Metabolic reprogramming in macrophages and dendritic cells in innate immunity

Macrophages and dendritic cells (DCs) are the frontline cells of innate immunity. They sense and immediately respond to invading pathogens, thus providing an early defense against external attack. Pattern recognition receptors (PRRs) on the surfaces of these cells recognize pathogen-associated molecular patterns (PAMPs) present in the invaders and, as a result, activate intracellular signaling cascades, leading to the induction of a general pro-inflammatory response 1 . This includes the release of antimicrobial mediators, which target the invading pathogen, chemokines, which recruit more immune cells to the site of infection, and pro-inflammatory cytokines, which drive further inflammation, and induce the adaptive immune response, which is mediated by T and B lymphocytes and is more specific for the particular invading pathogen. PRRs are also capable of detecting endogenous danger-associated molecular patterns (DAMPs) 2 , which indicate damage to host cells. There are several families of PRRs, the best characterized being the toll-like receptors (TLRs) and the NOD-like receptors (NLRs). The key output of PRRs is increased gene expression. However, cells activated by PAMPs also undergo profound metabolic changes. These changes are required for biosynthesis and energy production, and in addition are involved in signaling processes. Here, we describe recent findings in the field of metabolic reprogramming in innate immunity and place them in the context of our current understanding of the complex events occurring during innate immune cell activation.

Activation of macrophages or DCs with a range of stimuli, including LPS 10 , the TLR3 ligand poly(I:C) 11 , and type I interferon (IFN) 11 , induces a metabolic switch from OXPHOS to glycolysis, in a phenomenon similar to the Warburg effect 10 . TCA cycle activity is decreased 10 , 12 , while lactate production and flux through the PPP are increased 13 , 14 . Enhanced PPP activity boosts production of purines and pyrimidines, which can then be used for biosynthesis in the activated cell. It also provides NADPH for the NADPH oxidase enzyme, which produces ROS 15 . This metabolic shift occurs in mature activated immune cells even though they are not highly proliferative. The increase in glycolysis could be a mechanism to rapidly generate ATP. It is an inefficient means of generating ATP when compared to the TCA cycle, but it can be quickly induced and could be a particularly useful means of synthesizing ATP when cellular glucose uptake capacity is high 16 . Furthermore, in activated macrophages, mitochondrial ROS production is increased, serving as a mechanism of bacterial killing 17 . Glycolytic ATP production may compensate for this shift in mitochondrial metabolism away from ATP production and towards ROS production by the electron transport chain 18 . Substantial insights have been made into the underlying mechanisms of this metabolic switch. There are at least four main processes leading to Warburg metabolism in LPS-activated macrophages and DCs ( Figure 2 ). As the majority of studies investigating this mechanism use LPS as a stimulus, this review focuses on the metabolic alterations induced by LPS in macrophages and DCs. Other innate immune cell stimuli also alter metabolic parameters in innate immune cells, including IFNγ 11 , poly(I:C) 11 , and infection with bacterial species such as Listeria monocytogenes , Mycobacterium bovis BCG 19 , and Salmonella typhimurium 17 . NO and metabolic changes in macrophages and DCs Stimulation of macrophages 20 and DCs 21 with LPS and IFN-γ increases the expression of inducible nitric oxide synthase (iNOS), which generates nitric oxide (NO), a reactive nitrogen species that can inhibit mitochondrial respiration. NO achieves this by nitrosylating iron-sulfur proteins present in electron transport chain complexes, e.g., Complex I 22 , 23 , as well as cytochrome c oxidase 24 . The metabolic switch in macrophages and DCs activated by LPS is therefore dependent on iNOS and NO-mediated inhibition of mitochondrial metabolism. Inhibition of iNOS after LPS stimulation restores normal mitochondrial respiration, and treatment of iNOS-deficient DCs with LPS fails to shut down mitochondrial metabolism. Simultaneously, the LPS-induced increase in glycolysis in these cells is blunted. Convincingly, re-addition of NO, using an exogenous NO