Cytokines in atherosclerosis: Key players in all stages of disease and promising therapeutic targets

1Cardiovascular disease (CVD) is responsible for most mortality worldwide and accounted for about 31.9% of all deaths in 2010 in the United States alone. The total direct and indirect costs from CVD in 2010 were estimated at $315.4 billion. Although morbidity and mortality from CVD has decreased in the last two decades, at least in the western world, it is expected that this will reverse in the future because of a global increase in diabetes and obesity along with an alarming rise in CVD in developing countries in part due to acquisition of a westernized lifestyle.

2Atherosclerosis, the underlying cause of myocardial infarction, cerebrovascular accident or peripheral vascular disease, is the major cause of deaths from CVD. Atherosclerosis is now recognized as an inflammatory disorder of medium and large arteries initiated by risk factors such as high plasma cholesterol levels and hypertension. Atherosclerosis develops during the lifespan of an individual and involves a number of steps: activation of the endothelium and recruitment of immune cells; monocyte differentiation and foam cell formation; development of fibrotic plaques due to death of foam cells and migration and proliferation of smooth muscle cells (SMCs); and plaque rupture and thrombosis (Fig. 1). Both the innate and adaptive immune response in atherosclerosis is orchestrated by a range of cytokines, which regulate all stages of the disease,,.

3Cytokines are a diverse group of low-molecular weight proteins with over 100 identified so far. Cytokines are clustered into several classes such as the interleukins (IL), chemokines, colony-stimulating factors (CSF), tumor necrosis factors (TNF), the interferons (IFN) and transforming growth factors (TGF),,. Many cytokines are expressed in atherosclerotic plaques and all cells involved in the disease are capable of producing cytokines and responding to them. They can be generally classified as pro- or anti-atherogenic though the roles of some are not as clear-cut and often context-dependent,,. This review will discuss the roles of key cytokines in different stages of atherosclerosis. Although in vitro studies using cell culture model systems have made a major contribution in advancing our understanding of the roles of cytokines in the cellular processes associated with atherosclerosis, the major focus of this review will be on the outcome from studies using animal model systems. In particular, two mouse models, the apolipoprotein (Apo) E-deficient mice and the low-density lipoprotein receptor (LDLr)-deficient mice,, have been particularly useful in advancing our understanding of the molecular basis of atherosclerosis and the roles of various cytokines in the disease,,. The use of bone marrow transplantation (BMT) approaches in such models also informs on whether a particular phenotype is driven by hematopoietic or non-hematopoietic cells,. These mice can spontaneously form atherosclerotic lesions on a standard chow diet but feeding of a high fat, western-type diet can markedly speed up the development of the disease,. It should be noted that caution needs to be exerted in the extrapolation of outcomes from such mouse models to humans because of many differences between the two species, including in lipoprotein metabolism and the inflammatory response,. In addition, existing mouse models are not particularly useful for investigating the steps involved in the clinical complications of the disease, plaque rupture. It is therefore important that wherever possible key findings are analyzed in the human context.

4Atherosclerotic plaques tend to form particularly at the inner curvatures and branch points of arteries that are often associated with disturbed blood flow, and is augmented by other factors such as high plasma low-density lipoprotein (LDL) concentration, hypertension and toxins from cigarette smoke. The mechanical forces associated with blood flow have a profound effect on the properties of endothelial cells (ECs) of the arteries. Thus, shear stress generally triggers an anti-atherogenic gene expression and signal transduction profile that is lost at sites of disturbed blood flow. In addition, sites with disturbed blood flow are associated with changes in the morphology of ECs, increase in the permeability to macromolecules such as LDL, and accumulation of extracellular matrix (ECM) that causes the retention of such particles. Cytokines can modulate EC permeability,. For instance, IFN-γ and TNF-α cause reorganization of the actin and tubulin cytoskeletons in ECs, thereby opening up gaps between adjacent cells.

5The activated ECs release a range of chemokines and other cytokines that then cause the recruitment of circulating immune cells, particularly monocytes and T lymphocytes. In addition, the ECs express adhesion proteins, such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), that participate in the recruitment of immune cells.

