Macrophages play essential roles in immunity and lipid. Regulation of Macrophage Functions by PPAR-, PPAR-, and LXRs in Mice and Men

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1 ATVB In Focus Metabolic Syndrome and Atherosclerosis Series Editor: Marja-Riitta Taskinen Preview Brief Reviews in this Series: Deprés JP, Lemieux I, Bergeron J, Pibarot P, Mathieu P, Larose E, Rodés-Cabau J, Bertrand OF, Poirier P. Abdominal obesity and the metabolic syndrome: contribution to global cardiometabolic risk. Arterioscler Thromb Vasc Biol. 2008;28: Grundy, SM. Metabolic syndrome pandemic. Arteroscler Thromb Vasc Biol. 2008;28: Barter PJ, Rye KA. Is there a role for fibrates in the management of dyslipidemia in the metabolic syndrome. Arteroscler Thromb Vasc Biol. 2008;28: Kotronen A, Yki-Järvinen. Fatty liver: a novel component of the metabolic syndrome. Arteroscler Thromb Vasc Biol. 2008;28: Gustafson B, Hammarstedt A, Andersson CX, and Smith U. Inflamed adipose tissue: a culprit underlying the metabolic syndrome and atherosclerosis. Arteroscler Thromb Vasc Biol. 2007;27: Regulation of Macrophage Functions by PPAR-, PPAR-, and LXRs in Mice and Men Elena Rigamonti, Giulia Chinetti-Gbaguidi, Bart Staels Abstract Peroxisome proliferator-activated receptors (PPARs) and (liver X receptors) LXRs are ligand-activated transcription factors that control lipid and glucose metabolism, as well as the inflammatory response. Because the macrophage plays an important role in host defense and immunoinflammatory pathologies, particular attention has been paid to the role of PPARs and LXRs in the control of macrophage gene expression and function. Research over the last few years has revealed important roles for PPAR-, PPAR-, and LXRs in macrophage inflammation and cholesterol homeostasis with consequences for atherosclerosis development. In this review we will discuss the role of these transcription factors in the control of macrophage activities, with particular attention to species-differences in macrophage function control by PPARs and LXR between rodents and humans. (Arterioscler Thromb Vasc Biol 2008;28: ) Key Words: nuclear receptors atherosclerosis lipid metabolism inflammation Macrophages play essential roles in immunity and lipid homeostasis. As professional scavengers, macrophages phagocytose microbes and apoptotic and necrotic cells. Although macrophages play important roles in injury responses and tissue remodeling, it is generally considered that sustained activation of these responses may precipitate pathological states. For instance, when macrophages take up excess modified lipoprotein particles, this results in the development of macrophage foam cells, whose accumulation in the arterial wall is a hallmark of the early atherosclerotic lesion. Cholesterol-loading of macrophages stimulates the production of inflammatory mediators, such as cytokines and reactive oxygen species (ROS), resulting in the recruitment of other cell types and hence contributing to the development of a complex lesion. 1 Moreover, macrophages are a heterogeneous cell population, whose activation state and functions are profoundly affected by different cytokines and microbial products. 2 Whereas Th1 cytokines, such as interferon (INF), interleukin (IL) 1-, or lipopolysaccharide (LPS), induce a classical proinflammatory activation profile (M1), Th2 cytokines, such as IL-4 and IL-13, induce an alternative anti-inflammatory phenotype (M2) in macrophages. 3 Altered macrophage functions Original received November 6, 2007; final version accepted February 22, From the Institut Pasteur de Lille, Inserm, U545, and Université de Lille 2, Faculté de Pharmacie et de Médecine, Lille, France. Correspondence to Bart Staels, UR 545 INSERM, Institut Pasteur de Lille, 1, rue Calmette BP245, Lille, France. bart.staels@pasteur-lille.fr 2008 American Heart Association, Inc. Arterioscler Thromb Vasc Biol is available at DOI: /ATVBAHA

2 Rigamonti et al Role of PPAR- and LXRs in Macrophage Homeostasis 1051 contribute to the pathogenesis of many infectious, immunologic, and inflammation-related diseases, including sepsis, rheumatoid arthritis, atherosclerosis, insulin resistance, and the metabolic syndrome. 4,5 Therefore, pharmacological modulation of macrophage function represents an important strategy for the prevention and treatment of these diseases. Several nuclear receptors (NRs) have been identified to be expressed in macrophages, including receptors for steroid hormones, such as the estrogen (ER) and glucocorticoid (GR) receptors, receptors for nonsteroidal ligands, such as the vitamin D (VDR) and retinoic acid (RAR) receptors, as well as receptors binding diverse products of lipid metabolism, such as peroxisome proliferator-activated (PPAR), /,, and liver X receptors (LXR) and. 6 In macrophages, NRs (such as GR-, ER-, PPARs and LXRs) negatively modulate inflammatory responses regulated by pathways such as AP-1 and NF- B. 7 9 Second, a smaller subset of nuclear receptors, such as PPAR-, GR-, and VDR, influences the macrophage differentiation program. 