donor, to DCs in which iNOS is still inhibited by a specific inhibitor again induces mitochondrial dysfunction and the switch towards glycolytic metabolism. This suggests that iNOS-generated NO is the mediator of the LPS-induced mitochondrial functional collapse and increase in glycolysis 25 . Tumor necrosis factor (TNF)-α-induced protein 8-like 2 (TIPE2) is a recently described factor negatively regulating LPS-induced iNOS expression and NO production in macrophages 26 . The amino acid arginine is the substrate of iNOS. In macrophages activated by LPS and IFN-γ or bacterial infection (e.g., with M. bovis BCG), extracellular arginine is imported into the cell and is metabolized to NO and citrulline by iNOS. The produced citrulline is then exported from the cell. When extracellular arginine is depleted, citrulline can be imported into the cell and recycled to arginine via argininosuccinate synthase (Ass1) and argininosuccinate lyase (Asl) 19 . This optimum NO production sustained by citrulline recycling via Ass1 and Asl may be important for controlling mycobacterial infections, as Ass1-deficient mice were less well able to control M. tuberculosis infection 19 . In fact, some pathogens themselves have mechanisms for depleting arginine. For example, Helicobacter pylori expresses an arginase that inhibits NO production by activated macrophages, leading to less effective bacterial killing by these macrophages 27 . A previous study used mass spectrometry to identify nitrosylated proteins in various mouse tissues and revealed that many metabolic enzymes are S-nitrosylated on cysteine residues by NO, including enzymes involved in glycolysis, the TCA cycle and fatty acid metabolism 28 . It is likely that cysteine nitrosylation could affect the activity of these enzymes. Indeed, nitrosylation of the liver enzyme very long-chain acyl-CoA dehydrogenase (VLCAD) was reported to increase its activity, thereby boosting fatty acid metabolism, as VLCAD catalyzes the first step in β-oxidation of fatty acids 28 . It remains to be investigated whether nitrosylation of these metabolic enzymes is involved in the metabolic switch in activated innate immune cells. In sum

Another LPS-regulated target involved in the switch to glycolysis is the pfkfb3 gene encoding u-PFK2 (PFKFB3), an isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) 52 , 53 . PFK-2 regulates intracellular levels of the glycolytic intermediate fructose-2,6-bisphosphate (F-2,6-BP), as it has dual kinase:bisphosphatase activities and thus catalyzes both the synthesis and degradation of this intermediate. F-2,6-BP allosterically activates 6-phosphofructo-1-kinase, thereby increasing flux through the glycolytic pathway 54 . u-PFK2 is the isoform of this enzyme that has the highest kinase:bisphosphatase activity ratio, and therefore maintains the highest levels of the biphosphorylated form of the metabolite, F-2,6-BP 55 , 56 . Interestingly, pfkfb3 is a HIF-1α target gene in response to hypoxia in human glioblastoma cell lines and mouse embryonic fibroblasts 57 , and thereby provides another mechanism by which HIF-1α promotes glycolysis. The purine nucleoside adenosine, a metabolite of ATP, can synergize with LPS to boost glycolysis in macrophages 58 . LPS stimulation of macrophages increases the expression of the A 2A and A 2B cell surface receptors (A 2A R and A 2B R) for adenosine 58 . Adenosine binds to these receptors, which further increases LPS-induced expression of pfkfb3 . This synergistic induction of pfkfb3 is not mediated by HIF-1α, but by the transcription factor Sp1 58 . Adenosine also synergizes with LPS to boost lactate production, indicating that adenosine enhances the LPS-induced increase in glycolysis 58 . In general, however, adenosine signaling often acts in an anti-inflammatory manner. For example, following LPS stimulation, A 2B R-deficient macrophages showed increased production of NO and pro-inflammatory cytokines 59 . Adenosine boosts E. coli -induced production of the anti-inflammatory cytokine IL-10 in peritoneal macrophages, and E. coli is unable to induce IL-10 production in macrophages lacking A 2A R 60 . Interestingly, inosine, a breakdown product of adenosine, exerts anti-inflammatory effects by decreasing the LPS-induced production of TNF-α and IL-1 in mouse peritoneal macrophages 61 . Taken together, increasing u-PFK2 expression level is a critical step in the induction of glycolysis in activated innate immune cells.

