Metabolic control of innate lymphoid cells in health and disease

Abstract

Innate lymphoid cells (ILCs) are a family of predominantly tissue-resident lymphocytes that critically orchestrate immunity, inflammation, tolerance, and repair at barrier surfaces of the mammalian body. Heterogeneity among ILC subsets is comparable to adaptive CD4⁺ T helper cell counterparts, and emerging studies demonstrate that ILC biology is also dictated by cellular metabolism that adapts bioenergetic requirements during activation, proliferation, or cytokine production. Accumulating evidence in mouse models and patient-derived samples indicate that ILCs exhibit profound roles in shaping states of metabolic health and disease. Here we summarize and discuss our current knowledge of the cell-intrinsic and cell-extrinsic metabolic factors controlling ILC responses, as well as highlight contributions of ILCs to organismal metabolism. It is expected that continued research in this area will advance our understanding of how to manipulate ILCs or their metabolism for therapeutic strategies that benefit human health.

Editor summary:

Innate lymphoid cells (ILCs) are an integral part of the innate immune system. This review discusses how ILC function is regulated by both intrinsic and extrinsic metabolic pathways, and how ILCs contribute to metabolic disease.

Introduction

Innate lymphoid cells (ILCs) are a heterogeneous population of immune cells that have lymphoid morphology but do not express rearranged antigen receptors and are predominantly enriched at barrier surfaces^1–3. ILCs function primarily through the production of cytokines to orchestrate immune responses, promote barrier integrity, and maintain tissue homeostasis, but if dysregulated, have the potential to drive chronic inflammation^1–9. This family of innate lymphocytes are further classified into three groups based on their expression of lineage-specifying transcription factors, including T-bet⁺ group 1 ILCs (ILC1s), GATA3⁺ group 2 ILCs (ILC2s), and RAR-related orphan receptor-γt⁺ (RORγt⁺) group 3 ILCs (ILC3s), which exhibits striking similarities to the heterogeneity observed in CD4⁺ T helper cells^1–3. ILC1s comprise cytotoxic NK cells and non-cytotoxic ILC1s. Following stimulation with proinflammatory cytokines interleukin (IL)-12 and IL-18, NK cells produce perforin, granzyme and IFN-γ to protect from intracellular pathogens and tumors while ILC1s promote type 1 immune response against intracellular pathogens via the production of IFN-γ and TNF. ILC2s are capable of producing IL-4, IL-5, IL-9, IL-13 and amphiregulin in response to IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) stimulation, which promote type 2 immune response in the context of anti-helminth immunity, allergic inflammation, and tissue repair. ILC3s exhibit more diversity than ILC1s and ILC2s, including fetal lymphoid-tissue inducer (LTi) cells, CCR6⁺ LTi-like ILC3s, T-bet⁺ ILC3s and CCR6⁻ T-bet⁻ ILC3s in adult mice. LTi cells are essential for the formation of secondary lymphoid tissues during development through production of lymphotoxin. Upon engagement of microbiota-derived IL-1β, IL-23 or aryl hydrocarbon receptor (AhR) ligands, ILC3s critically mediate type-17 immune response against extracellular pathogen infection, strengthen intestinal barrier integrity, maintain intestinal regulatory T cell homeostasis, and promote tissue repair through production of IL-17, IL-22, GM-CSF, IL-2 and HB-EGF^1–7. Among these, IL-22, which predominantly produced by ILC3s, acts almost exclusively on non-hematopoietic cells at barrier surfaces of the mammalian body, such as the gut, airway, and skin. In these locations, IL-22 is an essential regulator of epithelial homeostasis and function, including regulation of epithelial cell proliferation, regeneration, permeability, as well as production of mucus and antimicrobial proteins^10,11. IL-22 also acts on non-hematopoietic cells at metabolically active sites, such as adipose tissue and liver.

As an integral component of barrier immunity, ILCs are predominantly tissue-resident and rapidly respond to perturbations through activation, proliferation, and cytokine production, thus providing signals that coordinate homeostasis and repair during infection or injury^1–7. Further, subsets of “inflammatory” ILC2s and ILC3s can be uniquely derived from the circulation and mediate airway inflammation or neuroinflammation, respectively^12,13. Numerous studies have described the metabolic requirements of tissue resident and circulatory CD4⁺ T cells at various stages of activation^14–16, but how its innate counterpart, ILCs, are controlled by specific metabolic programs remains elusive and in early stages of investigation. This review summarizes recent findings in ILC immunometabolism focusing on the cell-intrinsic metabolic pathways that are utilized by ILCs during homeostasis or activation, the cell-extrinsic environmental metabolic factors that impact ILC metabolism or functional potential, and the contribution of ILCs to metabolic health or associated diseases.

