Metabolites derived from intermediary metabolism play an important role in epigenetics and can mediate important health outcomes such as immunity. T cells undergo metabolic reprogramming following activation to develop a metabotype that is not only conducive to the bioenergetic and substrate needs of the cell, but also contributes to the epigenetic landscape. Herein, we studied the metabolic and epigenetic effects of disruption of PDC in T cells in vitro. PDC deficiency leads to widespread perturbations in glycolysis, mitochondrial metabolism, and the epigenome. The result is defects in T cell differentiation and changes in the response to extracellular metabolites. Our results indicate that glycolysis is a significant contributor to histone acetylation, and PDC serves as an important metabolic and epigenetic node in T cell differentiation.
Following engagement of the T cell receptor, pyruvate dehydrogenase kinase 1 (PDK1) becomes activated in T cells, leading to the phosphorylation and subsequent inhibition of PDC 30,31. As a result, a smaller fraction of pyruvate (~ 40% by our stable isotope studies) is metabolized in the mitochondria, and T cells adopt a glycolytic metabotype. In our current model, TPdh−/− T cells lack a critical component of PDC, resulting in a deficiency of this enzyme complex. As a result of this block, pyruvate is not fully oxidized and subsequently, OXPHOS is downregulated (by ~ 48%). In response, TPdh−/− undergo metabolic rewiring and upregulate glycolysis as evidenced by our extracellular flux and metabolomic studies. Consequently, total cellular ATP levels are maintained via substrate level phosphorylation. This upregulation of aerobic glycolysis is dependent upon the regeneration of NAD+, a process which occurs in the cytoplasm via the conversion of pyruvate to lactate via lactate dehydrogenase (LDH)32. Indeed, our extracellular flux analyses support increased activity of LDH. Not only does this lead to an upregulation of glycolysis, but also a metabotype where glycolysis is operating at its maximum, unable to be pushed further. As a result, TPdh−/− T cells become functionally dependent upon glycolysis, as indicated by our proliferation studies with 2DG.
In aerobic organisms, the TCA cycle is a sequence of chemical reactions used to produce energy through the oxidation of acetyl-CoA derived from glycolysis, fatty acid oxidation or amino acid metabolism33. In our TPdh−/− T cell model, the TCA cycle undergoes metabolic rewiring involving anaplerosis due to a deficiency of acetyl-CoA from glycolysis. As a mechanism to maintain homeostasis, glutamine becomes essential in this case of loss of glycolytic carbon sources for the TCA cycle 34. In our study, TPdh−/− T cells showed a depletion of multiple ketogenic amino acids (isoleucine, phenylalanine, tyrosine), as well as increased incorporation of glutamine into the TCA cycle as measured by stable isotopes. However, glutamine incorporation did not result in enhanced OXPHOS, indicating that its function lies beyond bioenergetics. One such important function may be the synthesis of aspartate from oxaloacetate seen in TPdh−/−. Aspartate synthesis in the setting of OXPHOS deficiency becomes an important pathway for producing DNA, RNA and protein in proliferating cells35. Furthermore, anaplerosis may also be enhanced by OXPHOS deficiency, leading to excessive anaplerosis36.
Since metabolism is intricately tied to T cell differentiation, it was not surprising to find abnormalities in TPdh−/− CD8+ T cells. CD8+ T cells are highly energetic and have a requirement for intact OXPHOS. Unlike CD4+ T cells, activation of CD8+ T cells does not result in a complete shift to aerobic glycolysis37. In fact, OXPHOS levels increase and are an important source of ATP needed for cell proliferation. Therefore, impaired OXPHOS and enhanced glycolysis seen in PDC deficiency are more consistent with TE cells and may partially account for this distinct phenotype seen in TPdh−/− TM.
Beyond metabolic reprogramming, CD8+ T cell differentiation also involves epigenetic and subsequently, transcriptional reprogramming 2–4. PDC deficiency leads to a deficiency of acetyl-CoA, an important substrate for histone modification38. Histone modification results in the activation and repression of key genetic loci involved in differentiation. The importance of acetyl-CoA derived from glycolysis in differentiation has also been reported in a number of cellular systems. For example, glycolysis-mediated changes in acetyl-CoA and histone acetylation control differentiation in embryonic stem cells38. In TPdh−/− TM cells, histone acetylation was markedly depressed as shown in our proteomic and ChIP studies, suggesting that glycolysis is a significant source of acetyl-CoA in these cells. Therefore, the deficits seen in TPdh−/− differentiation are mediated by metabolic and epigenetic perturbations.
Interestingly, our findings presented herein were in contrast to a recent paper utilizing genetic and pharmacologic inhibition of the mitochondrial pyruvate carrier (MPC) 39. Wenes et al. described a metabolic-epigenetic axis that enables CD8+ memory T cell formation39. Histone H3 acetylation at lysine 27 (H3K27ac) is a marker of active chromatin regions associated with memory CD8+ T cell differentiation 28,29. Inhibition of the MPC resulted in H3K27 acetylation, however the carbon source switched to glutamine, instead of glucose. In our study, not only was overall acetylation depressed, but H3K27 acetylation was also absent in our histone proteomic study. Although we do not have a direct explanation for these findings, it is worthwhile to point out the differences between these two models. Inhibition of the MPC via pharmacologic or genetic means may have different effects on metabolism when compared to PDC inhibition. For example, MPC1+/− mice employ fatty acid oxidation (FAO) to meet their bioenergetic needs40. TPdh−/− T cells displayed depressed FAO. Beyond its effects on metabolism, MPC1 also engages in signaling transduction, interacting with mitoSTAT3 41. Therefore, targeting different aspects of pyruvate metabolism may result in divergent phenotypes.
In summary, our data demonstrate that PDC deficiency leads to metabolic and epigenetic perturbations, affecting CD8+ memory T cell differentiation in mice. Based on our findings, we propose that PDC occupies a major node in T cell intermediary metabolism by mediating both biochemical and epigenetic responses in activation and differentiation.