Regulating metabolism to shape immune function: Lessons from Drosophila

Abstract

Infection with pathogenic microbes is a severe threat that hosts manage by activating the innate immune response. In Drosophila melanogaster, the Toll and Imd signaling pathways are activated by pathogen-associated molecular patterns to initiate cellular and humoral immune processes that neutralize and kill invaders. The Toll and Imd signaling pathways operate in organs such as fat body and gut that control host nutrient metabolism, and infections or genetic activation of Toll and Imd signaling also induce wide-ranging changes in host lipid, carbohydrate and protein metabolism. Metabolic regulation by immune signaling can confer resistance to or tolerance of infection, but it can also lead to pathology and susceptibility to infection. These immunometabolic phenotypes are described in this review, as are changes in endocrine signaling and gene regulation that mediate survival during infection. Future work in the field is anticipated to determine key variables such as sex, dietary nutrients, life stage, and pathogen characteristics that modify immunometabolic phenotypes and, importantly, to uncover the mechanisms used by the immune system to regulate metabolism.

1. Introduction

Immune responses are not metabolically inert. Mobilizing the immune system to fight infection stimulates a host of energetically demanding processes: cell proliferation and migration, phagocytosis, and synthesis and secretion of immune effectors [1,2]. Metabolic phenotypes of immune cells themselves influence immune function [3]. Prolonged immune responses can be pathological: chronic inflammation drives metabolic diseases such as type 2 diabetes and non-alcoholic steatohepatitis [4,5]. Immune signaling pathways control not only processes such as antibody production or pathogen engulfment, but also metabolism that supports and directs these functions.

Immune signaling pathways are pleiotropic. In fact, the core components of the Toll signaling pathway that fights bacterial and fungal infections were discovered over thirty years ago in large-scale forward genetic screens in Drosophila melanogaster for genes that control dorsal-ventral patterning in the embryo [6]. A decade later, recognition of sequence homology between Toll and mammalian interleukin 1-beta receptor suggested an immune role for the Toll pathway that was soon confirmed using fly genetics [7–9]. The fly continues to contribute to our understanding of immune function, and work over the last fifteen years has demonstrated the close and complex connections between immunity and host metabolism. This work is the subject of this review.

2. The Drosophila melanogaster immune system

2.1. Environmental exposure of fruit flies to beneficial and harmful microbes

Fruit fly larvae and adults consume rotting fruit, food that is rich not only in carbohydrates but also protein due to the presence of microbes such as the yeast Saccharomyces cerevisiae that ferment fruit sugars. Lab-reared flies also consume yeast or yeast extract, with molasses, sucrose or glucose serving as a carbohydrate source in most diets. Both wild and lab-reared fruit flies consume microbes that comprise the microbiome, with stable associations occurring for at least some commensal species [10,11]. Two major bacterial genera, Acetobacter spp. and Lactobacillus spp., are dominant members of the Drosophila melanogaster microbiome [12], with wild flies exhibiting increased microbial complexity compared with lab-reared animals [13]. Commensal bacteria have dominant effects on fruit fly growth and metabolism [14,15], influence host gut morphology and gene expression [16,17], promote harvest of specific nutrients from the diet, and generate metabolic intermediates that are used by the host [18].

The food that flies eat exposes them not only to beneficial microbes but also to pathogens that infect and sicken flies and cause death. Flies encounter entomopathogenic fungi and parasitic wasps and nematodes on the surface of food, and they consume entomopathogenic bacteria [19]. Adult flies and larvae can detect some pathogens via odorant cues and avoid contaminated food [20,21]. Flies that do ingest contaminated food can learn to avoid it in a process that requires immune signaling in neurons and associative memory formation [22]. Pathogen avoidance is also a parental trait, as female flies prefer to lay their eggs on citrus fruits that endoparasitoid wasps avoid [23].

