Intermittent hyperlipidaemia induced trained immunity in macrophage and promote atherosclerotic plaque progression via CXCL10-CXCR3 axis

Background Intermittent hyperlipidaemia exacerbates atherosclerosis, conferring greater cardiovascular risk than sustained hyperlipidaemia. The mechanisms of intermittent hyperlipidaemia influencing trained immunity in macrophages remain to be elucidated. Purpose We hypothesized intermittent hyperlipidaemia drives atherosclerosis via CXCL10-CXCR3-mediated macrophage trained immunity. We aimed to elucidate the metabolic-epigenetic mechanisms and evaluate a novel CXCR3-targeted nanobody. Methods Apoe−/− and Apoe−/−/Cx3cr1-EGFP/Ucp1-tdTomato mice were fed an intermittent (4-week high-fat diet (HFD), 4-week normal diet and 4-week HFD) or sustained HFD to assess aortic plaque burden and macrophage-adipocyte crosstalk via two-photon microscopy. Aortic plaques tissue were subjected to scRNA-seq, and a public human carotid plaque scRNA-seq dataset was also analysed. Furthermore, macrophage-specific Cxcr3 knockout (Lyz2-Cre;Cxcr3fl/fl;Apoe−/−) mice were generated and received bone marrow transplantations, while a CXCR3-targeted nanobody (screened from a phage display library) was used to assess in vivo efficacy. Finally, activated macrophages were subjected to functional assays, RNA-seq, Seahorse extracellular flux analysis, targeted metabolomics, lactylome profiling, and mitochondrial tRNA bisulfite sequencing to delineate metabolic and epigenetic reprogramming. Results Compared with sustained HFD, intermittent HFD exacerbated aortic plaque burden in Apoe−/− mice. Two-photon microscopy of Apoe−/−/Cx3cr1-EGFP/Ucp1-tdTomato mice confirmed intermittent HFD amplified intraplaque cellular crosstalk. Plaque scRNA-seq revealed marked CXCR3+ macrophage enrichment exclusively in intermittent lesions, validating this in a human carotid plaque scRNA-seq dataset. Macrophage Cxcr3 ablation, marrow replacement, and CXCR3 nanobody blockade reduced aortic lesions by 46.8%, 41.4%, and 37.1% (all P<0.05) and reversed cellular crosstalk. In vitro, CXCL10-CXCR3 activation promoted pro-inflammatory and proatherogenic traits. RNA-seq showed gene enrichment in OXPHOS and PI3K-AKT pathways (Padj<0.001; Padj=0.035). Immunoblotting revealed CXCL10-CXCR3 ligation increased PI3K, AKT, and mTOR phosphorylation, upregulating HIF1α. Metabolically, this elevated extracellular acidification rates and lactate production, but decreased oxygen consumption rates. CXCR3 stimulation downregulated MYC and NSUN3, reducing mitochondrial tRNA m5C methylation. Lactylome profiling demonstrated CXCL10-CXCR3 elevated nuclear histone H3K18 lactylation. Conclusion Intermittent hyperlipidaemia drives atherosclerosis via CXCL10-CXCR3-mediated macrophage trained immunity, sustained by enhanced glycolysis, H3K18 lactylation, and impaired tRNA m5C modification. Targeted CXCR3 nanobody blockade attenuates plaque progression, offering a novel cardiovascular immunotherapy.