Hexokinase is the first enzyme involved in glycolysis, catalyzing the phosphorylation of glucose to glucose 6-phosphate (G6P) (Figure 1). Induction of HK-II, one of four isoforms of hexokinase, appears to be tightly linked to activation of inflammatory programs in immune cells (11, 12) and tumorigenic programs in cancer cells (10). Phosphorylated AKT stabilizes the localization of HK-II to the outer mitochondrial membrane (OMM). At this location, mitoHK-II has increased access to mitochondrially derived ATP, which it can then use to phosphorylate glucose to G6P, thereby trapping glucose in the cell (13). MitoHK-II also plays an anti-apoptotic role, preventing the formation of the mitochondria permeability transition pore by Bcl-2 family proteins like Bax (14, 15). The mechanism behind this process involves PI3K-AKT-mediated phosphorylation of Thr473 in HK-II, a modification that prevents G6P-mediated dissociation of HK-II from the mitochondria (16). Thus, posttranslational modifications to HK-II both facilitate its activity as a glycolytic enzyme and promote its anti-apoptotic functions.

Upon activation, immune cells upregulate HK-II (17) as well as other HK family members (18). HK-targeted interventions block glycolysis, effector function, and survival of cells involved in driving inflammatory responses (6), and for myeloid cells, this is especially true in the context of gram-negative bacterial challenges (19). However, this may not be true of all inflammatory responses. N-acetylglucosamine, a peptidoglycan derivative from the cell wall of Gram-positive bacteria, has recently been shown to bind HK-II and promote its dissociation from the OMM. This dissociation results in the accumulation of mitochondrial DNA in the cytosol and NLRP3 inflammasome-dependent production of mature IL-1β and IL-18 in macrophages (20). Thus, while dissociation of HK-II from the OMM might, on the one hand, abrogate the efficiency of flux through the glycolytic cascade and thus block inflammation, on the other hand, it may potentiate signals that promote secretion of major soluble transducers of inflammation depending on context. Inflammasome components (21, 22), hexokinase (6), and mitochondrial dynamics (23) are all known modulators of T cell functions; however, whether or not HK relocalization can induce inflammasome activity in T cells, and what consequences this may have, remains unclear.

Glyceraldehyde 3-phosphate dehydrogenase is the enzyme that catalyzes conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate in glycolysis (Figure 1). GAPDH is well known for its numerous non-metabolic functions. In many bacteria, GAPDH is a major component of the cell surface. Multiple mechanisms are involved in this localization of GAPDH, including active transport (24) and lysis-mediated release of GAPDH which then decorates the surface of neighboring bacterial cells (25). Cell surface GAPDH binds fibronectin, plasminogen, and other tissue components (24–26) and is an important facilitator of bacterial adherence to and invasion of host tissues. These findings translate to eukaryotic systems. In response to inflammatory cues, macrophages recruit GAPDH to the cell surface where it functions as a plasminogen receptor. In this paradigm, plasminogen bound to GAPDH digests extracellular matrix thereby facilitating macrophage migration (27). GAPDH can also localize to numerous other subcellular compartments (28). For example, oxidative stress, as occurs during neutrophil respiratory burst, drives S-nitrosylation of GAPDH (29), redistributing it from the cytoplasm to the nucleus and mitochondria where it is broadly implicated as a regulator of cell survival [reviewed in Ref. (28)]. GAPDH itself has been shown to have anti-inflammatory properties, as systemic administration of GAPDH prior to LPS-induced sepsis reduces cytokine storm and mortality (30), though the mechanism of this immunomodulatory effect remains unknown.

Recent work in T cells implicates GAPDH as an energy sensor that regulates translation of inflammatory cytokine mRNA in response to the availability of glucose in the cell. When glucose concentrations are low, GAPDH binds to the AU-rich elements in the 3′-untranslated region (UTR) of mRNA, including those encoding interferon gamma (IFN-γ) and IL-2 (31, 32). Binding of GAPDH to these transcripts represses their translation, thus restricting cytokine production during glucose deprivation. 3′AU-rich elements are not unique features of IFN-γ and IL-2 mRNA, and it is likely that GAPDH can regulate translation beyond these two cytokines (33). The glycolytic reaction catalyzed by GAPDH requires nicotinamide adenine dinucleotide (NAD⁺), an essential indicator of cellular redox state, and intriguingly, Nagy and colleagues identified the NAD⁺ binding fold of GAPDH as its RNA-binding domain (34). This finding suggests any NAD⁺-dependent enzyme [in glycolysis, this is GAPDH and lactate dehydrogenase (LDH)] may be endowed with RNA-binding capabilities. Glucose deprivation, however, increases levels of intracellular NAD⁺ which might be expected to compete with GAPDH for RNA binding (35). Thus, there are likely additional layers of regulation governing the role of GAPDH as a translational repressor that functions during glucose deprivation and or in response to fluctuations in NAD⁺. Context-specific nuances that influence how NAD⁺ affects the mRNA-binding functions of glycolytic machinery offer an intriguing line of inquiry into the interplay between metabolism and the many fundamental processes (36, 37) regulated by NAD⁺.

