Metabolic control of T-cell immunity via epigenetic mechanisms

Metabolism has been increasingly considered to play an important role in inflammation and immunity via modulating cellular signaling and transcriptional networks.¹ Dysregulation of metabolic enzymes or mediators is increasingly linked to clinical diseases, such as type I diabetes, gliomas and breast cancer.^{2, 3} Numerous metabolic sensors, such as AMP-mediated protein kinase, mammalian target of rapamycin complex1 and hypoxia-induced factor 1α (HIF-1α), have been shown to regulate immunological responses, such as T-cell activation and macrophage polarization, via modulating the property and activity of signaling pathways.^{4, 5} Moreover, emerging evidence suggests that intermediates from multiple metabolic pathways are important in orchestrating transcriptional and epigenetic programs. This interaction between metabolic and epigenetic pathways is crucial for coordinating cellular immunological events in response to extracellular stimuli, such as infection, injury and stress, leading to varied biological and pathological outcomes.

Epigenetic mechanisms include multi-level intracellular events that influence chromatin structure and gene expression without altering DNA sequences, such as DNA methylation, post-translational modifications of histones, non-coding RNAs and chromatin remodeling. Importantly, intermediary metabolites can serve as the substrates, cofactors or inhibitors of epigenetic enzymes to affect gene expression and thus influence variable biological processes, such as long-term memory, muscle function, stem cell differentiation and tumorigenesis.^{6, 7, 8} Acetyl-CoA is produced in various metabolic pathways and serves as an acetyl group donor of histone acetyltransferase, which is the critical step of acetylation.⁶ Nicotinamide adenine dinucleotide (NAD⁺) produced from nicotinamide mononucleotide is required for deacetylation mediated by Sirtuin family histone deacetylases.⁷ Likewise, S-adenosylmethionine and α-ketoglutarate (α-KG) are respectively required for DNA/histone methylation (catalyzed by DNA methyltransferase and histone methyltransferases) or demethylation (catalyzed by ten-eleven translocation and jumonji C -domain-containing enzymes).⁸ Other examples of metabolites that regulate epigenetic pathways include crotonyl-CoA for histone crotonylation⁹ and UDP-GlcNAc for histone GlcNAcylation.¹⁰ These connections between cellular metabolism and epigenetic mechanisms lead to multi-level modulation of gene transcription and cell signaling, exerting important roles in cell development and function. While cellular immunological responses are accompanied by dynamic alterations and interactions of cellular metabolic and epigenetic pathways, how these two key biological pathways interplay to feedback modulate immune cell function during infectious or stress conditions is attracting more and more attention.

CD4⁺ T lymphocytes, including T helper 1 (Th1), Th2, Th9, Th17, regulatory T cells (Treg) and other possible unknown subsets, are critical for host adaptive immunity against infection and tumor. The specification of T-cell subsets, which is characterized by distinct cytokine production and expression of lineage-associated genes, is tightly regulated by the coordinated transcriptional network of lineage-determining transcriptional factors and multiple epigenetic modulators.¹¹ Metabolites play a profound impact on T-cell function, not only by meeting the bioenergetic and biosynthetic demands to support cell survival and activation, but also by interconnecting with epigenetic enzymes to regulate gene expression (Figure 1). Lactate dehydrogenase A induced in aerobic glycolysis can promote IFN-γ expression by maintaining high levels of acetyl-CoA to enhance histone acetylation and transcription of Ifng.¹² Histone deacetylase SIRT1 can inhibit IL-9 production of Th9 cells by negatively regulating the activity of mTOR and HIF-1α pathways in an NAD⁺-dependent way.¹³ Another study demonstrated that the methylcytosine dioxygenase TET2 can promote DNA demethylation and activation of cytokine encoding genes in T cells, such as Il17 for Th17, Ifng for Th1 and Foxp3 for Treg cells.¹⁴ Therefore, it is intriguing to depict the interplay network between metabolism and epigenetics in controlling T-cell biology and the development of T-cell-mediated inflammatory diseases.¹⁵ In the latest issue of Nature, Tao Xu and colleagues identified an important role for metabolic intermediates in regulating Th17 and induced Treg cell balance via epigenetically control of Foxp3 expression.

By screening the potential small molecules that regulate T-cell specification using a library of 10 000 small molecules, Xu et al. identified that a small molecule, (aminooxy) acetic acid (AOA), can inhibit Th17 cell differentiation but promote iTreg cell differentiation in a dose-dependent manner. The observation that AOA selectively reduced the mRNA levels of Il17a and Il17f, but not Rorc, in Th17 cells while it promoted the transcription of Foxp3 in Th17 and iTreg cells indicated that AOA may be involved in the transcriptional regulation of Treg cell differentiation. AOA is the inhibitor of glutamic-oxaloacetic transaminase 1 (GOT1), which transfers glutamine to α-KG. Consistent with the effect of AOA, knockdown of GOT1 by short hairpin RNA (shRNA) in differentiating Th17 cells reduced Th17 cells and increased iTreg cells, suggesting that AOA reprograms Th17 cell differentiation toward iTreg cells by inhibiting GOT1. A balanced network of Th17 cells and Treg cells is critical for the maintenance of immune homeostasis and is closely associated with various inflammatory diseases when uncontrolled. To investigate the in vivo biological significance of their findings, the authors also examined the effect of AOA by using the mouse model of experimental autoimmune encephalomyelitis (EAE). As expected, AOA treatment ameliorated EAE pathogenesis effectively by reducing the percentage of Th17 cells and increasing the percentage of iTreg cells. Consistently, GOT1 knockdown led to decreased infiltration of Th17 cells and increased iTreg cells into the central nervous system in a Th17-polarized transfer EAE model.