2.2. Physical and biochemical barriers that prevent systemic infection

Physical and biochemical barriers to infection are the first lines of defense against systemic infection in Drosophila. In fly embryos, larvae, pupae and adults, the tough exoskeleton, composed of layers of chitin and cuticular proteins, protects against microbe entry into the hemocoel [24]. Surface epithelia that line tracheae, portions of the larval and adult gut, and the adult reproductive system are also protected by cuticle and by expression of antimicrobial peptides (AMPs) that kill microbes [25, 26].

Multiple defenses in the gut protect flies from enteric infections [27] (Fig. 1). The gut lumen is surrounded by the peritrophic matrix, a sleeve-like lattice of chitin polymers and glycoproteins that lines the midgut and separates food and microbes from the gut epithelium. A layer of mucus, composed of polysaccharides and mucins, forms a second physical barrier between the peritrophic matrix and the gut epithelium [28]. Mutations that disrupt peritrophic matrix formation increase susceptibility to bacterial and viral infection and lead to early adult lethality [29–31]. Ingestion of pathogenic microbes activates biochemical defenses in the gut: the production of reactive oxygen species (ROS) and induction of AMPs, both of which kill invaders. Pathogenic bacteria, but not commensal bacteria, secrete uracil that stimulates expression and activation of dual oxidase (Duox), an enzyme that generates ROS [32]. Duox-derived ROS is microbicidal, and loss of Duox in gut renders flies susceptible to enteric infection [33]. Ingestion of Gram-negative bacteria activates the Imd signaling pathway in enterocytes (see Section 2.3) to stimulate production of microbe-killing AMPs [34]. Loss of Imd pathway signaling sensitizes flies to enteric infection [35,36].

Fig. 1.

Immune and metabolic interactions in the gut. Epithelial immunity in the gut requires expression of AMPs that is directed not only by the NF-κB protein Relish, but also by Foxo in enterocytes. Relish also controls expression of digestive enzymes (not shown). Pathogenic bacteria in the gut stimulate ROS production by the NAPDH-consuming enzyme Duox. Inhibition of mTORC1 and consequently the NADPH-consuming process of fatty acid synthesis frees substrate for the Duox reaction. Blocking mTORC1 also reduces protein synthesis, which becomes pathological when gut tissue is damaged during infection and in need of repair. Commensal microbes stimulate Imd in enteroendocrine cells to secrete tachykinin. Tachykinin signals in a paracrine manner to inhibit lipid storage in enterocytes and thus prevents intestinal steatosis. LDs, lipid droplets; PM, peritrophic matrix; TG, triglyceride.

Flies induce clotting and melanization reactions to quickly repair wounds and limit infection. Clots curtail hemolymph loss, create barriers to opportunistic infection, and trap bacteria that breach the cuticle, facilitating their killing. Clots consist of procoagulant proteins including hexamerin, fat body protein 1, lipophorin, the clotting factor fondue, and hemocyte-derived proteins such as hemolectin and tiggrin that are cross-linked by the enzyme transglutaminase. These cross-linked fibers trap microbes, cell debris and hemocytes, the circulating blood cells in the fly [37] (see Section 2.4). At wound sites and on the surfaces of parasites in the larval body cavity, the lamellocyte and crystal cell lineages of hemocytes release prophenoloxidase, which is cleaved by serine proteases to yield active phenoloxidase. Phenoloxidase-dependent polymerization of orthoquinones leads to deposition of melanin that encapsulates pathogens. Furthermore, intermediates in the melanization reaction are toxic to microbes [38].

2.3. Detecting and responding to infection

Systemic infections occur when pathogens breach physical and biochemical barriers and invade the body cavity [19]. Entomopathogenic fungi such as Beauvaria bassiana penetrate the cuticle directly [39], as does the entomopathogenic nematode Heterorhabditis bacteriophora with its gut bacterial symbiont Photorhabdus luminescens, a Drosophila pathogen [40]. Parasitoid wasps such as Leptopilina boulardi directly inject their eggs into Drosophila larvae [41]. Opportunistic infections can occur when bacteria enter the body cavity via open wounds. Enteric infections can become systemic infections when ingested pathogens such as Pseudomonas entomophila damage the peritrophic matrix and breach the gut epithelium [42]. In the laboratory, larval and adult flies can be infected with entomopathogens using infection routes described above or by septic injury caused by direct injection of microbes or by cuticle puncture that permits access of microbes to the body cavity.