10,11 Third, PPARs and LXRs are also critical orchestrators of macrophage lipid homeostasis, identifying these nuclear receptors as interesting molecular targets for atherosclerosis treatment On ligand-activation, PPARs and LXRs act as a heterodimer with the retinoid X receptor (RXR) and bind to specific DNA response elements in the promoter region of target genes, thus regulating their expression. PPARs can be activated by natural as well as synthetic ligands, such as the hypolipidemic fibrates (clinically used PPAR- activators), glitazones (a class of antidiabetic PPAR- ligands), and high-affinity ligands for PPAR- /, such as GW and GW PPAR- is expressed preferentially in tissues where fatty acids are catabolized, PPAR- is highly present in adipose tissue and modulates crucial aspects of adipocyte differentiation and glucose metabolism, and PPAR- / is ubiquitously expressed. Furthermore, PPARs are expressed in most cell types of the vascular wall as well as in atherosclerotic plaques. 15 LXRs can be activated by oxysterols as well as by synthetic ligands. LXR- is highly expressed in the liver and at lower levels in the adrenal glands, intestine, adipose tissue, macrophages, lung, and kidney, whereas LXR- is ubiquitously expressed. 16 Over the last 5 years, important progress has been made in understanding the role of these nuclear receptors in the control of macrophage functions. In this review we will provide a brief overview of the role of PPAR-, PPAR-, and LXRs in the control of cholesterol homoeostasis, inflammation, and immunity in human and mouse macrophages, highlighting species-specific differences of potential clinical relevance. Because the mouse is the most commonly studied model in functional genomics, we will highlight humanmouse species differences. Macrophage Cholesterol Metabolism The control of macrophage cholesterol homeostasis is of critical importance in the pathogenesis of atherosclerosis, as dysregulation of the balance of cholesterol influx and cholesterol efflux will lead to excessive accumulation of cholesterol in macrophages and their transformation into foam cells. Indeed, macrophage scavenger receptors, including scavenger receptor A (SR-A) and CD36, mediate the uptake of modified lipoproteins, exemplified by oxidized LDL (oxldl). Within the cells, modified LDL-derived cholesteryl esters are hydrolyzed in lysosomes, and cholesterol is probably initially transported to the plasma membrane where it integrates the cell membrane. 17 This process is controlled by a network of proteins of at least 2 components, namely the Niemann Pick type C (NPC) 1 and 2 proteins. 18,19 Excess cholesterol is transported back to the endoplasmic reticulum where it is reesterified by acyl-coenzyme A (CoA):cholesterol acyltransferase 1 (ACAT1) and stored as lipid droplets. 20 Cellular cholesterol at the plasma membrane is available for efflux. Cholesterol efflux is a highly regulated process mediated by specific molecules, including ATP-binding cassette (ABC) transporters (ABCA1, ABCG1, and ABCG4) 21,22 and the scavenger receptor B1 (SR-B1). 23 In addition, caveolin-1, the main structural protein of caveolae, is also involved in the regulation of cholesterol metabolism and efflux. 24 Role of PPAR- A number of studies have provided direct evidence for a critical role of PPAR- in the regulation of cholesterol and fatty acid homeostasis in macrophages. In human macrophages PPAR- activation does not influence acetylated LDL-induced lipid accumulation, indicating that PPAR- does not promote foam cell formation. 25 In contrast, PPAR- reduces macrophage uptake of glycated LDL, 26 a subtype of proatherogenic particles containing glycated apolipoprotein (apo) B, abundant in diabetic patients and taken up by macrophages through a lipoprotein lipase (LPL)-dependent mechanism. 27 Interestingly, PPAR- induces LPL gene expression, but decreases its secretion and enzyme activity. 26 In addition, PPAR- activators reduce triglyceride-rich lipoprotein accumulation, as a consequence of the suppression of apob-48 receptor expression. 28 Consistent with these results, macrophage transfer and bone marrow reconstitution experiments demonstrated that PPAR- inhibits macrophage-foam cell and atherosclerotic lesion formation in vivo in LDL receptor-deficient (LDLR / ) mice. 29,30 Recently, the role of PPAR- in intracellular lipid transport and metabolism has been studied in more detail in human macrophages. PPAR- activation stimulates the postlysosomal mobilization of cholesterol by inducing NPC1 and NPC2 gene and protein expression, leading to an enrichment of cholesterol in the plasma membrane. 31 Moreover, activation of PPAR- leads to a decrease of cholesteryl ester (CE) levels. 