A key functional outcome for the switch to glycolysis is increased ATP production and PPP activity for biosynthesis. However, it has also recently been found that another metabolic event occurs in response to LPS earlier in the time course of macrophage activation (before NO accumulation limits OXPHOS), which involves citrate. Citrate is a TCA cycle intermediate formed from oxaloacetate and acetyl-CoA by citrate synthase in the mitochondria. LPS increases expression of the mitochondrial citrate carrier, solute carrier family 25 member 1 (Slc25a1), via NFκB in macrophages 71 . This carrier transports citrate out of the mitochondrion in exchange for malate, causing cytosolic citrate accumulation. When ATP is present, the enzyme ATP-citrate lyase (ACLY) in the cytosol converts citrate back to oxaloacetate and acetyl-CoA. Acetyl-CoA is a source of acetyl groups for acetylation of histones 72 . Interestingly, the transcription of genes encoding glycolytic enzymes HK 2, phosphofructokinase-1 (PFK1) and LDHA is regulated downstream of ACLY. Silencing of ACLY suppressed expression of these genes, thereby decreasing glucose consumption. This effect was rescued by the addition of acetate, which can provide the acetyl groups needed for histone acetylation on these genes. Citrate metabolism can therefore influence the induction of glycolysis 72 . The increase in acetyl-CoA levels after TLR stimulation has another function in activated macrophages and DCs. Acetyl-CoA has been shown to help activated macrophages meet their increased biosynthetic demands, particularly in lipid biosynthesis. For example, ACLY is needed for the production of the eicosanoid prostaglandin E2 (PGE 2 ) in response to LPS or TNF-α stimulation in phorbol-12-myristate-13-acetate (PMA)-differentiated U937 cells, human monocytic cells from histiocytoma 73 . The function of acetyl-CoA in promoting lipid biosynthesis depends on acetyl-CoA carboxylase (ACC), which converts acetyl-CoA to malonyl-CoA, a metabolite that is used for fatty acid synthesis. In LPS-activated DCs, citrate is preferentially removed from the TCA cycle and used for de novo synthesis of fatty acids, and increased glycolytic flux is needed to support this metabolic shift 74 . This increase in glycolysis is mediated by the kinases TANK-binding kinase 1 (TBK1), inhibitor of κB kinase ε (IKKε) and Akt, which drive the association of the glycolytic enzyme HK 2 with the mitochondria. Genetic silencing of Slc25a1 or inhibition of fatty acid synthesis by blocking the activity of ACC both result in the impairment of LPS-induced DC activation. The decreased fatty acid biosynthesis limits membrane production needed for the expansion of endoplasmic reticulum and Golgi, which then decreases protein production, including the generation of cytokines needed for T-cell activation by DCs 74 . Consistent with this, liver DCs with a high lipid content are immunogenic, inducing T-cell and NK-cell activation, while those with lower lipid concentrations are more tolerogenic and induce regulatory T cells 75 . ACLY-catalyzed citrate metabolism also contributes to the production of inflammatory mediators such as NO and ROS. Oxaloacetate, the other product of citrate metabolism, is further metabolized to pyruvate with the concomitant generation of NADPH from NADP + . NADPH and molecular oxygen can be used by NADPH oxidase to generate ROS. NADPH is also required as a substrate for the conversion of L-arginine to NO and citrulline. NO could then participate in the LPS-induced metabolic switch by inhibiting mitochondrial OXPHOS, as could ROS, as they stabilize HIF-1α 76 . Activation of macrophages by either LPS, TNF-α, or IFNγ alone, or a combination of TNF-α and IFNγ induces ACLY expression, and inhibition of ACLY activity decreases the production of NO and ROS 73 . Inhibition of Slc25a1 or its silencing also results in decreased production of NO, ROS, and PGE 2 in U937 cells 71 . These changes in citrate transport and metabolism ( Figure 3 ) would therefore appear to be critical for the activation of macrophages and DCs by LPS, the latter in turn triggers T-cell activation and adaptive immunity.

