Metabolic regulation of epigenetic remodeling in immune cells

Abstract

Immune cells are capable of sensing various signals in the microenvironment and turning on specific immune functions in response. The appropriate transition of immune cells into diverse functional states, which is crucial for immunity, involves complex and well-regulated changes in transcriptional program. Accumulating evidence shows that epigenetic remodeling plays a central role in mediating the transcriptional program for immune cell activation and immunological memory. Concurrently, immune cells undergo significant metabolic reprogramming during immune response. Here we review recent studies that demonstrate shifts in metabolic state can orchestrate immune cell functions through its impact on epigenetic remodeling, and the microenvironment can exert its influence on immune cells through the metabolic regulation of epigenetics. We also discuss the systems biology approaches that enabled these discoveries.

Introduction

It has been well recognized that metabolism has a significant impact on epigenetic regulation via a variety of molecular mechanisms: Key metabolites can act as substrates, inhibitors, or activators of the covalent modifications of histones, DNA and RNA, and some metabolic enzymes can directly participate in epigenetic remodeling. These molecular mechanisms have been intensively discussed in recent reviews [1–4], and therefore are not discussed in detail here.

This connection between metabolism and epigenetics allows cells to integrate information about metabolic state, which reflects nutrient availability, environmental condition, and cellular health, into the cell fate decision. The regulatory role of metabolism on epigenetics has wide implications in physiological transitions, such as development [5,6], as well as pathological conditions, such as cancer [7,8]. Immune responses are tightly controlled processes coupled with massive reprogramming in both metabolism and epigenetics. High-throughput technologies have allowed quantitative characterization of the diverse epigenetic landscape and metabolic modes in immune cells in various states, and opened up active research on the impact of metabolism on epigenetics and the function of immune cells. This metabolism-epigenetics regulatory axis allows precise and dynamic control of immunity, and dysregulation of this process can contribute to many diseases. Here we discuss recent discoveries in this area.

Epigenetics in the activation and memory of immune cells

Innate immune cells, such as macrophages and neutrophils, can rapidly respond to molecular signals, including cytokines and pathogen-associated molecular patterns (PAMPs), and become activated accordingly. Epigenetic remodeling, here referred to as dynamics in gene regulation, for example due to changes in chromatin status, plays a crucial role in the activation process. Modification of histones creates new accessible chromatin sites and allows binding of specific transcription machinery, which permits the expression of specific genes, many functioning in pathogen killing, inflammation, or tissue remodeling. Recent research has revealed that innate immune cells can also form immunological memory, where epigenetic remodeling is an important underlying mechanism as well. In macrophages following lipopolysaccharide (LPS) stimulation, many genes become activated and have increased histone acetylation and H3K4 trimethylation. Some genes retain the H3K4 trimethylation but lose the acetylation mark quickly, and remain inaccessible upon secondary stimulation; while other genes retain the acetylation on their promoter, and can be turned on even faster upon secondary stimulation [9]. Using ChipIP-sequencing analysis, it has been revealed that the modifications of latent enhancers (enhancers that can only be bound by transcription factors after stimulation) are also an important mechanism for transcriptional memory in macrophages. Latent enhancers do not have activating histone marks such as H3K27 acetylation and H3K4 monomethylation in unstimulated macrophages, but acquire these marks after stimulation. After the first stimulation, these marks can persist, allowing faster and amplified responses upon re-stimulation [10,11]. The immunological memory in macrophages is not only mediated by acquiring new epigenetic marks, but also by the decrease of some epigenetic marks in comparison to unstimulated macrophages. For instance, upon LPS stimulation, the repressive H3K9 dimethylation is decreased in macrophages through an ATF7-dependent mechanism [12]. This chromatin remodeling de-represses a set of genes and can be maintained for a long time.

The control of innate immunity through epigenetic remodeling is not limited to immune cells with a long lifetime such as macrophages. Neutrophils are highly abundant, short-lived leukocytes that function as the front line of immunity. While the epigenetic remodeling in neutrophils is relatively less understood, several recent papers have demonstrated its role in neutrophil activation, recruitment, and function. For instance, an increase of H3K4 trimethylation and H3K27 and H4 acetylation upon stimulation promotes binding of transcription machinery and IL-6 production in neutrophils [13]. Histone acetylation promotes migration, phagocytosis and neutrophil extracellular trap formation, and loss of deacetylase HDAC11 leads to hyperacetylation and increased neutrophil activation, making animals more susceptible to LPS-induced sepsis [14,15]. Precise control of histone acetylation is a mechanism for controlling neutrophil activation. The transcription factor PU.1 prevents neutrophil overactivation by recruiting a histone deacetylase and inhibiting the accessibility of enhancers [16]. Dynamic chromatin remodeling has also been demonstrated in natural killer (NK) cells. Using ATAC-seq and RNA-seq, Lau et al. revealed that during early viral infection, NK cells show a change in chromatin accessibility that coordinates with transcriptional changes, and that memory NK cells have a distinct epigenetic landscape from naїve NK cells [17].

