INTRODUCTION

Hypercholesterolemia is a major risk factor in the development of atherosclerosis, and it has been postulated that excessive accumulation of cholesterol in vascular macrophages leads to inflammation, plaque instability and clinical manifestations of atherosclerosis. However, recent advances in single cell analyses allowed for identification of several distinct populations of atherosclerotic lesion macrophages, not all of them presenting a foam cell phenotype. These new findings suggest that lipid-laden macrophage foam cells are not inflammatory, likely because they have excess cholesterol removed from the cell surface, esterified and stored as cholesteryl esters in intracellular lipid droplets. In contrast, nonfoamy macrophages in atherosclerotic lesions are characterized by increased abundance of cholesterol-rich lipid rafts in the plasma membrane, fewer lipid droplets and expression of inflammatory genes. There is likely a causative association between increased plasma membrane cholesterol and lipid rafts and the inflammatory activation of macrophages.

Cholesterol and many receptors governing inflammatory responses colocalize in the ordered membrane microdomains – lipid rafts. Upon activation, lipid raft resident and recruited proteins assemble and initiate signaling cascades leading to inflammation. We define inflammarafts as enlarged, clustered lipid rafts containing increased amounts of cholesterol and harboring activated receptors and adaptor molecules, thus serving as a scaffold to organize cellular inflammatory responses [1]. Inflammarafts were found to be surprisingly stable in macrophages isolated from atherosclerotic lesions and in stimulated bone-marrow-derived macrophages (BMDM) in vitro[2▪▪]. The purpose of this review is to summarize current evidence in support of the inflammaraft hypothesis in atherosclerosis. 

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MACROPHAGE HETEROGENEITY IN ATHEROSCLEROTIC LESIONS

Macrophages in atherosclerotic lesions originate from resident vascular macrophages or circulating monocytes and are exposed to a complex of various stimuli, from oxidized lipids to microbial pathogens, which results in a remarkable macrophage heterogeneity and plasticity. Earlier macrophage classifications, starting from the ‘classically’ and ‘alternatively’ activated M1 and M2 macrophages to M4 (activated by CXCL4), Mox (exposed to oxidized phospholipids), or M(Hb) and Mhem (hemoglobin–haptoglobin and heme) [3], have been now updated by a more systematic characterization based on single cell RNA-seq. A recent meta-analysis of 12 scRNA-seq datasets [4▪▪] proposed to refine the nomenclature of macrophages [Adgre1(F4/80)+C5ar1(CD88)+Dpp4(CD26)-] in mouse atherosclerotic lesions to include:

1) resident ‘TLF’ macrophages that highly express Timd4, Lyve1, and Folr2, which further divided into TLF-Cd209hi and TLF-Cd209low clusters; 2) inflammatory macrophages that constitute two clusters, Nlrp3/Il1b (i.e. with active inflammasome) and Ccr2int/MHCII+; 3) foamy, that is, lipid-laden, macrophages that express high levels of Trem2 and Itgax (CD11c), but low MHC-II, and in turn, can be separated into Slamf9+ and Gpnmb+; 4) MAC-AIR (aortic intimal resident macrophages) that are Trem2lowMHC-IIhi; 5) IFNIC macrophages that express signature genes of type I interferon response; and 6) SPMs/cavity macrophages that have an expression profile characteristic of small peritoneal macrophages (Itgax+Cd226+Ccr2+ MHCII+). Additional mononuclear phagocytic cells include monocytes and at least three groups of dendritic cells [4▪▪]. Transcriptomic analysis of macrophages from human atherosclerotic plaques identified populations similar to those described for mouse lesional macrophages, emphasizing the relevance of mouse atherosclerosis models to the human disease [4▪▪]. It is important to realize that the cells with a macrophage phenotype in atherosclerotic lesions can be derived from myeloid as well as trans-differentiated smooth muscle cells [5].

The macrophage classification [4▪▪] reflects gene expression at the mRNA level, which offers an important but still limited view on cellular function. Integrating scRNA-seq with singe cell ATAC-seq, proteomics, lipidomics and metabolomics will add further granularity to understanding the remarkable macrophage heterogeneity in atherosclerosis. Alternatively, a comprehensive view on the intracellular machinery may suggest that macrophages with seemingly distinct gene expression programs have common functional traits, leading the way to a different approach to macrophage classification.

