Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation.

Activating the macrophage NLRP3 inflammasome can promote excessive inflammation with severe cell and tissue damage and organ dysfunction. Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and interleukin-1ß (IL-1ß) secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves crista ultrastructure, and attenuates mitochondrial reactive oxygen species (ROS) production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. Our study suggests a non-canonical role of mitochondrial PDHK in promoting mitochondrial stress and supporting NLRP3 inflammasome activation during acute inflammation.

Sepsis, pyruvate, and mitochondria energy supply chain shortage.

Balancing high energy-consuming danger resistance and low energy supply of disease tolerance is a universal survival principle that often fails during sepsis. Our research supports the concept that sepsis phosphorylates and deactivates mitochondrial pyruvate dehydrogenase complex control over the tricarboxylic cycle and the electron transport chain. StimulatIng mitochondrial energetics in septic mice and human sepsis cell models can be achieved by inhibiting pyruvate dehydrogenase kinases with the pyruvate structural analog dichloroacetate. Stimulating the pyruvate dehydrogenase complex by dichloroacetate reverses a disruption in the tricarboxylic cycle that induces itaconate, a key mediator of the disease tolerance pathway. Dichloroacetate treatment increases mitochondrial respiration and ATP synthesis, decreases oxidant stress, overcomes metabolic paralysis, regenerates tissue, organ, and innate and adaptive immune cells, and doubles the survival rate in a murine model of sepsis.

Dichloroacetate improves systemic energy balance and feeding behavior during sepsis.

Sepsis is a life-threatening organ dysfunction caused by dysregulated host response to an infection. The metabolic aberrations associated with sepsis underly an acute and organism-wide hyperinflammatory response and multiple organ dysfunction; however, crosstalk between systemic metabolomic alterations and metabolic reprogramming at organ levels remains unknown. We analyzed substrate utilization by the respiratory exchange ratio, energy expenditure, metabolomic screening, and transcriptional profiling in a cecal ligation and puncture model to show that sepsis increases circulating free fatty acids and acylcarnitines but decreases levels of amino acids and carbohydrates, leading to a drastic shift in systemic fuel preference. Comparative analysis of previously published metabolomics from septic liver indicated a positive correlation with hepatic and plasma metabolites during sepsis. In particular, glycine deficiency was a common abnormality of the plasma and liver during sepsis. Interrogation of the hepatic transcriptome in septic mice suggested that the septic liver may contribute to systemic glycine deficiency by downregulating genes involved in glycine synthesis. Interestingly, intraperitoneal injection of the pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate reversed sepsis-induced anorexia, energy imbalance, inflammation, dyslipidemia, hypoglycemia, and glycine deficiency. Collectively, our data indicated that PDK inhibition rescued systemic energy imbalance and metabolic dysfunction in sepsis partly through restoration of hepatic fuel metabolism.

Hematopoietic Cell-Specific SLC37A2 Deficiency Accelerates Atherosclerosis in LDL Receptor-Deficient Mice.

Macrophages play a central role in the pathogenesis of atherosclerosis. Our previous study demonstrated that solute carrier family 37 member 2 (SLC37A2), an endoplasmic reticulum-anchored phosphate-linked glucose-6-phosphate transporter, negatively regulates macrophage Toll-like receptor activation by fine-tuning glycolytic reprogramming in vitro. Whether macrophage SLC37A2 impacts in vivo macrophage inflammation and atherosclerosis under hyperlipidemic conditions is unknown. We generated hematopoietic cell-specific SLC37A2 knockout and control mice in C57Bl/6 Ldlr-/- background by bone marrow transplantation. Hematopoietic cell-specific SLC37A2 deletion in Ldlr-/- mice increased plasma lipid concentrations after 12-16 wks of Western diet induction, attenuated macrophage anti-inflammatory responses, and resulted in more atherosclerosis compared to Ldlr-/- mice transplanted with wild type bone marrow. Aortic root intimal area was inversely correlated with plasma IL-10 levels, but not total cholesterol concentrations, suggesting inflammation but not plasma cholesterol was responsible for increased atherosclerosis in bone marrow SLC37A2-deficient mice. Our in vitro study demonstrated that SLC37A2 deficiency impaired IL-4-induced macrophage activation, independently of glycolysis or mitochondrial respiration. Importantly, SLC37A2 deficiency impaired apoptotic cell-induced glycolysis, subsequently attenuating IL-10 production. Our study suggests that SLC37A2 expression is required to support alternative macrophage activation in vitro and in vivo. In vivo disruption of hematopoietic SLC37A2 accelerates atherosclerosis under hyperlipidemic pro-atherogenic conditions.

