Macrophage regulatory mechanisms in atherosclerosis: The interplay of lipids and inflammation - Chapter 2: Macrophage polarization: The epigenetic point of view

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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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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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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-12}low_{), 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].

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24

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

25

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-12}low_{), 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].

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Epigenetic mechanisms characterize and control enhancer activity

Alterations in gene expression regulate the macrophage phenotype and polarization state. These transcriptional responses require close collaboration between gene promoters and regulatory enhancer elements. Promoter regions, located proximal to gene transcription start sites (TSS) and TSS-distal cis-regulatory enhancer regions, contain particular DNA motifs that are recognized by specific transcription factors. In promoter regions, these transcription factors are mostly general transcription factors, although enhancer regions are bound by lineage determining transcription factors (LDTFs), mediating cell fate and identity. The formation of enhancers is therefore much more cell type-specific than the presence of promoter elements [7]. The LDTF PU.1 is commonly present on macrophage-specific enhancers and together with IRF-8 regulates macrophage differentiation [8]. These LDTFs also control the recruitment of signal-dependent transcription factors (SDTFs) upon macrophage stimulation. Upon M1 activation, SDTFs such as C/EBPβ, Nuclear Factor kappa-B, Signal Transducer and Activator of Transcription 1 (STAT1), Activator Protein 1 and IRFs are recruited [9–11]. Correspondingly, alternative activation with IL-4/IL-13 recruits SDTFs such as Signal Transducer and Activator of Transcription 6 and PPARγ [3].

Epigenetic features of the enhancer landscape

It is now becoming clear that enhancer accessibility and activity are tightly regulated by epigenetic mechanisms [8,9,12●,13●●,14●●,15]. Epigenetic processes alter gene expression by regulating the conversion of densely packed heterochromatin into transcription factor-accessible euchromatin, without affecting the DNA sequence itself. Although DNA methylation generally results in gene silencing, histone modifications can either mark an open or a closed state. Acetylation on lysine (K) residues by histone acetyltransferases (HATs) usually increases transcriptional activity. The activity of HATs can be counteracted by repressor complexes with Hdac activity removing lysine acetylation. Regulation of gene expression by histone methylation can be associated with either transcriptional activation or repression, depending on the sites of methylation and the number of methyl groups. Dimethylation or trimethylation of histone H3 at lysine-4, lysine-36 and lysine-79 is associated with activation of transcription, whereas H3K9me2/3 and H3K27me3 constitute repressive marks. The histone methylation status at these different positions is regulated by histone methyltransferases (HMTs) and the opposing histone demethylases (HDMs). A large number of histone-modifying enzymes (HMEs) cooperate to set the so-called histone code in enhancer regions, and thereby regulate macrophage differentiation, activation and polarization.

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In general, enhancers are characterized by H3K4me1 marks [16,17] and additional histone modifications determine the type of enhancer (Fig. 1). Poised enhancers in heterochromatin regions are inactive and show a combination of H3K4me1 and H3K27me3 marks. Upon macrophage activation and polarization, particular HATs and the HDMs are recruited, resulting in histone acetylation at different positions and removal of repressive H3K27me3 marks. As such, inactive regions with poised enhancers are turned into active regions with transcription factor accessible enhancers with H3K27ac marks and no repressive H3K27me3 marks [9,17–20]. These active enhancers subsequently drive gene transcription through acting on promoter regions.

Recently, two independent research groups identified a new type of macrophage enhancers and termed them latent or de-novo enhancers [13●●,14●●]. These regions in the genome initially lack histone marks and are not bound by transcription factors but gain histone modifications and transcription factor binding upon macrophage activation [13●●,14●●]. Latent enhancers gain and retain the enhancer mark H3K4me1/2 upon TLR4 ligation by a multiple-step process. TLR4-induced NF-κB activation of macrophages results in PU.1/ CEBP binding and recruitment of NF-κB p65 to the enhancer element. In turn, activation results in the recruitment of HATs causing acetylation and subsequently RNA polymerase II (Pol II) binding and transcription of the enhancer, generating the so-called enhancer RNAs [13●●]. Finally, the HMTs MLL1, MLL2/4 and MLL3 are recruited and are responsible for induced H3K4 methylation at the enhancer [13●●], resulting in a fully active enhancer. The enhancer elements generated through these mechanisms are stable and remain present upon loss of the stimuli (i.e. imprinted H3K4me1). Restimulation of these macrophages results in a faster and stronger response, thus suggesting enhancer memory in response to stimuli [14●●].

