Metabolism and acetylation in innate immune cell function and fate

Abstract

Innate immunity is the first line of defense against invading pathogens. Changes in both metabolism and chromatin accessibility contribute to the shaping of these innate immune responses, and we are beginning to appreciate that cross-talk between these two systems plays an important role in determining innate immune cell differentiation and function. In this review we focus on acetylation, a post-translational modification important for both regulating chromatin accessibility by modulating histone function, and for functional regulation of non-histone proteins, which has many links to both immune signaling and metabolism. We discuss the interactions between metabolism and acetylation, including the requirement for metabolic intermediates as substrates and co-factors for acetylation, and the regulation of metabolic proteins and enzymes by acetylation. Here we highlight recent findings, which demonstrate the role that the metabolism-acetylation axis has in coordinating the responses of innate immune cells to the availability of nutrients and the microenvironment.

1. Introduction

Innate immunity is generally considered to be mediated by cells in the immune system that are not conventional T cells or B cells, and which do not express clonally restricted antigen-specific receptors. Within this large group of cells are included NK cells, innate lymphoid cells, neutrophils, mast cells, basophils, eosinophils, macrophages and dendritic cells (DCs). Most innate immune cells are hematopoietically derived, but cells of non-hematopoietic origin, such as epithelial cells, can also participate in innate immunity. Innate immune cells are the first line of defense against invading pathogens. Most of these cells express pattern recognition receptors (PRRs), which recognize highly conserved pathogen-associated molecular patterns (PAMPs), or danger-associated molecular patterns (DAMPs) exposed during infection or injury. Innate immune cells can also respond to cytokines made by other cells responding to PAMPs or DAMPs. Stimulation through PRRs or cytokine receptors initiates changes in the expression of many genes and it is these changes that shape the function and fate of the activated cells and allow them to participate in host-protective processes.

2. Metabolism in innate immune cells

Based on observations first made 40 years ago and rediscovered and explored more deeply of late, we know that as cells of the innate immune system become activated in response to PAMPS, DAMPS and or cytokines, they undergo significant metabolic remodeling. Metabolic changes linked to activation have been studied most extensively in macrophages and DCs. Early observations focused on macrophages activated by bacteria, a process that we now know is largely mediated by the Toll like receptor (TLR) subset of PRRs. We now refer to macrophages activated in this way as being classically or M1 activated. In early studies on M1 macrophages, increased glucose uptake was associated with increased pentose phosphate pathway (PPP) activity [1]. This made sense because this pathway is necessary to produce nicotinamide adenine dinucleotide phosphate (NADPH), which is a required cof actor for NADPH oxidases, the effector enzymes that generate bactericidal superoxide [2,3]. Contemporary analyses have confirmed increased flux through the PPP in M1 cells [4]. However, recent work has emphasized that glycolysis, the core glucose metabolism pathway in cells that is responsible for converting glucose into pyruvate (Fig. 1), is greatly accentuated in M1 macrophages as well as in conventional DCs that have been stimulated with TLR agonists [5].

Fig. 1.

Metabolic pathways utilized in pro-inflammatory versus anti-inflammatory innate immune cells. Pro-inflammatory innate immune cells such as classically activated macrophages or immunogenic dendritic cells primarily employ anabolic metabolism, utilizing aerobic glycolysis and the pentose phosphate pathway. In contrast, anti-inflammatory cells, including alternatively activated macrophages and tolerogenic dendritic cells, primarily utilize catabolic pathways, such as fatty acid oxidation (FAO or β-oxidation).

In terms of macrophages, another major development that has occurred since the seventies is the realization that these cells can be activated in many different ways to assume distinct fates and functions. At the opposite end of the activation spectrum from M1 macrophages, is the M2 or alternative activation state that is adopted by macrophages that have been stimulated with IL-4 [6]. We now know that in contrast to M1 macrophages, where glycolysis is accentuated, M2 macrophage metabolism is more biased towards fatty acid β-oxidation (FAO; Fig. 1) [7]. Compared to unpolarised macrophages, M2 cells have high basal mitochondrial O2 consumption and moreover have large spare respiratory capacity that can be collapsed by inhibiting FAO. In these cells, FAO is fuelled by fatty acids released by lysosomal lipolysis of acquired triacylglycerols, but the cells can to some extent also utilize denovo synthesized fatty acids to support FAO [8]. Fatty acid synthesis requires glucose carbon and glucose consumption is greater in M2 macrophages than in M0 macrophages [9], and it is reasonable to assume that this reflects the use of glucose for fatty acid synthesis. However, recent work has shown that increased glucose uptake in M2 macrophages also supports accentuated UDP-GlcNAc synthesis, and more importantly from the perspective of this article, regulated increases in glycolysis in these cells has been linked to the acetylation of histone 3 (H3) and the association of acetylated H3 and histone 4 (H4) with some of the genes that when expressed define the M2 activation state [9].