Cell-intrinsic metabolic control of ILC responses

It has been well documented that T cells adapt their cellular metabolism to fulfil bioenergetic requirements during activation, differentiation, and cytokine production. Key metabolic processes such as glycolysis, fatty acid and mitochondrial metabolism, as well as metabolic signal hubs such as mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are recognized as crucial regulators in T cells during various stages. For example, naïve CD4⁺ T cells are generally thought to be quiescent and depend on mitochondrial oxidative phosphorylation (OXPHOS) to generate adenosine triphosphate (ATP), nevertheless, upon activation, these cell quickly reprogram cellular metabolic pathways from OXPHOS toward aerobic glycolysis to meet higher biosynthetic demand^14–16. As an innate counterpart to CD4⁺ T cells, ILCs share similar signaling pathways, transcriptional programs and effector cytokine profiles, raising the possibility that the key metabolic pathways utilized by CD4⁺ T cells can be applied to ILCs. Nevertheless, in contrast to T cells, the metabolic pathways that ILCs utilize during steady state and activation remain at early stages of investigation.

Group 1 ILC-intrinsic metabolism

Numerous studies have highlighted the importance of metabolic regulation of NK cell development, activation, and effector function, which has been recently reviewed elsewhere^17,18. In contrast to the extensive body of literature regarding NK cell-intrinsic metabolism, little is known about the cell-intrinsic metabolic pathways during steady state or activation stage of non-cytolytic ILC1s. Recent RNA-sequencing analyses of intestinal ILC1s from naïve mice revealed enrichment of transcripts associated with mTOR signaling¹⁹, suggesting an important role of glycolysis in their function, yet the functional significance of this observation requires further investigation. As non-cytotoxic ILC1s exhibit similar transcriptome and effector functions to NK cells, and transcriptional analyses indicate an activation of mTOR in ILC1s, it is possible to speculate that the metabolic profile of ILC1s resemble that of NK cells (Fig. 1a). This area of research requires additional extensive investigation.

Figure 1. Cell-intrinsic metabolic pathways employed by the ILC family.

a. IL-12, IL-18 or IL-15 signals induce ILC1 activation and function potentially through mTOR-dependent glycolysis. b. In response to IL-25 or IL-33 stimulation, activated ILC2s employ Arg1 to metabolize extracellular L-arginine into polyamines to fuel increased glycolysis. In addition, Atg5-mediated autophagy or PD-1 signaling negatively regulate glycolysis in ILC2s. ILC2s can also uptake extracellular fatty acids, and either utilize mitochondrial FAO to fuel proliferation and effector function during helminth infection, or transiently form lipid droplets (LD) and convert into phospholipids to promote cell proliferation and fuel pathogenic response in the context of allergen-induced chronic airway inflammation. c. In response to IL-1β and IL-23, ILC3s increase glucose metabolism and display increased mitochondrial oxygen consumption through mTORC1-HIF1α axis. ILC3s also require intact PD-1 signaling to adapt to increased glycolysis and inhibit excessive FAO during activation and effector cytokine production.

Group 2 ILC-intrinsic metabolism

ILC2s utilize both glycolysis and fatty acid oxidation (FAO) to fuel effector responses in various contexts (Fig. 1b). Both ILC2 precursors and mature ILC2s highly express arginase-1 (Arg1)²⁰, an amino acid catabolic enzyme that metabolizes the L-arginine into urea and ornithine to generate downstream metabolites that fuel bioenergetic pathways. Genetic deletion of Arg1 in lymphocyte-lineage cells impairs ILC2 proliferation and cytokine production, thereby protecting mice from allergen-induced airway inflammation²⁰. Mechanistically, inhibition of Arg1 results in ILC2-intrinsic defects of polyamine synthesis and aerobic glycolysis, suggesting a role for glycolysis in the pathogenicity of ILC2s in the lung²⁰. Interestingly, another study found that deletion of Arg1 in mature ILC2s did not impact ILC2 proliferation or cytokine production during helminth infection²¹, suggesting that Arg1 regulation of ILC2s might be tissue or context dependent. Emerging evidence suggested that fatty acid associated metabolic pathways are also employed by ILC2s. Intestinal ILC2s can uptake abundant exogenous fatty acids from the environment during homeostasis²². Systemic inhibition of FAO, but not glycolysis, impairs ILC2 accumulation, IL-5 and IL-13 production, and anti-helminth response²². In addition, a recent study demonstrated that ILC2s increase uptake of both external fatty acid and glucose to induce transient lipid droplets formation, which is required to promote ILC2 proliferation and fuel pathogenic ILC2 response in the context of allergen-driven airway inflammation²³. The pathogenic function of ILC2s in the lung requires the coordination of glucose and fatty acid metabolism, and a glucose restricted ketogenic diet might be an intervention strategy to treat airway inflammation²³. In contrast to murine ILC2s, human ILC2s utilize dichotomous metabolic pathways to support survival, proliferation, and effector function²⁴. At steady state, peripheral circulating ILC2s uptake abundant branched chain amino acids and arginine to fuel high level of OXPHOS, following activation, ILC2s do not undergo a metabolic switch from OXPHOS to glycolysis but rather maintain a dichotomous metabolism with persistent OXPHOS and concomitant increase in glucose uptake and glycolysis, to support their fitness and cytokine production, respectively²⁴.

