Control of hepatic T cell responses in biliary atresia

项目摘要/摘要 胆道闭锁(BA)是指新生儿胆道树的特发性闭锁。不幸的是，高达50%的 接受肝门肠吻合术(HPE)恢复胆汁流量的婴儿仍然存在 胆汁淤积，并迅速发展为终末期肝病。潜在的治疗靶点包括肝脏NK细胞， CD8和Th17-淋巴细胞已被确认为炎性导管梗阻的驱动因素 进行性肝内胆管上皮损伤。我们小组报告了调节性T细胞(Tregs) 都能够抑制肝淋巴细胞反应，保护胆管损伤和肝纤维化的启动。 因此，我们探讨了胆汁淤积条件下Treg/Th17动态平衡的调控机制。 概括地说，进步的学士学位。我们的初步研究结果表明，结合胆汁酸(CBA)抑制 体外表达Treg转录因子Foxp3并抑制Treg功能。在mdr2-/-小鼠模型中 对“有毒胆汁”引起的进行性胆汁淤积，抑制肠道胆汁酸再摄取导致戏剧性 血清胆汁酸浓度的降低改变了库普弗细胞细胞因子的产生 Tregs的肝脏快速扩张。在轮状病毒诱导的小鼠BA中，FXR激动剂治疗诱导 IL-10在肝单个核细胞、扩增树突状细胞中的表达，并减弱BA表型。我们 假设胆汁酸是表观遗传调节剂，控制Treg动态平衡并决定 通过激活胆汁酸受体TGR5和FXR，肝脏中的炎症微环境。我们将测试 通过研究胆汁酸诱导Foxp3沉默的分子机制，提出了这一假说 并通过删除急、慢性白血病小鼠CD4淋巴细胞中CBA的候选分子靶点 胆汁淤积症(目标1)。在目标2中，TGR5和FXR的激活对巨噬细胞衍生的调节的影响 细胞因子的产生和Treg动态平衡，以及对纤维性胆管病表型的研究将在 骨髓移植实验和条件性胆汁酸受体基因敲除小鼠。用于在人类中进行验证 我们将研究肝脏T淋巴细胞反应是否与不同的临床相关 通过分析肝脏RNAseq和临床数据并进行免疫荧光和图像分析来进行表型 203例多中心儿童期BA患者的初始队列中的肝活检档案 肝病研究网络(AIM 3)。在初步研究中，我们生成了与Th17相关的基因列表 来自体外极化新生儿Th17淋巴细胞的基因。对这些实验数据进行层次聚类分析 137例BA患者的肝脏RNAseq数据中衍生的Th17候选基因揭示了聚集的 相似的基因表达谱。这些集群之间的血清结合胆红素水平不同，支持 我们的假设。未来的研究将询问由整合的RNAseq-和 具有BA表型和HPE结局的肝脏浸润性T细胞的免疫荧光计数。

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.