6Chemokines are a large family of structurally related, chemoattracting cytokines that are divided into subgroups on the basis of the position of the amino terminal cysteine residues (CC, CXC, CX3C, XC),,. Chemokines interact with receptors that activate heterotrimeric G proteins and associated intracellular signaling pathways,. The roles of chemokines in atherosclerosis, particularly in the recruitment of monocytes, has been reviewed extensively,,, and hence is only briefly addressed here. Monocytes in the blood are generally classified in mice as the most abundant classical Lys6Chigh [express high levels of CC-chemokine receptor (CCR)-2, believed to be pro-inflammatory in nature and their levels increase in hyperlipidemia) and less frequent Ly6Clow [express CX3C-chemokine receptor 1 and perceived to carry out a homeostatic function),. The recruitment of monocytes occurs in a number of stages: capture and rolling; arrest; and extravasation,,. The capture and rolling phase for Lys6Chigh monocytes involves immobilization of CCL5 and CXCL1 on proteoglycans and P-selectins on ECs,,. These chemokines interact with receptors expressed on the surface of invading monocytes,,. The firm adhesion of monocytes to ECs requires binding of adhesion molecules on ECs to integrins on monocytes: VCAM-1 to integrin α4β1 (very late antigen 4 or VLA4) and ICAM-1 to αLβ2 (lymphocyte function-associated antigen 1 or LFA1),,. The transmigration of monocytes across the endothelium is mediated by chemokines produced by not only ECs but other cells present in the lesion such as SMCs and emigrated leukocytes along with VCAM-1 and platelet endothelial cell adhesion molecule-1 (PECAM-1),,.

7The roles of various chemokines and their receptors in atherosclerosis have been analyzed in mouse model systems, and the outcomes are summarized in Table 1, Table 2. It should be noted that different studies have not always produced a consistent outcome (e.g. CCR7) (Table 1, Table 2). In addition, there is often a marked functional redundancy in the action of various chemokines with further complexity produced by the ability of some of them to interact with multiple receptors together with individual receptors having many ligands,,. In addition, there are likely to be temporal expression pattern and function of some chemokines during different stages of atherogenesis,,. Overall, the three most prominent chemokine:chemokine receptors involved in the transmigration of Ly6Chigh monocytes include CCL2:CCR2, CX3CL1:CX3CR1, and CCL5-CCR5,. Deficiency of these three-chemokine action leads to almost complete attenuation of atherosclerosis in mouse model systems. The entry of patrolling Ly6Clow monocytes occurs less frequently and tends to rely on the CX3CL1:CX3CR1 axis.

8Although chemokines were originally discovered in relation to their roles in directing leukocytes to sites of inflammation, they are now known to play a number of other important functions in the disease,,. For example, CX3CL1 can also serve as an adhesion molecule. CCL2 also impairs reverse cholesterol transport (RCT) by repressing the expression of key proteins implicated in the efflux of cholesterol from cells. Manipulating the chemokine:chemokine receptor axis represents a promising therapeutic avenue,,. Indeed, existing therapies such as statins have been found to attenuate the expression of chemokine and their receptors. Other approaches that have shown promise, at least in mouse model systems, are: manipulating sialylation that affects the interaction of chemokines with their cognate receptors; antagonists/inhibitors of specific receptors,,; nano-particle-facilitated gene silencing; broad spectrum inhibitors such as viral proteins,,; blocking antibodies against chemokines or their receptors,,; decoy ligands that bind to the receptor but does not cause activation (e.g. met-RANTES),,; and molecules that target chemokine oligomerization,,. Targeting multiple chemokines will be necessary given the independent roles of some chemokines,. It is also important to point out that not all chemokines are pro-atherogenic. For example, CXCL5 limits macrophage foam cell formation in mouse model systems.

9Deficiency of many cytokines (e.g. IL-1RA, IL-10, IL-13, IL-18, IL-19) is also associated with reduced macrophage recruitment (see Table 3, Table 4 for a summary of the outcome of studies on the roles interleukins and other cytokines respectively in mouse model systems). Induced expression of chemokines and adhesion molecules by ECs represents a major mechanism for such an action of these cytokines,.

10The normal function of monocyte recruitment in inflammation is to participate in its resolution in order to ultimately decrease further entry of immune cells, cause egress of existing cells from inflammatory sites and efferocytosis of apoptotic cells,. However, these homeostatic mechanisms are impaired in atherosclerosis resulting in a continuous recruitment of monocytes and reduced egress and efferocytosis of existing cells,. In addition, local proliferation dominates macrophage accumulation in atherosclerotic plaques.

