Distinct surveillance pathway for immunopathology during acute infection via autophagy and SR-BI.

The mechanisms protecting from immunopathology during acute bacterial infections are incompletely known. We found that in response to apoptotic immune cells and live or dead Listeria monocytogenes scavenger receptor BI (SR-BI), an anti-atherogenic lipid exchange mediator, activated internalization mechanisms with characteristics of macropinocytosis and, assisted by Golgi fragmentation, initiated autophagic responses. This was supported by scavenger receptor-induced local increases in membrane cholesterol concentrations which generated lipid domains particularly in cell extensions and the Golgi. SR-BI was a key driver of beclin-1-dependent autophagy during acute bacterial infection of the liver and spleen. Autophagy regulated tissue infiltration of neutrophils, suppressed accumulation of Ly6C+ (inflammatory) macrophages, and prevented hepatocyte necrosis in the core of infectious foci. Perifocal levels of Ly6C+ macrophages and Ly6C- macrophages were unaffected, indicating predominant regulation of the focus core. SR-BI-triggered autophagy promoted co-elimination of apoptotic immune cells and dead bacteria but barely influenced bacterial sequestration and survival or inflammasome activation, thus exclusively counteracting damage inflicted by immune responses. Hence, SR-BI- and autophagy promote a surveillance pathway that partially responds to products of antimicrobial defenses and selectively prevents immunity-induced damage during acute infection. Our findings suggest that control of infection-associated immunopathology can be based on a unified defense operation.

Scavenger receptor CD36 mediates uptake of high density lipoproteins in mice and by cultured cells.

The mechanisms of HDL-mediated cholesterol transport from peripheral tissues to the liver are incompletely defined. Here the function of scavenger receptor cluster of differentiation 36 (CD36) for HDL uptake by the liver was investigated. CD36 knockout (KO) mice, which were the model, have a 37% increase (P = 0.008) of plasma HDL cholesterol compared with wild-type (WT) littermates. To explore the mechanism of this increase, HDL metabolism was investigated with HDL radiolabeled in the apolipoprotein (¹²5I) and cholesteryl ester (CE, [³H]) moiety. Liver uptake of [³H] and ¹²5I from HDL decreased in CD36 KO mice and the difference, i. e. hepatic selective CE uptake ([³H]¹²5I), declined (-33%, P = 0.0003) in CD36 KO compared with WT mice. Hepatic HDL holo-particle uptake (¹²5I) decreased (-29%, P = 0.0038) in CD36 KO mice. In vitro, uptake of ¹²5I-/[³H]HDL by primary liver cells from WT or CD36 KO mice revealed a diminished HDL uptake in CD36-deficient hepatocytes. Adenovirus-mediated expression of CD36 in cells induced an increase in selective CE uptake from HDL and a stimulation of holo-particle internalization. In conclusion, CD36 plays a role in HDL uptake in mice and by cultured cells. A physiologic function of CD36 in HDL metabolism in vivo is suggested.

Brown adipose tissue activity controls triglyceride clearance.

Brown adipose tissue (BAT) burns fatty acids for heat production to defend the body against cold and has recently been shown to be present in humans. Triglyceride-rich lipoproteins (TRLs) transport lipids in the bloodstream, where the fatty acid moieties are liberated by the action of lipoprotein lipase (LPL). Peripheral organs such as muscle and adipose tissue take up the fatty acids, whereas the remaining cholesterol-rich remnant particles are cleared by the liver. Elevated plasma triglyceride concentrations and prolonged circulation of cholesterol-rich remnants, especially in diabetic dyslipidemia, are risk factors for cardiovascular disease. However, the precise biological role of BAT for TRL clearance remains unclear. Here we show that increased BAT activity induced by short-term cold exposure controls TRL metabolism in mice. Cold exposure drastically accelerated plasma clearance of triglycerides as a result of increased uptake into BAT, a process crucially dependent on local LPL activity and transmembrane receptor CD36. In pathophysiological settings, cold exposure corrected hyperlipidemia and improved deleterious effects of insulin resistance. In conclusion, BAT activity controls vascular lipoprotein homeostasis by inducing a metabolic program that boosts TRL turnover and channels lipids into BAT. Activation of BAT might be a therapeutic approach to reduce elevated triglyceride concentrations and combat obesity in humans.

Inhibition of cholesteryl ester transfer protein by torcetrapib modestly increases macrophage cholesterol efflux to HDL.