Dichloroacetate reverses sepsis-induced hepatic metabolic dysfunction.

Metabolic reprogramming between resistance and tolerance occurs within the immune system in response to sepsis. While metabolic tissues such as the liver are subjected to damage during sepsis, how their metabolic and energy reprogramming ensures survival is unclear. Employing comprehensive metabolomic, lipidomic, and transcriptional profiling in a mouse model of sepsis, we show that hepatocyte lipid metabolism, mitochondrial tricarboxylic acid (TCA) energetics, and redox balance are significantly reprogrammed after cecal ligation and puncture (CLP). We identify increases in TCA cycle metabolites citrate, cis-aconitate, and itaconate with reduced fumarate and triglyceride accumulation in septic hepatocytes. Transcriptomic analysis of liver tissue supports and extends the hepatocyte findings. Strikingly, the administration of the pyruvate dehydrogenase kinase (PDK) inhibitor dichloroacetate reverses dysregulated hepatocyte metabolism and mitochondrial dysfunction. In summary, our data indicate that sepsis promotes hepatic metabolic dysfunction and that targeting the mitochondrial PDC/PDK energy homeostat rebalances transcriptional and metabolic manifestations of sepsis within the liver.

Solute Carrier Family 37 Member 2 (SLC37A2) Negatively Regulates Murine Macrophage Inflammation by Controlling Glycolysis.

Increased flux of glucose through glycolysis is a hallmark of inflammatory macrophages and is essential for optimal effector functions. Solute carrier (SLC) 37A2 is an endoplasmic reticulum-anchored phosphate-linked glucose-6-phosphate transporter that is highly expressed in macrophages and neutrophils. We demonstrate that SLC37A2 plays a pivotal role in murine macrophage inflammatory activation and cellular metabolic rewiring. Toll-like receptor (TLR) 4 stimulation by lipopolysaccharide (LPS) rapidly increases macrophage SLC37A2 protein expression. SLC37A2 deletion reprograms macrophages to a hyper-glycolytic process and accelerates LPS-induced inflammatory cytokine production, which partially depends on nicotinamide adenine dinucleotide (NAD+) biosynthesis. Blockade of glycolysis normalizes the differential expression of pro-inflammatory cytokines between control and SLC37A2 deficient macrophages. Conversely, overexpression of SLC37A2 lowers macrophage glycolysis and significantly reduces LPS-induced pro-inflammatory cytokine expression. In conclusion, our study suggests that SLC37A2 dampens murine macrophage inflammation by down-regulating glycolytic reprogramming as a part of macrophage negative feedback system to curtail acute innate activation.

Myeloid atg5 deletion impairs n-3 PUFA-mediated atheroprotection.