3. Epigenetics, acetylation & innate immune responses

Innate immune cell differentiation, activation and function is controlled by changes in gene expression, and epigenetic mechanisms are key regulators of this [10–12]. Epigenetic regulation modifies gene expression without altering the DNA sequence, through heritable changes to the chromatin state. The chromatin state controls DNA accessibility, and thus regulates the access of transcription factors to DNA, ultimately modulating transcription and gene expression.

The most well-studied form of epigenetic regulation is DNA methylation, which involves the addition of a methyl group directly to a cytosine in a CG dinucleotide (a CpG site). DNA methylation is generally associated with gene silencing, as methylated CpGs prevent transcription factor binding to DNA promoters and recruit transcriptional repression complexes [13]. The second fundamental mechanism of epigenetic regulation is the post-translational modification of histones. Histones form the core of nucleosomes, with each nucleosome comprised of DNA wrapped around a histone octamer, composed of two copies each of histone 2A (H2A), histone 2B (H2B), histone 3 (H3) and histone 4 (H4). Histone modifications take a variety of forms, including methylation, acetylation, phosphorylation, SUMOylation and ubiquitination of a range of amino acid residues on the N-terminal tails of chromatinized histones [14]. These moieties are added and erased by specialized enzymes and complexes, and histone modifications are both highly dynamic and tightly regulated [13]. Unlike direct methylation of DNA, modification of histones can both positively and negatively regulate gene expression. For example, histone methylation at H3 lysine 9 (H3K9) is associated with gene repression, while methylation at H3K36 is associated with activation [15]. Furthermore, the number of methyl groups added to the methylation site (mono-, dior trimethylation) ascribes different outcomes for gene regulation [14]. The site of histone modification and importantly the combination of modifications at any site within the genome, can lead to differential gene expression. This is called the histone code hypothesis, first proposed by Allis and Jenuwein [16]. Modifications to histones can interfere with DNA-histone interactions to increase or decrease the affinity with which DNA is bound to histones. A crucial part of this hypothesis is the idea that histone modifications can also be read by certain protein domains and this leads to the recruitment of proteins that have important function in gene expression or suppression. This model explains the importance of histone modifications in gene regulation.

3.1. Histone acetylation

Many reviews have been written about epigenetics in innate immune cell activation and function recently [17]. Our key area of interest is innate immune cell metabolism, and here we will focus not on epigenetics but rather will discuss the interplay between metabolism and acetylation, highlighting how this impacts innate immune cell function.

As previously mentioned, histones can undergo a number of post-translational modifications modifying DNA accessibility, including histone acetylation. This modification occurs post-translationally with an acetyl group added to the ε-amino group of lysine residues at the N-terminal histone “tail” (ε-acetylation), and is a reversible reaction [14,18]. The reversibility of lysine acetylation makes this post-translational modification useful in the regulation of proteins in response to metabolic changes, allowing dynamic regulation of protein function [18]. In general, histone acetylation is correlated with gene expression, while histone deacetylation is associated with gene repression [19]. Acetylation neutralizes the positive charge of histones, loosening the interaction between the histone and positively charged DNA, and improving transcription machinery access [14]. Correspondingly, histone deacetylation results in more tightly compacted, and inaccessible chromatin. The dynamic process of histone acetylation is catalyzed by the opposing activities of lysine acetyltransferases (KATs; previously termed histone acetyltransferases or HATs) and lysine/histone deacetylases (KDACs/HDACs). These enzymes respectively add or remove acetyl groups, and require specific metabolic intermediates to function efficiently [20]. The functional effect of histone acetylation is dependent on the site of modification. Table 1 lists some of the transcriptional outcomes associated with acetylation of histones 3 and 4, which are more commonly regulated by acetylation than histone 2A/2B [13].

Table 1.

Histone 3 and 4 acetylation sites and effects.

