The Metabolic Signature of Macrophage Responses

Antonella Viola, Fabio Munari, Ricardo Sánchez‐Rodríguez, Tommaso Scolaro, Alessandra Castegna•View original

OverviewBalancedalloy voice

Imagine a cell that can flip personalities. One day, it's a street fighter, spewing toxic molecules to stop an infection. The next, it's a cleanup crew and a contractor, clearing debris and rebuilding tissue. That's a macrophage. And here's the key idea that ties the story together: those personalities aren't just about which cytokine hit the cell first; they're powered by different engines under the hood. As Viola and colleagues laid out, macrophage identity tracks with metabolism. The pro-inflammatory edge of the spectrum—the M1 pole—runs on glycolysis and a disrupted Krebs cycle. The pro-resolving edge—the M2 pole—leans on a fully intact tricarboxylic acid cycle and oxidative phosphorylation, with fatty acids feeding the mitochondria. It's a tidy picture, but it's not a cage. In living tissues, macrophages slide along that spectrum as cues change, and their fuel mix changes with them. You can see the scale of this system in human fat. In lean adipose tissue, about one in ten to one in seven cells are macrophages. In obesity, that fraction can jump to roughly half, often skewed toward a pro-inflammatory stance that worsens insulin resistance. Zoom out further and remember the churn of life: about one million cells are turned over every second in the human body, and macrophages are central to that recycling. They engulf dying cells—what immunologists call efferocytosis—and, when they do it well, they release anti-inflammatory mediators like interleukin-10 and transforming growth factor beta to keep the peace. On the inflammatory side, lipopolysaccharide and interferon-gamma push macrophages into a fast-burning state. Glycolysis ramps up to generate quick ATP—just two per glucose molecule, but fast—while feeding the pentose phosphate pathway to make NADPH, the reducing currency for both antimicrobial bursts and lipid synthesis. Tannahill and others showed that this shift is locked in by HIF-1 alpha, the hypoxia-responsive transcription factor that, in these cells, is stabilized even in normal oxygen. Akt and mechanistic target of rapamycin, or mTOR, signaling, together with nuclear factor kappa B, or NF-kappaB, keep HIF-1 alpha high; mTORC1 also tunes mitochondrial capacity. Two enzymes sit on the throttle: PFKFB3, which drives glycolytic flux, and pyruvate kinase M2. PKM2 is a shapeshifter—its inactive forms can move into the nucleus and partner with HIF-1 alpha to boost inflammatory genes, while its active tetramer form pushes glycolysis forward and supports M1 polarization. Here's where the plot thickens. In M1 cells, the Krebs cycle isn't a smooth loop; it's intentionally interrupted. Citrate accumulates because isocitrate dehydrogenase wanes and the citrate carrier—CIC, described by Infantino and colleagues—pumps it into the cytosol. Once there, citrate does double duty. It inhibits early glycolytic checkpoints, nudging flux control, and it feeds ATP-citrate lyase to make acetyl-CoA, the precursor for fatty acids and a donor for histone acetylation. That means more prostaglandins and lipid mediators, and it means an epigenetic nudge on inflammatory genes. The same export also fuels nitric oxide and reactive oxygen species through malic enzyme-driven NADPH production, linking a carbon shuttle to the cell's antimicrobial punch. Succinate becomes a signal as much as a metabolite. It accumulates when the cycle stalls, then slips into the role of messenger. Inside the cell, high succinate inhibits prolyl hydroxylases, further stabilizing HIF-1 alpha and boosting interleukin-1 beta. It can even mark up proteins post-translationally; succinylation of PKM2 promotes its nuclear partnership with HIF-1 alpha, tightening the loop between metabolism and gene expression. Outside the cell, succinate can be released and sensed by the receptor SUCNR1, also called GPR91, on neighboring cells—including other macrophages—creating an autocrine and paracrine amplifier of inflammation. This same pathway shows up beyond infection. Elevated succinate associates with metabolic disease, and SUCNR1 signaling has been tied to inflammatory arcs in obesity and ischemia-reperfusion injury. There's a third molecule in this triangle that's almost cinematic in its role. Itaconate, made from the tricarboxylic acid intermediate cis-aconitate by the enzyme ACOD1—also known as IRG1—has a split personality. Lampropoulou and Mills traced its roots as an antimicrobial weapon; it directly hampers bacterial growth. At the same time, itaconate reaches back into host metabolism, inhibiting succinate dehydrogenase in the mitochondrion. That keeps succinate levels high, reinforcing the HIF-1 alpha state. Yet itaconate is also electrophilic, modifying the KEAP1 protein and freeing NRF2, a master regulator of antioxidant defenses. When NRF2 rises, transcription of genes that dampen inflammation—like those curbing interleukin-1 beta and interleukin-6—goes up. So you get a paradox: a cell roaring with pro-inflammatory outputs, but with a built-in brake that prevents the engine from burning out. All of these nodes point to the same effectors. NADPH oxidase fires off reactive oxygen species. Inducible nitric oxide synthase turns arginine into nitric oxide. The cytokine mix skews toward interleukin-1 beta, tumor necrosis factor, and interleukin-6. And crucially, these aren't separate pathways running in parallel; they're stitched together by carbon flow. Citrate export sets up lipid mediator synthesis and chromatin changes. Succinate stabilizes the transcriptional program. Itaconate restrains mitochondrial respiration and tempers the flames through NRF2. Now, swing to the other side of the spectrum. In M2-like macrophages, the mitochondria hum. The Krebs cycle runs clean, electrons move down the respiratory chain, and ATP is made efficiently by oxidative phosphorylation. Fatty acids are not just stored—they're burned. This fatty acid oxidation depends on shuttling long-chain fats into mitochondria through carnitine palmitoyltransferase, and it's supported by transcription factors like peroxisome proliferator-activated receptor gamma, or PPAR gamma, and its