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

 描述(申请人提供)：肝纤维化是慢性肝病的后果，是死亡率和发病率的主要原因。在小鼠模型上的研究表明，白介素33(IL-33)驱动的第二组天然淋巴细胞(IL2C)的增殖和激活在肝纤维化中起着不可或缺的作用。在人类中，ILC的作用还没有很好的定义，而且以前还没有对人类肝脏ILC2群体进行研究。我们提供的初步数据证实，在正常人和肝硬变患者的肝脏中存在表达IL-13的ILC2群体，并提示自然杀伤T(NKT)细胞参与了该部位ILC2的调节。我们推测：炎症肝脏中活化的NKT细胞产生的IL-33驱动肝脏ILC2s的扩张和激活，而ILC2s通过对肝星状细胞(HSCs)和巨噬细胞的IL-13依赖作用促进纤维化。有三个特定目的旨在验证这一假说：在特定目标1中，我们将检测正常人和肝硬变患者肝脏中ILC2s的水平、表型和功能特征。除了直接的体外流式细胞术分析外，还将使用一种既定的方案从组织中分离和扩增ILC2。IL-33对细胞迁移、细胞因子/趋化因子产生、基因调控和IL-33(ST2)受体表达的影响将通过趋化、多重-ELISA、流式细胞术和RT-PCR来检测。特异靶2旨在探讨ILC2s对HSCs激活和单核细胞成熟的直接影响。免疫组织化学、流式细胞术和共培养实验将被用来将ILC2水平与纤维化标志物/分期以及促纤维化的库普弗细胞和/或交替激活的巨噬细胞的聚集/激活联系起来。在具体目标3中，我们将探讨IL-33、NKT和ILC2对体内纤维化的调节作用。我们将使用AFC8-Hu HSC/HEP模型，该模型包含人类肝细胞和人类免疫系统。在这个模型中，纤维化是可诱导的，我们建议在纤维化的不同阶段检测ILC2、NKT、HSC的激活和人类纤维化基因的表达。由于这一模型需要造血细胞的共移植才能形成纤维化，我们将选择性地在Sst2存在和不存在的情况下转移ILC2s/NKT，以分析这些成分中的每一种在肝纤维化中的作用。深入研究人类ILC2在疾病和健康肝脏中的特征，将勾勒出它们在病理学中的作用，并确定以这一人群为目标是否代表了预防/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.