Innate and Adaptive Immunity in Atherosclerosis

Abstract

Atherosclerosis, a chronic inflammatory disorder, involves both the innate and adaptive arms of the immune response that mediate the initiation, progression, and ultimate thrombotic complications of atherosclerosis. Most fatal thromboses, which may manifest as acute myocardial infarction or ischemic stroke, result from frank rupture or superficial erosion of the fibrous cap overlying the atheroma, processes that occur in inflammatorily active, rupture-prone plaques.

Appreciation of the inflammatory character of atherosclerosis has led to the application of C-reactive protein as a biomarker of cardiovascular risk, and the characterization of the anti-inflammatory and immunomodulatory actions of the statin class of drugs. An improved understanding of the pathobiology of atherosclerosis and further studies of its immune mechanisms provide avenues for the development of future strategies directed toward better risk stratification of patients as well as the identification of novel anti-inflammatory therapies. This review retraces leukocyte subsets involved in innate and adaptive immunity and their contributions to atherogenesis.

Introduction

Atherosclerosis, a chronic inflammatory disease, involves both innate and adaptive arms of immunity which modulate lesion initiation, progression, and potentially devastating thrombotic complications ^{1 2}. Thrombosis often complicates physical disruption of the protective collagen-rich fibrous cap overlying the atheroma, exposing circulating clotting factors to procoagulants expressed within lesions as a result of inflammatory activation and initiation of the coagulation cascade ³. Importantly, inflammation also decisively influences the propensity of a given plaque disruption to lead to a sustained and occlusive thrombus that may manifest clinically as an acute coronary syndrome or ischemic stroke by controlling the balance between fibrinolytic enzymes and their endogenous inhibitors ^{4 5}.

Appreciation of the inflammatory character of atherosclerosis has spawned new avenues in basic, translational, and clinical research. CRP (C-reactive protein), an acute-phase reactant released during inflammatory processes, adds to the predictive power of traditional markers of cardiovascular risk ⁶. Basic research suggests that treatment with statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) — initially developed to decrease low density lipoprotein (LDL) cholesterol levels — also reduces leukocyte adhesion, accumulation of macrophages, protease production, procoagulant and pro-inflammatory mediator expression, antigen presentation, and T-cell activation ⁷. Additional support for the anti-inflammatory and immunomodulatory actions of statins came from clinical research. The magnitude of risk reduction associated with statin therapy may exceed that expected on the basis of LDL-cholesterol lowering alone. The CARE (Cholesterol And Recurrent Events) trial first demonstrated that statin therapy lowers plasma levels of CRP in addition to LDL-cholesterol ⁸. Retrospective evidence supported the utility of targeting the inflammatory marker CRP with statins in normocholesterolemic patients in both primary ⁹ and secondary prevention ¹⁰ of adverse cardiovascular events. Prospective evidence provided by the JUPITER trial (Justification for the Use of Statins in Primary Prevention: an Intervention Trial Evaluating Rosuvastatin) demonstrated that patients with LDL-cholesterol levels considered near optimal but elevated CRP levels benefit significantly from statin treatment in the prevention of adverse cardiovascular events. This direct result of the clinical application of the science of inflammation in atherosclerosis has potentially far reaching implications for everyday medical practice and public health.

To portray the chronic inflammation in atherothrombosis, we review here the leukocytes involved in innate and adaptive immunity with established or emerging roles in the disease process, and detail their cellular and molecular contributions. Beyond macrophages and CD4⁺ T-cells, new research highlights the important role of regulatory T-cells, dendritic cells, and mast cells in the disease process. Most of the cited experimental work relies on genetically altered atherosclerosis-prone mice, namely apolipoprotein E (ApoE)-deficient mice, which develop hypercholesterolemia and atherosclerotic disease spontaneously ¹¹, and low-density lipoprotein receptor-deficient mice, which require a high-fat diet to develop hypercholesterolemia and atherogenesis ¹².

The innate immune response in atherosclerosis

Monocytes and macrophages — the most numerous leukocytes at all stages of atherosclerosis — comprise the central cellular effectors of disease progression

Accumulation of lipid-laden macrophage-derived foam cells characterizes fatty streaks, the initial asymptomatic lesion of atherosclerosis ¹. The precursor lesion of atherosclerotic plaques, fatty streaks, have focal increases in the content of lipoproteins within regions of the intima where they associate with constituents of the extracellular matrix such as proteoglycans, slowing their egress ¹³. This retention sequesters lipoproteins within the intima, protecting them from plasma antioxidants, thus favoring their oxidative modification. Laboratory and clinical data suggest that oxidized or glycated LDL evokes an inflammatory response in the artery wall, unleashing many of the biological processes thought to participate in atherosclerosis initiation, progression, and complication ¹⁴.

Endothelial cells (ECs) normally resist leukocyte adhesion. Pro-inflammatory stimuli that include hypercholesterolemia, hyperglycemia, hypertension, and smoking trigger the endothelial expression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and P-selectin that mediate the attachment of circulating monocytes and other leukocytes ^{15 16 17 18}. Chemoattractant factors, including monocyte chemoattractant protein-1 (MCP-1) produced by vascular wall cells in response to modified lipoproteins, direct the migration and diapedesis of adherent monocytes. MCP-1 binds to CCR2 on the surface of migrating monocytes to exert this effect. Genetic absence of MCP-1 in LDLR-deficient mice dramatically decreases atherosclerotic disease with marked inhibition of monocyte recruitment ¹⁹. Similar results hold true upon deletion of CCR2 expression in ApoE-deficient mice ²⁰. During diapedesis, monocytes release the matrix metalloproteinase MMP-9 that can degrade type IV collagen in the intimal basement membrane to help them enter the growing intimal atherosclerotic lesion ²¹. Experimental evidence and human observations support the involvement of several other chemokines in monocyte recruitment into the nascent atherosclerotic lesion, including IL-8, which binds CXCR2 ^{22 23}, and fractalkine (CX₃CL1), which binds CX₃CR1 ^{24 25}. Importantly, low shear stress also induces expression of MCP-1, fractalkine, and other chemokines involved in atherogenesis ²⁶. Monocytes infiltrate the lesion and localize particularly in the shoulder region where the atheroma grows. In addition, neovessels arising from the artery's vasa vasorum provide another entry route for monocytes into established atherosclerotic lesions ²⁷.