11In the arterial intima, monocytes can differentiate to macrophages or dendritic cells (DCs) depending on the cytokine signal received with macrophage colony-stimulating factor (M-CSF) a major facilitator of macrophage differentiation. Recent studies are highlighting the polarization and plasticity of macrophages that is in part dictated by the cytokine signals,,. A number of macrophage phenotypes have been identified (e.g. M1, M2, M4) with the two major ones being classically activated M1 (thought to be derived from Ly6Chigh-monocytes and produces pro-inflammatory cytokines such as IL-6, IL-12 and TNF-α) and the alternatively activated M2 (thought to be derived from Ly6Clow-monocytes and produces anti-inflammatory cytokines such as IL-10 and TGF-β) that aid in the resolution of inflammatory responses,,,. T-helper (Th)-1 cytokines such as IFN-γ and IL-1β favor M1 differentiation whereas Th2 cytokines such as IL-4 and IL-13 are required for M2 differentiation,,. The pro-atherogenic roles of IFN-γ and IL-1β are well established (Table 3, Table 4) and recent studies have shown that administration of IL-13 in the LDLr−/− model system drives M2 macrophage polarization and inhibits atherosclerosis progression. However, the role of IL-4 in atherosclerosis is not clear-cut,.

12LDL tends to diffuse passively through EC junctions and its retention is aided by interaction of its apoB moiety with components of the extracellular matrix such as proteoglycans. The accumulated LDL is subject to modification such as aggregation and oxidation. The latter to form oxidized LDL (OxLDL) is in response to exposure to oxidants from the activated endothelium and due to the action of enzymes such as 12/15-lipooxygenase, myeloperoxidases and NADPH oxidases,. The effect of some cytokines on the oxidation of LDL by monocytes/macrophages or other cells involved in atherosclerosis along with the expression/activation of enzymes implicated in this process has been investigated though the outcome of these studies have not often been consistent,,,,. For example, TNF-α, IL-4 and IL-13 promoted LDL oxidation,, though contradictory findings were obtained with IFN-γ,.

13Macrophages express pattern recognition receptors (PRRs), such as scavenger receptors (SRs), toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLR), that participate in foam cell formation and/or elicit an inflammatory response against foreign particles or endogenously produced danger signals,. The SRs and to a certain extent TLRs play critical roles in foam cell formation. For example, minimally modified LDL (mmLDL) and its components stimulate TLR4-dependent fluid phase uptake of lipoproteins by macrophages. SRs, such as SR-A1 and CD36, recognize modified LDL and uptake these particles to convert into lipid-laden foam cells. The uptake of LDL via the LDLr is under negative feedback regulation through the sterol response element binding protein (SREBP) regulatory pathway. In contrast, the uptake of modified LDL by SRs in not under such negative feedback regulation and therefore causes uncontrolled uptake. Mechanisms other than SRs-mediated endocytosis, such as pinocytosis, phagocytosis and macropinocytosis, also participate in the uptake of lipoproteins, including native and modified LDL.

14As shown in Table 3, Table 4, several cytokines modulate foam cell formation in vivo; for example, IFN-γ and TNF-α promote this whereas IL-1RA and IL-33 are inhibitory. The formation of foam cells involves dysfunction of a homeostatic mechanism that controls the uptake, intracellular metabolism and efflux of cholesterol by macrophages. Several cytokines modulate macrophage foam cell formation by disrupting such a homeostatic mechanism via regulation of the expression and/or activity of key genes implicated in these processes. For example, IFN-γ promotes modified LDL uptake by inducing the expression of the SR that binds phosphatidyl serine and oxidized lipoproteins (SR-PSOX) and our recent studies have shown that the extracellular signal-regulated kinase (ERK)/signal transducer and activator of transcription-1 (STAT1) signaling is required for the macrophage uptake of modified LDL by this cytokine. In addition, the cytokine decreases cholesterol efflux by inhibiting the expression of key proteins involved in this process, such as ATP-binding cassette transporter (ABC)A1 and ApoE, and increasing intracellular levels of chostereyl esters by augmenting the expression/activity of acyl-CoA acyl transferase-1 (ACAT-1), which is involved in the esterification of cholesterol,,. The TNF superfamily members LIGHT (homologous to lymphotoxin, exhibits inducible expression, and competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes), TWEAK and TNF-like protein 1A (TL1A) also promote the uptake of modified LDL. For instance, we have shown that TL-1A stimulates macrophage foam cell formation by increasing the uptake of modified LDL and intracellular cholesteryl ester content and decreasing cholesterol efflux. The cytokine induced the expression of several SRs (SR-A, CD36, SR-PSOX) and decreased the expression of ApoE, ABCA1 and ABCG1. In contrast to these pro-inflammatory cytokines, TGF-β1, IL-10 and IL-33 inhibit macrophage foam cell formation. For example, IL-33 reduces foam cells in the ApoE−/− mice in vivo and macrophages in vitro by decreasing modified LDL uptake, reducing intracellular content of cholesteryl esters and stimulating cholesterol efflux. These changes were associated with reduced expression of key genes involved in the uptake and intracellular storage of cholesteryl esters, such as SR-A1, CD36, SR-BI, adipocyte differentiation related protein (ADRP) and ACAT-1, and increased expression of those implicated in the intracellular trafficking and efflux of cholesterol [e.g. ApoE, ABCA1, ABCG1, (Niemann-Pick disease, type C (NPC)-1, NPC-2]. TGF-β acts in a similar manner,,,. Additionally, TGF-β and IL-33 attenuate macropinocytosis. On the other hand, IL-10 inhibits foam cell formation by enhancing both modified LDL uptake and cholesterol efflux.

