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 HL-49373, HL-094525, and AT-27820 (J.S.P.) Ϫ / Ϫ , total Abca1 knockout; ABCG1, ATP-binding cassette transporter G1; BMDM, bone marrow-derived macrophage; CT-B, cholera toxin B; FC, free cholesterol; fPEG-chol, fl uorescein ester of polyethylene glycol-derivatized Macrophage ABCA1 reduces MyD88-dependent Toll-like This work was supported by National Institutes of Health Grants

In humans, the plasma concentration of HDLs has been repeatedly shown to be inversely correlated with the risk of developing coronary heart disease ( 1, 2 ). The concentrations of plasma HDL has been shown to be largely dependent on hepatic ATP-binding cassette transporter A1 (ABCA1) ( 3-5 ), which transports and promotes the effl ux of glycerophosphocholine (PC), free cholesterol (FC), and sphingomyelin (SM) to wild-type apolipoprotein A-I (apoA-I), resulting in the formation of nascent HDLs (nHDLs). Functional mutations in human ABCA1 cause Tangier disease ( 6, 7 ), which is characterized by very low levels of plasma HDL apoA-I. Tangier disease is believed to alter a process termed "reverse cholesterol transport." Although defects in ABCA1 function have been identifi ed by Abstract This report details the lipid composition of nascent HDL (nHDL) particles formed by the action of the ATP binding cassette transporter A1 (ABCA1) on apolipoprotein A-I (apoA-I). nHDL particles of different size (average diameters of ‫ف‬ 12, 10, 7.5, and <6 nm) and composition were purifi ed by size-exclusion chromatography. Electron microscopy suggested that the nHDL were mostly spheroidal. The proportions of the principal nHDL lipids, free cholesterol, glycerophosphocholine, and sphingomyelin were similar to that of lipid rafts, suggesting that the lipid originated from a raft-like region of the cell. Smaller amounts of glucosylceramides, cholesteryl esters, and other glycerophospholipid classes were also present. The largest particles, ‫ف‬ 12 nm and 10 nm diameter, contained ‫ف‬ 43% free cholesterol, 2-3% cholesteryl ester, and three apoA-I molecules. Using chemical cross-linking chemistry combined with mass spectrometry, we found that three molecules of apoA-I in the ‫ف‬ 9-14 nm nHDL adopted a belt-like conformation. The smaller (7.5 nm diameter) spheroidal nHDL particles carried 30% free cholesterol and two molecules of apoA-I in a twisted, antiparallel, double-belt conformation. Overall, these new data offer fresh insights into the biogenesis and structural constraints involved in forming nascent HDL from ABCA1 . -Sorci-Thomas, M. G., J. S. Owen, B. Fulp, S. Bhat, These studies were supported by grants from the National Institutes of Health grants and Abbreviations: BMDM, bone marrow-derived macrophage; CE, cholesteryl ester; DSP, dithiobis(succinimidylpropionate) ; EM, electron microscopy; FC, free cholesterol; FPLC, fast protein liquid chromatography; GP, glycerophospholipids; HEK, human embryonic kidney; HexCer; hexosylceramides; LPC, lyso-glycerophosphocholine; MSCE, mass spectrometer collision energy; NDGGE, nondenaturing gradient gel electrophoresis; nHDL, nascent HDL; PC, glycerophosphocholine; PE, glycerophospho ethanolamine; PG, glycerophosphoglycerol; PI, glyerophos phoinositol; PM, plasma membrane; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PS, glycerophosphoserine; rHDL, recombinant HDL; RT, room temperature; SL, sphingolipid; TC, total cholesterol.1 To whom correspondence should be addressed. e-mail...