Within the intima, monocytes mature into macrophages under the influence of macrophage colony-stimulating factor (M-CSF), overexpressed in the inflamed intima ^{28 29}. Importantly, M-CSF stimulation also leads to increased macrophage expression of scavenger receptors, pattern-recognition receptors involved in innate immunity, which engulf modified lipoproteins and apoptotic bodies through receptor-mediated endocytosis, leading to their lysosomal degradation. Scavenger receptors involved in macrophage foam cell formation include CD36, CD68, CXCL16, LOX1 (lectin-type oxidized low-density lipoprotein receptor 1), SR (scavenger receptor)-A, and SR-B ¹. Accumulation of cholesteryl esters in the cytoplasm produces the characteristic change of macrophages into foam cells. Another type of pattern-recognition receptor, Toll-like receptors (TLRs), directly elicit inflammatory responses ¹. In particular, monocytes in human atherosclerotic plaques have markedly enhanced expression of TLR1, TLR2, and TLR4 ³⁰. A majority of these monocytes show nuclear translocation of the transcription factor NF-κB (nuclear factor-κB), consistent with their inflammatory activation in lesions ³⁰. Within atherosclerotic lesions, IκB kinase 2 (IKK-2, or IKK-β) phosphorylates IκBα, leading to its ubiquitination and degradation. IKK-2 thereby terminates the inhibitory action of IκBα on NF-κB, and allows the transcription of pro-inflammatory cytokines and proteases ³¹. A large number of pathogen-associated molecular patterns can activate TLRs. Heat shock proteins (hsp60) ³² and oxLDL ^{33 34} mediate at least part of their effects within plaques through TLR4 binding. In support of the notion that TLR4 and downstream effectors such as MyD88 (myeloid differentiation primary-response gene 88) mediate inflammatory activation in atherosclerosis, their genetic abrogation reduces disease burden ^{35 36}. Apolipoprotein CIII, a constituent of certain atherogenic triglyceride-rich lipoproteins, can activate cells involved in atherogenesis through TLR2 ³⁷. TLR2, expressed on cells not derived from bone marrow, also appears to promote atherogenesis in mice ^{38 39}.

Macrophages proliferate and amplify the inflammatory response through the secretion of numerous growth factors and cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). These 2 key cytokines are central mediators of inflammatory pathways relevant to atherosclerosis (Figure 1). Among a myriad of actions, they induce expression of adhesion molecules such as VCAM-1, chemokines such as MCP-1, growth factors such as M-CSF, and proteases such as MMPs by cellular effectors present within lesions ¹. In human lesions, IL-18 colocalizes with mononuclear phagocytes while ECs, SMCs, and macrophages all express the IL-18 receptor ⁴⁰. Importantly, IL-18 signaling evokes essential effectors involved in atherogenesis, e.g., adhesion molecules (VCAM-1), chemokines (IL-8), cytokines (IL-6), and matrix metalloproteinases (MMP-1/-9/-13) ⁴⁰. Recent evidence supports selective mobilization during hypercholesterolemia in mice of a pro-inflammatory subset of monocytes that bear high levels of a surface marker denoted Ly6C. These Ly-6C^hi monocytes increase dramatically in the blood of fat-fed apoE-deficient mice, and preferentially adhere to activated endothelium, infiltrate lesions, and become lesional macrophages ⁴¹. Importantly, pro-inflammatory Ly-6C^hi monocytes are CCR2⁺ and CX₃CR1⁺ and rely on these chemokine receptors to enter lesions ⁴².

Figure 1. Leukocytes and platelets release mediators that control inflammation in atherosclerotic plaques and determine lesion fate.

Abbreviations: T_H1 T Cell; T helper 1 T cell, Treg; Regulatory T Cell, ROS; reactive oxygen species, IFN-γ; interferon-γ, TNF-α; tumor necrosis factor-α, IL; interleukin, TGF-β; transforming growth factor-β.

Advanced atheromata may exhibit a paucity of SMCs and abundant macrophages, key histological characteristics of plaques that have ruptured and caused fatal thrombosis. The fibrous cap covering the atherosclerotic plaque owes its biomechanical strength to interstitial collagens (types I and III). Inflammation interferes with the integrity of the fibrous cap by limiting the creation of new collagen by SMCs ⁴³ and by stimulating the destruction of existing collagen fibers (Figure 2). Indeed, CD40L as well as IL-1 produced by T-cells induce macrophages to release interstitial collagenases, including MMP-1 ⁴⁴, MMP-8 ⁴⁵, and MMP-13 ^{44 46}, which mediate the initial attack on interstitial collagen. MMP-14, a membrane-associated MMP, activates MMP-13 and also appears to contribute to collagenolysis ⁴⁷. Macrophage-derived foam cells contain MMP-9, a member of the gelatinase class of the metalloproteinase family, in human plaques ⁴⁸. The catalytically active MMP-9 may contribute to the dysregulation of extracellular matrix that leads to plaque rupture. MMP activity overwhelms regulation mediated by TIMPs (tissue inhibitors of metalloproteinases) produced by macrophages and other cells in atheromata, hence influencing plaque stability ^{49 50}. In addition, macrophages in lesions constitutively produce the serine protease neutrophil elastase, and release this enzyme upon CD40 ligation ⁵¹. By inactivating TIMP-1, neutrophil elastase favors MMP activity and collagen breakdown ⁵¹. Direct in vivo evidence in collagenase-resistant atherosclerosis-prone mice confirms the role of MMPs in plaque collagen turnover ⁵².

