CD36: linking lipids to the NLRP3 inflammasome, atherogenesis and atherothrombosis

A recent study published in Nature Immunology by Sheedy et al.¹ indicates that uptake of modified low-density lipoprotein (LDL) by the scavenger receptor CD36 primes and activates the NLRP3 inflammasome, providing an early pathogenic pathway that links cholesterol accumulation to the chronic inflammatory process of atherosclerosis.¹ Atherosclerosis arises from chronic vascular inflammation elicited by lipids. During the past few years, several studies have sought to identify the mechanistic link between lipids and inflammation in atherogenesis. Foam cell formation involves the phagocytosis of matrix-retained lipoproteins and the fluid-phase pinocytosis of aggregated lipoproteins by macrophages; however, a substantial body of evidence also suggests that oxidative modification of subendothelially accumulated LDL triggers the inflammatory process underlying atherosclerosis.

The scavenger receptors CD36 and SR-A mediate the majority of modified LDL uptake by macrophages in vitro. In Apoe^−/− mice, these receptors have been implicated in atheroprogression by inducing pro-inflammatory gene expression and macrophage apoptosis.² The inflammatory response of macrophages to atherogenic lipids depends on the cooperation of Toll-like receptor 4 (TLR4) and TLR6 with CD36. Oxidized LDL (oxLDL) sequestered by CD36 induces intracellular CD36–TLR4–TLR6 heteromerization, actiating NF-κB.³ Atherogenic lipid mediators, such as oxLDL, oxidized phospholipids, lipoproteins and fatty acids, also trigger an oxidative burst through the CD36–TLR2–TLR6 pathway, leading to apoptosis of endoplasmic reticulum-stressed cholesterol-overloaded foam cells.⁴ Defective efferocytosis of apoptotic cells then causes secondary necrosis, contributing to necrotic core formation in advanced plaques.

Cholesterol crystals are found in human and mouse atherosclerotic lesions, both in the extracellular spaces and in macrophages. Intracellular cholesterol crystals can exert proatherogenic effects by stimulating IL-1β production by macrophages through NLRP3 inflammasome activation.^5,6 Before the seminal study by Sheedy et al.,¹ it was thought that cholesterol crystals form in the extracellular space before being engulfed by macrophages and that NLRP3 inflammasome activation would result from dysfunctional phagocytosis or lysosomal damage. Sheedy et al. found that uptake of oxLDL by CD36 results in the formation of intracellular cholesterol crystals that cause lysosomal destabilization and NLRP3 activation. Recognition of sterile ligands by CD36 and transformation of these soluble ligands into their pathogenic particulate forms achieves both priming and activation of the NLRP3 inflammasome, which is an event involved not only in atherosclerosis but also in Alzheimer's disease and type 2 diabetes. The heterotrimeric CD36–TLR4–TLR6 signaling complex, acting via NF-κB and reactive oxygen species, primes the NLRP3 inflammasome in response to oxLDL. Macrophages isolated from Apoe^−/− mice that had been fed a high-fat, high-cholesterol Western diet had a greater abundance of Il1a, Il1b and Nlrp3 mRNA than macrophages from normolipidemic wild-type mice, which indicated that these cells were primed in vivo for NLRP3 activity. The abundance of Il1a, Il1b and Nlrp3 mRNA in macrophages from Cd36^−/−Apoe^−/− mice fed a Western diet was similar to that in macrophages from wild-type mice fed regular chow. The NLRP3 activator ATP induced secretion of IL-1β by macrophages obtained from Apoe^−/− mice that had been fed a Western diet, whereas macrophages from Cd36^−/−Apoe^−/− mice treated similarly did not exhibit enhanced secretion of IL-1β. These data demonstrate both the requirement for CD36 in atherogenic stimulus-mediated NLRP3 priming in macrophages in vivo and the contribution of CD36 to ATP-mediated inflammasome activation in hyperlipidemia. Thus, CD36 is the first upstream regulator of NLRP3 that has been described to function in sterile inflammation and can cooperate with the P2X₇ receptor in ATP-mediated NLRP3 activation.⁷ During NLRP3 inflammasome activation, supramolecular assembly of apoptosis-associated speck-like protein containing a caspase-associated recruitment domain (ASC) requires priming and activating signals. The authors observed that treatment with oxLDL alone induces the formation of ASC complexes, consistent with the idea that oxLDL not only primes but also activates NLRP3. Treatment with ATP after priming with oxLDL also induces the formation of such complexes, supporting a cooperative relationship between CD36 and P2X₇ receptor signaling.

In agreement with the critical role for CD36 in both priming and activation of the NLRP3 inflammasome, Cd36^−/−Apoe^−/−, Tlr4^−/−Apoe^−/− and Tlr6^−/−Apoe^−/− mice fed a Western diet had significantly less aortic plaque formation than Apoe^−/− control mice, which was characterized by a lower cholesterol crystal burden and less IL-1β. Accordingly, the inhibition of CD36 expression in macrophages with an antisense oligonucleotide prevented the increase in IL-1β in Apoe^−/− mouse serum. However, the authors did not report the effects of these oligonucleotides on atherogenesis.