The regulation of ILC2-intrinsic metabolism is nuanced. Autophagy has been shown to promote the homeostasis and effector functions of activated ILC2s through metabolic reprogramming. Deletion of Atg5 results in increased cell death and impaired cytokine production in ILC2s, which is mediated by shifting fuel dependency from FAO to glycolysis, thereby limiting ILC2-mediated airway hyperreactivity²⁵. In addition, programmed cell death protein-1 (PD-1) has been suggested as an important metabolic checkpoint in ILC2s. Pulmonary ILC2s upregulate PD-1 expression during activation, which is critical to regulate their survival, proliferation and cytokine production. Further, deletion of PD-1 exacerbates ILC2-mediated airway inflammation²⁶. Mechanistically, PD-1 deficiency shifts ILC2 metabolism toward glycolysis, glutaminolysis and methionine catabolism²⁶. Additionally, a recent study reported that transcription factor STAT3 controls ILC2 effector function and promotes ILC2-driven airway inflammation by regulating ILC2-intrinsic mitochondrial respiratory capacity and methionine metabolism²⁷. In response to IL-33, STAT3 undergoes mitochondrial translocation, facilitating ATP synthesis to fuel the methionine cycle and generation of S-adenosylmethionine, which further supports ILC2 effector cytokine production through epigenetic reprogramming²⁷. ILC2s are also uniquely primed to import amino acids via the transporters Slc7a5 and Scl7a8, and deletion of those transports impairs ILC2 expansion in part by augmenting mTOR activation, which collectively controls immunity to intestinal helminths²⁸.

Taken together, these studies suggest that ILC2-intrinsic metabolism is complicated and context-dependent, being influenced by numerous intrinsic and extrinsic factors. Future studies should carefully consider heterogeneity of ILC2 subsets, including canonical tissue resident ILC2s versus inflammatory ILC2s that are derived from the circulation¹². Consistent with this, inflammatory ILC2s potently upregulate the enzyme Tryptophan hydrolase 1 (Tph1) to drive immunity and inflammation to helminth infection²⁹, but the cell-intrinsic metabolic pathways engaged by this remain poorly understood. Further, ILC2s exhibit tissue-specific signatures that may translate into unique bioenergetic pathways, such as a higher responsiveness to IL-33 in the airway and adipose tissues, a higher responsiveness to IL-25 in the intestine, and a selective responsiveness to IL-18 in the skin³⁰. These should be considered as the field moves forward towards more carefully delineating the cell-intrinsic metabolic requirements of ILC2 responses.

Group 3 ILC-intrinsic metabolism

ILC3s have been shown to utilize glycolysis to fulfill metabolic requirements through mTOR signaling (Fig. 1c). By utilizing mTORC1, ILC3 mount a protective response against the enteric pathogen C. rodentium, in an IL-22-dependent manner³¹. Additional metabolic pathways employed by ILC3s may also mirror their adaptive counterpart, Th17 cells. For example, hypoxia-inducible factor 1α (HIF1α) serves as an important orchestrator of glycolytic metabolism that promotes the differentiation of Th17 cells by stabilizing the expression of RORγt, thereby facilitating IL-17 and IL-22 production in inflammatory contexts^32,33. Similarly, both murine and human ILC3s utilize glycolysis and mitochondrial production of reactive oxygen species (ROS) to fuel proliferation, activation and cytokine production through mTORC1-HIF1α axis³¹. However, it should be noted that the conclusions were largely supported by in vitro cell line culture experiments, thus the role of glycolysis and mitochondrial ROS in in vivo ILC3s requires further investigation.

In addition, mTORC2 is also required for the maintenance and proliferation of ILC3s, and promotes pathological immune response in the context of an innate model of colitis induced by an agonistic antibody to CD40³⁴. While the mTORC1-HIF1α axis sustains ILC3 responses toward glycolysis under normal conditions, HIF1α mediated ILC3 proliferation and activation in hypoxia does not depend on glycolysis or mitochondrial respiration³⁵. Furthermore, HIF1α controls the plasticity of NKp46⁺ ILCs in the gut by promoting an ILC3-to-ILC1 conversion and inhibiting ILC1-to-ILC3 conversion. Intriguingly, this occurs through direct transcriptional regulation of Tbx21 in ILC3s³⁶. Another study identified that c-Maf maintains ILC3 stability and limits physiological ILC1 conversion by supporting RORγt activity and restraining T-bet expression, which is potentially dependent on metabolic pathways related to cholesterol biosynthesis in ILC3s³⁷. As a metabolic checkpoint, PD-1 critically regulates the cellular metabolism of LTi-like ILC3s. In contrast to PD-1 deletion in ILC2s that promote cell activation and effector function through rewiring towards glycolysis and glutaminolysis, LTi-like ILC3s require intact PD-1 signaling to maintain increased glycolysis and lipid metabolism, subsequently supporting IL-22 production during activation. Deletion of PD-1 in ILC3s results in metabolic reprogramming towards excessive FAO and impaired IL-22 production, and thus, PD-1 serves as a metabolic checkpoint in LTi-like ILC3s by restraining FAO to facilitate activation and function³⁸.