Figure 2. T_H1 cells and macrophages promote atherosclerosis progression.

T_H1-biased T lymphocytes (lower left) express IFN-γ and CD40L within plaques. IFN-γ inhibits the de novo production of collagen in response to TGF-β and PDGF by vascular smooth muscle cells (middle left). In parallel, CD40L induces the synthesis and release of proteases by macrophages (center), including the interstitial collagenases MMP-1, MMP-8, and MMP-13, which mediate the initial proteolytic attack of intact collagen fibrils. In addition, CD40L also simulates the production of the gelatinases MMP-2 and MMP-9 as well as other proteases that continue the proteolytic degradation of collagen fibrils. These combined effects orchestrated by T_H1 cells and macrophages weaken the fibrous cap covering the atherosclerotic plaque — which owes its biomechanical strength to interstitial collagen (types I and III) fibrils — and render the lesion rupture-prone.

Abbreviations: CD40L; CD40 Ligand, IFN-γ; interferon-γ, MMP; matrix metalloproteinase, PDGF; platelet-derived growth factor, TGF-β; transforming growth factor-β. (Figure reproduced with kind permission of Springer Science and Business Media ²¹⁵.)

Cathepsins, members of the cysteine protease family, also participate in plaque evolution and destabilization ⁵³. Macrophages in human atheromata express most of the proteolytically active cathepsins S and cathepsins K, which display elevated elastolytic activities ⁵⁴. Experimental atherosclerotic lesions also express cathepsins L, and B ⁵⁵. Cathepsin S ⁵⁶, cathepsin L ⁵⁷, or cathepsin K ⁵⁸ deficiency in atherosclerosis-prone mice reduces collagen and elastin degradation as well as CD4⁺ T-cell, macrophage, and smooth muscle cell accumulation and overall plaque burden. Importantly, arteries under physiologic conditions abundantly express cystatin C, an endogenous inhibitor of cathepsins, whereas human atheromata exhibit very low cystatin C levels ⁵⁹.

Recently, proteases of the ADAMTS (a disintegrin and metalloprotease with thrombospondin motif) family have also garnered attention for their role in matrix protein turnover and progression of atherosclerosis ^{60 61}. The combined effects of all these classes of proteases, released mostly by macrophage foam cells within lesions, favors matrix and fibrous cap remodeling that may lead to plaque rupture with ensuing thrombosis and clinical manifestations ⁶².

Mast cells

Mast cells although numerically minor constituents of the leukocytic population in the atherosclerotic intima can also populate the adventita ^{63 64}. In human coronary artery specimens, mast cell numbers rise in parallel to the severity of clinical presentation ⁶⁵, and these leukocytes accumulate in the shoulder region of plaques, where they degranulate and release proteases and cytokines ⁶⁶. Recent studies suggest they can contribute to lesion progression in mice (Figure 1). These data require cautious interpretation in regard to the human disease, as rodents rely more on innate immunity and possess a more complex variety of mast cell functions and proteases ⁶⁷.

The chemokine eotaxin, found in atherosclerotic plaques, can mediate lesional mast cell recruitment by binding to cellular CCR3 ⁶⁸. Utilization of a mast cell-deficient mouse demonstrates decreased lesion size, lipid deposition, T-cell and macrophage numbers, and cell proliferation and apoptosis, but increased collagen content and fibrous cap development in a mast cell-derived IFN-γ and IL-6-dependent manner ⁶⁹. Periadventitial injury leads to lesion progression and activation of mast cells with ensuing intraplaque hemorrhage, macrophage apoptosis, vascular permeabilization, and recruitment of further leukocytes ⁷⁰. Importantly, treatment with the mast cell stabilizer chromoglycate prevents all these adverse events elicited by mast cell degranulation ⁷⁰.

Mast cells produce certain MMPs, including MMP-1 ⁷¹ and MMP-9 ⁷². In addition, the mast cell serine proteases tryptase and chymase can activate MMPs in human carotid endarterectomy samples, and MMP-1 and -3 colocalize with degranulated mast cells in the shoulder regions of atherosclerotic plaques ⁷³. Mast cell chymase also processes pro-MMP-2 and -9 into their active forms ⁷⁴. These results suggest that the direct release of certain MMPs and the activation of MMPs by mast cell-derived proteases may promote atherosclerotic plaque rupture.

The proportion of intimal mast cells expressing basic FGF (fibroblast growth factor) — a potent angiogenic mediator — rises with increasing severity of atherosclerosis, suggesting a role for these leukocytes in angiogenesis, plaque neo-vascularization, and disease progression ⁷⁵. Mast cell chymase also functions as an angiotensin-converting enzyme (particularly in rodents) and may thus contribute to the local regulation of vascular tone ⁷⁶.

Activated mast cells induce endothelial death by chymase-mediated inactivation of focal adhesion kinase (FAK) and Akt-dependent cell survival signaling, as well as TNF-α-mediated apoptosis, functions that contribute to plaque erosion ⁷⁷. Mast cell chymase can also inhibit smooth muscle cell proliferation and collagen expression ⁷⁸ and induce SMC apoptosis ^{79 80}. Histamine — abundantly present in mast cells — reportedly induces tissue factor expression in human aortic endothelial and vascular smooth muscle cells ⁸¹, promoting the plaque's thrombotic potential.

