High-density lipoprotein mediates anti-inflammatory reprogramming of macrophages via the transcriptional regulator ATF3.

High-density lipoprotein (HDL) mediates reverse cholesterol transport and is known to be protective against atherosclerosis. In addition, HDL has potent anti-inflammatory properties that may be critical for protection against other inflammatory diseases. The molecular mechanisms of how HDL can modulate inflammation, particularly in immune cells such as macrophages, remain poorly understood. Here we identify the transcriptional regulator ATF3, as an HDL-inducible target gene in macrophages that downregulates the expression of Toll-like receptor (TLR)-induced proinflammatory cytokines. The protective effects of HDL against TLR-induced inflammation were fully dependent on ATF3 in vitro and in vivo. Our findings may explain the broad anti-inflammatory and metabolic actions of HDL and provide the basis for predicting the success of new HDL-based therapies.

CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation.

Particulate ligands, including cholesterol crystals and amyloid fibrils, induce production of interleukin 1ß (IL-1ß) dependent on the cytoplasmic sensor NLRP3 in atherosclerosis, Alzheimer's disease and diabetes. Soluble endogenous ligands, including oxidized low-density lipoprotein (LDL), amyloid-ß and amylin peptides, accumulate in such diseases. Here we identify an endocytic pathway mediated by the pattern-recognition receptor CD36 that coordinated the intracellular conversion of those soluble ligands into crystals or fibrils, which resulted in lysosomal disruption and activation of the NLRP3 inflammasome. Consequently, macrophages that lacked CD36 failed to elicit IL-1ß production in response to those ligands, and targeting CD36 in atherosclerotic mice resulted in lower serum concentrations of IL-1ß and accumulation of cholesterol crystals in plaques. Collectively, our findings highlight the importance of CD36 in the accrual and nucleation of NLRP3 ligands from within the macrophage and position CD36 as a central regulator of inflammasome activation in sterile inflammation.

NLRP3 Inflammasome and the IL-1 Pathway in Atherosclerosis.

Inflammation is an important driver of atherosclerosis, the underlying pathology of cardiovascular diseases. Therefore, therapeutic targeting of inflammatory pathways is suggested to improve cardiovascular outcomes in patients with cardiovascular diseases. This concept was recently proven by CANTOS (Canakinumab Anti-Inflammatory Thrombosis Outcomes Study), which demonstrated the therapeutic potential of the monoclonal IL (interleukin)-1ß-neutralizing antibody canakinumab. IL-1ß and other IL-1 family cytokines are important vascular and systemic inflammatory mediators, which contribute to atherogenesis. The NLRP3 (NOD [nucleotide oligomerization domain]-, LRR [leucine-rich repeat]-, and PYD [pyrin domain]-containing protein 3) inflammasome, an innate immune signaling complex, is the key mediator of IL-1 family cytokine production in atherosclerosis. NLRP3 is activated by various endogenous danger signals abundantly present in atherosclerotic lesions, such as oxidized low-density lipoprotein and cholesterol crystals. Consequently, NLRP3 inflammasome activation contributes to the vascular inflammatory response driving atherosclerosis development and progression. Here, we review the mechanisms of NLRP3 inflammasome activation and proinflammatory IL-1 family cytokine production in the context of atherosclerosis and discuss treatment possibilities in light of the positive outcomes of the CANTOS trial.

Danger signaling in atherosclerosis.

All aspects of the pathogenesis of atherosclerosis are critically influenced by the inflammatory response in vascular plaques. Research in the field of innate immunity from the past 2 decades has uncovered many novel mechanisms elucidating how immune cells sense microbes, tissue damage, and metabolic derangements. Here, we summarize which triggers of innate immunity appear during atherogenesis and by which pathways they can contribute to inflammation in atherosclerotic plaques. The increased understanding gained from studies assessing how immune activation is associated with the pathogenesis of atherosclerosis has provided many novel targets for potential therapeutic intervention. Excitingly, the concept that inflammation may be the core of cardiovascular disease is currently being clinically evaluated and will probably encourage further studies in this area.

Ursodeoxycholic acid impairs atherogenesis and promotes plaque regression by cholesterol crystal dissolution in mice.

Atherosclerosis is a chronic inflammatory disease driven primarily by a continuous retention of cholesterol within the subendothelial space where it precipitates to form cholesterol crystals (CC). These CC trigger a complex inflammatory response through activation of the NLRP3 inflammasome and promote lesion development. Here we examined whether increasing cholesterol solubility with ursodeoxycholic acid (UDCA) affects vascular CC formation and ultimately atherosclerotic lesion development. UDCA mediated intracellular CC dissolution in macrophages and reduced IL-1ß production. In ApoE(-/-) mice, UDCA treatment not only impaired atherosclerotic plaque development but also mediated regression of established vascular lesions. Importantly, mice treated with UDCA had decreased CC-depositions in atherosclerotic plaques compared to controls. Together, our data demonstrate that UDCA impaired CC and NLRP3 dependent inflammation by increasing cholesterol solubility and diminished atherosclerosis in mice.

Weekly Treatment of 2-Hydroxypropyl-ß-cyclodextrin Improves Intracellular Cholesterol Levels in LDL Receptor Knockout Mice.