Phenotypic and functional changes in immune cells are generally accompanied by cell-intrinsic metabolic reprograming. A recent study uncovered that a subset of ILC3s can adopt a long-term “activated” phenotype in response to C. rodentium infection, termed “trained ILC3s”³⁹. These ILC3s exhibit enhanced proliferation and cytokine production and have a superior capacity to control infection upon pathogen rechallenge relative to naïve ILC3s. Mechanistically, ILC3s undergo significant metabolic shift from glycolysis and glutaminolysis to increased TCA cycle, OXPHOS and fatty acid synthesis upon infection. This durable metabolic rewiring may contribute to the generation of “trained ILC3s” and long-term mucosal defense³⁹, but this requires additional mechanistic investigation. Finally, beyond glycolysis, one study suggested that intestinal ILC3s can uptake abundant fatty acids from the environment²², yet the functional significance remains elusive. Future studies are needed to determine the bioenergetics pathways employed by ILC3 over time, across distinct tissue locations, and in a subset specific manner including conventional ILC3s, LTi-like ILC3s and recently described inflammatory ILC3s.

Cell-extrinsic metabolic control of ILC responses

Emerging evidence suggests that ILC-mediated immune responses are regulated by external environmental cues, such as dietary- and microbial-derived metabolites. Further, nutrition and microbiota contribute to the transcriptional and epigenomic imprinting of specific metabolic pathways in an ILC subset specific manner¹⁹, likely occurring over time in a circadian manner^40–42. A greater understanding of how these external metabolic cues shape the metabolic fitness and function of ILCs will provoke additional opportunities to modulate these responses in human health and disease. This could occur by targeting microbiota, dietary interventions, or employing small molecules strategies, and remains at early stages of investigation.

Vitamins.

Vitamins are essential micronutrients obtained from diet, which have long been known to exert beneficial roles on human health through regulating innate and adaptive immune responses. Metabolites of vitamins A and D critically regulate ILC responses (Fig. 2a). Vitamin A deprivation results in significantly reduced frequency and number of ILC3s, as well as IL-22 production, which render mice more susceptible to Citrobacter rodentium-induced intestinal infection and inflammation. Conversely, delivery of exogenous retinoic acid (RA), the main biologically active metabolite of vitamin A, drives ILC3-intrinsic IL-22 production, which in turn protects against C. rodentium infection and ameliorates the pathology of DSS-induced colitis^43,44. The profound effect of vitamin A on ILC3s is also essential for the development of host immune system, as inhibition of maternal uptake of retinoids during pregnancy impairs LTi cells and subsequent development of secondary lymphoid organs of the offspring⁴⁵. Mechanistically, RA engages with RA receptor, and activated RA receptor directly binds to Il22 and Rorc promoter regions, thereby controlling the development and function of ILC3s⁴⁵. Interestingly, vitamin A plays an opposite role on ILC2 biology relative to ILC3s. Vitamin A deficiency results in increased proportion and number of ILC2s, as well as effector cytokine production, which enhances ILC2-mediated immunity to helminth infections⁴³. The underlying mechanism by which vitamins A suppresses ILC2 response is unclear, potentially through downregulation of IL-7Rα⁴³. Thus, vitamin A critically control the adaptation of the intestinal immune response by adjusting the responses of ILC2 and ILC3 in the context of parasite infections or bacterial infections. Furthermore, RA has been suggested to control the migration of ILC1s and ILC3s, but not ILC2s, to the intestine through regulation of homing receptors, CCR9 and integrin α4β7⁴⁶. Beyond vitamin A, two studies have suggested that vitamin D is crucial for the development and effector function of ILC3s and host defense against pathogens^47,48.

Figure 2. Metabolic regulation of ILC responses by external environmental cues.

a. Vitamin A-derived RA critically controls ILC responses through promoting ILC3 development, migration and IL-22 production through binding of retinoic acid receptor (RAR) and retinoic X receptor (RXR) to Rorc and Il22 loci. In contrast, RA inhibits ILC2 development, proliferation, and effector cytokine production potentially via suppression of IL-7R. Vitamin D is also crucial for the development and effector function of ILC3s with unknown mechanisms. b. Dietary or tryptophan derived AHR ligands are sensed by ILCs. While promoting ILC3 development and ILC3-intrinsic IL-22 expression by directly binding to xenobiotic response elements in the Il22 locus, the AHR signaling suppresses ILC2 response through inhibition of IL-33R transcription. c. SCFAs regulate ILC responses through multiple mechanisms. Butyrate inhibits the development, proliferation, and effector cytokine production of ILC2s in the lung by inhibition of HDACs activity and glycolytic metabolism and OXPHOS. In contrast, it promotes ILC3 proliferation and IL-22 production through either STAT3-HIF1α axis or mTOR-AHR axis. In addition, acetate acts through GPR43 to regulate the binding of phosphorylation of STAT3 to Rorc locus, thereby promoting ILC3 proliferation and IL-22 production.

