Innate immune signals in atherosclerosis

Clinical Immunology (2010) 134, 5–24

available at www.sciencedirect.com

Clinical Immunology www.elsevier.com/locate/yclim

REVIEW

Innate immune signals in atherosclerosis Anna M. Lundberg ⁎, Göran K. Hansson Center for Molecular Medicine, L8:03, Department of Medicine, Karolinska Institute, Stockholm SE-17176, Sweden

Received 15 May 2009; accepted with revision 31 July 2009 Available online 9 September 2009 KEYWORDS Atherosclerosis; Innate immunity; Pattern-recognition receptors; Toll-like receptors; Inflammation; Infection

Abstract Atherosclerosis is a chronic disease characterised by lipid retention and inflammation in the arterial intima. Innate immune mechanisms are central to atherogenesis, involving activation of pattern-recognition receptors (PRRs) and induction of inflammatory processes. In a complex tissue, such as the atherosclerotic lesion, innate signals can originate from several sources and promote atherogenesis through ligation of PRRs. The receptors recognise conserved molecular patterns on pathogens and endogenous products of tissue injury and inflammation. Activation of PRRs might affect several aspects of atherosclerosis by acting on lesion resident cells. Scavenger receptors mediate antigen uptake and clearance of lipoproteins, thereby promoting foam cell formation. Signalling receptors, such as Toll-like receptors (TLRs), lead to induction of pro-inflammatory cytokines and antigen-specific immune responses. In this review we describe the innate mechanisms present in the plaque. We focus on TLRs, their cross-talk with other PRRs, and how their signalling cascades influence inflammation within the atherosclerotic lesion. © 2009 Elsevier Inc. All rights reserved.

Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pattern-recognition receptors in atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . Infection and pathogenic TLR ligands in atherosclerosis . . . . . . . . . . . . . . . . . . . . . . TLR recognition of endogenous inflammatory ligands . . . . . . . . . . . . . . . . . . . . . . . TLRs in experimental atherosclerosis — role in sterile inflammation . . . . . . . . . . . . . . . TLR polymorphisms and association with cardiovascular disease . . . . . . . . . . . . . . . . . TLR signalling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inflammatory signalling pathways involved in atherosclerosis . . . . . . . . . . . . . . . . . . . Crosstalk between TLRs, scavenger receptors and the inflammasome . . . . . . . . . . . . . . Effects of TLR stimulation and infection on cholesterol metabolism and foam cell formation. Innate TLR-dependent mechanisms involved in atherosclerotic plaque formation . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ⁎ Corresponding author. E-mail addresses: [email protected] (A.M. Lundberg), [email protected] (G.K. Hansson). 1521-6616/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2009.07.016

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Introduction Atherosclerosis is a chronic inflammatory disorder of the vessel wall, where both innate and adaptive immune responses influence disease progression [1]. It involves accumulation of lipids in the arterial intima resulting in formation of vascular lesions, or plaques, which are characterised by inflammation, cell death and fibrosis. The innate signals within the lesion can originate from several sources and promote atherosclerosis through inflammatory processes. Accumulation of oxidised low-density lipoprotein (oxLDL), starting in the fatty streaks, promotes the inflammatory response and this most likely continues throughout lesion development. Furthermore, pathogenic infection and endogenous danger signals increased during tissue injury have been implicated as inducers of lesion inflammation (Fig. 1). Endothelial cells are activated by accumulation of modified LDL and increase their expression of adhesion molecules, mainly vascular cell adhesion molecule (VCAM-1) [2,3]. Activation of the endothelial cells predominantly occurs at atherosclerosis prone sites with increased hemodynamic strain and this leads to migration of monocytes and T cells into the artery wall [4,5]. The recruitment of immune cells is a crucial early step in atherogenesis and is potentiated by local release of chemokines. Monocyte chemotactic protein-1

A.M. Lundberg, G.K. Hansson (MCP-1) appears to be particularly important for attracting monocytes, whereas T helper type 1 (Th1) cells are recruited to the lesion by production of regulated upon activation normal T cell expressed and secreted (RANTES), interferoninducible protein (IP)-10 and interferon-inducible T cell alpha chemoattractant (I-TAC) [6–9]. The infiltrated cells further propagate the local inflammation through production of several cytokines that increase the recruitment and activation of additional immune cells. Monocytes differentiate into macrophages in the presence of macrophage colony-stimulating factor (M-CSF) produced in the lesion by endothelial cells and smooth muscle cells. This is a crucial step in atherogenesis and is accompanied by upregulation of innate immune receptors necessary for phagocytosis and induction of inflammatory processes [10]. Modified LDL is taken up by macrophages and cholesterol accumulates in the cytoplasm turning the macrophages into foam cells (Fig. 1). The foam cells are the prototypic cells of the atherosclerotic plaque and when they die, debris and cholesterol form an extracellular pool that cannot be removed. As the disease progresses, the lesions mature and become necrotic and calcified. Certain plaques are unstable and increasing evidence suggest that inflammatory activation of cells within the plaque can initiate processes that culminate in plaque rupture [11,12]. Exposure of the prothrombotic material from the lesion core can trigger athero-

Figure 1 Toll-like receptors (TLRs) contribute to atherosclerotic progression and this process could be stimulated by both endogenous and exogenous TLR ligands. Atherosclerosis is initiated when low-density lipoprotein (LDL) is deposited in the arterial intima. LDL is oxidised (oxLDL) and stimulates local chemokine production (e.g. macrophage chemotactic protein-1) and upregulation of adhesion molecules (e.g. vascular cell adhesion molecule-1) on endothelial cells. This results in recruitment of monocytes, which differentiate to macrophages in the presence of locally produced macrophage colony-stimulating factor. During this process macrophages upregulate expression of scavenger receptors (SRs) and TLRs. oxLDL is taken up by SRs which leads to intracellular accumulation of cholesterol, turning the macrophages into foam cells. The TLRs can be activated by a multitude of TLR ligands to induce production of proinflammatory mediators. Several virus and bacteria have been implicated in atherogenesis and their products can trigger TLRs on both macrophage and endothelial cells. TLR activation can also dysregulate cholesterol transporters and intracellular lipid trafficking in macrophages, which increases cholesterol content and contribute to foam cell formation. Endogenous TLR ligands can be found in the atherosclerotic lesion at various stages during disease progression. Examples are; minimally oxidised LDL, heat shock proteins (hsps) and nuclear proteins, most likely released from dead cells in the necrotic core, and degradation products of extracellular matrix macromolecules that are indicative of tissue injury, or tissue remodelling.

