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Macrophage regulatory mechanisms in atherosclerosis
The interplay of lipids and inflammation
Neele, A.E.
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2018
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Neele, A. E. (2018). Macrophage regulatory mechanisms in atherosclerosis: The interplay of
lipids and inflammation.
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Macrophage regulatory mechanisms
in atherosclerosis:
the interplay of lipids and inflammation
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Macrophage regulatory mechanisms in atherosclerosis: the interplay of lipids and inflammation
PhD thesis, University of Amsterdam, The Netherlands Author: Annette Neele
Cover Design: Robin Tuijnenburg & Annette Neele Layout: Annette Neele
Printing: Ipskamp printing ISBN: 978-94-028-0877-3
Copyright 2018 © Annette E. Neele
Financial support by the Dutch Heart Foundation for the publication of this thesis is gratefully acknowledged.
The research described in this thesis was supported by a grant of the Dutch Heart Foundation (CVON 2011B019 Genius).
Further financial support for printing this thesis was kindly provided by the department of Medical Biochemistry (AMC) and Sanofi.
Macrophage regulatory mechanisms
in atherosclerosis:
the interplay of lipids and inflammation
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus
prof. dr. ir. K.I.J. Maex
ten overstaan van een door het College voor Promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel
op vrijdag 19 januari 2018, te 14.00 uur door Annette Elise Neele
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Macrophage regulatory mechanisms in atherosclerosis: the interplay of lipids and inflammation
PhD thesis, University of Amsterdam, The Netherlands Author: Annette Neele
Cover Design: Robin Tuijnenburg & Annette Neele Layout: Annette Neele
Printing: Ipskamp printing ISBN: 978-94-028-0877-3
Copyright 2018 © Annette E. Neele
Financial support by the Dutch Heart Foundation for the publication of this thesis is gratefully acknowledged.
The research described in this thesis was supported by a grant of the Dutch Heart Foundation (CVON 2011B019 Genius).
Further financial support for printing this thesis was kindly provided by the department of Medical Biochemistry (AMC) and Sanofi.
Macrophage regulatory mechanisms
in atherosclerosis:
the interplay of lipids and inflammation
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus
prof. dr. ir. K.I.J. Maex
ten overstaan van een door het College voor Promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel
op vrijdag 19 januari 2018, te 14.00 uur door Annette Elise Neele
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Promotores: prof. dr. M.P.J. de Winther AMC-Universiteit van Amsterdam
prof. dr. E. Lutgens AMC-Universiteit van Amsterdam
Co-promotores: dr. M.J.J. Gijbels AMC-Universiteit van Amsterdam dr. ir. J. Van den Bossche AMC-Universiteit van Amsterdam Overige leden: prof. dr. C.J.M. de Vries AMC-Universiteit van Amsterdam prof. dr. W.J. de Jonge AMC-Universiteit van Amsterdam
prof. dr. M.J.A.P. Daemen AMC-Universiteit van Amsterdam
prof. dr. N.P. Riksen Radboud Universiteit Nijmegen
prof. dr. J. Kuiper Universiteit Leiden
prof. dr. T.K. van den Berg Vrije Universiteit Amsterdam
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Promotores: prof. dr. M.P.J. de Winther AMC-Universiteit van Amsterdam
prof. dr. E. Lutgens AMC-Universiteit van Amsterdam
Co-promotores: dr. M.J.J. Gijbels AMC-Universiteit van Amsterdam dr. ir. J. Van den Bossche AMC-Universiteit van Amsterdam Overige leden: prof. dr. C.J.M. de Vries AMC-Universiteit van Amsterdam prof. dr. W.J. de Jonge AMC-Universiteit van Amsterdam
prof. dr. M.J.A.P. Daemen AMC-Universiteit van Amsterdam
prof. dr. N.P. Riksen Radboud Universiteit Nijmegen
prof. dr. J. Kuiper Universiteit Leiden
prof. dr. T.K. van den Berg Vrije Universiteit Amsterdam
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Chapter 1 General introduction 9 Chapter 2 Macrophage polarization: the epigenetic point of view 23 Chapter 3 Epigenetic pathways in macrophages emerge as novel 39
targets in atherosclerosis
Chapter 4 Inhibiting epigenetic enzymes to improve atherogenic 67 macrophage functions
Chapter 5 Macrophage Kdm6b controls the pro-fibrotic transcriptome 89 signature of foam cells
Chapter 6 Myeloid Kdm6b disruption results in advanced atherosclerosis 107
Chapter 7 Myeloid Ezh2 deficiency limits atherosclerosis 125
Chapter 8 Fine tuning of DNA methylation during the differentiation and 143 activation of human macrophages
Chapter 9 PCSK9 monoclonal antibodies reverse the pro-inflammatory 165 profile of monocytes in familial hypercholesterolemia
Chapter 10 Discussion and future perspectives 199
Appendixes Summary 213
Nederlandse samenvatting 219
PhD portfolio 225
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Chapter 1 General introduction 9 Chapter 2 Macrophage polarization: the epigenetic point of view 23 Chapter 3 Epigenetic pathways in macrophages emerge as novel 39
targets in atherosclerosis
Chapter 4 Inhibiting epigenetic enzymes to improve atherogenic 67 macrophage functions
Chapter 5 Macrophage Kdm6b controls the pro-fibrotic transcriptome 89 signature of foam cells
Chapter 6 Myeloid Kdm6b disruption results in advanced atherosclerosis 107
Chapter 7 Myeloid Ezh2 deficiency limits atherosclerosis 125
Chapter 8 Fine tuning of DNA methylation during the differentiation and 143 activation of human macrophages
Chapter 9 PCSK9 monoclonal antibodies reverse the pro-inflammatory 165 profile of monocytes in familial hypercholesterolemia
Chapter 10 Discussion and future perspectives 199
Appendixes Summary 213
Nederlandse samenvatting 219
PhD portfolio 225
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Chapter 1
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Chapter 1
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10 11
Atherosclerosis
Cardiovascular disease is still the leading cause of mortality worldwide (1). The main underlying cause is atherosclerosis, a lipid driven chronic inflammatory disorder of the arteries. Elevated levels of circulating low density lipoprotein (LDL) are associated with increased risk for atherosclerosis and cardiovascular disease (2). Within the arterial wall LDL can be modified (e.g. oxidized) resulting in immune cell activation and initiation of inflammatory responses. In response, blood monocytes are attracted to the site of inflammation (3). Once monocytes enter the arterial intima they differentiate into macrophages which make up the majority of immune cells present in atherosclerotic lesions and are important regulators of the disease (3). Atherosclerotic macrophages engulf modified lipids and turn into foam cells (Figure 1A). Besides foam cell characteristics, macrophages can adopt to a range of activation states (4). Foam cell formation and macrophage activation results in ongoing inflammation and disease progression. Also neutrophils, mast cells, dendritic cells, B cells and T cells are present in atherosclerotic lesions and contribute to lesion inflammation (5, 6). Excessive accumulation of lipids results in foam cell cytotoxicity, cell death and necrotic core formation. Atherosclerotic lesion growth causes smooth muscle cells (SMCs) to migrate from the media to the intima where activated SMCs produce collagen, elastin and proteoglycans, which together make up the fibrous cap that stabilize lesions (Figure 1B). Macrophages also contribute to plaque stabilisation by the production of matrix metalloproteinases that degrade the fibrous cap matrix components (7). Atherosclerotic lesion growth results in narrowing of the lumen, which can cause an occlusion. Unstable lesions are characterized by a large necrotic core and thin fibrous cap and these can ultimately rupture and initiate thrombosis (Figure 1C). This can result in a myocardial infarction or stroke. Patients at high cardiovascular risk are currently mainly treated with lipid-lowering agents, but besides these lipid-lowering actions, patients still have a strong residual risk for cardiovascular events (8). Since atherosclerosis is an inflammatory disorder, clinical trials with anti-inflammatory therapeutics (e.g. CANTOS and CIRT trial) have been performed in cardiovascular patients and the first results are considered to be predominantly beneficial (9, 10). Atherosclerosis is thus a multifactorial disease driven by lipids and inflammation. A better understanding of both the lipid metabolism and inflammatory processes in atherosclerosis (and the link between the two) is necessary to identify regulatory pathways and novel targets for atherosclerosis treatment.