coactivator, peroxisome proliferator-activated receptor gamma coactivator 1 alpha, or PGC-1 alpha. Liver X receptor, a lipid-sensing regulator, leans in here too, dialing down NF-kappaB and restraining inflammatory tone, as the review by Viola and colleagues emphasized. Amino acids feed the same identity. Arginine metabolism flips: instead of inducible nitric oxide synthase chewing it up to make nitric oxide, arginase 1 diverts arginine into ornithine and polyamines, building blocks for collagen and tissue repair. Glutamine becomes a hub. Through glutaminolysis, it generates alpha-ketoglutarate, which does two things. It fuels the tricarboxylic acid cycle and fatty acid oxidation, and it supplies the cofactor that demethylases use to remodel chromatin at M2 gene loci—think of loosening the methyl lock at H3K27 so repair programs can run. Glutamine also supplies UDP-GlcNAc for protein glycosylation, a post-translational hallmark of many M2 surface markers. And there's a feedback loop: glutamine synthetase is high in M2 cells, allowing them to make and hold onto glutamine locally. Inhibiting glutamine synthetase, as shown in tumor-associated macrophages, can tip the cells back toward an M1-like phenotype and reduce metastasis in models. If you're wondering whether fatty acid oxidation is absolutely required for M2 identity, the answer is: it's complicated. Pharmacologically blocking fatty acid oxidation, or FAO, often blunts M2 traits. But genetic deletion of carnitine palmitoyltransferase 2, or CPT2, one of the transport enzymes, doesn't always prevent interleukin-4 from inducing an M2 program. That tells you this network has redundancy and flexibility. In real tissues, M2-like macrophages can dial up glycolysis when needed, and M1 cells aren't always fully glycolytic. Context matters. Tumors are a masterclass in context. The microenvironment is acidic, hypoxic, and rich in lactate. In that space, macrophages—now called tumor-associated macrophages—often adopt a metabolism that feeds the tumor's agenda. Viola and colleagues describe a loop where tumor-associated macrophages run glycolysis, produce a lot of lactate, and that lactate feeds back as a signal. It induces vascular endothelial growth factor and arginase 1, tilting the cells toward angiogenesis and immune suppression, and it activates Akt and mTOR, which further entrenches glycolysis. But when oxygen is scarce, another switch flips. Tumor-associated macrophages upregulate the protein REDD1, a brake on mTOR. With mTOR down, their glucose uptake and glycolysis tighten. Here's the twist: that restraint in the macrophage leaves more glucose for endothelial cells, which then drive chaotic, leaky vessel growth. The net effect is worse vasculature and a niche that tumors exploit. On top of glucose and lactate, amino acid metabolism sharpens immunosuppression. The lactate-arginase 1 axis depletes extracellular arginine, which T cells need to proliferate and function. Meanwhile, macrophages with high cyclooxygenase activity ramp up indoleamine 2,3-dioxygenase, catabolizing tryptophan. Starved of tryptophan, T cells stall. The result isn't a macrophage that mindlessly burns sugar; it's a cell that uses carbon and nitrogen handling to rewrite the immune script in the tumor's favor. Step back, and a larger pattern emerges. M1 programs put carbon to work as a weapon—fast glycolysis, pentose phosphate-powered NADPH, citrate export for lipids and acetylation, succinate and itaconate as metabolic signals that steer transcription. M2 programs turn metabolism into a scaffold—intact oxidative cycles, fatty acid oxidation, glutaminolysis and alpha-ketoglutarate shaping both energy and epigenetics, arginase routing nitrogen into repair. And in tissues like adipose or tumor, environmental cues—oxygen, lactate, nutrients—nudge macrophages along that axis. The caveats are not footnotes; they're the main lesson. The M1 and M2 labels are a helpful map, not a street-by-street GPS. In some wounds, M2-like macrophages show surprising glycolytic dependence. In certain infections, the pentose phosphate pathway flux changes in ways that don't fit the cartoon. Fatty acid oxidation can be important without being absolutely required. And metabolites that act as on switches in one tissue can be brakes in another. Itaconate's NRF2 activation can suppress inflammatory gene expression, yet it coexists with high antimicrobial outputs. Succinate stabilizes HIF-1 alpha and fuels inflammation, but it also signals outside the cell to shape a broader tissue response through SUCNR1. There's also the metabolic disease thread weaving through. In obesity, adipose tissue macrophages skew toward M1-like states, and succinate rises systemically, correlating with body mass and type 2 diabetes. That's not just a correlation to memorize; it's a clue that the same molecules orchestrating a macrophage's internal state are whispering across tissues and organs, coordinating whole-body inflammation and metabolism. What do we do with this? The research points to pressure points. Block the succinate receptor SUCNR1, and you might dampen sterile inflammation in metabolic disease. Nudge the itaconate–NRF2 axis, and you might cool an overactive interleukin-1 beta loop without switching off host defense. Target glutamine synthetase or the glutamine–alpha-ketoglutarate pipeline in tumors, and you might destabilize the M2-like, pro-angiogenic tumor-associated macrophage phenotype that helps cancers grow. Even fatty acid oxidation, while nuanced, remains a lever in repair contexts. None of these are silver bullets; the context dependence we've been talking about is the caution label on every one of them. But that's the deeper payoff of seeing immunity as biochemistry with purpose. When you watch how macrophages allocate carbon and nitrogen—where they break a cycle, where they export a metabolite, when they flip a transcription factor—you're not just collecting trivia about enzymes. You're seeing how a cell decides who it is. And once you can see that, you can start to imagine therapies that don't just shout "more inflammation" or "less inflammation," but instead retune the engine—enough glycolysis here, a dash of alpha-ketoglutarate there—to help the cell choose a better role for the tissue it lives in.

