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The handle http://hdl.handle.net/1887/58107 holds various files of this Leiden University dissertation
Author: Heijden, Thomas van der
Title: The interplay between lipids and the immune system in atherosclerosis
Date: 2017-12-19
Chapter 1. General introduction
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chapter 1
general introduction
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introduction
Cardiovascular diseases (CVD) are among the most frequent causes of death in the world and a recent study showed that despite a decrease of 39% in age-specific death rates between 1990 and 2013, CVD related death increased with 41%.1 Furthermore, there is a shift in the global burden of diseases from death early in life to an increased number of years lived with disability due to the consequences of CVD.2
There is a large variation between various regions, as for in low-income countries the risk-factor burden to develop CVD is lower as compared to high-income countries.3 However, low-income countries have increased major CVD, such as myocardial infarction or stroke, and death rate compared to high-income countries.4 Factors, which can contribute to these differences are for example the affordability for health care and more frequent use of medication. In low-income countries, there is a decline in drug use compared to that of high-income countries despite the accessibility of effective drug treatments, such as statins.5 In high-income countries, there is an increased incidence of non-major CV event (like hypertension and angina).4 The main underlying pathology of CVD is the development of atherosclerosis in the medium and large-sized arteries, which can eventually cause myocardial infarction or ischemic stroke, the leading causes of death worldwide.2,6 atherosclerosis
Atherosclerosis is a chronic inflammatory disease, which proceeds in the context of high levels of blood cholesterol. Cholesterol circulates in the lipoproteins very- low density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL). These different lipoproteins are thought to be either atherogenic (VLDL and LDL) or anti-atherogenic (HDL).7 Besides high cholesterol levels, innate and adaptive immune responses contribute to the development of atherosclerosis.8 Because of the major consumption of sugary drinks and fatty food, mostly in Western countries, the prevalence of atherosclerosis and related CVD is high.9,10 However, also non-modifiable factors such as age, gender and family history can contribute to atherosclerotic lesion development. Interestingly, already during fetal development, atherogenesis occurs in human aortas.11 To determine the contribution of maternal hypercholesterolemia to lesion development in the offspring, fetuses (spontaneous abortions or premature newborns that died within 12 hours) from hypercholesterolemic and normocholesterolemic mothers have been investigated11, and it appeared that only during the first 6 months of fetal development, levels of fetal and maternal cholesterol levels were correlated. In this study, a systemic assessment of lesion formation was performed in human fetal aortas from these mothers. This revealed that fetuses from hypercholesterolemic mothers have increased atherosclerotic lesion development compared to those from normocholesterolemic mothers.11 Also in the developing countries levels of atherosclerosis do not always correlate with traditional risk factors such as high cholesterol levels and smoking, suggesting that other processes may be involved in lesion development, like the immune system, which will be described.12,13
atherosclerotic lesion development
A healthy arterial wall consists of three layers: the endothelial inner lining, the media, and the adventitia. The tunica intima is the innermost layer of the artery, which consists of a monolayer of endothelial cells that is in contact with the blood, lying upon a basement membrane consisting of the extracellular matrix. The media, which is the middle layer, is the thickest layer and consists of smooth muscle cells, which are embedded in elastin layers. The outer layer of the artery is called the
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adventitia and consists mainly of connective tissue, vasa vasorum (network of small blood vessels), and autonomic and sympathetic nerves.14
Atherosclerosis develops primarily at sites in the cardiovascular system with a low endothelial shear stress, for example at the branching points of the aorta.15,16 Atherosclerotic lesions are asymmetric focal thickenings, which can develop in the intima of medium and large-sized arteries. Most lesions consist of (immune) cells, lipids, connective-tissue elements, and debris.17 After endothelial damage, lipids, as well as inflammatory cells, accumulate in the intimal layer of large and medium- sized arteries. In 1904 the German pathologist Felix Marchand proposed for the first time the term “atherosclerosis”, which is derived from the Greek “athero”
(meaning gruel) and “sclerosis” (meaning hardening) to described the fatty streak he observed in an artery.18 Below the development of an atherosclerotic lesion from the initial phase up to arterial thrombosis is described.
initiation
The initiation of the atherosclerotic lesion starts with a change in the endothelial cells of the arterial wall.19 Upon different stimuli (such as pro-inflammatory mediators or dyslipidemia) endothelial cells upregulate their expression of adhesion molecules, which can subsequently capture leukocytes.20 Changes in the permeability of the endothelium can lead to migration of leukocytes such as monocytes into the intimal layer, upon which these monocytes can mature into macrophages. Cholesterol containing particles can be taken up by monocyte- derived macrophages. In a hyperlipidemic environment, the uptake of cholesterol is a process that is normally regulated by the LDL receptor.21. There are several mechanisms (e.g. endothelial cell induced modification of LDL) which can lead to the oxidative modification of LDL.22
The uptake of oxidized LDL (oxLDL) by macrophages in the atherosclerotic plaque is mediated by scavenger receptors (like CD36 and scavenger receptor A), which can lead to unlimited cholesterol uptake and the formation of foam cells.23 Besides macrophages, also DCs can become foam cells by accumulating oxLDL.24 Cholesterol efflux transporter (such as ABCA1 and ABCG1) are present on the cell membrane maintaining the cellular cholesterol homeostasis.25 Despite these cholesterol efflux transporters, massive uptake of cholesterol occurs26,27, which in turn results in the initiation of an atherosclerotic lesion called a fatty streak. This initial lesion or intimal layer consists mainly of foam cells, located within the subendothelial space.