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10 11
Atherosclerosis
Cardiovascular disease is still the leading cause of mortality worldwide (1). The main underlying cause is atherosclerosis, a lipid driven chronic inflammatory disorder of the arteries. Elevated levels of circulating low density lipoprotein (LDL) are associated with increased risk for atherosclerosis and cardiovascular disease (2). Within the arterial wall LDL can be modified (e.g. oxidized) resulting in immune cell activation and initiation of inflammatory responses. In response, blood monocytes are attracted to the site of inflammation (3). Once monocytes enter the arterial intima they differentiate into macrophages which make up the majority of immune cells present in atherosclerotic lesions and are important regulators of the disease (3). Atherosclerotic macrophages engulf modified lipids and turn into foam cells (Figure 1A). Besides foam cell characteristics, macrophages can adopt to a range of activation states (4). Foam cell formation and macrophage activation results in ongoing inflammation and disease progression. Also neutrophils, mast cells, dendritic cells, B cells and T cells are present in atherosclerotic lesions and contribute to lesion inflammation (5, 6). Excessive accumulation of lipids results in foam cell cytotoxicity, cell death and necrotic core formation. Atherosclerotic lesion growth causes smooth muscle cells (SMCs) to migrate from the media to the intima where activated SMCs produce collagen, elastin and proteoglycans, which together make up the fibrous cap that stabilize lesions (Figure 1B). Macrophages also contribute to plaque stabilisation by the production of matrix metalloproteinases that degrade the fibrous cap matrix components (7). Atherosclerotic lesion growth results in narrowing of the lumen, which can cause an occlusion. Unstable lesions are characterized by a large necrotic core and thin fibrous cap and these can ultimately rupture and initiate thrombosis (Figure 1C). This can result in a myocardial infarction or stroke. Patients at high cardiovascular risk are currently mainly treated with lipid-lowering agents, but besides these lipid-lowering actions, patients still have a strong residual risk for cardiovascular events (8). Since atherosclerosis is an inflammatory disorder, clinical trials with anti-inflammatory therapeutics (e.g. CANTOS and CIRT trial) have been performed in cardiovascular patients and the first results are considered to be predominantly beneficial (9, 10). Atherosclerosis is thus a multifactorial disease driven by lipids and inflammation. A better understanding of both the lipid metabolism and inflammatory processes in atherosclerosis (and the link between the two) is necessary to identify regulatory pathways and novel targets for atherosclerosis treatment.
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12
From Libby, P., Ridker, P.M. and Hansson, G.K., Nature. 2011;473(7347):317-25.(11)
LDL as a risk factor in atherosclerosis
It is well established that elevated levels of LDL and other apolipoprotein B (ApoB) containing lipoproteins, like very low-density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and lipoprotein(a) (Lp(a)) are associated with increased risk for atherosclerosis (12). ApoB100 is the main structural apolipoprotein of LDL and is mainly produced by the liver. ApoB100 is necessary for the synthesis and secretion of VLDL, which consist mainly of triglycerides (TG). The triglycerides of VLDL particles are hydrolyzed by lipoprotein lipase and hepatic lipase, which results in the formation of cholesteryl ester enriched particles IDL and LDL. LDL particles make up the majority of ApoB containing lipids in the blood and are cleared by the liver via the LDL receptor (LDLR). LDL, but also VLDL, IDL and Lp(a) from the bloodstream can enter the arterial intima (13). Having normal LDL-levels in the blood, this retention of lipids in the intima is of relative low risk to cardiovascular disease. However, increased levels of circulating LDL cause increased retention of LDL in the arterial intima contributing to the initiation of lesion formation (13). Recently, the European Atherosclerosis Society Consensus Panel stated that there is a causal link between LDL and atherosclerosis cardiovascular
Figure 1: Atherosclerotic lesion development.
(A) The initial steps of atherosclerosis include
adhesion of blood leukocytes to the activated endothelial monolayer, directed migration of leukocytes into the intima, differentiation of monocytes into macrophages, and their uptake of lipids, yielding foam cells. (B) Lesion progression
involves the migration of SMCs from the media to the intima, the proliferation of SMCs and the synthesis of extracellular matrix macromolecules. Extracellular lipid derived from dead and dying cells can accumulate in the central region of a plaque, often denoted the lipid or necrotic core. (C)
Thinning of the fibrous cap can result in rupture and thrombus formation.
13 disease based on evidence from genetic, epidemiologic and clinical studies (2). They state that there is a consistent dose-dependent log-linear association between the absolute magnitude of LDL exposure and the risk of atherosclerosis.
Familial hypercholesterolemia (FH) is a genetic disorder associated with increased risk of atherosclerosis. FH-patients have elevated LDL-levels in circulation and are marked by premature atherosclerosis (14). Loss-of-function mutations in the LDLR are the most common mutations causing FH. Other mutations include loss-of-function mutations in the APOB gene or gain-of-function mutation in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene, all resulting in elevated circulating LDL-levels (14). FH-patients are mainly treated with statins, which are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors that block the synthesis of cholesterol in the liver, thereby effectively lowering plasma LDL levels and the risk for cardiovascular disease (15). Nevertheless, a part of these patients will not reach effective LDL lowering by statins (16). Besides, there are also patients that are statin-intolerant, mainly due to statin-associated muscle symptoms (SAMS) (17). In recent years, monoclonal antibodies against PCSK9 have emerged as novel drugs to lower LDL cholesterol levels (18). LDL is cleared from the blood via hepatocyte endocytosis by the LDLR and these receptors are recycled to the surface. PCSK9 binds to the LDLR resulting in lysosomal degradation (Figure 2A). Using antibodies that inhibit PCSK9, LDL receptors are more recycled to the cell surface enhancing clearance of LDL from the circulation (Figure 2B) (19). PCSK9 monoclonal antibodies significantly lower LDL cholesterol, alone or added on top of standard statin therapy (20-24). Recently it was also shown that PCSK9 antibody treatment combined with statins effectively lowers cardiovascular events (25) making them valuable for patients who do not reach desired LDL lowering by statins alone. The link between lipids and immune cell activation should be further addressed since both play a key role in atherosclerosis development.
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12
From Libby, P., Ridker, P.M. and Hansson, G.K., Nature. 2011;473(7347):317-25.(11)
LDL as a risk factor in atherosclerosis
It is well established that elevated levels of LDL and other apolipoprotein B (ApoB) containing lipoproteins, like very low-density lipoproteins (VLDL), intermediate density lipoproteins (IDL) and lipoprotein(a) (Lp(a)) are associated with increased risk for atherosclerosis (12). ApoB100 is the main structural apolipoprotein of LDL and is mainly produced by the liver. ApoB100 is necessary for the synthesis and secretion of VLDL, which consist mainly of triglycerides (TG). The triglycerides of VLDL particles are hydrolyzed by lipoprotein lipase and hepatic lipase, which results in the formation of cholesteryl ester enriched particles IDL and LDL. LDL particles make up the majority of ApoB containing lipids in the blood and are cleared by the liver via the LDL receptor (LDLR). LDL, but also VLDL, IDL and Lp(a) from the bloodstream can enter the arterial intima (13). Having normal LDL-levels in the blood, this retention of lipids in the intima is of relative low risk to cardiovascular disease. However, increased levels of circulating LDL cause increased retention of LDL in the arterial intima contributing to the initiation of lesion formation (13). Recently, the European Atherosclerosis Society Consensus Panel stated that there is a causal link between LDL and atherosclerosis cardiovascular
Figure 1: Atherosclerotic lesion development.
