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.