α-Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate (PEP) in glycolysis (Figure 1). The gene that encodes α-enolase (Eno1) produces a single transcript with two translational start sites. Depending on the site of translation initiation, Eno1 can generate a full-length canonical α-enolase (48 kDa) enzyme that participates in glycolysis, or a truncated version of α-enolase (37 kDa), also known as Myc promoter-binding protein 1 (MBP-1) that represses the pro-proliferative transcription factor c-myc (38–41). Wang and colleagues identified c-myc as the master regulator of metabolic adaptation in T cells (17), demonstrating impaired growth and proliferation in c-myc deficient T cells treated with mitogenic stimuli. MBP-1 represses c-myc by binding to and inhibiting formation of the transcription initiation complex at the c-myc promoter (40, 41). Whereas α-enolase localizes to the cytoplasm, MBP-1 preferentially traffics to the nucleus where it serves these repressive functions (38). The signals that influence differential translation of α-enolase versus MBP-1 are unclear, though hypoxia may be one cue that favors translation of full-length α-enolase (42). The internal translation start site that generates MBP-1 off of Eno1 is not present in β or γ-enolase, potentially providing an added layer of specificity for future MBP-1 modulating interventions.

Intriguingly, it seems that the induction of MBP-1 functionally impacts T cell inflammatory responses in the context of autoimmunity. A recent study (43) revealed that an anti-inflammatory population of human CD4⁺ T cells, known as regulatory T cells (Tregs), expresses high levels of MBP-1. Moreover, MBP-1 in Tregs potentiates transcription of a specific spliced isoform of FoxP3 known to potently suppress inflammatory immune responses, particularly those mediated by the transcription factor RAR-related orphan receptor gamma T (RORγT). RORγT is a known driver of IL-17A (44) and granulocyte macrophage colony stimulating factor (GM-CSF) (45), pro-inflammatory cytokines strongly associated with auto-inflammatory diseases (46–48), and the therapeutic potential of its inhibition is under investigation for numerous inflammatory indications (49, 50). Interestingly, Tregs seem to elevate expression of both Eno1 gene products, suggesting that the suppressive effects of MBP-1 may dominate over metabolic contributions to inflammation facilitated by full-length α-enolase or elevated glycolysis (43, 51). Thus, inducing transcriptional activity at Eno1 may be sufficient to increase MBP-1 protein levels to immunosuppressive levels without blocking glycolysis. How the α-enolase/MBP-1 axis affects conventional T cell responses is unclear. Taken together, whereas Hk2 encodes a single protein that can play metabolic and non-metabolic roles in a cell, Eno1 encodes two gene products that differ drastically in their contributions to metabolism and inflammation (38, 39).