MACROPHAGE FOAM CELLS AND NONFOAMY MACROPHAGES

From a morphological viewpoint, formation of macrophage-derived foam cells is a hallmark of atherosclerosis. Macrophages take up excessive amounts of LDL accumulated in the vascular wall via various mechanisms. Fluid-phase endocytosis of LDL, irrespective of its native or oxidized state, can be constitutive and receptor-independent [6] or activated by minimally oxidized LDL via TLR4/SYK signaling [7]. Macrophage uptake of oxidized LDL (OxLDL) is driven by OxLDL recognition by many pattern-recognition receptors, with CD36 and SR-A playing a major role [8].

However, foam cells constitute only between 20 and 60% of all intimal macrophages depending on a mouse atherosclerosis model [2▪▪,9], raising the question of functional differences between foam cells and nonfoamy macrophages. A landmark study by Kim et al.[9] characterized macrophages sorted out from mouse atherosclerotic lesions based on the abundance of lipid droplets stained with the lipophilic dye BODIPY. The bulk RNA-seq of BODIPYhi (foam cells) and BODIPYlow (nonfoamy) macrophages identified very different gene expression profiles. Foam cells were characterized by increased expression of cholesterol uptake genes, including Cd36 and Msr1 (SR-A), and fatty acid and cholesterol transport genes, including the gene encoding a major cholesterol efflux transporter ABCA1 [9]. This lipid-trafficking profile is likely due to activation of LXR–RXR and PPARγ transcriptional programs [9,10], mediated at least in part via TREM2 [11]. Desmosterol, the most abundant cholesterol biosynthetic intermediate accumulated in foam cells, activates LXR and suppresses inflammation by reducing mitochondrial ROS production and NLRP3-dependent inflammasome activation [12,13▪]. In addition, efferocytosis, a process of apoptotic cell internalization by phagocytes, which contributes to foam cell formation, also activates LXR and suppresses inflammation [3]. Remarkably, deletion of LXR in myeloid cells switches macrophage foam cells to an inflammatory phenotype and accelerates atherosclerosis [14]. In contrast to foam cells, lesional nonfoamy macrophages highly expressed inflammatory genes, including Ccl2, Cxcl2, Tnf, and Nlrp3, as well as a small set of anti-inflammatory genes, such as Arg1 and Cd163[9].

Taken together, these data suggest that while lipid-accumulating foam cells may drive atherosclerotic lesion growth, nonfoamy macrophages are responsible for the plaque inflammatory phenotype.

MACROPHAGE INFLAMMARAFTS IN ATHEROSCLEROTIC LESIONS

‘Metaphorically, lipid rafts are islands of order in a sea of chaos’ [15]. In a simplified way, lipid rafts provide an ordered environment (often designated as liquid-ordered, Lo) for protein complexes to assemble in an otherwise liquid-disordered (Ld) membrane where protein–protein and protein–lipid interactions are less thermodynamically favorable. Lipid rafts are formed because of molecular interactions between cholesterol and sphingolipid [16], but in quiescent cells, lipid rafts are highly dynamic and transient [17]. Upon cell activation, lipid rafts cluster and become more stable to accommodate agonist-induced receptor assembly into functional complexes and initiation of signaling [17]. For example, in LPS-stimulated macrophages, TLR4 dimer-hosting lipid rafts last for approximately 15 min and then diminish because of internalization of the LPS-TLR4 complex [18]. However, under chronic inflammatory conditions, such as chemotherapy-induced peripheral neuropathy (CIPN), TLR4 dimers and increased lipid raft levels in spinal microglia can be detected as late as 3 weeks after a chemotherapeutic intervention [19▪▪]. The realization that the lipid rafts hosting inflammatory receptor complexes can be surprisingly stable led to the introduction of an inflammaraft hypothesis.