32,33 PPAR- increases the expression of carnitine palmitoyl transferase (CPT)-I, an enzyme located in the mitochondrial outer membrane controlling FA -oxidation. 34 Increased expression of CPT-I may result in a reduced availability of FAs as substrate for ACAT1, which could explain the decreased CE formation. 32 In addition, PPAR- activation may influence CE hydrolysis by increasing neutral cholesteryl ester hydrolase (NCEH) gene expression. 35 Finally, PPAR- controls the expression of genes involved in cholesterol efflux pathways. In human macrophages, PPAR- activators enhance the expression of CLA-1/SR-B1 by a posttranslational, not yet elucidated, mechanism. 36 In addition, PPAR- activation upregulates ABCA1 expression

3 1052 Arterioscler Thromb Vasc Biol June 2008 Table. Roles of PPAR-, PPAR-, and LXRs in Lipid Homeostasis and Immune Response in Macrophages Highlighting Species-Specific Differences Function Cholesterol PPARalpha PPARgamma LXR Metabolism Human Mouse Mechanism Human Mouse Mechanism Human Mouse Mechanism Cholesterol accumulation 1. AcLDL-cholesterol n.d. loading 2. glyldl-cholesterol 2 2 LPL activity 2 2 LPL activity loading 3. Tg-rich 2 2 apob-48r 2 2 apob-48r lipoprotein-uptake 4. In vivo model of 2 n.d. 2 1 CD36 2 SR-A foam-cell formation Cholesterol trafficking 1 X 1 NPC1 n.d. n.d. n.d. 1 X 1 NPC1 1 NPC2 1 NPC2 Cholesterol esterification 2 X 1 CPT-I ACAT1 (only H) 2 n.d. 2ACAT1 activity 2 ACAT1 activity 1 NCEH (only H) 1 NCEH n.d. (M) Cholesterol efflux 1 X 1 SR-BI SR-BI (only H) ABCA1 1 ABCA1 1 ABCA1 1 ABCG1 1 LXRalpha 1 LXRalpha 1 ABCG4 1 ApoE 1 PLTP 1 ABCG1 1 ApoE 1 Caveolin (only H) 1 ApoC cluster 1 Caveolin (only H) Immune Response Human Mouse Mechanism Human Mouse Mechanism Human Mouse Mechanism Production of inflammatory mediators 2 2 1) NF-kB pathway 2) AP-1 pathway Antibacterial activity ROS production 1) 1 NADPH oxidase subunits 2 2 1) NF-kB pathway 2) AP-1 pathway 3) SUMOylation (only M) 2 2 1) NF-kB pathway 2) AP-1 pathway 3) SUMOylation (only M) 4) 1 IL-1Ra (only H) 4) 1 arginase II (only M) 5) 1 macrophage alternative activation apoptotic neutrophil uptake (only H) (p47 and gp91) 1 CD36 f parasite phagocytosis (only M) 2) 1 MPO (only H) X indicates not regulated; n.d., not determined; H, shown for human; M, shown for mouse TLR-4 (only H) 1 MPO (only H) 1 NADPH oxidase subunits -MR f Candida Albicans uptake (only M) (p47 and gp91) (only H) 1 macrophage survival (only M) by an indirect mechanism involving the induction of LXR expression. 25,37 The increase in ABCA1 expression promotes apoai-mediated cholesterol efflux, 25 thus facilitating cholesterol removal and its transport back to the liver. Taken together, these data indicate that PPAR- is a key controller of macrophage cholesterol homeostasis. However, species-specific differences exist between human and murine macrophages and cannot be overlooked (Table). PPAR- regulation of cholesterol trafficking appears to occur in a species-specific manner, because no induction of NCP1 and NCP2 is observed in murine bone marrow derived macrophages on stimulation with PPAR- ligands. 31 Moreover, in murine macrophages, PPAR- does not affect the rate of cholesterol esterification, and the molecular basis for this

4 Rigamonti et al Role of PPAR- and LXRs in Macrophage Homeostasis 1053 difference is still unclear. 29 Finally, PPAR- agonists regulate apoai-dependent efflux in a species-specific manner because neither LXR nor ABCA1 are induced in cholesterolloaded peritoneal macrophages on PPAR- activation. 29 Role of PPAR- The involvement of PPAR- in regulating lipid metabolism in macrophages was initially suggested by the discovery of the scavenger receptor CD36 as a PPAR- target gene in macrophages. 38 The critical role of PPAR- in CD36 regulation was demonstrated by using PPAR- -null embryonic stem cells, 39,40 as well as PPAR- conditional gene knockout macrophages. 41 However, despite increased CD36 expression, glitazones do not induce significant cellular cholesterol accumulation in either wild-type or PPAR- -deficient mouse macrophages or human monocyte-derived macrophages, indicating that PPAR- does not promote foam cell formation. 25,40 In addition, PPAR- activation leads to repression of scavenger receptor (SR)-A. 40 Consistent with these results, PPAR- agonists were shown to inhibit macrophage-foam cell formation in vivo. 29 Finally, in human macrophages PPAR- ligands reduce triglyceride accumulation on incubation with triglyceride-rich lipoproteins via the of apob-48 receptor expression 28 and cholesterol accumulation on incubation with glycated LDL by decreasing LPL secretion and activity. 26 PPAR- also plays a role in macrophage intracellular lipid metabolism. Indeed, in THP-1 macrophages PPAR- activation reduces ACAT1 mrna levels, thus decreasing the rate of cholesterol esterification. 