Sheedy et al. identified a new mechanism of lipid-mediated inflammasome activation that depends on the recognition and endocytosis of oxLDL by macrophage CD36 and its subsequent nucleation into insoluble cholesterol crystals inside the cell (Figure 1). This particulate ligand activates NLRP3 via lysosomal destabilization. Therefore, the previously proposed model of NLRP3 activation via frustrated phagocytosis of extracellular particulate materials in sterile inflammatory diseases such as atherosclerosis should be revisited. It has been proposed that extracellular crystals released by dying endoplasmic reticulum-stressed and cholesterol-overloaded macrophages in the necrotic lipid core could activate the inflammasome through this mechanism at a later stage of plaque progression. The study by Sheedy et al indicates that CD36-mediated inflammasome activation provides an early pathogenic pathway that links cholesterol accumulation to the chronic inflammatory process of atherosclerosis. Engagement of CD36 by oxLDL would provide both NLRP3 priming and activating signals, thereby playing a crucial role in the long-lasting and low-grade inflammation-dependent process of atherogenesis. Priming of NLRP3 involves NF-κB-driven upregulation of its expression⁸ and deubiquitinating post-transcriptional events.^9,10 Sheedy et al. confirmed NF-κB activation downstream of the heterotrimeric CD36–TLR4–TLR6 complex. The question of whether this signaling complex plays a role in NLRP3 deubiquitination is currently unknown. The other stimuli usually described as being required for NLRP3 activation in TLR-activated cells include extracellular ATP, the ionophore nigericin and crystalline particles such as alum, silica and asbestos. Given the varied biochemical characteristics of NLRP3 stimuli, it was thought that these stimuli affect the homeostatic concentration of secondary messengers leading to NLRP3 activation. These secondary messengers include K⁺ efflux, lysosomal destabilization, membrane permeabilization, mitochondrial damage and release of oxidized DNA, the production of reactive oxygen species, Ca²⁺ influx and cell swelling.⁸ In a recent study, Munoz-Planillo et al.¹¹ concluded that most of the proposed second messengers are dispensable for NLRP3 activation. These authors identified K⁺ efflux as the sole common denominator induced by all tested NLRP3 stimuli, including crystalline and particulate molecules (silica, alum and calcium pyrophosphate dihydrate crystals). Uptake of these molecules by lipopolysaccharide-primed macrophages was accompanied by a significant drop in the intracellular K⁺ concentration that preceded IL-1β secretion. The phagocytosis inhibitors cytochalasin B and latrunculin B impaired crystalline molecule-induced NLRP3 activation. Bafilomycin A, which prevents lysosomal and endosomal acidification, also inhibited this activation. Although Munoz-Planillo et al. did not test cholesterol crystals, it is tempting to speculate that K⁺ efflux, concomitantly with lysosomal destabilization, could form part of the mechanism leading to oxLDL-induced NLRP3 inflammasome activation. If this speculation is correct, particulate materials and extracellular ATP would act through a common secondary messenger to activate NLRP3. During atherogenesis, activated or injured endothelial cells, leucocytes and platelets release ATP that acts in a paracrine manner to transduce sterile inflammatory signals. Among these signals, P2X₇ receptors mediate K⁺ efflux leading to NLRP3 activation in TLR-activated macrophages.⁷ Because ATP assembles ASC complexes in oxLDL-treated macrophages, P2X₇ receptors and CD36 may cooperate in vivo to activate the NLRP3 inflammasome, contributing to plaque formation. Interestingly, a common missense variant in P2X₇ receptors has been associated with reduced risk of ischemic stroke and ischemic heart disease.¹² Importantly, aside from macrophages, CD36 is also expressed on platelets where it mediates oxLDL-dependent platelet activation and release of granule content, including IL-1β.¹³ CD36 contributes to platelet hyperactivity and atherothrombosis in mouse models of hyperlipidemia. Atherothrombosis occurs in advanced plaques when active proteinases continuously released by macrophages/foam cells digest interstitial collagen fibers, reducing fibrous cap thickness and ultimately leading to plaque rupture and exposure of the plaque prothrombotic content to the blood. In macrophages, it has been shown that P2X₇ receptors have a tissue factor-dependent prothrombotic function, defining a link between inflammation and thrombosis.¹⁴ Thus, targeting CD36 and/or P2X₇ may represent a novel approach to anti-inflammatory and anti-thrombotic therapy in metabolic diseases commonly associated with an increased risk of cardiovascular events. However, the question of whether mutation of the CD36 gene protects against or increases the risk of hypercholesterolemia and atherosclerosis and its complications is still unclear.¹⁵ Furthermore, the molecular mechanisms underlying inflammasome activity in human disease remain to be elucidated.

Figure 1.

Linking hyperlipidemia to inflammation in atherogenesis and atherothrombosis. Uptake of the atherogenic lipid mediator oxLDL by CD36 results in the formation of intracellular cholesterol crystals that cause lysosomal destabilization and NLRP3 activation. oxLDL both primes NLRP3 via an NF-κB-dependent pathway and activates NLRP3. CD36-mediated inflammasome activation provides an early pathogenic pathway that links cholesterol accumulation to the chronic inflammatory process of atherosclerosis. During atherogenesis, activated or injured endothelial cells, leucocytes and platelets release ATP that acts in a paracrine manner to transduce sterile inflammatory signals. Among these signals, P2X₇ receptors mediate K⁺ efflux, leading to NLRP3 activation. Because ATP assembles ASC complexes in oxLDL-treated macrophages, P2X₇ receptors and CD36 may cooperate in vivo to activate the NLRP3 inflammasome, contributing to plaque formation. In addition to macrophages, CD36 is expressed on platelets, where it mediates oxLDL-dependent platelet activation and potentially further IL-1β release. P2X₇ receptors contribute to PDI TF-dependent thrombosis. Consequently, both CD36 and P2X₇ receptors may be involved in atherothrombosis upon plaque rupture. oxLDL, oxidized low-density lipoprotein; PDI, protein disulfide isomerase; TF, tissue factor.