All of the above events have been shown to be critical in macrophages and DCs activated by classical stimuli such as LPS, IFNγ and bacteria including E. coli . Such classically activated macrophages have been termed “M1 macrophages” and are positively associated with inflammation. Intriguingly, the so-called M2 or alternatively activated macrophages have a different metabolic profile and inflammatory phenotype 87 , 88 . These two phenotypes are not rigid and there may in fact exist a “spectrum” of macrophage activation, involving macrophages with different metabolic and inflammatory signatures, and with different roles in host defense against various pathogens, as well as in wound healing and the resolution of inflammation 89 , 90 . M2 macrophages exhibit increased flux through OXPHOS and higher expression of anti-inflammatory cytokines such as IL-10, but have decreased production of NO and TNF-α 88 . They are involved in immune responses to parasites 91 , as evidenced by their role in the induction of T h 2-type responses in disease models of parasitic infection 92 . Why glycolysis is needed for M1 macrophage activation, whilst OXPHOS promotes the M2 macrophage phenotype, is not known. The cytokines IL-4 and IL-13 are key inducers of M2 macrophage polarization. Many of the processes that drive the glycolytic switch in M1 macrophages are downregulated in M2 macrophages ( Figure 4 ). First, arginase 1 (Arg-1) is highly expressed in M2 macrophages, while iNOS expression is decreased 93 . M1 macrophages have increased levels of citrulline and the inflammatory mediator NO, while in M2 macrophages, arginine is preferentially metabolized to urea and ornithine 94 . Ornithine can be further metabolized to polyamines and proline. Schistosome eggs induce Arg-1 expression in infected mice, and the combination of IL-4 and IL-13 increases proline production by macrophages 93 . This increased proline production could contribute to granuloma formation and liver fibrosis in infected mice, as both of these are enhanced when proline synthesis is boosted 93 . However, there is also conflicting evidence suggesting that Arg-1-expressing macrophages suppress schistosome-induced fibrosis during chronic infection 95 . Interestingly, Arg-1 can be induced in classically activated macrophages by intracellular infection with mycobacteria, and functions in this context to suppress NO production 96 . M. tuberculosis -infected mice lacking Arg-1 expression in macrophages are better able to clear the bacteria 96 . Secondly, IL-4 and IL-13 induce oxidative metabolism by inhibiting mTOR via activation of its upstream negative regulators TSC1 and TSC2. Macrophages lacking these negative regulators cannot switch towards OXPHOS, and Arg-1 expression is reduced 41 . Inhibition of mTOR by rapamycin treatment or transient TSC2 expression 43 can also lead to a decrease in HIF-1α levels, and therefore could result in reduced HIF-1α-dependent glycolytic and inflammatory gene expression 97 . In contrast to M1 macrophages, expression of the HIF-2α isoform of HIF is induced in IL-4- and IL-13-stimulated macrophages, while its levels are reduced in macrophages treated with LPS and IFNγ 39 . HIF-2α-knockout macrophages have decreased levels of Arg-1, while iNOS is unaffected 39 . Interestingly, HIF-2α may still have some role in M1-like macrophages, particularly those in hypoxic conditions 98 . IL-1β, IL-12, and TNF-α levels are decreased in LPS-stimulated mice with macrophages lacking HIF-2α, while IL-10 levels are increased. However, HIF-2α may not have a role in induction of glycolysis and maintenance of ATP levels 98 . Thirdly, M2 macrophages predominantly express PFKFB1, an isoform of PFK2 52 . PFKFB1 has higher bisphosphatase activity than u-PFK2 55 , 56 , and therefore can more readily break down F-2,6-BP into fructose-6-phosphate, decreasing glycolytic activity. Furthermore, AMPK activity is high in M2 macrophages, and drives production of the anti-inflammatory cytokine IL-10 62 . M2 macrophages are fueled by fatty acid uptake and oxidation in the mitochondria, a metabolic program controlled by AMPK and its downstream targets, which include PPARs. IL-4 and IL-13 increase the expression of PPARs and their coactivator protein PGC1β, leading to increased expression of proteins of the mitochondrial respiratory chain. Knockdown of PGC1β antagonizes this anti-inflammatory skewing of macrophage metabolism 63 . IL-4 also enhances PPARγ activity via signal transducer and activator of transcription 6 (STAT6) in macrophages and DCs 99 . PPARγ boosts the ability of IL-4 to induce arginase expression in macrophages, and also has a role in the IL-4-stimulated increase in fatty acid β-oxidation in these macrophages 100 . AMPK also inhibits mTOR activity by activating TSC2 65 , as well as by directly phosphorylating the mTOR complex subunit Raptor 66 . Pharmacological activation of AMPK supports the view that it has anti-inflammatory activity. Treatment with the