That's a macrophage. And here's the key idea that ties the story together: those personalities aren't just about which cytokine hit the cell first; they're powered by different engines under the hood. As Viola and colleagues laid out, macrophage identity tracks with metabolism.

The pro-inflammatory edge of the spectrum—the M1 pole—runs on glycolysis and a disrupted Krebs cycle. The pro-resolving edge—the M2 pole—leans on a fully intact tricarboxylic acid cycle and oxidative phosphorylation, with fatty acids feeding the mitochondria. It's a tidy picture, but it's not a cage.

In living tissues, macrophages slide along that spectrum as cues change, and their fuel mix changes with them.

You can see the scale of this system in human fat. In lean adipose tissue, about one in ten to one in seven cells are macrophages. In obesity, that fraction can jump to roughly half, often skewed toward a pro-inflammatory stance that worsens insulin resistance.

Zoom out further and remember the churn of life: about one million cells are turned over every second in the human body, and macrophages are central to that recycling. They engulf dying cells—what immunologists call efferocytosis—and, when they do it well, they release anti-inflammatory mediators like interleukin-10 and transforming growth factor beta to keep the peace.

On the inflammatory side, lipopolysaccharide and interferon-gamma push macrophages into a fast-burning state. Glycolysis ramps up to generate quick ATP—just two per glucose molecule, but fast—while feeding the pentose phosphate pathway to make NADPH, the reducing currency for both antimicrobial bursts and lipid synthesis. Tannahill and others showed that this shift is locked in by HIF-1 alpha, the hypoxia-responsive transcription factor that, in these cells, is stabilized even in normal oxygen.

Akt and mechanistic target of rapamycin, or mTOR, signaling, together with nuclear factor kappa B, or NF-kappaB, keep HIF-1 alpha high; mTORC1 also tunes mitochondrial capacity. Two enzymes sit on the throttle: PFKFB3, which drives glycolytic flux, and pyruvate kinase M2. PKM2 is a shapeshifter—its inactive forms can move into the nucleus and partner with HIF-1 alpha to boost inflammatory genes, while its active tetramer form pushes glycolysis forward and supports M1 polarization.