Innate immune signals in atherosclerosis thrombosis. This, in turn, can cause sudden occlusion of the artery and lead to myocardial infarction or stroke. The activating agents that promote plaque rupture are not yet known but have been proposed to be lipids, microbial products or other endogenous substances produced during inflammation. The precise mechanisms linking endothelial dysfunction and lipid accumulation remain poorly understood. However, the last decades have seen an enormous increase in our understanding of the molecular pathways initiating and controlling inflammation within the atherosclerotic lesion. Many of these findings depend on data obtained from experimental mouse models of atherosclerosis, which have been critical for uncovering the basic pathogenic processes of the disease. Nevertheless, even though these mice develop lesions with striking similarities to human atherosclerotic plaques it is important to remember that they also differ in some critical aspects, such as the lipoprotein metabolism and plaque rupture with development of the clinical complications that can be observed in humans. Activation of the innate immune system is important for inducing an inflammatory response and relies on a set of pattern-recognition receptors (PRRs) for detection and clearance of harmful material [13]. This family of receptors consists of soluble, membrane-bound and cytoplasmic receptors, all of which are encoded in the germline DNA. In this review we will describe the activation mechanisms of the PRRs, focusing on the Toll-like receptors (TLRs), and discuss the consequence of their immunological roles on the outcome of atherosclerosis.

Pattern-recognition receptors in atherosclerosis Cells of the innate immune system express a wide range of PRRs and they are involved in multiple aspects of atherogenesis. The PRRs recognise highly conserved molecular patterns present both on pathogenic components and endogenously derived products [13]. Two principal classes of PRRs have been proposed; endocytic receptors that mainly mediate antigen uptake, and signalling receptors necessary for activation of proinflammatory pathways. Included among the signalling receptors are TLRs and cytoplasmic receptors, and ligand binding to these receptors results in increased expression of a multitude of proinflammatory genes. In contrast, endocytic receptors mediate clearance of blood lipoproteins, apoptotic cell fragments, and pathogen elimination, as well as antigen uptake for subsequent presentation by antigen-presenting cells (APCs). Included in this group are a wide variety of receptors, such as scavenger receptors (SRs), C-type lectins, and opsonic receptors. Ligand binding to these receptors does usually not induce production of proinflammatory cytokines. In atherosclerosis, endocytic receptors are involved in clearance of oxLDL and thereby participate in the formation of foam cells. The receptors SR-A and CD36 are suggested to play central roles in this process. However, their exact contribution to atherogenesis remains unclear since inconsistent results from several studies showed that these receptors can both protect and exacerbate atherosclerotic development [14–16]. Crosstalk between different groups of PRRs exists, where simultaneous triggering of receptors can activate distinct or

7 shared pathways. This interplay between signalling pathways is generated by the particular inflammatory environment and eventually determines the specific immune response directed at clearing pathogens or responding to tissue injury. The atherosclerotic lesion is a complex tissue containing a mixture of possible PRR ligands that are likely to activate several pathways either simultaneously or consecutively (Fig. 1). TLRs are the major signal-generating PRRs, coordinating innate immunity and shaping adaptive immunity [13]. So far, 10 human and 13 mouse TLRs have been characterised and found to recognise a broad spectrum of microbial structures. Triggering of the TLRs initiates acute inflammatory responses by induction of cytokines, chemokines and co-stimulatory molecules necessary for efficient antigen presentation. The TLRs are therefore essential in controlling and directing antigen-specific adaptive immune responses. Furthermore, TLR stimulation increases expression of adhesion molecules on endothelial cells and release of chemotactic factors that attract additional effector cells to the inflamed site. TLRs are type I transmembrane receptors that belong to the TLR/IL-1 receptor superfamily, which also includes receptors for the proinflammatory cytokines IL-1 and IL-18 [13]. All members of this family have a TLR/IL-1 receptor homology (TIR) domain, a conserved cytoplasmic domain that is essential for signalling [17]. TLRs initiate conserved signalling pathways involving mitogen-activated protein kinases (MAPKs) and other phosphorylation cascades that culminate in activation of nuclear factor (NF) κB, activating protein (AP)-1 and IFN-regulatory factor (IRF) transcription factors (Fig. 2). These factors drive the expression of proinflammatory genes and initiate the production of signals that initiate adaptive immunity. TLRs are expressed throughout the body and are mainly found on professional innate immune cells, including macrophages, dendritic cells, and mast cells, but also on nonprofessional immune cells, such as endothelial cells and smooth muscle cells. All of these cells are present in the atherosclerotic lesion and contribute to the inflammatory response. Most TLRs can be found in the normal human vessel wall at varying expression levels, however, selective expression patterns can be observed depending on the specific vessel type [18,19]. The expression of several TLRs is increased in atherosclerotic lesions, and are identified on macrophages, endothelial cells and smooth muscle cells [18,20]. Interestingly, TLR2 and TLR4 have the highest expression, both under healthy conditions and in lesions [18,19], suggesting that they may play dominant roles in controlling vessel inflammation. In addition, out of the six TLR mRNA species identified in human lesions, TLR1 and TLR5 also demonstrated high protein expression, whereas only weak levels were detected for TLR3 and TLR6 [18,21]. TLR4 is also increased on circulating monocytes from patients with coronary artery disease, when compared with healthy controls, possibly a consequence of an ongoing inflammation or a sign of an increased state of alert [22–24]. TLR expression has also been studied in two atherosclerotic mouse models; mice lacking apolipoprotein E (apoe)−/− that spontaneously develop hypercholesterolemia, and LDL receptor deficient (ldlr)−/− mice, which only develop lesions

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Figure 2 Toll-like receptor (TLR)/Interleukin (IL)-1 Receptor signalling pathways. TLRs activate common and unique signalling cascades that culminate in the activation of several families of transcription factors, including nuclear factor (NF) κB, activating protein (AP)-1 and interferon regulating factors (IRFs), critical for transcription of inflammatory genes. Stimulation with their respective TLR ligands induces dimerisation of the receptors, which results in recruitment of Toll-IL-1 receptor (TIR)-domaincontaining adaptors to the intracellular TIR domain of the receptors. While most TLRs homodimerise, TLR2 can heterodimerise with TLR1 or TLR6 to discriminate between different ligands and initiate signalling (represented here as TLR2). With the exception of TLR3, all TLRs, including TLR5 (not shown) and IL-1R/IL-18R recruit the adaptor myeloid differentiation factor 88 (MyD88), which initiates signalling and subsequent formation of a complex consisting of IL-1 receptor-associated kinase (IRAK)1 and IRAK4, TNF-associated factor (TRAF)6, as well as transforming growth factor β-associated kinase (TAK)1. Activated TAK1 in this complex phosphorylates the IκB kinase (IKK)β, which results in activation of the IKK complex (IKKα, IKKβ, IKKγ). Active IKKβ in turn phosphorylates inhibitor of NFκB (IκB)α, marking it for degradation by the proteasome and enables released NFκB to translocate into the nucleus. Simultaneously, TAK1 activates the mitogen-activated protein kinase (MAPK) cascades (including kinases p38, p42/44, p46/54), leading to activation of AP-1. In addition, TLR4 and TLR3 trigger a TIR domain containing adaptor-inducing interferon β (TRIF)-dependent signalling pathway from the endosome to NFκB/MAPK activation. TRIF recruits TRAF3 and the kinase TANK-binding kinase (TBK)1 that phosphorylates and activates IRFs, such as IRF-3, which then dimerise and translocate to the nucleus. The adaptors MyD88 adaptor-like (MAL) and TRIF-related adaptor molecule (TRAM) participate by facilitating recruitment of MyD88 and TRIF to the receptors. Furthermore, endosomal TLR7/8/9 can also activate IRFs, such as IRF-7, via a MyD88/TRAF3/TBK1-dependent pathway.

when fed a high cholesterol diet. TLR4 expression was found elevated in lesions from apoe−/− mice, and TLR2 was preferentially detected in vessel areas of disturbed flow in the aorta of ldlr−/− mice [21,25]. TLR2 expression was increased with disease progression and was found to contribute to early atherosclerotic processes [25]. The presence of TLRs in both healthy vessels and in atherosclerotic lesions suggests that they can modulate key processes both prior to and during disease progression. Therefore they could have a major impact on atherogenic events.

