Nuclear receptors in macrophages: A link between metabolism and inflammation

FEBS Letters 582 (2008) 106–116

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Nuclear receptors in macrophages: A link between metabolism and inflammation Attila Szantoa,*, Tama´s R} oszera,b b

a University of Debrecen, Medical and Health Science Center, Research Center for Molecular Medicine, Life Science Building, Department of Biochemistry and Molecular Biology, Debrecen, Egyetem ter 1 H-4032, Hungary Apoptosis and Genomics Research Group of the Hungarian Academy of Sciences, Debrecen, Egyetem ter 1 H-4032, Hungary

Received 15 August 2007; accepted 6 November 2007 Available online 20 November 2007 Edited by Laszlo Nagy and Peter Tontonoz

Abstract Subclinical inflammation is a candidate etiological factor in the pathogenesis of metabolic syndrome and in the progression of atherosclerosis. A central role for activated macrophages has been elucidated recently as important regulators of the inflammatory process in atherosclerosis. Macrophage differentiation and function can be modulated by a class of transcription factors termed nuclear receptors. These are activated by intermediary products of basic metabolic processes. In this review the contribution of peroxisome proliferator-activated receptors and liver X receptors to macrophage functions in inflammation and lipid metabolism will be discussed in light of their roles in macrophages during atherosclerosis. In the past decade much effort has been made to understand the mechanisms how lipids are handled by macrophages and how inflammation could promote the atherogenic process. Here, we also provide an overview of these two fields.  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Nuclear receptor; Inflammation; PPAR-c; LXR; Atherosclerosis

1. Introduction Disturbances in the basic metabolism lead to several disease states in western societies. Altered lipid turnover in the body results in pathological abnormalities. Among these, cardiovascular disease is the leading cause of death therefore understanding the progression of atherosclerosis has placed lipid metabolism and inflammation in the limelight. Atherosclerosis is a degenerative disease of the tunica intima within arteries characterized by lipid accumulation in the wall initially observed by the development of fatty streaks within the subintimal tissues [1]. Development of fatty streaks is considered to be driven by a pathogenic interaction between circu*

Corresponding author. Fax: +36 52 314989. E-mail addresses: [email protected] (A. Szanto), [email protected] (T. R} oszer). Abbreviations: PPAR-c, peroxisome proliferator-activated receptor c; LXR, liver X receptor; DC, dendritic cell; IFN, interferon; IL, interleukin; LDL, low density lipoprotein; LDLR, LDL receptor; SR, scavenger receptor

lating lipoproteins, hemodynamic factors, endothelial cells, vascular smooth muscle cells, macrophages, T lymphocytes and a constellation of metabolic abnormalities including insulin resistance, hypertension, dyslipidemia and central obesity collectively referred to as metabolic syndrome [2]. Handling of lipids by lesion macrophages is an important metabolic process in the context of hypercholesterolemia and the development of atherosclerotic lesions [3–5]. As a first step during atherogenesis a disturbance in endothel function results in the accumulation of low-density lipoprotein (LDL) in the sub-endothelial matrix. It is not clear how LDL gets modified but this leads to the appearance of minimally oxidized/modified (mmLDL) and subsequently fully oxidized LDL (oxLDL) containing multiple oxidized lipid molecules. Modified lipoproteins activate both endothelial cells and monocytes/macrophages resulting in further monocyte migration into the sub-endothelial space in response to locally produced chemoattractant molecules and cytokines like interferon c (IFN-c), interleukin-1b (IL-1-b), IL-6, tumor necrosis factor a (TNF-a) [6]. Macrophages are the first cellular components in lesion formation, they take up lipid particles through special cell surface proteins, scavanger receptors (SRs), as SR-A and CD36 that are not subjected to downregulation via a feed-back mechanism like LDL receptor. These processes evoke a characteristic inflammatory response by releasing inflammatory molecules to the extracellular environment such as monocyte chemoattractant protein-1 (MCP-1), which attract additional macrophages and other cells to the lesion [7,8]. Macrophages accumulate lipids from oxLDL leading to lipid-loaded foam cell formation, the characteristic cells of the early, cellular phase of lesions (Fig. 1B). Foam cells can also eliminate lipids from the sub-endothelial space through ATP-binding cassette transporters such as ABCA1, ABCG1 towards high-density lipoprotein (HDL) but if the transport is inhibited they accumulate lipids continuously resulting in increased cell death of inflammatory cells and the release of intracellular molecules that lead to a sustained chronic inflammation [4,9,10]. This chronic inflammation with secreted mediators and growth factors make smooth muscle cells migrate from the tunica media, proliferate and rearrange extracellular matrix (Fig. 1B,C). This results in the formation of the late, fibrous atherosclerotic plaques. This late lesion is characterized by calcification (sclerosis), which makes artery wall rigid and fragile. Finally, the originally stable lesion may change into

