Cholestatic Liver Injury

项目摘要 总体目标是确定导致胆汁淤积性肝损伤的细胞机制。导管 胆汁淤积性肝损伤中产生的反应性（DR）细胞群促进肝纤维化。我们的建议是 关注DR细胞凋亡作为减轻胆汁淤积性肝损伤的潜在治疗策略， 纤维化为此，我们提出了几个关键的意见。我们观察到肿瘤坏死因子- 相关凋亡诱导配体（TRAIL）受体（TR），一种死亡受体，限制了DR细胞凋亡的程度。 通过诱导DR细胞凋亡，在胆汁淤积的鼠模型（Mdr 2-/-小鼠）中体内培养DR细胞群。这 观察结果表明DR细胞可被认为是细胞凋亡的“启动”细胞。当巨噬细胞表达 我们接下来检查DR细胞是否通过代偿性反调节过程可以促进巨噬细胞 TRAIL表达从而限制了它们的扩增。EpCAM阳性反应性胆管细胞（ERC）类器官 来自Mdr 2-/-小鼠的细胞作为DR细胞模型。ERC类器官释放细胞外囊泡 (EVs)其含有促进巨噬细胞TRAIL表达的货物。具体地，EV包含S100 A11， 损伤相关分子模式（DAMP）蛋白，其结合清道夫受体（SR）， 巨噬细胞因此，称为Mer受体酪氨酸激酶（MerTK）的SR抑制剂减弱了 EV在巨噬细胞中的TRAIL诱导。针对TRAIL细胞毒性致敏的细胞对BH 3模拟物敏感 靶向骨髓细胞白血病1（Mcl 1），Bcl-2蛋白家族的促生存成员。一致性，ERC 类器官通过靶向Mcl 1的BH 3模拟物S63845经历凋亡。根据这些初步数据， 我们提出了一个中枢假说，即DR细胞促进了一种代偿性的反调节作用， 通过诱导巨噬细胞TRAIL表达来限制它们的扩增，并为细胞 死亡，其可以在治疗上用于减轻胆汁淤积性肝损伤。我们现在将雇用 目前和补充的实验方法，和临床前模型来检验这一假设。我们 具体目标将检验三个假设。首先，我们将检验TRAIL/TRAIL受体 使用条件性基因敲除小鼠，通过以下方式直接抑制胆汁淤积中的DR细胞群： 依赖于以下的机制：a）DR细胞的TRAIL受体表达;和B）DR细胞的TRAIL表达。 巨噬细胞其次，我们将测试ERC类器官释放诱导TRAIL的EV的假设 通过依赖于以下机制的机制在巨噬细胞中表达：a）EV货物S100 A11;和B）MerTK 激活巨噬细胞。最后，我们将检验ERC类器官和DR细胞被启动的假设。 对于细胞死亡，使得：a）存活依赖于Mcl 1在体外的抗凋亡功能;和B） 体内Mcl 1的药理学靶向诱导DR细胞凋亡，从而减轻肝损伤和纤维化。 这种技术和概念上的创新应用也很重要，因为它可以识别 有目的地诱导DR细胞凋亡以限制胆汁淤积性肝和纤维化的治疗策略。

PROJECT SUMMARY The overall objective is to define the cellular mechanisms that contribute to cholestatic liver injury. The ductular reactive (DR) cell population arising in cholestatic liver injury promotes hepatic fibrosis. Our proposal is focused on DR cell apoptosis as a potential therapeutic strategy to attenuate cholestatic liver injury and fibrosis. To this end, we have made several pivotal observations. We observed that tumor necrosis factor- related apoptosis-inducing ligand (TRAIL) receptor (TR), a death receptor, limits the extent of the DR cell population in vivo in a murine model of cholestasis (Mdr2-/- mice) by inducing DR cell apoptosis. This observation indicates that DR cells may be considered “primed” for apoptosis. As macrophages express TRAIL, we next examined if DR cells by a compensatory counter regulatory process may promote macrophage TRAIL expression thereby limiting their expansion. EpCAM positive reactive cholangiocyte (ERC) organoids from Mdr2-/- mice were developed by us as a DR cell model. ERC organoids release extracellular vesicles (EVs) which contain cargo promoting macrophage TRAIL expression. Specifically, the EVs contain S100A11, a damage-associated molecular pattern (DAMP) protein, which binds scavenger receptors (SR) on macrophages. Accordingly, an inhibitor of the SR termed Mer receptor tyrosine kinase (MerTK) attenuated TRAIL-induction in macrophages by the EVs. Cells primed for TRAIL cytotoxicity are sensitive to BH3 mimetics targeting myeloid cell leukemia 1 (Mcl1), a pro-survival member of the Bcl-2 protein family. Consistently, ERC organoids underwent apoptosis by a BH3 mimetic targeting Mcl1, S63845. Based on these preliminary data, we formulated the CENTRAL HYPOTHESIS that DR cells promote a compensatory counter regulatory process to limit their expansion by inducing macrophage TRAIL expression, and are primed for cell death which can be exploited therapeutically to attenuate cholestatic liver injury. We will now employ current and complementary experimental approaches, and preclinical models to examine this hypothesis. Our SPECIFIC AIMS will test three hypotheses. FIRST, we will test the hypothesis that TRAIL/TRAIL receptor signaling, using conditional knockout mice, directly restrains the DR cell population in cholestasis by mechanisms dependent upon: a) TRAIL receptor expression by DR cells; and b) TRAIL expression by macrophages. SECOND, we will test the hypothesis that ERC organoids release EVs inducing TRAIL expression in macrophages by mechanisms dependent upon: a) the EV cargo S100A11; and b) MerTK activation on macrophages. FINALLY, we will test the hypothesis that ERC organoids and DR cells are primed for cell death such that: a) survival is dependent upon the anti-apoptotic function of Mcl1 in vitro; and b) pharmacologic targeting of Mcl1 in vivo induces DR cell apoptosis thereby attenuating liver injury and fibrosis. This technically and conceptually innovative application, is also significant because it may identify therapeutic strategies for purposefully inducing DR cell apoptosis to limit cholestatic liver and fibrosis.