Infection and pathogenic TLR ligands in atherosclerosis TLRs recognise diverse molecular structures called pathogen-associated molecular patterns (PAMPs), ranging from lipids and proteins to nucleic acids, that are common to many microorganisms [13,26]. TLR4 is the receptor for the gramnegative cell wall component lipopolysaccharide (LPS), but also other bacterial toxins, heat shock proteins and viral

Innate immune signals in atherosclerosis envelope glycoproteins [27–31]. TLR2 interacts with the largest variety of pathogenic structures, including bacterial lipoproteins, fungal cell wall components and viral products [31–42]. Unlike the other TLRs, TLR2 increases its specificity for ligands through heterodimerisation with either TLR1 or TLR6, allowing it to discriminate between subtle differences in the ligands [32,34,43,44]. Activation of both TLR4 and TLR2 is mediated through the cooperation of co-receptors. TLR4 is an essential receptor for LPS, however, several accessory proteins participate in its recognition. LPS is captured by the serum protein LPSbinding protein (LBP), which delivers LPS to CD14 [45,46]. The latter is a glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein that lacks a transmembrane domain and cytoplasmic regions, rendering it incapable of transducing signals (Fig. 2) [47]. CD14 is also present in serum as a soluble GPI-tail-less receptor that facilitates LPS-induced signal transduction in cells lacking membrane-bound CD14, such as dendritic cells, endothelial cells and smooth muscle cells [48]. In addition, the secreted molecule MD2 associates with the extracellular portion of TLR4 and specifically interacts with the acylated lipid A core of LPS to enhance its responsiveness [49–52]. TLR2 also associates with CD14 to bind specific ligands [38,39]. In contrast to TLR2 and TLR4, the other TLRs are only described to recognise one type of microbial structure. TLR3 recognises viral double-stranded RNA (dsRNA) [53,54], TLR5 bacterial flagellin [55,56], TLR7/TLR8 single-stranded RNA (ssRNA) [57–59], and TLR9 hypomethylated CpG motifs in microbial DNA [60–62]. While most TLRs are expressed on the cell surface, the group recognising nucleic acids (TLR3, 7, 8, 9) are found almost exclusively in intracellular compartments, such as endosomes. Their ligands require internalisation into the endosomal compartment to induce signalling processes. Through the recognition of pathogenic structures TLRs are central receptors of innate immunity and crucial in host defence against invading pathogens. In atherosclerosis, systemic administration of LPS in hypercholesterolemic rabbits or apoe−/− mice demonstrated the pro-atherosclerotic role of TLR4 stimulation [63,64]. Similarly, injection of a TLR2 ligand into hypercholesterolemic ldlr−/− mice resulted in a significant increase in lesion severity, indicating that pathogenic products are able to promote atherogenesis [65]. Viral and bacterial infections have been implicated in the pathogenesis of atherosclerosis, as possible mechanisms for induction or perpetuation of lesion inflammation. Pathogenic infections promote atherosclerotic events and both myocardial infarctions and stroke are increased during acute infections [66]. In addition, several epidemiological studies have reported an elevated risk of atherosclerosis associated with a large number of pathogens; including the bacteria Chlamydophila pneumoniae (also known as Chlamydia pneumoniae), Helicobacter pylori, and Porphyromonas gingivalis, and the viruses Cytomegalovirus (CMV), Epstein–Barr-virus (EBV), Human immunodeficiency virus (HIV), Herpes simplex virus (HSV) 1 and 2, Hepatitis A and B, and Influenza A virus [67–70]. Several studies have also shown the presence of infectious agents within human atherosclerotic lesions [71–73]. In addition to being controversial, due to the possibility of contamination, it is also unclear whether the presence of these pathogens causes

9 plaque activation or is just a consequence of increased migration of immune cells to the site of inflammation. Several of these pathogens are able to infect and survive in circulating leukocytes and can thereby enter the plaque through cell migration from the blood. Interestingly, recent studies have shown decreased risk of acute coronary syndrome in individuals vaccinated against influenza virus [66,74–76]. The ability of antibiotic treatment against common bacteria has also been investigated. However, epidemiological studies evaluating the use of antibiotic prior to an acute cardiac event have either produced inconclusive results or have failed to show significant reductions in cardiovascular events [77–80]. Although these studies raise questions about the direct contribution of pathogens to atherosclerotic development, it is by no means indicative of a lack of involvement. A major disadvantage with intervention approaches is the difficulty in determining when during atherogenesis an intervention is needed; at the progression from fatty streaks to advanced plaques, or at later stages in preventing plaque rupture in patients with already established coronary artery disease (CAD). Several attempts have been made to confirm the epidemiological association studies through infection of atherosclerotic prone mice with several pathogens, such as CMV, C. pneumoniae, H. pylori, and P. gingivalis, and this have in most cases exacerbated atherosclerosis [81–92]. The precise mechanism whereby pathogens are able to accelerate atherosclerosis is unclear, but TLRs are probably involved in detection and initiation of a subsequent inflammatory response. As such, the pro-atherogenic effects of C. pneumoniae infection in apoe−/− mice are mainly mediated via a TLR2/TLR4 driven pathway [93]. Other TLRs may also participate in atherosclerosis, such as TLR3 and TLR9 in detecting infection by CMV and HSV [62,94,95]. However, this remains to be determined. Even though evidence for a role of infection in atherosclerosis is increasing, several questions remain to be answered to fully understand this association. It needs to be established if the suggested pathogens have a direct causative effect within the lesion or if the effect on lesion inflammation is instead mediated through a general TLRinduced expression of proinflammatory cytokines, resulting in an increase in systemic inflammation. The possibility also exists that pathogenic infections may affect existing vulnerable plaques and thereby lead to an acute myocardial infarction without being involved in plaque development. On the whole, there is no definitive proof that a single pathogen can cause atherosclerosis. Instead it is more likely that it is the total infectious burden leading to frequent TLR activation and a subsequent inflammation that promotes atherosclerosis.