OBJECTIVE: This study examines the effects of pharmacological inhibition of cholesteryl ester transfer protein (CETP) on the ability of high-density lipoprotein particles (HDL) to promote net cholesterol efflux from human THP-1 macrophage foam cells. METHODS AND RESULTS: Two groups of 8 healthy, moderately hyperlipidemic subjects received the CETP inhibitor torcetrapib at 60 or 120 mg daily for 8 weeks. Torcetrapib increased HDL cholesterol levels in both groups by 50% and 60%, respectively. Compared with baseline, torcetrapib 60 mg daily increased HDL-mediated net cholesterol efflux from foam cells primarily by increasing HDL concentrations, whereas 120 mg daily torcetrapib increased cholesterol efflux both by increasing HDL concentration and by causing increased efflux at matched HDL concentrations. There was an increased content of lecithin:cholesterol acyltransferase (LCAT) and apolipoprotein E (apoE) in HDL-2 only at the 120 mg dose. ABCG1 activity was responsible for 40% to 50% of net cholesterol efflux to both control and T-HDL. CONCLUSIONS: These data indicate that inhibition of CETP by torcetrapib causes a modest increase in the ability of HDL to promote net cholesterol efflux at the 60 mg dose, and a more dramatic increase at the 120 mg dose in association with enhanced particle functionality.

Selective uptake of HDL cholesteryl esters and cholesterol efflux from mouse peritoneal macrophages independent of SR-BI.

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL cholesteryl esters (CEs) and facilitates the efflux of unesterified cholesterol. SR-BI expression in macrophages presumably plays a role in atherosclerosis. The role of SR-BI for selective CE uptake and cholesterol efflux in macrophages was explored. Macrophages and HDL originated from wild-type (WT) or SR-BI knockout (KO; homozygous) mice. For uptake, macrophages were incubated in medium containing 125I-/3H-labeled HDL. For lipid removal, [3H]cholesterol efflux was analyzed using HDL as acceptor. Selective uptake of HDL CE ([3H]cholesteryl oleyl ether - 125I-tyramine cellobiose) was similar in WT and SR-BI KO macrophages. Radiolabeled SR-BI KO-HDL yielded a lower rate of selective uptake compared with WT-HDL in WT and SR-BI KO macrophages. Cholesterol efflux was similar in WT and SR-BI KO cells using HDL as acceptor. SR-BI KO-HDL more efficiently promoted cholesterol removal compared with WT-HDL from both types of macrophages. Macrophages selectively take up HDL CE independently of SR-BI. Additionally, in macrophages, there is substantial cholesterol efflux that is not mediated by SR-BI. Therefore, SR-BI-independent mechanisms mediate selective CE uptake and cholesterol removal. SR-BI KO-HDL is an inferior donor for selective CE uptake compared with WT-HDL, whereas SR-BI KO-HDL more efficiently promotes cholesterol efflux.

Annexin A6 stimulates the membrane recruitment of p120GAP to modulate Ras and Raf-1 activity.

Annexin A6 is a calcium-dependent membrane-binding protein that interacts with signalling proteins, including the GTPase-activating protein p120GAP, one of the most important inactivators of Ras. Since we have demonstrated that annexin A6 inhibits EGF- and TPA-induced Ras signalling, we investigated whether modulation of Ras activity by annexin A6 was mediated via altered subcellular localization of p120GAP. First, we exploited our observation that high-density lipoproteins (HDL) can activate the Ras/MAP kinase pathway. Expression of annexin A6 caused a significant reduction in HDL-induced activation of Ras and Raf-1. Annexin A6 promoted membrane binding of p120GAP in vitro, and plasma membrane targeting of p120GAP in living cells, both in a Ca(2+)-dependent manner, which is consistent with annexin A6 promoting the Ca(2+)-dependent assembly of p120GAP-Ras at the plasma membrane. We then extended these studies to other cell types and stimuli. Expression of annexin A6 in A431 cells reduced, while RNAi-mediated suppression of annexin A6 in HeLa cells enhanced EGF-induced Ras and Erk activation. Importantly, the enhancement of Ras activation following RNAi-mediated reduction in p120GAP levels was more marked in annexin A6-expressing A431 cells than controls, indicating that the effect of annexin A6 on Ras was mediated via p120GAP. Finally, we demonstrated that annexin A6 promotes plasma membrane targeting of p120GAP in A431 cells in response to a variety of stimuli, resulting in colocalization with H-Ras. These findings demonstrate an important role for annexin A6 in regulating plasma membrane localization of p120GAP and hence Ras activity.

