Senescent hepatocytes mediate reprogramming of immune cells in acute liver failure

项目总结 急性肝功能衰竭(ALF)的特点是凝血障碍、肝性脑病、黄疸和多器官损害。 失败了。在美国和英国，50%的ALF病例是由于扑热息痛(APAP)过量所致。 超生理剂量的APAP压倒了肝脏中的安全解毒途径，导致过量 将APAP生物活化为高活性代谢物N-乙酰-对苯二酚亚胺(NAPQI)。 这引发了一系列细胞内事件，导致肝细胞死亡。广泛的工作内容包括 阐明了APAP过量时肝细胞坏死的分子机制，但详细的机制 在ALF的发展中所起的作用在很大程度上是未知的。肝损伤后的先天免疫反应 在APAP过量的人相关小鼠模型中，损伤在肝脏恢复中起着关键作用。然而， 免疫细胞功能障碍参与了ALF的发生发展。尽管压力过大之间的串扰 肝细胞和免疫细胞积极支持肝脏恢复，持续和长期的肝细胞应激 可能会改变这种通讯网络，导致免疫细胞重新编程 促进ALF的表型。过度的细胞应激可引起暂时或永久性的细胞周期停滞，细胞 命运的决定叫衰老。一些细胞获得衰老相关的分泌表型(SASP) 它可以对附近的细胞和组织微环境产生多效性效应。我们的初步数据 证明KLF6和p21在APAP严重过量后诱导肝细胞衰老，并且这些 肝细胞有一种活跃的SASP，富含在循环中持续存在的细胞因子CxCl14。这些关键字 研究结果使我们假设，在严重的APAP诱导的肝损伤时，肝细胞衰老延长 导致持续接触富含CxCl14的分泌体，导致免疫细胞重编程受阻 组织修复和损伤解决。这一假设将通过追求两个具体目标来检验。实验在 目的1研究KLF6对p21和Cxcl14的诱导作用及体内沉默的影响。 APAP诱导的衰老和损伤后肝脏恢复的研究。AIM 2将使用单细胞RNA测序 与湿实验室方法相结合，定义关键SASP组件Cxcl14如何改变全球 严重APAP过量后衰老的肝细胞和免疫细胞之间的通讯网络。 总的来说，这些数据将确定持续诱导肝细胞的分子机制。 衰老及其与诱导衰老相关分泌表型的关系，以及信号转导 衰老肝细胞与先天免疫反应的相互作用。这项拟议的工作将提供 对APAP严重过量后发生ALF的有价值的见解，将有助于解决这一未满足的问题 临床需要。

PROJECT SUMMARY Acute liver failure (ALF) is characterized by coagulopathy, hepatic encephalopathy, jaundice and multi-organ failure. Fifty percent of all ALF cases in the United States and UK are due to acetaminophen (APAP) overdose. Supraphysiological doses of APAP overwhelm safe detoxification pathways in the liver leading to excessive bioactivation of APAP by Cyp2e1 into the highly reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). This initiates a cascade of intracellular events driving hepatocyte death. An extensive body of work has elucidated the molecular mechanisms of hepatocyte necrosis during APAP overdose, but detailed mechanisms involved in the development of ALF have been largely unexplored. The innate immune response after liver injury plays a critical role in liver recovery in the human relevant mouse model of APAP overdose. However, immune cell dysfunction has been implicated in the development of ALF. Though crosstalk between stressed hepatocytes and immune cells actively supports liver recovery, persistent and prolonged hepatocyte stress may alter this communication network, resulting in immune cell reprogramming towards detrimental phenotypes which facilitate ALF. Excessive cell stress can elicit temporary or permanent cell cycle arrest, a cell fate decision called senescence. Some cells acquire a senescent associated secretory phenotype (SASP) which can elicit pleotropic effects on nearby cells and the tissue microenvironment. Our preliminary data demonstrate that Klf6 and p21 induce hepatocyte senescence after a severe APAP overdose and that these hepatocytes have an active SASP, enriched in the cytokine Cxcl14 which persists in circulation. These key findings lead us to hypothesize that upon severe APAP-induced liver injury, prolonged hepatocyte senescence results in a sustained secretome exposure enriched in Cxcl14 causing immune cell reprogramming impeding tissue repair and injury resolution. This hypothesis will be tested by pursuit of two specific aims. Experiments in Aim 1 are designed to evaluate how Klf6 induces p21 and Cxcl14 and the influence of in vivo silencing of Klf6 on APAP-induced senescence and liver recovery after injury. Aim 2 will use single-cell RNA sequencing in combination with wet-lab approaches to define how the critical SASP component Cxcl14 alters the global communication network between senescent hepatocytes and immune cells after a severe APAP overdose. Collectively, these data will define the molecular mechanisms responsible for sustained induction of hepatocyte senescence, its relationship to the induction of a senescent associated secretory phenotype, and the signaling interactions between senescent hepatocytes and the innate immune response. This proposed work will provide valuable insights into why ALF develops after severe APAP overdose and will assist in addressing this unmet clinical need.