Toll-like receptor signaling: Common pathways that drive cardiovascular disease and rheumatoid arthritis

Authors


Inflammation: a common defining hallmark of rheumatoid arthritis and cardiovascular disease

Rheumatoid arthritis (RA) is a chronic and debilitating autoimmune disease that affects 1% of the population worldwide. It is characterized by synovial inflammation with proliferation of synovial fibroblasts and infiltration of immune cells into the synovium and synovial fluid. Persistent synthesis of proinflammatory cytokines, chemokines, and tissue-degrading enzymes by these cells leads to destruction of cartilage and bone and progressive loss of joint function (1).

Individuals with RA also exhibit a significantly higher incidence of cardiovascular disease (CVD) than individuals with no joint disease (2). RA patients have a >2-fold risk of myocardial infarction (MI) and are twice as likely to experience sudden cardiac death (3). They are also more likely to experience their first cardiovascular event at an earlier age (4) and exhibit subclinical atherosclerosis with high carotid artery intima-media thickness ratios (5, 6) and coronary artery disease affecting multiple vessels (7). The risk of CVD is strongly associated with RA disease duration; patients with longstanding RA have a higher Framingham risk score and more coronary artery calcifications than those with earlier disease (8). Moreover, in certain populations of RA patients the number of swollen joints is predictive of death from CVD (9).

RA patients commonly present with one or more risk factors for CVD. In particular, they have altered lipid profiles with high levels of proatherogenic lipoprotein(a) (10), and often display hypertension, obesity, physical inactivity, and smoking (11). However, CVD risk factors alone do not entirely explain the increased prevalence of CVD in the RA population, and RA itself is recognized as an independent risk factor for CVD (3, 7, 11, 12).

The precise molecular mechanisms that mediate the increased incidence or accelerated onset of CVD in patients with RA are not yet clear. There is evidence to suggest that inflammation may be a key link. Recently, the European League Against Rheumatism expressed a consensus view that the increased inflammatory burden in RA is probably the major contributor to the increased CVD risk in patients with arthritis (13).

The pathogenesis and etiology of RA and CVD share many common cellular and molecular themes (14). The same molecular and cellular mediators drive localized inflammation of the synovium and the arterial wall. Furthermore, high systemic levels of proinflammatory molecules in RA are likely a major contributing factor to the increased incidence and accelerated onset of CVD that occurs in patients with arthritis. Atherosclerosis, the principal cause of coronary artery and cerebrovascular disease (15), is increasingly recognized as an inflammatory disease of the arterial wall (16). The atherosclerotic plaque is characterized by proliferation of vascular smooth muscle cells and migration into the intima, accompanied by infiltration of blood-borne immune myeloid cells enabled by endothelial dysfunction or activation. Lipid accumulation and matrix deposition lead to atheroma formation. Proteolytic degradation of the plaque fibrous cap mediates lesion rupture leading to thrombus formation, potentially causing MI. Therefore, localized inflammation is key to both plaque formation and destabilization in atherosclerosis (14, 17–20). Moreover, systemic inflammation is also associated with both RA and CVD, and the high levels of inflammatory mediators and cells in RA have been proposed to act on the vasculature to induce or accelerate atherosclerosis.

Here we discuss the role of one group of proinflammatory mediators, the Toll-like receptors (TLRs), which are emerging as pivotal molecules and key mediators of inflammation in the pathogenesis of both RA and CVD. We focus on the common signaling pathways that drive inappropriate local inflammation at each distinct site, in particular the role of endogenous activators of TLRs in perpetuating a nonresolving, sterile inflammatory loop. We review the evidence supporting their role in driving inflammation in each disease focusing on how this has enhanced our understanding of disease progression, as well as how the systemic bioavailability of these ligands in RA may provide the long sought-after missing link between joint disease and CVD. We also highlight the implications this may have on the therapeutic management of these conditions.

