Atherosclerosis is the underlying cause of cardiovascular disease. Recent studies have shown that N6-methyladenosine (m6A) modification in macrophages is associated with atherosclerosis progression. However, there is a lack of systemic research on the role of m6A modification in macrophage differentiation and activation during atherosclerosis. Here we conducted multiomics analysis (MeRIP-seq and RNA-seq) of macrophages during their differentiation and activation to elucidate the regulatory network of the m6A spectrum at different stages. Western blot, quantitative PCR (qPCR), RNA-seq and RNA immunoprecipitation (RIP)-qPCR results demonstrated that m6A modification modulates KDM6B expression during macrophage activation. Through co-immunoprecipitation, RIP‒qPCR and genetic perturbation experiments, we revealed that Mettl3/Rbm15 regulates the stability of Kdm6b mRNA and that Kdm6b is required for interacting with and demethylating Jak1 to induce its phosphorylation-mediated macrophage activation. Next, through the analysis of single-cell RNA-seq data and coculture experiments, we revealed that Kdm6b-mediated macrophage activation promoted cytotoxic T lymphocyte cytotoxicity following atherosclerosis progression. Moreover, the systemic use of STM2457, a METTL3 inhibitor, revealed the importance of m6A modification in immune cell infiltration and plaque activation. Finally, we utilized macrophage-specific Kdm6b-knockout mice to determine whether Kdm6b facilitates macrophage and cytotoxic T lymphocyte activation and atherosclerosis. Our findings revealed that m6A modification plays a pivotal role in the upregulation of Kdm6b in response to IFN-γ stimulation, which is essential for the phosphorylation of Stat1-induced macrophage activation-mediated atherosclerosis development.
© 2025. The Author(s).