Recently, the importance of lysosomes in the context of the metabolic syndrome has received increased attention. Increased lysosomal cholesterol storage and cholesterol crystallization inside macrophages have been linked to several metabolic diseases, such as atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Two-hydroxypropyl-ß-cyclodextrin (HP-B-CD) is able to redirect lysosomal cholesterol to the cytoplasm in Niemann-Pick type C1 disease, a lysosomal storage disorder. We hypothesize that HP-B-CD ameliorates liver cholesterol and intracellular cholesterol levels inside Kupffer cells (KCs). Hyperlipidemic low-density lipoprotein receptor knockout (Ldlr(-/-)) mice were given weekly, subcutaneous injections with HP-B-CD or control PBS. In contrast to control injections, hyperlipidemic mice treated with HP-B-CD demonstrated a shift in intracellular cholesterol distribution towards cytoplasmic cholesteryl ester (CE) storage and a decrease in cholesterol crystallization inside KCs. Compared to untreated hyperlipidemic mice, the foamy KC appearance and liver cholesterol remained similar upon HP-B-CD administration, while hepatic campesterol and 7α-hydroxycholesterol levels were back increased. Thus, HP-B-CD could be a useful tool to improve intracellular cholesterol levels in the context of the metabolic syndrome, possibly through modulation of phyto- and oxysterols, and should be tested in the future. Additionally, these data underline the existence of a shared etiology between lysosomal storage diseases and NAFLD.

Cholesterol crystals and inflammation.

Chronic vascular inflammation is regarded as a crucial aspect of cardiovascular disease. However, the elicitors of this inflammatory response in the vessel wall are currently not well understood. Excessive amounts of cholesterol, an abundant and fundamental lipid molecule in mammalian cells, can initiate the development and progression of atherosclerosis. Accumulation of cholesterol in early atherosclerotic lesions results in the formation of macrophage foam cells, and crystalline cholesterol is found as a characteristic of advanced atherosclerotic plaques. Cholesterol crystals can activate the NLRP3 inflammasome, a multimolecular signaling complex of the innate immune system, resulting in caspase-1 mediated activation and secretion of proinflammatory interleukin-1 family cytokines. Furthermore, crystalline cholesterol is believed to induce plaque rupture by physical disruption of the fibrous cap covering atherosclerotic lesions. Here we review the effect of cholesterol deposition and crystallization on inflammatory responses in cardiovascular diseases.

Non-linear optical imaging of atherosclerotic plaques in the context of SIV and HIV infection prominently detects crystalline cholesterol esters.

Chronic HIV infection may exacerbate atherosclerotic vascular disease, which at advanced stages presents as necrotic plaques rich in crystalline cholesterol. Such lesions can catastrophically rupture precipitating myocardial infarct and stroke, now important causes of mortality in those living with HIV. However, in this population little is known about plaque structure relative to crystalline content and its chemical composition. Here, we first interrogated plaque crystal structure and composition in atherosclerotic SIV-infected macaques using non-linear optical microscopy. By stimulated Raman scattering and second harmonic generation approaches both amorphous and crystalline plaque lipid was detected and the crystal spectral profile indicated a cholesterol ester (CE) dominated composition. Versus controls, SIV+ samples had a greater number of cholesterol crystals (CCs), with the difference, in part, accounted for by crystals of a smaller length. Given the ester finding, we profiled HIV+ plaques and also observed a CE crystalline spectral signature. We further profiled plaques from Ldlr-/- mice fed a high fat diet, and likewise, found CE-dominate crystals. Finally, macrophage exposure to CCs or AcLDL induced auto-fluorescent puncta that co-stained with the LC3B autophagy sensor. In aggregate, we show that atheromatous plaques from mice, macaques and humans, display necrotic cores dominated by esterified CCs, and that plaque macrophages may induce autophagic vesicle formation upon encountering CCs. These findings help inform our knowledge of plaque core lipid evolution and how the process may incite systemic inflammation.

Cyclodextrin promotes atherosclerosis regression via macrophage reprogramming.

Atherosclerosis is an inflammatory disease linked to elevated blood cholesterol concentrations. Despite ongoing advances in the prevention and treatment of atherosclerosis, cardiovascular disease remains the leading cause of death worldwide. Continuous retention of apolipoprotein B-containing lipoproteins in the subendothelial space causes a local overabundance of free cholesterol. Because cholesterol accumulation and deposition of cholesterol crystals (CCs) trigger a complex inflammatory response, we tested the efficacy of the cyclic oligosaccharide 2-hydroxypropyl-ß-cyclodextrin (CD), a compound that increases cholesterol solubility in preventing and reversing atherosclerosis. We showed that CD treatment of murine atherosclerosis reduced atherosclerotic plaque size and CC load and promoted plaque regression even with a continued cholesterol-rich diet. Mechanistically, CD increased oxysterol production in both macrophages and human atherosclerotic plaques and promoted liver X receptor (LXR)-mediated transcriptional reprogramming to improve cholesterol efflux and exert anti-inflammatory effects. In vivo, this CD-mediated LXR agonism was required for the antiatherosclerotic and anti-inflammatory effects of CD as well as for augmented reverse cholesterol transport. Because CD treatment in humans is safe and CD beneficially affects key mechanisms of atherogenesis, it may therefore be used clinically to prevent or treat human atherosclerosis.