Role of IL-18 and Its Signaling in Atherosclerosis

REVIEW

Role of IL-18 and its signaling in atherosclerosis

Owais Mohammad Bhat, Veena Dhawan

Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh

Correspondence: Veena Dhawan E-mail: [email protected] Received: March 11, 2015 Published online: April 22, 2015

Cardiovascular diseases (CVD) including coronary artery disease (CAD) and stroke are the largest cause of worldwide morbidity and mortality, where atherosclerosis is the underlying pathology. Recent investigations of atherosclerosis have focused on the role of inflammation, providing new insights into the mechanism of the disease. Macrophages and T-lymphocytes present in the atherosclerotic lesions produce a wide array of cytokines that can exert both pro- and anti-inflammatory effects. Pro-inflammatory cytokines of the interleukin category are considered to be key players in the chronic vascular inflammation that is typical for atherosclerosis. Various studies support the concept that interleukin-18 (IL-18) is a pro-inflammatory cytokine with pro-atherogenic properties. Previous data in Apo E-/- mice demonstrated that IL-18 accelerates atherosclerosis via interferon gamma (IFN- γ) and CXCL16 expression. IL-18 binds to its receptor IL-18R complex which is a heterodimer with α (IL-1Rrp) chain responsible for extracellular binding of IL-18 and a nonbinding, signal-transducing β (AcPL) chain. By binding to IL-18Rα, IL-18 upregulates IL-1R-associated kinase (IRAK) and TRAF-6 thus, results in nuclear translocation of nuclear factor kappa-B (NF-κB). Activated NF-κB is shown to be present in coronary arteries of pigs fed a hypercholesterolemic diet, in rat arterial smooth muscle cells after balloon injury and in unstable coronary atherectomies. Peroxisome proliferator-activated receptor-γ (PPAR-γ) and Liver-X-receptor-α (LXR-α) genes are involved in the lipid uptake and cholesterol efflux in macrophages and regulate the expression of many key genes that are involved in the development and progression of atherosclerosis e.g. cytokines, matrix metalloproteinases (MMPs) and scavenger receptors (CD36, SR-A, SR-B1). MMP-9/MMP-2 (members of gelatinase family) are shown to be one of the effector genes of these nuclear receptors (PPAR-γ and LXR-α). MMP-9 is specifically implicated in atherosclerosis, plaque instability and rupture during arterial lesion progression. Thus IL-18 can be strongly viewed as a proatherogenic and pro-inflammatory cytokine, as IL-18 signaling results in upregulation of various pro-inflammatory genes and development of atherosclerotic lesions, thus could be envisaged as a target for therapeutics. Keywords: Atherosclerosis; inflammation; Recombinant IL-18;Apo E -/-; CD36; NF-κB To cite this article: Owais Mohammad Bhat, et al. Role of IL-18 and its signaling in atherosclerosis. Inflamm Cell Signal 2015; 2: e707. doi: 10.14800/ics.707.

Cardiovascular diseases (CVD) such as coronary artery Atherosclerosis is a complex multifactorial arterial disease disease (CAD) and stroke are the leading cause of death in of medium and large-size arteries. Several factors appear the developed world and are likely to attain this status likely to have contributed to the acceleration of the CAD worldwide that accounts for 16.7 million deaths each year epidemic in modern times, which are convergence of both [1,2]. The most common forms of CVD are coronary artery conventional and non-conventional risk factors [3]. disease (CAD) and cerebrovascular disease where atherosclerosis is the underlying pathology.

