To elucidate the spatiotemporal dynamics of Kupffer cells (KCs) in acetaminophen (APAP)-induced acute drug-induced liver injury and their regulatory mechanisms governing regional hepatocyte regeneration.

A Clec4f-iCre & Rosa26-tdTomato lineage-tracing mouse model was established to specifically label KCs. Acute liver injury was induced via intraperitoneal injection of acetaminophen (APAP, 400 mg/kg). Liver tissues and blood samples were collected at 0, 1, 2, 3 and 7 days post-APAP administration. Histopathological examinations (H & E staining, TUNEL assay for apoptosis detection), serum biochemical analyses (ALT and AST), and immunofluo-rescence staining were employed to find out about the spatiotemporal migration patterns of KCs, their phenotypic transition, and spatiotemporal correlations between KC dynamics and hepatocyte regeneration.

On day 1 (D1) post-APAP injury, extensive hepatocyte necrosis and apoptosis were observed in the pericentral (PC) zone, accompanied by a reduction in KCs count and their migration from the periphery toward the necrotic core. From D2 onward, repair started, with the necrotic area progressively decreasing. By D3, inflammatory cell infiltration was pronounced in the PC zone, concurrent with a peak in hepatocyte proliferation (Ki67⁺ cells: approximately 22%, P < 0.05). During repair, monocytes (CD11b⁺ IBA-1^-)differentiated into monocyte-derived macrophages (CD11b⁺ IBA-1⁺), which synergistically promoted the regeneration of cytochrome P450 2E1 (CYP2E1⁺) hepatocytes in the pericentral zone. By D7, the hepatic lobular architecture was largely restored, indicating the completion of reconstruction of metabolic zonation.

KCs can regulate hepatocyte regeneration through spatiotemporal dynamics and phenotypic transitions of monocyte-derived macrophages. These findings underscore the pivotal role of macrophage spatiotemporal reprogramming in liver injury repair and may provide data for developing macrophage-targeted therapeutic interventions in hepatic injury.