Similar to innate immune cells, epigenetic regulation is necessary in T cell development, activation, and mainte- nance of subtypes. Many of these aspects have been thoroughly reviewed recently [18^•]. High-throughput sequencing technologies have allowed systematic characterization of epigenetic remodeling during these processes, and contributed to the discovery of gene clusters controlled by specific epigenetic mechanisms (Table 1). For instance, using ATAC-seq, an approach to analyze open chromatin, it was revealed that chromatin remodeling in T cell development occurs in waves, and the lineage-determining transcription factor T cell factor 1 (TCF-1) controls T cell identity through its ability to create new open chromatin [19]. RNA-sequencing and ChIP-seq revealed that permissive chromatin configurations, which are installed after initial stimulation, are partially maintained in memory T cells, allowing for rapid responses upon reactivation [20].

Table 1.

Cutting edge technologies and approaches to study metabolism-epigenetic interactions in immune cells

Approach	Description	Examples for application
ChlP-sequencing	Analyzes protein-DNA interaction. ChlP-seq is a valuable tool to identify the enrichment of specific histone marks or transcription factor binding in the genome.	ChlP-seq analysis revealed increased H3K4me3 and H3K27Ac at promoters of metabolic genes induced by β-glucan training in monocytes [57]. In neutrophils, ChiP was used to identify genomic sites bound by important transcription factor PU.1 [16].
ATAC-seq	Analyzes genome-wide chromatin accessibility.	ATAC-seq was used to identify chromatin regions with increased accessibility caused by acetate supplementation in T cells grown in glucose restricted environment [43••].
Bisulfite sequencing	Analyzes DNA méthylation pattern by converting cytosine to thymidine, which methylated cytosine is resistant to.	Whole genome bisulfide sequencing identified genomic regions that are differentially methylated in different T cell populations, depending on their specific tissue environment [40].
RNA-seq	High-throughput method to quantify RNA levels. mRNA- seq is widely used to determine the changes in gene expression during immune response.	Using RNA-seq, Novakovic et al. characterized the clusters of genes whose dynamic expression patterns differ in response to LPS exposure or β-glucan exposure, and revealed the relationship between exposure-dependent expression pattern and epigenetic remodeling by integrating RNA-seq with ChlP-seq results [58].
Metabolomics	High-throughput method to quantify metabolite levels. Can be achieved by NMR or mass spectrometry.	Jha et al. used metabolomics to systematically characterize how the metabolic profile of naive macrophages and macrophages in M1 or M2 activation state differ, and identified metabolic modules important in macrophage polarization by integrating metabolomics with RNA-seq data using a network analysis method [21].
Isotopie tracing	By following the incorporation of labeled substrate into downstream metabolite or epigenetic modifications, it can be determined how metabolism is rewired during immune response and how various substrate or pathways contribute to epigenetic remodeling.	Using methionine labeling, Sinclair et al. probed changes in methionine uptake, metabolism, and methionine-driven méthylation inT cells in response to TCR [24•]. Using acetate labeling, Qiu et al. investigated the contribution of acetate in histone acétylation, and how it is dependent on ACSS activity in T cells [43••].

Metabolism orchestrates immune cell function via epigenetic mechanisms

As the epigenetic landscape undergoes significant remodeling during immune response, cellular metabolism also exhibits dynamic reprogramming specific to the activation signal [21–23]. Metabolism provides substrates for the highly active epigenetic modifications. Therefore the levels of epigenetic-modulating metabolites can affect functions in immune cells.

During T cell activation, the demand of methyl donor S-adenosyl methionine (SAM) increases as cells increase the methylation rate of DNA, RNA and histones. Using metabolomics, proteomics, and isotopic labeling approaches, Sinclair et al. found that the main regulation point controlling the flux for SAM supply is the import of extracellular methionine, the main substrate for SAM, in CD4+ T cells. Rapid upregulation of the methionine transporter is required for sustaining methylation activity and allowing full activation of T cells [24^•]. In macrophages, high SAM/S-adenosyl homocysteine (SAH) ratio promotes LPS-stimulated production of pro-inflammatory cytokine IL-1β, by supporting H3K36 trimethylation. Inhibiting SAM synthesis or SAH degradation alters cytokine profile [25]. Similar to methylation, the acetylation of histones and other proteins during immune response is under control of the level of its main substrate, acetyl-CoA. Upon activation, CD4+ T cells increase aerobic glycolysis to maintain high acetyl-CoA levels, which promotes histone acetylation and interferon-γ (IFNγ) expression. Deletion of lactate dehydrogenase A reduces aerobic glycolysis, decreases IFNγ production in activated T cells, and protects mice against autoinflammatory disease [26]. In addition to glucose, CD8+ memory T cells have been shown to take up acetate from the serum to increase the acetyl-CoA pool and promote acetylation upon infection [27]. In macrophages, IL-4 stimulation promotes glucose driven acetyl-CoA production by regulating ATP-citrate lyase. This leads to increased histone acetylation and induction of M2 genes [28]. Conversely, M1 activation, induced by LPS stimulation, suppresses mitochondrial metabolism, and at the same time decreases overall histone acetylation [29]. This effect is particularly strong after prolonged LPS stimulation, which contributes to LPS tolerance by limiting the histone acetylation and the activation of inflammatory genes (Figure 1) [30^•]. The limitation of acetyl-CoA availability in M1 macrophages can occur through modulation of glycerol phosphate shuttle and inhibition of pyruvate dehydrogenase [30^•,31].

