HIF1α and metabolic reprogramming in inflammation (original) (raw)
Metabolic reprogramming is crucial to both macrophage and DC activation. DC activation by LPS under normoxia promotes production of HIF1α mRNA and protein to greater levels than the levels induced by hypoxia alone (20). Inhibition of glycolysis using the glucose analogue 2-deoxyglucose (2-DG) blocks DC maturation in response to LPS, as measured by the reduced expression of the costimulatory molecules CD80 and CD86. HIF1α is implicated in this process, as the transcription of classic hypoxic response genes such as GLUT1 is increased and HIF1α deficiency similarly reduces expression of costimulatory molecules (20).
Macrophage activation following LPS stimulation is also dependent on this metabolic shift towards glycolysis. Inhibition of glycolysis with 2-DG blunts the inflammatory response in LPS-treated macrophages. Inhibitors of oxidative phosphorylation have no effect following LPS treatment, indicating that this process is already downregulated by LPS (46). IL-1β production in response to LPS treatment has been shown to be inhibited by 2-DG, and HIF1α has been implicated in this process (17, 47).
Stimuli such as LPS cause macrophage polarization towards a classically activated phenotype (termed M1), which exhibits inflammatory activities, or an alternatively activated phenotype (termed M2), which exhibits antiinflammatory activities (48). These polarization events are accompanied by HIF-dependent metabolic changes that provide macrophages with the functional ability to respond adequately to their respective stimuli (Figure 1). LPS in combination with IFN-γ induces metabolic reprogramming in M1 macrophages via HIF1α. Metabolism in M1 macrophages is characterized by increased glycolysis and pentose phosphate pathway (PPP) activity. LPS boosts HIF1α mRNA transcription in phagocytes via induction of NF-κB activity, further increasing signaling through the HIF pathway (49–51). M2 macrophages display a more oxidative metabolic profile compared with M1 macrophages, using oxidative phosphorylation and exhibiting high levels of fatty acid oxidation (FAO) (52, 53). While recent studies have questioned whether FAO has a correlative role or causal role in macrophage polarization (54), it is clear that FAO is essential to energetically support effector functions over a prolonged period of time, as required by M2 macrophages in the defense against parasites and in the resolution phase of infection and injury (55, 56).
Metabolic reprogramming in macrophage polarization. LPS and IFN-γ induce M1 macrophages. Metabolism in M1 macrophages is characterized by increased glycolysis and PPP activity, and a broken TCA cycle that leads to metabolite accumulation. M2 macrophages display a more oxidative metabolic profile, with a high reliance on the TCA cycle, utilizing OXPHOS and exhibiting high levels of FAO. Inhibition of glycolysis by 2-DG leads to an oxidative M2 phenotype (48).
Interestingly, HIF1α and its closely related isoform HIF2α may exert opposing effects in macrophage polarization. While depletion of both HIF1α and HIF2α is broadly antiinflammatory, HIF2α does not appear to have a role in the regulation of NO production or the expression of costimulatory molecules. HIF2α is suggested to be critical for the production of proinflammatory cytokines under hypoxic conditions (19, 57). It has been shown that HIF1α and HIF2α are differentially expressed in M1 and M2 macrophages, with HIF1α induced in M1 macrophages, and HIF2α increased in M2 macrophages (58). However, in models of sterile inflammation during muscle regeneration, HIF1α and HIF2α have been identified as redundant, as macrophage polarization and effector functions were largely unaltered in mice depleted of myeloid HIFs (59).
The importance of HIF1α in acute innate cell function was dramatically demonstrated by murine HIF1α deletion studies. Conditional deletion of HIF1α in the myeloid lineage is not embryonically lethal in a murine model, nor do the mice exhibit any deficiencies in monocyte or neutrophil development and differentiation. However, these mice exhibit impaired inflammatory responses, specifically a defect in cell metabolism that renders the myeloid cells unable to adequately upregulate glycolytic metabolism upon inflammatory stimulation. In the absence of HIF1α, macrophages exhibit decreased glycolytic rates and energy generation, as well as reduced motility and migration (19). HIF1α deletion in macrophages causes defects in phagocytic uptake and killing in various bacterial infection models (19, 60). Importantly, mice with myeloid cell–specific deletion of HIF1α are resistant to LPS-induced lethality, confirming the importance of HIF1α in a classic in vivo model of innate immune activation (61).
