Acetaminophen is a common pain reliever. It is safe and effective within recommended doses, but an overdose can cause liver injury and failure (AILI) as a result of hepatocyte necrosis and associated inflammation. Kupffer cells (KCs), the resident macrophages of the liver are hypothesized to sense signals from dying hepatocytes and initiate the immune response in AILI, however their precise roles in this are unknown. I have recently identified that KCs exist in two distinct states in AILI, with one population being specifically located in damaged zones. Two KC states are also reflected in the transcriptional profiles of KCs, as assessed by single cell RNA-sequencing with one state characterized by increased expression of genes related to metabolic activation. Thus, here, I aim to investigate the precise functions of these distinct KC states. To this end, I will first perform an in-depth characterization of KCs in AILI in both the murine and human liver. By combining the single cell RNA-seq with spatial transcriptomics approaches, I will then investigate the signals inducing this second state of KCs and investigate their specific origins. Finally, I aim to harness the knowledge gained alongside KC-specific mouse models to specifically assess the functions of these activated KCs in AILI, potentially enabling the development of novel strategies for the treatment of AILI, through the manipulation of these cells.