Targeting epigenetic modifiers to reprogramme macrophages in non-resolving inflammation-driven atherosclerosis

Abstract

Epigenomic and epigenetic research has been providing several new insights into a variety of diseases caused by non-resolving inflammation, including cardiovascular diseases. Atherosclerosis (AS) has long been recognized as a chronic inflammatory disease of the arterial walls, characterized by local persistent and stepwise accelerating inflammation without resolution, also known as uncontrolled inflammation. The pathogenesis of AS is driven primarily by highly plastic macrophages via their polarization to pro- or anti-inflammatory phenotypes as well as other novel subtypes recently identified by single-cell sequencing. Although emerging evidence has indicated the key role of the epigenetic machinery in the regulation of macrophage plasticity, the investigation of epigenetic alterations and modifiers in AS and related inflammation is still in its infancy. An increasing number of the epigenetic modifiers (e.g. TET2, DNMT3A, HDAC3, HDAC9, JMJD3, KDM4A) have been identified in epigenetic remodelling of macrophages through DNA methylation or histone modifications (e.g. methylation, acetylation, and recently lactylation) in inflammation. These or many unexplored modifiers function to determine or switch the direction of macrophage polarization via transcriptional reprogramming of gene expression and intracellular metabolic rewiring upon microenvironmental cues, thereby representing a promising target for anti-inflammatory therapy in AS. Here, we review up-to-date findings involving the epigenetic regulation of macrophages to shed light on the mechanism of uncontrolled inflammation during AS onset and progression. We also discuss current challenges for developing an effective and safe anti-AS therapy that targets the epigenetic modifiers and propose a potential anti-inflammatory strategy that repolarizes macrophages from pro- to anti-inflammatory phenotypes.

Keywords: Atherosclerosis; Epigenetic modifier; Inflammation; Macrophage; Polarization.

Graphical Abstract

Targeting epigenetic modifiers to repolarize macrophages in non-resolving inflammation-driven atherosclerosis. Based on an alternative model proposed for macrophage polarization, targeting epigenetic modifiers (e.g. for DNA methylation and histone methylation, acetylation, and lactylation) may repolarize macrophages straight from pro-inflammatory (M1) to anti-inflammatory/pro-resolving (M2) phenotype by transcriptional reprogramming of gene expression via epigenetic remodelling and metabolic rewiring, which thus represents a therapeutic strategy for developing novel anti-inflammatory therapy to treat non-resolving inflammation-driven diseases such as atherosclerosis.

Figure 1

Dysregulated macrophage polarization and its related epigenetic modifiers in non-resolving inflammation of AS. In AS lesions, macrophages display heterogeneous phenotypes due to their high plasticity, which allows them to polarize towards either pro-inflammatory (M1) or anti-inflammatory and pro-resolving (M2) phenotype (the traditional model), or inflammatory (M1-like), TREM2⁺ foamy, and other subsets of macrophages (recently identified by scRNAseq; note: another major subset named resident-like macrophages with uncertain functions in AS are likely located in the adventitia, thus not shown in this graph) in response to different microenvironmental stimuli (e.g. oxLDL, hypoxia, ROS, etc.). In general, predominant polarization of macrophage to M1, together with the impaired capability to repolarize to M2 (homeostasis), leads to an imbalance between these two phenotypes of macrophages with multiple pro- (red box) vs. anti-AS functions (green box), which in turn drives atherogenesis and disease progression until plaque rupture. The direction of macrophage polarization is determined via transcriptional reprogramming of gene expression by the epigenetic machinery, including epigenetic modifications of DNA and histones (e.g. methylation, acetylation, and lactylation) or their regulatory enzymes (named epigenetic modifiers). The epigenetic modifiers identified thus far to be involved in the regulation of macrophage polarization and functions are listed in the grey box, while those with unknown functions in AS are indicated by question marks. Based on the inter-phenotypic transition (or trans-differentiation) described in the updated Waddington’s epigenetic landscape, a possibility then arises that targeting these or other epigenetic modifiers (remaining to be explored) may repolarize M1 macrophages straight to M2 phenotype, which provide a strategy for developing anti-inflammatory therapy in AS.

