Metabolism in type 2 immune responses

Abstract

The immune response is tailored to the environment in which it takes place. Immune cells sense and adapt to changes in their surroundings, and it is now appreciated that in addition to cytokines made by stromal and epithelial cells, metabolic cues provide key adaptation signals. Changes in immune cell activation states are linked to changes in cellular metabolism that support function. Furthermore, metabolites themselves can signal between as well as within cells. Here we discuss recent progress in our understanding of how metabolic regulation relates to type 2 immunity firstly by considering specifics of metabolism within type 2 immune cells, and secondly by stressing how type 2 immune cells are integrated more broadly into the metabolism of the organism as a whole.

Introduction

Different functional states of immune cells, for example differentiated, quiescent or activated, are underpinned by metabolic adaptations¹. Furthermore, as immune cells circulate, migrating from lymphoid organs to tissues, they adjust metabolically to their new environments². Complexity is added by the fact that within any given tissue, conditions are not immutable; tissues experiencing inflammation or undergoing repair are likely to have a different metabolic landscape compared to those in homeostasis.

Type 2 immune responses, the focus of this review, are linked to resistance to helminth parasites, allergy and asthma, tissue homeostasis and repair. The notable tissue integration of key elements of type 2 immunity, discussed herein, raises particularly interesting questions about the metabolism of type 2 immune cells, especially since they are engaged in the homeostasis of central metabolic tissues including adipose tissue, pancreas, and liver^{3, 4, 5}, and in responses that occur at barrier sites where metabolic conditions are dynamic. Type 2 immune cells include innate elements such as group 2 innate lymphoid cells (ILC2) and eosinophils, and an adaptive component consisting of type 2 helper T cells (Th2 cells), which all share the ability to make cytokines that are a defining feature of type 2 immunity, including interleukin (IL)-4, IL-13 or IL-5. Type 2 inflammation leads to the formation of Th2 memory, including long-lived resident memory Th2 cells (Th2_RM), which have recently emerged as key perpetuators of chronic allergic diseases^{6, 7, 8}. IL-4 or IL-13 play critical roles in directing macrophages and B cells to assume type 2 immunity-specific functions. In macrophages, this involves expression of a set of genes, that define a so-called alternative activation (or “M2”, or M(IL-4)) state, and in B cells it involves a switch to IgE or IgG1 (mouse)/IgG4 (human) production.

In this review we first discuss what is known about cell-intrinsic metabolism linked to cellular fate and function in the major type 2 immune cell populations that have been investigated in this regard. In the second part, we consider how type 2 immune functions are an integral component of the sensory system that responds to and regulates whole-body metabolism. These functions are coordinated by cytokines, hormones, neurotransmitters, nutrients and metabolites and we focus on how type 2 immune cells interpret cues in the gastrointestinal tract and adipose tissue to facilitate host protection and metabolic homeostasis.

Metabolic pathways required for Th2 cell development from priming to tissue residency

The polarization of naïve CD4⁺ T cells towards the Th2 lineage occurs in the lymph nodes (LN), where they begin to express GATA3 and IL-4. However, full differentiation, which includes the capacity to secrete IL-13 and IL-5, occurs only after cells leave the lymphatics and receive tissue-derived signals IL-33, IL-25 and thymic stromal lymphopoietin (TSLP)⁹. Furthermore, like ILC2, Th2 cells elicited by parasitic infections can disseminate to distant barrier sites^{10, 11, 12}. Th2_RM cells exhibit innate-like properties that mirror ILC2, including antigen-independent cytokine secretion and neuropeptide expression^{13, 14, 15, 16, 17, 18}. Our understanding of Th2_RM cell metabolism is fragmented, but recent studies have revealed distinct metabolic adaptations to tissue residency.

Early work on in vitro polarized Th cells showed that activated Th2 cells increase expression of the glucose transporter GLUT1 and have the highest glucose uptake compared to Th1 or Th17 cells¹⁹ (see Figure 1 for metabolic pathway information). In contrast to Th1 cells, and similarly to T regulatory (Treg cells), in vitro Th2 cells can differentiate in the absence of glutaminolysis²⁰. Like other Th cells and activated ILC2, Th2 cells require signalling through mTORC1 for the upregulation of glycolysis and cell cycle entry²¹. However, Th2 cells, more than other Th subtypes, also depend on mTORC2 engagement, through pathways involving GTPase RhoA and AGC kinases²². Since mTORC2 signals promote cell survival, and migratory cytoskeleton rearrangement, mTORC2 could facilitate Th2 maturation and the motile behavior presumably required for tissue residency. Activated CD4⁺ T cells secrete IL-2, and autocrine signalling through the high affinity IL-2 receptor (IL-2Rα) has broad effects on metabolic remodeling in activated Th cells that depend on mTORC1²³, but are also directly downstream of STAT5, which drives transcription of multiple genes encoding enzymes in glycolysis and amino acid synthesis pathways²⁴. Interestingly, Th2_RM cells in lung, intestine, and adipose tissue have high expression of Il2ra^{6, 14, 18} and depend on IL-2 signalling for establishing tissue residency⁶. We speculate that this requirement could be linked to the metabolic changes that are induced through IL-2Rα, which perhaps are essential for supporting the final Th2 differentiation steps that occur within tissues.

Figure 1. Overview of core metabolic pathways used by type 2 immune cells.

The tricarboxylic acid (TCA) cycle is a central metabolic pathway. Localized to mitochondria and fuelled by glucose, fatty acids and amino acids, it utilizes a series of reactions to generate NADH and FADH which are then oxidized by the electron transport chain to create a proton gradient across the inner mitochondrial membrane. This gradient drives the ATP synthase to generate ATP through OXPHOS. Glucose carbon enters mitochondria as pyruvate, generated by glycolysis, and is there converted to Acetyl-CoA. Intermediates of the glycolysis pathway can be redirected for nucleotide synthesis in the pentose-phosphate pathway, or metabolised to amino sugar uridine diphosphate N-acetylglucosamine (UDP-GlcNac), which is the major sugar donor for glycosylation. Another side branch of glycolysis pathway is the serine synthesis and one carbon metabolism pathway, which generates not only serine but nucleotides and S-adenosyl methionine, the methionine donor for DNA and histone methylation. Glutamine and branched chain amino acids (BCAA) have various points of entry into the TCA cycle. Fatty acids (FA) fuel the TCA cycle after being transported into mitochondria by carnitine palmitoyltransferase 1A (CPT1a) and cleaved in the cycles of β-oxidation to generate Acetyl-CoA (the process of fatty acid oxidation, FAO). FA (and sterols) can be synthesized from citrate (by fatty acid synthesis, FAS), imported from the extracellular space as FA, or generated by lipolysis within lysosomes from triacylglycerols (TAG) taken up from the exterior. FA can be either used for FAO or membrane synthesis, or used for TAG synthesis, for storage along with cholesterol esters in lipid droplets (LD). LD TAG can be reconverted to FA by regulated lipolysis or autophagy. Acetyl-CoA made from citrate exported from mitochondria serves as the acetyl donor for protein (including histone) acetylation. αKG - alpha-ketoglutarate, MCT - Monocarboxylate transporters, LAA – system L amino acids transporters; ASCT – alanine/serine/cysteine transporters; HMG-CoA - β-Hydroxy β-methylglutaryl-CoA; ACC1 - Acetyl-CoA carboxylase1; DGAT1 - Diacylglycerol O-acyltransferase 1. Created with BioRender.com.