BACKGROUND AND AIMS: Dietary long-chain (≥20 carbons) n-3 polyunsaturated fatty acids (PUFAs) reduce atherosclerosis and enhance macrophage autophagy activation. How macrophage autophagy impacts atherosclerotic progression, particularly when comparing dietary n-3 PUFA supplementation vs. saturated fat feeding, is unknown. METHODS: We generated myeloid-specific autophagy-deficient and control mice in the Ldlr-/- background by transplanting bone marrow from myeloid-specific autophagy-related (atg) 5 knockout mice and wild type controls into irradiated Ldlr-/- recipients. After 7 weeks for recovery from radiation, mice were fed an atherogenic diet containing 0.2% cholesterol and 20% calories as palm oil (PO diet), or 10% calories as PO plus 10% calories as fish oil (FO diet) for 16 weeks. RESULTS: Compared to PO, FO significantly reduced plasma cholesterol, triglyceride, hepatic neutral lipid, and aortic caspase-1 cleavage, but increased aortic efferocytosis, leading to attenuated atherosclerosis in Ldlr-/- mice receiving wild type bone marrow. Myeloid atg5 deletion had little impact on plasma lipid concentrations and hepatic neutral lipid content, regardless of diet. Myeloid atg5 deletion increased aortic caspase-1 cleavage, decreased aortic efferocytosis and worsened atherosclerosis only in the FO-fed Ldlr-/- mice. CONCLUSIONS: Deficient myeloid autophagy significantly attenuated FO-induced atheroprotection, suggesting that dietary n-3 PUFAs reduce atherosclerosis, in part, by activation of macrophage autophagy.

Stimulating pyruvate dehydrogenase complex reduces itaconate levels and enhances TCA cycle anabolic bioenergetics in acutely inflamed monocytes.

The pyruvate dehydrogenase complex (PDC)/pyruvate dehydrogenase kinase (PDK) axis directs the universal survival principles of immune resistance and tolerance in monocytes by controlling anabolic and catabolic energetics. Immune resistance shifts to immune tolerance during inflammatory shock syndromes when inactivation of PDC by increased PDK activity disrupts the tricarboxylic acid (TCA) cycle support of anabolic pathways. The transition from immune resistance to tolerance also diverts the TCA cycle from citrate-derived cis-aconitate to itaconate, a recently discovered catabolic mediator that separates the TCA cycle at isocitrate and succinate dehydrogenase (SDH). Itaconate inhibits succinate dehydrogenase and its anabolic role in mitochondrial ATP generation. We previously reported that inhibiting PDK in septic mice with dichloroacetate (DCA) increased TCA cycle activity, reversed septic shock, restored innate and adaptive immune and organ function, and increased survival. Here, using unbiased metabolomics in a monocyte culture model of severe acute inflammation that simulates sepsis reprogramming, we show that DCA-induced activation of PDC restored anabolic energetics in inflammatory monocytes while increasing TCA cycle intermediates, decreasing itaconate, and increasing amino acid anaplerotic catabolism of branched-chain amino acids (BCAAs). Our study provides new mechanistic insight that the DCA-stimulated PDC homeostat reconfigures the TCA cycle and promotes anabolic energetics in monocytes by reducing levels of the catabolic mediator itaconate. It further supports the theory that PDC is an energy sensing and signaling homeostat that restores metabolic and energy fitness during acute inflammation.

Sirtuins and Immuno-Metabolism of Sepsis.

Sepsis and septic shock are the leading causes of death in non-coronary intensive care units worldwide. During sepsis-associated immune dysfunction, the early/hyper-inflammatory phase transitions to a late/hypo-inflammatory phase as sepsis progresses. The majority of sepsis-related deaths occur during the hypo-inflammatory phase. There are no phase-specific therapies currently available for clinical use in sepsis. Metabolic rewiring directs the transition from hyper-inflammatory to hypo-inflammatory immune responses to protect homeostasis during sepsis inflammation, but the mechanisms underlying this immuno-metabolic network are unclear. Here, we review the roles of NAD+ sensing Sirtuin (SIRT) family members in controlling immunometabolic rewiring during the acute systemic inflammatory response associated with sepsis. We discuss individual contributions among family members SIRT 1, 2, 3, 4 and 6 in regulating the metabolic switch between carbohydrate-fueled hyper-inflammation to lipid-fueled hypo-inflammation. We further highlight the role of SIRT1 and SIRT2 as potential "druggable" targets for promoting immunometabolic homeostasis and increasing sepsis survival.

Pyruvate dehydrogenase complex stimulation promotes immunometabolic homeostasis and sepsis survival.

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.