As LDTFs are critical for enhancer generation, natural variation in DNA-binding motifs for LDTFs may greatly influence their binding, subsequent enhancer formation and transcriptional responses. Indeed, using a very elegant approach comparing the enhancer repertoire of C57Bl/6 and BALB/c mice, the group of Glass recently showed that genetic variation in LDTF motifs in the two strains highly contributes to the difference in enhancer formation [12●]. Variation in the LDTF motifs influenced for instance NF-κB recruitment to enhancers and NF-κB dependent gene expression. Natural variation in LDTF motifs had even more pronounced effects than variation in NF-κB binding motifs themselves. These data show that natural variation in the enhancer landscape contributes to transcriptional responses and it is tempting to speculate that such variation may also underlie human disease.

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Epigenetic mechanisms characterize and control enhancer activity

Alterations in gene expression regulate the macrophage phenotype and polarization state. These transcriptional responses require close collaboration between gene promoters and regulatory enhancer elements. Promoter regions, located proximal to gene transcription start sites (TSS) and TSS-distal cis-regulatory enhancer regions, contain particular DNA motifs that are recognized by specific transcription factors. In promoter regions, these transcription factors are mostly general transcription factors, although enhancer regions are bound by lineage determining transcription factors (LDTFs), mediating cell fate and identity. The formation of enhancers is therefore much more cell type-specific than the presence of promoter elements [7]. The LDTF PU.1 is commonly present on macrophage-specific enhancers and together with IRF-8 regulates macrophage differentiation [8]. These LDTFs also control the recruitment of signal-dependent transcription factors (SDTFs) upon macrophage stimulation. Upon M1 activation, SDTFs such as C/EBPβ, Nuclear Factor kappa-B, Signal Transducer and Activator of Transcription 1 (STAT1), Activator Protein 1 and IRFs are recruited [9–11]. Correspondingly, alternative activation with IL-4/IL-13 recruits SDTFs such as Signal Transducer and Activator of Transcription 6 and PPARγ [3].

Epigenetic features of the enhancer landscape

It is now becoming clear that enhancer accessibility and activity are tightly regulated by epigenetic mechanisms [8,9,12●,13●●,14●●,15]. Epigenetic processes alter gene expression by regulating the conversion of densely packed heterochromatin into transcription factor-accessible euchromatin, without affecting the DNA sequence itself. Although DNA methylation generally results in gene silencing, histone modifications can either mark an open or a closed state. Acetylation on lysine (K) residues by histone acetyltransferases (HATs) usually increases transcriptional activity. The activity of HATs can be counteracted by repressor complexes with Hdac activity removing lysine acetylation. Regulation of gene expression by histone methylation can be associated with either transcriptional activation or repression, depending on the sites of methylation and the number of methyl groups. Dimethylation or trimethylation of histone H3 at lysine-4, lysine-36 and lysine-79 is associated with activation of transcription, whereas H3K9me2/3 and H3K27me3 constitute repressive marks. The histone methylation status at these different positions is regulated by histone methyltransferases (HMTs) and the opposing histone demethylases (HDMs). A large number of histone-modifying enzymes (HMEs) cooperate to set the so-called histone code in enhancer regions, and thereby regulate macrophage differentiation, activation and polarization.

27

In general, enhancers are characterized by H3K4me1 marks [16,17] and additional histone modifications determine the type of enhancer (Fig. 1). Poised enhancers in heterochromatin regions are inactive and show a combination of H3K4me1 and H3K27me3 marks. Upon macrophage activation and polarization, particular HATs and the HDMs are recruited, resulting in histone acetylation at different positions and removal of repressive H3K27me3 marks. As such, inactive regions with poised enhancers are turned into active regions with transcription factor accessible enhancers with H3K27ac marks and no repressive H3K27me3 marks [9,17–20]. These active enhancers subsequently drive gene transcription through acting on promoter regions.

Recently, two independent research groups identified a new type of macrophage enhancers and termed them latent or de-novo enhancers [13●●,14●●]. These regions in the genome initially lack histone marks and are not bound by transcription factors but gain histone modifications and transcription factor binding upon macrophage activation [13●●,14●●]. Latent enhancers gain and retain the enhancer mark H3K4me1/2 upon TLR4 ligation by a multiple-step process. TLR4-induced NF-κB activation of macrophages results in PU.1/ CEBP binding and recruitment of NF-κB p65 to the enhancer element. In turn, activation results in the recruitment of HATs causing acetylation and subsequently RNA polymerase II (Pol II) binding and transcription of the enhancer, generating the so-called enhancer RNAs [13●●]. Finally, the HMTs MLL1, MLL2/4 and MLL3 are recruited and are responsible for induced H3K4 methylation at the enhancer [13●●], resulting in a fully active enhancer. The enhancer elements generated through these mechanisms are stable and remain present upon loss of the stimuli (i.e. imprinted H3K4me1). Restimulation of these macrophages results in a faster and stronger response, thus suggesting enhancer memory in response to stimuli [14●●].