Metabolomics has emerged as a powerful technical tool to identify the systemic and dynamic cellular metabolic response to internal and external stimuli and has greatly advanced immunological research in investigating the interactions among metabolism, epigenetics and immunity.¹⁵ By liquid chromatography–mass spectrometry metabolomics, the authors found higher expression of tricarboxylic acid (TCA) cycle intermediates, such as α-KG, succinate, fumarate, malate and citrate, in differentiating Th17 cells compared to iTreg cells, and their levels are reduced by AOA treatment in both differentiating conditions. 2-hydroxyglutarate (2-HG) is the most significantly increased metabolite in Th17 cells relative to iTreg cells. [U-¹³C] glutamine flux analysis confirmed that more glutamine/glutamate carbon contributes to the TCA cycle and 2-HG synthesis. The newly synthesized 2-HG, α-KG and TCA cycle intermediates are higher in Th17 cells than in iTreg cells, and all these increases can be reduced by AOA treatment. Moreover, α-KG and 2-HG can rescue Th17 toward iTreg, reprograming the effect of AOA. In the absence of AOA, 2-HG (but not α-KG) promotes Th17 cell differentiation by downregulating the transcription of Foxp3 and inhibits Foxp3 expression under iTreg cell conditions. Consistently, knockdown of IDH1 and IDH2 (which mediate the metabolism of 2-HG) in differentiating Th17 cells obviously inhibits Th17 cell differentiation but promotes iTreg cell generation, which is rescued by exogenous R-2-HG. Therefore, central carbon metabolic pathways involving α-KG and 2-HG functionally regulate Th17 and Treg cell balance, which is responsive to AOA treatment.

Since 2-HG has been reported as an antagonist of TET1–TET3, demethylating the Foxp3 promoter and its intronic CpG island, the authors wondered whether 2-HG inhibits Foxp3 expression and Th17/Treg balance via epigenetic modulation of Foxp3 expression. Indeed, 2-HG markedly increased the methylation levels of the hypomethylated regions in both differentiating Th17 cells and iTreg cells, whereas AOA markedly decreased the methylation level at the Foxp3 gene locus in both Th17 and iTreg cells. The subsequent hydroxymethylated or methylated DNA immunoprecipitation followed by high-throughput sequencing (hMeDIP-seq or MeDIP-seq, respectively) confirmed that the Foxp3 locus is included in the increased 5mC signal resulting from 2-HG treatment and in the increased 5hmC signal resulting from AOA treatment. In addition, other important lineage-specific gene loci, especially the direct Foxp3 targets, showed no difference in the 5hmC or 5mC signal. Therefore, AOA and 2-HG regulate Th17 cell differentiation by altering the expression of Foxp3 via epigenetically promoting methylation of the Foxp3 locus. Coincidentally, another study observed an accumulation of 2-HG in CD8⁺ T cells after TCR stimulation; however, it showed that S-2-HG, another enantiomer of 2-HG, predominantly modulates histone and DNA demethylation and HIF-1α stability in CD8⁺ T cells.¹⁶ S-2-HG greatly enhances the in vivo proliferation, differentiation and anti-tumor function of CD8⁺ T cells.¹⁶ Whether 2-HG and other metabolites of the TCA cycle and glutaminolysis may be involved in other aspects of T-cell biology, such as T-cell memory, immune tolerance, cell death and repair, still requires further investigation. Additionally, 2-HG significantly accumulates in cancer cells with IDH1/2 mutation. How 2-HG is dramatically induced to high levels in TCR-activated CD8⁺ T cells or differentiating Th17 cells merits further study.

In summary, this study sheds new light on the mechanisms of metabolic control of T-cell fate by epigenetic mechanisms and opens up new opportunities for the prevention and treatment of EAE and other T-cell-mediated inflammatory diseases. With the rapid progress in metabolomics, epigenomes and epitranscriptomics, studies over the past decade have revealed a multitude of metabolites involved in genome transcription and immune response. There is an urgent need to further understand how cellular metabolic pathways integrate with epigenetic pathways to affect immune system development and function. Many challenging issues remain to be addressed in this field, for example, further investigations into the metabolic control of innate immune cells such as dendritic cells and macrophages,^{17, 18} identification of metabolic and epigenetic interactions in highly specialized immune cells at a single cell level, detection of dynamic metabolic and epigenetic interactions at subcellular organelles, and development of chemical and genetic therapies targeting metabolic and epigenetic pathways for treatment of immunological disorders and cancer.