Aryl hydrocarbon receptor (AHR) ligands.

AHR ligands, including phytochemical indole-3-carbinol that from cruciferous vegetables and the endogenous tryptophan-derived metabolite indole-3-aldehyde, directly modulate ILC response (Fig. 2b). AHR supports the development of NKp46⁺ ILC3s and associated IL-22 production via directly binding to xenobiotic response elements in the Il22 locus. Mice lacking ILC3-specific AHR, or mice fed diet that results in reduced generation of AHR ligands, exhibit substantial reductions in ILC3 responses and impaired IL-22-dependent immunity to C. rodentium infection^49–51. Another study suggested that gut commensal Lactobacilli utilize tryptophan as an energy source to produce metabolite indole-3-aldehyde, which protects against pathogenic infection by Candida albicans through the induction of IL-22⁵². In contrast to ILC3s, AHR suppresses the development and effector function of ILC2s exclusively in the gut. This inhibition of ILC2s is cell-intrinsic and potentially dependent on IL-33 signaling through suppression of IL-33R transcription⁵³. ILC2-specific AHR ablation enhances anti-helminth immunity, and conversely, overexpression of AHR suppresses ILC2 function. AHR overexpression also protects the host against C. rodentium infection by driving enhanced ILC3 activity, suggesting a central role for gut adaptation of AHR expression in regulating the ILC2-ILC3 balance in the context of various pathogens⁵³. In addition to ILC2s and ILC3s, one study reported that AHR is required for maintaining the homeostasis and function of liver-resident NK cells, though the mechanism is unclear⁵⁴. Therefore, through the AHR, nutritional intake and microbiota substantially shape mucosal immunity by regulating disparate ILC responses.

Short chain fatty acids (SCFAs).

Among the most abundant metabolites produced by gut microbiota are SCFAs, which have been found to play an important role in regulating ILC responses (Fig. 2c). Gut anaerobic microbiota-mediated fermentation of dietary fiber results in the generation of SCFA, such as acetate, propionate, and butyrate, which can be detected by their receptors GPR41, GPR43 or GPR109a on ILC subsets. It has been reported that acetate engages with its cognate receptor GPR43 on ILC3s and induces the phosphorylation of STAT3 via AKT and ERK signaling, thereby promoting colonic ILC3 expansion and IL-22 production⁵⁵. Further, GPR43-deficient mice are more susceptible to colonic injury and C. rodentium or Clostridium difficile infections^55,56. In addition to acetate, butyrate promotes IL-22 production through GPR41 and histone deacetylase (HDAC) inhibition. Mechanistically, butyrate upregulates HIF-1α expression and increases HIF1α binding to the Il22 promoter through histone modification, as well as promotes AHR activity⁵⁷. In contrast, two recent studies revealed that butyrate inhibits pulmonary ILC2 activation and effector cytokine production, and subsequent airway hyperreactivity and inflammation. Interestingly, butyrate suppresses ILC2 proliferation and GATA3 expression potentially through histone deacetylase (HDAC) inhibition or suppression of ILC2-intrinsic glycolysis and OXPHOS pathways, which is GPR independent^58,59. Taken together, these findings suggest SCFAs are key mediators of microbial and nutritional cues that shape ILC responses.

ILC regulation of metabolic health and disease

Accumulating evidence in mouse models and patient based translational studies, indicate that in addition to shaping a rapid response to tissue injury or infection, ILCs are also essential regulators of metabolic homeostasis. Perturbations of ILC responses result in altered metabolic states and have been associated with multiple metabolic disorders including obesity, type 2 diabetes (T2D), nonalcoholic fatty liver disease (NAFLD) and other metabolic syndromes. Defining the mechanisms by which ILCs regulate metabolic health and disease will shape our understanding of immune-metabolism interactions and create a foundation for developing preventative or therapeutic treatments for metabolic-associated diseases by harnessing ILCs.