TLRs mediating physiologic and pathologic inflammation

TLRs are a family of highly conserved pattern-recognition receptors (PRRs) that are key triggers of immunity (21). Ten functional human TLRs and 12 functional mouse TLRs have been identified. These receptors are type I transmembrane proteins that have different expression patterns and locations within the cell; TLR-3, -7, -8, and -9 are localized in intracellular compartments, while the other TLRs reside on the plasma membrane. TLRs detect invading microbes as well as sense tissue damage, and in response induce the expression of proinflammatory genes.

TLR activators.

TLRs recognize an enormously diverse range of ligands, including exogenous molecules derived from invading microbes, pathogen-associated molecular patterns (PAMPs), and endogenous molecules created or up-regulated upon tissue injury, damage-associated molecular patterns (DAMPs). PAMPs are conserved molecules from bacteria, viruses, and parasites, and include lipoproteins, lipopolysaccharides (LPS), flagellin, and nucleic acids. DAMPs include molecules released or secreted from activated or necrotic cells, fragments of extracellular matrix (ECM) molecules created upon tissue injury, or ECM molecules that are specifically induced upon tissue injury. DAMPs also comprise a wide range of type of molecules, including proteins, peptides, nucleic acids, fatty acids, lipoproteins, and glycosaminoglycans (22). Each member of the TLR family recognizes a different subset of ligands. Tables 1 and 2 list some of these exogenous and endogenous activators together with their cognate TLR.

Table 1. TLRs and exogenous activators*
 LigandSource
  • *

    TLR = Toll-like receptor; dsRNA = double-stranded RNA; LPS = lipopolysaccharide; ssRNA = single-stranded RNA.

TLR-1Triacyl lipoproteinBacteria
TLR-2LipoproteinBacteria, viruses, parasites
TLR-3dsRNAViruses
TLR-4LPSBacteria, viruses
TLR-5FlagellinBacteria
TLR-6Diacyl lipoproteinBacteria, viruses
TLR-7/-8ssRNABacteria, viruses
TLR-9CpG DNABacteria, viruses, protozoa
TLR-10UnknownUnknown
TLR-11Profilin-like moleculeProtozoa
Table 2. TLRs and endogenous activators*
 LigandSource
  • *

    TLR = Toll-like receptor; HMGB-1 = high mobility group box 1; mRNA = messenger RNA; LDL = low-density lipoprotein; ED-A = extra domain A; ssRNA = single-stranded RNA.

TLR-1β-defensin 3Released from activated/necrotic cells
TLR-2Hsp60, 70, gp 96, HMGB-1, HMGB-1 nucleosome complexes, β-defensin 3, surfactant proteins A and D, eosinophil-derived neurotoxin, antiphospholipid antibodies, serum amyloid AReleased from activated/necrotic cells
Biglycan, versicanInduced upon tissue damage
Hyaluronic acid fragmentsDegradation of tissue
TLR-3mRNAReleased from activated/necrotic cells
TLR-4HMGB-1, surfactant proteins A and D, β-defensin 2, Hsp60, 70, 72, 22, gp 96, S100A8, S100A9, neutrophil elastase, antiphospholipid antibodies, lactoferrin, serum amyloid A, oxidized LDL, saturated fatty acidsReleased from activated/necrotic cells
Biglycan, fibronectin ED-A, fibrinogen, tenascin-CInduced upon tissue damage
Heparin sulfate fragments, hyaluronic acid fragmentsDegradation of tissue
TLR-5UnknownUnknown
TLR-6UnknownUnknown
TLR-7/-8Antiphospholipid antibodies, ssRNAReleased from activated/necrotic cells
TLR-9IgG-chromatin complexes, mitochondrial DNAReleased from activated/necrotic cells
TLR-10UnknownUnknown
TLR-11UnknownUnknown

TLR activation.

In response to ligand recognition, each TLR induces distinct signaling pathways that stimulate the synthesis of proinflammatory mediators. Structural studies are beginning to reveal precisely how specific binding of PAMPs to the TLR ectodomain, or associated coreceptors, is mediated (23). Moreover, events downstream of TLR activation are becoming increasingly well characterized. Upon ligand binding, TLR dimerization allows the recruitment of intracellular adaptor proteins to the cytoplasmic Toll/interleukin-1 receptor domain. This in turn induces signaling pathways that culminate in the expression of a wide variety of proinflammatory genes, including cytokines, interferon, reactive oxygen species, and proteases (24) (Figure 1). It is also becoming clear that PAMPs and DAMPs activate the same TLR in a very different manner in order to stimulate different signaling pathways. This specificity enables the induction of distinct subsets of inflammatory genes designed to ensure an appropriate response to each insult; for example, viral infection, bacterial invasion, or sterile tissue injury (22).