(A) The initial steps of atherosclerosis include
adhesion of blood leukocytes to the activated endothelial monolayer, directed migration of leukocytes into the intima, differentiation of monocytes into macrophages, and their uptake of lipids, yielding foam cells. (B) Lesion progression
involves the migration of SMCs from the media to the intima, the proliferation of SMCs and the synthesis of extracellular matrix macromolecules. Extracellular lipid derived from dead and dying cells can accumulate in the central region of a plaque, often denoted the lipid or necrotic core. (C)
Thinning of the fibrous cap can result in rupture and thrombus formation.
13 disease based on evidence from genetic, epidemiologic and clinical studies (2). They state that there is a consistent dose-dependent log-linear association between the absolute magnitude of LDL exposure and the risk of atherosclerosis.
Familial hypercholesterolemia (FH) is a genetic disorder associated with increased risk of atherosclerosis. FH-patients have elevated LDL-levels in circulation and are marked by premature atherosclerosis (14). Loss-of-function mutations in the LDLR are the most common mutations causing FH. Other mutations include loss-of-function mutations in the APOB gene or gain-of-function mutation in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene, all resulting in elevated circulating LDL-levels (14). FH-patients are mainly treated with statins, which are 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors that block the synthesis of cholesterol in the liver, thereby effectively lowering plasma LDL levels and the risk for cardiovascular disease (15). Nevertheless, a part of these patients will not reach effective LDL lowering by statins (16). Besides, there are also patients that are statin-intolerant, mainly due to statin-associated muscle symptoms (SAMS) (17). In recent years, monoclonal antibodies against PCSK9 have emerged as novel drugs to lower LDL cholesterol levels (18). LDL is cleared from the blood via hepatocyte endocytosis by the LDLR and these receptors are recycled to the surface. PCSK9 binds to the LDLR resulting in lysosomal degradation (Figure 2A). Using antibodies that inhibit PCSK9, LDL receptors are more recycled to the cell surface enhancing clearance of LDL from the circulation (Figure 2B) (19). PCSK9 monoclonal antibodies significantly lower LDL cholesterol, alone or added on top of standard statin therapy (20-24). Recently it was also shown that PCSK9 antibody treatment combined with statins effectively lowers cardiovascular events (25) making them valuable for patients who do not reach desired LDL lowering by statins alone. The link between lipids and immune cell activation should be further addressed since both play a key role in atherosclerosis development.
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Figure 2: PCKS9. (A) Extracellular proprotein convertase subtilisin kexin 9 (PCSK9; red) binds the low-density
lipoprotein receptor (LDLR; blue), driving internalization of the receptor and its lysosomal degradation. LDL cholesterol (LDL-C; green) continues to circulate. (B) PCSK9-targeted monoclonal antibodies (mAbs) block the
interaction between PCSK9 and LDLR. LDLR, as a result, remains available to remove LDL-C from the bloodstream and incorporate it into cells. From Mullard. Nature Reviews Drug Discovery 11, 817-819, 2012 (19)
Monocytes, macrophages and foam cells in atherosclerosis
Monocytes develop from myeloid precursors in the bone marrow and fetal liver, where after they circulate in the blood (26). In addition, monocytes can be mobilized from the splenic hematopoietic stem and progenitor cells under inflammatory conditions like atherosclerosis (27, 28). Two major monocyte subsets are described in mice and three in humans. In mice these are the classical (Ly6Chi) and non-classical (Ly6Clow) monocytes, whereas in humans these are the classical (CD14++, CD16-), intermediate (CD14+, CD16+) and non-classical (CD14low, CD16++) monocytes (29). In both mice and humans, classical monocytes are characterized by high C-C chemokine receptor type 2 (CCR2) expression, whereas non-classical monocytes are characterized by high CX3C chemokine receptor 1 (CX3CR1) expression (30). The classical monocytes are described to be inflammatory monocytes that respond strongly to bacterial stimuli via toll like receptor (TLR) 4 and are recruited to sites of inflammation where they extravasate and differentiate into macrophages. Non-classical monocytes on the other hand respond to viral stimuli via TLR7 and TLR8 (31) and crawl and patrol the endothelium (32, 33).
15 In atherosclerosis, especially classical monocytes are recruited to sites of inflammation (34). During lesion progression there is continuous recruitment of monocytes that differentiate into macrophages in the presence of colony-stimulating factor 1 (CSF1; M-CSF). Besides by recruitment of blood monocytes, lesional macrophages increase in number by local proliferation, thereby contributing to the pool of macrophages present in atherosclerotic lesions (35). The lesional macrophages take up modified LDL (oxLDL) mainly via the scavenger receptors cluster of differentiation 36 (CD36) and SR-A1 (36, 37). OxLDL is intracellularly hydrolyzed into free cholesterol and fatty acids. Free cholesterol is subsequently re-esterified into cholesterol-esters via acyl-CoA cholesterol ester transferase (ACAT) eventually turning macrophages into foam cells (Figure 3). Excessive cholesterol uptake can lead to lipotoxicity resulting in foam cell apoptosis and combined with defective efferocytosis (clearance of apoptotic cells by macrophages), this results in the formation of a necrotic core. Lipid accumulation in macrophages also induces the expression of the cholesterol efflux genes ATP binding cassette transporters A1 and G1 (ABCA1 and ABCG1). Both promote cholesterol efflux to HDL particles, directly (ABCG1) and indirectly (ABCA1) for reverse cholesterol transport (Figure 3).
Besides obtaining foam cell characteristics, macrophages also contribute to atherosclerotic lesion inflammation by responding to danger signals (e.g. cholesterol crystals and oxLDL) and inflammatory triggers such as cytokines (Figure 3). OxLDL and cholesterol crystals can induce the NLR family pyrin domain containing 3 (NLRP3) inflammasome, which results in the secretion of the pro-inflammatory cytokine IL-1β (38, 39). In addition, oxLDL is recognized by various TLR receptors causing secretion of cytokines like TNF, IL-6 and IL-10 (40, 41). In contrast, oxLDL blocks the cytokine response via TLR2/4 signaling in human monocytes and macrophages (42, 43). In a mouse model of peritoneal foam cell formation it was further shown that foam cell formation dampens inflammatory responses, an effect which is mediated by desmosterol and liver X receptor (LXR) ligand (44). Macrophage foam cell formation is thus associated with various inflammatory responses. In human atherosclerotic lesions both inflammatory as well as anti-inflammatory macrophages are present (45). Pro-inflammatory macrophages are mainly found in rupture-prone shoulder regions. On the other hand, adventitial tissue contains more anti-inflammatory macrophages (45). Accordingly, monocytes, macrophages and foam cells are the central players in atherosclerosis where they contribute to lesion development in all stages of atherosclerosis.