As LDTFs are critical for enhancer generation, natural variation in DNA-binding motifs for LDTFs may greatly influence their binding, subsequent enhancer formation and transcriptional responses. Indeed, using a very elegant approach comparing the enhancer repertoire of C57Bl/6 and BALB/c mice, the group of Glass recently showed that genetic variation in LDTF motifs in the two strains highly contributes to the difference in enhancer formation [12●]. Variation in the LDTF motifs influenced for instance NF-κB recruitment to enhancers and NF-κB dependent gene expression. Natural variation in LDTF motifs had even more pronounced effects than variation in NF-κB binding motifs themselves. These data show that natural variation in the enhancer landscape contributes to transcriptional responses and it is tempting to speculate that such variation may also underlie human disease.

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Figure 1: Chromatin features of enhancers and active promoters. Enhancers are characterized by

H3K4me1/me2 marks and additional modifications determine the type of enhancer. Closed or poised enhancers contain the H3K27me3 mark. Stimulation with lipopolysaccharide results in active enhancers through SDTF binding, loss of H3K27me3 marks, increased H3K27 acetylation and PolII binding and transcription. Latent or de-novo enhancers do not contain any histone modifications or TF binding but gain these modifications upon stimulation. Active enhancers drive promoter activity and gene transcription.

Epigenetic enzymes control macrophage activation and polarization

The described changes in promoter and enhancer activity through histone modifications are carried out by a broad range of HMEs and different HMEs are now associated with distinct macrophage polarization states [21–25,26● ,27–40] (Table 1). For example, the H3K4 HMT Myeloid Lymphoid Leukaemia (MLL) was recently shown to be a marker and regulator of M1 activation [21]. MLL expression increases upon treatment of macrophages with IFNγ/LPS and pharmacological blockade of MLL reduces the induction of the M1 gene Chemokine (C-X-C motif) ligand CXCL10. Here, we focus on Jmjd3 KDM1 lysine (K)-specific demethylase 6B and Hdac3, two HMEs that in the last years emerged as principle regulators of macrophage differentiation, activation and polarization.

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Table 1: Epigenetic enzymes that affect macrophage polarization

Jmjd3 enables macrophage responses to various environmental stimuli

Repressive H3K27me3 marks are generated by the Polycomb repressive complex 2 (PRC2) containing Ezh2, Eed and Suz12. H3K27me3 helps to recruit the PRC1 complex and together PRC1 and PRC2 repress many genes during diverse biological processes including development and homeostasis. In blood monocytes, Ezh2 is rhythmically

Target Method* Function Ref.

Mll Inhib. Inhibition of the histone methyl transferase MLL abrogates CXCL10

expression in M1 macrophages. [21]

siRNA The histone methyl transferases MLL1-4 mediate H3K4 methylation at

de novo enhancers. [13●●]

Smyd5 RNAi H4K20 trimethylation by SMYD5 restricts the expression of TLR4 target

genes in macrophages. [22]

Jmjd3 RNAi/KO JMJD3 fine-tunes gene expression in LPS-activated macrophages. [23;24] RNAi JMJD3 promotes serum amyloid A (SAA)-induced expression of

inflammatory cytokines. [25]

Inhib. JMJD3/UTX inhibition reduces LPS-induced inflammatory cytokine

production in human macrophages. [26●]

RNAi JMJD3 is induced by IL-4 and regulates the expression of Arg1 and Fizz1

in M2 macrophages. [27]

KO JMJD3 promotes M2 polarization and immune responses against

helminth infections through IRF4. [28]

RNAi H3K27me3 demethylation in the Nfatc1 locus by JMJD3 promotes

RANKL-induced osteoclast differentiation. [29]

RNAi JMJD3 activates gene expression during macrophage differentiation by

promoting transcriptional elongation. [30]

Hdac3 KO Hdac3KO macrophages are hyper-M2 and limit Schistosoma mansoni

egg- induced inflammation in vivo. [31]

KO Hdac3 plays is crucial for the LPS-induced expression of about half of

the inflammatory genes in M1. [32]

Hdac Inhib. Broad spectrum HDAC inhibitors dampen inflammatory responses. [33-35]

RNAi Hdac1, -2 and -3 deacetylate MPK-1 and thereby promote MAPK

signaling and inflammatory responses. [36]

