S (2011) Toll-like receptors, their ligands, and atherosclerosis

Atherosclerosis is a disease characterized by inflammation in the arterial wall. Atherogenesis is dependent on the innate immune response involving activation of Tolllike receptors (TLRs) and the expression of inflammatory proteins. TLRs, which recognize various pathogen-associated molecular patterns, are expressed in various cell types within the atherosclerotic plaque. Microbial agents are associated with an increased risk of atherosclerosis and this is, in part, due to activation of TLRs. Recently considerable evidence has been provided suggesting that endogenous proteins promote atherosclerosis by binding to TLRs. In this review, we describe the role of TLRs in atherosclerosis with particular emphasis on those atherogenic endogenous proteins that have been implicated as TLR ligands. KEYWORDS: Toll-like receptors, innate immunity, atherosclerosis, heart disease INTRODUCTION Coronary artery disease predominantly results from atherosclerosis, a process involving the buildup of modified lipids, cholesterol, and cellular debris within the inner lining of the arterial wall. Atherosclerosis begins with damage to the endothelium and is typically caused by high cholesterol levels, elevated blood pressure, diabetes, and smoking. Activated endothelial cells then express various adhesion molecules and cytokines, which promote the migration of circulating leukocytes, such as monocytes, T lymphocytes, and dendrites, into the subendothelial space of the artery. Once recruited, the monocytes develop into macrophages, which then ingest cholesterol and modified lipids, becoming foam cells on the basis of their appearance. This is the earliest detectable stage of atherosclerosis and is termed a fatty streak. Impaired emigration of the foam cells leads to their accumulation in the subendothelial space. Eventually, these cells die by necrosis, leaving a core of crystalline cholesterol and cell debris. Smooth muscle cells migrate into the area and deposit extracellular matrix proteins to form a protective cap over the lesion. Instability in the cap, arising from expression of matrix metalloproteinases, for example, may lead to rupture and subsequent thrombus formation Persistent and prolonged inflammatory mechanisms are believed to underpin atherosclerosis Activation of the immune system is an important component of the chronic inflammatory state in atherosclerosis TLRs AND ATHEROSCLEROSIS Several lines of evidence suggest that TLRs are involved in atherosclerosis. TLR1, TLR2, and TLR4 are upregulated in human atheroma with active NF-κB colocalizing with TLR2 and TLR4 in the plaque The TLR4 polymorphism Asp299Gly is associated with a decreased response to inhaled LPS [19]. Several studies have suggested that this polymorphism in the human TLR4 gene is associated with atherosclerosis. A reduced risk of carotid artery atherosclerosis development [20] and the appearance of acute coronary events [21,22] have all been observed in association with the Asp299Gly polymorphism. This polymorphism also appears to act synergistically with statin treatment; in a study of 655 men with angiographically documented coronary atherosclerosis, pravastatin reduced the risk of cardiovascular events by about 1.6-fold, whereas in the 299G carriers, the reduction of risk was more striking at about 15-fold [22]. Holloway et al. also found that compared with 299Asp homozygotes, 299Gly carriers had a significantly lower risk of myocardial infarction only when they were receiving statins [23]. Inhibition of the LPS/TLR4 proinflammatory signaling pathway by statins appears to involve either Rho [24] or reduced expression of the TLR4 protein [25]. TLR SIGNALING Ligand binding to TLRs stimulates expression of various inflammatory molecules, including 26,27]. Once activated, the TLR recruits adaptor proteins, including MyD88, TRIF, TRAM, and TIRAP, via TIR-domain interactions. MyD88, with the exception of TLR3, is common to all TLR signaling pathways and is involved in MAPK (JNK, ERK, p38α) and proinflammatory transcription factor (NF-κB, AP1, Elk1) activation. Mice lacking MyD88 do not activate MAPK and proinflammatory Hodgkinson/Ye: Toll-Like Receptors, Their Ligands, and Atherosclerosis TheScientificWorldJOURNAL (2011) 11, 437-453 439 transcription factors in response to TLR2-, TLR5-, TLR7-, TLR8-, and TLR9-specific ligands [28,29,30,31,32,33]. TLR3 and TLR4 signal through a MyD88-independent pathway. Cells deficient in MyD88 do not produce inflammatory cytokines in response to LPS; however, activation of the transcription factor IRF3 and increased expression of type-I IFN is unaffected [34]. Similarly, polydI.dC, the TLR3 ligand, stimulates IFN expression in the absence of MyD88 [35,36]. TRIF was subsequently identified as the adaptor molecule that regulated this MyD88-independent pathway [37,38]. The adaptors TRAM and TIRAP confer specificity; TRAM participates in the TRIF pathway of TLR4, but not that of TLR3 [26], and TIRAP is involved in MyD88-dependent signaling for some TLRs (TLR1, TLR2, TLR4, and TLR6), but not others (TLR5 and TLR9) TLR EXPRESSION IN CELLS OF THE ATHEROSCLEROTIC LESION The cells of the atherosclerotic lesion (macrophages, lymphocytes, dendritic cells, smooth muscle cells, and endothelial cells) all express TLRs [41]. Macrophages express a large number of TLRs, including TLR1, TLR2, TLR4, TLR5, TLR6, TLR8, and TLR9 [42,43]. Increased expression of TLR2 and TLR4 has been observed in monocytes from human subjects and ApoE-/-mice with coronary artery disease [44,45,46]. Once recruited