0014-5793/$32.00  2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.11.020

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There is growing appreciation that a special group of ligandactivated transcription factors play key roles in both the metabolic as well as the inflammatory processes.

2. Lipid metabolism of lesion macrophages: the pros and cons for peroxisome proliferator-activated receptor c (PPAR-c) in atherogenesis

Fig. 1. The normal and pathological histology of the aortic arch. In mammals, as in mice (A) and humans (B and C) the aorta and the large arteries are built up from three tissue layers, the tunica intima (TI) covering the luminal surface (lu) of the vessels, the tunica media (TM) containing elastic layers and the tunica adventitia (TA). Human large arteries often have a subendothelial layer, which grows with age or atherosclerosis. Both connective tissue and smooth muscle cells are present in the intima. The border of the intima is delineated by the internal elastic membrane, which may not be conspicuous because of the abundance of elastic material in the tunica media (see inset in B). The tunica adventitia is a relatively thin connective tissue layer. In mice, mediastinal brown adipose cells (mf) can be involved in the formation of the tunica adventitia. Fibroblasts are the predominant cell type and many macrophages are also present. In mice, atherosclerotic plaques naturally cannot evolve, but in human lipid (lp) accumulation in the so-called fatty streaks of the subendothelial layer can occur with aging (B). In parallel with the accumulation of modified LDL, macrophages invade the tunica intima and differentiate into foam cells (fc). The vascular smooth muscle cells start to proliferate and form a hyalinized fibrous layer (hf) in the luminal surface of the subendothelial layer. The normal structure of elastic lamellae can be also degenerated. At the late phase of atheroprogression the subintimal layer is highly infiltrated by foam cells and the extracellular matrix proteins are degraded and show an eosinophilic hyalinic appearance (fibrous cap). Remnants of the apoptotic foam cells form a cholesterol (chol) rich necrotic core. Sections A, B, C are stained with hematoxylin–chromotrope, the inset is stained with orcein. Magnifications: 1000· (A), 250· (B and C), 400· (insert).

an unstable vulnerable plaque due to an imbalance between factors producing and degrading extracellular elements and this can easily rupture the covering endothelium leading to the formation of thrombus and intravascular coagulation.

Nuclear receptors are ligand-activated transcription factors. Small lipid-soluble molecules activate them. Members of this family can be located in the cytosol like classic steroid receptors (e.g. glucocorticoid receptor) or in the nucleus binding to the response elements of their target genes. Upon ligand binding receptors induce transcription of their target genes. Here, we are focusing on PPAR-c and liver X receptor (LXR) since they have been shown to play a substantial role in lesion macrophages during atherosclerosis. Both of them form heterodimer with a common partner, retinoid X receptor (RXR). PPAR-c is most prominently expressed in adipose tissue and myelomonocytic cells, as macrophages and dendritic cells (DCs). PPAR-c promotes uptake of oxLDL and subsequent differentiation of the macrophages to foam cells [11]. It is well known, that PPAR-c is expressed in foam cells of atherosclerotic lesion [11,12]. Oxidized but not native LDL promotes its own uptake via scavenger receptor CD36 by PPAR-c [11]. Two components from oxLDL, 9-hydroxy octadecadienoic acid (9-HODE) and 13-HODE were identified as endogenous activators and bona fide ligands for PPAR-c [13]. There are also other scavenger receptors involved in LDL/oxLDL uptake, which will be discussed later. Thus, the consequence of oxLDL internalization is the activation of PPAR-c that enhances further expression of CD36. To provide genetic evidence that PPAR-c is required for macrophage development and CD36 expression PPAR-c-deficient embryonic stem (ES) cells were generated. Surprisingly, these can be differentiated into macrophages in vitro. In chimeric mice generated with mutant ES cells PPAR-c-deficient cells are also able to contribute to the macrophage lineage in vivo [14]. However, retroviral transfer of the receptor facilitates induction of CD36 and oxLDL uptake in response to PPAR-c-specific agonists. There is no change in the mRNA level of CD36 when PPAR-c-deficient macrophages are treated with synthetic agonists such as thiazolidinediones (TZDs). These suggest that PPAR-c is not necessary for macrophage development but required for the induction of CD36 and oxLDL uptake. The model above would suggest that PPAR-c is a pro-atherogenic factor. To define the in vivo role of PPAR-c in atherogenesis PPAR-c null bone marrow was transplanted into LDL receptor (LDLR) knockout mice, a murine model of atherosclerosis. Although animals received cholesterol-rich diet for 8 weeks, plasma total cholesterol levels were similar in control and PPAR-c null bone marrow transplanted mice, however the degree of atherosclerosis was significantly increased in PPAR-c-deficient bone marrow transplanted recipients [15]. TZDs were also reported to inhibit the development of atherosclerosis in LDLR-deficient male mice [16]. Chen and colleagues showed similar results in ApoE null mice, another murine model of atherosclerosis [17]. Targeted disruption of the PPAR-c gene in macrophages resulted in reduced total