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One of the original concepts for the pathogenesis of chemokines, cytokines, and cell adhesion molecules [15]. atherosclerosis was proposed by Virchow (1856), who suggested that inflammation plays a primary role in initiating Interleukin-18 and cardiovascular disease the atherogenic process [4]. Any kind of injury to the vascular wall results in endothelial injury thereby, leading to In two large studies carried out in 1111 randomly (Perth, endothelial dysfunction (Russell Ross, 1973) [5]. Due to Australia) selected community subjects and 2231 subjects endothelial dysfunction and presence of oxidative stress, (Dallas County, USA), elevated levels of IL-18 were found excess lipids and lipoproteins are oxidized resulting in the to be associated with carotid intima-media thickness in formation of oxidized low density lipoprotein (Ox-LDL). univariate analyses, but not after adjustment for traditional risk factors [16, 17]. Two large prospective studies, which Atherosclerotic plaques are characterized by accumulation included 10,600 healthy European men [18] and 253 healthy of lipids in the arterial wall together with the infiltration of women [19] demonstrated that elevated IL-18 levels were immunocytes. Monocytic recruitment, egress of macrophage, associated with future cardiovascular disease. and the balance among proliferation, survival and apoptosis in the arterial walls determines the degree of influx of Interleukin-18 and atherosclerosis inflammatory cells to atherosclerotic lesions [6].The up-regulation of scavenger receptors in plaque-activated IL-18 is shown to be highly expressed in unstable macrophages results in the uptake of modified lipoprotein atherosclerotic lesions, mainly in the plaque macrophages particles (mLp), and transforms them into cholesterol-laden [20]. IL-18 exerts its pro-atherogenic effects through IFN-γ foam cells, characteristic of fatty-streak type lesion, that production that amplifies the inflammatory process leading progressively may evolve to advanced fibro-lipid plaque and to thinning of the fibrous cap formation causing advanced lesions [7] . Therefore, understanding the principle rupture-prone plaques [21, 22]. Further, IL-18 is shown to of the inflammatory processes is crucial for deciphering the indirectly cause plaque destabilization by increasing the complex mechanism involved in atherosclerosis progression. expression of matrix metalloproteinases in macrophages and vascular cells [14, 21]. IL-18 overexpression is shown to Cytokines and atherosclerosis enhance the collagenolytic activity of smooth muscle cells resulting in reduced intimal collagen and thickness of the Atherosclerosis is not only a disorder of lipid metabolism, fibrous cap, leading to vulnerable plaques in an Apo E-/- but is also considered as a chronic inflammatory disease [8]. mice [23]. Increased mRNA expression of cardiac IL-18 and a Presence of inflammatory cells, pro- and anti-inflammatory subsequent reduction in myocardial contractility was cytokines and their receptors in the atheromatous plaque reported in a mouse model with myocardial infarction [24]. demonstrates a positive correlation with coronary artery We also observed significantly augmented IL-18 gene disease and its consequences. A critical balance between expression and circulating levels in the IL-18-treated Apo pro-inflammatory and anti-inflammatory cytokines in E-/- mice [25]. In an earlier study, IL-18 administration atherosclerotic plaque is essential for the development and increased serum cholesterol and lipoprotein-cholesterol progression of the lesion. The pro-inflammatory cytokines distribution in these mice. secreted by type 1 CD4+ T-helper cells (TH1 cells) include interleukin-2 (IL-2), IL-12, IFN-γ, TNF-α and TNF-β that Chronic systemic inflammation through IL-18 may give exacerbate the atherosclerotic process, whereas TH2 rise to dysfunctional or pro-inflammatory HDL and therefore cytokines e.g. IL-4, IL-5, IL-10 and IL-13 are known to exert may cause reverse cholesterol transport dysregulation leading atheroprotective actions and can counteract TH1 cytokine to increased cholesterol levels. Increase in cholesterol levels activity [9-11]. Animal studies in mouse have revealed the could also be attributed to isoprenoid generation by pivotal role of interleukins, the macrophage-associated endogenous cellular cholesterol synthesis as well as by cytokines and the colony stimulating factors in atherogenesis. cholesterol synthesis in activated monocytes during the inflammatory response. Isoprenoids are an integral Interleukin-18 component of the signaling pathway for IL-6-mediated inflammation and there is plenty of evidence in the literature IL-18, a potent pro-inflammatory cytokine with pleotropic demonstrating that IL-18 induces IL-6 production both in properties is a member of the IL-1 family of cytokines and vitro and in vivo [26, 27]. Increased IL-6 production may was originally described as an IFN- γ inducing factor [12]. induce C-reactive protein (CRP) that may also trigger Produced constitutively in many different cell types [13, 14]. cholesterol levels and in this regard statin like drugs are Evidence shows that IL-18 is involved in T cell and natural shown to lower LDL-C and non-HDL-C and thereby lower killer cell maturation, and production of other inflammatory levels of CRP.