Acetylation site		Functional outcome	Ref
Histone 3	H3K9	open promoter regions, DNA damage response	[26], [27]
	H3K14	increased transcription, DNA damage response	[28], [29]
	H3K18	positioned at active/poised genes, cancer progression	[30], [31]
	H3K23	high gene expression	[32]
	H3K27	open chromatin regions, active/poised genes	[33], [30]
	H3K56	DNA damage response, Genomic stability	[34]
	H3K64	nucleosome stability, active transcription	[35]
	H3K122	active transcription	[26]
Histone 4	H4K5	poised genes	[36]
	H4K8	active promoters	[30]
	H4K12	active promoters, telomere dynamics	[30], [37]
	H4K16	active genes, DNA damage response	[38], [39]
	H4K91	chromatin assembly, active/poised genes	[40], [30]

Histone acetylation has been shown to regulate macrophage activation and DC differentiation. Epigenomic profiling of monocytes that were stimulated with human serum as a source of macrophage colony stimulating factor (M-CSF) to differentiate into macrophages revealed major acetylation changes upon stimulation [11]. Histone 3 lysine 27 acetylation (H3K27ac) was the most dynamic mark, with changes observed at 7611 distal regions and at 3063 annotated transcription start sites (TSSs). These changes in H3K27 acetylation levels correlated with changes in gene expression, and gene ontology analysis of expression data revealed that macrophage differentiation was associated with enrichment of metabolic genes, specifically those involved in monocarboxylic acid and cellular ketone metabolism. Upregulated enzymes included dehydrogenases implicated in peroxisomal β-oxidation and in peroxisome proliferator-activated receptor (PPAR) signaling, as well enzymes in the TCA cycle. This implies that acetylation has a role in the reprogramming of cellular metabolism during macrophage differentiation. Building on this, in the same study epigenomic analysis was performed on macrophages stimulated with LPS to induce long-term tolerant cells, which have dampened production of pro-inflammatory mediators, or with β-glucan to induce “trained” cells that have increased inflammatory responsiveness. While many epigenomic marks were shared, β-glucan trained cells also had a strong exclusive acetylation signature, with 40% of sites gaining acetylation only in trained cells. Induction of aerobic glycolysis is an essential process in the development of macrophages exhibiting trained immunity [21]. Thus it is possible that acetylation may also mediate the metabolic switch that allows development of trained immunity. These papers indicate that histone acetylation plays a major role in macrophage reprogramming, including metabolic shifts. This is also supported by previous studies, which have shown HDACs are both positive and negative regulators of TLR and interferon (IFN) signaling, modulating both macrophage and DC production of inflammatory mediators through a variety of mechanisms [22,23].

Histone acetylation and deacetylation is a highly dynamic process, which contributes to the chromatin state and would be expected to impact many genes involved in cellular metabolic processes. One instance of this is acetylation of H3K9 controlling the expression of multiple glycolytic genes, including HIF1α. It is known that SIRT6 deacetylation of H3K9 is required to slow expression of these genes, and accordingly, cells lacking in SIRT6 display high glycolytic metabolism and low TCA cycle activity [24]. There appears to be little work examining the role of histone acetylation regulating innate immune cell metabolism, but recent work indicates that histone acetylation and deacetylation may play a role in the regulation of M2 macrophage polarization and concurrent metabolic reprogramming. The deacetylase HDAC3 has been shown to negatively regulate expression of genes associated with IL-4 and IL-13 stimulation and downstream regulate the development of an M2 phenotype [25]. In HDAC3-deficient macrophages stimulation with IL-4 and IL-13 potentiated the induction of a panel of genes upregulated in alternative activation, including Arg1 and Retnla, indicating that HDAC3 limits the expression of IL-4 induced genes, thereby limiting the alternative activation phenotype. Interestingly, this is HDAC3-specific, as when cells were treated with a pan-HDAC inhibitor Arg1 expression was decreased both basally and following IL-4 stimulation.

3.2. Non-histone protein acetylation

Although, protein acetylation was initially discovered on histones, it has since been shown to occur on a wide range of non-histone proteins, modulating their functions significantly [18,41,42]. Proteins can undergo two different forms of acetylation; co-translational acetylation or post-translational acetylation. Acetylation of the N-terminal of proteins (Nt-acetylation) is a common co-translational modification, which is irreversible. Nt-acetylation has roles in controlling protein synthesis, stability and localization, and the majority of eukaryotic proteins are subjected to this form of acetylation [43]. The second form of protein acetylation is the post-translational ε-acetylation of the ε-amino group of lysine residues, which, like histone acetylation, is a reversible reaction [18]. The reversibility of lysine acetylation makes this post-translational modification useful in the regulation of proteins in response to metabolic changes, allowing dynamic modulation of proteins.