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plasma and HDL cholesterol levels and cholesterol efflux was significantly decreased from macrophages elicited by thioglycolate in mutant mice [18,19]. To summarize, these observations suggest that PPAR-c in macrophages is rather anti-atherogenic, it induces cholesterol removal and its synthetic agonists also exert anti-atherogenic effects.

3. Coupling of the signaling events of PPAR-c and LXR: role for endogenous ligands As detailed above, PPAR-c enhances uptake of its own ligand via oxLDL but it can also induce cholesterol efflux from macrophages via several mechanisms. An important one is the induction of another nuclear receptor, LXR-a [15], which in turn activates expression of a set of genes involved in cholesterol efflux and transport. Two LXR proteins (a and b) are known to exist in mammals. LXR-a is highly expressed in the liver but significant levels are present in macrophages, kidney, intestine, spleen and adrenal glands. LXR-b is more ubiquitously expressed and it has been found in nearly every tissue examined [20]. Several oxysterols have been identified as endogenous ligands for LXR. LXRs have been implicated in the regulation of cholesterol metabolism and clearance [21,22] and recently in inflammation [23,24]. Mice lacking LXR-a lose their ability to respond normally to dietary cholesterol and are unable to tolerate any amount of cholesterol in excess of that they synthesize de novo [25–27]. These mice develop severe atherosclerosis. Serum and hepatic cholesterol levels and lipoprotein profiles of cholesterol-fed animals showed no significant differences between LXR-b / and wild-type mice. LXR-b / mice – in contrast to LXR-a / mice – maintain their resistance to dietary cholesterol. Synthetic LXR agonist was shown to reduce atherosclerosis in two mouse models, in ApoE / and in LDL receptor / mice [28]. Among LXR target genes there are lipid transporters, such as ABCA1 and ABCG1, members of the ATP-binding cassette family of transporter proteins that are likely to be responsible for cholesterol efflux [29]. They are highly expressed in lipidloaded macrophages [29]. Cholesterol clearance is impaired in fibroblasts isolated from patients with Tangier-disease, a disease characterized by mutations in ABCA1 gene and marked cholesterol accumulation in macrophages and other reticuloendothelial cells [30–32] which suggests that ABCA1 has a pivotal role in cholesterol efflux. Several studies reported that LXRs mediate cholesterol efflux by inducing cholesterol transporters ABCA1, ABCG1 and later ABCG5 and ABCG8 [21,29,33–35]. It is also known that oxLDL has cytotoxic effects on macrophages [36]. Induction of apoptosis inhibitor of macrophages (AIM) by LXR in mouse supports macrophage survival in the lesion, which allows macrophages to harbor large amount of cholesterol [37–39]. These observations highlight the importance of LXR activation in foam cells. A number of oxysterols were identified as potential endogenous ligands for LXR [22,25,40–42]. One of these, 27-hydroxycholesterol is produced by a p450 enzyme CYP27, which is a mitochondrial enzyme involved in alternative bile acid synthesis [43,44] and was reported to be associated with atherosclerotic lesions [45,46]. Mutation of the

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enzyme leads to a human disease cerebrotendinous xanthomatosis (CTX), a rare sterol-storage disease characterized by xanthomas in tendons and also in the central nervous system leading to ataxia, spinal cord paresis, neurological dysfunctions, normolipidemic xanthomatosis and accelerated atherosclerosis [47–49]. We found that retinoid receptors and PPAR-c induce the expression of CYP27 resulting in increased production 27-hydroxycholesterol in macrophages which activates LXR and enhances cholesterol efflux from macrophages through ABC transporters [50]. Thus, it seems that oxLDL uptake leads to the activation of PPAR-c in macrophages, which in turn enhances cholesterol efflux via the induction of LXR and CYP27.

