MD ‐1 Deficiency Mitigates Atherosclerosis by Inhibiting NLRP3 Inflammasome Activation via cGAS ‐ STING Dependent Pathway in Macrophages

ABSTRACT MD‐1 plays significant roles in various pathological processes closely associated with cardiovascular diseases through its functions in different cell types. However, the critical effects and molecular mechanisms mediated by MD‐1 in macrophages involved in the development of atherosclerosis remain elusive. In this study, we observed that the expression of MD‐1 was upregulated in atherosclerotic plaques derived from human carotid arteries, as analysed through the Gene Expression Omnibus database. Additionally, MD‐1 expression was elevated in the aortas of hyperlipidemic ApoE‐deficient mice and in oxidised low‐density lipoprotein‐stimulated bone marrow‐derived macrophages, as demonstrated by RT‐PCR and western blot analyses. Furthermore, MD‐1 expression exhibited strong co‐localisation with macrophages, as confirmed by double immunofluorescence staining. Notably, MD‐1 −/− ApoE −/− mice subjected to a high‐fat diet displayed a significant reduction in atherosclerotic plaques throughout the entire aorta, aortic root, and brachiocephalic arteries. Moreover, the plaques in MD‐1 −/− ApoE −/− mice were more stable, characterised by a decreased necrotic core size, reduced macrophage infiltration, and lower lipid deposition, alongside an increased accumulation of collagen and smooth muscle cells. Bone marrow transplantation from MD‐1 −/− ApoE −/− mice to ApoE −/− mice markedly attenuated the development of atherosclerosis. This attenuation was associated with an enhanced anti‐inflammatory macrophage phenotype and a reduction in pro‐inflammatory macrophage polarisation. Similar effects of MD‐1 ablation on macrophage behaviour were also observed in vitro. Mechanistically, MD‐1 deficiency inhibited the cyclic GMP‐AMP synthase‐stimulator of interferon genes‐TANK‐binding kinase 1‐IRF3 pathway, thereby attenuating NLRP3 inflammasome activation in macrophages. However, the use of the STING agonist DMXAA counteracted the effects of MD‐1 deficiency on macrophage polarisation. Our findings reveal MD‐1 promotes pro‐inflammatory (M1‐like) macrophage polarisation in atherosclerosis by inhibiting NLRP3 inflammasome activation via a cGAS‐STING dependent pathway. These results suggest that MD‐1 neutralisation could be a potential therapeutic strategy for atherosclerosis management.