lesion progression
After the initial formation of the atherosclerotic lesion, smooth muscle cells migrate from the media to the intima and start proliferating upon mediators like platelet-derived growth factors.28 Differentiated smooth muscle cells in the intima produce collagen and elastin which covers the plaque in the form of a fibrous cap.20 During this process, immune cells such as T cells, mast cells and neutrophils start to accumulate within the plaque.
In the progressing lesion, immune cells like macrophages but also non-immune cells such as smooth muscle cells can undergo apoptosis or necrosis, thereby releasing their (lipid) content, which leads to the formation of a necrotic core.29 Advanced lesions also contain cholesterol crystals.30
the vulnerable lesion and thrombosis
Upon progression to an advanced stage, the fibrous cap of a plaque can become thinner. This cap serves as a barrier between the lumen of the vessel and the necrotic core. Normally this cap protects the lesion from breaking but upon activation of
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proteases such as MMPs and the release of inflammatory cytokines produced by macrophages, T cells and mast cells, the fibrous cap becomes thinner, thus increasing the risk of plaque rupture.31
Eventually, upon rupture, the contents of the lesion are released into the vessel, which can activate the blood coagulation system. Tissue Factor (TF) that has accumulated in the lesional core can come into contact with blood coagulation components present in the blood vessel. This can lead to the formation of a thrombus that can occlude the artery.20 Thrombosis can also occur upon plaque erosion, which is described as thrombus formation without plaque rupture.32 Plaque erosion is mainly characterized by the loss of the endothelial cell layer, while the plaque itself seems fairly stable.32 Upon the complete occlusion of the blood vessel by the thrombus, an acute cardiovascular event such as a myocardial infarction or a stroke can occur.
mouse models
Atherosclerosis is a complex disease and to understand the processes involved in the development of atherosclerotic plaques, preclinical studies are helpful. One of the most frequently used animal models in atherosclerosis research is the mouse.
There are several mouse models that are used to investigate atherosclerotic lesion development.33 Already more than 25 years ago two mouse models, LDLr-/-34 and apoE-/-35,36, were described, which are up to now frequently used to investigate atherosclerosis. In both models, atherosclerosis is driven by non-HDL hyperlipidemia (VLDL and LDL). A mouse model which has a more human like lipoprotein profile is the ApoE3-Leiden-CETP mice which express human cholesteryl ester transfer protein (CETP).37 Below the different mouse models are discussed in more detail.
apoE-/- mice
In normal lipoprotein metabolism, apoE is primarily involved in the transport and delivery of lipids. It thereby serves as a ligand for receptors to clear remnants of both chylomicrons and VLDL.36 In C57Bl/6 mice and heterozygous apoE mice on a regular chow diet, cholesterol levels in the serum are approximately 100 mg/dL38,39. In mice completely lacking apoE, cholesterol levels in the serum are elevated to 400 to 800 mg/dL.36,39 The age or sex of the mice does not affect the increased cholesterol levels.36 Circulating lipoprotein profiling of these mice revealed that nearly 80%
of the cholesterol is carried by the lipoproteins VLDL and LDL, while in wild-type mice HDL is the major lipoprotein carrier in the circulation.36 ApoE-/- mice on chow diet spontaneously develop atherosclerotic lesions.36 Accumulation of foam cells is observed at the age of 8 weeks on a chow diet while the first lesions are detected at the age of 10 weeks.35 Younger mice (age 11–21 weeks) show early atherosclerotic lesion development and there is a progressive development of plaques in these mice between 12 and 38 weeks of age.39 Feeding apoE-/- mice a Western-type diet increases the cholesterol levels in the serum to levels ranging from 1085 to 4402 mg/dL.38 In apoE-/- mice on Western-type diet foam cell accumulation is observed already at the age of 6 weeks and the first lesions are found at the age of 8 weeks.35
LDLr-/- mice
The LDLr is a surface receptor expressed primarily on mammalian liver cells, which can clear lipoproteins such as LDL from the circulation by binding apoE.21,40 This contributes to the regulation of plasma cholesterol levels. Feeding LDLr-/- mice a normal chow diet results in plasma cholesterol levels of, approximately 200 mg/
dL, which is about twice as high as in wildtype mice, both in females and males.34 There are no differences in non-fasting triglyceride levels in LDLr-/- mice compared
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to wildtype.34 In LDLr-/- mice fed a high cholesterol diet (1% cholesterol and 4.4%
fat) plasma cholesterol levels increase to 800 mg/dL, which is primarily caused by elevation of LDL levels.41 Western-type diet (0.06% cholesterol and 21% milk fat) induced extreme hypercholesterolemia with cholesterol levels of 1600 mg/dL and lipoprotein analysis showed an enrichment in LDL and VLDL particles in the blood.41
apoE3-Leiden and apoE3-Leiden-CETP mice
A mouse model more closely to the human situation is the apoE3-Leiden mouse.