4. Alternative mechanisms for PPAR-driven lipid transport in macrophages Besides the PPAR-c-induced LXR activation there are further possible mechanisms that could modulate nuclear receptor-regulated lipid transport in macrophages. Another mechanism of transcriptional regulation exists at the promoter level of target genes. Activity of the nuclear receptors depends on the recruitment and exchange of corepressors and co-activators. Recently, a co-factor, adipocyte enhancer-binding protein 1 (AEBP1) was shown to act as a transcriptional repressor that impedes macrophage cholesterol efflux, promotes foam cell formation via PPAR-c and LXR-a down-regulation. Contrary to AEBP1-deficiency, AEBP1 overexpression in macrophages is accompanied by decreased expression of PPAR-c, LXR-a and their target genes with concomitant elevation of pro-inflammatory cytokines [51]. It was also reported that activation of PPAR-c reduced cholesterol esterification, induced expression of ABCG1 and stimulated HDL-dependent cholesterol efflux in an LXR-independent manner [52] suggesting that PPAR-c can both directly and indirectly (through LXR) induce transcription of ABCG1.

5. Summary of the roles of PPARs and LXR in lipid metabolism of the macrophages According to the current model, oxLDL activates transcription on multiple levels. It transports ligand for PPAR-c and ligand precursor for LXR. These lipid molecules turn on a specific transcription program within the macrophages resulting in further synthesis of PPAR-c and induction of its target genes: scavenger receptors, metabolic enzymes, LXR. Consequently, further oxLDL is taken up and reverse cholesterol transport is also activated through the LXR. Furthermore, the survival of macrophages is also influenced by LXR. The existence of such transcriptional cascade predicts that modulation of one of the elements will affect all the others and the net effect on cellular cholesterol level depends on the balance between the influx and efflux. In the context of macrophages, an alteration in the balance between the two processes and the subsequent overloading of macrophages with lipids results in foam cell formation. Mutation of PPAR-c, CD36 or other scavenger receptors, LXR, ABC-transporters result in increased atherosclerosis in both men and mice. However, there are species-specific differences: Cholesterol metabolism of the men and mice are different: the main lipoprotein is the HDL

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in mice resulting in a protection against atherosclerosis. The regulation of lipid influx and efflux by nuclear receptors is also different. CYP27 can be induced by retinoids and PPAR-c only in human macrophages [50]. Deletion of CYP27 does not induce atherosclerosis in mice [53–55] only in men [45,46]. LXR can induce its own transcription only in human cells [26,56,57]. AIM is regulated by LXR only in mice [37]. There are reports suggesting that PPAR-a and b/d can also participate in regulatory processes in foam cells. When PPARa, c and b/d agonists were compared, a PPAR-a-specific agonist strongly inhibited atherosclerosis, whereas a PPARb/d-specific agonist failed to inhibit lesion formation. The PPAR-b/d agonist, GW0742X has potent anti-atherogenic activity in the LDLR knockout mice [58]. PPAR-b/d functions as a sensor for VLDL [59] and promotes VLDL-derived fatty acid catabolism. Unliganded PPAR-b/d suppresses fatty acid utilization through active repression, which is reversed upon ligand binding [60,61]. PPAR-b/d agonist reduces expression of Niemann-Pick C1-like 1 protein (Npc1l1) in the intestine resulting in lower cholesterol absorption [62]. Based on these data we can conclude that all three PPAR isotypes have anti-atherogenic effects. Analyzing nuclear receptors in macrophages revealed that PPAR-c and LXR-a have key functions in the regulation. Nevertheless, many questions remain to be answered about nuclear receptor activation.

