Pathobiology of Liver Injury

项目摘要。我的长期职业目标是确定肝脏炎症的机制， 非酒精性脂肪性肝炎（NASH），美国最流行的慢性肝病。 NASH的特征在于内质网（ER）应激，其导致促炎性细胞因子的释放。 细胞外囊泡（EV）来自脂质过载（脂毒性）肝细胞。被征聘人员的贡献 单核细胞源性巨噬细胞（MDM）与肝内巨噬细胞（IHM）池的比例增加， 募集的MDM在炎症中起关键作用。目前的建议审查了机械联系 肝细胞衍生EV和特定MDM亚群之间的关系。在初步研究中，我们已经确定， 脂毒性ER应激激活的核糖核酸内切酶，肌醇需要酶1 α（IRE 1 α）和 其靶转录因子X-box结合蛋白1（X-box binding protein 1，XBP 1）上调S100 A11的表达。这 S100 A11的上调与组蛋白3赖氨酸27的激活、增强子区域的乙酰化有关 S100A11脂毒性肝细胞释放含S100 A11的EV（S100 A11-EV）。S100 A11-EV激活 晚期糖基化终产物（AGEs）信号转导的巨噬细胞受体。肝细胞表达 NASH中S100 A11和MDM表达上调。沉默S100 A11降低MDM- NASH相关肝脏炎症。根据这些原始的初步数据，我们制定了 脂毒性肝细胞释放S100 A11-EV，其激活一个亚群， 促炎因子表达促进NASH发病的IHM。因此，这一目标 建议是了解：i）S100 A11-EV如何被肝细胞释放; ii）S100 A11-EV如何被肝细胞激活。 巨噬细胞亚群中的脂毒性EV;和iii）肝脏炎症可以通过抑制 S100 A11-EV-1000信令？拟议的实验将采用互补的体外和体内 脂肪毒性和NASH模型，以及药理学、分子和遗传学方法来解决这三个问题。 综合假设。首先，我们将直接检验脂毒性肝细胞释放S100 A11的假设。 a）通过结合和募集组蛋白乙酰化，由XBP 1驱动的S100 A11的转录上调 B）通过S100 A11选择性货物分选进入脂毒性EV。第二，我们将 直接检验巨噬细胞亚群中的Ev被脂毒性EV激活的假设a）通过 产生多价的信号感受态寡聚体，和B）导致以下的亚组的积累 肝脏中表达促炎性细胞因子的巨噬细胞。第三，我们将直接检验假设， 中断S100 A11-EV诱导的NASH活化减弱鼠NASH a）通过减少 S100 A11-EV，和B）当巨噬细胞吞噬细胞缺失时。此R 01补助金申请将产生机械 深入了解NASH中肝细胞与巨噬细胞的相互作用，从而鉴定潜在的药物靶点，例如， S100 A11或S100 A12的抑制剂。

PROJECT ABSTRACT. My long term career objective is to define the mechanisms of liver inflammation in nonalcoholic steatohepatitis (NASH), the most-prevalent chronic liver disease in the United States of America. NASH is characterized by endoplasmic reticulum (ER) stress, which results in release of proinflammatory extracellular vesicles (EVs) from lipid overloaded (lipotoxic) hepatocytes. The contribution of recruited monocyte-derived macrophages (MDMs) to the intrahepatic macrophage (IHM) pool increases in NASH and the recruited MDMs play a pivotal role in inflammation. The current proposal examines the mechanistic link between hepatocyte-derived EVs and specific MDM subsets. In preliminary studies we have identified that activation of the lipotoxic ER stress activated endoribonuclease, inositol requiring enzyme 1 alpha (IRE1α) and its target transcription factor X-box binding protein 1 (XBP1), upregulates the expression of S100A11. This upregulation of S100A11 is associated with activating, histone 3 lysine 27, acetylation in the enhancer region of S100A11. Lipotoxic hepatocytes release S100A11 containing EVs (S100A11-EVs). S100A11-EVs activate macrophage receptor for advanced glycation endproducts (RAGE) signaling. Hepatocellular expression of S100A11 and MDM expression of RAGE are upregulated in NASH. Silencing S100A11 decreases MDM- associated hepatic inflammation in NASH. Based on these original preliminary data, we have formulated the CENTRAL HYPOTHESIS that lipotoxic hepatocytes release S100A11-EVs which activate a subset of proinflammatory RAGE expressing IHMs promoting NASH pathogenesis. Therefore, the goals of this proposal are to understand: i) how S100A11-EVs are released by hepatocytes; ii) how RAGE is activated by lipotoxic EVs in a subset of macrophages; and iii) can hepatic inflammation be attenuated by inhibiting S100A11-EV-RAGE signaling? The proposed experiments will employ complementary in vitro and in vivo models of lipotoxicity and NASH, and pharmacological, molecular and genetic approaches to address three integrated hypotheses. First we will directly test the hypothesis that lipotoxic hepatocytes release S100A11- EVs a) by XBP1 driven transcriptional upregulation of S100A11 by binding to and recruiting histone acetylating factors to an enhancer region, and b) by selective cargo sorting of S100A11 into lipotoxic EVs. Second we will directly test the hypothesis that RAGE is activated in a subset of macrophages by lipotoxic EVs a) by generating multivalent signal competent RAGE oligomers, and b) leads to the accumulation of a subset of proinflammatory RAGE expressing macrophages in the liver. Third we will directly test the hypothesis that interrupting S100A11-EV induced RAGE activation attenuates murine NASH a) by reducing the release of S100A11-EVs, and b) when macrophage RAGE is deleted. This R01 grant application will yield mechanistic insights into hepatocyte-to-macrophage crosstalk in NASH, thus identifying potentially druggable targets, e.g., inhibitors of S100A11 or RAGE.