Interplay Among NKT Cell Subsets in Alcoholic Liver Disease

描述(申请人提供)：本提案旨在了解导致慢性酒精摄入后肝损伤的关键先天免疫机制(S)。细胞间的相互作用 在肝脏中的天然免疫细胞中，包括NKT细胞对肠道或代谢抗原具有耐受性，同时对病原体的免疫知之甚少。NKT细胞由两个不同的亚群组成，I型和II型，识别CD1d分子呈递的不同脂类抗原。我们鉴定的II型NKT细胞的一个主要亚群识别一种自我糖脂，硫脂。在两种不同的肝损伤模型中，我们发现硫脂介导的II型NKT细胞的激活导致了一种新的免疫调节途径的参与，导致了I型NKT细胞的无能诱导，阻断了炎症级联反应，抑制了组织损伤。在这里，我们假设NKT细胞亚群的差异激活在肝损伤中扮演相反的角色，它们与包括DC和髓系细胞在内的其他固有免疫细胞的相互作用在协调导致ALD的炎症事件中起决定性作用。因此，我们发现，在慢性加暴食Lieber-DeCarli流食的雄性C57BL/6小鼠ALD的临床前阶段，I型而不是II型NKT细胞被部分激活，导致炎症细胞重新进入肝脏。我们的一个中心发现是，给予硫脂或全反式维甲酸(ATRA)后，I型NKT细胞失活可阻断ALD。此外，缺乏I型NKT细胞的Jα18-/-小鼠显著免受肝脏损伤。值得注意的是，肝脏基因表达谱显示骨桥蛋白(OPN)的表达依赖于I型NKT，而硫脂增加了肝脏中过氧化物酶体增殖物-激活物受体-α(PPARα)的水平。分别用αGalCer/CD1d-和硫脂/CD1d-四聚体、胞浆内染色和流式细胞术检测ALD不同时期细胞表面活化标志物和细胞因子分泌的变化。IL-23/IL-17途径和腺苷受体A2aR在I型NKT细胞激活中的作用将被检测。实时定量聚合酶链式反应和酶联免疫吸附试验将被用来检测在酒精喂养后的临床前期和临床阶段肝脏中其他固有细胞的激活情况。过继转移实验或耗竭常规CDC或CD11b+Gr-1+细胞分别在NKT细胞激活和ALD中进行研究。给予全反式维甲酸或硫脂抑制I型活化导致阻断肝损伤的机制将被调查。硫脂、全反式维甲酸、维甲酸 受体γ(RARγ)或其与其他抑制性细胞因子的组合将被用于有效治疗正在进行的肝病。NKT介导的肝脏表达骨桥蛋白和PPARα基因的机制在人类和实验性ALD中都是常见的，将使用siRNA和基因缺陷动物来研究。建议的研究将更好地了解以酒精摄入后NKT细胞亚群激活为中心的关键先天免疫机制，并将为ALD潜在的治疗干预识别新的免疫靶点。

DESCRIPTION (provided by applicant): This proposal aims to understand key innate immune mechanism(s) leading to liver injury following chronic alcohol ingestion. How cellular interactions among innate immune cells in liver, including NKT cells provide both tolerance to gut or metabolic antigens and at the same time immunity against pathogens is poorly understood. NKT cells are comprised of two distinct subsets, type I and type II, and recognize different lipid antigens presented by CD1d molecules. A major subset of type II NKT cells that we identified recognizes a self-glycolipid, sulfatide. In two different models of liver injury we found that sulfatide-mediated activation of type II NKT cells leads to engagement of a novel immune regulatory pathway resulting in anergy induction in type I NKT cells, blockade of the inflammatory cascade, and inhibition of tissue damage. Here we hypothesize that differential activation of NKT cell subsets play opposing roles in liver injury and their interactions with othe innate immune cells, including DCs and myeloid cells, are decisive in orchestrating inflammatory events leading to ALD. Accordingly, we found that during the preclinical phase of ALD following chronic plus binge feeding of Lieber-DeCarli liquid diet in male C57BL/6 mice, type I but not type II NKT cells are partially activated leading to recruitment of inflammatory cells into liver. A central finding we have made is that inactivation of type I NKT cells following administration of sulfatide or all-trans retinoic acid (ATRA) blocks ALD. Furthermore, Jα18-/- mice deficient in type I NKT cells are significantly protected from liver injury. Notably, hepatic gene expression profiling revealed type I NKT-dependence of osteopontin (OPN) expression, while sulfatide enhances peroxisome proliferator-activator receptor-α (PPARα) levels in liver. Cell surface activation markers and the cytokine secretion profile of type I and type II NKT cell subsets during different phases of ALD will be examined using αGalCer/CD1d- and sulfatide/CD1d-tetramers respectively, and intracytoplasmic staining and flow cytometry. A role for IL-23/IL-17 pathway and adenosine receptor A2aR in activation of type I NKT cells will be examined. Real time PCR and Elisa assays will be used to examine activation of other innate cells in liver during preclinical and clinical phases following ethanol feeding. Adoptive transfer experiments or depletion of conventional cDC or CD11b+Gr-1+ cells, respectively in NKT cell activation and in ALD will be studied. Mechanisms by which administration of ATRA or sulfatide inhibits type I activation leading to blockade of liver damage will be investigated. Sulfatide, ATRA, retinoic acid receptor-γ (RARγ) or their combinations along with other inhibitory cytokines will be used to effectively treat ongoing liver disease. Mechanisms of NKT- mediated hepatic expression of OPN and PPARα genes common in both human and experimental ALD will be examined using siRNA and gene-deficient animals. The proposed studies will provide a better understanding of the key innate immune mechanisms centered on activation of NKT cell subsets following ethanol ingestion and should allow identification of novel immune targets for potential therapeutic intervention in ALD.