Figure 1.

Toll-like receptor (TLR) signaling pathways. TLRs are activated by exogenous and endogenous ligands, either at the cell membrane or within endosomal compartments. Upon activation, TLR dimerization induces 2 major downstream signaling pathways: the NF-κB and the interferon regulatory factor (IRF) pathway. Signaling adaptors couple TLRs to downstream signaling and gene transcription. Myeloid differentiation factor 88 (MyD88)–dependent signaling relies on recruitment of MyD88 adaptor–like protein (MAL), which leads to the recruitment of the interleukin-1 (IL-1) receptor–associated kinase (IRAK). Phosphorylation of IRAK signals to tumor necrosis factor (TNF) receptor–associated factor 6 (TRAF6) and subsequent nuclear translocation of NF-κB and translation of inflammatory cytokines is driven by phosphorylation of the IκB kinase (IKK) complex upon activation of TRAF6. MyD88-independent signaling is via TRAM and TRIF, and can activate both NF-κB and IRF, inducing interferon (IFN) synthesis. ECM = extracellular matrix; LPS = lipopolysaccharide; TBK1 = TANK-binding kinase 1; dsRNA = double-stranded RNA; ssRNA = single-stranded RNA; MCP-1 = monocyte chemotactic protein 1; MMP-1 = matrix metalloproteinase 1; pDCs = plasmacytoid dendritic cells.

Aberrant TLR activity.

The identification of the TLR family uncovered the molecular mechanisms by which inflammation is initiated during infection and injury. However, with the discovery of the importance of TLRs in host defense also came the realization that aberrant activation of TLRs drives pathologic inflammation (25–28).

TLR signaling driving cytokine production in RA and CVD

An increasing body of evidence demonstrates that TLR activation plays a key role in the progression of both RA and atherosclerosis. Here we discuss how studies with both diseased human tissue and in vivo animal models have led to the implication of TLRs in each of these diseases.

TLRs in the rheumatoid joint.

Expression of TLRs in RA.

Both TLR-2 and TLR-4 protein is expressed at high levels in the synovial lining and sublining layer of RA synovium compared to low levels observed in synovial tissue from patients with osteoarthritis and healthy donors (29). Stimulation of a mixed population of cells isolated from RA synovial membranes with TLR-2 ligands, including lipoteichoic acid, and the TLR-4 ligand LPS induced increased tumor necrosis factor (TNF), interleukin-6 (IL-6), and IL-8 above that spontaneously produced (30), demonstrating that both TLRs are functional in this ex vivo model of RA. Inhibition of myeloid differentiation factor 88 (MyD88) or MyD88 adaptor–like protein (Mal), intracellular adaptor proteins used only by TLR-2 and -4, inhibited spontaneous TNF, IL-6, and vascular endothelial growth factor release and synthesis of matrix metalloproteinase 1 (MMP-1), -2, -3, and -13 in RA membrane cultures (31), indicating that these TLRs contribute to proinflammatory cytokine synthesis in human RA. Moreover, highly purified LPS from the gram-negative bacterium Bartonella quintana, a potent antagonist of TLR-4, inhibited cytokine synthesis induced by LPS derived from Escherichia coli in human monocytes (32). This TLR-4 antagonist also blocked spontaneous IL-1 synthesis in ex vivo RA synovial tissue (33). These data confirm the importance of TLR-4 in contributing to cytokine synthesis in human RA tissue.