Sirt1 Multiple Activation of the class III histone deacetylase sirtuin 1 has

anti-inflammatory and anti-atherogenic effects. [37;38]

BET Inhib. The BET protein inhibitors I-BET and JQ1 blocks macrophage

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Figure 1: Chromatin features of enhancers and active promoters. Enhancers are characterized by

H3K4me1/me2 marks and additional modifications determine the type of enhancer. Closed or poised enhancers contain the H3K27me3 mark. Stimulation with lipopolysaccharide results in active enhancers through SDTF binding, loss of H3K27me3 marks, increased H3K27 acetylation and PolII binding and transcription. Latent or de-novo enhancers do not contain any histone modifications or TF binding but gain these modifications upon stimulation. Active enhancers drive promoter activity and gene transcription.

Epigenetic enzymes control macrophage activation and polarization

The described changes in promoter and enhancer activity through histone modifications are carried out by a broad range of HMEs and different HMEs are now associated with distinct macrophage polarization states [21–25,26● ,27–40] (Table 1). For example, the H3K4 HMT Myeloid Lymphoid Leukaemia (MLL) was recently shown to be a marker and regulator of M1 activation [21]. MLL expression increases upon treatment of macrophages with IFNγ/LPS and pharmacological blockade of MLL reduces the induction of the M1 gene Chemokine (C-X-C motif) ligand CXCL10. Here, we focus on Jmjd3 KDM1 lysine (K)-specific demethylase 6B and Hdac3, two HMEs that in the last years emerged as principle regulators of macrophage differentiation, activation and polarization.

29

Table 1: Epigenetic enzymes that affect macrophage polarization

Jmjd3 enables macrophage responses to various environmental stimuli

Repressive H3K27me3 marks are generated by the Polycomb repressive complex 2 (PRC2) containing Ezh2, Eed and Suz12. H3K27me3 helps to recruit the PRC1 complex and together PRC1 and PRC2 repress many genes during diverse biological processes including development and homeostasis. In blood monocytes, Ezh2 is rhythmically

Target Method* Function Ref.

Mll Inhib. Inhibition of the histone methyl transferase MLL abrogates CXCL10

expression in M1 macrophages. [21]

siRNA The histone methyl transferases MLL1-4 mediate H3K4 methylation at

de novo enhancers. [13●●]

Smyd5 RNAi H4K20 trimethylation by SMYD5 restricts the expression of TLR4 target

genes in macrophages. [22]

Jmjd3 RNAi/KO JMJD3 fine-tunes gene expression in LPS-activated macrophages. [23;24] RNAi JMJD3 promotes serum amyloid A (SAA)-induced expression of

inflammatory cytokines. [25]

Inhib. JMJD3/UTX inhibition reduces LPS-induced inflammatory cytokine

production in human macrophages. [26●]

RNAi JMJD3 is induced by IL-4 and regulates the expression of Arg1 and Fizz1

in M2 macrophages. [27]

KO JMJD3 promotes M2 polarization and immune responses against

helminth infections through IRF4. [28]

RNAi H3K27me3 demethylation in the Nfatc1 locus by JMJD3 promotes

RANKL-induced osteoclast differentiation. [29]

RNAi JMJD3 activates gene expression during macrophage differentiation by

promoting transcriptional elongation. [30]

Hdac3 KO Hdac3KO macrophages are hyper-M2 and limit Schistosoma mansoni

egg- induced inflammation in vivo. [31]

KO Hdac3 plays is crucial for the LPS-induced expression of about half of

the inflammatory genes in M1. [32]

Hdac Inhib. Broad spectrum HDAC inhibitors dampen inflammatory responses. [33-35]

RNAi Hdac1, -2 and -3 deacetylate MPK-1 and thereby promote MAPK

signaling and inflammatory responses. [36]

Sirt1 Multiple Activation of the class III histone deacetylase sirtuin 1 has

anti-inflammatory and anti-atherogenic effects. [37;38]

BET Inhib. The BET protein inhibitors I-BET and JQ1 blocks macrophage

inflammatory responses. [39;40]

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recruited to the promoters of the Ccl2 and Ccl8 genes, resulting in cyclic gene silencing and the control of diurnal rhythms in inflammatory monocyte numbers [41●●]. PRC2 recruitment and its repressive activity depend on the interaction with the circadian regulator BMAL1. Accordingly, BMAL1 deficiency impairs Ezh2 recruitment and induces a more active chromatin state of Ccl2 and Ccl8 with more H3K4me3 activation marks and constitutive recruitment of Pol II to the promoter. The actual effect of Ezh2 deletion on macrophage differentiation and polarization remains to be investigated, but its deletion in CD4+ T cells was found to augment Th1 and Th2 cell differentiation and plasticity [42].