In this transgenic mouse, a form of the human apoE3 has been introduced in the genome.42 The apoE3-Leiden mouse displays a plasma lipid profile comparable to that in humans.42 Atherosclerotic lesion development in these mice is a relatively slow process which resembles the human situation.43 Up to 16 weeks on high fat/
low cholesterol diet this mouse develops no lesion and between 18 and 27 weeks, there is only initial lesion formation as foam cell formation occurs.43 After 29 weeks atherosclerotic lesions are formed and in 2 weeks progressed lesion have been observed.43 ApoE3-Leiden mice can synthesize endogenous apoE and are therefore less susceptible for atherosclerosis compared to apoE-/- mice.38 A variant of the ApoE3-Leiden mouse is the ApoE3-Leiden-CETP mice which express human CETP.37 CETP transfers cholesterol esters from HDL to triglyceride rich particles such as VLDL.44 This shift from HDL to VLDL resulted in ApoE3-Leiden-CETP mice to 2-fold increased VLDL levels and 7-fold increased atherosclerotic lesion development compared with the apoE3-Leiden mice.37
comparison of the apoE-/-, LDLr-/-, apoE3-Leiden and apoE3-Leiden-CETP mice LDLr-/- and apoE-/- mice are the most frequently used models to investigate atherosclerotic lesion development. Most studies use one of these models to investigate a certain therapeutic approach or genetic influence on the development or progression of atherosclerosis. There are some important differences between these two models which should be taken into account when designing a research strategy. For example, while in apoE-/- mice VLDL levels are increased, in LDLr-/- increased plasma cholesterol levels are primarily caused by elevated LDL.
In a study to compare between both models, apoE-/- and LDLr-/- mice were put on a high-cholesterol diet (1.25%) containing cholic acid. After 3 months, apoE-/- mice had higher cholesterol levels and increased atherosclerotic lesion development in the aortic root compared to the LDLr-/- mice.45 In addition, necrotic cores in atherosclerotic lesions of apoE-/- mice were larger. On the other hand, LDLr-/- mice displayed more T lymphocytes in the lesions.45 In general, lesion development in apoE-/- and LDLr-/- mice are less representative with atherosclerotic lesion development in humans.46 In contrast, apoE3-Leiden mice develop lesions with more comparable characteristic of the human atherosclerotic lesion from the formation of fatty streaks to moderate and severe lesions.47 Furthermore, apoE3-Leiden show reduced cholesterol levels and anti-atherogenic effect upon statin treatment (in line with humans) while in apoE-/- mice cholesterol levels are not decreased and in LDLr-/- mice results are varying.48–50 This makes the apoE3-Leiden mice suitable for testing of combination therapies. The apoE3-Leiden-CETP mice can be used to investigate the effect of HDL- increasing therapies, as modulation of HDL cholesterol levels is possible through the induced CETP.
Similar to the apoE-/- and LDLr-/- mice, apoE3-Leiden mice do not develop plaque rupture or thrombus formation which is a major difference with the human situation.20,47 However, by using these models, there are many advantages, such as the ability to create specific gene-knockout models, which make the mouse the model of choice in atherosclerosis research.46
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translation from mice to humans
Experimental studies in mice are important to find novel targets for further investigation, but there are still challenges to translate these findings to humans.20 Especially the lack of plaque rupture and thrombosis in mice, which are complications causing acute cardiovascular syndromes as regularly seen in humans, is a disadvantage of using mice.51 Also, the very high cholesterol levels, which are needed to develop atherosclerosis in mice, are not representative of the levels encountered in humans.52 Despite these disadvantages, there are many overlapping pathways, which illustrate the importance of mouse models in atherosclerosis research53, and can be used for mechanistic studies to help finding novel targets.20
Due to the complex and multifactorial processes that contribute to the development of atherosclerosis, the design of a study to investigate a novel therapeutic in humans is complicated. The slow onset and progression of the disease further frustrate such clinical trials. One group of patients, which is frequently used to investigate the effect of dyslipidemia on atherosclerosis are patients with familial hyperlipidemia (FH). Deficiency in the LDL receptor, one of the underlying causes of FH, is an autosomal dominant inherited disease which is characterized by high plasma cholesterol/LDL levels and premature coronary heart disease.54,55 Untreated FH patients can develop coronary heart disease already before the age of 20.56
Another disease with severe hyperlipidemia is type III hyperlipoproteinemia and this is associated with familial apoE deficiency.57,58 Deficiency of apoE in humans results in high VLDL levels with normal LDL, HDL and triglyceride levels.57 Humans with apoE deficiency develop premature coronary artery disease, in the Western society mostly before the age of 60.56
Besides high cholesterol levels, atherosclerosis is an inflammatory disease.59 In 2008, a multicenter trial showed how C-reactive protein (CRP), which is an inflammatory biomarker, could identify apparently healthy men and women with low LDL cholesterol levels but high CRP levels who were at risk for CVD events.60 In these patients, statin treatment reduced the inflammation and moreover reduced major cardiovascular events like myocardial infarction and stroke compared to placebo treatment.60 This indicates that not only high cholesterol levels contribute to atherosclerosis but that also the immune system is involved.