Expression of the endosomal TLRs, TLR-3, -7, -8, and -9, has also been demonstrated at both the messenger RNA (mRNA) and protein levels in RA synovium (34–36). Stimulation of RA synovial fibroblasts with the TLR-3 ligand poly(I-C) promoted synthesis of high levels of interferon-γ, CXCL10, CCL5, and IL-6 (35), suggesting that functional TLR-3 is expressed in RA. However, stimulation of TLR-3 and -8, but not TLR-7 or -9, induced TNF synthesis in a mixed population of cells isolated from RA synovial membranes (34). The selective serotonin reuptake inhibitors fluoxetine and citalopram, which also inhibit signaling by TLR-3, -7, -8, and -9, suppressed inflammatory cytokine production in human RA tissue (37). Moreover, imiquimod, an inhibitor of TLR-8 function, reduced spontaneous TNF synthesis in RA membrane cultures (34). These data indicate that TLR-8 contributes to driving inflammatory cytokine production in RA.

A role for TLRs in experimental arthritis.

TLR ligands such as bacterial DNA (38), streptococcal cell wall preparations (39), and LPS (40) can induce joint inflammation upon intraarticular injection in mice or rats. Injection of LPS together with type II collagen also promotes a more chronic, progressive arthritis than collagen alone (40). Mice with targeted deletions in TLR-2 exhibit reduced inflammation upon streptococcal cell wall injection (41), and mice bearing a loss of function mutation of the TLR-4 gene (Tlr-4lps-d) exhibited only mild disease in collagen antibody–induced arthritis (42). More compelling evidence derives from studies using serum transfer-induced arthritis, a model where disease induction is not dependent on TLR activation. Here, joint inflammation was not sustained in TLR-4 null mice (43). Similarly, deletion of TLR-4 in IL-1 receptor antagonist null mice, which develop spontaneous arthritis, conferred protection against severe arthritis. Conversely, in this model, TLR-2 deletion promoted disease severity, whereas deletion of TLR-9 had no effect on disease progression (44). TLR-4, but not TLR-2, null mice were also protected from IL-1–induced joint tissue damage (45). The TLR-4 antagonist, LPS derived from B quintana, can reduce joint destruction in mice when given intraperitoneally before the onset of collagen-induced arthritis. This LPS had no effect on serum levels of collagen antibodies, suggesting that disease induction was unaffected. It was also efficacious in treating spontaneous arthritis observed in IL-1 receptor antagonist knockout mice (33). Together, these data implicate TLR-4 in driving joint inflammation in vivo and provide evidence that interfering with TLR-4 activation may have therapeutic potential.

TLRs in CVD.

Expression of TLRs in the vasculature and atherosclerotic plaques.

TLRs are differentially expressed by vessels within normal human vasculature. For example, TLR-2 and -4 are ubiquitously present, whereas TLR-3 is detected specifically in the aorta and TLR-8 in the temporal and iliac arteries. More marked expression of TLRs is observed in arteries than in veins; the latter are almost devoid of any TLR expression (46). CD11c+ myeloid dendritic cells at the intima–neointima junction are reported to be the only cells clearly stained for TLRs in healthy human vessels (46). However, primary endothelial and smooth muscle cells respond to TLR ligands (47), and cultured human vascular smooth muscle cells (SMCs) constitutively express TLR-1, -3, -4, and -6 at the mRNA level (48–51), of which TLR-3 and -4 have been shown to be functional (49–53).

Human atherosclerotic intima, compared to normal intima, highly expresses TLR-1, -2, and -4 (54). These TLRs are expressed by endothelial cells and CD68+ macrophages (54, 55), and to a lesser extent by T lymphocytes (54), SMCs (54, 56), and adventitial fibroblasts (57). Patients with arterial disease also have higher expression of TLR-4 and -2 on circulating monocytes compared to healthy individuals (58–61). However, this does not always correlate with enhanced TLR signaling, which seems to be restricted to patients with acute coronary syndromes (62–64).

TLRs play a pivotal role in the activation of human atherosclerotic lesions. Colocalization of TLR-2 and -4 expression with the expression of NF-κB family member p65 in endothelial cells and macrophages is observed in human lesions (54). We have recently shown that TLR-2 and MyD88 play a predominant role in NF-κB–mediated production of inflammatory mediators CCL2/monocyte chemotactic protein 1, IL-6, IL-8, and matrix degrading enzymes MMP-1, -2, -3, and -9 in human carotid endarterectomies (65). Conversely, signaling though TLR-4 and the downstream TLR-4 signaling adaptor TRAM was not rate limiting for cytokine production in human atherosclerotic plaques, but had a selective role in MMP-1 and -3 production. This provides evidence for a dual role of TLRs in plaque inflammation as well as matrix degradation and plaque vulnerability to rupture (65).