Several lipid metabolism pathways regulate Th2 cell effector functions. Fatty acid synthesis (FAS) contributes to Th2 cell differentiation, although Th2 cells are less dependent than Th17 cells on this pathway²⁵. FAS supports membrane biogenesis in rapidly proliferating cells, but might also facilitate T cell activation through increased production of ROS, as FAS limits NADPH availability for antioxidant responses. Consistent with this, deletion or inhibition of ACC1 (the rate limiting enzyme for FAS, Figure 1) in Th cells increases NADPH/NADP⁺, decreases cellular ROS, and protects from apoptosis²⁶. ACC1 inhibition also increases the abundance of tricarboxylic acid (TCA) cycle intermediates, promotes mitochondrial spare respiratory capacity (SRC), and, consistent with this being a feature of memory T cells²⁷, increases commitment towards Th2 memory²⁶. Thus, decreasing FAS might be an important metabolic switch in transiting towards the memory phenotype. IL-33-driven reactivation of memory Th2 cells is again associated with an increase in FAS: IL-33R⁺ memory Th2 cells express more ACC1 compared to IL-33R^- Th2 cells, which is driven by mTORC1 activation and corelates with increased IL-5 and IL-13 production²⁸.

In addition to its use for FAS, citrate from the TCA cycle is also utilized as a substrate for cholesterol and sterol hormone synthesis in the mevalonate pathway. In helminth infection, Th2 cells that express Cyp11a1^{18, 29}, an enzyme that converts cholesterol to pregnenolone, a precursor for steroid synthesis, are able to inhibit Th proliferation in vitro²⁹, and in this way exert regulatory effects that remain to be fully explored. Interestingly, lipid metabolism gene expression is enriched in Th2_RM cells compared to circulating memory Th2 cells⁷. Indeed, in the HDM-induced asthma model, lung Th2 cells express genes associated with FA storage and oxidation^{7, 13}, a signature also observed in Th2 cells from the small intestine and adipose tissue of mice infected with a helminth parasite^{14, 18} (Figure 2). Th2 cell maintenance in the lungs is negatively affected by the pharmacological inhibition of not only FAS and FA oxidation (FAO), but also glycolysis, suggesting that all three pathways are critical, although glycolysis inhibition has the most potent effect¹³. This may reflect a need to augment glycolysis to reach full effector potential³⁰. In line with this, during enteric infection with the helminth parasite Heligmosomoides polygyrus, expression of several glycolysis pathway enzymes is increased in Th2 cells in the small intestine compared to Th2 cells in mesenteric adipose tissue, which is a more distal responsive tissue in this infection (Figure 2).

Figure 2. Metabolic adaptation to different tissues of residence.

Infection with the helminth parasite H. polygyrus is restricted to the small intestine and induces a strong type 2 immune response that protects against reinfection. The mesenteric lymph node (mLN) is the draining LN for this infection, in which the adaptive immune response is initiated. Th2 cells then enter the intestine, where they mediate a protective response that includes promoting alternatively activated macrophages. In this infection, the mesenteric adipose tissue (mAT) also becomes populated by resident memory Th2 cells. For illustrative purposes, fraction dot plot representation is shown for expression of selected genes encoding transcription factors, cytokines, enzymes of glycolytic and lipid metabolism pathways, amino acid (aa) transporters, and genes involved in metabolite sensing in the intestine. These single-cell RNA-sequencing (scRNAseq) data are from an analysis available in Gene Expression Omnibus (GEO) under accession number GSE157314, as previously described14. The schematic was created with BioRender.

PPARγ is a lipid-activated transcription factor that regulates expression of genes in lipid metabolism pathways³¹. Its role in Th2 responses during helminth infection and lung inflammation has been established in mouse models with T cell-specific PPARγ deletion^{32, 33}. PPARγ function in Th2 cells appears multifaceted. PPARγ expression is downstream of mTORC1 in naïve CD4⁺ T cells activated in vitro and promotes lipid uptake necessary for proliferation³⁴. Beyond this role in early stages of Th2 differentiation^{34, 35}, PPARγ activity appears essential for Th2_RM cell function, since PPARγ-deficient Th2 cells have diminished IL-33R expression and effector cytokine production^{32, 33, 34}. Mice lacking PPARγ in T cells are less protected from re-infection with H. polygyrus²⁸, which is Th2_RM cell-dependent¹¹. PPARγ likely links Th2_RM cell maturation with lipid metabolism by modulating expression of several genes associated with differentiated Th2 cells (Gata3, Bhlhe40, Il2ra, Il5, Il13), as well as genes linked to lipid uptake (Fabp5, Ldlr, Scarb1)^{34, 35}. In line with this, pharmacologically inhibiting PPARγ decreases FA uptake and proliferation in re-activated memory Th2 cells³⁴. Furthermore, CRISPR/Cas9 screening and chromatin accessibility assays have highlighted a crucial role for cross-regulation between PPARγ and Bhlhe40 in the Th2 differentiation programme³⁵. Bhlhe40 is a transcription factor that is highly expressed during inflammation by Th2_RM cells in the lung and small intestine, but not in adipose tissue (Figure 2)^{13, 18}. Bhlhe40 transcriptionally represses most target genes through both histone deacetylase-dependent and independent mechanisms, but in other contexts can also activate transcription³⁶. Bhlhe40 activity in Th2 cells is required for the production of the common β-chain cytokines IL-5 and GM-CSF during H. polygyrus infection and is necessary for protective responses after re-challenge, highlighting its crucial role for functional Th2 memory¹⁸. Interestingly, Bhlhe40 is also expressed by CD8⁺ T_RM, but not circulating memory CD8⁺ T cells, where it regulates the expression of several mitochondrial genes, contributing to mitochondrial fitness³⁷. Bhlhe40 deficiency in CD8⁺ T_RM cells decreases TCA cycle intermediate levels, which might be linked with the impaired gene acetylation observed in these cells³⁷. It remains to be investigated whether Bhlhe40 expression in Th2_RM cells similarly contributes to mitochondrial homeostasis.

In atopic individuals, high PPARγ expression in Th2 cells is seen in several settings, including in circulating Th2 cells from individuals with allergy and asthma^{33, 38, 39}, Th2 cells isolated from nasal polyps⁴⁰, esophagus⁴¹, lungs of asthmatic individuals⁴², and skin of dermatitis patients⁴³. Consistent with the known functions of PPARγ, human PPARγ+ Th2 cells isolated from nasal polyps have increased expression of lipid storage genes compared to other T cell subsets isolated from this tissue⁴⁰. Human studies also link PPARγ expression with IL-9 production in Th9 cells, which are argued to be a subpopulation of memory Th2 cells present in atopic individuals^{38, 40, 43}, and are crucial for allergic airway recall responses in a mouse model⁴⁴. CD8⁺ T_RM cells also express PPARγ along with Bhlhe40³⁷, and depend on FAO, which is fuelled by exogenous FA, and promotes their long-term survival in the tissue⁴⁵. Together with the essential role of PPARγ and Bhlhe40 in tissue resident macrophages⁴⁶ and of PPARγ in ILC2^{47, 48, 49, 50}, PPARγ and Bhlhe40 expression emerges as a shared adaptation for long-lasting metabolic fitness of tissue resident immune cells, that in Th2 cells is also intertwined with the core regulatory network that governs their differentiation and acquisition of effector functions^{35, 51}. In adipocytes, Bhlhe40 expression is induced by hypoxia and downregulates FAO through PPARγ repression⁵², so it is tempting to speculate that cross-regulation of PPARγ and Bhlhe40 in resident immune cells fine tunes mitochondrial metabolism in hypoxic tissue environments to balance survival with effector function.

Metabolic adaptations necessary for ILC2 differentiation and persistence

ILC2 are the innate counterparts of Th2 cells. They do not express the T cell receptor (TCR) and hence are unable to react in an antigen-specific manner or form adaptive memory, but instead rapidly release type 2 cytokines in response to tissue derived signals, primarily IL-25, IL-33 and TSLP, which can be made by epithelial cells and certain types of stromal cells⁵. Circulating human ILC2 from healthy donors express a range of system L amino acid (LAA) transporters, which import neutral amino acids, including branched chain amino acids (BCAA). In these cells, arginine and BCAA are the main fuels used to support the TCA cycle and mitochondrial respiration⁵³, which are required for survival (Figure 3). This conclusion is supported by findings in mice, in which the LAA transporters Slc7a5 (LAT1) and Slc7a8 (LAT2) are highly expressed in tissue-resident ILC2^{54, 55} and their deletion results in fewer of these cells in lung, intestine, and adipose tissue, and reduced type 2 responses in models of lung inflammation and helminth infection^{55, 56}. While the requirements for amino acid transport and metabolism have not been comprehensively explored in Th2_RM cells, it is worth noting that they have distinct expression patterns of amino acid transporters, including expression of Slc7a8¹⁸ and Slc38a1, depending on the tissue within which they reside (Figure 2).