Upon TLR engagement, Jumonji-C domain H3K27 demethylases such as Jmjd3, Utx and Uty are induced [23]. LPS-induced transcription of pro-inflammatory genes is strongly decreased in Jmjd3-deficient macrophages [24]. However, this correlates with minimal changes in H3K27me3, suggesting that Jmjd3 has no or low enzymatic activity and exerts its inflammatory regulation through other mechanisms. Recently, also pro-inflammatory cytokine induction by the acute-phase protein serum amyloid A (SAA) was shown to depend on Jmjd3 expression [25]. Accordingly, targeting both Jmjd3 and Utx H3K27me3 demethylases with small molecule inhibitors impairs inflammatory responses in human primary macrophages and could thus be of high pharmacological interest for the treatment of inflammatory diseases [26●]. At the other side of the macrophage polarization spectrum, Jmjd3 is also induced by IL-4 [27] and was shown to be crucial for M2 polarization in helminth infection and responses to chitin [28]. The role of Jmjd3 in both M1 and M2 polarization is not necessarily enigmatic and probably reflects the need for Jmjd3 to enable responses to various environmental stimuli.

In agreement with this hypothesis, Chen et al. [30] shed light on the molecular mechanism underlying Jmjd3-mediated induction of transcription during macrophage differentiation. Jmjd3 associates with the H3K27me2 demethylase KIAA1718 and directly binds to and regulates the expression of multiple genes during macrophage differentiation. Both demethylases cooperate to resolve H3K27me3 repressive marks on genes poised for activation with a bivalent H3K4me3/H3K27me3 mark and promoter-proximal paused Pol II. As such, Jmjd3 and KIAA1718 allows Pol II travelling and activation of gene transcription. Although these observations are valid for macrophage differentiation, they might also explain the crucial role of Jmjd3 during seemingly heterogeneous types of macrophage activation. Overall, Jmjd3 is required to allow various responses to different external stimuli, including IL-4, Receptor activator of nuclear factor kappa-B ligand (RANKL), Macrophage colony stimulating factor, SAA and LPS, but is not associated with a single macrophage polarization state [23–25,26● ,27–30].

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Hdac3 is an epigenetic break of M2 polarization

In contrast to Jmjd3, which is not strictly associated with M1 or M2 polarization, Hdac3 promotes M1 responses and at the same time acts as a brake for M2 polarization. Mullican et al. [31] were the first to demonstrate that macrophages lacking Hdac3 show an M2-like phenotype in the absence of external stimuli and are hyper-responsive to IL-4. Hdac3 deacetylates histone marks in regulatory regions of IL-4 induced genes and thereby represses alternative macrophage activation. Releasing this epigenetic M2 brake creates ‘hyper-M2’ anti-inflammatory macrophages. As such, pharmacological blockade of Hdac3 could be beneficial as a therapy in inflammatory diseases. Indeed, following exposure to Schistosoma mansoni eggs, an in vivo model of Th2 cytokine-mediated disease that is limited by M2 macrophages, lung inflammation was improved in mice lacking Hdac3 in macrophages [43]. Moreover, Hdac3 is required for the activation of hundreds of, mainly STAT1-dependent, inflammatory genes in M1 macrophages and this was shown to be dependent on defective IFNβ signalling in macrophages [32]. Overall, Hdac3 is a key regulator of the macrophage polarization and an interesting target for intervention, as Hdac3 loss has anti-inflammatory effects.

Epigenetic enzymes are a potential link between metabolism and polarization

It is now clear that distinct environmental stimuli affect the expression and activity of epigenetic enzymes and the resultant macrophage’s epigenetic landscape. Yet, it remains unstudied to what extent variations in cofactor availability affect epigenetic enzyme activity, histone marks and cellular phenotype. Epigenetic enzymes utilize metabolites generated by cellular metabolism, thereby providing a potential link between environment, metabolism, gene regulation and the resulting macrophage polarization status [44●]. For example, acetyl-CoA is used by HATs as an acetyl donor, the deacetylation activity of Sirtuin (SIRT) family deacetylases requires NAD+, S-adenosylmethionine (SAM) is used by methyltransferases for DNA and histone methylation and some HDMs use α-ketoglutarate as the substrate. Thus, histones could act as metabolic sensors that translate alterations in metabolism into changes in gene expression and phenotype.