During a systemic infection, the fruit fly’s innate immune system detects the presence of microbes or parasites in the hemocoel and initiates humoral and cellular responses that fight infection. In bacterial and fungal infections, pathogen-associated molecular patterns that are components of microbial cell walls are detected by host pattern recognition receptors that activate immune signaling. Bacterial cell walls contain layers of peptidoglycan, a polymer of glycan strands with alternating N-acetylglucosamine and N-acetylmuramic acid residues that are crosslinked by peptide bridges containing L-lysine (Lys) or diaminopimelic acid (DAP). Lys-type peptidoglycan characteristic of Gram-positive bacteria and ß-1,3-glucan in fungal cell walls activates the Drosophila Toll signaling pathway, while DAP-type peptidoglycan in Gram-negative bacteria (and Gram-positive Bacillus) activates the Imd pathway [43]. For many microbial infections, however, both the Toll and Imd pathways contribute to host survival (reviewed in [44]). Molecular, biochemical, and genetic characterization of the Toll and Imd pathways from the mid 1990s to the late 2000s provided a clear picture of the signal transduction events that allow the host to detect and respond to microbial infections. The Toll and Imd pathways are briefly described below, and the reader is referred to literature reviews for detailed descriptions of work by multiple labs that led to the discovery of individual signaling events in each pathway.

Flies fight Gram-positive and fungal infections by activating the Toll signaling pathway (see additional details and references in [45]). Detection of Lys-type peptidoglycan by the secreted peptidoglycan recognition protein SA (PGRP-SA) and Gram-negative bacteria binding protein 1 (GNBP1) [46,47] or detection of fungal wall ß-1,3-glucan by GNBP3 [48] activates the serine proteases Grass and modSP [49–51]. Proteases released by pathogenic microbes activate the serine protease Persephone [52]. These serine proteases and others are required for activation of Spatzle Processing Enzyme, a protease that cleaves the cytokine spatzle to generate the mature ligand capable of binding to and activating the transmembrane Toll receptor [53]. The activated Toll receptor binds to the cytoplasmic adaptor protein MyD88 [54,55], leading to recruitment of the proteins tube and pelle via interactions through their death domains [56]. The kinase activity of pelle is required for phosphorylation of cactus [57], leading to its degradation and freeing the NF-κB family transcription factors Dif and dorsal to translocate into the nucleus where they bind to and activate expression of genes encoding AMPs such as Drosomycin and Bomanins [58]. While dorsal is required for Toll-dependent dorsal-ventral patterning in Drosophila embryos [59], Dif plays the dominant role in the innate immune response [60,61]. The critical role of this immune pathway is evidenced by the failure of flies with loss of function mutations in spatzle, Toll, tube, and pelle to induce expression of AMPs that neutralize and kill microbes, leading to near complete lethality in response to fungal infection [7].

Infection with Gram-negative bacteria activates the Imd pathway (see additional details and references in [62]). In response to infection with Gram-negative bacteria, binding of DAP-type peptidoglycan fragments to cell surface PGRP-LC receptors [43,63–66] and cytosolic PGRP-LE receptors [67] recruits a signaling complex that includes Imd [68,69], the adaptor protein dFadd, and the caspase-8 homolog Dredd [70]. Ubiquitination by the E3-ligase Iap2 activates Dredd, which cleaves Imd, promoting its ubiquitination by Iap2 [71]. Subsequently, Tab2 and the kinase Tak1 are recruited to the complex [72], and Tak1 phosphorylates and activates the IκB kinase complex, consisting of the proteins Ird5 (IKKß) and Kenny (IKKγ) [73–75]. This complex plays two roles in activation of the NF-κB family transcription factor Relish: it promotes cleavage of Relish by Dredd [76,77], and it directly phosphorylates Relish [75,78]. Cleavage of Relish exposes a nuclear localization sequence and a dimerization domain, and phosphorylation stimulates transcriptional activation by Relish. Like Dif, Relish binds to and activates genes encoding AMPs to drive humoral immunity. Loss of imd blocks induction of AMPs that kill Gram-negative bacteria, leading to increased reduced survival after septic infection [8].