Figure 3. Metabolism changes significantly as ILC2 transition between resting and activated states.

Resting ILC2 predominantly use mitochondrial respiration fuelled by arginine and BCAA taken up through LAAs transporters. Upon activation with IL-33, ILC2 express PPARγ and increase GLUT1 expression and glycolysis, as well as FA uptake and mitochondrial respiration. Further, glutamine transporter expression is upregulated and glutamine becomes a major substrate for mitochondrial respiration, while FA are transiently stored in LD and subsequently used for membrane biogenesis needed for proliferation. The autophagy pathway is induced upon activation and my facilitate utilisation of lipids from LD. Upregulated glycolysis is necessary for proliferation and effector cytokine secretion. BCAA – branch chain amino acids; LAA – system L amino acids transporters; FA – fatty acids; LD – lipid droplets; FAO – fatty acid oxidation. Created with BioRender.com

The use of pathway selective inhibitors indicated that IL-33-driven human ILC2 activation is dependent on glutamine oxidation⁵³. Furthermore, arginine is depleted in these cells⁵³ and in mouse ILC2 during lung inflammation⁵⁷, possibly for polyamine synthesis. Based on work in macrophages, the important step here may be the use of the polyamine spermidine to hypusinate the eukaryotic translation initiation factor (eIF)5A. Hypusinated eIF5A (eIF5A^H) is required for the efficient translation of a subset of proteins that are involved in the TCA cycle and OXPHOS⁵⁸. Human ILC2 also increase FA uptake upon IL-33 stimulation, but this may not be required to sustain OXPHOS, as inhibition of FAO has no effect on mitochondrial mass or membrane potential⁵³. Mouse ILC2 in the skin, adipose and intestinal tissues also show a propensity for FA uptake⁵⁹. Furthermore, in a model of lung inflammation, chronically activated ILC2 increase FA uptake, and store these as triacylglycerides (TAG) in lipid droplets (LD), from which they are released to support membrane synthesis⁴⁹. LD production and cellular activation in the context of IL-33 stimulation are dependent on PPARγ, which regulates many facets of fatty acid metabolism, including the expression of DGAT1 which is important for TAG synthesis. Interestingly, expression of DGAT1 is also increased in small intestinal Th2 cells compared to mesenteric adipose Th2 cells during H. polygyrus infection (Figure 2). The importance of PPARγ is supported by recent studies linking it to ILC2 effector function in lung, adipose tissue, and colorectal cancer^{47, 48, 49, 50}. Utilization of intracellular lipid stores is mediated through organized LD-intrinsic lipolysis, or through autophagy, which is also upregulated in activated ILC2⁶⁰; deletion of Atg5, an essential autophagy gene, results in impaired cytokine production and increased apoptosis in these cells⁶⁰. This is in contrast to Th2 cells, which are able to expand in the absence of functional autophagy pathways⁶¹.

Chronic activation of mouse ILC2 is characterized by increased glucose uptake. In these cells, mTORC1 activation requires the sensing of sufficient glucose, and glucose deprivation consequently also limits chronic ILC2 activation. This may be a therapeutically tractable finding, since mice on a ketogenic diet were shown to develop less severe chronic allergic pulmonary disease associated with diminished ILC2 activation⁴⁹. Similarly, inhibition of glucose uptake in activated human ILC2 results in decreased proliferation and IL-13 production, although has no significant effect on mitochondrial function⁵³. However, enhanced glycolysis, which results from decreased proteasomal degradation of Hif1α, attenuates mitochondrial respiration, leading to reduced permissive H3K4me3 methylation at the Gata3 and Il5 loci, as well as at the Il1rl1 (IL-33R) locus, which effectively diminishes IL-33 responsiveness and ILC2 numbers in non-lymphoid tissues⁶². Furthermore, hypoxia-driven upregulation of glycolysis in human ILC2 also results in decreased GATA3 and IL-33R expression⁵³. Thus, while glucose is needed for ILC2 activation, it appears that the ratio between glycolysis and mitochondrial respiration must be tightly regulated for ILC2 tissue residence⁶².

Overall, a picture of ILC2 differentiation governed by progressive metabolic rewiring is emerging where initial activation increases glycolytic flux and mitochondrial respiration, and the balance between these two metabolic modules is crucial for subsequent maturation. Glycolysis is required for initial proliferation and pro-inflammatory cytokine production, but must be decreased for ILC2 to acquire tissue resident properties and rewire towards a metabolic state that favors longevity.

The metabolism of alternatively activated macrophages

Macrophages sense a broad range of signals within their microenvironment and in response express genes that confer distinct functional attributes cued to enforce normal physiology, but which can cause disease when inappropriately elicited⁶³. There are a range of signals to which macrophages can respond in transcriptionally distinct ways^{64, 65, 66}, but the metabolic changes, which clearly accompany stimulus-induced changes in biology, have been investigated in the context of only a subset of stimuli, including IL-4 and IL-13, which both signal through IL-4Rα and STAT6, to induce alternative activation. Alternative activation is broadly associated with efferocytosis and tissue homeostasis, the control of inflammation, resistance to helminth parasites, and wound healing^{67, 68, 69, 70}.

IL-4 induces expression of genes encoding enzymes in the TCA cycle, and involved in fatty acid metabolism, including PPARγ, which promotes FAO to support the TCA cycle, OXPHOS and mitochondrial biogenesis^{71, 72, 73, 74, 75}; these findings have been confirmed over multiple studies (reviewed in^{76, 77, 78}). An active and complete TCA cycle in IL-4 stimulated macrophages is a striking contrast to the decline in mitochondrial activity, increase in Warburg-type metabolism, and interruption and repurposing of the TCA cycle to produce the metabolite itaconate, observed in macrophages that have become proinflammatory in response to stimulation by IFNγ plus TLR agonists⁷⁹. The inflammatory metabolic state is resistant to reversal by subsequent IL-4 signalling⁸⁰, possibly due to the ability of itaconate to post-translationally modify, and in so doing to inhibit, STAT6⁸¹. In contrast though, alternatively activated macrophages can become inflammatory in response to IFNγ plus TLR agonists⁸⁰. Integrating RNAseq and metabolomics data revealed an important role for glutamine and an emphasis on UDP-GlcNAC synthesis during alternative activation⁷⁹. Mechanistically, the need for glutamine reflects a critical role for αKG, produced by glutaminolysis, in the activation of the histone demethylase Jmjd3⁸². Expression of Jmjd3 increases following stimulation with IL-4 and is required for the demethylation of genes associated with alternative activation, such as Retnla, Mrc1, and Arg1⁸². Glutamine is also required for increased FAO in response to IL-4⁸². Further, flux through the UDP-GlcNAc synthesis pathway, which can be promoted by Hedgehog signalling, which can also potentiate alternative activation⁸³, contributes to alternative activation because UDP-GlcNAc is a donor for the O-GlcNAcylation of STAT6, an activating posttranslational modification⁸⁴.Adequate TCA cycle flux is also required to produce acetyl-CoA from citrate, which is important for histone acetylation and gene expression to support alternative activation⁸⁵.

