Additional Relevant Details Acetaminophen (APAP, paracetamol) poisoning is a leading cause of acute liver failure (ALF) in humans and induces hepatocyte necrosis, followed by the activation of the innate immune system further aggravating liver injury. The role of infiltrating monocytes during the early phase of ALF is still ambiguous. Upon experimental APAP overdose in mice, monocyte-derived macrophages (MoMF) massively accumulate in injured liver within 12-24h, while the number of tissue-resident macrophages (Kupffer cells) decreases. The influx of MoMF is dependent on the chemokine receptor CCR2, since Ccr2-/- mice display reduced infiltration of monocytes and attenuated liver injury after APAP overdose at early time-points. As evidenced by intravital multiphoton microscopy of Ccr2-reporter mice, CCR2+ monocytes infiltrate liver as early as 8-12h after APAP overdose and form dense cellular clusters around necrotic areas. The CCR2+ MoMF express a distinct pattern of inflammatory but also repair-associated genes in injured livers. Adoptive transfer experiments revealed that MoMF primarily exert pro-inflammatory functions early after APAP, thereby aggravating liver injury. Consequently, early pharmacological inhibition of either CCL2 (by the inhibitor mNOX-E36) or CCR2 (by the orally available dual CCR2/CCR5 inhibitor cenicriviroc, CVC) reduces monocyte infiltration and APAP-induced liver injury in mice. Importantly, neither the early nor continuous inhibition of CCR2 impair repair processes during resolution from injury. In line, human livers of ALF patients requiring liver transplantation reveal increased CD68+ hepatic macrophage numbers with massive infiltrates of periportal CCR2+ macrophages that display a pro-inflammatory polarization. Conclusion: Infiltrating monocyte-derived macrophages aggravate APAP hepatotoxicity, and the pharmacological inhibition of either CCL2 or CCR2 might bear therapeutic potential by reducing the inflammatory reaction during the early phase of APAP-induced liver injury. This article is protected by copyright. All rights reserved.