Degranulation of mast cells may facilitate SMC and macrophage uptake of LDL and their development into foam cells ^{82 83}, mediated in part by chymase-dependent degradation of the ApoB moiety of LDL ⁸⁴. In addition, mast cells inhibit cellular cholesterol efflux and reverse cholesterol transport, in part by chymase-mediated degradation of certain apolipoproteins such as ApoE ⁸⁵ and tryptase-mediated degradation of HDL ⁸⁶.

Natural Killer cells

Nature killer (NK) cells, cellular effectors of innate immunity, play a critical role in the defense against infectious organisms, particularly viruses ⁸⁷. Contrary to T-cells and B-cells, these bone marrow-derived lymphocytes do not require antigen receptor gene rearrangement during cellular development and do not express T-cell receptors or surface immunoglobulins. NK cells receive dual signals from inhibition and activation surface receptors ⁸⁷. NK cell inhibitory receptors such as Ly49A are specific for MHC class I molecules on target cells and prevent NK cell activation, cytotoxicity, and cytokine secretion. Cells that have lost their expression of MHC-I molecules — typically virus-infected cells — are susceptible to NK cell attack. NK cells also express activation receptors such as Ly49D and Ly49H, structurally related to the inhibitory receptors, which recognize target cell ligands and can trigger perforin-dependent natural killing. In addition, NK cells express a receptor that binds the Fc portion of antibodies known as FcγRIII (CD16). Cross-linking of Fc receptors by IgG antibody-coated target cells may constitute a second form of activation that signals the NK cell to kill the target.

Limited direct evidence supports NK cell involvement in atherogenesis. The shoulder region of human plaques contains modest numbers of CD56⁺NK cells ⁸⁸. Given their presence within lesions, and the abundance of cytokines known to activate NK cells such as IFN-α/β, IL-12, IL-15, and IL-18, these cells plausibly contribute to lesion progression. Upon activation, NK cells in turn secrete numerous cytokines and growth factors including IFN-γ, TNF-α, GM-CSF, and trigger perforin-mediated natural killing ⁸⁷. Indeed, atherosclerosis-prone mice with genetically impaired NK inhibitory signaling have decreased plaque burden ⁸⁹.

The possible role of neutrophils in atherothrombosis

Neutrophils comprise a minority of the inflammatory cell composition of atherosclerotic lesions. However, neutrophil numbers rise in ruptured human coronary plaques ⁹⁰, consistent with their role of endocytosing and clearing damaged tissue, but also raising the possibility of their presence before clinical events. In parallel, high circulating neutrophil counts predict myocardial infarction better than any other leukocyte subset, including total white blood cell, lymphocyte, or monocyte count ⁹¹. Intraplaque hemorrhage contributes importantly to the progression of atherothrombosis ⁹², and analyses of human carotid endarterectomy samples suggest intraplaque hemorrhage as an entry route for neutrophils — which constitute 60% of circulating leukocytes — into lesions ⁹³. Atheromatous lesions demonstrate markers of neutrophil degranulation (α1-antitrypsin/elastase complexes, myeloperoxidase, and α-defensins) and the presence of proteases preferentially released by these leukocytes [NGAL (neutrophil gelatinase-associated lipocalin)/MMP-9 heterodimers, and HLE (human leukocyte elastase)], suggesting the presence of active neutrophils within lesions ⁹³ (Figure 1). Atherosclerotic plaques contain NGAL that inhibits MMP-9 inactivation and thereby promotes its proteolytic and matrix-degrading capabilities ⁹⁴. Another report confirms the colocalization of neutrophils with myeloperoxidase in lesions, suggesting a source for this enzyme beyond a macrophage subset ⁹⁵. Myeloperoxidase generates the reactive oxygen species hypochlorous acid, which contributes to endothelial apoptosis and tissue factor expression and lesion advancement ⁹⁶. In addition to mediating chlorination of tyrosyl residues, myeloperoxidase also leads to LDL protein nitration and lipid peroxidation, facilitating the uptake of these modified LDL particles by macrophages and contribution to foam cell formation ⁹⁷.

Under physiologic circumstances, neutrophils arise exclusively in the bone marrow, and the chemokine ligand CXCL12 (SDF-1) expressed on bone marrow stromal cells and its receptor CXCR4 expressed on neutrophils allow not only their retention but also the homing of senescent neutrophils back to the bone marrow ⁹⁸. Experimental antagonism of CXCR4 leads to increased circulating neutrophil levels and their enrichment within atherosclerotic lesions in response to CXCL1 expressed within lesions interacting with CXCR2 on neutrophils ⁹⁹. Neutrophil recruitment also depends on the expression of neutrophilic Mac-1 and endothelial P-selectin for leukocyte rolling ¹⁰⁰. These recruited neutrophils can secrete numerous enzymes including proteases. In addition, recruited neutrophils increase lesion size and enhance intraplaque IFN-γ, tissue factor, and CXCL1 levels, further amplifying their recruitment ⁹⁹. These results suggest intraplaque hemorrhage and the more classic transendothelial route as sources of neutrophil recruitment, enriching the oxidative and proteolytic content and overall inflammatory activation of lesions.

