Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol

Supplementary key words free cholesterol cytokines immunology lipid droplets lymphocytes retinoids ATP-binding cassette transporter A1 (ABCA1) is a plasma membrane protein that functions to eliminate excess free cholesterol (FC) from tissues by effl uxing cellular FC and phospholipid (PL) to lipid-free apolipoprotein AI, forming nascent HDL particles ( 1, 2 ). Therefore, ABCA1 plays a critical role in the movement of cholesterol from peripheral tissues to the liver in a process known as reverse cholesterol transport. Mutations that inactivate the human ABCA1 gene result in Tangier disease, which is characterized by extremely low plasma HDL cholesterol concentrations, mildly elevated plasma triglyceride levels, and accumulation of cholesterol in macrophages ( 3-5 ). ABCA1 protein is expressed to a variable extent in most cells in the body, and its expression is regulated by transcriptional activation and protein degradation ( 6, 7 ), making it diffi cult to determine Abstract We previously showed that macrophages from macrophage-specifi c ATP-binding cassette transporter A1 (ABCA1) knockout ( Abca1 -M/-M ) mice had an enhanced proinfl ammatory response to the Toll-like receptor (TLR) 4 agonist, lipopolysaccharide (LPS), compared with wild-type (WT) mice. In the present study, we demonstrate a direct association between free cholesterol (FC), lipid raft content, and hyper-responsiveness of macrophages to LPS in WT mice. Abca1 -M/-M macrophages were also hyper-responsive to specifi c agonists to TLR2, TLR7, and TLR9, but not TLR3, compared with WT macrophages. We hypothesized that ABCA1 regulates macrophage responsiveness to TLR agonists by modulation of lipid raft cholesterol and TLR mobilization to lipid rafts. We demonstrated that Abca1 -M/-M vs. WT macrophages contained 23% more FC in isolated lipid rafts. Further, mass spectrometric analysis suggested raft phospholipid composition was unchanged. Although cell surface expression of TLR4 was similar between Abca1 -M/-M and WT macrophages, signifi cantly more TLR4 was distributed in membrane lipid rafts in Abca1 -M/-M macrophages. Abca1 -M/-M macrophages also exhibited increased traffi cking of the predominantly intracellular TLR9 into lipid rafts in response to TLR9-specifi c agonist (CpG). Collectively, our data suggest that macrophage ABCA1 dampens infl ammation by reducing MyD88-dependent TLRs traffi cking to lipid rafts by selective reduction of FC content in lipid rafts.