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14
Figure 2: PCKS9. (A) Extracellular proprotein convertase subtilisin kexin 9 (PCSK9; red) binds the low-density
lipoprotein receptor (LDLR; blue), driving internalization of the receptor and its lysosomal degradation. LDL cholesterol (LDL-C; green) continues to circulate. (B) PCSK9-targeted monoclonal antibodies (mAbs) block the
interaction between PCSK9 and LDLR. LDLR, as a result, remains available to remove LDL-C from the bloodstream and incorporate it into cells. From Mullard. Nature Reviews Drug Discovery 11, 817-819, 2012 (19)
Monocytes, macrophages and foam cells in atherosclerosis
Monocytes develop from myeloid precursors in the bone marrow and fetal liver, where after they circulate in the blood (26). In addition, monocytes can be mobilized from the splenic hematopoietic stem and progenitor cells under inflammatory conditions like atherosclerosis (27, 28). Two major monocyte subsets are described in mice and three in humans. In mice these are the classical (Ly6Chi) and non-classical (Ly6Clow) monocytes, whereas in humans these are the classical (CD14++, CD16-), intermediate (CD14+, CD16+) and non-classical (CD14low, CD16++) monocytes (29). In both mice and humans, classical monocytes are characterized by high C-C chemokine receptor type 2 (CCR2) expression, whereas non-classical monocytes are characterized by high CX3C chemokine receptor 1 (CX3CR1) expression (30). The classical monocytes are described to be inflammatory monocytes that respond strongly to bacterial stimuli via toll like receptor (TLR) 4 and are recruited to sites of inflammation where they extravasate and differentiate into macrophages. Non-classical monocytes on the other hand respond to viral stimuli via TLR7 and TLR8 (31) and crawl and patrol the endothelium (32, 33).
15 In atherosclerosis, especially classical monocytes are recruited to sites of inflammation (34). During lesion progression there is continuous recruitment of monocytes that differentiate into macrophages in the presence of colony-stimulating factor 1 (CSF1; M-CSF). Besides by recruitment of blood monocytes, lesional macrophages increase in number by local proliferation, thereby contributing to the pool of macrophages present in atherosclerotic lesions (35). The lesional macrophages take up modified LDL (oxLDL) mainly via the scavenger receptors cluster of differentiation 36 (CD36) and SR-A1 (36, 37). OxLDL is intracellularly hydrolyzed into free cholesterol and fatty acids. Free cholesterol is subsequently re-esterified into cholesterol-esters via acyl-CoA cholesterol ester transferase (ACAT) eventually turning macrophages into foam cells (Figure 3). Excessive cholesterol uptake can lead to lipotoxicity resulting in foam cell apoptosis and combined with defective efferocytosis (clearance of apoptotic cells by macrophages), this results in the formation of a necrotic core. Lipid accumulation in macrophages also induces the expression of the cholesterol efflux genes ATP binding cassette transporters A1 and G1 (ABCA1 and ABCG1). Both promote cholesterol efflux to HDL particles, directly (ABCG1) and indirectly (ABCA1) for reverse cholesterol transport (Figure 3).
Besides obtaining foam cell characteristics, macrophages also contribute to atherosclerotic lesion inflammation by responding to danger signals (e.g. cholesterol crystals and oxLDL) and inflammatory triggers such as cytokines (Figure 3). OxLDL and cholesterol crystals can induce the NLR family pyrin domain containing 3 (NLRP3) inflammasome, which results in the secretion of the pro-inflammatory cytokine IL-1β (38, 39). In addition, oxLDL is recognized by various TLR receptors causing secretion of cytokines like TNF, IL-6 and IL-10 (40, 41). In contrast, oxLDL blocks the cytokine response via TLR2/4 signaling in human monocytes and macrophages (42, 43). In a mouse model of peritoneal foam cell formation it was further shown that foam cell formation dampens inflammatory responses, an effect which is mediated by desmosterol and liver X receptor (LXR) ligand (44). Macrophage foam cell formation is thus associated with various inflammatory responses. In human atherosclerotic lesions both inflammatory as well as anti-inflammatory macrophages are present (45). Pro-inflammatory macrophages are mainly found in rupture-prone shoulder regions. On the other hand, adventitial tissue contains more anti-inflammatory macrophages (45). Accordingly, monocytes, macrophages and foam cells are the central players in atherosclerosis where they contribute to lesion development in all stages of atherosclerosis.
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16
Figure 3: Macrophage foam cell formation. Macrophages internalize LDL, VLDL and oxLDL via macropinocytosis,
phagocytosis and scavenger receptor-mediated uptake. OxLDL is intracellularly hydrolyzed intro free cholesterol and fatty acids. Free cholesterol is then re-esterfurcated into cholesterol-esters via ACAT turning macrophages into foam cells. The accumulation of cellular cholesterol upregulates expression of the cholesterol efflux genes ABCA1 and ABCG1. This mediates the transfer of free cholesterol to lipid-HDL. Excessive free cholesterol accumulation can induce cholesterol crystal formation to activate the NLRP3 inflammasome. OxLDL is not only a ligand for the scavenger receptors, but can also be recognized by various TLR receptors. This signalling results in the activation of nuclear factor-κB (NF-κB) and in the production of pro-inflammatory cytokines and chemokines.
from Moore KJ, Sheedy FJ, Fisher EA. Nat rev Immunol. 2013;13(10):709-21.(4).
Epigenetic mechanisms in macrophages as targets for atherosclerosis
Since macrophages are central regulators in atherosclerosis, skewing monocytes and macrophages to cells with anti-atherogenic properties could be envisaged as an athero-protective treatment. It is therefore essential to identify regulatory pathways that are important for macrophage functioning in atherosclerosis. Epigenetic pathways are now identified to play a key role in monocyte-to-macrophage differentiation and activation (46, 47). Epigenetic regulation of gene transcription refers to changes in DNA accessibility without changing the DNA itself. Several modes of epigenetic regulation are described where DNA methylation and histone modifications are most common. Histone modifications are modifications at histone tails and the type and
17 position of modification determines whether genes are transcribed or repressed. Histone modifications, histone modifying enzymes, their role in macrophage function and their potential as a novel therapeutic target for atherosclerosis are discussed and reviewed in detail in chapter 2 and chapter 3.
Aim and outline of the thesis
The overall aim of this thesis is to identify novel regulators in macrophages to combat atherosclerosis. The first part of my thesis focuses on the identification and validation of histone modifying enzymes in macrophages as a target for atherosclerosis in mice. The second part of this thesis focuses on regulatory mechanisms in human monocytes and macrophages (Figure 4).
We hypothesize that epigenetic regulators and histone modifications regulate the transcriptional profile of macrophages. Chapter 2 is a review on epigenetic
mechanisms controlling macrophage activation and polarization. This review highlights histone modifications and histone modifying enzymes as important regulators of macrophage function. As macrophages are key immune cells in atherosclerosis, interfering with epigenetic mechanisms in these cells is an interesting approach to treat atherosclerosis. In chapter 3 we further discuss epigenetic mechanisms as a
novel target for atherosclerosis. In chapter 4 we performed an in vitro screening assay
with pharmacological inhibitors for histone modifying enzymes to identify epigenetic enzymes and their classes that modulate macrophage activation.
Kdm6b (also known as Jmjd3) is a histone H3K27 demethylase, which removes the repressive H3K27 methyl marks and has been a well-studied enzyme in macrophages. It was shown in literature that Kdm6b controls both the inflammatory and anti-inflammatory properties of macrophages. In chapter 5 we therefore studied the role
of Kdm6b in foam cells, an important macrophage subset in atherosclerosis. We found that Kdm6b regulates the pro-fibrotic transcriptional profile of foam cells. To study the functional consequence for disease, we further studied macrophage Kdm6b in atherosclerosis. In chapter 6 we found that kdm6b deficiency in myeloid cells results in
more advanced atherosclerosis. Since the H3K27 methyltransferases have opposite effects on this histone mark, we hypothesized that inhibition of the H3K27me3 methyltransferase Ezh2 in myeloid cells might be beneficial in case of atherosclerosis which is studied in chapter 7.