atherosclerosis and the immune system
High plasma (LDL) cholesterol levels contribute to atherosclerotic lesion development. Accumulation of lipids in the arterial wall is one of the primary processes in atherogenesis, however, also immune cells are involved (Figure 1).59 The immune system can be classically divided into two immune responses: the innate and the adaptive. The identification of a number of immune cell subsets, from both the innate and the adaptive immune system, within the plaque led to the hypothesis that the immune system contributes of the atherogenesis process.61 Interestingly, the fatty streak, which is the first visible lesion in atherosclerotic lesion development, consists mainly of monocyte-derived macrophages and T cells.62
The innate immune response is characterized by a rapid but non-specific reaction to foreign pathogens, which can result in an inflammatory response. Cells belonging to the innate immune system are also antigen presenting cells (such as macrophages and DCs), which can internalize pathogens and present them to lymphocytes (such as CD4+ and CD8+ T cells), which are part of the adaptive immune system. Immune responses of cells from the adaptive immune system are thus initiated via antigen presentation. In atherosclerosis one of the antigens is modified LDL and human atherosclerotic lesions were observed to contain CD4+ T cells, which respond specifically to oxLDL fragments.63
monocytes/macrophages
In mice, circulating monocytes are classified by the expression of CD11b/Ly-6C and in humans by CD14/CD16.64,65 Recruitment of monocytes to the atherosclerotic lesion is induced when endothelial cells become activated, leading to increased expression of adhesion molecules like VCAM-1.66 After entering the arterial wall, monocytes differentiate into macrophages. Macrophage colony-stimulating factor (m-CSF) is a cytokine, secreted by several cell types including endothelial cells and fibroblasts, that induces the differentiation of monocytes into macrophages.14,67 Macrophages contribute to early lesion development as macrophages can accumulate lipids and become foam cells which lead to fatty streak formation. In progressed lesions, these foam cells undergo apoptosis or necrosis and lead to the formation of a necrotic core.64 Macrophages in the atherosclerotic lesion can process local antigens, such as oxLDL. Expression of these antigens on their cell surface via MHC molecules leads to antigen-specific T cell activation.68
Macrophages are classified in several different subsets but the most well-known subsets are M1 and M2.69 Classically M1 is described as pro-inflammatory and M2 as anti-inflammatory,70 and both subsets have been described in atherosclerosis.71 M1 macrophages are described as pro-atherogenic and produce pro-inflammatory cytokines such as TNF-α and IL-1ß.72 In contrast, M2 macrophages produce anti- inflammatory cytokines like IL-10 and dampen the inflammatory response.72 However, recent studies have shown that there are many other subsets and therefore a new nomenclature has been proposed.73 It is suggested to describe the activator (e.g. IL-4, TNF-α), instead of using M1, M2 and the more recently described M2a, M2b macrophages. Furthermore, to avoid misinterpretation a description of used markers should be indicated, such as transcription factors in CD4+ T helper subsets.
figure 1. Overview of the interplay between lipids and the immune system in the development of atherosclerosis. Figure adapted from Libby P, Ridker PM, Hansson GK. Progress and
challenges in translating the biology of atherosclerosis. Nature.
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Vasa vasorum Lipid
core B cell
Antigen- presenting cell T cell
oxLDL
Scavenger receptor
Apo-CIII
VLDL
LDL VLDL
TLR2
TLR2 Antibody
LDL
Pro-inflammatory monocyte
Endothelial cell
Treg cell TH1
cell
TGF-β IL-10
ABCA1 Apo-AI
HDL Chol
Chol Chol
Lipid-laden macrophage
Pre-β HDL
CETP ABCG1
cell
ABCG1 C Foam-cell formation
TH1 IFN-γ
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neutrophils
Although abundantly present in the circulation, neutrophils do not accumulate in very high numbers in either mouse or human lesions.74 Neutrophils have a relatively short lifespan of a few days and remains are cleared by macrophages, which may explain their low numbers in atherosclerotic lesions.75 Hyperlipidemia, for example, causes neutrophilia and in apoE-/- mice it correlated with early lesion formation.76 Furthermore, depletion of neutrophils reduced atherosclerotic lesion development.