42 Identification of NCEH as a PPAR- target gene also provides molecular evidences for a role of PPAR- in cholesterol distribution. 42 Finally as PPAR-, PPAR- positively controls cholesterol efflux in macrophages by enhancing the expression of CLA-1/SR-B1 and ABCA1. 25,36 ABCG1 and apoe as well as caveolin-1 33,43,44 were also identified as PPAR- target genes by pharmacological studies or by using conditional PPAR- -deficient macrophages, 37,41 thus corroborating the role of PPAR- in cholesterol removal from macrophages. Collectively, the studies highlighted above point to a central role for PPAR- in governing cholesterol homeostasis both in human and murine macrophages. A couple of differences exist in terms of mechanism (Table). For example, PPAR- activation decreases cholesterol esterification in murine macrophages without affecting ACAT1 or NCEH mrna levels as in humans. 29 This may result from indirect effects on transfer of cholesterol for esterification or posttranslational effects on ACAT1 activity. Role of LXRs A primary function of LXRs in macrophages is to maintain cellular cholesterol homeostasis. LXR activation does not induce lipid accumulation of acetylated LDL, 45 but rather leads to induction of genes involved in the cholesterol efflux pathway in an attempt to reduce the intracellular cholesterol overload. Expression of ABCA1 is strongly induced by natural and synthetic LXR ligands because of the presence of LXR response elements (LXREs) in the proximal promoter of the ABCA1 gene LXRs are essential for lipid-inducible ABCA1 expression, as induction is lost in macrophages from LXR- and LXR- double-knockout mice (LXR- / mice). 49 Conversely, LXRs do not stimulate cholesterol efflux to lipid-poor lipoproteins in fibroblasts from Tangier disease patients, demonstrating that ABCA1 is essential for the LXR-mediated efflux pathway. 48 ABCG1 and ABCG4 have also been identified as direct targets of LXRs in mouse and human cells ABCG1 is thought to function as a homodimer, 51 although a functional partnership with ABCG4 has been also suggested. 53 Additional mechanisms that may contribute to the LXR-driven reverse cholesterol transport are the induction of caveolin-1 44 and of a subset of apolipoproteins that may serve as cholesterol acceptors. Specifically, LXRs induce ApoE in human macrophages. 54,55 Moreover, phospholipid transfer protein (PLTP), another modulator of HDL metabolism with a potential role in reverse cholesterol transport, is also a direct target gene for LXR in lipid-loaded macrophages within human atherosclerotic lesions. 56 Recently, the role of LXRs in intracellular lipid transport and metabolism has been studied in more detail in human macrophages. LXR activation increases the expression of NPC1 and NPC2, leading to an enrichment of cholesterol in the plasma membrane accompanied by a redistribution of cholesterol within the plasma membrane to the outer layer. 45 LXRs also modulate the intracellular distribution of cholesterol. In fact, LXR activation decreases cholesterol esterification rates and reduces cholesteryl ester levels in human macrophage foam cells. These actions of LXR are not attributable to a decreased gene expression of ACAT1. Thus, it is likely that the enhanced cholesterol mobilization to the plasma membrane by LXR activators, results in a reduced availability of cholesterol as substrate for ACAT1. 45 Clear evidence has been provided that LXRs are key regulators of cholesterol efflux in macrophages (Table). However, differences in gene regulation exist between human and murine macrophages. Although the role of LXRs in intracellular lipid transport and metabolism has been studied in more detail in human macrophages, 45 LXR regulation of cholesterol trafficking may occur in a species-specific manner, because stimulation with LXR ligands does not result in the induction of NCP1 and NCP2 in murine bone marrow derived macrophages. 45 No data are still available concerning the role of LXR in the regulation of foam cell formation and cholesterol esterificaton in murine macrophages. Macrophages in the Inflammatory and Immune Response By participating in innate and adaptive immune responses, macrophages play a crucial role in host defense. 57 Ligation of cell-surface recognition receptors, such as Toll-like receptors (TLR), initiates a signaling cascade in macrophages that stimulates ROS production and culminates with the activation of both the nuclear factor NF- B and the activator protein AP-1, which together transcribe the immune response genes. 58 This program of classical activation not only enables the macrophage to phagocytose and dispose of invading pathogens efficiently, but also promotes the activation of adaptive immune responses. To maintain homeostasis and to prevent damage to nearby tissues, however, this respiratory burst must be tightly restricted both spatially and temporally.

