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

 描述（由申请人提供）：慢性肝病导致的肝纤维化是死亡和发病的主要原因。小鼠模型研究表明，白细胞介素 33 (IL-33) 驱动的第 2 组先天淋巴细胞 (IL2C) 扩张和激活在肝纤维化中发挥着不可或缺的作用。在人类中，ILC 的作用尚不明确，并且之前也没有研究过人类肝脏 ILC2 群体。我们提供了初步数据，确定正常和肝硬化人类肝脏中存在表达 IL-13 的 ILC2 群体，并表明自然杀伤 T (NKT) 细胞参与该位点 ILC2 的调节。我们假设：发炎肝脏中激活的 NKT 细胞产生的 IL-33 驱动肝脏 ILC2 的扩张和激活，从而通过 IL-13 对肝星状细胞 (HSC) 和巨噬细胞的依赖性作用促进纤维化。三个具体目标旨在检验这一假设： 在具体目标 1 中，我们将检查健康和肝硬化人类肝脏中 ILC2 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，还将使用既定方案从组织中分离和扩增 ILC2。 IL-33 对迁移、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性、多重 ELISA、流式细胞术和 RT-PCR 进行测量。具体目标 2 旨在探索 ILC2 对 HSC 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用于将 ILC2 水平与纤维化标记物/阶段以及促纤维化库普弗细胞和/或替代激活的巨噬细胞的积累/激活相关联。在具体目标 3 中，我们将探索体内 IL-33、NKT 和 ILC2 对纤维化的调节。我们将使用 AFC8-hu HSC/Hep 模型，其中包含人类肝细胞和人类免疫系统。纤维化在该模型中是可诱导的，我们建议检查 ILC2、NKT、HSC 激活和人类纤维化基因在纤维化各个阶段的表达。由于该模型需要共同移植造血来源的细胞来发展纤维化，因此我们将在存在和不存在 sST2 的情况下选择性地转移 ILC2/NKT，以剖析这些成分中的每一个对肝脏纤维化的贡献。与健康肝脏相比，对患病肝脏中的人类 ILC2 进行深入表征将描述它们在病理学中的作用，并确定针对该人群是否代表了预防/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.