Dendritic cells link the innate and adaptive arms of the immune response

Dendritic cells (DCs), an innate immune cell type, populate atherosclerotic plaques, particularly in the rupture-prone shoulder region of lesions ¹⁰¹, in part under the control of CX₃CR1 ¹⁰². Granulocyte-macrophage colony-stimulating factor (GM-CSF), produced locally in response to oxidized low-density lipoprotein cholesterol (oxLDL), also regulates DC numbers within lesions ¹⁰³, probably by controlling their differentiation from monocyte precursors. Importantly, stimuli known to accelerate atherogenesis, such as oxLDL or TNF-α, increase DC adhesion to the endothelium and their subsequent transmigration ¹⁰⁴. Nicotine increases DC expression of MHC-II, costimulatory molecules, and adhesion molecules, and promotes the production of IL-12 by DCs, thereby promoting a Th1 response ¹⁰⁵. In addition, CD11c⁺ leukocytes with dendritic processes inhabit regions of the normal arterial intima predisposed to atherosclerosis ¹⁰⁶. In human lesions, DC numbers increase in parallel to lesion complexity ¹⁰⁷, as does the expression of CD83, a marker of DC activation ¹⁰⁸.

Hypercholesterolemia may impede the emigration of a proportion of DCs, retaining them in peripheral tissues ^{109 110} where they locally promote immune responses, for example by re-stimulating effector CD4⁺ T-cells ¹¹¹. Contrary to macrophages ¹¹², DCs retain antigen presenting function under conditions typical of atherosclerotic plaques ¹¹¹. This property may result from superior DC defenses against oxidative stress, displayed by elevated levels of superoxide dismutase and peroxiredoxin-1 ¹¹³, and an apparent resistance to cholesterol-induced cytotoxicity, displayed by the absence of expression of the transcription factor CHOP (C/EBP-homologous protein), a marker of unfolded protein response (UPR) induction, after loading of DCs with unesterified cholesterol ¹¹¹. Indeed, DCs embedded in ‘artificial arteries’ made of ECs, VSMCs, and type I collagen stimulate CD4⁺ T-cells upon exposure to lipopolysaccharide robustly and superiorly to embedded monocytes and macrophages ¹¹⁴.

DCs constitute a heterogeneous family, with different subsets characterized by varying tissue distributions, surface markers, cytokine profiles, and ensuing functions in the orchestration of immune responses ¹¹⁵. Plasmacytoid dendritic cells (pDCs) specialize in sensing bacterial and viral products and produce IFN-α abundantly ¹¹⁵. Through the release of key cytokines, PDCs contribute to the regulation of VSMC numbers within lesions. Upon microbial stimulation, pDCs release IFN-α, inducing tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) on the surface of CD4⁺ T-cells ¹¹⁶. TRAIL binds to death receptor 5 (DR-5) and thereby mediates VSMC death in an alternate pathway to the canonical CD8⁺ T-cell mediated cytotoxicity ¹¹⁷. Plasmacytoid-derived IFN-α also amplifies the inflammatory response by enhancing the production of TNF-α and IL-12 by ‘classic’ CD11c⁺ DCs ¹¹⁸. PDCs may thus provide a potential link between infections and disease progression, an association often evoked in atherosclerosis ^{119 120}.

DCs prime T-cells in secondary lymphoid organs, enabling T-cell antigen-specific differentiation into effectors and the targeting of select tissues in the periphery such as atherosclerotic vessels. To emigrate toward regional lymph nodes, where they regulate adaptive immune responses, DCs must induce expression of CCR7 ¹²¹ and CCR8 ¹²² in tissues such as plaques.

Co-stimulatory and co-inhibitory molecule expression patterns by DCs drive antigen-dependent activation of naïve T-cells and the initiation of adaptive immunity (Figure 3) ¹²³. Importantly, overlapping co-stimulatory and co-inhibitory molecules control both effector T-cell and regulatory T-cell responses, a complexity discussed elsewhere ¹²³. Two major families comprise the costimulatory molecules: the B7 ¹²⁴ and TNF ¹²⁵ families that bind to the CD28 and TNF receptor families, respectively ¹²³.

Figure 3. Dendritic cells express co-stimulatory and co-inhibitory molecules involved in T cell activation.

Dendritic cells (DCs) deliver co-stimulatory (denoted by a (+) sign) and co-inhibitory (denoted by a (-) sign) signals to T cells through molecules belonging to the B-7 or TNF family. In the B-7 family, co-stimulatory molecules include CD80 (B7-1), CD86 (B7-2), and ICOS-L, and co-inhibitory molecules include PD-L1, PD-L2. T cells can render a co-stimulatory signal delivered by CD80 or CD86 co-inhibitory by replacing the CD28 receptor with CTLA-4. In the TNF family, co-stimulatory molecules include OX40-L and CD137-L. T cells also activate DCs by CD40-L, which increases DC expression of CD80 and CD86.

Abbreviations: CTLA-4; cytotoxic T-lymphocyte antigen 4, ICOS; inducible co-stimulatory molecule, ICOS-L; inducible co-stimulatory molecule-ligand, PD-L1; programmed death-ligand 1, PD-1; programmed death-1, TNF; tumor necrosis factor.

CD80 and CD86 (B7-1 and B7-2), the prototypical and best described co-stimulatory molecules on DCs/APCs, initially deliver their signals by binding to CD28 on T-cells. CD80/CD86 deficiency in atherosclerosis-prone mice reduces lesion development and decreases IFN-γ production by CD4⁺ T-cells upon presentation of the atherosclerosis-associated antigen hsp60 ¹²⁶, suggesting inefficient priming by DCs. Following their activation, T-cells express CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4). CTLA-4 then binds CD80 and CD86 on DCs, and turns the co-stimulatory signal initially delivered by CD28 into a co-inhibitory one, dampening the T-cell response ¹²⁴.