Although enhanced FAO is a feature of IL-4 stimulated macrophages, it is not required for alternative activation^{86, 87} (and discussed in^{77, 88}) because macrophages can utilize disparate substrates to support this pathway⁷⁷. It is nevertheless the case that inhibition or enhancement of OXPHOS and/or FAO often go hand in hand with the inhibition or enhancement of alternative activation. For example, increased FAO in IL-4 stimulated macrophages is accompanied by the regulated expression of numerous lipid metabolism genes including Cd36, which is important for fatty acid uptake, and Lipa, which encodes the lysosomal TAG lipase, and inhibition of these pathways limits the extent of alternative activation⁸⁹. Moreover, FAS is also increased in IL-4 stimulated macrophages, in an SREBP1-dependent fashion, and inhibition of this pathway also limits alternative activation⁹⁰. FAS may be important here because it contributes FA for FAO (in a futile cycle), and/or because it results in the depletion of NADPH, an essential cofactor in lipid synthesis, thereby limiting NADPH availability for antioxidant defences, allowing the accumulation of sufficient ROS to support the alternative activation process^{91, 92}. Despite increased FAS, IL-4 activated macrophages do not store FA as TAG in lipid droplets (LD), an indication that FA are being utilized for other purposes, presumably including FAO. Further, since IL-4 drives macrophage proliferation in vivo⁹³, which requires phospholipids for membrane synthesis, it is possible that induced FAS is needed to support this process. It is of interest that LD accumulate in IL-4-stimulated macrophages when lipase activity is pharmacologically inhibited⁸⁹, suggesting that FA are fluxing through the TAG pathway in these cells. Increased FAO in alternatively activated macrophages is linked to their ability to mitigate atherosclerosis^{94, 95}, a disease associated with lipid droplet accumulation in macrophages in the arterial intima. In this context it is intriguing that respiration is significantly inhibited by endogenous nitric oxide production in inflammatory macrophages, and that these cells have a high LD content⁹⁶. In this setting, LD serve a pro-inflammatory role as a platform for prostaglandin production⁹⁶, and prevention of LD accumulation in alternatively activated macrophages may therefore serve to guard against the activation of this pathway. It is of interest to speculate that the observed association of LD with pathogenic ILC2⁴⁹ might reflect a capacity of these cells to produce inflammatory lipid mediators.

There is great interest in the role of macrophages in tumor growth and metastasis^97, 98, and while tumor associated macrophage (TAM) populations are heterogeneous they contain cells that share transcriptional and metabolic features with alternatively activated macrophages^{99, 100, 101}. Indeed, mice in which IL-4 signaling is blocked are more capable of controlling cancer growth and metastasis than are control mice^102, 103. Recent findings have described a population of Trem2-positive “lipid associated macrophages (LAM)” in adipose tissue, that are essential for healthy tissue homeostasis and glucose metabolism¹⁰⁴. Cells with a similar transcriptional signature have been identified in multiple tumor subtypes¹⁰⁰, and these cells share transcriptional similarities, including expression of Lipa, with alternatively activated macrophages. It is possible that FA uptake in LAM promotes FAO and OXPHOS, and that this synergizes with STAT6-dependent processes to drive an alternative activation state. Interestingly, OXPHOS is required for the persistence of tissue resident macrophages in niches such as the alveoli, where they are exposed to extracellular lipids and cholesterol, which in the absence of OXPHOS induce cellular stress and apoptosis¹⁰⁵. What might be an analogous situation is seen during efferocytosis, in which macrophages engulf cell corpse-associated lipids through a process that has evolved to not be inflammatory, and consequently synergizes with IL-4 to promote alternative activation¹⁰⁶. Metabolically, this synergy may relate to the fact that both stimulation with IL-4 and exposure to the tumor microenvironment promote the PERK arm of the unfolded protein response¹⁰⁷. This has two effects. First, PERK promotes mitochondrial function and FAO. Second, downstream of PERK, the induction of ATF-4 leads to increased PSAT1 activation and serine synthesis, which is coupled to a transamination reaction that converts glutamate to αKG. As described above, αKG is critical for Jmjd3-dependent histone demethylation at alternative activation genes and genes related to FAO, and consistent with this, loss of PSAT1 function inhibits IL-4 induced FAO. Moreover, serine synthesis inhibition can increase IGF1 production, which antagonizes STAT6 signaling, and simultaneously promotes classical activation by activating p38-dependent JAK-STAT1 signalling¹⁰⁸.

Other metabolic factors can also synergize with IL-4 to potentiate alternative activation. For example, extracellular adenosine signaling through A_B2 adenosine receptor (A_2BAR)¹⁰⁹, and the TCA cycle intermediate succinate, sensed through the G protein coupled succinate receptor SUCNR1¹¹⁰. During helminth infection, adenosine generated by the CD39-mediated breakdown of ATP in the extracellular environment, presumably due to cell damage, plays a role in broadly promoting type 2 immunity by stimulating epithelial cells, via A_B2AR, to release IL-33 and IL-25. This process is critical for the subsequent development of Th2_RM cells and immunity to secondary infection¹¹¹. Moreover, through SUCNR1, succinate from intestinal microbes or intestinal helminth parasites activates Tuft cells in the intestinal epithelium to produce IL-25, which in turn activates local ILC2 (reviewed in¹¹²). Additionally, in settings of strong type 2 immune response-mediated inflammation, the successful establishment of recruited monocytes into tissue-resident macrophages, which assume an alternatively activated state, is vitamin A-dependent¹¹³. These data indicate broad links between the cellular sensing of succinate (in contrast to intracellular succinate, which can be pro-inflammatory), adenosine and vitamin A, with effective type 2 immunity.

Another interesting feature of a lipid rich-environment is that, at least within tumors, it induces the activation of PI3Kγ in macrophages. This is important because targeted inhibition of macrophage PI3Kγ has shown promise in cancer therapy¹¹⁴. Mechanistically, this reflects the role of PI3kγ in the activation of Akt and mTORC2 to induce the C/EBPβ-dependent expression of anti-inflammatory genes. Intriguingly, the induction of Arg1 by IL-4 is regulated by a response element containing STAT6 and C/EBP binding sites and C/EBPβ has been directly implicated in IL-4 induced alternative activation¹¹⁵, raising the possibility that PI3Kγ synergizes with IL-4 in the development of tumor promoting TAM. There is a further link here with TAM lipid metabolism, in that membrane cholesterol efflux through ABC transporters, and associated lipid raft depletion, potentiate IL-4-induced alternative activation through increased STAT6 signaling and the PI3K-mTORC2-Akt pathway¹⁰³; mTORC2 is critical for alternative activation^{116, 117}. The importance of cholesterol efflux for full alternative activation is supported by the fact that TAM from mice with a myeloid deficiency in ABC cholesterol efflux transporters exhibit diminished expression of IL-4-inducible genes and enrichment of tumoricidal genes, and in this way they phenocopy PI3K-deficient TAM.

The expression of Arg1 by IL-4 activated macrophages results in these cells being able to diminish extracellular arginine levels, thereby depriving adjacent cells of this amino acid, with negative effects on their biology¹¹⁸. This is associated with increased tumor growth (e.g.¹¹⁹), most probably because of T cells’ sensitivity to arginine depletion¹²⁰. Sensing arginine sufficiency is one of the permissive cues for mTORC1 activation¹²¹, a process required for the type of anabolic metabolism that typifies fast-proliferating T lymphocytes¹¹⁸. However, arginine depletion by macrophages expressing Arg1 can also serve protective functions as it can limit immunopathology¹²², and play direct roles in the killing of parasitic helminths¹²³. Arginase is important not only as an effector enzyme, but also in the production of ornithine, a precursor for polyamine synthesis and thereby eIF5A hypusination, which is critical for enhanced TCA cycle activity and OXPHO associated with alternative activation, as discussed above⁵⁸. Thus, arginase is required for both the establishment and effector functions of alternatively activated macrophages (Figure 4).

Figure 4. Alternatively activated macrophages manipulate amino acid metabolism to impact neighbouring cells.