The immune system is complex, with multiple layers of regulation that serve to prevent the production of self-antigens. One layer of regulation involves regulatory T cells (Tregs) that play an essential role in maintaining peripheral self-tolerance. Patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis have decreased levels of HDL, suggesting that apoA-I concentrations may be important in preventing autoimmunity and the loss of self-tolerance. In published studies, hypercholesterolemic mice lacking HDL apoA-I or LDLr ؊/؊ , apoA-I ؊/؊ (DKO), exhibit characteristics of autoimmunity in response to an atherogenic diet. This phenotype is characterized by enlarged cholesterol-enriched lymph nodes (LNs), as well as increased T cell activation, proliferation, and the production of autoantibodies in plasma. In this study, we investigated whether treatment of mice with lipid-free apoA-I could attenuate the autoimmune phenotype. To do this, DKO mice were first fed an atherogenic diet containing 0.1% cholesterol, 10% fat for 6 weeks, after which treatment with apoA-I was begun. Subcutaneous injections of 500 g of lipid-free apoA-I was administered every 48 h during the treatment phase. These and control mice were maintained for an additional 6 weeks on the diet. At the end of the 12-week study, DKO mice showed decreased numbers of LN immune cells, whereas Tregs were proportionately increased. Accompanying this increase in Tregs was a decrease in the percentage of effector/effector memory T cells. Furthermore, lipid accumulation in LN and skin was reduced. These results suggest that treatment with apoA-I reduces inflammation in DKO mice by augmenting the effectiveness of the LN Treg response.

The collapse of thymic stromal cell microenvironment with age and resultant inability of the thymus to produce naïve T cells contributes to lower immune-surveillance in the elderly. Here we show that age-related increase in ‘lipotoxic danger signals’ such as free cholesterol (FC) and ceramides, leads to thymic caspase-1 activation via the Nlrp3 inflammasome. Elimination of Nlrp3 and Asc, a critical adaptor required for inflammasome assembly, reduces age-related thymic atrophy and results in an increase in cortical thymic epithelial cells, T cell progenitors and maintenance of T cell repertoire diversity. Using a mouse model of irradiation and hematopoietic stem cell transplantation (HSCT), we show that deletion of the Nlrp3 inflammasome accelerates T cell reconstitution and immune recovery in middle-aged animals. Collectively, these data demonstrate that lowering inflammasome-dependent caspase-1 activation increases thymic lymphopoiesis and suggest that Nlrp3 inflammasome inhibitors may aid the reestablishment of a diverse T cell repertoire in middle-aged or elderly patients undergoing HSCT.

Objective-The purpose of this study was to determine the effects of an atherogenic diet on immune function in LDLr Ϫ/Ϫ , ApoA-I Ϫ/Ϫ mice. Methods and Results-When LDLrϪ/Ϫ , ApoA-I Ϫ/Ϫ (DKO), and LDLr Ϫ/Ϫ (SKO) mice were fed an atherogenic diet, DKO had larger peripheral lymph nodes (LNs) and spleens compared to SKO mice. LNs were enriched in cholesterol and contain expanded populations of T, B, dendritic cells, and macrophages. Expansion of all classes of LN cells was accompanied by a Ϸ1.5-fold increase in T cell proliferation and activation. Plasma antibodies to dsDNA, ␤2-glycoprotein I, and oxidized LDL were increased in DKO, similar to levels in diet-fed Fas lpr/lpr mice, suggesting the development of an autoimmune phenotype. Both LN enlargement and cellular cholesterol expansion were "prevented" when diet-fed DKO mice were treated with helper dependent adenovirus expressing apoA-I. Independent of the amount of dietary cholesterol, DKO mice consistently showed lower plasma cholesterol than SKO mice, yet greater aortic cholesterol deposition and inflammation. Conclusions-ApoA-I prevented cholesterol-associated lymphocyte activation and proliferation in peripheral LN of diet-fed DKO mice. A Ϸ1.5-fold increase in T cell activation and proliferation was associated with a Ϸ3-fold increase in concentrations of circulating autoantibodies and Ϸ2-fold increase in the severity of atherosclerosis suggesting a common link between plasma apoA-I, inflammation, and atherosclerosis.