Contrary to resting naïve T-cells, effector and memory T-cells express ICOS (inducible co-stimulatory molecule), another CD28 family member ¹²⁷. ICOS-ligand activates ICOS — which figures critically in Treg function — on DCs, with decreased Treg suppressive function and increased lesional CD4⁺ T-cell content in ICOS-deficient mice ¹²⁸.

Programmed death-ligand 1 (PD-L1) and PD-L2, B7 family members expressed on several cell types including DCs, inhibit T-cell activation by binding to programmed death-1 (PD-1) on T-cells ¹²⁹. PD-L1/PD-L2-deficient mice have increased atherosclerotic burden in conjunction with exaggerated systemic immune responses with lymphadenopathy and elevated numbers of activated T-cells due to enhanced stimulation by DCs ¹³⁰.

DCs and APCs express OX40Ligand (OX40L), a member of the TNF receptor family that provides co-stimulatory signals to T-cells through the TNF family member OX40, enabling long-lasting T-cell responses ¹²⁵. Genetic studies identified OX40L as an atherosclerosis-susceptibility locus in mice ¹³¹. OX40L-deficient mice fed an atherogenic diet have decreased lesion size, whereas transgenic over-expression of OX40L demonstrates opposite effects ¹³¹. Antagonist anti-OX40L antibody treatment of atherosclerosis-prone mice also decreases plaque burden ¹³². Moreover, polymorphisms in the OX40L gene increase the risk of myocardial infarction ¹³¹, further highlighting the possible contribution of OX40L/OX40-mediated co-stimulation in promoting atherosclerosis.

CD137Ligand (CD137L), a TNF family member, also belongs to the TNF receptor family ¹²⁵. The wide cellular distribution of these molecules complicates the elucidation of their contribution to atherosclerotic disease, but antigen recognition induces CD137 expression on T-cells that receive co-stimulatory signals from CD137L expressed on DCs ¹²⁵. CD137L/CD137 participates importantly in CD8⁺ T-cell responses ¹³³. Indeed, agonistic anti-CD137 antibody treatment increases CD8⁺ T-cell infiltration, pro-inflammatory cytokine expression, and overall lesion size ¹³⁴.

DCs maintain their antigen-processing and -presentation functions and co-stimulatory capabilities under hypercholesterolemic conditions present in experimental atherosclerosis ¹¹¹. DCs thereby activate the adaptive immune response and efficiently generate monoclonal and polyclonal effector CD4⁺ T-cells that may subsequently leave secondary lymphoid organs and reach atherosclerotic vessels ¹¹¹.

The adaptive immune response in atherosclerosis

CD4⁺ T_H1 T-cells promote atherothrombosis

IFN-γ-producing T_H1 CD4⁺ T-cells with αβ T-cell receptors constitute the majority of T lymphocytes present in human ¹³⁵ and experimental ¹³⁶ atherosclerotic lesions ^{1 2} (Figure 1). T-cells enter lesions in response to the chemokines inducible protein-10 (IP-10), monokine induced by IFN-γ (MIG), and IFN-inducible T-cell α-chemoattractant (I-TAC), which bind CXCR3, highly expressed by T lymphocytes in the plaque ¹³⁷. CD4⁺ T-cells undergo oligoclonal expansion within lesions, suggesting the occurrence of antigen-driven T-cell proliferation ¹³⁸. Indeed, CD4⁺ T-cell clones in plaques recognize oxLDL ¹³⁹ and hsp60 ¹⁴⁰.

Experimental evidence supports an important role for CD4⁺ T-cells in atherosclerosis. Immunodeficient RAG (recombinase activating gene)-deficient ¹⁴¹ or DNA-PK (DNA-dependent protein kinase)-knockout ¹⁴² atherosclerosis-prone mice have no lymphocytes and reduced lesions compared to immunocompetent atherosclerosis-prone mice. Adoptive transfer of CD4⁺ T-cells into immunodeficient animals, however, greatly increases lesion size in parallel to increased T-cell recruitment and MHC-II expression in plaques ¹⁴².

In addition to the histopathologic features of macrophage and T-cell accumulation within lesions, several lines of evidence support T_H1 predominance in atherosclerosis. Different strains of mice have varying susceptibilities to atherosclerosis ¹⁴³. T_H1-biased C57BL/6 mice develop significantly more atherosclerosis in association with increased serum levels of IL-6 and the acute-phase protein serum amyloid A (SAA) than T_H2-biased BALB/c mice ¹⁴⁴. Genetic T-bet deficiency (a transcription factor required for T_H1 differentiation) ¹⁴⁵ or treatment with pentoxifylline (an inhibitor of T_H1 differentiation) ¹⁴⁶ protects against atherosclerosis. While administration of IL-12 ¹⁴⁷, the central cytokine driving T_H1-cell differentiation, enhances atherosclerosis, genetic deficiency of IL-12 ¹⁴⁸ or IL-12 blockade through vaccination ¹⁴⁹ attenuates the disease. In addition, the genetic deficiency ¹⁵⁰ or inhibition ¹⁵¹ of IL-18, which drives T_H1-cell differentiation synergistically with IL-12, also decreases disease progression. Deficiency of IFN-γ ¹⁵², the prototypical T_H1-cytokine, or of the IFN-γ receptor ¹⁵³, greatly decreases plaque burden, macrophage content, and MHC-II expression within lesions. Administration of recombinant IFN-γ has opposite effects ¹⁵⁴. IFN-γ inhibits the proliferation and differentiation of vascular smooth muscle cells ¹⁵⁵ and also decreases collagen production by these cells ⁴³, functions that could contribute to the thinning of the collagen-rich fibrous cap (Figure 2). IFN-γ also inhibits endothelial cell proliferation ¹⁵⁶ and potentiates the production of pro-inflammatory cytokines by macrophages as well as MHC-II expression ¹. Further supporting these observations, human plaques contain an abundance of cells producing the T_H1-type cytokines IFN-γ, IL-12, IL-15, IL-18, and TNF-α, but few cells producing the T_H2 cytokine IL-4 ¹⁵⁷. In vivo, IFN-γ appears to augment arteriosclerosis, based on observations on the effects of this cytokine on small human arteries dwelling in immunodeficient mice ¹⁵⁸.