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IL-18 Receptor and atherosclerosis acute complications of atherosclerosis, which includes acute coronary syndrome. Nuclear NF-κB binding activity has Upon binding of IL-18 to its receptor IL-18Rα, IL-18Rα is been found in peripheral blood mononuclear cells and recruited to form a high-affinity complex-inducing signaling myocardial biopsies of patients with unstable angina [33, 34]. pathway shared with other IL-1R family members. Gerdes et Nuclear translocation of relA is reported to be higher in al [14], reported increased expression of functional IL-18 and unstable coronary atherectomies, and is also present instable IL-18 receptor on endothelial cells, SMCs and macrophages angina patients [35]. The majority of our knowledge is present in the human-atheroma, and induced IFN-γ gathered from studies using animal models of atherosclerosis expression in SMCs, thereby suggesting simmering despite evidence in human atherosclerosis. Activated NF-κB inflammation during atherogenesis. The induction of was detected in coronary arteries of pigs fed a IL-18Rα/β by IFN-γ in association with the IL-18-induced hypercholesterolemic diet and in arterial SMCs after balloon IFN-γ expression in the vasculature via IL-18 signaling injury in a rat model [36, 37]. In our study, we also suggests the presence of a positive feedback loop that might demonstrated a significant augmentation of NF-κB in contribute to the dysregulated inflammatory response in IL-18-treated mice [25]. Further, a significant downregulation atheroma [14]. of IL-18-induced molecules like IL-18 Rα, CD36, MMP-9, NF-κB and upregulation of LXR-α was observed with PDTC In our study, a significantly augmented IL-18 Rα demonstrating that IL-18 acts through NF-κB pathway. expression was observed on peripheral blood mononuclear Significant neutralization of IL-18-induced signaling by cells and circulatory levels of IL-18 in IL-18-treated Apo E-/- PDTC as observed via blockage of IL-18 as well as NF-κB mice as compared to the controls [25]. Our study also activation was found to be responsible for these observations. demonstrated that IL-18 induced IL-18 Rα expression via Also, PDTC displayed robust hypolipidemic effects in our NF-κB, as a significant downregulation IL-18 and IL-18 Rα study [25]. PDTC is shown to attenuate plasma TG and was observed using a specific NF-κB inhibitor i.e. VLDL-C and restore HDL-C levels in obese db/db mice via pyrrolidine dithiocarbamate (PDTC) [25, 28], Various in vitro reduction in TNF-α and IL-6 levels, thus reduces studies reported that IL-18 binding to IL-18 Rα results in inflammation [38]. Our findings are further supported by the upregulation of IL-1R-associated kinase (IRAK) and finding of Jawień et al [28], who observed that inhibition of TRAF-6 resulting in nuclear translocation of NF-κB and also NF-κB activity reduces atherosclerosis in Apo E/LDLR- activates p38 mitogen activated protein kinase (p38 MAPK) double knockout (KO) mice model. signaling [29- 31]. Bone marrow transfers from p50-deficient mice to Nuclear Factor-kappa B (NF-κB) and atherosclerosis LDL-R-deficient mice demonstrated an astonishing shift towards a different plaque phenotype, characterized by Activation of NF-κB signaling is initiated by extracellular reduced number of foam cells, increased macrophages and T stimuli which are recognized by the receptors and transmitted cells, and the appearance of B cells (traditionally not present into the cell, where the adaptor signaling proteins initiate a at the lesion site besides reduction in atherosclerotic lesion signaling cascade culminating in IκB kinase (IKK) size) [39]. These targeted gene deletion studies have activation. IKK phosphorylates the inhibitory IκB subunit of highlighted that NF-κB is involved in atherogenesis in a far the NF-κB·IκB complex in the cytoplasm that marks IκB for more complex manner than already predicted. In order to degradation by the proteasome and releases NF-κB from the identify suitable therapeutic targets, that may likely to inhibitory complex [32]. The freed NF-κB proteins are then interfere with the whole pathway, future research is therefore translocated into the nucleus and binds to the promoter needed to identify the key stimuli leading to the activation of regions of various genes thereby activates their transcription. NF-κB signaling.

Atherosclerosis and inflammatory diseases involve Cholesterol efflux regulators activation of NF-κB, which is now considered to be a major transcription factor and a master regulator regulating many Expression of genes involved in lipid uptake and functions of the vessel wall. Moreover, NF-κB activation is cholesterol efflux in macrophages is controlled by nuclear thought to lie downstream of many of the stimuli proposed to receptors i.e. peroxisome proliferator-activated receptors be involved in atherosclerosis, such as mLp, cytokines and (PPARs) like PPAR-α, PPAR-δ, PPAR-γ and liver x receptor infectious agents. alpha (LXR-α), which are not only implicated in the modulation of macrophage inflammatory gene expression in Activated NF-κB has been identified in situ in human vascular diseases, but are also involved in the cholesterol atherosclerotic plaques and seems to be more prominent in efflux via macrophages [40, 41].