5 1054 Arterioscler Thromb Vasc Biol June 2008 Bacterial and viral pathogens as well as proinflammatory cytokines have long been suspected to contribute to cardiovascular disease risk based on epidemiological studies and on experimental models of infections in atherosclerosis-prone mice. 1,59,60 Several studies on PPARs and LXRs indicate that these nuclear receptors are also important modifiers of macrophage inflammatory and immune responses. Role of PPAR- The first evidence for a potential role of PPAR- in inflammation came from the observation that PPAR- -deficient mice display a prolonged inflammation response to the proinflammatory leukotriene B4 (LTB4). 61 Indeed, binding of LTB4 to PPAR- results in the activation of PPAR- -mediated transcription of enzymes implicated in the -oxidation pathway. Via such feedback mechanism, LTB4 and other fatty acid-derived compounds may induce their own catabolism leading to the resolution of inflammation. PPAR- is furthermore activated by oxldl, suggesting that PPAR- mediates certain of the antiinflammatory activities of oxidized phospholipids. 62 Subsequent studies have demonstrated the antiinflammatory properties of PPAR- both in human and in murine macrophages. In classically activated M1 macrophages, PPAR- activation inhibits the production of various proinflammatory molecules by negatively interfering with the AP1 and NF- B signaling pathway, such as metalloproteinase-9 (MMP-9) 63 and tissue factor (TF). 64,65 Recently, PPAR- has been shown to inhibit the expression of osteopontin, a proinflammatory cytokine implicated in the development of atherosclerosis, via AP-1 pathway. 66 An additional antiinflammatory property of PPAR- in human macrophages may consist in the of tumor necrosis factor (TNF) -induced sphingomyelinase activity, 32 a pathway leading to the generation of ceramide, a second messenger involved in several cellular processes, including apoptosis. Taking the antiinflammatory properties of PPAR- into consideration, several studies have addressed the role of PPAR- in the control of macrophage redox signaling. Quite surprisingly, PPAR- activation upregulates the expression of NADPH oxidase 67 and myeloperoxidase (MPO), 68 leading to an increase in ROS production with antibacterial properties. The (patho)physiological consequences of these regulations are currently unclear. Taken together, these observations provide a basis for understanding how PPAR- ligands may exert their antiatherogenic effects not only by regulating cholesterol homeostasis but also by limiting inflammation and by improving the immune response. The anti-inflammatory properties of PPAR- have been observed both in human and murine macrophages; however, a number of genes are regulated in a species-specific manner (Table). For instance, MMP-9 63 and TF 65 are specifically inhibited by PPAR- ligands in human macrophages and MPO is not regulated in murine macrophages, probably because the PPARresponse element (PPRE) is lacking in the promoter of the murine MPO gene. 68 Role of PPAR- The anti-inflammatory effects of PPAR- activation are observed both in human and murine monocyte/macrophages. In human activated macrophages, PPAR- reduces MMP-9 activity and inhibits IL-1, IL-6, TNF- and osteopontin expression. 63,69 72 In murine macrophages, PPAR- activation represses the induction of several inflammatory response genes by LPS and IFN, including inos, COX-2, and IL-12. 8,73,74 Several distinct mechanisms may contribute to the repression of inflammatory genes by PPAR-. The preponderance of evidence points to an intranuclear crosstalk between PPAR- and transcription factors on the promoters of inflammatory genes, a phenomenon known as trans-repression. 75 In contrast to transcriptional activation, trans-repression does not involve binding to typical receptor-specific response elements, but PPAR- and transcription factors such as NF- B, AP-1, C/EBP, and STAT bind each other via protein-protein interactions thus modulating their transcriptional activity. 76 PPAR- can also inhibit inflammatory responses by blocking the signal-dependent clearance of NCoR corepressor complexes. This mechanism involves SUMO modification of PPAR-, thus facilitating interactions with the NCoR complex and, as a consequence, preventing inos induction by LPS. 77 Moreover, PPAR- exerts anti-inflammatory activities by inducing the production of IL-1 receptor antagonist (IL-1Ra) in THP-1 macrophages. 72 Interestingly, pharmacological blockade of IL-1 signaling using recombinant IL-1Ra resulted in improved -cell function, ameliorated glycemia, and reduced markers of systemic inflammation. 78 More recently, PPAR- has been shown to enhance the differentiation of monocytes into alternative macrophages, induced by Th2 cytokines, such as IL-4 and IL ,80 Such alternatively differentiated macrophages display a more pronounced anti-inflammatory phenotype. IL-4 also induces PPAR- expression and stimulates cellular generation of natural PPAR- ligands by activation of the 12/15-lipoxygenase pathway, thus enhancing the of inos expression. 