TLR recognition of endogenous inflammatory ligands Aside from pathogens, TLRs also recognise endogenous ligands that can be found at sites of inflammation. These are mainly products of tissue stress that are released during necrotic cell death or are derived from the degradation of extracellular matrix (ECM) (Table 1). The endogenous TLR ligands have been termed damage-associated molecular

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patterns (DAMPs) and serve as danger signals to alert the innate immune system of tissue injury in the absence of infections [96,97]. The atherosclerotic plaque is characterised by accumulation of cholesterol and debris from dead cells in the necrotic core, and increased ECM turnover during tissue remodelling. The majority of the proposed endogenous ligands can therefore be found in the atherosclerotic lesion at various stages during disease progression and could contribute to atherogenesis through transduction of inflammatory signals (Fig. 1). Heat shock proteins (hsps), most likely released from necrotic cells, were the first of this group of ligands to be described, and they induce the production of proinflammatory cytokines in a TLR2- and TLR4-dependent pathway [31,98–102]. Under normal conditions, hsps are expressed at low levels, but their expression is upregulated in response to a wide variety of stress stimuli and their release into the extracellular milieu is an indication of cell death. Interestingly, hsp60 can be found expressed on the endothelial surface of athero-prone vessel areas, probably induced by altered shear stress [103]. In addition, certain hsps such as hsp60 are released in the plaque and they have been implicated in the pathogenesis of atherosclerosis in both clinical as well as experimental studies [104,105]. Another intracellular protein functioning as an extracellular TLR ligand is high-mobility group box 1 (HMGB1), which is detected by TLR2 and TLR4 [106]. HMGB1 is a DNA-binding protein that also functions as a proinflammatory cytokine, after being actively secreted by stimulated macrophages or released through passive diffusion from necrotic cells Table 1 Endogenous TLR ligands associated with inflammation. Ligand Hsp60

Function

Stress inducible cytosolic heat shock protein Hsp70 Stress inducible cytosolic heat shock protein Gp96 Stress inducible ER heat shock protein HMGB1 Chromosomal binding protein ApoCIII Apolipoprotein in VLDL mRNA Intracellular nucleic acid Fibrinogen Acute-phase protein Fibronectin EDA ECM component Heparan sulfate ECM component Hyaluronan fragment ECM component β-defensin 2 Cationic antimicrobial peptide Oxidised Component of oxLDL phospholipid mmLDL Lipoprotein modified by mild oxidation Nucleic acids RNA/DNA-containing immune complex

TLR TLR2/TLR4 TLR2/TLR4 TLR2/TLR4 TLR2/TLR4 TLR2 TLR3 TLR4 TLR4 TLR4 TLR4 TLR4 TLR4 TLR4 TLR7/TLR9

Abbreviations: ECM (extracellular matrix), EDA (extra domain A), ER (endoplasmatic reticulum), HMGB1 (High mobility group box 1 protein), mmLDL (minimally modified low-density lipoprotein), and very low-density lipoprotein (VLDL).

[107–110]. Increased extracellular levels of HMGB1 have been observed in atherosclerotic lesions. It promotes a local inflammatory response, and its release originates from endothelial cells, foam cells, and smooth muscle cells [111,112]. Furthermore, increased serum levels of HMGB1 is associated with coronary artery disease (CAD) and was reported to be predictive of an increased risk for developing a subsequent acute cardiac event [113,114]. Several host-specific nucleic acids are also recognised by TLRs, including mRNA via TLR3 [115]. TLR7 and TLR9 instead bind RNA/DNA-containing immune complexes that together with synergistic stimulation of either Fc-receptors or the B cell receptor (BCR) induce cell activation [116–118]. The recognition motif in the chromatin complex was suggested to be unmethylated CpG motifs, which are common features of bacterial DNA, but are also present in mammalian DNA promoter elements [119]. Degradation products of extracellular matrix macromolecules are indicative of tissue injury, or tissue remodelling, and have been found to function as TLR ligands (Table 1). One such component is fibronectin, which undergoes alternative splicing and one of the spliced exons is the extra domain A (EDA) that has been shown to signal through TLR4 [120]. The level of EDA in the atherosclerotic lesion increases with disease progression in apoe−/− mice and it was shown to promote lesion development [121–123]. Hyaluronan (HA), one of the major glycosaminoglycans of the extracellular matrix, is another ligand of TLR2 and TLR4 [124–127]. HA is a linear polysaccharide that undergoes rapid degradation at sites of inflammation and accumulates in human atherosclerotic lesions. HA degradation products induce activation of DCs and chemokine production by ECs through TLR4dependent mechanisms [125,126]. Furthermore, they can act as adjuvants and promote activation of antigen-specific T cells in vivo via a mechanism controlled by TLR2 [124]. Heparan sulfate is a polysaccharide found on all cell surfaces and in the ECM and it is rapidly shed as a result of inflammation induced by tissue injury or infection. Degradation products of heparan sulfate activate dendritic cells through a TLR4-dependent mechanism [127–129]. Other host-specific TLR ligands are extravascular fibrinogen, leaking from the vasculature at sites of inflammation, and the antimicrobial peptide β-defensin-2. Both are recognised by TLR4 and induce expression of proinflammatory cytokines [130–132]. TLR4 is also able to sense and respond to different types of fatty acids. Interestingly, saturated fatty acids (SFAs) are reported to be natural ligands for TLR4 and to induce inflammatory gene expression, while polyunsaturated fatty acids (PUFAs) were found to block the activation of TLR4 [133–138]. This suggests that an inflammatory disease, such as atherosclerosis, can be modulated by different types of dietary fatty acids via TLR4 signalling. In the context of atherosclerosis, the most interesting endogenous TLR ligand is probably minimally modified (mm) LDL, an early form of oxLDL that is induced by mild oxidation. Stimulation by mmLDL triggers a cellular response in macrophages and ECs, in part via a CD14/TLR4/MD-2 dependent pathway [139–141]. mmLDL treatment was recently found to also generate reactive oxygen species (ROS) in macrophages via TLR4, in a process that culminates in expression of the proinflammatory mediators IL-1β, IL-6, and RANTES [142]. mmLDL is a complex structure that contains a mixture of possible ligands, including apolipoproteins, cholesterols, and