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PPAR-γ is shown to induce the expression of LXR-α and cyclooxygenase (COX)-2, inducible nitric oxide synthase thereby stimulate ABCA1-dependent cholesterol efflux to (iNOS) and interleukin-6 (IL-6) whereas, in vivo, these apo A-I [42]. Allevaet al [43] reported that ligand-activated agonists decrease inflammation in a model of contact PPAR-γ complexes suppress murine macrophage production dermatitis and also inhibit inflammatory gene expression in of the proinﬂammatory cytokines by binding critical the aortas of atherosclerotic mice. This study concludes that transcription factors such as NF-κB, AP-1, STAT-1 and Ets genes inhibited by LXR are well known targets of NF-κB, as (E26 transformation-specific), suggesting that these ligands iNOS and COX-2 were inhibited through antagonism of use PPAR-γ-mediated suppressor pathway. A large number NF-κB [55]. of inﬂammatory responses are shown to be negatively regulated by PPAR-γ agonists [44]. These nuclear receptors In a previous study [25], we observed significant also regulate the expression of many genes involved in the downregulation of LXR-α expression in IL-18-treated Apo production of pro-inflammatory mediators e.g. cell adhesion E-/- mice. Studies carried out in the literature using LXR molecules, cytokines and scavenger receptors (CD36, SR-A, knockouts and LXR agonists suggest that LXR-α is an SR-B1) that control the native and modified LDL uptake that important regulator of cholesterol efflux in macrophages [51]. is an important step in the formation of foam cells [45]. The All these reports outlined above support our observations of pathogenic role of Ox-LDL in atherosclerosis is shown to be finding decreased LXR-α expression in IL-18-treated Apo largely dependent on CD36 expression. E-/- mice. Our observation that IL-18 attenuates LXR-α in a NF-κB dependent manner is the first report in the literature. Liver X receptor-α (LXR-α) Scavenger Receptor CD36 LXRs belong to the orphan nuclear receptor superfamily, first identified in the liver and hence their name [46]. LXR-α CD36, a transmembrane glycoprotein, is one of the most and LXR-β isoforms have already been well characterized. critical scavenger receptors on macrophages that cross the The latter is ubiquitously expressed [47], while the expression membrane twice. CD36 is expressed on multiple cells and of the former is more restricted in the skeletal muscle, has several physiologic functions that include its role as a adipose tissue, spleen, kidney, intestine, lung and high-affinity receptor for specific oxidized phospholipids macrophages [48]. Intracellular cholesterol levels stimulate the found within Ox-LDL [56]. An interesting aspect of CD36 production of LXR specific physiological ligands [45], such as biology is that its expression on macrophages is increased 24(S), 25-epoxycholesterol, 24(S)-hydroxycholesterol, and when the cells are exposed to Ox-LDL. Amongst the changes 22(R)-hydroxycholesterol as the most abundant and potent that occur in the lipid components of LDL, subsequent to oxysterols capable of activating LXRs in the cell [49]. Similar oxidation, is the formation of oxidized fatty acids such as 9- to PPARs, ligand-bound LXRs tend to form heterodimers and 13-hydroxy octadecadienoic acid (HODE). These with their obligate partner RXR, and the activated LXR-RXR oxidized fatty acids are ligands for the PPAR-γ which heterodimers are capable of binding to specific DNA binding regulates expression of many genes, including CD36. Hence, sites known as LXR response elements (LXREs) [46]. LXREs Ox-LDL induces CD36 expression and further cellular have been recognized in the promoter regions of several uptake of Ox-LDL. This feed-forward loop, most likely genes regulated by LXRs including ABCA1 [50], ABCG1 [51], accelerates foam cell formation in the arterial neointima. The PPAR-γ [52] and Apo E [53]. pathogenic role of Ox-LDL in atherosclerosis is largely dependent on CD36. Studies employing macrophages from LXR agonists increase reverse cholesterol transport (RCT) genetically altered mice lacking CD36 have demonstrated from macrophages by increasing the expression of that the absence of CD36 expression was associated with a macrophage Apo E and cholesterol efflux transporters lack of foam cell formation in vitro when cells were treated ABCA1 and ABCG1 [40]. Excessive accumulation of with Ox-LDL. In contrast, formation of foam cells was cholesterol within macrophages at the sites of atherosclerotic observed after 12 to 24 hours in wild-type mice [57]. lesions converts them into foam cells and accounts for the major fraction of the cholesterol deposited in the lesions [54]. Though, we did not determine Ox-LDL levels, a Thus, by stimulating reverse cholesterol transport, LXR significantly increased scavenger receptor (CD36) expression reduces foam cell formation and cholesterol content in the with IL-18 administration in our study could be due to raised lesion directly. LXRs and their agonists act as negative Ox-LDL that may lead to foam cell formation and thus regulators of macrophage inflammatory gene expression. An development and progression of atherosclerosis [25]. in vitro study demonstrated that in response to bacterial Inflammatory cytokines such as granulocyte macrophage infection or lipopolysaccharide (LPS), LXR agonists reduce colony–stimulating factor (GM-CSF) and macrophage the expression of inflammatory molecules such as, colony–stimulating factor (M-CSF) upregulates