ILC1 regulation of tissue metabolic states

It has been well recognized that NK cells are essential for maintaining metabolic homeostasis and regulating the pathogenesis of metabolic disease such as obesity, T2D and NAFLD, which have been comprehensively reviewed previously⁶⁰. In contrast, the role of non-cytotoxic ILC1s in metabolic health and disease is only now being explored and initial findings suggest several key functions (Fig. 3a). One study revealed a subset of adipose tissue resident ILC1s that phenotypically and functionally distinct from adipose mature or immature NK cells, contributing to insulin resistance and tissue pathology during diet-induced obesity⁶¹. Mechanistically, high fat diet (HFD) consumption results in adipocytes production of IL-12, which induces the proliferation and accumulation of adipose-resident ILC1s, leading to the production of IFN-γ, thereby driving proinflammatory macrophage polarization or inhibiting IL-33-induced ILC2 response to promote obesity-associated insulin resistance and pathology^61,62. Interestingly, another study proposed an alternative mechanism in which adipose-resident ILC1s maintain tissue homeostasis through killing alternatively activated macrophage (termed M2 macrophage) via its cytotoxic activity at steady state⁶³. During HFD-induced obesity, both the number and the cytotoxicity of ILC1s are impaired, resulting in a pathogenic accumulation of pro-inflammatory macrophages and subsequently driving an exacerbated metabolic disorder⁶³. Adipose-resident ILC1s might be a pathogenic factor promoting the transition of obesity to T2D, and this is supported by a recent study that found the number of ILC1s in adipose tissue is increased within T2D patients and positively correlate with insulin resistance and induce adipose fibrosis through an IFN-γ dependent mechanism⁶⁴. ILC1s have also been recently shown to participate in the development of NAFLD. In the liver of obese mice, NK cells display a phenotypically and functionally shift toward ILC1s characterized with a decreased ability of degranulation, decreased cytotoxicity, and a transcriptional profile with characteristics of ILC1s, which is partially mediated by high levels of TGF-β produced in the obese liver. The conversion of NK cells to this less cytotoxic ILC1s may be protective against NAFLD⁶⁵. Thus, the pathogenic versus protective role of ILC1s in metabolic disease may vary by anatomical location or in a subset specific manner and requires extensive additional investigation to enable a mechanistic understanding how ILC1s could be harnessed to promote metabolic health. Advanced technologies, such as single cell RNA-sequencing, are revealing unexpected states and shared progenitors of ILC1s in obese versus lean human tissues⁶⁶, are paving the way towards achieving this goal.

Figure 3. Regulation of host metabolic homeostasis and disease by ILCs.

a. ILC1s promote the development of obesity and T2D through activation of inflammatory macrophages or inhibition of IL-33-induced ILC2 response via IFN-γ. b. ILC2s critically maintain adipose tissue metabolic homeostasis and limit obesity through multiple mechanisms, including regulate WAT lipolysis, beige conversion of white adipose tissue and beige fat biogenesis. Furthermore, ILC2s control the development of T2D and atherosclerosis. c. IL-17A-producing ILC3s promote obesity associated-airway hyperreactivity. In addition, ILC3s limit obesity, T2D and NAFLD through production of IL-22 via several distinct pathways and target cell types.

ILC2 regulation of tissue metabolic homeostasis and disease

ILC2s were originally identified as a unique immune cell population residing in the mesenteric fat⁶⁷, and subsequently have been found to critically orchestrate metabolic homeostasis (Fig. 3b). As one of the major immune cells in the lean adipose tissue, ILC2s are required to maintain tissue homeostasis and become disrupted in the context of HFD-induced obesity. Depletion of ILC2s in Rag1^−/− mice results in significant weight gain and glucose intolerance and transferring ILC2s into obese mice induces transient weight loss and stabilizes glucose homeostasis^68,69. The mechanisms by which ILC2s promote adipose tissue homeostasis are extensive, including coordinating M2 macrophage and eosinophil response, as well as regulating beige conversion of white adipose tissue (WAT) and beige fat biogenesis^69–72. In response to IL-25 and IL-33 signals, ILC2s produce IL-5 to recruit eosinophils to WAT, and eosinophils in turn produce IL-4 to sustain M2 macrophages, thereby controlling insulin sensitivity and regulating adipocyte metabolism^{69,70,72–74}. Besides the effect on M2 macrophages, ILC2 and eosinophil-derived IL-4 also directly act on adipocyte precursors and promote the differentiation of beige adipocytes⁷². In addition, ILC2s directly promote M2 macrophage response through production of IL-13. Consistently, IL-13 deficient mice exhibit increased weight gain, reduced eosinophils and M2 macrophages in adipose tissue⁶⁸. Finally, ILC2s can also promote beiging of WAT through the generation of methionine-enkephalin (Met-Enk) from opioid peptide proenkephalin A (PENK) via prohormone convertase 1, which is instructed by IL-33 produced from adipocyte progenitors and mesenchymal stromal cells in WAT^71,75–77. ILC2-derived Met-Enk acts directly on beige adipocytes and upregulates uncoupling protein 1 expression, thereby increasing energy expenditure by uncoupling of the electrochemical gradient of the respiratory chain in the mitochondria^71,75.