Innate immune signals in atherosclerosis phospholipids. Although the exact ligand in mmLDL that mediates receptor binding is not fully characterised, several of its components have been shown to induce multiple proatherogenic events in endothelial cells, macrophages and vascular smooth muscle cells. As such, oxidised cholesteryl esters found in mmLDL can be detected in murine atherosclerotic lesions and are, at least in part, responsible for its impact on macrophage activation [143]. There is also extensive evidence that specific oxidised phospholipid (oxPL) products promote vascular inflammation. OxPLs are created during oxidative modifications of LDL but can also be found within apoptotic and necrotic cell membranes. They accumulate at sites of inflammation, such as the atherosclerotic plaque where they induce several proatherogenic events, including upregulation of adhesion molecules and chemokines, thus promoting the recruitment of inflammatory cells to the vessel wall [144–147]. The major bioactive lipids in mmLDL are derived from oxidised products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC). They mediate their activation through TLR4, however, this is independent of CD14 and MD-2 involvement [148]. Interestingly, certain oxidation products of oxPAPC phospholipids also have inhibitory effects on TLR4 and TLR2 inflammatory signalling [149,150]. The mechanisms by which oxPAPCs inhibits TLR activation are under debate and two main models have been suggested, including interference with lipid raft formation, and blocking ligand binding to the receptor via competitive inhibition of the accessory molecules LBP, CD14, and MD-2 [149–152]. Further examination of the properties of specific oxPAPCs is needed to fully understand their anti-inflammatory roles. ApoCIII, a component of very-low-density lipoprotein (VLDL), was also found to be recognised by TLR2 and to induce proinflammatory signals in monocytes [153]. ApoCIII has been shown to exert several pro-atherogenic activities. ApoCIIIenriched lipoproteins, but not ApoCIII-deficient VLDL, induce endothelial cell expression of VCAM-1 that leads to increased adhesion of monocytes to endothelial cells [154,155]. It is not clear if TLRs can discriminate between microbial and endogenous ligands, and direct their activation mechanisms accordingly. Presumably, pathogenic infections and tissue damage present different challenges for the innate response. In the former case, pathogen clearance is of outmost importance, whereas tissue repair is needed in the latter situation. Perhaps this could be the reason why certain phospholipids exhibit anti-inflammatory properties. Thus, the differences reported between stimulation by pathogenic and natural TLR ligands could be an indication that infection and mmLDL require the tissue to respond in different ways.

TLRs in experimental atherosclerosis — role in sterile inflammation Perhaps the strongest evidence that TLRs play a role in detection of tissue injury comes from studies of proatherosclerotic mouse models, where knockouts of specific TLRs reduce disease severity. The exact characterisation of ligands in these models is still lacking, but since gnotobiotic hyperlipidemic mice appear to develop atherosclerosis, it is difficult to dispute a causative role of endogenous TLR ligands in these studies [156]. So far only TLR2 and TLR4 have

11 been studied for their roles in atherogenesis and they have been found to promote atherosclerotic progression in mouse models, in the absence of infection or exogenous ligands. Apoe−/− mice deficient in TLR4 exhibited reduced lesion area and macrophage recruitment after being fed a high cholesterol diet for 6 months [157]. The importance of TLR4 in atherogenesis is also supported by an early study in C3H/HeJ mice. These mice carry a point-mutation resulting in a nonfunctional TLR4, and this mutation rendered the mice resistant to atherosclerosis induced by high cholesterol diet [158]. Furthermore, TLR2 and TLR4 have also been implicated in additional vascular pathological processes such as neointima formation and outward arterial remodelling [159–161]. TLR2 was also shown to be involved in atherosclerosis, where TLR2 deficiency led to inhibition of disease development in ldlr−/− mice [65]. tlr2−/−/ldlr−/− mice on a high cholesterol diet had reduced lesion area and a decrease in inflammatory cytokines. In addition, a bone-marrow transplantation study showed that TLR2 expression on resident vascular non-haematopoietic cells, such as endothelial cells and smooth muscle cells, is important for atherogenesis in the absence of infection [65]. In contrast, TLR2 expression on bone-marrow derived hematopoietic cells, such as macrophages, was important for exacerbating disease in response to exogenous administration of a pathogenic TLR2 ligand [65]. This suggests that endogenous TLR ligands mediate their pro-atherogenic effect on non-haematopoietic cells, whereas infectious products aggravate atherosclerosis by acting on hematopoietic cells. Furthermore, atheroprotective laminar flow down-regulates TLR2 expression on endothelial cells and aortic TLR2 can therefore only be detected in areas of disturbed flow in hyperlipidemic ldlr−/− mice [25]. The involvement of TLR2 in atherogenesis was recently confirmed in a study by Liu et al, where tlr2−/−/apoe−/− mice exhibited reduced atherosclerotic lesion size and inflammatory markers compared with controls [162]. Surprisingly, CD14 that is a co-receptor for both TLR4 and TLR2 does not appear to be involved in this process, since no reduction in atherosclerosis could be observed in cd14−/−/apoe−/− mice [163]. This paradox might be reconciled if atherogenic endogenous TLR ligands act independently of CD14.

TLR polymorphisms and association with cardiovascular disease Genetic variants of specific TLRs have been investigated for their association with increased cardiovascular risk. However, the results remain inconclusive. The TLR4 single nucleotide polymorphism (SNP) Asp299Gly, was initially reported to be associated with a reduced risk of carotid atherosclerosis [164]. However, an increased risk for myocardial infarction in individuals carrying this genotype was found [165]. Subsequent studies have yielded conflicting data on the relationship between TLR4 gene variants, atherosclerosis and clinical disease in man [166]. Similarly, investigating the effect of TLR2 and TLR9 polymorphisms on CAD risk have shown either no association or generated inconsistent results [166,167]. Further studies using larger patient cohorts are needed to elucidate the role of TLRs in CAD.

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TLR signalling pathways TLR stimulation induces synthesis of proinflammatory mediators by activating complex cytoplasmic signal transduction pathways (Fig. 2) [13]. Four adaptors have been characterised and they regulate signalling from specific TLRs, including myeloid differentiation factor 88 (MyD88), MyD88 adaptor-like (MAL), TIR-domain-containing adaptor-inducing interferon β (TRIF), and TRIF-related adaptor molecule (TRAM) [168–175]. Different combinations of these adaptors can trigger unique or redundant pathways, leading to the activation of transcription factors, such as NFκB, AP-1 and IRFs [13,176]. These transcription factors regulate the expression of a many proinflammatory genes, such as those for cytokines, adhesion molecules, and co-stimulatory molecules. The adaptor protein MyD88 controls signalling from most TLRs, as well as the IL-1/IL-18 receptors, and initiates a common pathway that ultimately activates MAPKs and NFκB [177–190]. Certain TLRs, such as TLR2 and TLR4, also recruit MAL, whose principal function is to stabilise the interaction between MyD88 and the receptor complex [191,192]. In contrast, TRIF is used only by TLR3 and TLR4, where TRAM acts as a bridging adaptor between TRIF and TLR4 [175,193]. Activation of TRIF-dependent pathways result in activation of IRF transcription factors that are involved in anti-viral responses and regulate the production of type I interferons and the chemokines IP-10, RANTES and I-TAC [194,195]. TRIF also induces an alternative pathway to NFκB activation. It appears to be cellular localisation of the receptor that determines whether MyD88- or TRIF-dependent pathways are initiated upon TLR4 ligation (Fig. 2) [196,197]. Membranebound TLR4 activates MAL and MyD88 to induce NFκB activation, whereas endosomal TLR4 engages the TRIFdependent pathway, leading to IRF activation and induction of type I IFNs. In contrast, endosomal TLR7, TLR8, and TLR9 induce type I IFN production in a MyD88-dependent manner that is also dependent on location. When MyD88 localises to the endosome instead of the plasma membrane, it associates with TRAF3, facilitating the formation of a complex with TBK1 and the activation of IRFs [194,195].