Figure 2

Gene expression profiling for understanding transcriptional reprogramming of macrophage polarization. The analysis is based on a transcriptomic dataset [GSE5099; Exp Macrophage (Polarization)—Estrada—15—MAS5.0—u133a] public available on the R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl). The data were acquired by transcriptional profile analysis using the Human Genome U133 A and B arrays (HG-U133; Affymetrix, containing a total of 39 000 transcripts) from freshly isolated human monocytes (Mo), which were then cultured in the presence of M-CSF (100 ng/mL) for 7 days to differentiate into macrophages (M0), followed by incubation for an additional 18 h with IFN-γ (20 ng/mL) plus LPS (100 ng/mL) for M1 macrophages or with IL-4 (20 ng/mL) for M2 macrophages (see Reference⁵⁵). (A) Mo, M0 (a resting state), M1, and M2 phenotypes display distinct gene expression profiles (GEPs), containing numerous differentially expressed genes (DEGs), with the most DEGs in M1 when compared to either M0 (reflecting M1 polarization) or M2 (presumably reflecting M1 to M2 repolarization) but the least in M2 compared to M0 (reflecting M2 polarization). (B) Functionally, up-regulated DEGs involving M1 polarization are enriched for proteasome (1), NF-κB (2), JAK/STAT (3), and apoptosis (4), while down-regulated DEGs are enriched for PPAR (5), oxidative phosphorylation (6), and lysosome (7). However, these changes are reversed during M1 to M2 repolarization. (C) These pathways (1–7) are also involved in the differentiation of Mo to M0 macrophages, although to a lesser extent. (D) Among these DEGs, there are many histone epigenetic modifiers, which may represent candidate targets in epigenetic remodelling of macrophage phenotypes. (E) There are also extensive correlations among these epigenetic modifiers, suggest an extensive network across two different types of histone PTMs (e.g. methylation and acetylation) in transcriptional reprogramming of macrophage polarization and repolarization.

Figure 3

An alternative model for macrophage polarization proposed based on transcriptional reprogramming. (A) Following dynamic changes of the DEGs (mainly involving seven pathways) identified in Figure 2, a continuous process is hypothesized to illustrate the flow of transcriptional reprogramming for macrophages differentiation, polarization, and repolarization, which matches with metabolic rewiring during phenotypic transitions of macrophages from M0 (OXPHOS) to M1 (glycolysis) and M1 to M2 (OXPHOS). Values indicate the number of DEGs. (B) According to these changes of DEGs, an alternative model (middle) is thus proposed by integrating M1→M2 repolarization into the classical model of macrophage polarization (left) based on Waddington’s landscape. Furthermore, a comprehensive model (right) is presented to cover the full process of macrophage evolution from maturation through polarization to repolarization.

Figure 4

Current understanding of the molecular mechanisms underlying macrophage (re)polarization in AS. To date, many mechanisms have been described to address how macrophage polarization in response to various AS risk factors (including traditional and novel ones) and microenvironmental cues (e.g. oxLDL, hypoxia, and other pro-inflammatory factors) is regulated in inflammation and particularly non-resolving inflammation-driven diseases like AS. Briefly, macrophages are regulated via at least four mechanisms, including epigenetic remodelling, transcriptional reprogramming, metabolic rewiring, and phenotypic polarization. Among them, epigenetic remodelling links environmental stimuli or intracellular alterations (e.g. metabolic changes) to transcriptional reprogramming (either expression or silencing of specific genes), which in turn determine the direction of phenotypic (re)polarization directly or indirectly via metabolic rewiring. Owe to the central role of epigenetic remodelling in orchestrating these mechanisms, epigenetic modifiers that govern epigenetic remodelling by regulating DNA methylation and histone PTMs (e.g. methylation, acetylation, and lactylation) may therefore represent promising targets for the development of novel anti-inflammatory therapies against non-resolving inflammation-driven diseases, especially AS. However, extensive crosstalks among these mechanisms and potential ‘off-target’ effects (e.g. due to affecting PTMs of non-histone proteins, including acetylation and probably lactylation as well) may make this task difficult and complicated, especially in regards to the issue of effectiveness and safety. Thus, further understanding of these mechanisms and the precise roles of the epigenetic modifiers in AS is necessary to address such a challenge in this field.