There is a substantial body of evidence demonstrating that lysine acetylation can regulate proteins in immunological pathways. One fundamental example of this is the regulation of the transcription factor NF-κB by acetylation. NF-κB mediates pro-inflammatory cytokine production, such as the production of TNFα by macrophages. Acetylation of the RelA/p65 subunit of NFκB at lysine 30 has been shown to be required for transcriptional activity, and production of proinflammatory cytokines [44,45]. Interestingly, it is also now well-established that deacetylation of NF-κB and the resultant inactivation of NF-κB is regulated by SIRT1 [46], which as discussed in greater detail below links this acetylation to metabolism, through the requirement of sirtuins for metabolic intermediates as cofactors. A second key example of acetylation modulating proteins essential to immunological pathways, is the regulation of members of the interferon regulatory factor (IRF) family by acetylation. IRFs are required for TLR-induced production of type I IFNs as well as other inflammatory mediators, and several members of this family have been shown to be directly acetylated [23]. IRF7 plays a critical role in plasmacytoid DC (pDC) type IIFN production, and can be acetylated on lysine 92 within the DNA binding domain, resulting in impaired DNA binding [47,48]. Furthermore, HDAC inhibitors have been shown to reduce nuclear translocation of IRF7 in pDCs and have similar effects on IRF1-mediated type I IFN production in conventional DCs [48], indicating an important role for acetylation/deacetylation in the regulation of type I IFN production by DCs. These examples are far from exhaustive, with other proteins in immunological pathways also described as being regulated by acetylation. Moreover, global analysis of human protein acetylation has revealed 3600 acetylation sites on 1750 proteins, comparable to the number of proteins shown to be phosphorylated [49,50]. Based on this abundance of protein acetylation it is likely that acetylation also plays a role in the regulation of many more proteins involved in innate immune cell function and metabolism.

4. Metabolic processes play a role in the regulation of acetylation in innate immune cells

The first evidence for metabolism influencing gene transcription via epigenetic mechanisms was reported over ten years ago in yeast [51]. It is now clear that many chromatin-modifying enzymes require substrates or co-factors that also have roles in metabolism as intermediary metabolites (reviewed by Gut and Verdin in 2013 [52]). This is also true of both histone and non-histone protein acetylation, which is sensitive to the metabolic state of the cell, with metabolites serving as substrates for both acetyltransferase and deacetylation reactions.

4.1. Acetyl-coenzyme A; metabolite and acetate donor for acetylation

Acetyl-Coenzyme A (Acetyl-CoA) is a prime example of a metabolite regulating acetylation. Acetyl-CoA is a central carbon metabolite and the substrate for the tricarboxylic acid (TCA) cycle. However, it is also the primary acetyl donor for protein acetylation. KATs perform acetylation by transferring acetyl groups from acetyl-CoA to lysine residues on proteins. Therefore, acetylation is directly dependent on acetyl-CoA availability within the relevant sub-cellular compartment (Fig. 2).

Fig. 2.

The sources of acetyl-CoA for acetylation of proteins and histones. Acetyl-CoA can be produced in the mitochondria, cytosol, and also directly in the nucleus. The sources for acetyl-CoA in the mitochondria are imported pyruvate and fatty acids. Mitochondrial proteins can be acetylated using this substrate, or acetyl-CoA can be used through the TCA cycle. Acetyl-Co cannot pass through the mitochondrial membranes. However, citrate can be exported from the TCA cycle to the cytoplasm via SLC25a1, and then in cytoplasm can be converted back to acetyl-CoA by ATP-citrate lyase (ACL). Alternative sources for acetyl-CoA are actetate and N-acetylaspartate (NAA), which can be converted to acetyl-CoA by the enzymes acetyl-CoA synthase (ACS) and aspartoacylase (ASPA) respectively. Proteins in the cytoplasm are acetylated using this acetyl Co-A generated from any of these substrates. Cytoplasmic acetyl-CoA can directly enter the nucleus for histone acetylation. Pyruvate, acetate and NAA can also enter the nucleus and may serve as additional sources of acetyl-CoA in this organelle. The enzymes and carriers involved in these reactions are highlighted in blue.