Inflammatory signalling pathways involved in atherosclerosis In addition to the participation of individual TLR receptors, several of the downstream signalling components involved in TLR-induced pathways have been implicated in atherogenesis. MyD88 is the only TLR adaptor investigated in atherosclerosis so far, and it was shown to be important for disease development in apoe−/− mice [157,163]. MyD88deficient mice exhibited marked reduction in lesion size and the inflammatory cytokines IL-12 and MCP-1 [157,163]. It is the main adaptor controlling signal transduction downstream of all TLRs, with the exception of TLR3. However, MyD88 is also crucial for signalling via the IL-1 and IL-18 receptors and the observed effects could therefore be due to an inhibition of these receptors, particularly since IL-1/IL-18 stimulation has been shown to promote atherosclerosis [198–200]. IRAK4, the main kinase downstream of MyD88 has also been implicated in atherosclerosis (Fig. 2) [201]. A critical

A.M. Lundberg, G.K. Hansson role for IRAK4 in TLR- and IL-1-mediated inflammation was recently shown when functional deficiency of IRAK4 accelerated injury-induced carotid lesion development in apoe−/− mice [201]. Expression of the inactive form of IRAK4 resulted in down-regulation of several key proinflammatory genes, as well as inhibition of macrophage infiltration. NFκB is a major transcription factor that controls the inflammatory response through regulation of a multitude of immune genes, many of which have been implicated in atherosclerosis. Members of the NFκB family are dimers in their active form and the most abundant complex is the classical p65/p50 NFκB dimer, which is initiated by TLR stimulation, as well as activation by TNF and IL-1. The presence of activated NFκB has been demonstrated in atherosclerotic lesions, but not in unaffected vascular tissue [202]. Activated NFκB was shown in plaque macrophages, endothelial cells and smooth muscle cells and it co-localised with TLR expression [18,202]. Contradictory observations have been reported on the function of NFκB in atherosclerosis, and whether it has a pro- or anti-atherogenic role appears to be dependent on the cell type studied. Recent data showed that specific inhibition of NFκB in endothelial cells of apoe−/− mice led to reduction in atherosclerotic lesion development [203]. This was demonstrated in mice deficient in either IKKγ, or expressing a non-degradable IκBα, both of which block NFκB function by sequestering it in the cytoplasm and preventing it from translocating to the nucleus and induce gene transcription. These mice exhibited reduced expression of the adhesion molecule VCAM-1, thereby impairing recruitment of macrophages to the lesion [203]. In contrast, macrophage specific deletion of IKKβ in ldlr−/− mice led to increased lesion size [204]. In the absence of macrophage IKKβ, lesions exhibited increased cell death suggesting that IKKβ has a pro-survival role in murine macrophages. IKKβ has been shown to regulate NFκB activation in cells from human carotid lesions and control expression of inflammatory cytokines, therefore species-specific mechanisms might also exist [205]. The activities of the TLR/IL-1 induced signalling pathways are tightly controlled by a number of endogenous regulatory proteins. Their purpose is to limit excessive and prolonged production of proinflammatory mediators, which otherwise can cause tissue damage and chronic inflammation. Starting at the receptor complex, membrane-bound TIRdomain-containing proteins, such as ST2, are involved in negative regulation of TLR signalling. ST2 inhibits activation of NFκB induced by IL-1 and TLR4, probably via sequestering TLR adaptors, such as MyD88 and MAL, and by preventing them from participating in downstream signalling [206]. Both ST2 and its ligand IL-33 are present in the normal and atherosclerotic vasculature of mice and humans and administration of IL-33 can reduce atherosclerosis development in apoe−/− mice on a high-fat diet [207]. Downstream of the receptor complex, IRAK-1 activity is inhibited by IRAK-M, another negative regulator. IRAK-M lacks kinase activity and blocks signalling by enhancing the binding of MyD88 to IRAK-1 and IRAK-4 [208]. In this way, IRAK-M traps both IRAKs in the receptor complex, thereby preventing their dissociation and downstream signal transduction. IRAK-M expression in the atherosclerotic lesion increases with age and disease progression in apoe−/− mice [121].

Innate immune signals in atherosclerosis A20 is a broadly expressed cytoplasmic protein that can influence atherosclerosis susceptibility through inhibition of NFκB induced by TNF and IL-1/TLR activation. A20 is expressed in response to TLR stimulation as a negative feedback mechanism, and terminates TLR-induced NFκB activation and production of proinflammatory cytokines [209]. In this way, TLRs and NFκB inhibit their own activation by inducing the expression of A20, which in turn targets TRAF6 and blocks its activity [209]. Mice deficient in A20 show increased lesion size and transgenic overexpression of A20 in apoe−/− mice results in smaller lesions [210]. Restricting inflammatory signals is important for controlling the magnitude of immune responses. Since several of these proteins target signal conversion points for multiple pathways, such as those for IL-1R/IL-18R and TNF receptors, their participation in atherosclerotic development does not necessary equal TLR involvement. However, the fact that activation of downstream mediators in the TLR/IL-1 signalling pathway is controlled by several negative regulators present in lesions highlights the importance of these signalling pathways in vascular inflammation.

Crosstalk between TLRs, scavenger receptors and the inflammasome In a complex tissue, such as the atherosclerotic lesion, numerous innate immune mechanisms are in play simultaneously. They include several extracellular activation cascades and various intracellular signalling pathways, leading to effective clearance of harmful material and induction of inflammatory responses. Since numerous PRRs are expressed in the lesion, crosstalk between their signalling pathways is inevitable. Therefore TLR signalling can be modulated by other PRRs to tailor immune responses. The different PPRs can be activated simultaneously or sequentially to induce both distinct and shared signalling pathways. Several scavenger receptors can act as co-receptors for TLR2 and modulate its inflammatory response. TLR2 associates with the scavenger receptor CD36, which is involved in binding ligands to the TLR2/TLR6 complex [211,212]. TLR2 signalling can also be enhanced by co-stimulation through Dectin-1, a C-type lectin receptor important for recognising fungal cell wall products [213,214]. Moreover, TLR2 is proposed to associate and cooperate with lectin-like oxidised low-density lipoprotein receptor-1 (LOX-1) to induce downstream signalling [215]. LOX-1 is a scavenger receptor that recognises endogenous ligands such as oxLDL and apoptotic cells. It lacks a signalling domain and cannot propagate downstream events on its own. Overexpression of LOX-1 in apoe−/− mice results in increased inflammation in atherosclerotic lesions [216,217]. TLRs can influence phagocytosis indirectly by affecting the expression of phagocytic receptors. Stimulation with TLR3, TLR4, and TLR9 ligands induced the expression of the scavenger receptors SR-A, macrophage receptor with collagenous structure (MARCO) and LOX-1 on macrophages, leading to increased phagocytosis [218]. TLR activation in the lesion could therefore result in increased uptake by the scavenger receptors, which could promote processes such as foam cell development. Interestingly, SR-A cooperates with TLR4 to promote macrophage apoptosis [219]. SR-A does not signal on its own, but SR-A