Interestingly, polarized macrophages display a distinct regulation of cellular metabolism. M1 macrophages show a metabolic switch to glycolysis [45]. Conversely, enhanced fatty acid oxidation (FAO) and mitochondrial oxidative phosphorylation (OXPHOS) provides sustained energy in IL-4 induced M2 macrophages. Importantly, the inhibition of glycolysis or OXPHOS/FAO impairs M1 or M2 activation, respectively [46,47]. AMP-activated protein kinase (AMPK) senses cellular energy levels (AMP/ATP ratio), enhances mitochondrial respiration and is a key regulator that stimulates M2

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recruited to the promoters of the Ccl2 and Ccl8 genes, resulting in cyclic gene silencing and the control of diurnal rhythms in inflammatory monocyte numbers [41●●]. PRC2 recruitment and its repressive activity depend on the interaction with the circadian regulator BMAL1. Accordingly, BMAL1 deficiency impairs Ezh2 recruitment and induces a more active chromatin state of Ccl2 and Ccl8 with more H3K4me3 activation marks and constitutive recruitment of Pol II to the promoter. The actual effect of Ezh2 deletion on macrophage differentiation and polarization remains to be investigated, but its deletion in CD4+ T cells was found to augment Th1 and Th2 cell differentiation and plasticity [42].

Upon TLR engagement, Jumonji-C domain H3K27 demethylases such as Jmjd3, Utx and Uty are induced [23]. LPS-induced transcription of pro-inflammatory genes is strongly decreased in Jmjd3-deficient macrophages [24]. However, this correlates with minimal changes in H3K27me3, suggesting that Jmjd3 has no or low enzymatic activity and exerts its inflammatory regulation through other mechanisms. Recently, also pro-inflammatory cytokine induction by the acute-phase protein serum amyloid A (SAA) was shown to depend on Jmjd3 expression [25]. Accordingly, targeting both Jmjd3 and Utx H3K27me3 demethylases with small molecule inhibitors impairs inflammatory responses in human primary macrophages and could thus be of high pharmacological interest for the treatment of inflammatory diseases [26●]. At the other side of the macrophage polarization spectrum, Jmjd3 is also induced by IL-4 [27] and was shown to be crucial for M2 polarization in helminth infection and responses to chitin [28]. The role of Jmjd3 in both M1 and M2 polarization is not necessarily enigmatic and probably reflects the need for Jmjd3 to enable responses to various environmental stimuli.

In agreement with this hypothesis, Chen et al. [30] shed light on the molecular mechanism underlying Jmjd3-mediated induction of transcription during macrophage differentiation. Jmjd3 associates with the H3K27me2 demethylase KIAA1718 and directly binds to and regulates the expression of multiple genes during macrophage differentiation. Both demethylases cooperate to resolve H3K27me3 repressive marks on genes poised for activation with a bivalent H3K4me3/H3K27me3 mark and promoter-proximal paused Pol II. As such, Jmjd3 and KIAA1718 allows Pol II travelling and activation of gene transcription. Although these observations are valid for macrophage differentiation, they might also explain the crucial role of Jmjd3 during seemingly heterogeneous types of macrophage activation. Overall, Jmjd3 is required to allow various responses to different external stimuli, including IL-4, Receptor activator of nuclear factor kappa-B ligand (RANKL), Macrophage colony stimulating factor, SAA and LPS, but is not associated with a single macrophage polarization state [23–25,26● ,27–30].

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Hdac3 is an epigenetic break of M2 polarization

In contrast to Jmjd3, which is not strictly associated with M1 or M2 polarization, Hdac3 promotes M1 responses and at the same time acts as a brake for M2 polarization. Mullican et al. [31] were the first to demonstrate that macrophages lacking Hdac3 show an M2-like phenotype in the absence of external stimuli and are hyper-responsive to IL-4. Hdac3 deacetylates histone marks in regulatory regions of IL-4 induced genes and thereby represses alternative macrophage activation. Releasing this epigenetic M2 brake creates ‘hyper-M2’ anti-inflammatory macrophages. As such, pharmacological blockade of Hdac3 could be beneficial as a therapy in inflammatory diseases. Indeed, following exposure to Schistosoma mansoni eggs, an in vivo model of Th2 cytokine-mediated disease that is limited by M2 macrophages, lung inflammation was improved in mice lacking Hdac3 in macrophages [43]. Moreover, Hdac3 is required for the activation of hundreds of, mainly STAT1-dependent, inflammatory genes in M1 macrophages and this was shown to be dependent on defective IFNβ signalling in macrophages [32]. Overall, Hdac3 is a key regulator of the macrophage polarization and an interesting target for intervention, as Hdac3 loss has anti-inflammatory effects.