74,81 Consistent with these results, selective inactivation of PPAR- in macrophages in mice results in an impairment in the maturation of alternatively activated M2 macrophages and exacerbation of diet-induced obesity, insulin resistance, glucose intolerance, and expression of inflammatory mediators. 79,82 PPAR- also plays important roles in the regulation of the innate immune response. In humans, blocking PPAR- activation during monocyte differentiation leads to downregulation of molecular elements of the engulfment process (CD36, AXL, Transglutaminase [TG] 2 and prototypical tissue pentraxin [PTX] 3), thus resulting in decreased uptake of human apoptotic neutrophils. 83 In addition, PPAR- agonists increase the expression of MPO, 84 thus contributing to the generation of ROS and killing of microbes in the phagolysosome. Through its ability to upregulate the expression of scavenger receptor CD36, PPAR- has been found to enhance the phagocytosis of malarian parasites and to decrease malaria-induced TNF secretion in murine macrophages. 85,86 In addition, treatment of murine macrophages with IL-13 or PPAR- ligands promotes uptake and killing of Candida Albicans, and ROS production via upregulation of surface mannose receptor expression. 87 Collectively these results support a physiological role for PPAR- in the regulation of inflammation and the immune response both in human and murine macrophages (Table). Consistent with this idea, administration of glitazones atten-

6 Rigamonti et al Role of PPAR- and LXRs in Macrophage Homeostasis 1055 uates inflammation in murine models of atherosclerosis, 88 inflammatory bowel, 89 and autoimmune diseases. 90 However, certain anti-inflammatory properties of PPAR- ligands were also observed in PPAR- -deficient macrophages, thus suggesting that certain compounds could exert anti-inflammatory activities both in a PPAR- -dependent and independent manner. 39 As most anti-inflammatory effects of these compounds are observed at very high concentrations, it is possible that activation of the other PPARs expressed in these cells may also contribute to the observed anti-inflammatory activities. In this sense, PPAR- ligands have been shown to inhibit the induction of LPS and IFN -activated proinflammatory genes in a PPAR- -dependent manner at low concentrations and in a PPAR- -independent manner at higher concentrations, and this effect could be at least partly explained by the activation of PPAR /. 74 Role of LXRs Considerable evidence has identified LXRs as regulators of the inflammatory response both in human and murine macrophages. LXR activation represses a set of inflammatory genes after bacterial, LPS, TNF, or IL-1 stimulation. Examples of such genes include those involved in generation of bioactive molecules such as inos and COX2, IL-6 and IL-1, MCP-1 and MCP-3, MMP9, 9,91 TF, and osteopontin. 92,93 Both LXR isoforms possess anti-inflammatory activities, because repression of these genes is lost in macrophages from LXR / mice. 9,91 Recently, LXRs have also been shown to positively regulate the expression of the antiinflammatory arginase II in murine macrophages. Because arginase II and inos use a common substrate, induction of arginase II expression has the potential to exert antiinflammatory effects by shifting arginine metabolism toward polyamine synthesis at the expense of NO production. 94 The mechanism underlying the repression of inflammatory genes by LXRs is poorly understood. LXREs have not been identified in the proximal promoters of the repressed genes, thus pointing to an indirect mechanism. Certain lines of evidence suggest that of the NF- B pathway is involved. Inhibition of this pathway does not entail of NF- B translocation to the nucleus, binding to DNA, or degradation of the NF- B inhibitor I B. 9,91,93 Most likely, transrepression of NF- B by LXRs involves a nuclear event. In a recent study of transrepression of the inos and IL-1 promoters by LXRs, SUMOylation of LXRs was identified as a possible mechanism involved in this process. Sumoylated LXR was suggested to prevent LPS-dependent exchange of corepressors for coactivators, thus maintaining the inos promoter in a repressed state. 95 In addition to their ability to modulate the acute inflammatory response, LXRs are also key regulators of the innate immune response. In human macrophages, LXR agonists increase TLR-4 expression, which results in an enhanced responsiveness to LPS. 96 Moreover, LXR activation increases ROS generation in both resting and LPS-stimulated macrophages by enhancing the expression of the NADPH oxidase subunits. These results suggest a role of LXRs in the modulation of the human macrophage response against bacteria. 