Pyruvate kinase is the ATP-generating enzyme that catalyzes the conversion of PEP to pyruvate during glycolysis (Figure 1). Four isoforms of the PK enzyme exist, with the M1 (PKM1) and M2 (PKM2) isoforms being most predominant in leukocytes of the adult animal (52). PKM2 is the major isoform expressed at the protein level by lymphocytes (52). Interestingly, many cancer cell lines also exclusively express PKM2 (53), and cancer researchers have likewise identified many pro-proliferative and non-canonical functions that are specifically attributed to this particular isozyme (54–63). PKM1 and PKM2 are alternatively spliced isoforms of the PK enzyme that differ by inclusion of a single exon (exon 9 for PKM1 versus exon 10 for PKM2), of which only 22 amino acid residues differ (64). The structures of PKM1 and PKM2 are extremely similar (65), but importantly, the minute difference in amino acid sequence allows PKM2 to uniquely contribute to proliferative responses in cancer cells and inflammatory responses of immune cells (66–69). Whereas PKM1 exists solely as a tetramer that functions as a glycolytic enzyme, PKM2 can exist as a tetramer with similar functions as PKM1 or as a dimer that loses activity as a glycolytic enzyme, but can perform numerous other non-glycolytic functions in the cell. From the perspective of glycolysis, this dynamic feature of PKM2 reduces its efficiency as a glycolytic enzyme and allows for the accumulation of upstream glycolytic intermediates, thereby promoting de novo amino acid and lipid biosynthesis—processes that are critical for the production of a daughter cell (70). From the perspective of inflammation, the PKM2 dimer can localize to the nucleus (58) where it is a well-known co-activator of Hif-1α gene signatures (54, 66, 67). In macrophages, this interaction is critical for the appropriate transcriptional activation of metabolic machinery, such as lactate dehydrogenase A (LDH-A) and pro-inflammatory cytokines, such as IL-1β (66). Similarly, signal transducer and activator of transcription 3 (STAT3) (55) and the aryl hydrocarbon receptor (AhR) (71) also require interaction with PKM2 for appropriate DNA binding. Thus, the PKM2 dimer seems to play a unique role as a direct modulator of proliferative and inflammatory programs. Relating to T cells, AhR, STAT3, and Hif-1α are all well-known regulators of Th17 cell differentiation perhaps implicating PKM2 as a regulator of this cell type.

Many groups in cancer research (56, 57, 60) and immunology (66–69, 72) are exploring the therapeutic potential of enforcing PKM2 tetramerization with pharmacologic compounds (62, 73). The major endogenous driver of PKM2 tetramerization is fructose 1,6 bisphosphate (FBP) (65), the product of the phosphofructokinase-catalyzed step in glycolysis. Phosphotyrosine residues generated by growth factor signaling (57, 59) can bind to PKM2 and promote release of FBP, and along with posttranslational modifications, such as PKM2 phosphorylation (74), oxidation (61), acetylation (58), and succinylation (75, 76), are endogenous drivers of tetramer dissociation. Synthetic activators of PKM2 tetramerization, originally characterized in cancer models as tumor-blocking agents (62), also potently block inflammation in numerous disease models (66, 67, 77). Thus, enforcing PKM2 tetramerization shows promise as a metabolic machinery-based paradigm for controlling inflammatory responses without overtly inhibiting metabolism itself.

Lactate dehydrogenase is a tetrameric enzyme variably composed of A and B subunits that, when combined, form a complex with the capability of converting pyruvate to lactate (Figure 1). This reaction is the defining step of aerobic glycolysis (78), the form of metabolism engaged by activated immune cells, which increase their regeneration of NAD⁺ consumed during glycolysis by producing lactate regardless of environmental oxygen content (2, 3). Peng and colleagues (79) recently showed that T cells almost exclusively express the A subunits of LDH, which they further upregulate upon activation, and expression of LDH-A is critical for the proper production of the inflammation-promoting cytokine IFN-γ. They found that genetic ablation of LDH-A in T cells heightened consumption of glycolysis-derived acetyl-CoA through the tricarboxylic cylic acid cycle and depleting intracellular stores of this metabolic byproduct of glucose catabolism. This acetyl-CoA depletion impaired activation-induced permissive histone acetylations that are required for opening of the Ifng locus during T cell activation. These findings and others (80–82) suggest that metabolic adaptations like aerobic glycolysis are important (1) as a means of generating sufficient ATP and metabolic intermediates to support anabolic processes and (2) as drivers of the epigenetic changes that are responsible for facilitating engagement of the inflammatory program [reviewed in Ref. (83)]. In addition to its ability to indirectly modulate the epigenetic landscape of the activated T cell, there is evidence to suggest that LDH-A may also be capable of directly influencing inflammatory responses. In a manner reminiscent of direct repression of IFN-γ and IL-2 mRNA translation by the glycolytic enzyme GAPDH (31, 32), LDH-A has been reported to bind to 3′AU-rich elements in GM-CSF mRNA (84). It remains unclear how the mRNA-binding properties of LDH-A affects downstream protein expression and, additionally, if this non-metabolic function is related to the level of flux through the glycolytic cascade or enzymatic activity. Inflammatory T cells are major producers of GM-CSF, a prominent driver of autoimmune responses (45–47, 85, 86), and a detailed study elucidating the metabolic requirements for GM-CSF production in vivo, including how it may relate to LDH-A, is warranted.