15Cholesterol is toxic to cells and mechanisms exist to maintain homeostasis. For example, derivatives of cholesterol, such as oxysterols, activate liver X receptors (LXRs) that then stimulate cholesterol efflux by inducing the expression of ABCA1 and ABCG1. LXRs also attenuate gene expression associated with inflammation via transrepression. Cytokines, on the other hand, attenuate the actions of LXRs. For instance, IFN-γ inhibits LXR signaling and the expression of LXR-α is decreased during the hepatic acute phase response. Continued inflammation ultimately causes the cholesterol homeostatic mechanisms to become overwhelmed during atherosclerosis. This leads to endoplasmic reticulum (ER) stress in macrophages and, together with other insults, results in cell death by apoptosis and necrosis,. Defective clearance of such apoptotic cells (efferocytosis) continues to result in lipid accumulation in the atherosclerotic lesions,. Efferocytosis and components involved in the process are also subject to regulation by cytokines,.

16Defective lipid homeostasis is instrumental in the amplification of the inflammatory response and activation of the immune response. Thus, a number of “danger signals” are produced from modification of LDL and lysis of foam cells that activate PRR, such as SRs, TLRs and NLRs, expressed by macrophages. For example, the uptake of cholesterol crystals, which have been observed in early lesions, by macrophages via macropinocytosis activates the NLR family, pyrin domain containing 3 (NLRP3) inflammasome. The process involves lysosomal destabilization and release of reactive oxygen species (ROS) and proteases that then activate NLRP3 inflammasome leading to processing and secretion of IL-1β and IL-18, and subsequent amplification of the inflammatory response. In addition, increases in intracellular cholesterol by other mechanisms (e.g. SRs-mediated uptake) leads to the formation of intracellular cholesterol crystals and activation of NLRP3 inflammasome. Furthermore, mmLDL, which is not recognized by SRs, causes production of pro-inflammatory cytokines via a TLR4-dependent manner. Moreover, OxLDL uptake by CD36 causes expression of cytokines via a mechanism involving TLR2-TLR4.

17Antigen presenting cells, such as macrophages and DCs, are essential for the adaptive immune responses as they engulf and process antigens, and present them on the cell surface in association major histocompatibility complexes (MHC) for recognition by T-cells,. A range of T-cells exists and many studies have investigated their roles in atherosclerosis,. In general, Th1 cells are most abundant in atherosclerotic plaques, secrete cytokines such as IFN-γ, TNF-α and IL-2 and are pro-atherogenic, whereas Tregs, which produce TGF-β and IL-10, are anti-atherogenic,. In contrast, the roles of Th2 cells (secrete IL-4, IL-5 and IL-13) and Th17 cells (produce IL-17A/F along with IL-22 and IL-23) are not clear-cut,. The actions of some cytokines on atherosclerosis (e.g. IL-10, IL-18, IL-19, IL-33, TGF-β) are mediated, at least in part, by modulating T-cell activation/levels/actions (Table 3, Table 4). Th1 commitment is mainly triggered by IFN-γ and IL-12, IL-6 and IL-13 play an important role in Th2 differentiation, TGF-β and IL-6 are necessary for Th17 differentiation, and generation of Tregs depends on TGF-β. In addition to T-cells, other immune cells, such as DCs, natural killer (NK) cells, NKT cells, subsets of B cells, mast cells and neutrophils play an important role in atherosclerosis and contribute to the inflammatory responses in this disease,.

18SMCs also play an important role in atherosclerosis and more advanced lesions are generally covered with a fibrous cap, composed of SMCs and ECM components produced by them, and lipid-rich necrotic core. Migration of SMCs from the media to the intima along with their proliferation is controlled by various growth factors produced by macrophages, ECs and T-cells,. SMCs also express SRs on their cell surface and can uptake modified LDL to form foam cells,. Cytokines can modulate the process by regulating the expression of SRs in isolation or synergistically with growth factors,. In addition, IL-1β disrupts the cholesterol-mediated LDLr feedback regulation in these cells and produces increased expression of this receptor.