Acetyl-CoA exists both within mitochondria and in the cytoplasm, but it cannot move directly across the mitochondrial membranes, and therefore has to be independently synthesized in each compartment. In mitochondria acetyl-CoA is made from pyruvate by the pyruvate dehydrogenase complex (PDC), or from fatty acids by β-oxidation. It can then be used for acetylation of mitochondrial proteins, or can enter the TCA cycle where it is converted to citrate. Depending on demand, citrate can continue through the TCA cycle to allow the generation of NADH and FADH to fuel the electron transport chain, oxidative phosphorylation and adenine-triphosphate (ATP) production. Alternatively, citrate can be exported into the cytoplasm through the citrate transporter SLC25A1, where it can be converted back into acetyl-CoA by ATP-citrate lyase (ACL). Cytoplasmic acetyl-CoA can be used for both fatty acid synthesis and for protein acetylation. Thus it is reasonable to assume that acetylation of both cytoplasmic and mitochondrial proteins will have to compete with use of acetyl-CoA for bioenergetic purposes, depending on demand within each pathway and overall acetyl-CoA availability (Fig. 2).

Other pathways for acetyl-CoA production have also been shown to be utilized by immune cells. For example memory CD8⁺ T cells use acetyl-CoA synthase (ACS) to synthesize acetyl-CoA from acetate [53] (Fig. 2). While it is possible to generate acetyl-CoA from acetate, early studies by Wellen and colleagues stressed the importance of glucose-derived citrate for histone acetylation in immune cells in response to cellular stimulation with growth factors [54]. Under conditions of low glucose decreased histone acetylation was observed. In this situation cells were able to fuel bioenergetic needs through the alternative pathway of FAO, however this failed to rescue histone acetylation levels. A second alternative pathway for acetyl-CoA synthesis is the degradation of N-acetylaspartate (NAA), a metabolite abundant within the brain, which can be converted into aspartate and acetate by aspartoacylase (ASPA) [55] (Fig. 2). Whilst this pathway has traditionally been associated with lipid synthesis for myelination, more recently it has been shown to be a source of nucleo-cytoplasmic acetyl-CoA utilized for histone acetylation in brown adipocytes [56]. This implies more widespread use of the NAA pathway, and is intriguing because ASPA is expressed by resident peritoneal macrophages but not monocyte-derived peritoneal macrophages, indicating a dichotomy in the potential to use NAA in distinct innate immune cell subsets [57].

The pool of nuclear acetyl-CoA can either be derived from cytoplasmic acetyl-CoA, which is able to diffuse through nuclear pores, or generated directly within the nucleus; ACL and ACS are both present in the nucleus, facilitating nuclear acetyl-CoA production from citrate or acetate as previously described [54]. Additionally, nuclear acetyl-CoA can be produced from pyruvate by the PDC, which is traditionally associated with the mitochondria but has recently been identified in the nucleus [58]. The varied sources and uses of acetyl-CoA in different sub-cellular compartments are summarized in Fig. 2.

The dual role for acetyl-CoA as a central carbon metabolite and as the acetyl-donor for acetylation suggests that the dynamic process of histone acetylation links metabolism to transcriptional regulation of innate immune cells. Furthermore, acetylation of non-histone proteins connects metabolism to regulation of cell function at a post-transcriptional level. Collectively, this evidence supports that cells can sense nutrient availability and metabolic state via acetyl-CoA, and regulate histone and protein acetylation following metabolic perturbation to influence cell fate or adaptation.

4.2. Metabolite regulation of sirtuins

Metabolites can regulate acetylation via effects on acetyltransferase and deacetylase enzymes. Sirtuins are a family of proteins that primarily function as deacetylases, removing acetyl groups from lysine residues in an NAD⁺-dependent manner. NAD⁺ is a coenzyme in redox reactions, and the reduced form (NADH) is an essential intermediate for energy transfer in various metabolic pathways. The ratio of NAD⁺ to NADH is indicative of the cellular metabolic state and a high NAD⁺/NADH ratio occurs in low energy conditions. Therefore conditions of nutrient deprivation can promote deacetylation, by providing abundant NAD⁺ for sirtuin activity. Under conditions of prolonged catabolism, the rate of NADH oxidation surpasses the rate of NAD⁺ reduction, increasing the NAD⁺/NADH ratio. The resulting elevated level of NAD⁺ has been shown to activate SIRT1 [59]. Similarly, imbalance of the metabolic pathways that utilize NAD⁺ as a co-factor, including glycolysis, the TCA cycle and FAO can result in NAD⁺ depletion, leading to reduced sirtuin activity, and inhibition of deacetylation. Therefore, the metabolic state of innate immune cells will be reflected in cellular NAD⁺ levels and have downstream impacts on sirtuins. This has been demonstrated in macrophages, where high glucose use resulted in depletion of cellular NAD⁺ through oxidative stress, which was associated with reduced SIRT1 expression and activity [60]. SIRT1 has been implicated in the control of a number of cellular processes undertaken by macrophages, including NF-κB dependent pro-inflammatory cytokine production and COX-2 expression [61,62]. Thus, the increased glucose use and ROS production typical of classically activated macrophages likely impacts a range of downstream functions via regulation of SIRT1.