A role for TLRs in experimental CVD.

Atherosclerosis development in murine models has largely been shown to be associated with increased expression of TLR-2 and -4. Expression of TLR-4 appears to be confined to tissue macrophages (55), whereas TLR-2 is selectively expressed on endothelial cells in atheroprone regions exposed to nonlaminar flow (66). Increased surface expression of TLR-2 and -4 is also found on circulating monocytes of apoE−/− mice with advanced atherosclerotic disease (67). Aortic lesion development is inhibited in apoE−/− animals devoid of MyD88 (68, 69), TLR-4 (69), and TLR-2 (69–72), with reduced deposition of aortic lipids and macrophage infiltration. Atherosclerotic lesions in TLR-2–competent apoE+/− also exhibit a greater macrophage to SMC ratio, and increased apoptosis compared to their TLR-2–deficient counterparts (71). C3H/HeJ mice with a missense mutation in TLR-4 that causes loss of function are also resistant to atherosclerosis (73, 74). However, in the LPS-hyporesponsive strain C57BL/10ScN, no difference in the extent of atherosclerosis was observed (75). Mice lacking TLR-4 or -2 also develop smaller neointimal lesions after vascular injury (57, 76).

Stimulation of murine macrophages with TLR-2, -4, and -9 ligands promotes foam cell formation and lipid uptake (77–80). This may be mediated via a number of different mechanisms, including TLR-4–dependent fluid phase uptake of lipids (81) or TLR-2– and TLR-4–dependent engagement of scavenger receptors associated with lipid uptake and fatty acid binding, for example, scavenger receptor class A, macrophage receptor with collagenous structure, and 1-lipoxygenase (79, 82–85). Formation of lipid droplets and cholesterol efflux are also influenced by TLR signaling: agonists of TLR-2, -3, -4, and -7 increase macrophage cholesterol ester storage (86), and activation of TLR-3 and -4 reduces cholesterol efflux via activation of interferon regulatory factor 3 (87).

A convergence of molecular pathogenesis in RA and CVD.

These data highlight a role for specific TLRs in driving inflammation in CVD and RA. Both diseases involve common TLRs; TLR-2 and -4 appear to be important in the activation of signaling pathways that induce high levels of cell infiltration and mediate cytokine and protease synthesis in CVD and RA, in addition to enhancing lipid uptake in CVD. In contrast, while the endosomal TLRs also play a role in RA, it is not yet known if they exhibit parallel importance in CVD. One key area requiring further investigation arising from these studies focuses on the relevance of multiple TLRs in disease pathogenesis. Some studies examining this interplay are emerging, for example, TLR-2 and -4 may exert a synergistic effect on aortic lesion development in a rabbit hypercholesterolemia model (88).

TLRs and metabolic syndrome.

TLR signaling is also affected in metabolic disorder. Targeted mutations in TLR-4 provide protection from obesity-associated insulin resistance in rodents (89, 90). Adipocytes have been shown to have significantly up-regulated TLR-4 expression, thus actively contributing to the cardiovascular and metabolic complications of obesity (91). Mice lacking in the expression of CD14, a coreceptor for TLR-2 and -4, have significantly decreased lipid and macrophage content in hepatic and adipose tissues, suggesting a pivotal role of CD14-mediated signaling independent of TLR-2 and -4 gene expression (92). Mice deficient in TLR-5, which is mostly expressed on the gut mucosa, have altered gut microbiota that reflected several hallmarks of metabolic syndrome (93). Recent data have also demonstrated an unexpected role of MyD88, showing increased circulating levels of insulin, leptin, and cholesterol, as well as liver dysfunction, thus suggesting a higher risk of diabetes mellitus in mice lacking the receptor (94). However, the molecular mechanisms underlying metabolic syndrome are poorly understood and require further investigation. TLRs are perhaps becoming the best-defined targets in mediating cardiovascular and metabolic complications; however, the complexity of the molecular interactions cannot be underestimated and other factors are likely to actively contribute to the pathophysiology of these conditions.