13 ligation was found to inhibit a TRIF-dependent pathway leading to IFNβ production and cell survival, while simultaneously activating the MyD88-dependent apoptotic pathway [219]. Combined activation of both SR-A and TLR4 may therefore result in increased plaque cell death, which is thought to aggravate inflammation and promote lesion instability. Activation of TLRs triggers a signalling cascade via MyD88 and NFκB activation, which increases transcription pro-IL-1β and pro-IL-18. These are potent proinflammatory cytokines that operate to induce inflammation at sites of infection and tissue damage and play an important role in the pathogenesis of atherosclerosis [198–200]. However, production of these cytokines requires a second signal resulting in cleavage of the pro-forms to release the active molecules. This is regulated by a cytosolic protein complex called the inflammasome. It consists of an NLR (nucleotide-binding domain, leucine-rich repeat-containing) family member, the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD), and the protease caspase-1 [220]. The inflammasome plays a crucial role in this second step of proinflammatory cytokine activation, through caspase-1 mediated cleavage of IL-1β and IL-18 precursors into their mature secreted forms, IL-1β and IL-18. A sequential activation scenario has been proposed for TLRs and the inflammasome. According to it, TLRs provide a signal for transcription of pro-IL-1β and pro-IL-18, and the inflammasome provides a signal for cleavage and activation of these pro-cytokines. A variety of stimuli can result in the activation of NLR pathways and the inflammasome, including bacterial products and endogenous molecules from dying cells, such as uric acid and ATP [221,222]. The inflammasome was recently reported to have a role in cardiovascular disease through an ASC regulated mechanism [223]. ASC controls the recruitment and activation of caspase-1, thereby regulating the caspase-1-mediated maturation of IL-1β and IL-18. ASC deficiency attenuated neointimal formation after vascular injury via reduced expression of IL-1β and IL-18 in neointimal lesions, resulting in a decrease of vascular inflammation [223]. Retinoic acid-inducible gene-I (RIG-I) is another cytoplasmic receptor and a member of the RIG-like receptor (RLR) family that recognises viral RNA. RIG-I has been detected in intimal macrophages in human atherosclerotic lesions [224]. However, its role in vascular inflammation in cardiovascular disease remains to be determined, as does that of other inflammasome components, such as Nod-like receptors.

Effects of TLR stimulation and infection on cholesterol metabolism and foam cell formation The formation of foam cells in the arterial wall is a key event in atherosclerotic lesion formation. In the lesion, macrophages ingest oxLDL through scavenger receptors, which lead to accumulation of cholesterol in the cytoplasm and transformation of the macrophages into foam cells. Several molecular pathways contribute to foam cell development, not only oxLDL uptake via scavenger receptors, but also dysregulation of cholesterol transporters and intracellular lipid trafficking. TLR activation induces intracellular signal pathways that can

14 modulate several of these mechanisms in macrophages. Activation of murine macrophages by TLR ligands, such as those for TLR2 and TLR4 increase LDL uptake and cholesterol content, leading to foam cell formation [225–227]. The nuclear receptor lipid-X receptors (LXRs) are master regulators of cholesterol homeostasis. They are ligandactivated transcription factors that control cholesterol and fatty acid metabolism in macrophages through transcriptional regulation of members of the cholesterol efflux machinery. LXRs have been shown to prevent progression of atherosclerosis by increasing expression of genes involved in cholesterol efflux such as the ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1), which have potent anti-atherogenic activity [228–231]. The enhanced cholesterol efflux caused by stimulation of LXR can be inhibited by TLR activation. TLR3 and TLR4 stimulation block cholesterol efflux in macrophages by inhibiting LXR transcriptional activity, thereby reducing the expression of ABCA1 and several other genes [232]. Inhibition of LXR is independent of NFκB and MyD88 and is instead controlled by IRF-3, through an unknown mechanism [232]. Conversely, LXRs can repress the expression of certain inflammatory genes induced by TLR signalling via inhibition of NFκB activity. As such, stimulation of macrophages with TLR4 and LXR agonists reduces proinflammatory responses by a mechanism that is MyD88 dependent [233,234]. Fatty acids present in macrophages associate with cytoplasmic fatty acid binding proteins (FABPs) for intracellular transport. TLR2 and TLR4 stimulation can affect lipid accumulation in macrophages through increased expression of FABP4, which is normally expressed at very low levels [235]. The increased levels of FABP4 promote intracellular lipid accumulation by assisting with the transport of fatty acids needed for cholesteryl esterification and triglyceride accumulation. Therefore, FABP4 deficiency reduces lesion formation and progression in hypercholesterolemia-induced atherosclerosis [236–238]. Cross-signalling between LXR and TLR represents a direct link between innate immunity and macrophage cholesterol metabolism, and it is a potential way for pathogens to contribute to cardiovascular disease. Several studies have shown that C. pneumoniae can induce foam cell formation in macrophages in a TLR2- and TLR4-dependent manner [93,227,239,240]. This is mediated by signalling via activation of both NFκB and IRF-3 pathways [93,240]. In addition, C. pneumoniae infection is mediating pro-atherogenic effects by affecting LXR, probably through inhibition of cholesterol efflux by macrophages [93]. Further studies are needed to assess whether other pathogens can influence atherosclerosis in a similar way by altering the balance between cholesterol homeostatic pathways and stimulation of innate immune mechanisms. It would also be interesting to know if stimulation by endogenous TLR ligands can increase lipid accumulation in foam cells and thus lead to progression of atherosclerosis.

Innate TLR-dependent mechanisms involved in atherosclerotic plaque formation As outlined above, the presence of TLRs and their ligands might affect several aspects of the disease by acting on cells resident in the lesion. Activated macrophages and T cells are observed