An interesting consequence of the recognition of metabolic involvement in innate immune responses has been the identification of certain individual metabolites as signals in inflammation. The nonhypoxic stabilization of HIF1α in LPS-activated macrophages is mediated by metabolic intermediates with hitherto unknown roles in cell signaling. The TCA cycle intermediates succinate and citrate accumulate following LPS treatment in macrophages (17, 44, 62). Succinate accumulation leads to HIF1α stabilization and increased transcription of target genes such as Il1b (17). The mechanism of succinate accumulation may also involve the metabolite itaconic acid, which is synthesized by immune response gene 1 (IRG1) following citrate accumulation. IRG1 is induced by LPS and converts a citrate derivative, _cis_-aconitate, to itaconic acid. Itaconic acid inhibits an essential metabolic pathway, the glycoxylate shunt, which is necessary for the survival of some pathogenic bacteria (63). Itaconate is also a weak competitive inhibitor of SDH and may contribute to the accumulation of succinate in LPS-treated macrophages (64).
Citrate accumulation is a key event during the metabolic reprogramming process in M1 macrophages, and this reprogramming ultimately impacts HIF1α stability. Citrate accumulation leads to the production of three important proinflammatory mediators: NO, ROS, and prostaglandins (PGs) (65). Citrate also generates NADPH via pyruvate and malic enzyme. NADPH is required for expression of inducible NOS (iNOS), thereby playing a role in NO production. NO is an important inflammatory mediator that nitrosylates and inhibits components of the electron transport chain required for OXPHOS. ROS act to stabilize HIF1α and are produced in a similar fashion by NADPH oxidase (66).
Pyruvate kinase M2 (PKM2) has been shown to be a critical determinant of metabolic reprogramming in macrophages via HIF in response to LPS stimulation. PKM2 is the rate-limiting enzyme of glycolysis that converts phosphoenolpyruvic acid (PEP) to pyruvate. Enzymatically inactive PKM2 monomers or dimers exist in equilibrium with enzymatically active PKM2 tetramers. PKM2 dimers can translocate to the nucleus, where they directly interact with HIF1α to regulate expression of proglycolytic enzymes (67). The highly active PKM2 tetramers are retained in the cytosol, supporting the final step of glycolysis (68). PKM2 is upregulated in tumors and LPS-activated macrophages (69, 70). Following LPS activation of macrophages, PKM2 dimers stabilize HIF1α, thereby regulating HIF1α target genes such as Il1b and genes encoding the glycolytic machinery. Forcing PKM2 into its tetrameric form using the small-molecule activator DASA-58 or TEPP-46 prevents nuclear translocation. This impairs interaction with HIF1α and reverses the LPS-induced, HIF1α-mediated shift towards glycolysis and IL-1β production, reprogramming macrophages to an M2-like phenotype (69). These findings support the importance of the PKM2/HIF1α axis for M1 macrophage differentiation and function, with PKM2/HIF1α acting as a pivot in the process of M1/M2 differentiation.
It is important to note that the role of HIF in macrophages and other immune cells is neither definitive nor without controversies. The HIF signaling pathway modulates a significant number of crucial cellular processes and must be carefully controlled; thus, it follows that there exist multiple levels of regulation, including positive and negative feedback loops as well as extensive crosstalk with other signaling pathways. Responses may be dependent on time and context. For example, there are important differences in the regulation of HIF by hypoxia versus regulation by inflammatory stimuli such as LPS. HIF is required for the later shift to glycolysis that occurs in DCs, but the immediate metabolic switch is HIF independent and is mediated by the PI3K/Akt pathway (20). Many aspects have yet to be elucidated, but the primary model of HIF-mediated regulation is a multilevel regulatory network of great complexity that modulates responses such as proliferation, apoptosis, and differentiation (71).