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Figure 3: Macrophage foam cell formation. Macrophages internalize LDL, VLDL and oxLDL via macropinocytosis,
phagocytosis and scavenger receptor-mediated uptake. OxLDL is intracellularly hydrolyzed intro free cholesterol and fatty acids. Free cholesterol is then re-esterfurcated into cholesterol-esters via ACAT turning macrophages into foam cells. The accumulation of cellular cholesterol upregulates expression of the cholesterol efflux genes ABCA1 and ABCG1. This mediates the transfer of free cholesterol to lipid-HDL. Excessive free cholesterol accumulation can induce cholesterol crystal formation to activate the NLRP3 inflammasome. OxLDL is not only a ligand for the scavenger receptors, but can also be recognized by various TLR receptors. This signalling results in the activation of nuclear factor-κB (NF-κB) and in the production of pro-inflammatory cytokines and chemokines.
from Moore KJ, Sheedy FJ, Fisher EA. Nat rev Immunol. 2013;13(10):709-21.(4).
Epigenetic mechanisms in macrophages as targets for atherosclerosis
Since macrophages are central regulators in atherosclerosis, skewing monocytes and macrophages to cells with anti-atherogenic properties could be envisaged as an athero-protective treatment. It is therefore essential to identify regulatory pathways that are important for macrophage functioning in atherosclerosis. Epigenetic pathways are now identified to play a key role in monocyte-to-macrophage differentiation and activation (46, 47). Epigenetic regulation of gene transcription refers to changes in DNA accessibility without changing the DNA itself. Several modes of epigenetic regulation are described where DNA methylation and histone modifications are most common. Histone modifications are modifications at histone tails and the type and
17 position of modification determines whether genes are transcribed or repressed. Histone modifications, histone modifying enzymes, their role in macrophage function and their potential as a novel therapeutic target for atherosclerosis are discussed and reviewed in detail in chapter 2 and chapter 3.
Aim and outline of the thesis
The overall aim of this thesis is to identify novel regulators in macrophages to combat atherosclerosis. The first part of my thesis focuses on the identification and validation of histone modifying enzymes in macrophages as a target for atherosclerosis in mice. The second part of this thesis focuses on regulatory mechanisms in human monocytes and macrophages (Figure 4).
We hypothesize that epigenetic regulators and histone modifications regulate the transcriptional profile of macrophages. Chapter 2 is a review on epigenetic
mechanisms controlling macrophage activation and polarization. This review highlights histone modifications and histone modifying enzymes as important regulators of macrophage function. As macrophages are key immune cells in atherosclerosis, interfering with epigenetic mechanisms in these cells is an interesting approach to treat atherosclerosis. In chapter 3 we further discuss epigenetic mechanisms as a
novel target for atherosclerosis. In chapter 4 we performed an in vitro screening assay
with pharmacological inhibitors for histone modifying enzymes to identify epigenetic enzymes and their classes that modulate macrophage activation.
Kdm6b (also known as Jmjd3) is a histone H3K27 demethylase, which removes the repressive H3K27 methyl marks and has been a well-studied enzyme in macrophages. It was shown in literature that Kdm6b controls both the inflammatory and anti-inflammatory properties of macrophages. In chapter 5 we therefore studied the role
of Kdm6b in foam cells, an important macrophage subset in atherosclerosis. We found that Kdm6b regulates the pro-fibrotic transcriptional profile of foam cells. To study the functional consequence for disease, we further studied macrophage Kdm6b in atherosclerosis. In chapter 6 we found that kdm6b deficiency in myeloid cells results in
more advanced atherosclerosis. Since the H3K27 methyltransferases have opposite effects on this histone mark, we hypothesized that inhibition of the H3K27me3 methyltransferase Ezh2 in myeloid cells might be beneficial in case of atherosclerosis which is studied in chapter 7.
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
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18
Next to histone modifications, DNA methylation is another important epigenetic regulator. In chapter 8 we studied the DNA methylation profile in human
monocyte-to-macrophage differentiation and subsequent activation.
In chapter 9 we studied the link between LDL and monocyte activation, two key
players in atherosclerosis. We hypothesize that high levels of circulating LDL are associated with monocyte activation, which can be reversed by lipid lowering. We tested this hypothesis using novel PCSK9 monoclonal antibodies in patients with FH.
Chapter 10 is a general discussion of the thesis, which summarizes the findings and
discusses future research lines.
Figure 4: Schematic overview of the chapters in this thesis.
19
References
1. World health organization CD. http://www.who.int/cardiovascular_diseases/en/.
2. Ference BA, Ginsberg HN, Graham I, Ray KK, Packard CJ, Bruckert E, et al. Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel. European heart journal. 2017. 3. Woollard KJ, Geissmann F. Monocytes in atherosclerosis: subsets and functions. Nature reviews Cardiology. 2010;7(2):77-86.
4. Moore KJ, Sheedy FJ, Fisher EA. Macrophages in atherosclerosis: a dynamic balance. Nature reviews Immunology. 2013;13(10):709-21.
5. Hartwig H, Silvestre Roig C, Daemen M, Lutgens E, Soehnlein O. Neutrophils in atherosclerosis. A brief overview. Hamostaseologie. 2015;35(2):121-7.
6. Hansson GK, Hermansson A. The immune system in atherosclerosis. Nature immunology. 2011;12(3):204-12.
7. Newby AC. Metalloproteinases and vulnerable atherosclerotic plaques. Trends in cardiovascular medicine. 2007;17(8):253-8.
8. Ridker PM. How Common Is Residual Inflammatory Risk? Circulation research. 2017;120(4):617-9. 9. Everett BM, Pradhan AD, Solomon DH, Paynter N, Macfadyen J, Zaharris E, et al. Rationale and design of the Cardiovascular Inflammation Reduction Trial: a test of the inflammatory hypothesis of atherothrombosis. American heart journal. 2013;166(2):199-207 e15.
10. Ridker PM, Thuren T, Zalewski A, Libby P. Interleukin-1beta inhibition and the prevention of recurrent cardiovascular events: rationale and design of the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS). American heart journal. 2011;162(4):597-605.
11. Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature. 2011;473(7347):317-25.
12. Goldstein JL, Brown MS. A century of cholesterol and coronaries: from plaques to genes to statins. Cell. 2015;161(1):161-72.
13. Tabas I, Williams KJ, Boren J. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications. Circulation. 2007;116(16):1832-44.
14. Wiegman A, Gidding SS, Watts GF, Chapman MJ, Ginsberg HN, Cuchel M, et al. Familial hypercholesterolaemia in children and adolescents: gaining decades of life by optimizing detection and treatment. European heart journal. 2015;36(36):2425-37.
15. Besseling J, Hovingh GK, Huijgen R, Kastelein JJ, Hutten BA. Statins in Familial Hypercholesterolemia: Consequences for Coronary Artery Disease and All-Cause Mortality. Journal of the American College of Cardiology. 2016;68(3):252-60.
16. Ridker PM, Mora S, Rose L, Group JTS. Percent reduction in LDL cholesterol following high-intensity statin therapy: potential implications for guidelines and for the prescription of emerging lipid-lowering agents. European heart journal. 2016;37(17):1373-9.
17. Stroes ES, Thompson PD, Corsini A, Vladutiu GD, Raal FJ, Ray KK, et al. Statin-associated muscle symptoms: impact on statin therapy-European Atherosclerosis Society Consensus Panel Statement on Assessment, Aetiology and Management. European heart journal. 2015;36(17):1012-22.
