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.

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.

Atherogenic response to dietary fat and cholesterol challenge was evaluated in mice lacking both the LDL receptor (LDLr(-/-)) and apoA-I (apoA-I(-/-)) gene, LDLr(-/-)/apoA-I(-/-) or double-knockout mice. Gender- and age-matched LDLr(-/-)/apoA-I(-/-) mice were fed a diet consisting of 0.1% cholesterol and 10% palm oil for 16 weeks and compared to LDLr(-/-) mice or single-knockout mice. The LDLr(-/-) mice showed a 6- to 7-fold increase in total plasma cholesterol (TPC) compared to their chow-fed mice counterparts, while LDLr(-/-)/apoA-I(-/-) mice showed only a 2- to 3-fold increase in TPC compared to their chow-fed controls. This differential response to the atherogenic diet was unanticipated, since chow-fed LDLr(-/-) and LDLr(-/-)/apoA-I(-/-) mice began the study with similar LDL levels and differed primarily in their HDL concentration. The 6-fold diet-induced increase in TPC observed in the LDLr(-/-) mice occurred mainly in VLDL/LDL and not in HDL. Mid-study plasma samples taken after 8 weeks of diet feeding showed that LDLr(-/-) mice had TPC concentrations approximately 60% of their 16-week level, while the LDLr(-/-)/apoA-I(-/-) mice had reached 100% of their 16-week TPC concentration after only 8 weeks of diet. Male LDLr(-/-) mice showed similar aortic cholesterol levels to male LDLr(-/-)/apoA-I(-/-) mice despite a 4-fold higher VLDL/LDL concentration in the LDLr(-/-) mice. A direct comparison of the severity of aortic atherosclerosis between female LDLr(-/-) and LDLr(-/-)/apoA-I(-/-) mice was compromised due to the loss of female LDLr(-/-)/apoA-I(-/-) mice between 10 and 14 weeks into the study. Diet-fed female and, with time, male LDLr(-/-)/apoA-I(-/-) mice suffered from severe ulcerated cutaneous xanthomatosis. This condition, combined with a complete depletion of adrenal cholesterol, manifested in fatal wasting of the affected mice. In conclusion, LDLr(-/-) and LDLr(-/-)/apoA-I(-/-) mice showed dramatic TPC differences in response to dietary fat and cholesterol challenge, while despite these differences both genotypes accumulated similar levels of aortic cholesterol.

Limited understanding of the mechanisms responsible for life-threatening organ and immune failure hampers scientists' ability to design sepsis treatments. Pyruvate dehydrogenase kinase 1 (PDK1) is persistently expressed in immune-tolerant monocytes of septic mice and humans and deactivates mitochondrial pyruvate dehydrogenase complex (PDC), the gate-keeping enzyme for glucose oxidation. Here, we show that targeting PDK with its prototypic inhibitor dichloroacetate (DCA) reactivates PDC; increases mitochondrial oxidative bioenergetics in isolated hepatocytes and splenocytes; promotes vascular, immune, and organ homeostasis; accelerates bacterial clearance; and increases survival. These results indicate that the PDC/PDK axis is a druggable mitochondrial target for promoting immunometabolic and organ homeostasis during sepsis.