19The continued inflammatory response ultimately leads to the destabilization of atherosclerotic plaques via the action of pro-inflammatory cytokines. Indeed, studies in mouse model systems have shown that IFN-γ, IL-18, GDF-15 and TWEAK destabilize plaques whereas TGF-β causes stabilization (Table 3, Table 4). Cytokines modulate a number of steps in the control of plaque stability and rupture. For instance, some, such as IFN-γ, TNF-α and IL-1β, promote apoptosis of macrophages along with foam cells leading to enlargement of the lipid core. In addition, such cytokines stimulate apoptosis of SMCs leading to thinning of the fibrous cap,,. Pro-inflammatory cytokines also inhibit the synthesis of plaque stabilizing components of the ECM produced by SMCs,. For example, IFN-γ inhibits the synthesis of collagen by SMCs. Further remodeling of the ECM is controlled by a range of proteases, particularly matrix metalloproteinases (MMPs), and their inhibitors (tissue inhibitor of metalloproteinases—TIMPs) produced by macrophages and other vascular cells,. The expression and/or activities of MMPs and TIMPs are regulated by cytokines,. Vulnerable plaques have very few SMCs and high macrophage content, and are susceptible to rupture leading to thrombosis,,. Key components involved in thrombosis are also subject to regulation by cytokines. For example, studies in mouse model systems have shown that deficiency of IL-10 is associated with tissue factor (TF) activities together with markers of systemic coagulation and vascular thrombosis. In addition, the interaction of TWEAK with its receptor, fibroblast growth factor-inducible 14 (Fn14), stimulates the expression of plasminogen activator inhibitor (PAI)-1 and TF by SMCs. Furthermore, the production of TF is activated by a number of cytokines such as TNF-α, IL-1, IL-6, IL-8 and IFN-γ. Similarly, the expression of PAI-1 is modulated in inflammatory conditions,. Pro-inflammatory cytokines also suppress natural anticoagulant mechanisms, such as the protein C pathway.

20Inflammation plays a pivotal role in all stages of atherosclerosis: endothelial dysfunction; recruitment of immune cells; modification of LDL; foam cell formation; apoptosis of foam cells; plaque rupture; and thrombosis. The inflammatory response in atherosclerosis is regulated by both the innate and adaptive immune system via the action of cytokines. Our understanding of the roles of such cytokines has recently advanced considerably mainly via studies using mouse model systems (summarized in Table 1, Table 2, Table 3, Table 4). Attenuating cytokine-induced inflammation and promoting the actions of anti-inflammatory cytokines represent potential therapeutic avenues. Indeed, statins and athero-protective agonists of nuclear receptors attenuate signaling and gene expression mediated by certain cytokines,,, and this could be responsible, at least, in part for their beneficial effects. In addition, the athero-protective effects of certain agents (e.g. estradiol) on atherosclerosis in mouse model systems have been attributed to TGF-β.

21A number of anti-inflammatory therapies aimed at manipulating cytokine actions are currently being evaluated,,. For example, the Cardiovascular Inflammation Reduction Trial (CIRT) has been initiated to evaluate the efficacy of low doses of methotrexate, which has proved beneficial in the treatment of the inflammatory disorders rheumatoid arthritis and psoriasis, for the secondary prevention of myocardial infarction. In addition, a phase II clinical trial of canakinumab, IL-1β neutralizing antibody, reported lowering of inflammatory markers and formed the basis of a larger phase III, secondary prevention Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS),,. A monoclonal antibody (MLN1202) that targets interaction of CCL2 with its receptor has also been evaluated on a smaller scale with reduction in the levels of the acute phase reactant C-reactive protein.

22Therapeutic approaches need not be restricted to cytokines themselves. For instance, many studies in mouse model systems have demonstrated the protective roles of Tregs and cytokines produced by them (IL-10 and TGF-β),. Many studies are investigating the potential of approaches that augment the levels/action of Tregs or stimulate immune tolerance to antigens associated with atherosclerosis,. Manipulating cytokine signaling represents another approach and could include small molecule inhibitors that attenuate the action of pro-inflammatory components or enhance those that are present naturally to dampen inflammation (e.g. suppressors of cytokine signaling—SOCS),,. Noncoding RNA, particularly microRNA (miRNA), are emerging as future potential targets in dampening cytokine-mediated inflammation in atherosclerosis. For example, deficiency of mIR155, whose expression is stimulated by miR-342-5p, reduces inflammatory responses in mouse model systems. Pro-inflammatory cytokines induces miR-146a/b in ECs, which then acts as a negative feedback loop to control pro-inflammatory signaling in EC activation. Future research will inform on the potential of miRNA therapeutics and refine current approaches aimed at manipulating cytokine actions in atherosclerosis.

23None.