18. Giugliano RP, Sabatine MS. Are PCSK9 Inhibitors the Next Breakthrough in the Cardiovascular Field? Journal of the American College of Cardiology. 2015;65(24):2638-51.
19. Mullard A. Cholesterol-lowering blockbuster candidates speed into Phase III trials. Nature reviews Drug discovery. 2012;11(11):817-9.
20. Kastelein JJ, Ginsberg HN, Langslet G, Hovingh GK, Ceska R, Dufour R, et al. ODYSSEY FH I and FH II: 78 week results with alirocumab treatment in 735 patients with heterozygous familial hypercholesterolaemia. European heart journal. 2015;36(43):2996-3003.
21. Stroes E, Colquhoun D, Sullivan D, Civeira F, Rosenson RS, Watts GF, et al. Anti-PCSK9 antibody effectively lowers cholesterol in patients with statin intolerance: the GAUSS-2 randomized, placebo-controlled phase 3 clinical trial of evolocumab. Journal of the American College of Cardiology. 2014;63(23):2541-8. 22. Koren MJ, Lundqvist P, Bolognese M, Neutel JM, Monsalvo ML, Yang J, et al. Anti-PCSK9 monotherapy for hypercholesterolemia: the MENDEL-2 randomized, controlled phase III clinical trial of evolocumab. Journal of the American College of Cardiology. 2014;63(23):2531-40.
23. Blom DJ, Hala T, Bolognese M, Lillestol MJ, Toth PD, Burgess L, et al. A 52-week placebo-controlled trial of evolocumab in hyperlipidemia. The New England journal of medicine. 2014;370(19):1809-19.
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18
Next to histone modifications, DNA methylation is another important epigenetic regulator. In chapter 8 we studied the DNA methylation profile in human
monocyte-to-macrophage differentiation and subsequent activation.
In chapter 9 we studied the link between LDL and monocyte activation, two key
players in atherosclerosis. We hypothesize that high levels of circulating LDL are associated with monocyte activation, which can be reversed by lipid lowering. We tested this hypothesis using novel PCSK9 monoclonal antibodies in patients with FH.
Chapter 10 is a general discussion of the thesis, which summarizes the findings and
discusses future research lines.
Figure 4: Schematic overview of the chapters in this thesis.
19
References
1. World health organization CD. http://www.who.int/cardiovascular_diseases/en/.
2. Ference BA, Ginsberg HN, Graham I, Ray KK, Packard CJ, Bruckert E, et al. Low-density lipoproteins cause atherosclerotic cardiovascular disease. 1. Evidence from genetic, epidemiologic, and clinical studies. A consensus statement from the European Atherosclerosis Society Consensus Panel. European heart journal. 2017. 3. Woollard KJ, Geissmann F. Monocytes in atherosclerosis: subsets and functions. Nature reviews Cardiology. 2010;7(2):77-86.
4. Moore KJ, Sheedy FJ, Fisher EA. Macrophages in atherosclerosis: a dynamic balance. Nature reviews Immunology. 2013;13(10):709-21.
5. Hartwig H, Silvestre Roig C, Daemen M, Lutgens E, Soehnlein O. Neutrophils in atherosclerosis. A brief overview. Hamostaseologie. 2015;35(2):121-7.
6. Hansson GK, Hermansson A. The immune system in atherosclerosis. Nature immunology. 2011;12(3):204-12.
7. Newby AC. Metalloproteinases and vulnerable atherosclerotic plaques. Trends in cardiovascular medicine. 2007;17(8):253-8.
8. Ridker PM. How Common Is Residual Inflammatory Risk? Circulation research. 2017;120(4):617-9. 9. Everett BM, Pradhan AD, Solomon DH, Paynter N, Macfadyen J, Zaharris E, et al. Rationale and design of the Cardiovascular Inflammation Reduction Trial: a test of the inflammatory hypothesis of atherothrombosis. American heart journal. 2013;166(2):199-207 e15.
10. Ridker PM, Thuren T, Zalewski A, Libby P. Interleukin-1beta inhibition and the prevention of recurrent cardiovascular events: rationale and design of the Canakinumab Anti-inflammatory Thrombosis Outcomes Study (CANTOS). American heart journal. 2011;162(4):597-605.
11. Libby P, Ridker PM, Hansson GK. Progress and challenges in translating the biology of atherosclerosis. Nature. 2011;473(7347):317-25.
12. Goldstein JL, Brown MS. A century of cholesterol and coronaries: from plaques to genes to statins. Cell. 2015;161(1):161-72.
13. Tabas I, Williams KJ, Boren J. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications. Circulation. 2007;116(16):1832-44.
14. Wiegman A, Gidding SS, Watts GF, Chapman MJ, Ginsberg HN, Cuchel M, et al. Familial hypercholesterolaemia in children and adolescents: gaining decades of life by optimizing detection and treatment. European heart journal. 2015;36(36):2425-37.
15. Besseling J, Hovingh GK, Huijgen R, Kastelein JJ, Hutten BA. Statins in Familial Hypercholesterolemia: Consequences for Coronary Artery Disease and All-Cause Mortality. Journal of the American College of Cardiology. 2016;68(3):252-60.
16. Ridker PM, Mora S, Rose L, Group JTS. Percent reduction in LDL cholesterol following high-intensity statin therapy: potential implications for guidelines and for the prescription of emerging lipid-lowering agents. European heart journal. 2016;37(17):1373-9.
17. Stroes ES, Thompson PD, Corsini A, Vladutiu GD, Raal FJ, Ray KK, et al. Statin-associated muscle symptoms: impact on statin therapy-European Atherosclerosis Society Consensus Panel Statement on Assessment, Aetiology and Management. European heart journal. 2015;36(17):1012-22.
18. Giugliano RP, Sabatine MS. Are PCSK9 Inhibitors the Next Breakthrough in the Cardiovascular Field? Journal of the American College of Cardiology. 2015;65(24):2638-51.
19. Mullard A. Cholesterol-lowering blockbuster candidates speed into Phase III trials. Nature reviews Drug discovery. 2012;11(11):817-9.
20. Kastelein JJ, Ginsberg HN, Langslet G, Hovingh GK, Ceska R, Dufour R, et al. ODYSSEY FH I and FH II: 78 week results with alirocumab treatment in 735 patients with heterozygous familial hypercholesterolaemia. European heart journal. 2015;36(43):2996-3003.
21. Stroes E, Colquhoun D, Sullivan D, Civeira F, Rosenson RS, Watts GF, et al. Anti-PCSK9 antibody effectively lowers cholesterol in patients with statin intolerance: the GAUSS-2 randomized, placebo-controlled phase 3 clinical trial of evolocumab. Journal of the American College of Cardiology. 2014;63(23):2541-8. 22. Koren MJ, Lundqvist P, Bolognese M, Neutel JM, Monsalvo ML, Yang J, et al. Anti-PCSK9 monotherapy for hypercholesterolemia: the MENDEL-2 randomized, controlled phase III clinical trial of evolocumab. Journal of the American College of Cardiology. 2014;63(23):2531-40.
23. Blom DJ, Hala T, Bolognese M, Lillestol MJ, Toth PD, Burgess L, et al. A 52-week placebo-controlled trial of evolocumab in hyperlipidemia. The New England journal of medicine. 2014;370(19):1809-19.