The inflammaraft hypothesis posits that:

1) an inflammatory signal recruits receptors localized in small, isolated lipid rafts to form receptor complexes within a large, clustered inflammaraft, thus initiating intracellular signaling events, which lead to cell reprogramming; 2) in turn, this reprogramming changes cholesterol and sphingolipid synthesis and trafficking in a way that sustains inflammarafts; 3) inflammatory receptors remain in close proximity within the inflammaraft – the cell is now primed for chronic inflammatory gene expression and/or augmented response to next, not necessarily the same pathogen or an endogenous, tissue damage-associated molecular pattern (DAMP) agonist.

Similar to microglia in CIPN spinal neuroinflammation, atherosclerotic lesion macrophages express inflammarafts. In a recent study [2▪▪], single cell suspensions from the aortae of Ldlr-/- mice fed a 20-week western-type diet were gated for BODIPYhi foam cells and BODIPYlow nonfoamy F4/80 macrophages. The cells were further analyzed for the levels of lipid rafts and TLR4 dimers, which characterize inflammarafts [19▪▪]. In agreement with the RNA-seq inflammatory gene expression data [9], nonfoamy aortic macrophages maintained significantly higher levels of TLR4 dimers and lipid rafts, that is, expressed inflammarafts, than foam cells [2▪▪]. The study [2▪▪] then tested the hypothesis that in addition to TLR4, inflammarafts host a variety of other receptor complexes as illustrated in Fig. 1. Engagement of even one receptor by its ligand, for example, TLR4 by LPS, may result – by the virtue of fusing several small lipid rafts hosting distinct proteins – in recruitment to and proximity in inflammarafts of many other receptors. This hypothesis was tested using a proximity ligation assay (PLA) in which juxtaposition of oligonucleotide-labeled antibodies allows for fluorescent signal amplification [20]. In murine atherosclerotic lesions, nonfoamy macrophages had TLR4 localized to lipid rafts (TLR4-LR measured by PLA) and expressed TLR2-CD36, TLR2-TLR1 and IFNγR1-IFNγR2 complexes. The majority of these receptor complexes were at significantly lower numbers in foam cells. Further, TLR4-LR and IFNγR1-IFNγR2 in nonfoamy macrophages correlated with MCP-1, IL-1β and IL-6 but not TNFα plasma levels, and TLR2-CD36 correlated with MCP-1 only. There was a significant association between inflammaraft components and total plasma cholesterol. The size of necrotic cores correlated with TLR2-TLR1 [2▪▪]. Exploration of other protein–protein interactions and the localization of these proteins to lipid rafts (PLA between proteins and cholesterol or sphingolipids, which enriched in lipid rafts) will help better understand the molecular architecture of inflammarafts. For example, nicotine-induced NLRP3 inflammasome activation and nicotine-accelerated atherosclerosis depend on α1-nAchR localization to lipid rafts [21▪], and it would be interesting to examine α1-nAchR interactions with other components of its signaling cascade within inflammaraft.

FIGURE 1:

Macrophage inflammarafts. Macrophage foam cells accumulating large quantities of lipid droplets, which are considered a hallmark of atherosclerotic plaques, constitute a fraction of all macrophages in murine aortic lesions. Foam cells contain less cholesterol-rich lipid rafts per surface area than nonfoamy macrophages. Receptors mediating OxLDL uptake, such as CD36 and SR-A, are localized to these smaller lipid rafts, but fewer receptor complexes are formed. In contrast, larger lipid rafts in nonfoamy macrophages provide a platform for inflammatory receptors assembly into functional complexes, with an example of CD36 interacting with TLR2 and supporting oxidized phospholipid inflammatory signaling as reported. TLR4 homodimers, TLR2-TLR1 and IFNγR1/2 complexes are also present. These enlarged lipid rafts hosting large numbers of assembled or poised to assemble inflammatory receptor complexes are designated as inflammarafts. Cholesterol efflux (illustrated with ABCA1 transporter) is inhibited in inflammatory cells, leading to sustained inflammaraft expression in nonfoamy macrophages. In conjunction with overall inflammatory gene expression, the assembly of NOX in inflammarafts and inflammatory increase in mitochondrial ROS (reactive oxygen species) activate NLRP3 inflammasome and IL-1β production.

