Conjugated bile acid-driven CD14+CD16+ monocyte infiltration promotes cholestatic liver injury by enhancing hepatocyte necroptosis.

BACKGROUND & AIMS: Cell death and inflammation cause liver injury during cholestasis. Monocyte infiltration is crucial for the inflammatory response in tissues and organs. Thus, to study the role of monocytes in cholestatic liver disease, we used single-cell RNA sequencing (scRNA-seq) to analyze liver non-parenchymal cells (NPCs) from patients with obstructive cholestasis (OC).

METHODS

Hepatic monocyte infiltration in liver was assessed using scRNA-seq and multiplex immunofluorescence. In vivo functional validation was conducted through serum biochemistry, liver histopathology, RT-qPCR, and Western blotting after intermediate monocyte infusion and elimination in mice. In vitro, CD14+CD16+ monocytes were isolated by flow cytometry for analysis of tumor necrosis factor-alpha (TNFα) and factor-related apoptosis ligand (FASL) expression.

RESULTS

scRNA-seq and multiplex immunofluorescence revealed that CD14+CD16+ monocyte infiltration was increased in OC livers (n = 5-20; p <0.0001). The number of CD14+CD16+ monocytes was positively correlated with liver injury severity (alanine aminotransferase (ALT), n = 20, p = 0.00002; aspartate aminotransferase (AST), n = 20, p = 0.002). Infusion of human CD14+CD16+ monocytes (i.e. human intermediate monocytes) exacerbated cholestatic liver injury in mice fed 1% lithocholic acid (ALT, n = 5, p <0.01; AST, n = 5, p <0.01). Meanwhile, mice that received mouse intermediate monocytes also exhibited higher cholestatic liver injury (ALT, n = 5, p <0.01; AST, n = 5, p <0.01). Mechanistically, CD14+CD16+ monocyte infiltration increased hepatic levels of TNFα (n = 5, p <0.01) and FASL (n = 5, p <0.01), activating the receptor-interacting serine/threonine-protein kinase (RIPK)-1-RIPK3-mixed-lineage kinase domain-like protein (MLKL) axis to promote hepatocyte necroptosis. CD14+CD16+ monocytes isolated from peripheral blood mononuclear cells (PBMCs) from patients with OC expressed higher levels of TNFα (n = 3, p <0.05) and FASL (n = 3, p <0.05) compared with other PBMC populations. Bile acid-stressed hepatocytes drove CD14+CD16+ monocyte infiltration via JNK/c-Jun-mediated CCL2 (n = 5-20, p <0.0001) and CCL3 (n = 5-20, p <0.05) upregulation.

CONCLUSION

Infiltrated CD14+CD16+ monocytes promote hepatocyte necroptosis and exacerbate cholestatic liver injury. Thus, targeting their chemotaxis represents a promising therapeutic strategy against cholestasis.

IMPACT AND IMPLICATIONS: This study revealed that CD14+CD16+ monocyte-derived tumor necrosis factor-alpha (TNFα) and factor-related apoptosis ligand (FASL) drive hepatocyte necroptosis via the receptor-interacting serine/threonine-protein kinase (RIPK)-1-RIPK3-mixed-lineage kinase domain-like protein (MLKL) axis, establishing these infiltrating monocytes as crucial mediators of cholestatic liver injury. Bile acid-stressed hepatocytes promoted CD14+CD16+ monocyte infiltration by upregulating CCL2 and CCL3 via JNK/c-Jun signaling. These findings highlight the therapeutic potential of modulating monocyte infiltration or necroptosis pathways to alleviate disease progression, offering novel strategies for treating OC and related liver pathologies.