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24. Robinson JG, Nedergaard BS, Rogers WJ, Fialkow J, Neutel JM, Ramstad D, et al. Effect of evolocumab or ezetimibe added to moderate- or high-intensity statin therapy on LDL-C lowering in patients with hypercholesterolemia: the LAPLACE-2 randomized clinical trial. Jama. 2014;311(18):1870-82.
25. Sabatine MS, Giugliano RP, Keech AC, Honarpour N, Wiviott SD, Murphy SA, et al. Evolocumab and Clinical Outcomes in Patients with Cardiovascular Disease. The New England journal of medicine. 2017;376(18):1713-22.
26. Ginhoux F, Jung S. Monocytes and macrophages: developmental pathways and tissue homeostasis. Nature reviews Immunology. 2014;14(6):392-404.
27. Robbins CS, Chudnovskiy A, Rauch PJ, Figueiredo JL, Iwamoto Y, Gorbatov R, et al. Extramedullary hematopoiesis generates Ly-6C(high) monocytes that infiltrate atherosclerotic lesions. Circulation. 2012;125(2):364-74.
28. Swirski FK, Nahrendorf M, Etzrodt M, Wildgruber M, Cortez-Retamozo V, Panizzi P, et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science. 2009;325(5940):612-6. 29. Ziegler-Heitbrock L, Ancuta P, Crowe S, Dalod M, Grau V, Hart DN, et al. Nomenclature of monocytes and dendritic cells in blood. Blood. 2010;116(16):e74-80.
30. Shi C, Pamer EG. Monocyte recruitment during infection and inflammation. Nature reviews Immunology. 2011;11(11):762-74.
31. Cros J, Cagnard N, Woollard K, Patey N, Zhang SY, Senechal B, et al. Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors. Immunity. 2010;33(3):375-86.
32. Auffray C, Fogg D, Garfa M, Elain G, Join-Lambert O, Kayal S, et al. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science. 2007;317(5838):666-70.
33. Carlin LM, Stamatiades EG, Auffray C, Hanna RN, Glover L, Vizcay-Barrena G, et al. Nr4a1-dependent Ly6C(low) monocytes monitor endothelial cells and orchestrate their disposal. Cell. 2013;153(2):362-75. 34. Rahman MS, Murphy AJ, Woollard KJ. Effects of dyslipidaemia on monocyte production and function in cardiovascular disease. Nature reviews Cardiology. 2017;14(7):387-400.
35. Robbins CS, Hilgendorf I, Weber GF, Theurl I, Iwamoto Y, Figueiredo JL, et al. Local proliferation dominates lesional macrophage accumulation in atherosclerosis. Nature medicine. 2013;19(9):1166-72. 36. Kunjathoor VV, Febbraio M, Podrez EA, Moore KJ, Andersson L, Koehn S, et al. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. The Journal of biological chemistry. 2002;277(51):49982-8.
37. Kzhyshkowska J, Neyen C, Gordon S. Role of macrophage scavenger receptors in atherosclerosis. Immunobiology. 2012;217(5):492-502.
38. Sheedy FJ, Grebe A, Rayner KJ, Kalantari P, Ramkhelawon B, Carpenter SB, et al. CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation. Nature immunology. 2013;14(8):812-20.
39. Duewell P, Kono H, Rayner KJ, Sirois CM, Vladimer G, Bauernfeind FG, et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature. 2010;464(7293):1357-61.
40. Chavez-Sanchez L, Chavez-Rueda K, Legorreta-Haquet MV, Zenteno E, Ledesma-Soto Y, Montoya-Diaz E, et al. The activation of CD14, TLR4, and TLR2 by mmLDL induces IL-1beta, IL-6, and IL-10 secretion in human monocytes and macrophages. Lipids in health and disease. 2010;9:117.
41. Stewart CR, Stuart LM, Wilkinson K, van Gils JM, Deng J, Halle A, et al. CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer. Nature immunology. 2010;11(2):155-61.
42. Kannan Y, Sundaram K, Aluganti Narasimhulu C, Parthasarathy S, Wewers MD. Oxidatively modified low density lipoprotein (LDL) inhibits TLR2 and TLR4 cytokine responses in human monocytes but not in macrophages. The Journal of biological chemistry. 2012;287(28):23479-88.
43. Jongstra-Bilen J, Zhang CX, Wisnicki T, Li MK, White-Alfred S, Ilaalagan R, et al. Oxidized Low-Density Lipoprotein Loading of Macrophages Downregulates TLR-Induced Proinflammatory Responses in a Gene-Specific and Temporal Manner through Transcriptional Control. Journal of immunology. 2017;199(6):2149-57.
44. Spann NJ, Garmire LX, McDonald JG, Myers DS, Milne SB, Shibata N, et al. Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses. Cell. 2012;151(1):138-52. 45. Stoger JL, Gijbels MJ, van der Velden S, Manca M, van der Loos CM, Biessen EA, et al. Distribution of macrophage polarization markers in human atherosclerosis. Atherosclerosis. 2012;225(2):461-8.
46. Hoeksema MA, de Winther MP. Epigenetic Regulation of Monocyte and Macrophage Function. Antioxidants & redox signaling. 2016;25(14):758-74.
47. Amit I, Winter DR, Jung S. The role of the local environment and epigenetics in shaping macrophage identity and their effect on tissue homeostasis. Nature immunology. 2016;17(1):18-25.
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24. Robinson JG, Nedergaard BS, Rogers WJ, Fialkow J, Neutel JM, Ramstad D, et al. Effect of evolocumab or ezetimibe added to moderate- or high-intensity statin therapy on LDL-C lowering in patients with hypercholesterolemia: the LAPLACE-2 randomized clinical trial. Jama. 2014;311(18):1870-82.
25. Sabatine MS, Giugliano RP, Keech AC, Honarpour N, Wiviott SD, Murphy SA, et al. Evolocumab and Clinical Outcomes in Patients with Cardiovascular Disease. The New England journal of medicine. 2017;376(18):1713-22.
26. Ginhoux F, Jung S. Monocytes and macrophages: developmental pathways and tissue homeostasis. Nature reviews Immunology. 2014;14(6):392-404.
27. Robbins CS, Chudnovskiy A, Rauch PJ, Figueiredo JL, Iwamoto Y, Gorbatov R, et al. Extramedullary hematopoiesis generates Ly-6C(high) monocytes that infiltrate atherosclerotic lesions. Circulation. 2012;125(2):364-74.
28. Swirski FK, Nahrendorf M, Etzrodt M, Wildgruber M, Cortez-Retamozo V, Panizzi P, et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science. 2009;325(5940):612-6. 29. Ziegler-Heitbrock L, Ancuta P, Crowe S, Dalod M, Grau V, Hart DN, et al. Nomenclature of monocytes and dendritic cells in blood. Blood. 2010;116(16):e74-80.
30. Shi C, Pamer EG. Monocyte recruitment during infection and inflammation. Nature reviews Immunology. 2011;11(11):762-74.
31. Cros J, Cagnard N, Woollard K, Patey N, Zhang SY, Senechal B, et al. Human CD14dim monocytes patrol and sense nucleic acids and viruses via TLR7 and TLR8 receptors. Immunity. 2010;33(3):375-86.
32. Auffray C, Fogg D, Garfa M, Elain G, Join-Lambert O, Kayal S, et al. Monitoring of blood vessels and tissues by a population of monocytes with patrolling behavior. Science. 2007;317(5838):666-70.
33. Carlin LM, Stamatiades EG, Auffray C, Hanna RN, Glover L, Vizcay-Barrena G, et al. Nr4a1-dependent Ly6C(low) monocytes monitor endothelial cells and orchestrate their disposal. Cell. 2013;153(2):362-75. 34. Rahman MS, Murphy AJ, Woollard KJ. Effects of dyslipidaemia on monocyte production and function in cardiovascular disease. Nature reviews Cardiology. 2017;14(7):387-400.