6. Inflammation in atherosclerosis: another target for PPAR-c and LXR Macrophages are primary inflammatory cells regulating many steps of innate and adaptive immune responses. The questions how inflammation regulates nuclear receptor activity and how PPAR-c and LXR could influence inflammatory reactions are the most important ones in the context of atherogenesis. Early atherosclerosis is characterized by leukocyte recruitment and expression of pro-inflammatory cytokines [9,10]. Lesion formation starts with the attraction, recruitment and activation of monocytes, endothelial cells, T lymphocytes and smooth muscle cells accompanied with a local inflammation. Modulation of these inflammatory processes is very likely to affect progression of the disease. Macrophages are considered as the key cellular components of atherosclerotic lesions, therefore factors that influence their presence and inflammatory activity can also affect progression of the disease. A variety of macrophage functions related to immune regulation are known to be involved in atherogenesis. PPAR-c was described as a general negative regulator of macrophage activation and has also been shown to reduce inflammatory reactions based on data showing that PPAR-c agonists, like the prostanoid 15-deoxy-D12,14-prostaglandin J2 (15d-PGJ2) or synthetic activators like TZDs inhibited the expression of inflammatory cytokines and other molecules. It was also shown in PPAR-c-deficient macrophages that the receptor is not essential for PPAR-c ligands to exert antiinflammatory effects in macrophages [14]. Expression of proinflammatory genes is also inhibited by natural and synthetic ligands in both wild-type and macrophage-specific PPAR-c conditional knockout mice [63,64]. Nevertheless, recently a possible molecular mechanism responsible for PPAR-c-mediated inhibition of inflammatory gene expression has been

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reported demonstrating that ligand-induced SUMOylation of PPAR-c targets the receptor to a co-repressor complex on inflammatory gene promoters [65]. A reciprocal regulation was shown to exist between inflammation and LXR-mediated lipid metabolism. LXR activators inhibit the expression of inflammatory mediators [23] and microbial compounds block the induction of LXR target genes [24]. This model provides a possible scenario how the theory of pathogen-evoked atherosclerosis and the cholesterol metabolism can be integrated. LXR, similarly to PPAR-c has been reported as target for SUMOylation-mediated trans-repression [66]. So, both PPAR-c and LXR interfere with inflammatory processes and generally inhibit inflammation, which may be important during atherogenesis in the developing lesion.

7. Macrophage development, adhesion and migration: steps towards atherosclerosis Beside the general effects of PPAR-c and LXR on inflammation, several mechanisms are known to exist where nuclear receptors regulate various aspects of the inflammatory reactions. In this chapter we summarize these checkpoints during atherosclerosis and the possible involvement of PPAR-c and LXR in modulating these processes at the macrophage level. Development of macrophages is driven by monocyte colonystimulating factor (M-CSF). It also augments SR-A, cytokine and growth factor expression and serves as a survival stimulus. Mice deficient in M-CSF are relatively resistant to atherosclerosis [67,68]. Both granulocyte-macrophage and granulocyte colony-stimulating factors (GM-CSF and G-CSF) increase lesion formation when administered to ApoE knockout mice [69–71]. At the same time GM-CSF deficiency reduces PPAR-c expression and aggravates atherosclerosis in ApoE knockout mice [70]. It was reported that PPAR-c promotes differentiation of macrophages [11] but it is not required for macrophage development per se [14]. Pro-inflammatory cytokines and high cholesterol level induce adhesion molecule expression on endothelial cells, like vascular cell adhesion molecule-1 (VCAM-1) [72]. VCAM-1, P- and E-selectin contributes to atherosclerosis by recruiting monocytes to the lesion as it was demonstrated by smaller fatty streak formation in VCAM-1 or P/E-selectin and LDLR-deficient mice [73–75]. Following their adherence to endothelial cells, monocytes are attracted to inflammatory sites by a family of chemoattractant cytokines known as chemokines (CC). The major signal for monocytes is MCP-1 (Fig. 2). It is known that LDLR-deficient mice lacking also MCP-1 [76] or deletion of its receptor CCR2 in ApoE null mice make them highly susceptible to atherosclerosis [77]. IL-8 exerts similar role in attracting leukocytes to the lesion [78]. Atherosclerotic plaques also express CX3CL1, which stimulates migration of monocytes into the lesion [79,80]. Macrophage migration inhibitory factor (MIF) is upregulated during atherosclerosis [81,82] and deletion of it decreases intimal thickening in LDLR null mice [83]. MIF has been reported recently as a ligand for chemokine receptors CXCR2 and CXCR4 that control leukocyte recruitment during atherogenesis [84]. PPAR-c represses transcription of MCP-1 and its receptor CCR2 in macrophages regulating recruitment of these cells to inflammatory loci in the colon [19,85–88].