A.M. Lundberg, G.K. Hansson throughout atherogenesis. Chemokines and adhesion molecules, such as VCAM-1, expressed by the activated endothelium are critical for the recruitment of these cells to the lesions [2,241]. This activation of the endothelium can either be induced by direct stimulation of TLRs expressed on the endothelial surface, or indirectly by inflammatory cytokines produced as a consequence of TLR ligation (Fig. 3) [242,243]. A multitude of cytokines are detected within the atherosclerotic lesion, as discussed in a recent review [244]. Stimulation of all TLRs results in the secretion of proinflammatory cytokines including TNF, IL-12 and IL-6. These cytokines are mainly induced via NFκB activation through the MyD88dependent TLR pathway and they all have functional roles in atherogenesis. Several macrophage and Th1-specific chemokines are instead mainly induced via the TRIF-dependent TLR pathway, including RANTES and IP-10 [193]. RANTES controls the recruitment and trans-endothelial migration of leukocytes to inflammation sites, and inhibition of RANTES prevents progression of, new as well as established, atherosclerotic lesions in ldlr−/− mice [9,245,246]. In addition, IP-10 is also expressed in human atherosclerotic lesions and activation of its receptor is critically involved in recruitment of Th1 cells to the plaque [8,247,248]. Once in the circulation, the mediators induced by TLR stimulation may subsequently activate hepatic acute phase responses. As such, systemic IL-6 can induce expression of the acute phase proteins C-reactive protein (CRP) and serum amyloid A (SAA), whose elevated levels are associated with increased cardiovascular risk [249–251]. Interestingly, a recent study showed that treatment with statins (HMG-CoA reductase inhibitors) dramatically reduced the risk of myocardial infarction by 47% in patients with low serum cholesterol but elevated CRP [252]. This study provides strong evidence that inflammation plays a major role for atherosclerotic disease. Triggering of TLRs also leads to activation of specific antigen-driven immune responses (Fig. 3). This is mediated through increased antigen presentation and via synthesis and release of various cytokines that regulate T lymphocyte function. TLR-mediated stimulation is necessary to induce complete maturation of dendritic cells and is essential for activation of adaptive immune responses. Dendritic cell maturation is accompanied by upregulation of MHC class II and co-stimulatory molecules, as well as by secretion of T cell polarising cytokines, such as the Th1 cytokines IL-12 and IL-18. TLRs have therefore emerged as an essential link between activation of the innate immune response and the induction of adaptive immunity. CD4+ Th1 cells have been shown to be pro-atherogenic [253]. They are recruited to the lesion in an antigenindependent manner, but once there they are activated by local APCs in an antigen-specific way. Dendritic cells present antigens to naïve T cells and initiate primary immune responses. They have been found in arterial tissue and in atherosclerotic lesions, suggesting that local presentation of lesion antigens might be important for disease development [254–256]. T cells specific for oxLDL and hsp60 are found in the atherosclerotic plaques [257,258]. TLR stimulation of dendritic cells can also enhance presentation of lipid antigens and subsequent activation of Natural Killer T cells (NKT cells) [259,260]. NKT cells are a subset of non-conventional T cells that recognise endogenous and/or exogenous glycolipids presented by CD1d, a MHC-like

Innate immune signals in atherosclerosis

15

Figure 3 TLR-mediated activation of immune cells in the atherosclerotic plaque. TLR stimulation of macrophages results in release of proinflammatory cytokines, including tumour necrosis factor (TNF), Interleukin (IL)-1 and IL-6 that can have both local and systemic effects. TLR stimulation also induces expression of matrix-degrading matrix metalloproteinases (MMPs), which probably plays a role in weakening the fibrous cap and promoting plaque vulnerability. Several chemokines are expressed in response to TLR activation; such as monocyte chemotactic protein-1 (MCP-1), important for attracting monocytes, and regulated upon activation normal T cell expressed and secreted (RANTES), as well as interferon-inducible protein (IP)-10, involved in recruitment of T cells to the lesion. Increased levels of extravasated immune cells will contribute to the local inflammation and promote lesion formation. Local peptide antigens (e.g. oxidised low-density lipoproteins, heat shock proteins, or microbial antigens) will be presented to T helper type 1 cell (Th1) cells bound to major histocompatibility complex (MHC)II. Lipid antigens are instead recognised by natural killer T (NKT) cells in the context of CD1 molecules. TLR stimulation will enhance antigen presentation by inducing increased expression of co-stimulatory molecules and T cell polarising cytokines, such as IL-12 and IL-18, by antigen-presenting cells (APC). The activated Th1 and NKT cells will produce a host of proinflammatory mediators (e.g. TNF and interferon γ). They will potentiate the inflammatory process through acting back and further stimulate lesion resident cells, such as macrophages, endothelial cells and smooth muscle cells. The locally produced inflammatory mediators will together result in atherosclerotic disease progression and promote plaque vulnerability.

mole-cule. NKT cells can be detected in atherosclerotic lesions and their activation aggravates atherosclerosis [261]. TLRs might also affect later stages of the disease and perhaps be involved in acute vascular events (Fig. 3). Plaque rupture is associated with inflammatory activation of cells that secrete matrix-degrading enzymes, such as matrix metalloproteinases (MMPs). Local production of MMPs probably plays a significant role in weakening the fibrous cap and promoting plaque vulnerability. Deficiency of MMP-9 in apoe−/− mice results in reduced atherosclerosis, accompanied by decreased macrophage infiltration [262]. TLRs are involved in matrix turnover through induction of MMPs from macrophages [120,263]. TLR4 stimulation of macrophages mediates expression of MMP-9 that degrades collagen and may in this way promote plaque vulnerability [120]. In addition, TLR stimulation can lead to MMP expression indirectly via induction of cytokines. In this way, TLR9 induced expression of TNF results in MMP-9 expression [264]. Platelets also participate in atherosclerotic innate immune responses and activated platelets release a variety of pro-thrombotic and proinflammatory mediators. Adhesion of platelets to the exposed matrix is considered to be the initial step in thrombus formation in vascular lesions. The majority of TLRs are expressed by platelets and stimulation of TLR2 and TLR4 directly activates their thrombotic and inflamma-

tory responses [265–269]. In addition, a recent study reports that P. gingivalis can induce a proinflammatory response in platelets in a TLR2-dependent manner [265]. This is of particular interest since infection increases risk for thrombosis and acute cardiovascular events.

Conclusions Components of innate immunity contribute to atherosclerosis in several ways. Endocytosing pattern-recognition receptors on macrophages, including scavenger receptors, play key roles in cholesterol accumulation and foam cell formation in atherosclerotic lesions. Signalling pattern-recognition receptors, such as TLRs, are also expressed in lesions and promote atherosclerosis through induction of inflammatory processes. TLRs are key regulators of innate immunity and recognise both microbial products and endogenous ligands associated with tissue injury and inflammation. Several pathogens have been implicated in atherosclerosis and their products could trigger TLRs on lesion resident cells. Furthermore, endogenous TLR ligands are also present in the lesion at various stages of the disease progression. However, the exact nature and involvement of endogenous TLR ligands and the role of specific pathogens in the development of atherosclerosis remain to be understood.

16 TLRs operate by inducing production of inflammatory mediators and by promoting antigen presentation, resulting in activation and polarisation of the adaptive immunity. In addition, TLR signal transduction pathways can interact with signals activating nuclear receptors and can in this way affect lipid metabolism and influence foam cell formation. Data from mouse models and in vitro studies point to an important role for these mechanisms in disease initiation and progression. However, the importance of innate immune mechanisms for coronary artery disease and other clinical manifestations of atherosclerosis remains unclear. Before therapeutic targeting of these mechanisms can be developed, it is crucial to expand our knowledge of the roles of TLRs and their cross-talk with other PRRs, as well as metabolic pathways. Therefore, clinical and epidemiological investigations are warranted, as are further studies into the pathogenetic mechanisms by which innate immunity can affect atherosclerosis.

Acknowledgments This work was supported by the Swedish Research Council, the Swedish Heart-Lung Foundation, and the European Union (Framework VII). We thank Annika Röhl for the preparation of colour graphics and Daniel Johansson for critical reading of the manuscript.

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