The rate-limiting influence of NAD⁺ on sirtuin activity also has implications for DC differentiation and activity. PPARγ has been well-characterized as a key regulator of the differentiation of human monocytes into DCs. For example, granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4 induced differentiation of monocytes into DCs is marked by the upregulation of PPARγ expression, and this PPARγ expression is associated with the development of Type 2 immune responses. Conversely, PPARγ repression is generally associated with the development of DCs that can induce Type 1 immune responses [63,64]. Transcriptionally, PPARγ is critical in the regulation of both lipid metabolism and PPARγ co-activator 1α (PGC1α). PGC1α is a master regulator of mitochondrial biogenesis, and upregulation of PGC1α increases biogenesis to promote OXPHOS, which is essential for DC differentiation [65]. Significantly, SIRT1 has been reported to regulate both PPARγ and PGC1α activity in DCs [66,67]. SIRT1 regulation of PGC1α is through direct deacetylation, which has an activating effect [67]. In contrast SIRT1 activity has a repressive effect on PPARγ, however the mechanism by which acetylation modulates PPARγ in DCs remains unclear. In other cell types PPARγ has been shown to be positively regulated by direct acetylation at lysine residues 154 and 155 [68]. This SIRT1 regulation of PPARγ and PGC1α expression links DC differentiation to the redox balance and NAD⁺ availability.

In addition to regulating DC differentiation, sirtuins are also implicated in the modulation of DC cytokine production. Acetylation of the interferon-regulatory factor 1 (IRF1) protein negatively regulates the ability of DCs to produce IL-27 and interferon-β [69]. Deacetylation of IRF1 is mediated by SIRT1, and such deacetylation inhibits the ability of DCs to produce IL-27 and interferon-β. Likewise, SIRT1 has been reported to modulate IL-12p70 production, and the balance between IL-12p70 and TGF-β [70,71]. The first of these studies found treatment of DCs with a β-glucan increased nuclear concentrations of NAD⁺ and SIRT1 activity, resulting in deacetylation of H3K14 in the il12a nucleosome, and reducing IL-12p70 transcription [70]. The second study identified a non-histone mechanism of SIRT1 regulation of DC cytokine production, in which SIRT1 modulation of IL-12p70 and TGFβ production was via negative regulation of HIF1α [71]. Inhibition of SIRT1 activity increased HIF1α expression and resulted in decreased IL-12p70 and increased TGFβ, pushing T cells towards a Th1 phenotype and decreasing Treg generation. Collectively this suggests an important role for SIRT1 in regulation of DC function, and the distinct mechanisms of regulation may explain why sirtuins have been shown to drive both anti-and pro-inflammatory immune responses. The NAD⁺ requirement for sirtuin deacetylation activity links nutrient levels to chromatin accessibility and gene expression. It is also important to note that metabolite regulation of sirtuin deacetylase activity is not limited to NAD⁺. Free long chain fatty acids, such as oleic acid, have been demonstrated to stimulate SIRT6 deacetylase activity, specifically on H3K9Ac substrate in vitro [72]. The ubiquitous nature of sirtuins means that this axis of metabolic-acetylation crosstalk has wide-reaching implications for innate immune cell function and differentiation.

4.3. Regulation of non-sirtuin deacetylases by metabolites

In addition to the rate-limiting effects of NAD⁺ on sirtuins there is a growing list of metabolic intermediates that modulate other histone deacetylases, promoting the maintenance of acetylation. One such endogenous metabolite is lactate, a byproduct of glycolysis that accumulates in conditions when Warburg metabolism (or aerobic glycolysis) is active, which has been shown to be an endogenous HDAC inhibitor [73]. This was demonstrated in a human colon cancer cell line, where over a period of a few days cells were cultured until confluent and the media was acidified by accumulation of lactate, which was associated with an increase in acetylation of histones H3 and H4. While lactate was a relatively weak inhibitor compared to traditional HDAC inhibitors, such as trichostatin A, it regulated gene expression changes in a similar pattern, with comparable genes affected with directionally similar changes in expression. The ketone β-hydroxybutyrate (βOHB) can also bind to and inhibit class I and II HDACs [74]. βOHB is the major energy source for mammals following extended periods of fasting or exercise, carrying energy from adipocytes to peripheral tissues. Under these conditions human serum levels of βOHB increase from a low micromolar range to 1–2 mM or higher [75]. In vitro, exposure to βOHB resulted in a level of histone acetylation similarto that seen in fasting or calorie restriction, both of which induce ketogenesis [74]. Importantly, these effects were observed at physiologically relevant concentrations, within the range reached in serum in periods of fasting or exercise. Whilst these experiments were performed in non-innate immune cell models these concepts are relevant to innate immune cells, they are also exposed to these metabolites. M1 macrophages in particular are high producers of lactate and circulating innate immune cells would be expected to be exposed to βOHB during times of fasting or exercise. Thus it is likely that the regulation of acetylation by these endogenous metabolites impacts innate immune cell acetylation.