In human atherosclerotic lesions, neutrophils localize in rupture-prone lesions and are associated with lesion instability,77 which suggests that neutrophils contribute to plaque stabilization.
dendritic cells
Dendritic cells are professional antigen presenting cells and can take up antigens in the lesion. Migration and subsequent presentation of these antigens in lymph nodes, leads to the activation of predominantly antigen specific CD4+ T cells.78 DCs can get activated via pattern recognition receptors signaling caused by stimuli such as pathogen-associated molecular patterns.79 Upon activation, dendritic cells mature and increase their expression of several surface molecules, such as costimulatory molecules CD80 and CD86 and antigen presenting molecules like MHC-I and MHC-II.80 Activation leads to the downregulation of endocytic activity to reduce the presentation of encountered antigens after the first stimulus.81
In atherosclerosis, dendritic cells can present atherosclerosis-related antigens.
Dendritic cells have been found in both mouse and human atherosclerotic lesions,82,83 mainly in rupture-prone regions and are frequently clustering with T cells.83 Dendritic cells isolated from atherosclerotic lesions highly express MHC molecules and are thereby able to induce T cell proliferation.84 Furthermore, carotid artery lesions from patients with ischemic complications showed increased activation of dendritic cells compared to non-symptomatic patients.85 Dendritic cells in LDLr-/- and apoE-/- mice on a Western-type showed impaired migration to the lymph nodes possibly due to impaired response to TLR stimulation and thereby reduced activation of dendritic cells.86,87
Dendritic cells are the initiators of antigen-specific adaptive immune responses and pre-clinical data has illustrated the potential effect of vaccination with, for example, oxLDL-pulsed dendritic cells in LDLr-/- mice for reducing atherosclerotic lesion development.88 Currently dendritic cells based vaccination strategies are in development for potentially treatment of atherosclerosis.89,90
CD4+ T cells
The majority of T cells that are found in the atherosclerotic lesion are CD4+ T cells.91,92 Naïve CD4+ T cells have the ability to differentiate in several subsets of T helper cells, which can be either pro-inflammatory like Th1, Th2 and Th17 cells, or anti-inflammatory like and regulatory T cells (Tregs).93
Th1The majority of CD4+ T cells in atherosclerotic lesion development is of the Th1 subset.66 T-bet is the transcription factor leading towards differentiation the Th1 lineage of T cells94 and this subset is characterized by the production of IFNγ.95 The development and activation of Th1 cells from naïve CD4+ T cells is induced by the production of IL-12 by macrophages and DCs.96,97
Vaccination in LDLr-/- mice against IL-12 resulted in reduced lesion development, with an increased collagen content.98 In addition, IL-12 vaccination led to decreased serum IFNγ levels and resulted in a complete absence of IFNγ in the plaque itself.98
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IFNγ produced by Th1 cells in turn can activate macrophages and DCs, while inhibiting proliferation of vascular smooth muscle cells.99 LDLr-/- mice deficient in T-bet showed reduced atherosclerosis which indicates that Th1 cells induce atherosclerotic lesion development.100 Downregulation of the Th1 response using a phosphodiesterase inhibitor which blocks the polarization of Th1 cells, results in reduced atherosclerotic lesion development in apoE-/- mice.101 Administration of IFNγ leads to increased lesion size in apoE-/- mice.102 Patients with acute coronary syndromes display elevated levels of circulating Th1 cells compared to stable angina patients.103 Together, these studies illustrate the pro-atherogenic role of Th1 cells.
Th2Only a small part of the CD4+ T cells present in atherosclerotic plaque are Th2 cells, which is characterized by the production of IL-4, IL-5, and IL-10. The transcription factor GATA3 is important in the development of these Th2 cells.104 Some studies show that Th2 cells are pro-atherogenic, as for example, IL-4 deficiency results in reduced atherosclerotic lesion development.105,106 Another study showed that IL-4 deficiency did not affect atherosclerosis in either apoE-/- or LDLr-/- mice.107 T cells are activated via antigen stimulation but also co-stimulatory molecules, such as OX40, contribute to its activation. Blocking the OX40-OX40L pathway with an anti- OX40L antibody reduced Th2 activation in LDLr-/- mice fed a WTD for 2 weeks.108 Furthermore, atherosclerotic lesion development was reduced in LDLr-/- mice treated with anti-OX40L for 6 weeks during WTD.108 These studies indicate a pro- atherogenic role for Th2 cells. On the other hand, IL-5, which is also a Th2 related cytokine, appears to have atheroprotective properties. IL-5 was shown to stimulate the proliferation and antibody production of B1 cells which cells are mainly described as atheroprotective.109 IL-5 deficiency in LDLr-/- mice showed increased atherosclerotic lesion development.110 Thus, in contrast to the Th1 subset, the effect of Th2 cells is less outspoken in atherosclerosis.
Th17Th17 cells are IL-17 producing T cells and their role in atherosclerosis remains controversial.20 In many inflammatory diseases, Th17 cells are involved and this T cell subset has been observed in both mouse and human atherosclerotic plaques.111,112 Previously, IL-17 expression was found increased in atherosclerotic lesions of symptomatic patients as compared to those of non-symptomatic patients112 Also, blockade of IL-17 in apoE-/- mice reduced lesion development.111 In contrast, another study showed that IL-17 deficiency in apoE-/- mice accelerated early lesion development, which was partly caused by modulation of IFNγ and IL-5 production by CD4+ T cells.113 These studies indicate that the contribution of Th17 cells to atherosclerosis remains contradictive.