Secretion of IL-4 and IL-13 by ILC2 and Th2 cells induces a state of alternative activation in macrophages through IL-4Rα. This enhances expression of amino acid metabolising enzymes IL4i1 and Arg1. IL4i1 converts tryptophan into indole-3-pyruvate, which is a ligand for nuclear receptor Ahr. Signalling through Ahr induces exhaustion in effector T (Teff) cells, while promoting Treg cell development. IL4i1 also converts tyrosine into 4-hydroxy-phenylpuryvate. Both indole-3-pyruvate and 4-hydroxy-phenylpuryvate act as ROS scavengers (ROS is shown outside the cells but is actually intracellular). Depletion of arginine by Arg1 inhibits Teff cell activation. Arginine sufficiency is one of the permissive signals for mTORC activation. In macrophages, arginine is converted to ornithine and further used for polyamine synthesis, which is essential for alternative activation. Created with BioRender.com

The expression of distinct amino acid metabolizing enzymes is a feature of differentially activated macrophages¹²⁴. Arginase is one of these, but so too are inducible NO synthase (iNOS), which generates NO from arginine, and IDO1 and IDO2, which degrade tryptophan¹²⁵. These three enzymes are expressed in IFNγ−exposed macrophages. In contrast, IL-4 induces the expression of IL4i1, a tryptophan, phenylalanine, and tyrosine catabolizing secreted enzyme that was first identified as being expressed in IL-4-stimulated B cells¹²⁶, and which like IDO1 and IDO2 has been implicated in the suppression of T cell proliferation¹²⁷. Kynurenic acid, and in particular derivatives of indole-3-pyruvic acid produced in tryptophan breakdown by IL4i1, drive AhR dependent cancer cell motility and adaptive immune response suppression, promoting cancer progression¹²⁸. AhR-dependent suppressive effects of IL4i1 reflect enhanced Treg cell development by AhR signaling¹²⁹ and the induction of an exhausted phenotype in effector T cells (Figure 4). The broad significance of IL4i1 expression and tryptophan catabolism in cancer is supported by the finding that Il4i1 expressing monocyte-derived TAM, also expressing IDO1 and PD-L1, are common to cancers represented in multiple single cell RNAseq datasets across different human tissues¹⁰⁰.

While Il4i1 was originally defined as an IL-4-inducible gene, IL4i1⁺ TAM express genes indicative of stimulation by IFNγ and CD40L¹⁰⁰, which is consistent with reports that expression of Il4i1 is driven more by inflammatory signals in macrophages than by IL-4¹³⁰. However, Il4i1 expression is highest in myeloid cells¹³¹ and Il4i1 expression in macrophages is induced by IL-4, and indeed there is evidence that it promotes IL-4-driven alternative activation¹³². This is of interest from the context of a second function of IL4i1-its role in ferroptosis prevention. Specifically, 4-hydroxy-phenylpyruvate from tyrosine catabolism, as well as indole-3-pyruvic acid from tryptophan, are radical scavengers which activate protective pathways that suppress ferroptosis¹³³, an oxidative cell death program characterized by iron-dependent lipid peroxidation. One of the genes induced in this context is Slc7a11, which encodes a component of the cysteine importer Xc^- (xCT). Cysteine import is critical for glutathione synthesis, and in this way the activity of GPX4, which catalyzes the reduction of lipid peroxides to mitigate against ferroptosis and cell death in IL-4 activated macrophages, although not in resting or inflammatory macrophages, suggesting that regulation of this pathway is of particular importance for alternatively activated macrophage survival¹³⁴. It is conceivable that the tumor promoting properties of IL4i1 may reflect a role in protecting both alternatively activated TAM and tumor cells from ferroptosis. It is intriguing that in uniform manifold approximation and projection plots, Il4i1-expressing TAM cluster closely to TAM expressing genes more typical of LAM and IL-4-induced alternatively activated macrophages, including Trem2, ApoE, Fabp5 and Lipa¹⁰⁰, raising the possibility that IL4i1⁺ TAM preferentially support the survival of adjacent alternatively activated macrophages in the tumor environment. This would be expected to promote resilience in the anti-inflammatory macrophage TAM compartment.

In addition to acting as a substrate for Arg1 and iNOS, and a precursor for polyamine synthesis, arginine is required for creatine synthesis. Glycine amidinotransferase (Gatm), the rate limiting enzyme in this pathway, is expressed in alternatively activated macrophages, and Gatm deletion in macrophages diminishes the IL-4 induced expression of alternative activation genes, but has no effect on inflammatory activation¹³⁵. Moreover, addition of exogeneous creatine significantly inhibits STAT1 signaling and IFNγ responsiveness, but promotes alternative activation whereas deletion or inhibition of the creatine transporter Slc6a8 diminishes the IL-4 induced expression of certain alternative activation genes¹³⁶. Phosphocreatine donates phosphate for rapid regeneration of ATP from ADP and data indicate that creatine promotes expression of STAT6 target genes through ATP-dependent SWI-SNF-mediated chromatin remodeling¹³⁶.

In summary, increased OXPHOS accompanied by FAO is a feature of alternatively activated macrophages related to their pro-homeostatic function in catabolizing lipids in lipid-rich environments and during efferocytosis. Alternatively activated macrophages are also specialized to break down several amino acids through their expression of distinct amino acid degrading enzymes. Amongst these, arginase is also expressed by ILC2^{57, 137} and Th2 cells¹⁴, suggesting that type 2 immune cells exhibit functional overlap related to arginine depletion from the microenvironment to limit the metabolic potential and therefore functions of other cells. In the context of the regulation of type 1 immunity, this may underlie the effects of alternatively activated macrophages and type 2 immunity that are related to wound healing and tumor progression. Interestingly, alternative activation is enhanced by metabolites such as adenosine in the extracellular environment, suggesting a mechanism by which the appearance of damage-associated signals in the environment reinforce the development of macrophages associated with the regulation of inflammation and tissue repair.

Metabolic regulation of IgE production

Antibody production by plasma cells is an important part of adaptive type 2 responses and IgE production in particular contributes to protection from certain parasites, venoms and toxins^{138, 139, 140}. In healthy individuals, IgE is the least abundant antibody class in the circulation, with most of IgE being bound to high affinity receptor FcεRI on the surface of mucosa or skin residing mast cells. Class switching to IgE requires IL-4¹⁴¹ and high serum IgE levels are associated with many allergic disorders. Cross-linking of FcεRI, mediated by antigen binding, leads to mast cell degranulation and release of inflammatory mediators. In sensitized individuals these inflammatory signals trigger hypersensitivity reactions, including anaphylaxis¹³⁹. The role of IgE in many type 2 disorders makes IgE an attractive therapeutic target; anti-IgE antibodies are currently used for the treatment of asthma and chronic spontaneous urticaria. However, they require prolonged dosing to reach a desired reduction in circulating IgE levels¹⁴². A better understanding of the metabolic control of IgE⁺ humoral responses could contribute to the development of novel and more effective therapies.

In order to become antibody secreting plasma cells (PC), B cells transit through several stages of differentiation, each marked by rapid metabolic change. While short lived PC (SLPC) produce low affinity antibodies and are generated early in the humoral response through the extrafollicular pathway, long-lived plasma cells (LLPC) are mainly high affinity and are generated in a T cell dependent manner. Substantial progress has been made in understanding metabolic regulation of humoral responses^{143, 144}. Antigen-driven exit of B cells from a resting state results in a rapid increase in cell mass and proliferation. These changes are supported by anabolic reprogramming mediated by PKCβ and mTORC1 activity, and include increased glucose uptake, paralleled by mitochondrial remodeling and a progressive increase in mitochondrial respiration^{145, 146, 147}. Activated B cells responding to protein antigens subsequently form germinal canters (GC), where they undergo cycles of proliferation, somatic hypermutation, and affinity selection. Preferential use of OXPHOS is paramount for GC B cells^{148, 149, 150}, a metabolic requirement that can be compromised in bacterial infections, leading to decreased antibody production¹⁵¹. IL-4 signaling is important for B cell reprograming towards OXPHOS through epigenetic regulation, which in turn contributes to increased expression of Bcl6 through epigenetic remodeling of the Bcl6 locus¹⁵⁰. BCL6 is a central transcription factor for GC B cells, and its expression has previously been linked to glycolysis repression¹⁵². Indeed, a distinctive feature of GC B cell energetics is the reliance on mitochondrial and peroxisomal FAO and a minimal requirement for glucose¹⁴⁹. Overall, these data indicate that as GC B cells differentiate, the fuel choice is switched from glucose to FA. This raises an interesting possibility that GC B cells and Tfh cells utilize distinct metabolic pathways as a strategy to avoid nutrient competition, since Tfh cells use glucose and glutamine to fuel mitochondrial respiration¹⁵³. Such distinctive metabolic adaptations could facilitate targeted therapeutic manipulation of either GC B cells or Tfh cells in antibody mediated diseases. In this context, it is of interest that a subset of Tfh cells that selectively promotes anaphylactic IgE responses has recently been identified¹⁵⁴. Whether these cells are metabolically distinct from other Tfh cell subsets remains to be investigated, but it seems plausible, since metabolic differences have been shown between autoreactive and infection-driven Tfh cells¹⁵⁵.