In addition to the effects of endogenous metabolites on acetylation, exogenous metabolites can impact acetylation and gene expression. One prime example is the short-chain fatty acid (SCFA) butyrate, a compound closely related to βOHB. Butyrate is the most abundant SCFA in the gut and is produced by commensal bacteria when they ferment dietary fiber. Butyrate has been demonstrated to be a potent HDAC inhibitor, specific for most class I and II HDACs [76,77]. In DCs butyrate has been shown to block GM-CSF driven development in vitro [78]. Similar effects on DC maturation were seen with the SCFA propionate [78]. Furthermore, in response to TLR agonists butyrate simultaneously promoted IL-23 expression by DCs, while suppressing IL-12 production [79]. These effects of butyrate and propionate have been shown to be dependent on SLC5A8, a sodium-coupled monocarboxylate transporter, differentiating them from HDAC-independent effects that butyrate can have as agonist of the G-protein-coupled receptor GPR109A [78–80]. The ability of SCFAs to promote immature and tolerogenic DC phenotypes has implications for inflammatory bowel disease (IBD), in which immune responses are dysregulated. Interestingly, active IBD has been associated with elevated fecal levels of butyrate [81]. In homeostatic conditions the acetylation-mediated immunomodulatory effects of butyrate and propionate may represent one mechanism by which commensal bacteria suppress the development of immune activation, which may become dysregulated in IBD and colitis.

5. Acetylation controlling immunometabolism

There is a substantial body of evidence demonstrating that acetylation can regulate immunological pathways, as has been previously discussed. While acetylation can clearly modulate innate immune cell function at a transcriptional level, direct evidence showing a role for acetylation in controlling the metabolic program of innate immune cells is less well-established, however there are numerous links that deserve further exploration.

5.1. Acetylation can directly regulate metabolic enzymes

Acetylation can modulate protein function, as previously discussed, and there is a growing body of evidence to suggest that acetylation plays an important role in regulating the function of metabolic proteins and enzymes. Proteomic analysis has revealed there are 277 lysine acetylation sites on 133 mitochondrial-localized proteins [82]. Furthermore, almost all the metabolic enzymes involved in glycolysis, gluconeogenesis, the TCA, FAO, and urea cycles and in glycogen metabolism have been identified to be acetylated [83], and enzymes involved in oxidative phosphorylation are abundantly acetylated [82]. The presence of mitochondrial localized SIRTs suggest that acetylation and deacetylation of proteins is a dynamic process occurring within mitochondria. There is increasing evidence linking this process to nutrient levels, with mitochondrial protein acetylation levels demonstrated to be responsive to manipulations such as fasting, caloric restriction and high-fat diet [82,84–86].

Clearly numerous enzymes involved in metabolic pathways are functionally regulated by post-translational acetylation. Whilst there are few studies exploring the acetylation of these enzymes specifically in innate immune cells, many of these are key enzymes in pathways critical to innate immune cell function. Therefore, it is logical to assume that regulation of such enzymes by acetylation in other cell types will also be important in control of innate immune cell metabolism, and downstream of this, immune cell function, and future work in this area is likely to be valuable.