Regulatory CD4+ T cells (Tregs)
Tregs can inhibit the T cell-mediated immune responses via either suppressive cytokines (such as IL-10, TGF-ß, and IL-35) or by direct cell-cell contact with effector T cells.114,115 Foxp3 is the transcription factor of Tregs and in atherosclerosis, Tregs exert atheroprotective effects. In human atherosclerotic lesion, the number of Tregs has for example be associated with lesion stability.116 In patients with coronary artery disease, the number of circulating Tregs is decreased compared to controls.117 In addition, the suppressive capacity of Tregs was reduced in patients with acute coronary syndromes compared to stable angina patients.118
In isolated peritoneal macrophages from C57Bl/6 mice, treatment with Tregs reduced the accumulation of oxLDL.119 To investigate the effects of Treg depletion
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on atherosclerosis, transgenic mice termed “depletion of regulatory T cell” (DEREG) were generated.120 These mice express a diphtheria toxin (DT) receptor which is under the control of the Foxp3 gene locus, which enables specific depletion of Foxp3+ Treg upon DT administration.120 Treg depletion in DEREG/LDLr-/- mice increased lesion development.121 Furthermore, there was reduced infiltration of macrophages and Major Histocompatibility Complex (MHC) class II molecule expressing cells in the atherosclerotic lesions.121 Vice versa, expansion of Tregs in LDLr-/- mice suppressed atherosclerosis and the inflammatory response as demonstrated by reduced splenic Th1 (T-bet+) and CD4+IFNγ+ T cells.122
These studies show the potency of Tregs and their atheroprotective effects.
Therefore increasing the suppressive capacity or adoptive transfer of Tregs could be a novel therapeutic strategy for the treatment of atherosclerosis.
CD8+ T cells
Cytotoxic CD8+ T cells contribute to the host defense response. To become cytotoxic T cells, naïve CD8+ T cells are activated by antigen presenting cells that present antigens on MHC-I molecules. Upon activation CD8+ T cells can secrete large amounts of granzyme B and IFNγ inducing cell death of the target cells such as virus-infected antigen presenting cells.123
In atherosclerotic lesions of apoE-/- mice CD8+ T cells were observed, although in low numbers.91 The contribution of CD8+ T cells to atherosclerotic lesion development is more controversial than that of CD4+ T cells. In apoE-/- mice both lesion inflammation and vulnerability were shown to be increased by adoptive transfer of CD8+ T cells.124 Furthermore, CD8+ T cells show increased activation as demonstrated by increased proliferation and IFNγ secretion in WTD-fed apoE-/- mice compared to chow-fed apoE-/- mice.125
Other studies have made use of CD8+ T cell depleting strategies to establish its effects on atherogenesis. TAP1, for example, is an antigen transporter maintaining the MHC-I surface expression pathway and these TAP1-/- mice lack CD8+ T cells.126 In contrast to the pro-atherogenic effects of CD8+ T cells in the study mentioned above, lesion development in TAP1-/-apoE-/- mice was actually similar as in apoE-/- mice.127 To complicate the matter, a CD8+ T cells subset has been identified that can be anti-atherogenic:128 the regulatory CD8+CD25+ cell, which has been described both in mice and humans.129,130
Adoptive transfer of CD8+CD25+ T cells to apoE-/- mice reduced atherosclerotic lesion development and CD4+ T cell proliferation, while CD8+CD25- T cells did not exert these effects.131 This illustrates the importance of defining CD8+ T cells subsets in cardiovascular diseases.132 More studies will be necessary to determine the exact contribution of the CD8+ T cell and its subsets to atherosclerosis.
B cells
B cells mediate several immune and inflammatory responses. B cells have been detected in atherosclerotic lesions, although in smaller numbers as compared to T cells133 and its contribution to the development of atherosclerosis is controversial.
Mature B cell depletion using a specific CD20 antibody resulted for example in reduced atherosclerosis in both apoE-/- mice and LDLr-/- mice.134 On the other hand, removal of B cells via splenectomy increased atherosclerosis.135 Also, LDLr-/- mice which were B cell deficient showed increased lesion development which indicates a more protective function of B cells in atherosclerosis.136
Like T cells, these conflicting results could be caused by the several B cell subsets present. B cells are generally divided into either B1 cells or B2 cells.137 B1 cells can be further subdivided into B1a and B1b cells, whereas B2 cells are mostly
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subdivided into either follicular or marginal zone B cells. B1a and B1b cells showed atheroprotective effects by secreting natural antibodies which can induce the clearance of apoptotic cells and oxLDL lipoproteins.138–140 In contrast, B2 cells have been shown to be atherogenic in apoE-/- mice as depletion reduced atherosclerosis lesion development while adoptive transfer increased atherosclerosis.141 Recently, a regulatory type of B cells named Bregs has been identified.142 This subset represents a small population and participates in the suppression of immune responses via production of IL-10.143 This indicates that, like the T helper cell subsets, also B cells should be differentiated in their effects on atherosclerosis.