The first study of macrophage inflammarafts in atherosclerosis [2▪▪] introduces a new perspective on the components of an inflammatory milieu in atherosclerotic lesions. Its limitation is in sorting lesional macrophages only based on lipid droplet accumulation and not accounting for a greater macrophage diversity identified by RNA-seq. This will become feasible as robust surface markers are assigned to the macrophage populations classified by their mRNA expression profiles. Alternatively, new methods of interrogating inflammarafts may emerge, which will be compatible and can be integrated with scRNA-seq.

MECHANISMS OF INFLAMMARAFT FORMATION AND MAINTENANCE

Mechanisms behind formation and maintenance of inflammarafts in macrophages are underexplored. However, recent conceptual and technological advances in the study of membrane organization and lipid rafts in various cell types suggest novel hypotheses and chart new directions of research. Below are several such reports; the list is certainly not exhaustive.

An intriguing recent development is the discovery of thermodynamic coupling between protein condensates and lipid rafts [22▪▪]. In T cells, the transmembrane adaptor protein LAT localizes to lipid rafts and also, upon cell activation, forms biomolecular condensates with Grb2 and Sos1, facilitating signaling. Formation of LAT/Grb2/Sos1 condensates at the cytoplasmic membrane leaflet induces lipid raft clustering and stabilizes these enlarged lipid raft assemblies. Conversely, lipid rafts nucleate and stabilize LAT protein condensates [22▪▪]. LAT expression in macrophages is low, but other lipid raft resident proteins may have a similar function of coupling protein condensates with lipid rafts in macrophages. One candidate is the family of prohibitins, which mediate fatty acid transport and also function as a scaffold organizing protein complexes at different membrane compartments. Depletion of prohibitins in macrophages reduces lipid rafts and significantly inhibits the inflammatory response to LPS [23▪].

Also fascinating are the recent reports that small noncoding RNAs modified with sialylated glycans (glycoRNA) are expressed on cell surface and localized to lipid rafts [24▪▪,25▪▪]. Because glycoRNAs interact with many Siglecs, which play diverse roles in inflammation and atherosclerosis [26], the discovery of glycoRNA and their localization to lipid rafts may open a new window into inflammaraft biology.

INFLAMMARAFTS REFLECT HYPERINFLAMMATORY MACROPHAGE REPROGRAMMING

Lipid rafts are required for many cellular functions, not only for mediating inflammatory responses. For example, CD36 localization to lipid rafts is required for its function in OxLDL uptake and foam cell formation [27]. The difference between foam cells and nonfoamy macrophages is that inflammaraft-dependent clustering of CD36 with the inflammatory receptor TLR2 preferentially occurs in nonfoamy macrophages [2▪▪], where CD36 likely supports inflammatory response to OxLDL rather than its uptake. Another recent example is the discovery of a hyperhomocysteinemia-mediated increased expression of acid sphingomyelinase, which produces increased levels of ceramide and promotes lipid raft clustering that in turn activates NOX and ROS-dependent NLRP3 inflammasome and exacerbates atherosclerosis [28▪▪]. This nuanced receptor/oxidase trafficking to distinct membrane domains underscores the significance of inflammaraft-mediated macrophage reprogramming in atherosclerosis.

In cell culture, a short exposure of macrophages to the TLR4 ligand LPS not only induced TLR4 localization to lipid rafts (TLR4-LR) but also the TLR2-TLR1 assembly, and conversely, the TLR2/TLR1 ligand Pam3CSK4 induced TLR4-LR [2▪▪], suggesting that one agonist can prime macrophages for a hyperinflammatory response to a subsequent different stimulus. In a cellular model of trained immunity [29▪▪], BMDM were subjected to a 24 h treatment with LPS or OxLDL, followed by a 6-day washout period and then a 4 h stimulation with Pam3CSK4 or LPS [2▪▪]. At 24 h, OxLDL induced accumulation of lipid droplets but reduced overall surface TLR4 expression and TLR4 dimers, whereas the LPS treatment did not induce lipid accumulation but resulted in significant increases of TLR4 dimers and lipid rafts. Of note, the TLR4 inflammaraft phenotype persisted in BMDM for 6 days after LPS removal [2▪▪]. Even after a 6-day washout, the LPS-trained BMDM continued to express higher levels of Cxcl2, Il6 and Il1b mRNA. Predictably, BMDM treated with 100 ng/ml LPS were tolerant to a second LPS stimulation [2▪▪]. However, the LPS-trained cells displayed hyperinflammatory Cxcl2, Ccl2, Il6 and Il1b responses to Pam3CSK4 compared with cells exposed to vehicle or OxLDL [2▪▪]. These results indicate that inflammarafts are associated with hyperinflammatory macrophage reprogramming. Indeed, signaling from TRL4 within lipid rafts reprograms macrophages to an activated inflammatory phenotype by metabolic and epigenetic rewiring [30]. The results also suggest that one agonist can prime macrophages – via formation of inflammarafts that host a community of inflammatory receptors – for a hyperinflammatory response to a broad spectrum of secondary stimuli. Atherosclerotic lesions flooded with DAMPs and occasionally exposed to microbial pathogens are the fertile ground for maintaining inflammarafts by macrophages reprogrammed for a low grade but persistent expression of inflammatory genes and for an augmented inflammatory response to incoming stimuli.

