Role of Bone Marrow-Derived Myeloid Cells in Alcohol Liver Disease

描述(申请人提供)：晚期酒精性肝病(ALD)的唯一有效治疗方法是肝移植。为了促进治疗的发展，必须更好地了解其发病机制。有证据表明，肝巨噬细胞(MACs)起着重要的作用；然而，迄今为止的研究还没有区分常驻MACs，Kupffer细胞(KCs)和浸润性骨髓来源的髓样细胞(BMMCs)，后者在病理条件下补充KCs。我们检测了酒精处理小鼠肝脏中的骨髓单个核细胞(BMMC)，并将它们与KCs区分开来。我们假设在ALD期间招募到肝脏的BMMCs具有不同的表型和功能，这取决于ALD的微环境和严重程度。本研究的具体目的是：(1)探讨酒精处理后肝BMMC蓄积的机制。A)取乙醇喂养小鼠的肝脏，检测IL-1b、IL-6、S100A8/A9、HSP72和COX-2的表达水平。B)用中和抗体和IL-1R-/-小鼠研究IL-1b和S100A8/A9在BMMCs肝脏蓄积中的作用。C)STAT3hep/-小鼠将被用来研究肝细胞特异的STAT3信号在酒精暴露后BMMC在肝脏中积累的作用。目的2、检测轻、晚期ALD患者骨髓单个核细胞的表型和功能。A)用含5%乙醇的Lieber-DeCarli饮食长期喂养小鼠，模拟轻度ALD。利用该模型，我们将探讨BMMCs在促进酒精性微循环障碍修复中的作用，以及血管内皮生长因子和基质金属蛋白酶在其中的作用。在这些研究中，CD11b-DTR小鼠和抗Gr-1抗体将被用来耗尽骨髓单个核细胞。B)一种混合模型，在该模型中，小鼠随意喂食“西方饮食”，并灌胃注入乙醇，这种混合模型具有#年发现的酒精性脂肪性肝炎(ASH)和肝纤维化的特征 晚期酒精性肝病患者。该模型将用于检测BMMCs的表型，并探讨其在肝损伤中的作用。(目的3)剖析轻、晚期ALD患者BMMC表型的分子调控。A)在ALD的混合模型中，将测量全身和肝脏的内毒素和损伤相关分子模式(DAMP)分子的水平。B)在混合模型中，将通过给予小鼠利福昔明抗生素和DAMPS的抑制剂来研究内毒素和DAMPS在BMMCs的促炎激活和肝损伤中的作用。C)轻度ALD和ASH患者BMMC中STAT3的激活和STAT1/NF-kB的激活将分别被研究。

DESCRIPTION (provided by applicant): The only effective treatment for advanced alcohol liver disease (ALD) is liver transplantation. It is imperative to better understand the pathogenesi in order to advance therapeutic development. Evidence suggests that hepatic macrophages (Macs) play an important role; however research to date has not distinguished resident Macs, Kupffer cells (KCs), from infiltrating bone marrow-derived myeloid cells (BMMCs), which replenish KCs during pathological conditions. We detected BMMCs and distinguished them from KCs in the liver of mice treated with alcohol. We hypothesize that the BMMCs recruited into the liver during ALD assume different phenotypes and functions, depending on the microenvironment and severity of ALD. The Specific Aims of the proposed studies are: (Aim 1), investigate the mechanisms of hepatic BMMC accumulation as result of alcohol treatment. a) Livers from ethanol-fed mice will be harvested to examine message expression levels of IL-1b, IL-6, S100A8/A9, HSP72, and COX-2. b) The specific involvement of IL-1b and S100A8/A9 in hepatic accumulation of BMMCs will be investigated by using neutralizing antibodies and IL-1R-/- mice. c) The STAT3hep/- mice will be utilized to examine the role of hepatocyte-specific STAT3 signaling in BMMC accumulation in the liver after alcohol exposure. (Aim 2), Determine the phenotypes and functions of BMMCs in mild and advanced ALD. a) Chronic feeding of mice with 5% ethanol-containing Lieber-Decarli diet simulates mild ALD. Using this model, we will investigate the function of BMMCs in promoting the repair of alcohol-induced microcirculation disorder, and examine the roles of VEGF and MMPs in mediating such function. The CD11b-DTR mice and anti-Gr-1 antibody will be employed to deplete BMMCs in these studies. b) A hybrid model, in which mice are fed ad libitum with "Western diet" and intragastrically infused with ethanol, shares characteristics of alcoholic steatohepatitis (ASH) and liver fibrosis found in patients with advanced ALD. This model will be employed to examine the phenotype of BMMCs, and investigate their contribution to liver injury. (Aim 3), Dissect the molecular regulations of BMMC phenotypes in mild and advanced ALD. a) The systemic and hepatic levels of endotoxin and damage-associated molecular pattern (DAMP) molecules will be measured in the hybrid model of ALD. b) The contributions of endotoxin and DAMPs to pro-inflammatory activation of BMMCs and liver damage in the hybrid model will be investigated by administering mice with rifaximin antibiotic, and inhibitors of DAMPs. c) STAT3 activation and STAT1/NF-kB activation in BMMCs associated with mild ALD and ASH, respectively, will be investigated.