Cholestatic Liver Injury

项目总结 总体目标是确定导致胆汁淤积性肝损伤的细胞机制。肾小管 淤胆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 重点关注DR细胞凋亡作为减轻淤胆性肝损伤的潜在治疗策略 纤维化症。为此，我们提出了几点关键意见。我们观察到肿瘤坏死因子- 相关的凋亡诱导配体(TRAIL)受体是一种死亡受体，限制了DR细胞的活动范围 通过诱导DR细胞凋亡在胆汁淤积症小鼠模型(mdr2-/-小鼠)中的体内种群。这 观察表明，DR细胞可能被认为是“准备好”进行凋亡的。随着巨噬细胞表达 TRAIL，我们接下来检查了DR细胞是否通过代偿性逆调节过程促进巨噬细胞 TRAIL表达从而限制了它们的扩展。EpCAM阳性反应性胆管细胞有机物 Mdr2-/-小鼠是我们建立的DR细胞模型。ERC类有机物释放细胞外小泡 (EVS)含有促进巨噬细胞TRAIL表达的货物。具体地说，电动汽车包含S100A11，一款 损伤相关分子模式(DAMP)蛋白，与清道夫受体(SR)结合 巨噬细胞。因此，一种名为Mer受体酪氨酸激酶(MerTK)的SR抑制剂减弱 EVS对巨噬细胞TRAIL的诱导。准备进行TRAIL细胞毒性的细胞对BH3模拟物敏感 靶向髓样细胞白血病1(MCL1)，它是Bcl-2蛋白家族中支持生存的成员。始终如一地，ERC 通过针对MCL1的BH3模拟物S63845，有机物发生了凋亡。根据这些初步数据， 我们提出了一个中心假设，即DR细胞促进了代偿性的反调节 通过诱导巨噬细胞TRAIL表达来限制其扩张的过程，并为细胞做好准备 可用于治疗以减轻淤胆性肝损伤的死亡。我们现在将雇用 现有的和互补的实验方法，以及检验这一假设的临床前模型。我们的 具体目标将检验三个假设。首先，我们将检验TRAIL/TRAIL受体的假设 使用条件性基因敲除小鼠的信号，通过以下方式直接抑制胆汁淤积中的DR细胞数量 机制依赖于：a)DR细胞TRAIL受体的表达；b)TRAIL的表达 巨噬细胞。第二，我们将检验ERC类有机物释放EVS诱导TRAIL的假设 巨噬细胞的表达机制依赖于：a)EV Cargo S100A11；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：a)生存取决于MCL1在体外的抗凋亡功能；以及b) 体内药物靶向MCL1可诱导DR细胞凋亡，从而减轻肝损伤和肝纤维化。 这一技术和概念上的创新应用也具有重要意义，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脏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.