96 Indeed, LXRs may contribute to bacterial elimination through recruitment or activation of neighboring cells as well as through the production of antibacterial ROS. However, acute cotreatment of human macrophages with LXR agonists together with LPS or IFN- results in a reduction of TNF and MCP-1 secretion. 96,97 Thus, it appears that LXR activation prepares human macrophages to allow an enhanced antibacterial response via induction of the TLR-4 signaling pathway, whereas, once the inflammatory stimulus is present, LXRs exert anti-inflammatory actions to promote the resolution of inflammation. In murine macrophages, LXRs also modulate the innate immune response, primarily via promoting macrophage survival. Indeed, mice lacking LXRs are highly susceptible to infection with the Gram-positive intracellular bacteria Listeria monocytogenes. 98 This phenotype was recapitulated by transplantation of bone marrow from LXR / mice into WT mice, suggesting that altered macrophage function is a major contributor to susceptibility. Furthermore, the inability of LXR-null mice to mount an appropriate response to L monocytogenes infection correlated with accelerated rates of macrophage apoptosis. The increased susceptibility of LXRnull macrophages to pathogen-induced apoptosis results, at least in part, from the loss of regulation of the antiapoptotic gene AIM 99 (also known as SP and Api6) by LXR. 98 Similarly, Valledor et al 100 showed that activation of LXR/ RXR heterodimers by synthetic and natural ligands inhibits macrophage apoptosis in response to apoptotic stimuli (eg, cycloheximide), and infection with Bacillus anthracis, E coli, and Salmonella typhimurium. This activity was attributed to induction of AIM and other antiapoptotic factors, as well as to of a set of proapoptotic genes. Taken together, these studies point to a central role of LXRs in governing the diverse immune functions of macrophages. LXR activation has been reported to improve the immune response both in human and in murine macrophages, albeit via distinct mechanisms. In humans, LXR activation increases the LPS signaling pathway and the production of antibacterial ROS. 96 This regulation occurs in a species-specific manner, because no induction of TLR-4 and NADPH oxidase subunits is observed in murine bone marrow derived macrophages on stimulation with LXR ligands. 96 By contrast, in mice LXR agonists promote macrophage survival via induction of AIM and other antiapoptotic factors. 98 However, AIM is not detectable in human macrophages in culture 96 (Figure). Clinical Consequences of PPAR and LXR Activation Clinical trials using fibrates and glitazones provide indications regarding the clinical efficacy of PPAR agonists in the control of lipid metabolism and inflammation. The influence of fibrates on cardiovascular morbidity and mortality was studied in primary (Helsinki Heart Study [HHS] 101 and Fenofibrate Intervention and Event Lowering in Diabetes [FIELD] 102 ) and secondary (FIELD, Bezafibrate Infarction Prevention [BIP], 103 and Veterans Affairs High-density Lipoprotein Cholesterol Intervention trial [VA-HIT] 104 ) cardiovascular prevention studies, as well as in coronary angiography trials. A detailed discussion of these studies is beyond the scope of this review, and the readers are referred to a recent

7 1056 Arterioscler Thromb Vasc Biol June 2008 A Synthetic ligands LXRs TNFα IFNγ Human macrophage TNFα MCP-1 Activation of neighbouring cells TLR4 LPS NADPH oxidase ROS Bacterial clearance? TLR4 article in the same series on this topic. 105 Collectively, PPAR- agonists appear specifically efficacious in diabetic patients as well as in nondiabetic overweight insulin-resistant patients at high risk with dyslipidemia (high TG or low HDL) and chronic inflammation. These effects could only be partially explained by increased levels of HDL and are consistent with actions in peripheral tissues, including macrophages. Glitazones were demonstrated to be efficient in the management of insulin resistance and type 2 diabetes in a number of prospective clinical trials. 106,107 Although troglitazone was withdrawn, because of rare but severe idiosyncratic hepatotoxicity, glitazones are increasingly prescribed to patients with diabetes and the currently used glitazones may even be hepatoprotective against fatty liver disease and potentially nonalcoholic steatohepatitis. 108 However, results from meta-analysis studies suggested that rosiglitazone use may be associated with an increase in the risk of myocardial infarction from cardiovascular causes. 109,110 These observations raised questions on the cardiovascular safety of rosiglitazone in the treatment of type 2 diabetes. However, the increase in absolute cardiovascular risk after rosiglitazone treatment was very small in these studies on low-risk patients, such as DREAM and ADOPT. 