The burgeoning field of neuro-immune interactions also identified ILC2s as a central player that responds to neurotrophic signals, neuropeptides, or associated pathways^78,79, and this can also critically link to metabolic health and disease. A recent study delineated a brain-ILC2-adipose circuit that critically regulates systemic adipose tissue metabolism and obesity, in which sympathetic neurons integrate higher-order signal from paraventricular nucleus of the hypothalamus in the brain, induce gonadal adipose mesenchymal cells to produce glial-derived neurotrophic factor (GDNF) via the β2-adrenergic receptor, and further regulate ILC2 response via GDNF-RET axis, thereby controlling energy expenditure, insulin resistance and limiting obesity⁸⁰. This study provides additional insights on how neuro-immune interactions shape host metabolism and obesity. In contrast to WAT-ILC2 mediated obesity resistance, a recent study unexpected found that ILC2s from small intestine promote HFD-induced obesity⁸¹, suggesting the heterogeneity of tissue-resident ILC2s and the importance of adipose-resident ILC2s in regulating adipose tissue metabolism and limiting obesity.

Beyond the functional significance in adipose tissue, ILC2s also modulate insulin secretion in pancreatic islets. During acute metabolic stress, islet mesenchymal cells secret IL-33, induce myeloid cell-intrinsic RA release through islet-resident ILC2s production of IL-13 and GM-CSF, RA in turn acts on β cells to increase insulin secretion, suggesting that the immune-metabolic crosstalk between mesenchymal cells, ILC2s and myeloid cells is essential for maintaining pancreatic-islet function and preventing from T2D pathogenesis⁸². Furthermore, it has been reported that ILC2 perform a central role in controlling the progression of atherosclerosis, which is in part dependent on ILC2-derived IL-5 and IL-13⁸³. These findings complement emerging studies that have revealed key roles for ILC2s in regulating tissue homeostasis across multiple anatomical locations associated with nutrition and systemic metabolic homeostasis, notably including the stomach and biliary tract^84,85.

ILC3 regulation of tissue metabolic homeostasis and disease

Emerging evidence suggests that ILC3s may impact obesity or metabolic homeostasis through IL-17A or IL-22 (Fig, 3c). CCR6⁺ LTi-like ILC3-derived IL-17A promotes the pathogenesis of obesity-associated airway hyperreactivity in Rag1^−/− mice in a mechanism dependent of macrophage derived IL-1β that induced by HFD⁸⁶. IL-22, which is predominantly produced by ILC3s at steady state, also critically regulates host metabolism by acting on multiple non-hematopoietic cell types. IL-22 is required for the prevention of insulin resistance and obesity through regulation of triglyceride lipolysis and FAO in adipocytes. Mice lacking IL-22 receptor are more susceptible to HFD-induced obesity and insulin resistance, and treatment of obese mice with IL-22 suppressed TNF-α expression in adipose tissue and improved insulin resistance⁸⁷. IL-22 has also been shown to protect against diabetes, partly by modulating oxidative stress and inflammatory pathways in islet β cells, subsequently promoting the secretion of insulin⁸⁸. In addition, ILC3-derived IL-22 can protect against obesity-associated NAFLD through upregulation of hepatic lipid metabolism and inhibition of palmitate-induced primary hepatocytes apoptosis⁸⁹. Furthermore, IL-22 promotes metabolic homeostasis via regulating lipid metabolism in the gut, which is augmented by adaptive immune responses directed against the microbiota^87,90.

These functional roles for IL-22 are complex and context dependent, likely involving microbiota-dependent stimulation, circadian rhythms, and many potential modifiers, such as adaptive immunity⁹⁰. Further, with all these functions for IL-22 in promoting metabolic health, it is intriguing that IL-22 levels are substantially diminished in ILC3s and CD4⁺ T cells in some mouse models of obesity⁸⁷, indicating that an inherent disruption of the intestinal ILC3-IL-22 pathway is linked to microbiota dysbiosis, increased susceptibility to infections, and metabolic disease progression across these mouse models and potentially obese humans. Therefore, defining strategies to boost or restore this ILC3-IL-22 response in the gut could hold a key to metabolic health.

Extending the study of ILCs and metabolism

Cellular metabolism has emerged as a key driver of ILC development, maintenance, and function, with ILCs displaying plasticity and adaptability to metabolic perturbations in the contexts of immunity, inflammation, or tissue repair. The cellular and molecular mechanisms by which metabolic programs facilitate the fitness and function of ILCs in discrete microenvironments or inflammatory contexts are only recently being defined, however, many limitations exist and could potentially be addressed with more recent technological advances (Box 1). One major limitation is that most studies of ILC-intrinsic metabolic pathways are dependent of ex vivo culture, which may not accurately reflect physiologic conditions of oxygen, nutrient (i.e., glucose, glutamine, and fatty acids), and cytokine milieus that occur within tissues. The conclusions may be limited by how the differences in these conditions impact cellular metabolism in vitro versus in vivo, as well as challenges associated with obtaining large numbers of ILCs for these assays. Many studies also heavily rely on gene expression analysis to make conclusions about metabolic pathways in cells without accompanying functional validation. In addition to these caveats, several fundamental questions remain underexplored. First, the mechanisms that drive the metabolic reprogramming of ILCs from OXPHOS to aerobic glycolysis or FAO upon activation is yet to be determined. Second, it will be important to interrogate the mechanisms that dictate the metabolism-driven effects on ILC responses in different contexts. For example, how the cell-extrinsic metabolic factors alter cell-intrinsic ILC metabolism to fulfill its functional effects remains undetermined. Finally, current studies of ILC-mediated regulation of metabolic homeostasis and disease is unidirectional and tissue local microenvironment dependent, thus future prospects necessitate a deeper focus on a molecular understanding of the bidirectional immune-metabolic crosstalk, systemic, and perhaps microbiome-associated influences on these interactions or functional outcomes. These challenges can be addressed in part with recent technological advances.