B cells that successfully emerge from the GC reaction provide long lasting humoral immunity by becoming either memory B cells or LLPC. Continuous antibody production and remarkable longevity in this population are supported by a specific set of metabolic adaptations, including increased mitochondrial SRC, amino acid uptake, and activation of autophagy pathways¹⁴³. Moreover, in contrast to SLPC, LLPC have an increased requirement for glucose, which is at least in part mediated by CD28 signalling¹⁵⁶, reminiscent of the CD28 co-stimulation requirement in T cells for the upregulation of glycolysis and mitochondrial SRC¹⁵⁷. Interestingly, the majority of glucose acquired by LLPC is not used to fuel energetic demands, which are met by FAO and glutaminolysis¹⁵⁸, but is instead diverted into the hexosamine pathway to produce UDP-GlcNAc, a sugar donor required for antibody glycosylation^{159, 160}. N-glycosylation of the heavy chain is essential for antibody folding and, depending on the glycosylation pattern, modulates its interactions with Fc receptors, diversifying antibody functionality¹⁶¹. Alterations in antibody glycosylation have been observed in infections and autoimmune diseases. Recent characterization of IgE glycosylation revealed a disease-specific pattern in individuals with peanut allergy, marked by increased sialylation¹⁶². It would be of interest to investigate whether metabolic alterations in the hexosamine pathway in LLPC underlie allergy-associated modifications in glycosylation.

The majority of LLPC reside in the specialized bone marrow (BM) niches, where they continuously receive pro-survival signals from supporting cells, while SLPC localize to secondary lymphoid organs (SLO). With regard to IgE serological memory, it has been unclear whether IgE⁺ LLPC exist and if so, where they reside. The potentially life-threatening consequences of systemic IgE reactions necessitate tight control that limits longevity and anatomic localization of these responses. Previous research showed that IgE⁺ PC that are generated in physiological circumstances, such as in helminth infections, localize to SLO and are short lived¹⁶³. The limited lifespan of IgE⁺ PC was linked to mitochondrial apoptosis that is driven by persistent calcium signaling downstream of the BCR¹⁶⁴, suggesting that mitochondria remodeling that leads to reduced calcium buffering might control IgE⁺ PC longevity. These regulatory mechanisms are altered in individuals with chronic allergies, where clinical findings indicate the existence of long-lived IgE responses¹⁶⁵. In line with this, recent studies in allergic individuals and in mice chronically exposed to antigens revealed that PC producing high affinity IgE are found outside of the SLO, including in the BM and gastrointestinal tissues^{166, 167}. Understanding whether metabolic adaptations support the persistence of IgE⁺ PC in the BM and mucosal niches in allergic individuals could be of clinical relevance.

Sensing of the outside world – type 2 immunity in the intestine

As mentioned above, type 2 immunity plays an important role at barrier surfaces. This raises interesting questions regarding the intestine, where the need for continuous immune surveillance and reinforcement of barrier function must be balanced with the maintenance of adequate nutrient absorption. In homeostasis, the intestinal mucosa is dominated by type 1 and type 3 immune responses with critical contributions from Treg and myeloid cells that ensure tolerance towards food and commensal antigens. The homeostatic development and functions of mucosal immune cells are promoted by dietary- and microbiota-derived metabolites¹⁶⁸. In contrast, expansion of intestinal type 2 immune cells is often associated with metabolic perturbations, such as those caused by helminth infections or malnutrition^{169, 170, 171}.

It has been postulated that type 2 responses are engaged in food quality monitoring, protecting the host from ingesting noxious substances, for instance by changing absorption or promoting expulsion¹⁷². From this perspective, dysregulated protective mechanisms are proposed to underlie the development of food allergies and abdominal pain^{172, 173}. Part of such control might be to sense malnutrition, which is evolutionarily linked with persistent presence of intestinal helminths, prototypic examples of type 2 immune response inducers¹⁷⁴. Indeed, helminth parasite infections are associated with decreased blood glucose levels, decreased essential amino acids, including BCAA, in blood and tissues, and increased circulating lipids along with alterations in ketone body metabolism^{170, 175, 176}. Longitudinal metabolomic studies indicate that the chronic stage of helminth infection is characterized by the establishment of a new metabolic homeostatic setpoint, in which circulating metabolites (with the exception of lipids) are brought to pre-infection levels, but the metabolic state of several tissues remains altered¹⁷⁶.

SCFA (acetate, butyrate and propionate) are produced by anaerobic fermentation of complex carbohydrates by intestinal microbiota. Alterations in microbiota-derived metabolites, including SCFA and bile acid derivatives accompany helminth infections^{175, 177}. Moreover, acetate is produced by parasites such as Tritrichomonas and H. polygyrus and can promote parasite invasion through disruption of the epithelial barrier^{178, 179}. SCFA bind to GPR41, GPR109a, and GPR43¹⁶⁸, the latter of which is highly expressed by SI Th2 cells, but not mesenteric adipose tissue Th2 cells in H. polygyrus infected mice (Figure 2). SCFA signaling enhances human Th2 effector functions; GPR41 and IL-5 expression are strongly correlated in Th2 cells isolated from tissue biopsies of patients with eosinophilic esophagitis, and in vitro stimulation with butyrate enhances Th2 cytokine production⁴¹. Thus, alterations in SCFA levels could act as a danger signal that boosts intestinal type 2 responses during parasitic infection.

Bile acids are produced from cholesterol, where they are conjugated to taurine or glycine to increases their solubility¹⁸⁰. In addition to their role in the absorption of dietary lipids and fat-soluble vitamins, bile acids and secondary bile acids (produced when microbiota metabolize bile acids in the intestinal lumen) act as ligands for several GPR and nuclear receptors, including FXR, PXR, LXR, Rorγt, VDR, and TGR5¹⁸⁰. Bile acids regulate metabolic pathways in many tissues, and have emerged as modulators of innate and adaptive immune cells¹⁸¹. For example, microbiota-derived secondary bile acids directly regulate Th17 and Treg cell differentiation downstream of Rorγt or VDR¹⁸¹. Intestinal Th2 cells express VDR and LXR during H. polygyrus infection¹⁸, but whether they are also subjected to similar regulation remains to be tested.

Imbalance in secondary bile acid metabolism might be sensed as a danger signal that induces inflammation^{182, 183}. For example, a diet rich in inulin alters the intestinal microbiota with a resultant increase in the levels of circulating unconjugated bile acids. This in turn activates intestinal stromal cells, in an FXR-dependent manner, to secrete IL-33 that activates ILC2 and promotes eosinophilia, responses that are able to mediate resistance to an intestinal helminth¹⁸³. Taken together, changes in the abundance or composition of bile acid metabolites might act as a sensitive readout of metabolic perturbations that are associated with intestinal infection or malnutrition. Such cues could be interpreted by type 2 immune cells to mount the appropriate response. Alterations in the levels of micronutrients may synergize in such pathways. For example, ratios of conjugated to unconjugated bile acids are altered during acute vitamin A deficiency, which may contribute to observed microbiota shifts in this model¹⁸⁴. Consistent with this idea, deficiency in retinoic acid, a derivative of vitamin A, has been previously linked to enhanced IL-13⁺ ILC2 response in the intestine^{59, 169}.

