与IEC发育的基因， 屏障功能、新陈代谢和营养吸收。HNF4a基因座和HNF4a的遗传变异 人IBD的转录因子结合基序已在GWAs中被鉴定。肠特异性基因敲除 HNF4a在小鼠体内导致高渗透性自发性肠炎。然而，目前还不清楚 这种表型是由于IECS中HNF4a的内在抗炎作用或导致 固有层中免疫细胞的激活。本项目的总体目标是了解 HNF4a在维持肠道微生物区系动态平衡和调节微生物区系反应中的作用 IECS中的增强剂。我们的实验室最近有了一个关键发现，即IEC基因组中的HNF4a活性是 在小鼠和斑马鱼中被微生物群抑制。此外，我们发现HNF4a保护斑马鱼免受 微生物区系驱动的转录变化与人类IBD中的转录变化相关。我们也 确定了受小鼠IECS中微生物区系调控的增强子子集，这些增强子也受 HNF4a。然而，HNF4a通过作用于这些微生物群在维持肠道动态平衡中的作用 增强剂尚不清楚。我将测试HNF4a促进肠道内环境平衡的中心假设。 通过介导微生物区系在整个IEC基因组中诱导的增强子活性的变化来存在微生物区系。 在特定的目标1中，我将在IEC中缺乏HNF4a的小鼠品系(HNF4aΔIEC)衍生到无菌(GF)条件下 检测微生物区系在IBD模型小鼠肠道炎症表型中的作用。另外，我会 从这些小鼠的原代IEC获得肠样培养，以研究HNF4a在介导IEC内源性中的作用 在体外对微生物区系的反应。在具体目标2中，我将定义HNF4a在微生物群反应中的作用 使用GF和ex-GF的增强剂常规(CV)HNF4aΔIEC小鼠和野生型对照。其结果是 这项工作将提供创新的体内遗传学证据，确定HNF4a作为IEC的中介作用 转录程序保护免受微生物区系驱动的肠道炎症。这项研究针对的是 关于HNF4a在维持肠道与微生物区系内环境平衡中的作用的关键知识缺口。

Abstract Loss of homeostatic relationships with microbiota can result in inflammatory diseases such as the inflammatory bowel diseases (IBD). Maintaining homeostasis with microbiota is predicated on the ability of host cells to adjust their transcriptional programs in response to signals from microbiota. Intestinal epithelial cells (IECs) serve critical roles as a barrier against microbiota. IECs perform these roles by integrating external signals into various transcriptional programs which output appropriate physiologic responses. Hepatocyte nuclear factor 4 alpha (HNF4A) is a nuclear receptor transcription factor (TF) that is highly expressed in the vertebrate digestive tract. In IECs, HNF4A acts predominantly as a transcriptional activator regulating genes involved in IEC development, barrier function, metabolism, and nutrient absorption. Genetic variants at the HNF4A gene locus and HNF4A transcription factor binding motifs have been identified in GWAS for human IBD. Intestine specific knockout of Hnf4a in mice results in highly penetrant spontaneous intestinal inflammation. However, it is unknown whether this phenotype is due to intrinsic anti-inflammatory roles for HNF4A in IECs or barrier defects that result in activation of immune cells in the lamina propria. The overall objective of this project is to understand the role of HNF4A in maintaining homeostasis with microbiota in the intestine and in regulating microbiota-responsive enhancers in IECs. Our lab recently made the key discovery that HNF4A activity in the IEC genome is suppressed by microbiota in mice and zebrafish. Additionally we found that Hnf4a protects zebrafish from a microbiota-driven transcriptional shift which correlates with transcriptional shifts seen in human IBD. We also identified a subset of enhancers that are regulated by the microbiota in mouse IECs, which are also bound by HNF4A. However the role of HNF4A in maintaining intestinal homeostasis with microbiota by acting on these enhancers is unknown. I will test the central hypothesis that HNF4A promotes intestinal homeostasis in the presence of microbiota by mediating microbiota-induced alterations in enhancer activity across the IEC genome. In Specific Aim 1, I will derive a mouse strain lacking Hnf4a in IECs (Hnf4aΔIEC) into germ free (GF) conditions to test the role of microbiota in the intestinal inflammation phenotype of this mouse model of IBD. Additionally, I will generate enteroid cultures from primary IECs of these mice to study the role of HNF4A in mediating IEC intrinsic responses to microbiota in vitro. In Specific Aim 2, I will define the role of HNF4A at microbiota responsive enhancers using GF and ex-GF conventionalized (CV) Hnf4aΔIEC mice and wild-type controls. The outcomes of this work will provide innovative in vivo genetic evidence establishing the role of HNF4A as a mediator of IEC transcriptional programs protective against microbiota-driven intestinal inflammation. This research addresses a critical knowledge gap of the role of HNF4A in maintaining intestinal homeostasis with microbiota.