Bilirubin ameliorates murine atherosclerosis through inhibiting cholesterol synthesis and reshaping the immune system

Atherosclerosis is a chronic inflammatory disease caused mainly by lipid accumulation and excessive inflammatory immune response. Although the lipid-lowering and cardioprotective properties of bilirubin, as well as the negative relationship between bilirubin and atherosclerosis, were well documented, it is not yet clear whether bilirubin can attenuate atherosclerosis in vivo. In this study, we investigated the role of bilirubin in improving atherosclerosis. We found that mildly elevated bilirubin significantly reduced the risk factors of atherosclerosis, such as plasma glucose, total cholesterol, and low-density lipoprotein cholesterol, and the formation of atherosclerotic plaques, liver total cholesterol, and cholesterol ester concentration in apolipoprotein E-deficient (ApoE^−/−) mice fed a western-type (high fat) diet. It was further found that bilirubin could promote the degradation of 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR), a rate-limiting enzyme for endogenous cholesterol synthesis. Using mass cytometry-based high dimensional single cell analysis, we observed a decrease of natural killer cells and an increase of dendritic cells and myeloid-derived suppressor cells, which all are closely associated with atherosclerosis risk factors and contribute to the improvement of atherosclerosis, in ApoE^−/− mice treated with bilirubin. By in-depth analysis, modulation of multiple spleen or peripheral blood T cell clusters exhibiting either positive or negative correlations with total cholesterol or low-density lipoprotein cholesterol was detected after bilirubin treatment. In this study, we demonstrate that bilirubin serves as a negative regulator of atherosclerosis and reduces atherosclerosis by inhibiting cholesterol synthesis and modulating the immune system.