Eosinophils are a prominent component of type 2 immunity as a result of their dependence on IL-5, and play roles in tissue homeostasis and protection against helminth parasites, but also in a variety of immunopathologic conditions associated with type 2 immunity¹⁸⁵. Eosinophils are numerous in the GI tract and in the steady state contribute to an anti-inflammatory environment by inhibiting Th17 and Th1 responses^{186, 187}, and moreover contribute to the development of the architecture of the small intestine in a microbiota- and IL-33-dependent manner¹⁸⁸. Mice lacking eosinophils have altered extracellular matrix (ECM) turnover and maladaptive changes characterised by villous atrophy, altered muscle contractility, and decreased intestinal epithelial cell (IEC) migration along the basement membrane, which together results in impaired lipid absorption from the lumen, underscoring the homeostatic role of type 2 immunity and resident eosinophils in interpreting microbial signals to adjust intestinal absorptive functions¹⁸⁸. Signaling through AhR is needed to acquire the tissue resident phenotype by intestinal eosinophils¹⁸⁹. Within the GI tract, AhR in thought to be activated by diet or microbiota-derived tryptophan metabolites. A role of AhR signalling in promoting mucosal IEC, ILC3, and Th17 responses is well documented¹⁹⁰. In eosinophils, AhR activity promotes transcriptional reprograming needed to adapt to tissue residency, including induction of genes that regulate ECM interactions and remodeling, which affected eosinophil adhesion¹⁸⁹. In contrast, AhR negatively regulates intestinal ILC2¹⁹¹. AhR facilitates its own gene chromatin accessibility and transcription, in cooperation with the transcription factor Gfi1. This presumably sequesters Gfi1, which is a positive regulator of ILC2 development through its binding to Il1rl1 locus. AhR-deficient ILC2 have increased IL-33 expression and effector functions, including being able to mediate better protection against H. polygyrus infection¹⁹¹. It remains unclear which environmental signals regulate this self-enhancing mechanism of AhR expression, as germ free mice or mice on a diet deprived of AhR ligands had comparable chromatin remodeling around the Ahr locus¹⁹¹. One possibility would be that the activity of IL4i1 secreted by alternatively activated macrophages provides a local source of such ligands, as discussed above. Whether Ahr regulates intestinal Th2 cells is not known, but it is interesting that AhR expression in mouse SI Th2 cells (Figure 2), and in human Th2 cells from nasal polyps or endoscopic biopsies correlates with the expression of effector cytokines and the SCFA receptors^{40, 41}. Together, environmental sensing through AhR could affect type 2 immune cells in the intestine, favoring homeostatic functions, while inhibiting excessive inflammatory type 2 responses.

Intestinal type 2 immune cells participate in the monitoring of metabolic homeostasis and appear well equipped to respond to alterations related to malnutrition and parasitic infections. Their intracellular metabolic adaptability might enable them to mount a prolonged protective response in the presence of metabolic stress. As dietary interventions are being proposed for the treatment of chronic inflammatory disorders, including asthma¹⁹², a better understanding of the relationships between nutrients, microbiota, and mucosal type 2 immune responses could inform such therapeutical strategies.

Type 2 immune cells in adipose tissue and systemic metabolism

Adipose tissue plays a central role in regulating systemic metabolism. Composition and function of this highly plastic tissue differs depending on the type and anatomical location. The primary task of white adipose tissue (WAT) is energy storage, insulation and organ protection, with the main depots being visceral (VAT) and subcutaneous adipose tissues (scWAT). Brown adipose tissue (BAT), which is less abundant and developmentally distinct from WAT, is able to generate heat upon cold exposure through adaptive thermogenesis. Beige adipose tissue is developmentally related to WAT, but shares characteristics of thermogenic BAT, including high mitochondrial content and expression of uncoupling protein 1 (UCP-1), which permits dissipation of the mitochondrial proton gradient in a manner that generates heat¹⁹³. Beyond its well-recognized roles in lipid storage and body temperature regulation, adipose tissue participates in immune surveillance, host defense, and tissue regeneration. In addition to adipocytes, it hosts heterogeneous populations of immune cells, stromal cells in various stages of commitment towards mature adipocytes, as well as neurons, and endothelial cells. Cross-talk between these cell types impacts tissue homeostasis and function¹⁹⁴.

Metabolically healthy WAT responds to dynamically fluctuating levels of nutrients and contributes to circulating glucose homeostasis. Elevated levels of glucose and FA activate insulin production in the pancreas, to which adipocytes respond by increasing glucose uptake, TAG synthesis, and storage. However, chronic overnutrition leads to adipose tissue inflammation and expansion, and inflammatory signals, such as CCL2 and TNF, further recruit and polarize immune cells, e.g. macrophages, towards inflammatory phenotypes¹⁹⁵. The paradigm in which inflammatory macrophages promote obesity, while alternatively activated macrophages maintain healthy adipose tissue through phagocytosis of dead adipocytes and anti-inflammatory cytokine secretion, has provided a framework for conceptualizing how the balance between type 1 and type 2 inflammation contributes to adipose tissue physiology. However, our understanding of the versatile roles of macrophages in adipose tissues has expanded greatly in recent years, providing a more complex picture. Several macrophage populations, positioned in distinct anatomical niches, have been identified^{194, 196}. Understanding their roles in metabolic homeostasis is an important task for ongoing and future research.

Initial inflammatory responses associated with expanding adipose tissue are considered beneficial in that they can help the tissue to cope with prolonged nutritional stress by, for instance, enhancing angiogenesis to ameliorate hypoxia in the growing tissue¹⁹⁷. Gradually however, chronic inflammation contributes to resistance to anabolic signals provided by insulin, as well as catabolic signals provided by catecholamines and leptin, leaving adipocytes metabolically inflexible. In this state, both energy storage and expenditure are dysregulated. Ultimately, such disfunction contributes to the development of metabolic disorders including type 2 diabetes mellitus, non-alcoholic fatty liver disease (NAFLD) and cardiovascular disease¹⁹⁵.

Healthy WAT is enriched in type 2 immune cells, which participate in sensing and tuning nutritional and thermoregulatory signals. Mice deficient in components of type 2 immunity, including IL-13^{198, 199} and IL-33^{200, 201} have increased body weight compared to control animals. Catabolic processes that promote energy expenditure within adipose tissue are under control of the sympathetic nervous system, with the main neurotransmitters being catecholamines, including epinephrine and norepinephrine. These signal via β-adrenergic receptors to promote lipid mobilization and thermogenesis^{193, 202}. Research over the past decade has established a link between adipose tissue type 2 immune cells and sympathetic signals, and a major role for ILC2, which coordinate eosinophil and macrophage responses^{203, 204}. IL-5 expression by ILC2 maintains resident eosinophils, which are the main source of IL-4 in the adipose tissue. In turn, IL-4, together with ILC2-derived IL-13, skews adipose macrophages towards an alternatively activated phenotype, improving insulin sensitivity and resistance to HFD-induced obesity^{205, 206}. The eosinophil – macrophage axis is also thought to underlie beneficial effects on glucose homeostasis that are observed when type 2 inflammation is induced by helminth infections²⁰⁷. A consequence of helminth infection is also an establishment of an adipose Th2_RM population, which becomes a major source of type 2 cytokines and maintains eosinophils in the adipose tissue¹⁴. However, whether these cells take over homeostatic roles of ILC2 in promoting long-term metabolic homeostasis remains to be tested. Decreased frequencies of WAT eosinophils are observed in aged mice and humans, which may contribute to deteriorating metabolic health associated with aging²⁰⁸. Transfer of eosinophils from young animals decreases systemic low-grade inflammation and adipose hypertrophy observed in aged mice, partially in an IL-4 dependent manner²⁰⁸.