Epigenetic enzymes are a potential link between metabolism and polarization

It is now clear that distinct environmental stimuli affect the expression and activity of epigenetic enzymes and the resultant macrophage’s epigenetic landscape. Yet, it remains unstudied to what extent variations in cofactor availability affect epigenetic enzyme activity, histone marks and cellular phenotype. Epigenetic enzymes utilize metabolites generated by cellular metabolism, thereby providing a potential link between environment, metabolism, gene regulation and the resulting macrophage polarization status [44●]. For example, acetyl-CoA is used by HATs as an acetyl donor, the deacetylation activity of Sirtuin (SIRT) family deacetylases requires NAD+, S-adenosylmethionine (SAM) is used by methyltransferases for DNA and histone methylation and some HDMs use α-ketoglutarate as the substrate. Thus, histones could act as metabolic sensors that translate alterations in metabolism into changes in gene expression and phenotype.

Interestingly, polarized macrophages display a distinct regulation of cellular metabolism. M1 macrophages show a metabolic switch to glycolysis [45]. Conversely, enhanced fatty acid oxidation (FAO) and mitochondrial oxidative phosphorylation (OXPHOS) provides sustained energy in IL-4 induced M2 macrophages. Importantly, the inhibition of glycolysis or OXPHOS/FAO impairs M1 or M2 activation, respectively [46,47]. AMP-activated protein kinase (AMPK) senses cellular energy levels (AMP/ATP ratio), enhances mitochondrial respiration and is a key regulator that stimulates M2

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macrophages [48,49]. Thus, intervening with metabolic pathways in macrophages can be utilized to modulate their phenotype. It is tempting to speculate that epigenetics serves as a link between environment, macrophage metabolism and macrophage polarization and this hypothesis is now ripe for investigation.

Future directions

The observation that macrophage polarization is regulated by specific HMEs and requires particular histone modifications implies that a better understanding of this epigenetic layer of regulation will pave the way for multiple future applications. Pharmacological manipulation of epigenetic enzymes may become a new therapeutic tool to alter macrophage polarization and control excessive inflammation not only in acute but also chronic inflammatory diseases such as atherosclerosis, rheumatoid arthritis and multiple sclerosis. Broadspectrum Hdac inhibitors are the most extensively studied pharmaceuticals and are currently being thoroughly tested in clinical trials for the treatment of various cancers. Developing novel inhibitors against specific chromatin-modifying enzymes will be an important next step to manipulate particular epigenetic enzymes. Recently, small molecule Jmjd3/Utx inhibitors [26●] and entities that mimic acetylated histone tails and thereby fool bromodomain and extraterminal domain (BET) epigenetic readers (e.g. I-BET [40], JQ1 [39] and the influenza nonstructural proteins NS1 [50]) were shown to impair the inflammatory programme in macrophages.

Future diagnostics and identification of disease risk factors may also start focusing on the epigenome. The above-mentioned murine studies show that natural variation in enhancer elements highly contributes to their activity. Hence, polymorphisms in enhancer elements in the human population may influence the epigenetic landscape and cellular responses. Recent studies in several T cell-driven disorders indeed show that disease-associated Single-nucleotide polymorphism (SNPs) are linked with the enhancer landscape of T cells [51]. Future screening, beyond whole exome sequencing, may shed light on this novel group of potential modulators of disease and may elucidate the molecular mechanisms underlying disease.

In addition, lifestyle factors can influence disease development, as smoking and high caloric intake are established risk factors for obesity and atherosclerosis. These risk factors may influence the epigenetic landscape of immune cells and prime them to a pro-inflammatory state. Screening key immune cells to identify pathogenic epigenetic patterns associated with inflammatory priming and disease susceptibility could enlighten the molecular mechanisms of disease. This may help diagnostics of disease and lead to personalized lifestyle recommendations. Hence, the epigenetic landscape, not only partly driven by the genetic make-up of individuals but also highly influenced

33

by environmental risk factors, may prove an important future target for diagnostics and development of novel therapeutics to modulate chronic inflammatory disorders.

Conclusion

Inflammatory responses and macrophage polarization depend on the cell’s aptitude to respond to (micro)environmental changes and stimuli. Epigenetic mechanisms allow such ‘adaptive’ responses and enhance macrophage diversity and plasticity with the ultimate goal to protect the host against a plethora of threats. However, maladaptive epigenetic changes contribute to the persistence of (inflammatory) diseases. Therefore, pharmacological intervention to reprogram a ‘diseased’ epigenetic landscape into a healthy one is a prospective target for the treatment of inflammatory disorders. Equally, screening the epigenetic landscape of key immune cells could aid to diagnose pathogenic epigenetic signatures and predict disease susceptibility, even before other symptoms arise.