Expression of CD40Ligand (CD40L) by activated CD4⁺ T-cells induces the expression of the procoagulant tissue factor in endothelial cells ¹⁵⁹, VSMCs ¹⁶⁰, and macrophages ¹⁶¹. CD40L also stimulates production of the interstitial collagenases MMP-1 ⁴⁴, MMP-8 ⁴⁵, and MMP-13 ⁴⁴ by macrophages, which interfere with the integrity of the protective fibrous cap (Figure 2). Recent results suggest that CD40L mediates certain effects in atherosclerosis through Mac-1 binding ¹⁶². CD40L on T-cells also activates APCs/DCs ¹²³. Indeed, CD40 ligation increases co-stimulatory molecule expression by APCs, mainly CD80 and CD86, which then bind CD28 on the T-cell and transmit co-stimulatory signaling ¹⁶³. In addition to T-cells, endothelial cells, VSMCs, macrophages, and platelets all express CD40L and CD40 ^{164 165}. As CD40 ligation promotes the inflammatory activation of all the major cell types participating in atherosclerosis, genetic ¹⁶⁶ or antibody-mediated ¹⁶⁷ disruption of CD40L signaling significantly decreases lesion size, macrophage and T-cell content, as well as VCAM-1 expression, and promotes the formation of a fibrous collagen-rich plaque.

CD8⁺ T-cells also populate lesions ¹⁶⁸, but their relevance and contribution is less well understood ². Conditions that enhance CD8⁺ T-cell function and increase their numbers by modulating co-stimulation ¹³⁴ or co-inhibition ¹³⁰ promote atherosclerosis in experimental settings. Though CD8⁺ T-cells kill VSMCs in aortic aneurysms ¹⁶⁹, their role in atherosclerosis remains uncertain.

Natural Killer T-cells and γδ T-cells

A subpopulation of T-cells that include Natural Killer T (NKT) cells and γδ T-cells express semi-invariant TCRs. NKT cells constitute a heterogeneous family of cells that has characteristics of both NK cells and conventional T cells ¹⁷⁰. NKT cells express TCRs that recognize glycolipid antigens presented on CD1d, an MHC class I–like molecule expressed by antigen-presenting cells ¹⁷⁰. The normal development of NKT cells also requires CD1d, found in human lesions ¹⁷¹. CD1d deficiency results in reduction of plaque burden in atherosclerosis-prone mice ^{172 173 174}. Administration of a synthetic glycolipid that activates NKT cells via CD1d induces IFN-γ, MCP-1, TNF-α, IL-2, IL-4, IL-5, and IL-6 production within lesions ¹⁷². Lipid antigens present within plaques may activate NKT cells, which appear to participate in the early phases of atherogenesis ¹⁷⁵.

Although the majority of T-cells express the classic αβ TCR, a subset expresses γδ TCRs with limited diversity. γδ T-cells represent fewer than 1-5% of circulating T-cells, but are enriched in sites of chronic inflammation ¹⁷⁶. Importantly, γδ T-cells may recognize a wide array of antigens in the absence of MHC presentation by APCs ¹⁷⁶. Some γδ T-cells recognize lipid antigens presented by CD1. These cells reside in lesions ¹⁷⁷ and secrete IFN-γ and other cytokines, but the extent of their contribution to the pathobiology of atherosclerosis remains unclear ¹⁷⁸.

Regulatory T-cells and immunosuppression

Regulatory T-cells (Tregs) control other T-cell types and suppress their activation in secondary lymphoid organs, or their effector functions in peripheral tissues such as plaques, either directly or indirectly via actions on APCs (Figure 1). Tregs contribute to the maintenance of tolerance to self-antigens and the regulation of immunity. Of the different Treg subsets, CD4⁺CD25⁺FoxP3⁺ Tregs are the best characterized. ¹⁷⁹ FoxP3 serves as a lineage-specific transcription factor involved in Treg suppressive function. These well-characterized ‘natural’ Tregs, generated during thymic development, comprise 5-10% of peripheral CD4⁺ T-cells. However, naïve CD4⁺T-cells induce additional Tregs during antigen-specific immune responses in the presence of IL-2 and TGF-β ¹⁸⁰. Other surface molecules expressed by Tregs include CTLA-4, GITR (Glucocorticoid-Induced Tumor necrosis factor Receptor), and CD127. These antigen-specific Tregs inhibit effector T-cell activation either by direct contact inhibition or through the release of the anti-inflammatory cytokines transforming growth factor (TGF)-β and IL-10. These cytokines made by Tregs can mitigate atherogenesis in mice.

IL-10-deficient mice fed an atherogenic diet have increased lesion area, effector T-cell infiltration, and IFN-γ expression ¹⁸¹. IL-10-transfected mice demonstrate opposite results, with IL-10 also impeding the modified LDL-mediated endothelial recruitment of monocytes ¹⁸². Bone marrow transplantation of transgenic IL-10 overexpressing T-cells also decreases lesion size and inflammation ¹⁸³.