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Inflammation & Cell Signaling2015; 2: e707.doi: 10.14800/ics.707; © 2015 by Owais Mohammad Bhat, et al. http://www.smartscitech.com/index.php/ics mRNAexpression of CD36 in macrophages, and blocking CD36 expression or its downstream signaling is shown to inhibit Ox-LDL uptake and limit experimental atherosclerosis in mice [58, 59].

Further, studies have shown that the proatherogenic role of CD36 was likely mediated by CD36 on macrophages, since bone marrow transplantation from CD36-null mice into Apo E-null mice, had the same effect on atherosclerosis as seen in the Apo E/CD36-double-null mice [60].

Matrix Metalloproteinases (MMPs)

Macrophage-derived MMPs are involved in the thinning of the fibrous cap [61]. MMPs are a family of protease-activated enzymes that degrade extracellular matrix (ECM) proteins. Presently, 26 members of the MMP family Figure 1. Present hypothetical model represents the molecular targets of IL-18 and NF-κB mediated signaling. have been identified in vertebrates out of which 23 have been found in humans [62, 63]. inhibition of NF-κB downregulates IL-18-induced Among MMPs, MMP-2 and MMP-9 have stimulated great pro-inflammatory genes. interests for their potential roles in plaque rupture [64]. Scientific evidence suggests that local overexpression of Clinical and experimental studies clearly demonstrate that MMP-9 promotes intravascular thrombus formation through MMPs are directly involved in the atherosclerotic plaque increased tissue factor expression and tissue factor-mediated destabilization and show that members of the MMP family activation of the coagulation cascade [70]. Studies using have widely differing effects on atherogenesis. A prominent various genetic manipulations in animal models have also role of MMP-2/9 has been demonstrated in atherosclerosis, been used to determine which MMPs are relevant in the plaque instability as well as rupture during arterial lesion progression of atherosclerosis. Galis et al [71] used MMP-9 progression [62]. Galis et al [64] demonstrated that knockout mouse carotid artery model to demonstrate that over-expression of activated MMPs can lead to the MMP-9 deficiency lead to a decrease intimal hyperplasia, promotion of destabilization of atherosclerotic plaques. lumen loss and also caused accumulation of interstitial Gough et al [65] demonstrated that retroviral overexpression collagen. Inhibition of MMP-9 increases the mechanical of an active form of MMP-9 in macrophages induces stability of arteries by increasing their collagen content and morphological appearances interpreted as plaque disruption. decreasing lumen loss. Therefore, the MMPs may represent MMP-9 is known to process a number of inflammatory as potential prognostic markers as well as therapeutic targets chemokines (CXCL5, CXCL8) and cytokines (TNF-α, for the prevention of atherosclerotic lesion development and IL-1β) through the proteolysis involved in vascular progression. remodeling and reorganization of ECM [66]. IL-18 is shown Our study clearly demonstrated that exogenous to induce MMPs in murine peritoneal macrophages and administration of a pro-inflammatory cytokine such as IL-18 human monocytes [14, 67]. Another mechanism of lipid significantly upregulates CD36 and MMP-9 expression via independent atherosclerotic plaque development due to IL-18 NF-κB pathway and suggested that a significant crosstalk could be due to increased MMP-9 expression in an earlier involved in the progression and destabilization of study [25]. Our study revealed that atherosclerotic plaques atherosclerotic plaques (Figure 1). were rich in inflammatory cells, smooth muscle cells and collagen, thus induction of MMP-9 via IL-18 may be a In conclusion, this review highlights IL-18 and NF-κB as crucial link in the chain of events promoting plaque rupture, important therapeutic targets for prevention of atherosclerotic thrombosis and myocardial infarction [25]. Bond et al [68] plaque development and its complications. Our findings open reported that NF-κB activity was required for upregulation up a new horizon for future research in human subjects to and production of MMP-9 in rabbit and human VSMCs. explore not only the molecular targets of IL-18 and Both MMP-2 and MMP-9 were found to be significantly NF-κB-mediated signaling, but also that these targets could decreased with PDTC treatment in spontaneously be specifically manipulated for therapeutic benefits. hypertensive rats [69]. These studies support our findings that

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