into the lesion, these macrophage-borne TLRs are exposed to a wide array of ligands; subsequent activation of these TLRs is important in converting the macrophages into foam cells [47,48,49]. Dendritic cells, both myeloid and plasmacytoid, accumulate in the atherosclerotic plaque and enhance T-cell stimulation [50,51]. Similar to macrophages, a large repertoire of TLRs are expressed in both dendritic cell subsets [52,53,54,55]. The differentiation of naïve T lymphocytes into T H 1 or T H 2 cells, which are considered pro-or antiatherogenic, respectively, is affected by TLRs. Knockout of MyD88 switches differentiation of T cells from T H 1 to T H 2 [56,57]. Smooth muscle and endothelial cells respond to TLR ligands by producing proinflammatory cytokines [58,59,60,61]. Arterial endothelial and smooth muscle cells respond to a wider range of TLR ligands than the corresponding venous cell types [59]. In low-density lipoprotein receptor-deficient (LDLr(-/-)) mice, TLR2 expression is only found in aortic endothelial cells within regions of disturbed blood flow. These regions are particularly prone to the development of atherosclerotic lesions [62]. Expression of the receptor is important for leukocyte infiltration, foam cell formation, and endothelial cell injury within these disturbed flow areas [63]. TLR LIGANDS AND ATHEROSCLEROSIS The realization that TLRs are involved in atherosclerosis has prompted the search for the ligands involved in disease progression. Both exogenous and endogenous ligands have been shown to activate TLRs and many have been implicated in atherosclerosis (summarized in Exogenous Ligands Pathogen-borne TLR ligands have been shown to encourage the development of atherosclerosis. Administration of the TLR4 ligand LPS into ApoE-/-mice or hypercholesterolemic rabbits promotes atherosclerosis Acute infections are associated with a transiently increased risk of cardiac events 440 Endogenous Ligands A large number of structurally diverse endogenous molecules have been described as TLR ligands. In the context of atherosclerosis, perhaps the most pertinent of the proposed endogenous TLR ligands are the modified LDLs. LDL oxidation and glycation are both important for atherogenesis Certain oxidized phospholipids have been suggested as TLR4 ligands. Oxidized phospholipids can be generated during cell apoptosis and necrosis, as well as the oxidation of LDL. Accumulation of oxidized phospholipids within the plaque induces the expression of various proinflammatory proteins Apo-CIII directly activates monocytes, enhancing their adhesion to endothelial cells and stimulating VCAM-1 expression Inflammation substantially increases the expression of serum amyloid A, and a positive correlation exists between serum amyloid A levels and cardiovascular risk LPS is comprised of three parts: the O-antigen, the core oligosaccharide, and Lipid A. The toxicity of LPS is caused by Lipid A LPS-induced proinflammatory cytokine production in macrophages is suppressed by the consumption of n-3 polyunsaturated fatty acids 442 is clearly an important issue in inflammatory diseases. Fatty acid-induced insulin resistance, which promotes atherosclerosis, has been linked to TLR4 The heat shock protein HSP60, a protein implicated in atherosclerosis[119], has been described as a TLR4 ligand. HSP60 elicited strong inflammatory TNF-α cytokine release from C57BL/6 and C3H/HeN macrophages. However, macrophages from C3H/HeJ mice, which possess a mutation rendering TLR4 inactive, did not release any TNF-α in response to HSP60 treatment The heat shock protein gp96 activates dendritic cells to produce proinflammatory proteins. This effect is reduced in dendritic cells derived from C3H/HeJ mice and inhibited still further in dendritic cells from C3H/HeJ/TLR2(-/-) animals High-mobility group box protein 1 (HMGB1) was originally identified as a DNA-binding protein, but has subsequently been shown to act as a proinflammatory molecule Cellular fibronectins, which contain an alternatively spliced exon encoding a type-III repeat extra domain A (EDA), are produced during inflammation and may have tissue-remodeling properties Fibrinogen is a plasma glycoprotein that is converted into fibrin during blood coagulation. Elevated fibrinogen levels are associated with an increased risk of cardiovascular disease Glycosaminoglycans have been implicated in atherosclerosis β-Defensins are a family of small mammalian antimicrobial peptides that prevent the colonization of epithelial surfaces by bacteria TLR9 is activated by unmethylated CpG dinucleotides present in bacterial DNA Cautionary Tales Clearly, in studies investigating the potential of endogenous molecules as TLR activators, there is always the possibility of confounding results arising from contamination by microbial agents. Most of the atherogenic endogenous TLR ligands described above activate either TLR2 or TLR4. Considering that LPS will stimulate TLR4 at concentrations in the pg/mL range Despite the necessary caution, there are grounds for optimism regarding endogenous TLR ligands. Truly germ-free mice, devoid of any bacterial, viral, or fungal agents, develop atherosclerosis at the same rate as animals exposed to normal levels of microbial organisms CROSS-TALK BETWEEN TLRS AND OTHER RECEPTORS HMGB1, as discussed above, promotes inflammation via significant cross-talk between TLR2/4 and RAGE. Other examples exist whereby TLR signaling is modified by other receptors. The atheroprotective effect of sphingosine 1-phosphate occurs via attenuation of TLR2 signaling by the sphingosine 1-phosphate receptors class 1 and