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Fig. 2. Monocyte differentiation is an important step of atherogenesis. As a first step of lesion formation a disturbance in endothelium function results in the accumulation of oxidized LDL (oxLDL) in the sub-endothelial matrix. OxLDLs activate both endothelial cells and circulating monocytes resulting in monocyte migration into the subendothelial space in response to locally produced chemoattractant molecules and cytokines. The further differentiation of intimal macrophages is regulated by monocyte chemoattractant protein-1 (MCP1) and monocyte colony stimulating factor (M-CSF). The intimal macrophages express chemokine receptor 2 (CCR2) which forwards differentiation signals to cells. As a result macrophages take up oxLDL via scavenger receptors (SR) and start to produce metalloproteinases (MMPs), cytokines and reactive oxygen species (ROS). These processes evoke a characteristic inflammatory response around lipid accumulation by releasing inflammatory molecules to the environment like MCP-1, which attract additional macrophages and other cell types to the lesion. This process is accompanied with the induction of vascular cell adhesion molecules (VCAM) on endothelial cells, which facilitates the adhesion of monocytes.

Almost every factor affecting macrophage development or migration seems to affect atherogenesis and based on in vivo studies most of them function as an atherogenic factor. On one hand PPAR-c enhances macrophage/foam cell development and on the other hand it inhibits expression of molecules involved in macrophage chemotaxis.

8. Uptake of modified LDL by pattern recognition receptors There are special pattern recognition molecules (scavenger receptors [SR], Toll-like receptors [TLRs]) on the surface of the macrophages originally involved in pathogen uptake, which can also recognize and bind the altered structure of lipoproteins [89]. Several of these, SR-A and CD36, SR-B1 oxLDL receptor-1, collectin 2, CXCL16 [90], stabilin 1, 2 and SR-F on endothelial cells, are involved in the recognition of LDL or oxLDL. The literature is controversial about the consequence of deleting SR-A from mice. There are reports suggesting that it reduces atherosclerosis in ApoE [91,92] or LDLR null mice [93,94] although others did not detect significant effects in another model of murine atherosclerosis (ApoE3 Leiden) [95]. CD36-deficient macrophages are more resistant to foam cell formation as shown in ApoE knockout mice [96,97] and by using CD36 blocking antibodies [98]. Moore and colleagues showed that the lack of SR-A or CD36 results in decreased lipid accumulation in peritoneal macrophages whilst associated with increased lesion areas in ApoE null background [99]. This could be explained by the fact that lipid uptake by

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SRs is an important step in the elimination of cholesterol via reverse cholesterol transport. SR-B1 is expressed in the liver and also in macrophages. It binds HDL, LDL, modified LDL and if knocked out, atherosclerosis is increased in ApoE-deficient [100,101] or LDLR-deficient mice [102]. Deficiency of TLR-2 or 4 significantly reduced atherosclerosis in both ApoE and LDLR knockout animals [103–105]. As detailed above, PPAR-c was reported to upregulate expression of scavenger receptor CD36 [11,106,107] and downregulate SR-A in macrophages [108]. PPAR-c was also reported to induce oxLDL receptor-1 in adipocytes resulting in increased uptake of the lipoprotein [109]. PPAR-c agonists attenuate CXCL16 expression in macrophages [110]. Scavenger receptors are important in the uptake of lipoproteins; they enhance uptake in the periphery and protect cells from excess amount of oxidized lipids by scavenging them towards reverse cholesterol transport but once this system is overloaded lipids accumulate in macrophages and foam cells are formed. Therefore, the presence of SRs is beneficial under ‘‘normal’’ conditions but they turn into harmful molecules during atherogenesis.