Endogenous ligands driving sterile inflammation in RA and CVD

A key question arising from studies that implicate TLR activation in RA and CVD is: which factors activate these PRRs during disease? Accumulating evidence shows that endogenous molecules, or DAMPs, which can stimulate the immune system in order to mediate an inflammatory response even in the absence of infection, play a key role in both RA and CVD.

In atherosclerosis, minimally modified low-density lipoproteins (LDLs) activate the CD14/MD-2 TLR-4 receptor complex and signal both via MyD88-dependent and -independent pathways (95, 96). Extensively oxidized LDL can also initiate inflammatory responses through a receptor complex comprising TLR-4 and -6 and CD36 (97). Apolipoprotein C-III, a component of very LDL, is also recognized by TLR-2 and induces proinflammatory signals in monocytes (98). The ability of other lipid components of lipoprotein particles, such as saturated fatty acids, to directly induce TLR signaling has recently been questioned (99). However, the acute-phase protein serum amyloid A, which is deposited in murine atherosclerosis where levels significantly correlate with lesion size (100), induces TLR-1/-2 signaling to induce IL-12, IL-23, TNF, and IL-10 synthesis (101).

One key class of DAMPs common to both RA and CVD are ECM molecules that are specifically induced during tissue injury. Among these matrix components, fibrinogen, which activates TLR-4, is abundantly localized in human atherosclerotic plaques (102) and in the RA synovium (103). High levels of the alternatively spliced fibronectin isoform, which contains the type III repeat extra domain A, are also observed in the RA synovium (104) and in atherosclerotic plaques (105). Fibronectin extra domain A activates TLR-4 in an MD-2–dependent manner and can induce transient ankle joint inflammation upon intraarticular injection into mice (106). Tenascin-C (TN-C) is another ECM glycoprotein with an expression pattern restricted to sites of tissue injury (107). We recently showed that TN-C levels in the RA synovium correlate with disease activity and demonstrated that both immune myeloid cells and synovial fibroblasts act as the source of this DAMP in RA (108). We also showed that the fibrinogen-like globe of this molecule is able to induce TLR-4– and MyD88-dependent signaling and chronicity in a murine model of inflammatory arthritis (109). TN-C expression is also increased at sites of arterial injury. Interestingly, expression of TN-C was found to correlate with the inflammatory burden, but not plaque size, in humans. TN-C may also modulate gene expression of metalloproteinases in macrophages, thus stimulating rupture of the fibrous cap in atherosclerotic plaques (110).

Extracellular proteoglycans are also prime suspects for driving inflammation in RA and CVD. Biglycan, which activates TLR-2 and -4 MyD88-dependent signaling (111), is found at high levels in the RA synovium (112, 113), and both versican, which induces cytokines, including TNFα and IL-6, following stimulation of TLR-2 and -6 (78), and biglycan are up-regulated in human coronary atherosclerosis. Overexpression of biglycan in CVD leads to increased high-affinity lipoprotein binding and subsequent retention in the ECM (114). Increased versican accumulation by macrophages is also associated with 4.5-fold increase in lesion size in murine atherosclerotic lesions (115). Lesion development and instability have been associated with other proteoglycans. Heparan sulfate increases binding of LDL to the endothelial matrix and increases monocyte binding, and depletion of the heparan sulfate proteoglycan perlecan is associated with reduced atherosclerosis (116). Targeting proteoglycans could potentially impede both lipoprotein entrapment and inflammation (117).

Nonsulfated glycosaminoglycans, specifically hyaluronan, are also produced in the vasculature both by smooth muscle cells and endothelial cells. High levels of hyaluronan oligosaccharides are also observed in the RA joint (118). Hyaluronan signaling via MyD88 promotes chemokine production and inflammation (119–121). Hyaluronan in particular is responsible for the matrix alterations in the arterial wall in diabetes mellitus, as part of the diabetic macroangiopathy. Overproduction of hyaluronan in apolipoprotein E–deficient mice led to transformed aortic strength and stiffness, triggering atherosclerosis (122).