35. Robbins CS, Hilgendorf I, Weber GF, Theurl I, Iwamoto Y, Figueiredo JL, et al. Local proliferation dominates lesional macrophage accumulation in atherosclerosis. Nature medicine. 2013;19(9):1166-72. 36. Kunjathoor VV, Febbraio M, Podrez EA, Moore KJ, Andersson L, Koehn S, et al. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. The Journal of biological chemistry. 2002;277(51):49982-8.
37. Kzhyshkowska J, Neyen C, Gordon S. Role of macrophage scavenger receptors in atherosclerosis. Immunobiology. 2012;217(5):492-502.
38. Sheedy FJ, Grebe A, Rayner KJ, Kalantari P, Ramkhelawon B, Carpenter SB, et al. CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation. Nature immunology. 2013;14(8):812-20.
39. Duewell P, Kono H, Rayner KJ, Sirois CM, Vladimer G, Bauernfeind FG, et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature. 2010;464(7293):1357-61.
40. Chavez-Sanchez L, Chavez-Rueda K, Legorreta-Haquet MV, Zenteno E, Ledesma-Soto Y, Montoya-Diaz E, et al. The activation of CD14, TLR4, and TLR2 by mmLDL induces IL-1beta, IL-6, and IL-10 secretion in human monocytes and macrophages. Lipids in health and disease. 2010;9:117.
41. Stewart CR, Stuart LM, Wilkinson K, van Gils JM, Deng J, Halle A, et al. CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer. Nature immunology. 2010;11(2):155-61.
42. Kannan Y, Sundaram K, Aluganti Narasimhulu C, Parthasarathy S, Wewers MD. Oxidatively modified low density lipoprotein (LDL) inhibits TLR2 and TLR4 cytokine responses in human monocytes but not in macrophages. The Journal of biological chemistry. 2012;287(28):23479-88.
43. Jongstra-Bilen J, Zhang CX, Wisnicki T, Li MK, White-Alfred S, Ilaalagan R, et al. Oxidized Low-Density Lipoprotein Loading of Macrophages Downregulates TLR-Induced Proinflammatory Responses in a Gene-Specific and Temporal Manner through Transcriptional Control. Journal of immunology. 2017;199(6):2149-57.
44. Spann NJ, Garmire LX, McDonald JG, Myers DS, Milne SB, Shibata N, et al. Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses. Cell. 2012;151(1):138-52. 45. Stoger JL, Gijbels MJ, van der Velden S, Manca M, van der Loos CM, Biessen EA, et al. Distribution of macrophage polarization markers in human atherosclerosis. Atherosclerosis. 2012;225(2):461-8.
46. Hoeksema MA, de Winther MP. Epigenetic Regulation of Monocyte and Macrophage Function. Antioxidants & redox signaling. 2016;25(14):758-74.
47. Amit I, Winter DR, Jung S. The role of the local environment and epigenetics in shaping macrophage identity and their effect on tissue homeostasis. Nature immunology. 2016;17(1):18-25.
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Chapter 2
Macrophage polarization:
the epigenetic point of view
Jan Van den Bossche*, Annette E. Neele*, Marten A. Hoeksema,
Menno P.J. de Winther *Contributed equally
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Chapter 2
Macrophage polarization:
the epigenetic point of view
Jan Van den Bossche*, Annette E. Neele*, Marten A. Hoeksema,
Menno P.J. de Winther *Contributed equally
515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele 515552-L-bw-neele Processed on: 1-12-2017 Processed on: 1-12-2017 Processed on: 1-12-2017
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Abstract
Purpose of review
The first functions of macrophages to be identified by Metchnikoff were phagocytosis and microbial killing. Although these are important features, macrophages are functionally very complex and involved in virtually all aspects of life, from immunity and host defense, to homeostasis, tissue repair and development. To accommodate for this, macrophages adopt a plethora of polarization states. Understanding their transcriptional regulation and phenotypic heterogeneity is vital because macrophages are critical in many diseases and have emerged as attractive targets for therapy. Here, we review how epigenetic mechanisms control macrophage polarization.
Recent findings
It is becoming increasingly clear that chromatin remodelling governs multiple aspects of macrophage differentiation, activation and polarization. In recent years, independent research groups highlighted the importance of epigenetic mechanisms to regulate enhancer activity. Moreover, distinct histone-modifying enzymes were identified that control macrophage activation and polarization.
Summary
We recap epigenetic features of distinct enhancers and describe the role of Jumonji domain-containing protein 3 (Jmjd3) and Hdac3 as crucial mediators of macrophage differentiation, activation and polarization. We hypothesize that epigenetic enzymes could serve as the link between environment, cellular metabolism and macrophage phenotype. To conclude, we propose epigenetic intervention as a future pharmacological target to modulate macrophage polarization and to treat inflammatory diseases such as atherosclerosis.
Keywords
enhancers, epigenetics, histone modifications, histone-modifying enzymes, macrophage polarization
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Introduction
Macrophages are one of the most plastic cells of the haematopoietic system. In response to microenvironmental stimuli, they adopt different polarization states driving their functional repertoire in tissue homeostasis, host defense and disease. Although several efforts have been made to classify macrophages, the binary M1/M2 classification remains the most used and offers a useful reductionist framework to study and describe their function.
Classically activated macrophages (CAM or M1) are induced by Th1 cytokines such as interferon gamma (IFNγ), Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS) and danger signals. This well studied activation state produces pro-inflammatory cytokines [interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF), IL-12], chemokines (CXCL1–2, Chemokine (C-C motif) ligand (CCL)2–5), reactive oxygen and nitrogen intermediates. In addition, enhanced expression of Major Histocompatibility Complex (MHC)-I and MHC-II and co-stimulatory molecules promotes the induction of polarized Th1 adaptive immune responses. Although these pro-inflammatory macrophages are beneficial for host protection, they also can cause collateral tissue damage. Indeed, unrestrained inflammatory macrophage activity aggravates and sustains chronic inflammatory diseases such as atherosclerosis, multiple sclerosis and rheumatoid arthritis [1].
More recently, it became clear that macrophages are also altered by diverse non-inflammatory factors, including the Th2 cytokines IL-4 and IL-13, IL-10, transforming growth factor-beta (TGF-β), glucocorticoids and immune complexes. All these types of non-M1 macrophages are often grouped under the generic term M2 (mainly defined as IL-10high and IL-12low), which can be further subdivided as M2a, M2b and M2c. IL-4 and IL-13 are inducers of the so-called bona fide alternatively activated macrophages (AAMs or M2a), M2b are induced by the simultaneous presence of immune complexes and TLR or IL-1R ligands, and the anti-inflammatory cytokine IL-10 induces M2c macrophages. M2 macrophages are important in antiparasitic immune responses, promote tissue remodelling and wound healing, and have anti-inflammatory immunoregulatory functions that are beneficial during chronic inflammatory diseases such as atherosclerosis [2]. Molecularly, M2 macrophages are characterized by a large collection of marker genes. Although some commonly used mouse M2 markers have no human homologs (e.g. Fizz1/Retnla and Ym1/Chi3- l3) or are not regulated by M2-inducing cytokines in humans (e.g. arginase-1/Arg1), the surface markers macrophage mannose receptor (MRC1), E-cadherin (CDH1) and transferrin receptor CD71 (TFRC), and the enzyme transglutaminase 2 (TGM2) are excellent markers for both human and mouse IL-4/IL-13-induced M2 macrophages [3–6].