cholesterol crystals and NLRP3 inflammasome cholesterol crystals
Macrophages loaded with lipids become foam cells. These foam cells can die and thereby release intracellular cholesterol. Upon extracellular accumulation, cholesterol crystallizes, which leads to the formation of cholesterol crystals.26,144
Cholesterol crystals are present in atherosclerotic lesions and contribute to atherosclerotic lesion development.145 Large crystals are mostly observed in advanced lesions, however, also in earlier lesion development crystals have been detected.30 Using a combination of fluorescence confocal microscopy and laser reflection many small crystals were observed in apoE-/- mice already after two weeks of high cholesterol diet.30 Cholesterol crystals that accumulate in the plaque are able to activate the NLRP3 inflammasome and induce IL-1ß secretion from macrophages after LPS-priming.30,146
NLRP3 inflammasome
Inflammasomes are multi-protein complexes and are mainly expressed in myeloid cells like macrophages. Up to date, there are several inflammasomes described such as NLRP1, NLRP3 and NLRC4.147 One of the most extensively described inflammasomes is NLRP3, which is involved in the initiation and progression of several metabolic diseases. NLRP3 is also the inflammasome, which recognizes the most danger signals. Activation of the NLRP3 inflammasome requires two signals:
a priming signal, which leads to increased expression of NLRP3 and formation of pro-IL-1ß, and an activation signal. This leads to the formation of the multi-protein complex consisting of NLRP3, an apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1.148,149 Upon activation, the NLRP3 inflammasome secretes mature forms of caspase-1, which cleave biologically inactive pro-IL-1ß and this leads to the secretion of IL-1ß.150 The priming signal is mostly caused by microbial molecules or endogenous cytokines (TNF-α and IL-1ß) and the signal which activates the inflammasome can be several different stimuli like pore-forming toxins, ATP, cholesterol crystals.147 NLRP3 is described to be involved in atherosclerosis.30,146,151 NLRP3 and NLRP3-related genes were upregulated in human atherosclerotic lesions compared to non-atherosclerotic vessels.151 Furthermore, human atherosclerotic lesions stimulated with LPS and cholesterol crystals induced the release of IL-1ß.151 This shows that inflammasome activation might be a novel link between cholesterol accumulation and the inflammatory processes in atherosclerosis.
cytokines
Inflammatory and vascular cells contribute to the development of atherosclerosis amongst others by secreting anti- and pro-inflammatory cytokines.95 Cytokines are grouped into several subclasses such as chemokines, colony-stimulating factors (CSF), interleukins, interferons (IFN) and transforming growth factors (TGF). Examples of pro-atherogenic cytokines usually are the proinflammatory cytokines (e.g. IFNγ,
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TNF-α, IL-1ß, IL-6) while anti-atherogenic cytokines are usually anti-inflammatory cytokines (e.g. IL-10, TGF-ß, IL-35). In the human atherosclerotic plaque, a variety of these cytokines has been detected152, which can affect atherosclerotic lesion growth and stability. In the early stage of atherosclerosis, cytokines can, for example, alter the endothelial function. Recruitment of lymphocytes and monocytes in the vessel wall can be favored by the expression of adhesion molecules and specific chemokines by the vascular endothelium.153
therapeutic strategy in atherosclerosis
Statins were for a long time the most effective way to treat and/or prevent cardiovascular diseases.154 Besides the lipid lowering effects, statins also have anti- inflammatory properties. However, despite statin treatment, there is still a high residual risk.154
Currently, there are two patient groups which, despite statin therapy, have still high levels of circulating LDL cholesterol or high levels of high-sensitivity C-reactive protein (hs-CRP).155 Hs-CRP, a protein which is made by the liver upon inflammation, has been shown to be an important inflammatory biomarker for CVD.155
For the patient group with continuously high LDL levels, treatment with ezetimibe or PCSK9 inhibitors, possibly combined with statins, further reduces high cholesterol levels and the incidence of ACS.156,157 Recently, it was shown that the PCSK9 inhibitor alirocumab could strongly reduce LDL cholesterol levels.158
However, the group with lowered LDL cholesterol levels and remaining high hs- CRP levels would benefit from an anti-inflammatory treatment. This illustrates that besides the development of lipid lowering strategies, also new anti-inflammatory therapies are needed as a therapeutic strategy in atherosclerosis.
anti-inflammatory therapies
Currently, there are several clinical trials ongoing investigating the effect of an anti- inflammatory therapy to reduce atherosclerotic lesion development and below these trials or strategies are discussed.
Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS) One of the studies with a potential positive outcome is the CANTOS trial. This is a large clinical trial which investigates the effects of canakinumab in reducing recurrent MI, stroke, and cardiovascular death in patients with coronary artery disease.159 Canakinumab is a human antibody, which targets the pro-inflammatory cytokine IL-1ß. In human atherosclerotic coronary arteries, IL-1ß levels are increased.160 Several studies in humans and mice have shown that IL-1ß plays an important role in the development of atherosclerotic lesion development. Some data describing effects of canakinumab are already available. In well-controlled diabetes and high cardiovascular risk patients, canakinumab reduced IL-6 and hs-CRP levels.161 In a subset of patients with atherosclerosis, canakinumab also reduced these levels but on the vascular structure or function, no improvements were found.162 Furthermore, these patients had increased total cholesterol levels.