Lipid-Free Apolipoprotein A-I Reduces Progression of Atherosclerosis by Mobilizing Microdomain Cholesterol and Attenuating the Number of CD131 Expressing Cells: Monitoring Cholesterol Homeostasis Using the Cellular Ester to Total Cholesterol Ratio.

BACKGROUND: Atherosclerosis is a chronic inflammatory disorder whose development is inversely correlated with high-density lipoprotein concentration. Current therapies involve pharmaceuticals that significantly elevate plasma high-density lipoprotein cholesterol concentrations. Our studies were conducted to investigate the effects of low-dose lipid-free apolipoprotein A-I (apoA-I) on chronic inflammation. The aims of these studies were to determine how subcutaneously injected lipid-free apoA-I reduces accumulation of lipid and immune cells within the aortic root of hypercholesterolemic mice without sustained elevations in plasma high-density lipoprotein cholesterol concentrations. METHODS AND RESULTS: Ldlr-/- and Ldlr-/- apoA-I-/- mice were fed a Western diet for a total of 12 weeks. After 6 weeks, a subset of mice from each group received subcutaneous injections of 200 µg of lipid-free human apoA-I 3 times a week, while the other subset received 200 µg of albumin, as a control. Mice treated with lipid-free apoA-I showed a decrease in cholesterol deposition and immune cell retention in the aortic root compared with albumin-treated mice, regardless of genotype. This reduction in atherosclerosis appeared to be directly related to a decrease in the number of CD131 expressing cells and the esterified cholesterol to total cholesterol content in several immune cell compartments. In addition, apoA-I treatment altered microdomain cholesterol composition that shifted CD131, the common ß subunit of the interleukin 3 receptor, from lipid raft to nonraft fractions of the plasma membrane. CONCLUSIONS: ApoA-I treatment reduced lipid and immune cell accumulation within the aortic root by systemically reducing microdomain cholesterol content in immune cells. These data suggest that lipid-free apoA-I mediates beneficial effects through attenuation of immune cell lipid raft cholesterol content, which affects numerous types of signal transduction pathways that rely on microdomain integrity for assembly and activation.

Procollagen C-endopeptidase Enhancer Protein 2 (PCPE2) Reduces Atherosclerosis in Mice by Enhancing Scavenger Receptor Class B1 (SR-BI)-mediated High-density Lipoprotein (HDL)-Cholesteryl Ester Uptake.

Studies in human populations have shown a significant correlation between procollagen C-endopeptidase enhancer protein 2 (PCPE2) single nucleotide polymorphisms and plasma HDL cholesterol concentrations. PCPE2, a 52-kDa glycoprotein located in the extracellular matrix, enhances the cleavage of C-terminal procollagen by bone morphogenetic protein 1 (BMP1). Our studies here focused on investigating the basis for the elevated concentration of enlarged plasma HDL in PCPE2-deficient mice to determine whether they protected against diet-induced atherosclerosis. PCPE2-deficient mice were crossed with LDL receptor-deficient mice to obtain LDLr(-/-), PCPE2(-/-) mice, which had elevated HDL levels compared with LDLr(-/-) mice with similar LDL concentrations. We found that LDLr(-/-), PCPE2(-/-) mice had significantly more neutral lipid and CD68+ infiltration in the aortic root than LDLr(-/-) mice. Surprisingly, in light of their elevated HDL levels, the extent of aortic lipid deposition in LDLr(-/-), PCPE2(-/-) mice was similar to that reported for LDLr(-/-), apoA-I(-/-) mice, which lack any apoA-I/HDL. Furthermore, LDLr(-/-), PCPE2(-/-) mice had reduced HDL apoA-I fractional clearance and macrophage to fecal reverse cholesterol transport rates compared with LDLr(-/-) mice, despite a 2-fold increase in liver SR-BI expression. PCPE2 was shown to enhance SR-BI function by increasing the rate of HDL-associated cholesteryl ester uptake, possibly by optimizing SR-BI localization and/or conformation. We conclude that PCPE2 is atheroprotective and an important component of the reverse cholesterol transport HDL system.

