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.

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.

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.

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.

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.