Recently, the outcome of the CANTOS trial have been described and canakinumab reduces levels of hs-CRP without affecting cholesterol levels.163 More importantly, the rate of recurrent cardiovascular events, such as myocardial infarction, was reduced compared to placebo.163 This results indicate that canakinumab is effective for patients who have a residual risk for cardiovascular events due to their high levels of hs-CRP.
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methotrexate
Another clinical trial investigating the effect of reducing inflammation on CVD is the CIRT trial (Cardiovascular Inflammation Reduction Trial), in which the effects of methotrexate on reducing cardiovascular diseases, such as myocardial infarction and stroke, are investigated.164,165 Methotrexate is a folic acid antagonist, which mainly acts as an anti-proliferative and immunosuppressive compound. It is used as an anti-inflammatory treatment in rheumatoid arthritis and does not affect cholesterol levels.166 Furthermore, usage of low dose methotrexate in patients with rheumatoid arthritis showed a 21% reduction in risk of CV events.167 Therefore, the objective of CIRT is to investigate whether a low dose methotrexate can reduce the risk of recurrent CV events in patients with prior myocardial infarction.164
colchicine
An anti-inflammatory agent which already showed promising results in reducing CVD is colchicine.168 Colchicine mainly acts anti-inflammatory by inhibiting neutrophil function.169 Recently, in a short-term treatment study, acute coronary syndrome patients treated with colchicine showed reduced plasma levels of pro-inflammatory cytokines IL-1ß and IL-6 compared to untreated patients.170 As a secondary prevention, in combination with high-dose statins, it was also seen to prevent CV events in patients with stable coronary disease.168 These results appear promising but 11% of the patients receiving colchicine in this study withdrew because of intolerance to the drug.
So, although colchicine showed reduced pro-inflammatory cytokines and reduced CV events, it is more likely to be used as an acute suppressor of pro- inflammatory cytokines instead of anti-inflammatory treatment in reducing atherosclerosis.
tregs
Besides reducing pro-inflammatory or increasing anti-inflammatory cytokines, also therapies using specific cell subsets may have beneficial effects in the treatment of atherosclerosis. Tregs are important in maintaining self-tolerance and can exert atheroprotective effects.171 Adoptively transferred CD4+CD25+ T cells resulted in a reduced atherosclerotic lesion development in mice.172 Also, increasing the number of Tregs or induce their suppressive capacity could also be a novel strategy. In mice, expanding of Tregs in vivo has been achieved and reduced initial atherosclerotic lesion development in LDLr-/- mice.122
Results are promising but there are still some concerns about the efficacy and long term safety, and for maximal efficacy, large numbers of Tregs are necessary at multiple time points.173 Although the majority of adoptively transferred Tregs will maintain their suppressive capacity, loss of Foxp3 expression in only a few cells can lead to differentiation into conventional T cells which might cause severe diseases.174 Furthermore, excessive activity of Tregs can induce immune tolerance against infectious agents or increased rates of tumor formation.173 However, improvement of these experimental therapies may eventually lead to a clinical application in humans.
In conclusion, several clinical trials are investigating new therapeutic strategies to dampen the inflammatory response during atherosclerotic lesion development.
The outcome of these studies will demonstrate if targeted therapies to reduce residual inflammatory risk in cardiovascular patients will be beneficial.
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Chapter 1. General introduction
thesis outline
The aim of this thesis is to identify new therapeutic immune targets, which can limit the development of atherosclerosis. As one of the potential contributing cytokine families to atherosclerosis, Chapter 2 reviews the current knowledge of the IL-12 cytokine family to cardiovascular diseases, including both experimental and clinical studies. Furthermore, the potential use of cytokines from this specific family as a biomarker in acute cardiovascular syndromes is discussed. One of the cytokines belonging to this family is IL-35 and in Chapter 3 the role of IL-35 in atherosclerotic lesion development is investigated. In this study we show that IL-35 treatment can reduce lesion development in apoE deficient mice. In Chapter 4 we focused on the interplay between lipid-laden antigen presenting cells, like macrophages and dendritic cells, and CD4+ T cells. Our findings demonstrate that the antigen presentation pathway is affected by lipid loading and suggests that there is a lipid- associated immune regulation. In Chapter 5 we investigated the effect of NLRP3 inflammasome inhibition by a small molecule named MCC950 on atherosclerosis.
Previous studies have shown that NLRP3 is involved in atherosclerotic lesion development. Furthermore, IL-1ß, which is secreted upon NLRP3 activation, is a pro-inflammatory cytokine and blocking via a monoclonal antibody is currently investigated in clinical trials. Treatment with MCC950 reduced atherosclerotic lesion development in apoE deficient mice. The results of the described studies in this thesis, including future prospects, are discussed in Chapter 6.
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