9. Inflammatory cytokines: agitators of inflammatory milieu Most cells present in an atherosclerotic lesion produce pro-inflammatory cytokines like IFNc and TNF-a [10]. Cells responsible for pro-inflammatory cytokine production (TH1 polarized cells) are the dominant cell types in atheromas in contrast to TH2 cells that produce predominantly anti-inflammatory cytokines [9]. The dominant cytokines are IFNc, IL-12, IL-15, IL-18 and TNF-a [111]. Deficiency of IFNc signaling (IFNc receptor knockout) reduces [112] while administration of IFNc enhances atherogenesis in ApoE null mice [113]. Deletion of IFNc in LDLR null animals resulted in findings consistent with this scenario [114]. Inhibition of TH1 signaling with pentoxyphilline reduced lesion size [115]. IL-18deficient animals developed reduced atherosclerosis in ApoE knockout [116]. IL-12 has been reported as a pro-atherogenic cytokine [117–121]. Blockade of IL-12 signaling by vaccination attenuates atherosclerosis [122]. IL-12 also induces T-cell recruitment to the plaque [123]. The role of TH2 cytokines is more controversial as shown in both ApoE and LDLR knockout models [121,124]. This suggests that both TH1 and TH2 responses are involved in the pathogenesis of atherosclerosis with a predominant role for the pro-inflammatory molecules in the pathogenesis. This is also supported by the observation that C57Black/6 mice (prone to TH1 responses) can develop atherosclerosis while BALB/c mice (prone to TH2 immune responses) are relatively resistant to atherogenesis [125,126]. Targeted deletion of the transcription factor signal transducer and activator of transcription 6 (STAT6) through which IL-4 regulates gene expression in BALB/c mice makes them susceptible to atherogenesis [126]. Binding of CD40 ligand to its receptor CD154 induces inflammatory response along with expression of pro-inflammatory cytokines, MMPs, adhesion molecules and tissue factor while inhibition of CD40 ligation reduces plaque size [127–130]. PPAR-c agonists inhibited the expression of several proinflammatory molecules, e.g. inducible nitric oxide synthase, gelatinase B [12,16,131], inflammatory cytokines like TNF-a,

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IL-1-b, IL-6 [132–134] and IL-12 [135]. IL-4 induces expression of PPAR-c [106] and according to our observations PPAR-c signaling seems to be part of the anti-inflammatory response by intensifying processes driven by IL-4 and mitigating IFNc-, TNF-a-provoked pro-inflammatory reactions (Szanto et al., unpublished data). It was shown recently, that activation of LXR inhibits secretion of pro-inflammatory molecules like inducible nitric oxide synthase, IL-6, IL-1-b, TNF-a [23]. The anti-inflammatory cytokine IL-10 generates protective signals against atherosclerosis shown in IL-10 deficient mice by increased and in IL-10 transgenic mice by decreased lesion formation [136–138]. 15d-PGJ2 can inhibit IL-10 signaling in a STAT3 dependent and PPAR-c-independent way [139]. Another anti-inflammatory cytokine, transforming growth factor b (TGF-b) also inhibits atherosclerosis at several levels. By inducing collagen synthesis it stabilizes the plaque and prevents formation of the vulnerable plaque. Patients with atherosclerosis have less active TGF-b in their sera [140]. Stimulating TGF-b signaling in mice reduces the formation of fatty streaks, whilst blocking it with neutralizing antibody accelerates disease progression in ApoE-deficient mice [141]. PPAR-c agonists inhibit TGF-b-induced connective tissue growth factor expression via Smad3 [142] or fibronectin expression [143–145]. PPAR-c can repress TGF-b1 gene through induction of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) [146]. Inflammatory processes at the lesion site elaborate growth factors from macrophages, which stimulate smooth muscle cell proliferation and synthesis of extracellular matrix elements resulting in the formation of the fibrous cap. Narrowing of vessel lumen and disruption of this plaque are the major sources of clinical outcomes in atherosclerosis, such as angina pectoris, myocardial infarction and stroke. Destabilization of the fibrous cap is enhanced by secretion of matrix metalloproteinases (MMPs), which are type IV collagenases and degrade collagens of extracellular matrix in mammals. MMPs were also shown to be expressed in atherosclerotic lesion at high level [147–153]. There is evidence to suggest that PPAR-c is capable of inhibiting extracellular matrix remodeling in fatty streaks. It inhibits expression of MMP-9 secretion [154,155]. Interestingly, LXR can also repress MMP-9 expression [156]. These data suggest that nuclear receptors regulate matrix degradation during late atherosclerosis and also inhibit the formation of vulnerable plaque and subsequently thrombosis.