INFLAMMARAFTS AS A THERAPEUTIC TARGET

Lipid rafts can be disrupted by depleting the membrane of cholesterol or sphingolipids. Statins reduce overall cholesterol biosynthesis and have anti-inflammatory properties, in part because of decreasing the cholesterol inflow supporting lipid rafts in macrophages [31]. β-Cyclodextrins, which effectively sequester cholesterol, have been used in many in-vitro and in-vivo studies and in clinical trials to treat Niemann–Pick disease [28▪▪,32,33]. LXR agonists and reconstituted HDL or apoA-I mimetics facilitate cholesterol efflux and thus the reduction of lipid rafts [34▪▪,35]. Inhibition of ganglioside synthases also reduces lipid rafts [36]; however, activation of sphingomyelinases while hydrolyzing sphingomyelin, also produces lipid raft-clustering and inflammatory ceramides [28▪▪].

The challenge of using these methods for reducing lipid rafts is that they are nonselective and, in clinical settings, may cause adverse effects by interfering with normal physiological processes that rely on lipid rafts integrity. Apolipoprotein A-I binding protein (AIBP) is a new, selective regulator of inflammarafts. AIBP binds to TLR4 and thus facilitates cholesterol depletion primarily from cells expressing high surface levels of TLR4, such as dorsal root ganglia (DRG) nociceptive neurons and macrophages and activated spinal microglia [19▪▪,37]. Intrathecal administration of AIBP reverses established neuropathic pain in mouse models, with the therapeutic effect of a single dose lasting for over 2 months [18,19▪▪]. The prolonged therapeutic impact of AIBP is dissociated from its relatively short (hours) pharmacokinetics, suggesting a disease-modifying effect, likely via reprogramming of spinal and DRG cells involved in nociception [19▪▪]. The detailed characterization of AIBP effect on macrophage inflammarafts in atherosclerosis is yet to be conducted, but earlier studies found that HFD-fed Apoa1bp−/−Ldlr−/− mice had more atherosclerosis than Ldlr−/− mice and that atherosclerosis was reduced in mice infected with AAV-AIBP [38].

CONCLUSION

Among diverse macrophage populations in atherosclerotic lesions, nonfoamy macrophages expressing inflammatory genes have abundant lipid rafts and increased numbers of lipid raft-associated TLR4, TLR2, TLR1, CD36 and IFNRG receptor complexes, that is, these macrophages express inflammarafts. Lipid-laden foam cells are not inflammatory and express less inflammarafts. Formation of inflammarafts may serve as an effector mechanism for inflammatory macrophage reprogramming in atherosclerosis. Additional studies are required to test the inflammaraft hypothesis, as well as the therapeutic potential of targeting inflammarafts.

Acknowledgements

None.

Financial support and sponsorship

Authors’ studies are supported by NIH grants HL135737 and HL136275 (to Y.I.M.).

Conflicts of interest

Y.I.M. and S.-H.C. are inventors listed in patent applications related to the topic of this article. Y.I.M. is scientific co-founder of Raft Pharmaceuticals LLC. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. Other authors declare that they have no competing interests.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

▪ of special interest

▪▪ of outstanding interest

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