Acknowledgements

Jan Van den Bossche received a Junior Postdoc grant from the Netherlands Heart Foundation (2013T003). Menno de Winther is an established investigator of the Netherlands Heart Foundation (2007T067), is supported by a Netherlands Heart Foundation grant (2010B022) and holds an AMC-fellowship. We acknowledge the support from the Netherlands CardioVascular Research Initiative: the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development and the Atherosclerosis Royal Netherlands Academy of Sciences for the GENIUS project ‘Generating the best evidence-based pharmaceutical targets for atherosclerosis’ (CVON2011-19).

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macrophages [48,49]. Thus, intervening with metabolic pathways in macrophages can be utilized to modulate their phenotype. It is tempting to speculate that epigenetics serves as a link between environment, macrophage metabolism and macrophage polarization and this hypothesis is now ripe for investigation.

Future directions

The observation that macrophage polarization is regulated by specific HMEs and requires particular histone modifications implies that a better understanding of this epigenetic layer of regulation will pave the way for multiple future applications. Pharmacological manipulation of epigenetic enzymes may become a new therapeutic tool to alter macrophage polarization and control excessive inflammation not only in acute but also chronic inflammatory diseases such as atherosclerosis, rheumatoid arthritis and multiple sclerosis. Broadspectrum Hdac inhibitors are the most extensively studied pharmaceuticals and are currently being thoroughly tested in clinical trials for the treatment of various cancers. Developing novel inhibitors against specific chromatin-modifying enzymes will be an important next step to manipulate particular epigenetic enzymes. Recently, small molecule Jmjd3/Utx inhibitors [26●] and entities that mimic acetylated histone tails and thereby fool bromodomain and extraterminal domain (BET) epigenetic readers (e.g. I-BET [40], JQ1 [39] and the influenza nonstructural proteins NS1 [50]) were shown to impair the inflammatory programme in macrophages.

Future diagnostics and identification of disease risk factors may also start focusing on the epigenome. The above-mentioned murine studies show that natural variation in enhancer elements highly contributes to their activity. Hence, polymorphisms in enhancer elements in the human population may influence the epigenetic landscape and cellular responses. Recent studies in several T cell-driven disorders indeed show that disease-associated Single-nucleotide polymorphism (SNPs) are linked with the enhancer landscape of T cells [51]. Future screening, beyond whole exome sequencing, may shed light on this novel group of potential modulators of disease and may elucidate the molecular mechanisms underlying disease.

In addition, lifestyle factors can influence disease development, as smoking and high caloric intake are established risk factors for obesity and atherosclerosis. These risk factors may influence the epigenetic landscape of immune cells and prime them to a pro-inflammatory state. Screening key immune cells to identify pathogenic epigenetic patterns associated with inflammatory priming and disease susceptibility could enlighten the molecular mechanisms of disease. This may help diagnostics of disease and lead to personalized lifestyle recommendations. Hence, the epigenetic landscape, not only partly driven by the genetic make-up of individuals but also highly influenced

33

by environmental risk factors, may prove an important future target for diagnostics and development of novel therapeutics to modulate chronic inflammatory disorders.

Conclusion

Inflammatory responses and macrophage polarization depend on the cell’s aptitude to respond to (micro)environmental changes and stimuli. Epigenetic mechanisms allow such ‘adaptive’ responses and enhance macrophage diversity and plasticity with the ultimate goal to protect the host against a plethora of threats. However, maladaptive epigenetic changes contribute to the persistence of (inflammatory) diseases. Therefore, pharmacological intervention to reprogram a ‘diseased’ epigenetic landscape into a healthy one is a prospective target for the treatment of inflammatory disorders. Equally, screening the epigenetic landscape of key immune cells could aid to diagnose pathogenic epigenetic signatures and predict disease susceptibility, even before other symptoms arise.

Acknowledgements

Jan Van den Bossche received a Junior Postdoc grant from the Netherlands Heart Foundation (2013T003). Menno de Winther is an established investigator of the Netherlands Heart Foundation (2007T067), is supported by a Netherlands Heart Foundation grant (2010B022) and holds an AMC-fellowship. We acknowledge the support from the Netherlands CardioVascular Research Initiative: the Dutch Heart Foundation, Dutch Federation of University Medical Centers, the Netherlands Organization for Health Research and Development and the Atherosclerosis Royal Netherlands Academy of Sciences for the GENIUS project ‘Generating the best evidence-based pharmaceutical targets for atherosclerosis’ (CVON2011-19).

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