Here's where the plot thickens. In M1 cells, the Krebs cycle isn't a smooth loop; it's intentionally interrupted. Citrate accumulates because isocitrate dehydrogenase wanes and the citrate carrier—CIC, described by Infantino and colleagues—pumps it into the cytosol.

Once there, citrate does double duty. It inhibits early glycolytic checkpoints, nudging flux control, and it feeds ATP-citrate lyase to make acetyl-CoA, the precursor for fatty acids and a donor for histone acetylation. That means more prostaglandins and lipid mediators, and it means an epigenetic nudge on inflammatory genes.

The same export also fuels nitric oxide and reactive oxygen species through malic enzyme-driven NADPH production, linking a carbon shuttle to the cell's antimicrobial punch.

Succinate becomes a signal as much as a metabolite. It accumulates when the cycle stalls, then slips into the role of messenger. Inside the cell, high succinate inhibits prolyl hydroxylases, further stabilizing HIF-1 alpha and boosting interleukin-1 beta.

It can even mark up proteins post-translationally; succinylation of PKM2 promotes its nuclear partnership with HIF-1 alpha, tightening the loop between metabolism and gene expression. Outside the cell, succinate can be released and sensed by the receptor SUCNR1, also called GPR91, on neighboring cells—including other macrophages—creating an autocrine and paracrine amplifier of inflammation. This same pathway shows up beyond infection.

Elevated succinate associates with metabolic disease, and SUCNR1 signaling has been tied to inflammatory arcs in obesity and ischemia-reperfusion injury.

There's a third molecule in this triangle that's almost cinematic in its role. Itaconate, made from the tricarboxylic acid intermediate cis-aconitate by the enzyme ACOD1—also known as IRG1—has a split personality. Lampropoulou and Mills traced its roots as an antimicrobial weapon; it directly hampers bacterial growth.

At the same time, itaconate reaches back into host metabolism, inhibiting succinate dehydrogenase in the mitochondrion. That keeps succinate levels high, reinforcing the HIF-1 alpha state. Yet itaconate is also electrophilic, modifying the KEAP1 protein and freeing NRF2, a master regulator of antioxidant defenses.

When NRF2 rises, transcription of genes that dampen inflammation—like those curbing interleukin-1 beta and interleukin-6—goes up. So you get a paradox: a cell roaring with pro-inflammatory outputs, but with a built-in brake that prevents the engine from burning out.

All of these nodes point to the same effectors. NADPH oxidase fires off reactive oxygen species. Inducible nitric oxide synthase turns arginine into nitric oxide.

The cytokine mix skews toward interleukin-1 beta, tumor necrosis factor, and interleukin-6. And crucially, these aren't separate pathways running in parallel; they're stitched together by carbon flow. Citrate export sets up lipid mediator synthesis and chromatin changes.

Succinate stabilizes the transcriptional program. Itaconate restrains mitochondrial respiration and tempers the flames through NRF2.

Now, swing to the other side of the spectrum. In M2-like macrophages, the mitochondria hum. The Krebs cycle runs clean, electrons move down the respiratory chain, and ATP is made efficiently by oxidative phosphorylation.

Fatty acids are not just stored—they're burned. This fatty acid oxidation depends on shuttling long-chain fats into mitochondria through carnitine palmitoyltransferase, and it's supported by transcription factors like peroxisome proliferator-activated receptor gamma, or PPAR gamma, and its coactivator, peroxisome proliferator-activated receptor gamma coactivator 1 alpha, or PGC-1 alpha. Liver X receptor, a lipid-sensing regulator, leans in here too, dialing down NF-kappaB and restraining inflammatory tone, as the review by Viola and colleagues emphasized.

Amino acids feed the same identity. Arginine metabolism flips: instead of inducible nitric oxide synthase chewing it up to make nitric oxide, arginase 1 diverts arginine into ornithine and polyamines, building blocks for collagen and tissue repair. Glutamine becomes a hub.

Through glutaminolysis, it generates alpha-ketoglutarate, which does two things. It fuels the tricarboxylic acid cycle and fatty acid oxidation, and it supplies the cofactor that demethylases use to remodel chromatin at M2 gene loci—think of loosening the methyl lock at H3K27 so repair programs can run. Glutamine also supplies UDP-GlcNAc for protein glycosylation, a post-translational hallmark of many M2 surface markers.

And there's a feedback loop: glutamine synthetase is high in M2 cells, allowing them to make and hold onto glutamine locally. Inhibiting glutamine synthetase, as shown in tumor-associated macrophages, can tip the cells back toward an M1-like phenotype and reduce metastasis in models.