Macrophage apoAI protects against dyslipidemia-induced dermatitis and atherosclerosis without affecting HDL.

Tissue cholesterol accumulation, macrophage infiltration, and inflammation are features of atherosclerosis and some forms of dermatitis. HDL and its main protein, apoAI, are acceptors of excess cholesterol from macrophages; this process inhibits tissue inflammation. Recent epidemiologic and clinical trial evidence questions the role of HDL and its manipulation in cardiovascular disease. We investigated the effect of ectopic macrophage apoAI expression on atherosclerosis and dermatitis induced by the combination of hypercholesterolemia and absence of HDL in mice. Hematopoietic progenitor cells were transduced to express human apoAI and transplanted into lethally irradiated LDL receptor(-/-)/apoAI(-/-) mice, which were then placed on a high-fat diet for 16 weeks. Macrophage apoAI expression reduced aortic CD4(+) T-cell levels (-39.8%), lesion size (-25%), and necrotic core area (-31.6%), without affecting serum HDL or aortic macrophage levels. Macrophage apoAI reduced skin cholesterol by 39.8%, restored skin morphology, and reduced skin CD4(+) T-cell levels. Macrophage apoAI also reduced CD4(+) T-cell levels (-32.9%) in skin-draining lymph nodes but had no effect on other T cells, B cells, dendritic cells, or macrophages compared with control transplanted mice. Thus, macrophage apoAI expression protects against atherosclerosis and dermatitis by reducing cholesterol accumulation and regulating CD4(+) T-cell levels, without affecting serum HDL or tissue macrophage levels.

Nascent high density lipoproteins formed by ABCA1 resemble lipid rafts and are structurally organized by three apoA-I monomers.

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 ∼ 12, 10, 7.5, and <6 nm) and composition were purified 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, ∼ 12 nm and 10 nm diameter, contained ∼ 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 ∼ 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.

Dysfunctional HDL containing L159R ApoA-I leads to exacerbation of atherosclerosis in hyperlipidemic mice.

The mutation L159R apoA-I or apoA-I(L159R) (FIN) is a single amino acid substitution within the sixth helical repeat of apoA-I. It is associated with a dominant negative phenotype, displaying hypoalphaproteinemia and an increased risk for atherosclerosis in humans. Mice lacking both mouse apoA-I and LDL receptor (LDL(-/-), apoA-I(-/-)) (double knockout or DKO) were crossed>9 generations with mice transgenic for human FIN to obtain L159R apoA-I, LDLr(-/-), ApoA-I(-/-) (FIN-DKO) mice. A similar cross was also performed with human wild-type (WT) apoA-I (WT-DKO). In addition, FIN-DKO and WT-DKO were crossed to obtain WT/FIN-DKO mice. To determine the effects of the apoA-I mutations on atherosclerosis, groups of each genotype were fed either chow or an atherogenic diet for 12weeks. Interestingly, the production of dysfunctional HDL-like particles occurred in DKO and FIN-DKO mice. These particles were distinct with respect to size, and their enrichment in apoE and cholesterol esters. Two-dimensional gel electrophoresis indicated that particles found in the plasma of FIN-DKO mice migrated as large α(3)-HDL. Atherosclerosis analysis showed that FIN-DKO mice developed the greatest extent of aortic cholesterol accumulation compared to all other genotypes, including DKO mice which lack any apoA-I. Taken together these data suggest that the presence of large apoE enriched HDL particles containing apoA-I L159R lack the normal cholesterol efflux promoting properties of HDL, rendering them dysfunctional and pro-atherogenic. In conclusion, large HDL-like particles containing apoE and apoA-I(L159R) contribute rather than protect against atherosclerosis, possibly through defective efflux properties and their potential for aggregation at their site of interaction in the aorta. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Apolipoprotein A-I modulates regulatory T cells in autoimmune LDLr-/-, ApoA-I-/- mice.

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 µg 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.

Apolipoprotein A-I and its role in lymphocyte cholesterol homeostasis and autoimmunity.