Figure 1.

Coordinated changes in metabolism and epigenetics in macrophages/monocytes during polarization by different stimulation signals or formation of immunological memory. Abbreviations: LPS, lipopolysaccharide; IL-4, interleukin-4; AKG, α-ketoglutarate; OXPHOS, oxidative phosphorylation; H3K27Ac, acetylation of histone H3K27; H3K4me3, trimethylation of histone H3K4; H3K9me2, dimethylation of histone H3K9; H3K4me1 monomethylation of H3K4.

In addition to the installation of histone and DNA modifications, the epigenetic landscape is also controlled by the reactions removing these modifications. Particularly, demethylation of histones and DNA can be catalyzed by α-ketoglutarate (AKG)-dependent demethylases. Several metabolites that are structurally similar to AKG, including succinate, fumarate, and 2-hydroxy-glutarate (2HG) can competitively inhibit demethylation. Therefore, the ratio of AKG to these inhibitory metabolites modulates methylation levels. These metabolites are intermediates in the TCA cycle or directly derived from the TCA cycle, and a lot of these compounds are largely derived from glutamine. This allows TCA cycle activity and glutamine metabolism to regulate immune cells through modulation of demethylases. In macrophages, the AKG/succinate ratio is increased upon IL-4 stimulation (M2) but decreased upon LPS stimulation (M1). Liu et al. showed that manipulating AKG/succinate ratio altered macrophage polarization in a dose-dependent manner, with increasing AKG/succinate ratio promoting M2 gene expression while decreasing AKG/succinate ratio strengthening M1 phenotypes [32^••]. Memory in macrophages and monocytes is also regulated by TCA metabolism. Arts et al. revealed that induction of glucose and glutamine metabolism is crucial in trained immunity. Treating monocytes with fumarate was sufficient to induce an epigenetic program, including increased H3K4 trimethylation at the promoters of pro-inflammatory cytokines, similar to monocytes trained with β-glucan (Figure 1) [33^•]. If glycolysis or glutaminolysis is inhibited, monocytes are unable to induce trimethylation of histones at the promoters of pro-inflammatory cytokines IL-6 and TNFα, which is an important part of BCG-induced, trained immunity [34]. And glutaminolysis can modulate LPS-induced tolerance in macrophages [32^••]. Similarly, the ratio of AKG and metabolites that inhibit demethylation regulates the fate of T cells. In CD8+ T cells, 2HG accumulates in response to T cell receptor triggering. Increase of 2HG, particularly S-2HG, leads to an increase of global H3K27 trimethylation and decrease of its dimethylation, and alters T cell differentiation and proliferation [35]. In CD4+ T cells, transamination activity controls 2HG production. Increased 2HG level causes hypermethylation of Foxp3 gene, and shifts the balance between Th17 and induced Treg cells [36^•].

Besides the mechanisms discussed above where metabolites directly modulate epigenetic modifications by acting as substrates or inhibitors, metabolism can impact epigenetics through indirect mechanisms involving the production of metabolites with signaling properties. For instance, the accumulation of mevalonate, an intermediate in cholesterol metabolism, can induce trained immunity in monocytes through activation of IGF1 receptor and subsequent histone modifications [37]. The metabolism and release of other signaling molecules, such as eicosanoids, can also activate corresponding receptors and trigger downstream epigenetic remodeling [38].

The metabolism-epigenetic axis allows microenvironment to shape immune cells

Microenvironment has a significant impact on the epigenetic landscape of immune cells. For instance, studies that leverage multiple systematic sequencing approaches, including whole genome bisulfide sequencing (WGBS), ChIP-seq, ATAC-seq and RNA-seq, to compare the immune cells isolated from various tissues have demonstrated the epigenetic diversity of macrophages and T cells is dependent on the specific tissue they reside [39,40]. At the same time, metabolism of immune cells is also tissue-specific [41]. Two types of mechanisms link the specific metabolic environment with the epigenetic changes in immune cells: (1) nutrient availability and environmental condition shape the metabolism in immune cells, which in turn affect their epigenetics; (2) metabolites produced by other cells in the microenvironment can affect the epigenetics in immune cells.