Release of endogenous alarmins may also drive inflammation in RA and CVD. The best-studied alarmin is high mobility group box 1 (HMGB-1), a ubiquitous nonhistone DNA-binding protein that stabilizes the nucleosomal structure and facilitates gene transcription. Active and passive release of HMGB-1 is seen in RA (123), and intraarticular injection of HMBG-1 in mice caused synovitis (124). HMGB-1 is also abundantly secreted in atherosclerotic plaques by SMCs upon exposure to cholesterol. Moreover, extracellular HMGB-1 is a mitogenic and chemotactic factor for human SMCs, and in turn induces HMGB-1 expression and secretion in a positive feedback loop (125). HMGB-1 promotes the migration of rat arterial SMCs and foam cells (126). Signaling by HMGB-1 is not only mediated both via TLR-2 and -4, but also depends on activation of receptor for advanced glycation end products (RAGE). RAGE signaling in endothelial cells induces adhesion molecule expression, while RAGE genetic deletion reduces atherosclerosis development in murine models of hypercholesterolemia (127).

DAMPs: the missing link between RA and CVD?.

Created by the immensely destructive environment of the inflamed arterial wall and synovium, a concentrated reservoir of DAMPs is likely to contribute to disease by perpetuating a chronic cycle of inflammation. Here, tissue injury creates more mediators of tissue injury, thus prolonging local inflammation in the joint and vasculature (Figure 2). As such, both RA and CVD constitute sterile inflammatory diseases (128).

Figure 2.

Endogenous activation of Toll-like receptors (TLRs) drives a perpetual cycle of inflammation in rheumatoid arthritis (RA) and cardiovascular disease (CVD). Activation of TLRs either by exogenous (pathogen-associated molecular patterns [PAMPs]) or endogenous (damage-associated molecular patterns [DAMPs]) ligands induces proinflammatory signaling pathways within the arterial wall and the joint synovium. The resultant cell death and increased synthesis of cytokines and tissue-degrading enzymes creates high local levels of DAMPs, which then feedback to activate further TLR-mediated inflammation. This promotes a nonresolving loop of tissue destruction and inflammation that eventually causes loss of joint or vascular function. Furthermore, high systemic levels of DAMPs in RA patients may mediate arterial injury, inducing the more rapid or more frequent onset of CVD in these individuals.

This vicious cycle may also contribute to the increased incidence of CVD events in patients with inflammatory arthritis. In patients with RA, DAMPs circulate at high levels in the peripheral blood, and this raises the possibility of the induction of endothelial activation at sites susceptible to atherosclerosis. For example, expression of TLR-2 on the endothelium appears selectively on the atheroprone region of the lower aortic curvature almost immediately after high-fat diet, even before lesion formation in murine models of atherosclerosis (66). It is possible to envisage that the increased bioavailability of DAMPs might accelerate lesion formation at sites of endothelial activation and TLR expression (Figure 2). There are a number of implications of this hypothesis. First, DAMPS may act systemically to mediate CVD disease and may be more potent and far reaching than their more short-lived downstream mediators, including cytokines (129). Second, systemic levels of DAMPs may prove to be valuable biomarkers for CVD. Future studies should determine whether the systemic DAMP burden is indicative of the linear relationship between the number of swollen joints and extraarticular manifestations of RA and CVD incidence (9, 130). Further investigation is also warranted to determine precisely how the link between DAMPs, TLRs, and inflammatory disease is mediated, and rigorous clinical studies are required to determine if the degree of tissue damage can be classified as a risk factor for CVD. Finally, this hypothesis may inform future strategies to treat RA and CVD. Reducing tissue destruction could not only reduce joint inflammation but also maintain vascular integrity in these patients.

Implications for new therapeutic strategies for RA and CVD

In light of the evidence implicating common proinflammatory signaling pathways in RA and CVD, it follows that treatments that are beneficial for RA may also be useful in treating CVD. In addition, given the causative link between RA and CVD, improved treatment of RA should also lower the burden of CVD. There are now clinical data that support both of these views.