If you're wondering whether fatty acid oxidation is absolutely required for M2 identity, the answer is: it's complicated. Pharmacologically blocking fatty acid oxidation, or FAO, often blunts M2 traits. But genetic deletion of carnitine palmitoyltransferase 2, or CPT2, one of the transport enzymes, doesn't always prevent interleukin-4 from inducing an M2 program.

That tells you this network has redundancy and flexibility. In real tissues, M2-like macrophages can dial up glycolysis when needed, and M1 cells aren't always fully glycolytic. Context matters.

Tumors are a masterclass in context. The microenvironment is acidic, hypoxic, and rich in lactate. In that space, macrophages—now called tumor-associated macrophages—often adopt a metabolism that feeds the tumor's agenda.

Viola and colleagues describe a loop where tumor-associated macrophages run glycolysis, produce a lot of lactate, and that lactate feeds back as a signal. It induces vascular endothelial growth factor and arginase 1, tilting the cells toward angiogenesis and immune suppression, and it activates Akt and mTOR, which further entrenches glycolysis. But when oxygen is scarce, another switch flips.

Tumor-associated macrophages upregulate the protein REDD1, a brake on mTOR. With mTOR down, their glucose uptake and glycolysis tighten. Here's the twist: that restraint in the macrophage leaves more glucose for endothelial cells, which then drive chaotic, leaky vessel growth. The net effect is worse vasculature and a niche that tumors exploit.

On top of glucose and lactate, amino acid metabolism sharpens immunosuppression. The lactate-arginase 1 axis depletes extracellular arginine, which T cells need to proliferate and function. Meanwhile, macrophages with high cyclooxygenase activity ramp up indoleamine 2,3-dioxygenase, catabolizing tryptophan.

Starved of tryptophan, T cells stall. The result isn't a macrophage that mindlessly burns sugar; it's a cell that uses carbon and nitrogen handling to rewrite the immune script in the tumor's favor.

Step back, and a larger pattern emerges. M1 programs put carbon to work as a weapon—fast glycolysis, pentose phosphate-powered NADPH, citrate export for lipids and acetylation, succinate and itaconate as metabolic signals that steer transcription. M2 programs turn metabolism into a scaffold—intact oxidative cycles, fatty acid oxidation, glutaminolysis and alpha-ketoglutarate shaping both energy and epigenetics, arginase routing nitrogen into repair.

And in tissues like adipose or tumor, environmental cues—oxygen, lactate, nutrients—nudge macrophages along that axis.

The caveats are not footnotes; they're the main lesson. The M1 and M2 labels are a helpful map, not a street-by-street GPS. In some wounds, M2-like macrophages show surprising glycolytic dependence.

In certain infections, the pentose phosphate pathway flux changes in ways that don't fit the cartoon. Fatty acid oxidation can be important without being absolutely required. And metabolites that act as on switches in one tissue can be brakes in another.

Itaconate's NRF2 activation can suppress inflammatory gene expression, yet it coexists with high antimicrobial outputs. Succinate stabilizes HIF-1 alpha and fuels inflammation, but it also signals outside the cell to shape a broader tissue response through SUCNR1.

There's also the metabolic disease thread weaving through. In obesity, adipose tissue macrophages skew toward M1-like states, and succinate rises systemically, correlating with body mass and type 2 diabetes. That's not just a correlation to memorize; it's a clue that the same molecules orchestrating a macrophage's internal state are whispering across tissues and organs, coordinating whole-body inflammation and metabolism.

What do we do with this? The research points to pressure points. Block the succinate receptor SUCNR1, and you might dampen sterile inflammation in metabolic disease.

Nudge the itaconate–NRF2 axis, and you might cool an overactive interleukin-1 beta loop without switching off host defense. Target glutamine synthetase or the glutamine–alpha-ketoglutarate pipeline in tumors, and you might destabilize the M2-like, pro-angiogenic tumor-associated macrophage phenotype that helps cancers grow. Even fatty acid oxidation, while nuanced, remains a lever in repair contexts.

None of these are silver bullets; the context dependence we've been talking about is the caution label on every one of them.

But that's the deeper payoff of seeing immunity as biochemistry with purpose. When you watch how macrophages allocate carbon and nitrogen—where they break a cycle, where they export a metabolite, when they flip a transcription factor—you're not just collecting trivia about enzymes. You're seeing how a cell decides who it is.

And once you can see that, you can start to imagine therapies that don't just shout "more inflammation" or "less inflammation," but instead retune the engine—enough glycolysis here, a dash of alpha-ketoglutarate there—to help the cell choose a better role for the tissue it lives in.

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