In the tumor microenvironment, low availability of glucose, glutamine and oxygen is common, due to insufficient blood perfusion and high metabolic activity of cancer cells. Glucose restriction dampens effector T cell functions. One mechanism is through altering histone methylation [42]. Another mechanism is that low glucose availability restricts acetyl-CoA level, which is required for histone acetylation. Acetate supplementation can rescue effector CD8+ T cell functions in a low glucose environment by enhancing histone acetylation and chromatin accessibility [43^••]. In addition to glucose restriction, glutamine deprivation can promote the generation of Treg cells from a naїve CD4+ T cell population by limiting AKG availability, which is needed for DNA demethylation [44]. Hypoxia can also alter T cell phenotypes and anti-tumor capacity by influencing methylation [35]. In the gut microenvironment, the microbiome can regulate immune cells by producing regulatory metabolites, particularly short chain fatty acids. For instance, butyrate regulates local macrophage function by inhibiting histone deacetylases, and promotes tolerance to bacteria in such environments [45]. In B cells, short chain fatty acids regulate gene expression by increasing histone acetylation, which is likely due to both inhibition of histone deacetylation and increase in acetyl-CoA level [46]. The regulatory connection between metabolism and epigenetics also allows diet to influence immune function. A Western diet can trigger inflammation, and induce epigenetic reprogramming and trained immunity in granulocyte-monocyte progenitor cells [47].

Discussion and perspective

Increasing studies demonstrate that metabolism plays a crucial role in regulating immune cell functions. As discussed here, modulating the epigenetic landscape is an important mechanism for such regulation. Other mechanisms, including metabolism impacting signaling path-ways, metabolic enzymes directly regulating transcription, and metabolism influencing organelle function, also allow metabolism to regulate immunity. These are discussed in several recent reviews [48^•,49]. As a result, metabolic intervention is emerging as a promising strategy to modulate immunity [50].

While here we focused on immune cells, and mainly discussed how chromatin modification, in the form of acetylation and methylation, can be regulated by metabolism, the connection between metabolism and epigenetics is more ubiquitous. Other less characterized modifications can link metabolism with epigenetics as well. For instance, Zhang et al. demonstrated that a novel histone modification, lysine lactylation, is regulated by intracellular lactate level. In macrophages, lactate production from glycolysis induced by bacteria exposure causes increased histone lactylation, which then induces the expression of a set of genes during the late phase of M1 polarization [51^•]. Metabolic regulation of epigenetics also widely occurs in other types of cells besides immune cells. Altered metabolism is a hallmark in cancer. The important role of metabolic alteration in impacting cancer epigenome and disease progression has been shown in many studies over the last decade, and has been thoroughly reviewed [52,3,53]. For instance, accumulation of 2HG, the first identified oncometabolite produced in large quantity in glioma harboring mutant IDH, causes DNA and histone hypermethylation. Metabolism influences epigenetics during development and cell fate decision through a similar mechanism as well [3,5]. It is also worth noting that this metabolism-epigenetics connection is not limited to a mammalian system. For example, in yeast, acetyl-CoA level acts as a signal for carbon metabolism and can promote proliferation through histone acetylation [54].

It has been proposed and demonstrated in other biological systems that epigenetic modifications can act as a sink and/or reservoir for metabolites [55]. It is also likely that some of the metabolic rewiring observed in immune cells is caused by the increased metabolic demand due to highly active epigenetic remodeling during immune response. For instance, inflammatory macrophages become dependent on NAD⁺ salvage pathway due to high NAD⁺ consumption for poly-ADP-Ribosylation [56]. It is also possible that demands for metabolic substrates, such as acetate, would drive epigenetic changes in immune cells. Quantitative analysis of overall epigenetic remodeling flux during immune response, and how that compares to the production flux of required metabolite substrates, is still largely missing.

Many important open questions remain to be answered in this area: Which of the metabolism-influenced epigenetic changes are reversible whereas which persist for a longer period? What molecular mechanisms allow metabolism to regulate the chromatin of a specific set of genes at a specific time window? Furthermore, the crosstalk between metabolism and epigenetics during immune response is bidirectional: the metabolic alterations can be both a consequence and a driver for epigenetic remodeling. For instance, glycolysis enzymes are among the most upregulated genes by histone modifications in β-glucan trained macrophages [57]. Therefore, feed-forward and feed-back regulation may drive the dynamic transitions in both metabolism and epigenetics. Future studies working toward dynamic understanding of the metabolism-epigenetic regulation network would provide insights into these questions. Development of methods that integrate high-throughput analysis of epigenetic landscape and metabolism is needed to explore these intriguing interactions.