Effective treatment of RA has been shown to reduce symptoms of CVD. Anti-TNF therapy, widely used to treat RA, also improves arterial function (131), reduces subclinical atherosclerosis (132), and lowers the incidence of a first cardiovascular event (133). Anti-TNF responders also show a reduced incidence of MI compared to nonresponders (134). Similar results have been observed in a study of RA with methotrexate treatment (135). As such, better treatments for RA would by association improve mortality and morbidity caused by associated CVD.

Likewise, drugs traditionally used to treat CVD have also shown efficacy in treating RA. There is evidence to suggest that the reduction of cardiovascular comorbidity and mortality by statins can be attributed not only to their effects on lipid biosynthesis, but also to antiinflammatory properties (136–138). Indeed, trials with atorvastatin or simvastatin significantly reduced disease activity scores and C-reactive protein levels in patients with RA (139, 140). Conversely, a clinical trial to assess the outcome of simply reducing inflammation as an effective means of treating CVD (the Cardiovascular Inflammation Reduction Trial) is also underway using low-dose methotrexate (141).

Moreover, the emergence of TLRs in driving the pathogenesis of RA and CVD makes these PRRs and their associated ligands or signaling pathways very tractable targets for reducing inflammation in both conditions. Much effort has been directed toward therapeutic inhibition of the activation of TLRs to treat RA (28, 142). A number of strategies have been investigated, including inhibition of signaling pathways downstream of TLR stimulation and blockade of individual TLR function using natural TLR antagonists. In an exploratory, phase II, randomized, double-blind, multicenter RA trial, the TLR-4 antagonist chaperonin 10 improved symptoms of RA patients (143). Inhibition of the endosomal TLRs with short oligodeoxyribonucleotides, immunoregulatory sequences, have also proved effective in the prophylactic suppression of disease severity in mouse models of RA (144, 145). However, one potential drawback of global blockade of TLR function is that this approach may offer no improvement over the safety of currently approved treatments. Given their key role in physiologic inflammation during host defense, blanket suppression of TLR signaling may confer increased risk of opportunistic infection.

One way to overcome this hurdle is to block the activation of TLR by disease-specific stimuli. For example, blockade of HMGB-1 with polyclonal antibodies or with the DNA-binding box domain inhibited inflammation and reduced cartilage destruction in collagen-induced arthritis in mice (146, 147). However, monoclonal antibodies have thus far shown limited effect (148). Other DAMP antagonists have demonstrated efficacy in experimental arthritis, including the SNX-7081 inhibitor of HSP90 (149) and the neutrophil elastase inhibitor ONO-5046 (150). Specifically targeting endogenous TLR activators relevant to RA offers several advantages. Intervention upstream of the source of inflammatory mediators moves us even closer to the cause of the disease, and this approach would also avoid global immune suppression.

Translational studies directed at blockade of TLR action in CVD are less well developed. Our study demonstrates that TLR-2 blockade is efficacious in reversing cytokine, chemokine, and MMP production in human atherosclerosis, and that TLR-4 blockade can specifically limit MMP-1 and -3 production (65). Interestingly, a TLR-2 antagonistic antibody has also been shown to reduce infarct size and protect heart function in a murine model of myocardial ischemia/reperfusion injury (151). Furthermore, a link between other autoimmune diseases such as systemic lupus erythematosus and CVD has been established. Whether the pathogenesis of CVD in this case parallels CVD in RA should be investigated.

Discussion

TLR-mediated inflammation contributes to a variety of classic and nonclassic immune conditions, including RA and CVD. Investigating which TLRs contribute to each disease, how each TLR is activated during disease, and the resultant signaling pathways induced will further provide valuable insight into disease pathogenesis and reveal potential new therapeutic targets for reducing aberrant inflammation.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be submitted for publication. Drs. Monaco and Midwood had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Monaco, Midwood.

Acquisition of data. Monaco, Terrando, Midwood.

Analysis and interpretation of data. Monaco, Terrando, Midwood.

Ancillary