Study on the pro-inflammatory mechanism mediated by the RANK-SPP1 axis in macrophage immunomodulation during atherosclerosis.

Abstract

Atherosclerosis (AS) is a chronic inflammatory disease driven by macrophages. While the RANK/RANKL signaling axis and SPP1 (osteopontin) are individually known to contribute to vascular inflammation, whether they function within a coordinated regulatory hierarchy in plaque macrophages remains unknown. This study investigates whether RANK defines a pro-inflammatory macrophage subset and whether SPP1 acts as a critical downstream effector within the RANK signaling pathway to promote AS progression. Plaque tissue samples were obtained from databases of atherosclerotic patients, experimental atherosclerotic animal models, and in vitro cell experiments to analyze the role of the RANK molecule and macrophages in atherosclerosis and to investigate its association with disease severity. Single-cell RNA sequencing, histological analysis, co-culture of ox-LDL with macrophages, and gene knockout animal experiments were employed to study the mechanism of RANK high expression on macrophages in atherosclerosis progression. We found that high expression of RANK is closely correlated with the severity of atherosclerosis. Single-cell transcriptomic analysis revealed that RANK⁺ cells are primarily enriched in macrophages, and RANK⁺ macrophages exhibit active metabolic and immune response pathways in AS. Cell communication analysis showed that RANK⁺ macrophages are closely associated with SPP1, with SPP1 being a key gene in RANK⁺ macrophages. Furthermore, the number of RANK⁺/SPP1⁺ macrophages increases with AS progression. Mechanistically, SPP1 is a downstream gene of RANK. RANK promotes AS progression by upregulating SPP1, acting through macrophage immunomodulatory pathways. Knocking out RANK in macrophages suppressed SPP1 expression and inhibited the ability of macrophages to phagocytose ox-LDL. Additionally, inhibiting RANK in mouse and rat AS models similarly suppressed SPP1 expression and reduced plaque formation, thereby delaying AS progression. This study establishes a previously unrecognized RANK-SPP1 signaling axis that functions as a macrophage-autonomous inflammatory amplifier in atherosclerosis. We demonstrate that RANK defines a distinct pro-inflammatory macrophage subset characterized by high metabolic and inflammatory pathway activity. Mechanistically, RANK transcriptionally upregulates SPP1 via the NF-κB pathway, and the accumulation of RANK⁺SPP1⁺ macrophages correlates with disease severity. Pharmacological inhibition of RANK signaling attenuates plaque progression in preclinical models, highlighting this axis as a potential therapeutic target for AS.