10. Involvement of adaptive immune response in atherogenesis Dendritic cells (DCs) are professional antigen-presenting cells derived from monocytes or other myeloid progenitors and have been implicated in lesion progression. S-100 positive DCs are present at lesion sites [157]. CCR7 the main chemokine receptor regulating DC migration is expressed in the lesion [158]. Recently, a specific group of T-cells, natural killer T-cells (NKT-cells) have been detected in the atherosclerotic lesion. These cells are characterized by the expression of Va14Ja281-containing T-cell receptor (TCR) a-chains and recognize lipid antigens presented by CD1d molecules. Lack of CD1d in ApoE null mice decreases atherosclerosis [159,160]. This suggests a novel crosstalk between lipid molecules and the immune system at the level of DC function.

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PPAR-c has been implicated in several functions of DCs. Activation of the receptor change the expression of co-stimulatory molecules on DCs with reduced capacity to induce lymphocyte proliferation and inhibits migration by decreasing MCP-2, CCR7, EBI1 ligand chemokine [161,162]. Activation of PPAR-c leads to reduced stimulation of DCs by TLR agonists [163]. Furthermore, PPAR-c via induction of retinoic acid synthesis induces CD1d expression in DCs resulting in NKT cell proliferation and possibly altered lipid presentation by CD1 molecules [164,165]. Absence of PPAR-c in DCs increases their immunogenicity [166]. LXR also alters DC phenotype by decreasing IL-12, increasing IL-10 secretion and blocking its T-cell stimulatory ability [167]. A recent study demonstrated that regulatory T-cells (Tregs) reduced atherosclerosis when transferred, while depletion of these CD25+ cells increased pathogenesis [168,169]. Transfer of Tregs into ApoE knockout mice attenuates atherosclerosis [170]. PPAR-c-expressing, but not PPAR-c null Tregs were shown to prevent colitis suggesting a role for PPAR-c in regulating immune responses through Tregs [171].

11. Perspectives PPARs and LXRs are known as ligand-regulated transcription factors that play central regulatory roles in lipid uptake, metabolism and efflux. Importantly, they are also implicated in the regulation of inflammatory reactions. PPARs and LXRs are activated by fatty acids, oxLDL, arachidonic acid metabolites like eicosanoids, prostaglandins, leukotriens and oxidized cholesterol, respectively. Majority of these natural ligands are derived from the metabolism of unsaturated fatty acids and cholesterol, therefore disorders of lipid consumption and processing can influence transcriptional activity of PPAR and LXR-regulated genes. Here, we provided a synopsis of how the regulation of lipid homeostasis and inflammation is interlaced by macrophages within the artery wall during atherogenesis. PPAR-c is an inflammatory mediator controlling many aspects of the inflammatory program with a net anti-atherogenic consequence. However we have to note that some of the anti-inflammatory effects assigned to PPAR-c activators are receptor-independent and inflammatory processes positively regulated by PPAR-c are still elusive. Much less is known about LXR in inflammation but based on our current knowledge we can assume further important findings concerning the role of LXR in DC and lymphocyte functions. Although majority of PPAR- and LXR-induced cellular events result in anti-atherogenic processes, some effects are predicted to be atherogenic at the early stage of atherogenesis. Activation of PPARs and LXRs by exogenous ligands can be easily a ‘‘poison hath residence and medicine power’’, since it can result in multiple changes in gene expression profile of lesion macrophages and cells of the artery wall. Here, we should refer to a very recent report on increased risk of myocardial infarction and risk of death from cardiovascular causes in patients treated with a TZD, Rosiglitazone upon a meta-analysis of previous studies. However, as the authors claim there were limitations of this analysis and further comprehensive studies are required [172]. The main perspective of PPAR and LXR research is to elucidate the biological activities of each receptor subtype to facilitate the development of selective PPAR and LXR modulators

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that exhibit improved pharmaceutical benefits in therapy of atherosclerosis and metabolic syndrome. Also a huge challenge for the future is the characterization of the species-specificity of the identified processes, since many reported mechanisms are seemed to be human or murine-specific. Furthermore, macrophage biology has also been shown to be more complex than previously thought. The identification of various monocyte subtypes, differently activated macrophage subsets and the involvement of DCs in metabolic disorders predict that much remained to be discovered in this field. Acknowledgements: The authors are grateful for L. Nagy for critical reading of the manuscript and for insightful discussions. A.Sz. is supported by the Hungarian Academy of Sciences (Bolyai Scholarship) and by Grants from Hungarian Science Research Fund (OTKA/ 61814) and from the University of Debrecen (Mecenatura). T.R. is also supported by the Hungarian Academy of Sciences.

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