Background C-C motif chemokine ligand 2 (CCL2), the most upregulated chemokine in injured LSECs, leads to hepatic C-C motif chemokine receptor 2 (CCR2)⁺ monocyte-derived macrophage accumulation. Therefore, we performed pharmacological CCL2-CCR2 inhibition and examined how CCL2-CCR2 inhibition restored hepatic immune landscapes related to fibrogenesis.

Methods Liver injury and liver fibrosis were induced by carbon tetrachloride (CCl₄) in wild-type mice, LSEC-specific Ccl2 deficiency mice, and Ccr2 knockout mice. Murine intrahepatic leukocytes were isolated from olive oil/CCl₄-treated wild-type mice for mass cytometry by time-of-flight (CyTOF) analysis. Liver and primary mouse LSEC gene expression was analyzed using a PCR array and RNA sequencing. Cenicriviroc (CVC) was administrated to fibrotic mice in prevention and treatment models. Single-cell RNA sequencing was performed on the livers of olive oil/CCl₄/CVC-treated mice.

Results CCL2 was the most upregulated chemokine in injured LSECs compared to control LSECs. The CCL2 secreted by injured LSECs leads to CCR2⁺ monocyte-derived macrophage accumulation in CCl₄-induced murine livers as determined by CyTOF. However, CCl₄-mediated liver fibrosis and macrophage accumulation were reduced in LSEC-specific Ccl2 deficiency and CCR2 knockout mice. Thus, the CCL2-CCR2 angiocrine signaling axis is related to liver fibrogenesis, and the CVC was applied to block the CCL2-CCR2 angiocrine signaling axis. CVC was demonstrated to alleviate liver fibrosis by restoring the FSCN1⁺ macrophage and HERC6⁺ neutrophil landscape. Pathway analysis indicated that the STAT1, NFκΒ, and ERK signaling pathways might be involved in the anti-fibrotic effects of CVC. Consistently, both CVC treatment and Ccr2 knockout decreased the levels of phosphorylated STAT1, NFkB, and ERK in the fibrotic liver. In vitro, CVC could transcriptionally suppress crucial profibrotic genes (Xaf1, Slfn4, Slfn8, Ifi213, and Il1b) in macrophages by inactivating the STAT1/NFkB/ERK signaling pathways.

Conclusions In summary, activation of the angiocrine signal CCL2 in injured LSECs recruits CCR2⁺ monocyte-derived macrophages to the liver, leading to liver fibrosis. CVC restores the hepatic immune cell landscape by inhibiting profibrotic gene transcription via the CCR2-STAT1/NFkB/ERK signaling pathways.