Box 1. Technical challenges and recent advances in metabolic profiling that apply to ILCs.

Numerous metabolic programs control ILC responses, but a more comprehensive understanding of the interconnection between metabolic pathways and other cellular processes, for example epigenetic modifications, remains elusive and requires advanced approaches. Further, most studies of immunometabolism rely on in vitro manipulated cells, which do not reflect their physiological conditions in tissues. There is an urgent need to develop technological advances in order to analyze metabolic states directly ex vivo or in vivo. Several recent technical advances exist that could be applied to boost the study of ILC metabolism:

1. SCENITH (single-cell energetic metabolism by profiling translation inhibition)⁹⁶, a flow cytometry-dependent approach to examine cellular metabolism at single-cell resolution, which allows to effectively interrogate the rare but heterogeneous ILC populations, as well as avoid metabolic perturbation from culture media.

2. Mass cytometry or CyTOF (Cytometry by time-of-flight)⁹⁷, a real-time and high-throughput analyses of single cells method by using metal ion-conjugated probes instead of fluorescent antibodies, which maximizes the information from each sample and exhibits a major advantage over classic flow cytometry. This advanced approach allows to extensively analyze the phenotypic, metabolic and functional characteristics of ILC populations.

3. Single-cell metabolic regulome profiling (scMEP)⁹⁸, an approach to characterize the metabolic features of single cells through a high-dimensional antibody-based proteomic platforms, which enables the study of cellular metabolic states and phenotypic identity.

4. Spatial metabolomics and mass-spec based imaging of metabolic profiles^99,100, are another emerging technology that could rapidly advance our understanding of the cell-intrinsic metabolic pathways that are utilized by ILCs within tissues in state of health and disease.

Recently, the interplay between metabolism and epigenomics has been shown to critically control the development, activation, and function of immune cells. Metabolic regulation always correlates with changes in epigenetic landscapes, and many substrates and cofactors utilized by chromatin- and DNA-modifying enzymes are intermediates of metabolic pathways^91–93. While the metabolic and epigenetic coordination of CD4⁺T cell immunity has been extensive studied, it remains largely unexplored in the ILC field. One study revealed that ILC2-intrinsic methionine metabolism generated S-adenosylmethionine is capable of regulating Il5 and Il13 transcription through H3K4me3 modification, suggesting that the interconnection between methionine metabolism and epigenetic reprogramming critically shape ILC2 effector function²⁷. The formation of innate immune memory, termed “trained immunity”, is dependent of the epigenetic and metabolic reprogramming of cells^94,95. Though the metabolic rewiring endows the adaptation of “trained ILC3s” and long-term mucosal defense, the precise mechanism that underlie the induction and function of these cells remains to be determined, potentially through epigenetic reprogramming resemble that of innate immune cells³⁹. Beyond ILC-intrinsic metabolism and epigenome connection, extrinsic metabolites, such as butyrate, have been shown to modulate ILC effector function through inhibition of HDACs activity. Altogether, the interconnection between metabolism and epigenetic modifications in ILCs still remains elusive, and further studies are required. Nutritional status, microbiota, and diurnal oscillations may dynamically impact this epigenomic imprinting of specific metabolic pathways among ILC subsets and requires extensive consideration in future studies.

Taken together, additional research and more advanced metabolic approaches and technologies may help shape our understanding of metabolic regulation of ILC fitness and function, and a better understanding of how ILCs orchestrate metabolic homeostasis will be critical for understanding the pathogenesis of metabolic disease and for the development of preventative, therapeutic, and curative strategies against these diseases. Given currently available data and differential metabolic dependencies across subsets, it may be possible to favor group 2 versus group 3 ILC responses through intervention via targeting Ahr ligands, oxygen gradients or vitamin-A or -D bioavailability. These are broad and non-specific approaches, as the study of cellular metabolism or function of ILCs in metabolic diseases remains at early stages. However, fundamental results thus far are encouraging and indicate that more specific harnessing of ILCs is possible in the future and could lead to breakthroughs for metabolic health.