Control of hepatic T cell responses in biliary atresia

项目概要/摘要 胆道闭锁（BA）是指新生儿胆道系统的特发性闭塞。不幸的是，高达50%的 通过肝门肠吻合术（HPE）尝试手术恢复胆汁流动的婴儿仍然 胆汁淤积并迅速进展为终末期肝病。潜在的治疗靶点包括肝NK， CD 8和Th 17-淋巴细胞已被确定为炎症性导管阻塞的驱动因素， 肝内胆管上皮的进行性损伤。我们的小组已经报道，调节性T细胞（Tcells） 能抑制肝淋巴细胞反应，防止胆管损伤和肝纤维化的发生。 因此，我们探讨了胆汁淤积条件下调节Treg/Th 17稳态的机制 概括进行性BA。我们的初步研究结果表明，结合胆汁酸（CBA）抑制 表达Treg转录因子Foxp 3并在体外抑制Treg功能。Mdr 2-/-小鼠模型 在“毒性胆汁”诱导的进行性胆汁淤积中，肠胆汁酸再摄取的抑制导致了显著的 血清胆汁酸浓度降低，改变了枯否细胞的细胞因子产生， 肝硬化的快速肝扩张。在轮状病毒诱导的鼠BA中，用Fxr激动剂处理诱导了 IL-10在肝单个核细胞中的表达，扩增T细胞，并减弱BA表型。我们 假设胆汁酸是表观遗传修饰剂，其控制Treg体内平衡并决定 通过激活胆汁酸受体Tgr 5和Fxr来调节肝脏中的炎症微环境。我们将测试 通过研究介导胆汁酸诱导Foxp 3沉默的分子机制， 体外和通过删除急性或慢性白血病小鼠CD 4淋巴细胞中CBA的候选分子靶点， 胆汁淤积（目的1）。目的2：探讨Tgr 5和Fxr激活对巨噬细胞分化的调节作用。 细胞因子的产生和调节性T细胞稳态，以及对纤维化胆管病表型的影响将在 骨髓移植实验和条件性胆汁酸受体敲除小鼠。用于人体验证 疾病，我们将研究肝脏T淋巴细胞反应是否与不同的临床 通过分析肝脏RNAseq和临床数据并进行免疫荧光和图像分析， 对来自203例BA患者的初始队列的存档肝活检进行多中心儿童 肝病研究网络（Aim 3）。在初步研究中，我们生成了Th 17相关的基因列表， 来自体外极化的新生儿Th 17淋巴细胞的基因。系统聚类分析这些实验 来自137名BA患者的肝脏RNAseq数据中衍生的Th 17候选基因揭示了具有以下特征的患者群： 相似的基因表达谱。血清结合胆红素水平在这些聚类之间存在差异，支持 我们的假设未来的研究将询问由整合的RNAseq-和 基于免疫荧光计数具有BA表型的肝浸润性T细胞和HPE结果。

Project Summary/ Abstract Biliary atresia (BA) refers to idiopathic obliteration of the biliary tree in neonates. Unfortunately, up to 50% of infants who undergo attempts at surgical restoration of bile flow by hepatoportoenterostomy (HPE) remain cholestatic and rapidly progress to end-stage liver disease. Potential therapeutic targets include hepatic NK, CD8 and Th17-lymphocytes which have been identified as drivers of inflammatory ductal obstruction and progressive injury of the intrahepatic biliary epithelium. Our group has reported that regulatory T cells (Tregs) are able to restrain hepatic lymphocyte responses protecting from bile duct injury and initiation of liver fibrosis. Therefore, we explored the mechanisms controlling Treg/Th17 homeostasis under cholestatic conditions recapitulating progressive BA. Our preliminary findings demonstrate that conjugated bile acids (CBA) repress expression of the Treg- transcription factor Foxp3 and inhibit Treg-function in vitro. In the Mdr2-/- mouse model of “toxic bile” induced progressive cholestasis, inhibition of intestinal bile acid reuptake resulted in dramatic reduction in serum bile acid concentration which shifted cytokine production by Kupffer cells accompanied by rapid hepatic expansion of Tregs. In rotavirus induced murine BA, treatment with Fxr agonists induced expression of Il-10 in hepatic mononuclear cells, expanded Tregs, and attenuated the BA phenotype. We hypothesize that bile acids are epigenetic modifiers that control Treg homeostasis and determine the inflammatory microenvironment in the liver through activation of the bile acid receptors Tgr5 and Fxr. We will test this hypothesis by investigating the molecular mechanisms mediating bile acid induced silencing of Foxp3 in vitro and by deleting candidate molecular targets for CBA in CD4 lymphocytes in mice with acute or chronic cholestasis (Aim 1). In Aim 2, the effects of activation of Tgr5 and Fxr on regulation of macrophage derived cytokine production and Treg homeostasis, and on the fibrosing cholangiopathy phenotype will be studied in bone marrow transplant experiments and in conditional bile acid receptor knockout mice. For validation in human disease, we will investigate whether hepatic T lymphocyte responses are associated with distinct clinical phenotypes by analyzing liver RNAseq and clinical data and performing immunofluorescence and image analysis on archived liver biopsies from an inception cohort of 203 patients with BA followed by the multicenter Childhood Liver Disease Research Network (Aim 3). In preliminary studies we generated a gene list of Th17 associated genes from in vitro polarized neonatal Th17 lymphocytes. Hierarchical cluster analysis of these experimentally derived Th17 candidate genes in liver RNAseq data from 137 patients with BA revealed clusters of patients with similar gene expression profile. Serum conjugated bilirubin levels differed between these clusters, supporting our hypothesis. Future studies will interrogate segregation of clusters defined by integrated RNAseq- and immunofluorescence - based enumeration of liver infiltrating T cells with BA phenotype and HPE outcome.