106,107 Intermediary safety analysis of a trial assessing the cardiovascular effects of rosiglitazone combined with metformin or sulfonylurea, the Rosiglitazone Evaluated for Cardiac Outcomes and Regulation of glycemia in Diabetes (RECORD) study, reported nonsignificant changes in cardiovascular morbidity and mortality after rosiglitazone treatment. 111 The cardioprotective properties of pioglitazone have been demonstrated in a secondary prevention study (PROACTIVE) 112 as well as by a meta-analysis. 113 The ability of LXRs to promote reverse cholesterol transport, to limit inflammation, and to improve glucose tolerance makes them attractive targets for drug development. However, the finding that first-generation synthetic LXR ligands markedly increase hepatic lipogenesis and plasma triglyceride levels is an obstacle that needs to be cleared. 114 LXR- is the predominant LXR expressed in the liver, and the ability of LXR agonists to B LPS Murine macrophage Pro-inflammatory mediators Pro-apoptotic mediators Anti-apoptotic factors Bcl-XL AIM Bacterial Clearance? Synthetic ligands LXRs Anti-apoptotic paracrine effects? Figure. Species-specific regulation of the immune response against bacterial infections by LXR agonists. A, In human macrophages LXR activation enhances antibacterial response via induction of TLR-4 signaling pathway. B, In murine macrophages LXR agonists promote macrophage survival. stimulate hepatic lipogenesis is thought to result primarily from LXR- induction of SREBP1c and fatty acid synthase (FAS) expression. 115,116 This suggests that partial or gene-specific agonists designed to increase reverse cholesterol transport, but not to induce hepatic lipogenic gene expression, would be a better therapeutic strategy. An alternative approach to the undesirable effects of LXR agonists is to develop isoform-specific LXR ligands. The rationale is that LXR- -specific ligands may induce the desired reverse cholesterol transport but circumvent the hepatic complications attributed to LXR-. Indeed, Bradley et al 117 have recently demonstrated that ligand activation of LXR- reverses atherosclerosis and cellular cholesterol overload in mice lacking LXR- and apoe, providing in vivo evidence for drug development strategies targeting LXR-. Conclusions In the last few years, PPARs and LXRs have emerged as key regulators of macrophage biology. Clear evidence has been provided that these nuclear receptors control transcriptional programs involved in macrophage lipid homeostasis. In addition, PPARs and LXRs negatively regulate macrophage-mediated inflammation. However, several important issues need further exploration. For example, important differences exist between human and mouse in term of gene regulation by PPARs and LXRs, especially in the field of macrophage lipid homeostasis. These observations, which call to caution in the extrapolation of the results obtained in a given species to the other, can be attributable to different reasons. First, the expression levels of PPARs and LXRs can be different between human and mouse macrophages. As an example, PPAR- is abundant in human macrophages and is only barely detectable in mouse macrophages. Second, the expression of nuclear receptor cofactors (coactivators or corepressors) which participate in the regulation of target gene transcription, can be also different between human and mouse macrophages. Third, because the promoter regions of genes are not entirely conserved across species, the transcription factors that control gene expression in one species might not be crucial regulators in another. Finally, synthetic ligands used in the pharmacological studies can display different affinity and selectivity for the human and mouse PPARs or LXRs proteins. 118,119 To tackle species-specificity problems, humanized models represent an attractive option. Indeed, it would be interesting to develop knock-in mice for a given nuclear receptor, in which the coding region for the mouse protein is replaced by the equivalent human coding region, resulting in physiological expression levels of the human protein. At present, no murine model available is perfect for studying nuclear receptor ligands, and further work is required. The ideal model could be defined as a mouse model that displays a mixed dyslipidemia and insulin resistance, spontaneously develops atherosclerosis, expresses human forms of the nuclear receptor studied, and is not a knockout model. Finally, the ultimate validation of new pharmacological compounds will only be achieved by the assessment of their effects on humans in large-scale clinical trials. Sources of Funding This work was supported by grants from the EVGN (European Vascular Genomics Network) No. LSHM-CT , the Région Nord- Pas de Calais/FEDER, and the Fondation Coeur et Artères.

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