OBJECTIVE: The purpose of this study was to determine the effects of an atherogenic diet on immune function in LDLr(-/-), ApoA-I(-/-) mice. METHODS AND RESULTS: When LDLr(-/-), ApoA-I(-/-) (DKO), and LDLr(-/-) (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 approximately 1.5-fold increase in T cell proliferation and activation. Plasma antibodies to dsDNA, beta2-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 approximately 1.5-fold increase in T cell activation and proliferation was associated with a approximately 3-fold increase in concentrations of circulating autoantibodies and approximately 2-fold increase in the severity of atherosclerosis suggesting a common link between plasma apoA-I, inflammation, and atherosclerosis.

Inflammation and skin cholesterol in LDLr-/-, apoA-I-/- mice: link between cholesterol homeostasis and self-tolerance?

Diet-fed low density lipoprotein receptor-deficient/apolipoprotein A-I-deficient (LDLr-/-, apoA-I-/-) mice accumulate a 10-fold greater mass of cholesterol in their skin despite a 1.5- to 2-fold lower plasma cholesterol concentration compared with diet-fed LDLr-/- mice. The accumulation of cholesterol predominantly in the skin has been shown to occur in a growing number of other hypercholesterolemic double knockout mouse models sharing deficits in genes regulating cellular cholesterol homeostasis. Exploring the relationship between cholesterol balance and inflammation, we have examined the time course of cholesterol accumulation in a number of extrahepatic tissues and correlated with the onset of inflammation in diet-fed LDLr-/-, apoA-I-/- mice. After 4 weeks of diet, LDLr-/-, apoA-I-/- mice showed a significant increase in skin cholesterol mass compared with LDLr-/- mice. In addition, after 4 weeks on the diet, cholesterol accumulation in the skin was also found to be associated with macrophage infiltration and accompanied by increases in tumor necrosis factor-alpha, cyclooxygenase-2, and langerin mRNA, which were not seen in the liver. Overall, these data suggest that as early as 4 weeks after starting the diet, the accumulation of skin cholesterol and the onset of inflammation occur concurrently. In summary, the use of hypercholesterolemic LDLr-/-, apoA-I-/- mice may provide a useful tool to investigate the role that apoA-I plays in maintaining cholesterol homeostasis and its relationship to inflammation.

Quality control in the apoA-I secretory pathway: deletion of apoA-I helix 6 leads to the formation of cytosolic phospholipid inclusions.

From a total of 47 known apolipoprotein A-I (apoA-I) mutations, only 18 are linked to low plasma HDL apoA-I concentrations, and 78% of these map to apoA-I helices 6 and 7 (residues 143-186). Gene transfer and transgenic mouse studies have shown that several helix 6 apoA-I mutations have reduced hepatic HDL production. Our objective was to examine the impact of helix 6 modifications on intracellular biosynthetic processing and secretion of apoA-I. Cells were transfected with wild-type or mutant apoA-I, radiolabeled with [(35)S]Met/Cys, and then placed in unlabeled medium for up to 4 h. Results show that >90% of newly synthesized wild-type apoA-I was secreted by 60 min. Over the same length of time, only 20% of helix 6 deletion mutant (Delta 6 apoA-I) was secreted, whereas 80% remained cell associated. Microscopic and biochemical studies revealed that cell-associated Delta 6 apoA-I was located predominantly within the cytoplasm as lipid-protein inclusions, whereas wild-type apoA-I was localized in the endoplasmic reticulum/Golgi. Results using other helix deletions or helix 6 substitution mutations indicated that only complete removal of helix 6 resulted in massive cytoplasmic accumulation. These data suggest that alterations in native apoA-I conformation can lead to aberrant trafficking and accumulation of apolipoprotein-phospholipid structures. Thus, conformation-dependent alterations in intracellular trafficking and turnover may underlie the reduced plasma HDL concentrations observed in individuals harboring deletion mutations within helix 6.

Induction of fatal inflammation in LDL receptor and ApoA-I double-knockout mice fed dietary fat and cholesterol.

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.