Impaired recycling of apolipoprotein E4 is associated with intracellular cholesterol accumulation.

After internalization of triglyceride-rich lipoproteins (TRL) in hepatoma cells, TRL particles are immediately disintegrated in the early endosomal compartment. This involves the targeting of lipids and apoprotein B along the degradative pathway and the recycling of TRL-derived apoE through recycling endosomes. Re-secretion of apoE is accompanied by the concomitant association of apoE and cellular cholesterol with high-density lipoproteins (HDL). Since epidemiological data showed that apoE3 and apoE4 have differential effects on HDL metabolism, we investigated whether the intracellular processing of TRL-derived apoE4 differs from apoE3-TRL. In this study, we demonstrated by radioactive and immunofluorescence uptake experiments that cell-surface binding and internalization of TRL-derived apoE4 are increased compared with apoE3 in hepatoma cells. Pulse-chase experiments revealed that HDL-induced recycling, but not disintegration and degradation, of apoE4-enriched TRL is strongly reduced in these cells. Furthermore, impaired HDL-induced apoE4 recycling is associated with reduced cholesterol efflux. Studies performed in Tangier fibroblasts showed that apoE recycling does not depend on ATP-binding cassette transporter A1 activity. These studies provide initial evidence that impaired recycling of apoE4 could interfere with intracellular cholesterol transport and contribute to the pathophysiological lipoprotein profile observed in apoE4 homozygotes.

Hepatic lipase mediates an increase in selective uptake of HDL-associated cholesteryl esters by cells in culture independent from SR-BI.

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL cholesteryl esters (CEs) by the liver. Hepatic lipase (HL) promotes this lipid uptake independent from lipolysis. The role of SR-BI in this HL-mediated increase in selective CE uptake was explored. Baby hamster kidney (BHK) cells were transfected with the SR-BI cDNA yielding cells with SR-BI expression, whereas no SR-BI was detected in control cells. These cells were incubated in medium containing 125I [3H]cholesteryl oleyl ether-labeled HDL3 (d = 1.125-1.21 g/ml) and HL was absent or present. Tetrahydrolipstatin (THL) blocked lipolysis. In control BHK cells and in BHK cells with SR-BI, HDL3 selective CE uptake (3H-125I) was detectable and SR-BI promoted this uptake. In both cell types, HL mediated an increase in selective CE uptake from HDL3. Quantitatively, this HL effect was similar in control BHK cells and in BHK cells with SR-BI. These results suggest that HL promotes selective uptake independent from SR-BI. To investigate the role of cell surface proteoglycans on the HL-mediated HDL3 uptake, proteoglycan deficiency was induced by heparinase digestion. Proteoglycan deficiency decreased the HL-mediated promotion of selective CE uptake. In summary, the stimulating HL effect on HDL selective CE uptake is independent from SR-BI and lipolysis. Proteoglycans are a requisite for the HL action on selective uptake. Results suggest that (a) pathway(s) distinct from SR-BI mediate(s) selective CE uptake from HDL.

Recycling of apoprotein E is associated with cholesterol efflux and high density lipoprotein internalization.

After receptor-mediated endocytosis of triglyceride-rich lipoproteins (TRL) into the liver, TRL particles are immediately disintegrated in peripheral endosomal compartments. Whereas core lipids and apoprotein B are delivered for degradation into lysosomes, TRL-derived apoE is efficiently recycled back to the plasma membrane. This is followed by apoE re-secretion and association of apoE with high density lipoproteins (HDL). Because HDL and apoE can independently promote cholesterol efflux, we investigated whether recycling of TRL-derived apoE in human hepatoma cells and fibroblasts could be linked to intracellular cholesterol transport. In this study we demonstrate that HDL(3) does not only act as an extracellular acceptor for recycled apoE but also stimulates the recycling of internalized TRL-derived apoE. Furthermore, radioactive pulse-chase experiments indicate that apoE recycling is accompanied by cholesterol efflux. Confocal imaging reveals co-localization of apoE and cholesterol in early endosome antigen 1-positive endosomes. During apoE re-secretion, HDL(3)-derived apoA-I is found in these early endosome antigen 1, cholesterol-containing endosomes. As shown by time-lapse fluorescence microscopy, apoE recycling involves the intracellular trafficking of apoA-I to pre-existing and TRL-derived apoE/cholesterol-containing endosomes in the periphery. Thus, these studies provide evidence for a new intracellular link between TRL-derived apoE, cellular cholesterol transport, and HDL metabolism.