Signals derived from adipose stromal cells are essential in providing the niche for type 2 immune cells. Eosinophil recruitment to adipose tissue is influenced by the chemokine CCL11, which is secreted by stromal cells in response to IL-4 and IL-13²⁰⁹, or by mature adipocytes following sympathetic stimulation²¹⁰. Adipose tissue resident ILC2 also respond to stroma-derived signals, particularly IL-33^{206, 211}. A DPP4⁺ multipotent progenitor cell stromal population, analogous to universal fibroblast progenitors²¹², is the main source of IL-33 in adipose tissue^{14, 201, 209, 213, 214,} 215. These DPP4⁺ cells comprise the stem compartment for renewal of adipocytes within adipose tissues²¹⁶. In adipose, but also in other organs, these cells are enriched in interstitial niches, where they are in close proximity to blood vessels and lymphatics, positioning that may facilitate sensing of metabolic cues, and interactions with vasculature-associated macrophages, ILC2, and Th2 cells^{14, 217, 218, 219, 220}. Signals that promote IL-33 production in adipose tissue are not well understood, but TNF, IL-17A, as well as sympathetic stimulation, have been implicated^{214, 221}. Mesothelial cells also express IL-33 in adipose tissue, providing an additional source of this cytokine during inflammatory stress²⁰¹ or in aged animals²²². The switch in IL-33 source in aged adipose tissue is associated with a senescence-like phenotype of ILC2, indicating that the cellular context of IL-33 production might impact ILC2 functionality and contribute to age-related metabolic dysfunction²²². Glial-derived neurotrophic factor (GDNF) is another stromal-derived factor that maintains ILC2 in VAT²²³. Hypothalamus-derived signals via sympathetic neurons induce GDNF production in stromal cells, and this in turn supports ILC2 numbers and cytokine production through the tyrosine kinase receptor RET. Mice harbouring RET-deficient ILC2 have increased body weights and impaired glucose tolerance compared with control animals²²³. Stromal cells can also support ILC2 in a contact-dependent manner, where binding of LFA-1 expressed by ILC2 with ICAM-1 expressed by stromal cells induces ILC2 proliferation and IL-5 production²⁰⁹.

Type 2 inflammation expands DPP4⁺ multipotent progenitor cells in the VAT¹⁴, while signalling through IL-4Rα increases proliferation of adipocyte progenitors (broadly defined as PDGFRα+ Sca1⁺ stromal cells)²²⁴. On the other hand, adipose eosinophils, macrophages, and Th2 cells can produce TGFβ₁14, 225,226 - a cytokine that is essential for the maintenance of multipotent progenitor cells, as it inhibits their differentiation towards committed adipocytes²¹⁶. Therefore, the balance between different type 2 cytokines might regulate the pool of highly plastic progenitor cells, priming the tissue for rapid adaptation to changing conditions, i.e. by allowing for hypertrophic tissue expansion in response to calorie excess, and promoting beige adipocyte development when increased energy expenditure is required.

Early studies indicated that mice deficient in eosinophils or IL-5 show decreased oxygen consumption and heat production, indicating that these cells play a role in enhancing oxidative metabolism and energy expenditure²⁰⁶. Increased energy expenditure in the adipose tissue is promoted by the activity of brown and beige adipocytes. Signals derived from type 2 immune cells aid in this metabolic regulation mainly by facilitating beiging of WAT in the subcutaneous compartments^{200, 224, 227}, which is also known to be more prone to beiging compared to visceral WAT²²⁸. In particular, signaling through IL-4Rα on stromal cells, but not on mature adipocytes, promotes beiging of scWAT, which contributes to increased energy expenditure in a UCP-1 dependent manner²²⁴. Both ILC2- and eosinophil-derived IL-4 and IL-13 promote beiging of scWAT²²⁴. Eosinophils might contribute to beiging through factors beyond IL-4, as eosinophils deficient in the transcriptional repressor KLF3 express more of the beige fat-inducing factor meteorin-like^{229, 230} (Metrn), and mice with KLF3-deficiency have enhanced beiging in scWAT²³⁰. Several studies have implicated alternatively activated macrophages in beiging^{227, 229, 231, 232, 233}, however precisely how these cells perform this function is not well understood^{234, 235}. Additionally, ILC2 induce beiging at the later stages of adipocyte differentiation through the production of methionine-enkephalin (Met-Enk) from the opioid peptide proenkephalin A²⁰⁰.

The physiological importance of type 2 immune cells in the activation of thermogenesis within BAT of adult animals has not been convincingly demonstrated²³⁵. This suggests that the main impact of type 2 signals is on WAT beiging, which might serve functions beyond classical heat generation. Beige adipocytes, with high mitochondrial content compared to white adipocytes, are more efficient in utilizing glucose and lipids, likely contributing to improved glucose homeostasis that is associated with BAT activity²³⁶. Additionally, brown and beige adipocytes have endocrine functions distinct from WAT. Indeed, in recent years several brown adipokines (batokines), which regulate immune cell recruitment, as well as liver, pancreas, and muscle metabolism, have been identified²³⁷. For example, neuregulin 4 (Nrg4) expression is increased during cold acclimation in BAT and scWAT and acts on liver to downregulate lipogenesis. Nrg4-deficient mice have increased insulin resistance and develop hepatic steatosis when fed HFD²³⁸. Thus, through such mechanisms, beiging induced by type 2 signals might contribute to metabolic regulation in the homeostatic state, but the same pathways could also be utilized during prolonged type 2 inflammation, when perhaps a new energy homeostasis setpoint needs to be established.

While WAT is a central metabolic hub, other organs including pancreas, liver, and muscle participate in the maintenance of whole-body metabolic homeostasis. Mediators of type 2 immunity are emerging as important metabolic regulators also in these tissues^{3, 4, 239, 240}, indicating that much remains to be discovered about the roles that type 2 immune cells play in the regulation of metabolic health.

Concluding Remarks

Here we have discussed how type 2 immune cells adopt specific metabolic pathways to support their functional needs. Decisions about cellular differentiation and activation are significantly impacted by signals from cytokines along with intersecting signals from changes in nutrient and metabolite levels in the environment, which in turn drive emphasis on particular metabolic pathways that support or limit cellular function. This remains a rich and diverse area for investigation.

While advances have been made in understanding the roles of metabolic pathways in the biology of type 2 immune cells in an in vitro context, it is clear that these conditions do not fully capture the conditions in vivo, where cells are undoubtedly exposed to more complex stimuli, with consequences for their metabolic profiles. Moreover, scRNAseq has revealed the heterogeneity of activation states in vivo, and while transcriptional signatures can be informative about the metabolic status of individual cells, single cell metabolomics, ideally with matched scRNAseq, will be required for high-definition characterization. These approaches will hopefully become available in the near future. This would not only open the door to understanding metabolic reprogramming at the single cell level, but also to the metabolic analysis of cells that are integral to type 2 immunity, but which currently remain largely uncharacterized metabolically, such as eosinophils, cDC2, basophils and mast cells.

There are certain metabolic features that have been found to be common to multiple Type 2 immune cells. For example, tissue ILC2, Th2 cells and alternatively activated macrophages share increased expression of genes involved in fatty acid metabolism downstream of PPARγ activation. PPARγ binds medium to long-chain fatty acids and certain eicosanoids, and it is of particular interest in this light that type 2 immunity is so heavily implicated in adipose tissue homeostasis.

As a final point, it is noteworthy that rapid progress has been made in biologic therapies for allergic diseases and asthma, with multiple antibody therapies that target critical components of type 2 immunity, such as IL-4, IL-5, IL-33, and TSLP either already on the market or in trials²⁴¹. Many of these drugs have remarkably beneficial effects. An inevitable outcome of their extended use will be clarification of whether conclusions drawn from the study of specific aspects of type 2 immunity, often in mice, hold true in human biology over the long term. For example, how important are IL-4, IL-5 or IL-33 for adipose tissue homeostasis and consequently is weight gain or insulin sensitivity a concern in people treated with these biologics? Likewise, does IL-4 signaling inhibition, with the attendant loss of expression of genes such as Arg1 in alternatively activated TAMs, confer advantage in cancer? Insights from the use of these drugs thus promise to be incredibly informative and valuable.

Key elements of Type 2 immune responses are integrated within tissues and are part of tissue homeostasis and repair. Pearce and colleagues review how metabolic regulation relates to type 2 immunity, discussing both specifics of metabolism and metabolic adaptation within type 2 immune cells and how type 2 immune cells are integrated more broadly into the metabolism of the organism as a whole.