One example of a metabolic enzyme regulated by acetylation is the long-chain acyl-CoA dehydrogenase (LCAD) [87]. LCAD is a key enzyme involved in the oxidation of long-chain fatty acids. In contrast to previously discussed examples of proteins, which are activated by acetylation, LCAD is inhibited by this post-translational modification. The addition of acetyl groups to the Lys-318 and Lys-322 residues disrupts the LCAD active site, interrupting substrate binding and decreasing catalytic efficiency. The acetylation ofLys-318 and Lys-322 of LCAD is specifically regulated by SIRT3. Modulation of LCAD function by acetylation provides a possible mechanism for feedback inhibition, as LCAD function in FAO produces acetyl-CoA from fatty acids, potentially providing donor acetyl groups for lysine acetylation. Furthermore, acetylation modulates the function of other enzymes involved in fatty acid metabolism, such as pyruvate kinase M2 (PKM2) and PGC1a [88,89]. Based on the critical role for FAO in alternative (M2) macrophage activation [7,8] and in pDC activation [90], we would envisage that dynamic changes in acetylation could play a critical role in the regulation of these innate immune cell activation states.

5.2. Modulation of mitochondrial transporters by acetylation

Acetylation can also modulate cellular metabolism through regulation of transporter or channel proteins, which play an important role in metabolism. At least 15 mitochondrial transporters or channel proteins have been identified as being lysine-acetylated [82]. For some of these proteins how acetylation regulates their function is yet to be elucidated, but it is possible that this represents one way in which mitochondrial functions are tailored to meet the needs of specific and diverse metabolic programs. Exemplifying this point, acetylation has been shown to regulate the function of the mitochondrial citrate carrier SLC25A1 [91]. SLC25A1 catalyzes the exchange of mitochondrial citrate for cytosolic malate, and as previously mentioned has a crucial role in supplying acetyl-CoA to the cytosol and nucleus. Citrate within the cytosol can be cleaved to acetyl-CoA and oxaloacetate by ACL, and acetyl-CoA can then be used to fuel fatty acid synthesis or acetylation while oxaloacetate produces NADPH via malate dehydrogenase. Recently, TNFα and IFNγ have been shown to induce expression of SLC25A1 in activated macrophages, and this expression is required for production of the inflammatory mediators nitric oxide and prostaglandins, which are key mediators of inflammatory macrophage function [92]. Importantly, acetylation of SLC25A1 enhances citrate exchange for malate in activated inflammatory macrophages, modulating the carrier protein function to respond to increased demand for NADPH and acetyl-CoA [91]. Recently, acetylation has also been shown to regulate the activity of mitochondrial pyruvate carrier 1 (MPC1) [93]. MPC1 is one component of the mitochondrial pyruvate carrier, and along with MPC 2 facilitates pyruvate import into mitochondria, modulating mitochondrial pyruvate oxidation [94]. This transport of pyruvate into the mitochondria via MPC1 is required for early DC activation [95]. Acetylation of MPC1 decreases its activity, and deacetylation of this protein is mediated by SIRT3 [93], thus regulation of MPC1 by acetylation and sirtuin activity will affect early DC activation. Regulation of SLC25A1 and MPC1 by acetylation illustrates that acetylation can regulate metabolic transporters to directly impact their function, which will have downstream effects on metabolism. With numerous other transporters shown to be acetylated, it is likely that acetylation regulates mitochondrial transporter function more widely, and the role of this in innate immune cell metabolism and inflammatory functions requires further investigation.

6. Conclusion and outlooks

The critical role that metabolism and epigenetics play in determining cell fate and function is now widely recognized across many cell types, including innate immune cells. In this review we highlight the importance of the interplay between acetylation and metabolism in innate immune cells, focusing on macrophages and DCs. Enzymes involved in acetylation sense intermediary metabolic products and process this information into dynamic modifications of proteins that affect function. These adaptations help to coordinate homeostatic or transcriptional responses and can drive the activity of gene networks that control cell fate and function. Rapid alterations in chromatin and protein acetylation allow innate immune cells to respond quickly to pathogens, and sensing of metabolic signals co-ordinates such responses to the availability of nutrients and the microenvironment. Perturbations in either the metabolism or acetylation of innate immune cells can have significant impacts on their differentiation and function. This suggests that pharmacological intervention to specifically alter metabolism and/or acetylation in innate immune cells could have profound effects on immune responses in disease settings. Much remains to be understood about the links between metabolic pathways and epigenetic control of gene expression. While the current literature provides evidence that the metabolic-epigenetic axis is critical in determining both macrophage and DC differentiation and function much remains to be understood in these cell types. In other innate immune cells research into metabolism and interactions between metabolism and epigenetics is sparse. Many of the concepts discussed in this review have wide relevance to a variety of cell types and thus would likely also apply to other types of innate immune cells, however it is important that research is undertaken to address these gaps in our understanding. Further characterisation of metabolism and acetylation, and the interplay between these systems in all cells of the innate immune system will provide insight into the mechanisms by which innate immune responses are regulated.