Human Group 2 Innate Lymphocyte Cell (ILC2) populations contribute to Hepatic Fibrosis

 描述（由申请人提供）：肝纤维化是慢性肝病的后果，是死亡和发病的主要原因。小鼠模型研究已证明白细胞介素-33（IL-33）驱动的第2组先天淋巴细胞（IL 2C）扩增和激活在肝纤维化中发挥着不可或缺的作用。在人类中，ILC的作用尚未明确定义，并且先前未研究过人类肝脏ILC 2群体。我们提供了初步的数据，建立了IL-13表达ILC 2人群在正常和肝硬化的人肝脏中的存在，并建议，自然杀伤T（NKT）细胞参与调控ILC 2在这个网站。我们假设：由发炎肝脏中活化的NKT细胞产生的IL-33驱动肝ILC 2的扩增和活化，其通过对肝星状细胞（HSC）和巨噬细胞的IL-13依赖性作用促进纤维化。三个具体目标是针对测试这一假设：在具体目标1中，我们将检查ILC 2在健康和肝病人肝脏中的水平、表型和功能特征。除了直接离体流式细胞术分析之外，还将使用已建立的方案从组织中分离和扩增ILC 2。将采用趋化性、多重ELISA、流式细胞术和RT-PCR测量IL-33对迁移、细胞因子/趋化因子产生、基因调控和IL-33（ST 2）受体表达的影响。Specific Aim 2旨在探索ILC 2对HSC活化和单核细胞成熟的直接作用。将使用免疫组织化学、流式细胞术和共培养实验将ILC 2水平与纤维化标志物/阶段和促纤维化枯否细胞和/或替代活化的巨噬细胞的积累/活化相关联。在具体目标3中，我们将探索IL-33、NKT和ILC 2在体内对纤维化的调节。我们将使用AFC 8-hu HSC/Hep模型，该模型包含人肝细胞和人免疫系统。在该模型中纤维化是可诱导的，我们建议在纤维化的各个阶段检查ILC 2、NKT、HSC活化和人纤维化基因的表达。由于该模型需要造血来源的细胞的共移植以发展纤维化，我们将在存在和不存在sST 2的情况下选择性地转移ILC 2/NKT，以剖析这些组分中的每一种对肝脏纤维化的贡献。与健康肝脏相比，患病肝脏中人ILC 2的深入表征将描述其在病理学中的作用，并确定靶向该人群是否代表预防/治疗肝纤维化的有效新策略。

 DESCRIPTION (provided by applicant): Liver fibrosis a consequence of chronic liver disease is a major cause of mortality and morbidity. Studies in mouse models have demonstrated an integral role for interleukin-33 (IL-33) driven group 2 innate lymphocyte cell (IL2C) expansion and activation in hepatic fibrosis. In humans, the role of ILCs is not well defined and human hepatic ILC2 populations have not previously been investigated. We provide preliminary data that establishes the presence of IL-13 expressing ILC2 populations in normal and cirrhotic human liver and to suggest that natural killer T (NKT) cells are involved in the regulation of ILC2s at this site. We hypothesize that: IL-33 produced by activated NKT cells in the inflamed liver drives expansion and activation of hepatic ILC2s which promote fibrosis through IL-13-dependent effects on hepatic stellate cells (HSCs) and macrophages. Three specific aims are directed at testing this hypothesis: In Specific Aim 1 we will examine the levels, phenotype and functional characteristics of ILC2s in healthy and cirrhotic human liver. In addition to direct ex vivo flow cytometric analysis, ILC2s will be isolated and expanded from tissue using an established protocol. The effects of IL-33 on migration, cytokine/chemokine production, gene regulation, and IL-33 (ST2) receptor expression will be measured with chemotaxis, multiplex-ELISA, flow cytometry and RT-PCR. Specific Aim 2 is designed to explore the direct effects of ILC2s on the activation of HSCs and monocyte maturation. Immunohistochemistry, flow cytometry and co-culture experiments will be used to correlate ILC2 levels with fibrosis markers/stage and accumulation/ activation of pro-fibrogenic kupffer cells and/or alternatively activated macrophages. In Specific Aim 3 we will explore the regulation of fibrosis by IL-33, NKTs and ILC2s in vivo. We will use the AFC8-hu HSC/Hep model which contains human liver cells and a human immune system. Fibrosis is inducible in this model and we propose to examine ILC2s, NKTs, HSC activation and expression of human fibrogenic genes at various stages of fibrosis. As this model requires co-transplantation of cells of haematopoietic origin to develop fibrosis, we will selectively transfer ILC2s/NKTs in the presence and absence of sST2 to dissect the contribution of each of these components to fibrosis in the liver. In depth characterization of human ILC2s in diseased compared to healthy liver will delineate their role in pathology and determine if targeting this population represents an effective novel strategy to prevent/treat hepatic fibrosis.