Inhibition of TGF-β signaling using neutralizing antibodies ¹⁸⁴ or recombinant soluble TGF-β receptors ¹⁸⁵ accelerates the development of atherosclerotic lesions in ApoE-deficient mice and favors the development of lesions with increased monocyte and lymphocyte accumulation and decreased collagen content. TGF-β exerts its atheroprotective effect by dampening CD4⁺ T-cell effector function. Indeed, mice carrying dominant-negative TGF-β receptors on CD4⁺ T-cells (CD4dnTβRII) exhibit increased inflammation and a paucity of mature interstitial collagen fibers within vascular lesions ^{186 187}. CD4dnTβRII effector T-cells inhibit the production of lysyl oxidase, the extracellular enzyme needed for collagen cross-linking, limiting collagen maturation in the atherosclerotic plaque while having little effect on collagen degradation ¹⁸⁸. Although numerous cell types produce TGF-β and IL-10, including endothelial cells, smooth muscle cells, macrophages, and platelets, regulatory T-cells may constitute an antigen-specific source of these anti-inflammatory cytokines ¹⁸⁹.

In mice, decreased Treg numbers ¹⁸⁹ or Treg function secondary to absence of ICOS ¹²⁸ leads to increased lesional CD4⁺ T cells and macrophage numbers as well as amplified expression of the pro-inflammatory cytokines IFN-γ and TNF-α. Moreover, patients with acute coronary syndromes have reduced circulating Treg numbers and suppressive function ¹⁹⁰. Conversely, the adoptive transfer of Tregs ¹⁹¹ or Treg-induction secondary to measles virus nucleoprotein vaccination (known to induce immunosuppression) ¹⁹² inhibits macrophage and T-cell accumulation within lesions. In addition, leptin deficiency in atherosclerosis-prone mice reduces atherosclerotic lesion formation, in association with diminished Th1 responses in addition to a marked increase in the number and suppressive function of Tregs ¹⁹³. Given that the adipokine leptin increases in obesity, the possibility that leptin limits Treg function presents an additional link between obesity and inflammation. Interestingly, deficiency of the chemokine CXCL10 (IP-10) decreases atherogenesis not only by diminishing the recruitment of CD4⁺ T-cells, but also by increasing intra-plaque Treg numbers ¹⁹⁴.

Therapeutic modulation of Tregs has undergone scrutiny in experimental settings. Anti-CD3 antibody treatment decreases CD3/T-cell receptor complex expression and has immunosuppressive functions by increasing Treg numbers and TGF-β levels, thus reducing lesion initiation and progression ¹⁹⁵. Induction of tolerance through the oral administration of oxLDL ¹⁹⁶ or hsp60/65 ¹⁹⁷ to atherosclerosis-prone mice increases Treg numbers in secondary lymphoid organs as well as TGF-β and IL-10 levels, again attenuating experimental atherogenesis. These reports highlight the central role of Tregs in tolerance induction, which may constitute the mechanism of atheroprotection reported in earlier similar studies to hsp65 ^{198 199} and β2-glycoprotein I ²⁰⁰.

B-cells and humoral immunity

B-cells infiltrate the adventitial layer of human coronary lesions ²⁰¹ and atherosclerosis-prone mice ²⁰² where they may form lymphoid follicles. The plaques of ApoE-deficient mice contain B-cells at all stages of the disease ²⁰³ (Figure 1). Splenectomized mice have increased susceptibility to atherosclerosis, a situation reversed by the transfer of B-cells and the production of anti-oxLDL antibodies ²⁰⁴.

Both humans and atherosclerosis-prone animals have antibodies against oxLDL particles ^{205 206}. Germ-line encoded natural anti-oxLDL IgM antibodies produced by B1-cells bind the oxidized phospholipids on oxLDL and also recognize phosphorylcholine in the cell wall of Streptococcus pneumoniae ²⁰⁷. Taking advantage of this molecular mimicry, pneumococcal vaccination of atherosclerosis-prone mice decreases the extent of experimental atherosclerosis ²⁰⁸.

Anti-hsp60 antibodies cross-react between microbial and eukaryotic hsp60/65, a consequence of high sequence conservation. As such, infections, for example by Chlamydia pneumoniae, might result in breaking tolerance to self-hsp60 and promoting auto-immunity and atherogenesis ²⁰⁹. Indeed, experimental results using hsp65 as an immunogen for vaccination ^{210 211} mediate endothelial cytotoxicity ²¹² and promote atherogenesis. Over all, B-cells are considered to mediate protective immunity during the development of atherosclerosis, possibly by preventing antigens from reaching lesions.

Conclusion

The evidence reviewed here highlights the extensive role of innate and adaptive immunity in atherosclerosis, from its initiation to its final thrombotic complications. Our improved pathobiologic understanding of this disease allows the detection of patients at high risk and the design and development of novel treatment modalities targeting cellular and molecular mediators. These have already led to the identification of certain inflammatory indicators, such as CRP, as biomarkers of adverse cardiovascular events, allowing a better risk stratification of patients and targeting of therapy ²¹³. In addition, statins have emerged as powerful anti-inflammatory and immunomodulatory agents, with extensive clinical use in primary and secondary prevention. Despite these strides, death from cardiovascular disease continues to increase worldwide, with many patients experiencing cardiovascular events despite statin, anti-platelet and anti-hypertensive treatment ²¹⁴.

As a systemic and non-selective modulation of immune responses could lead to adverse effects including acquired immunodeficiency with ensuing infectious and oncologic complications, a more subtle and targeted approach of the immune response would probably prove advantageous. Based on the myriad immune mechanisms involved in atherogenesis, a wide range of potential therapies appear on the horizon, with the goal of diminishing the global cost paid by humanity to this scourge.