The microbicidal actions of AMPs, secreted from the fat body of infected larvae and adults into the hemolymph, underlie humoral immunity. AMPs fight infection by disrupting bacterial and fungal membranes and inhibiting spore germination, and AMPs exhibit specificity in the types of pathogens they kill. Fungal cell wall components activate Toll signaling and the production of Drosomycin, Daisho1, Daisho2, and BaramicinA; these peptides are required for resistance to fungal infections [79–82]. The Bomanin family of AMPs confers resistance to Gram-positive bacteria and fungi [83]. Diptericins and Drosocin defend against Gram-negative Providencia rettgeri and Enterobacter cloacae, respectively, with these interactions providing clear examples of AMP specificity. Other infections are fought by AMPs working synergistically [82].

Sterile wounding and tissue damage can also activate the humoral immune response. Damaged and dying cells release endogenous proteins that activate the innate immune system to stimulate wound healing and tissue repair [84]. In Drosophila, mechanical damage to larval tissues without cuticle puncture is sufficient to activate Drosomycin expression, in a manner partially dependent on Toll signaling pathway components [85]. Work is ongoing to identify molecules that serve as damage associated molecular patterns in the fruit fly [86,87].

Intracellular pathogens that invade host cells through phagocytosis and establish residence in the cytosol or vacuoles are protected from the humoral arm of the immune response [88]. Bacteria such as Mycobacterium marinum, Salmonella typhimurium, and Listeria monocytogenes establish intracellular infections in Drosophila [89–91], although in severe infections, L. monocytogenes can also be found extracellularly [92]. In flies, signaling from the intracellular DAP-type peptidoglycan receptor PGRP-LE [67] to the Imd pathway stimulates autophagy that suppresses intracellular growth of L. monocytogenes and promotes host survival [93]. Similarly, the ubiquitin ligase parkin, a positive regulator of mitochondria-specific autophagy (mitophagy) limits M. marinum and S. typhimurium growth in flies and promotes host survival [94].

2.4. Cell types that fight systemic infection

The bulk of AMPs produced during the immune response are synthesized and secreted into hemolymph by the Drosophila fat body, a multi-functional organ that is a major regulator of whole-animal energy metabolism and growth [95,96] (Figs. 2 and 3). Fat body cells synthesize and store glycogen and triglycerides, processes regulated by anabolic hormones such as Drosophila insulin-like peptides (Dilps) and catabolic hormones such as adipokinetic hormone. The larval fat body regulates animal size, with the master growth regulator mTORC1 acting in fat body of well-fed larvae to stimulate Dilp secretion from brain insulin-producing cells [97]. Larval fat body cells secrete Dilp6, a driver of whole-animal growth at late stages of larval development [98,99]. Fat body morphology differs considerably in Drosophila larvae and adults. The larval fat body comprises roughly two thousand large, flat, polyploid cells, arranged in a pair of single-cell-layer sheets situated between the gut and the body wall. The adult fat body is a collection of round cells that adhere to the inner body wall. In the adult fly, individual fat body cells can mobilize toward wounds, where they participate in wound healing processes and produce AMPs that kill invading microbes [100]. The fly fat body is functionally analogous to mammalian liver, an organ that produces immune effectors including acute phase proteins and cytokines, regulates whole-animal carbohydrate and lipid metabolism, and promotes whole-animal growth via secretion of the Dilp6 homolog insulin-like growth factor 1 in response to growth hormone signaling.

Fig. 2.

Growth and metabolic effects of infection and immune signaling in larval fat body. Activating Toll signaling in the larval fat body reduces insulin signaling by interfering with Pdk1-dependent phosphorylation of Akt, thereby reducing cell size. Signaling via the PGRP-LC/Imd pathway also reduces insulin signaling and fat body cell growth in larvae. Reduced TOR signaling in fat body facilitates AMP production through de-repression of the 5’-cap-dependent translation inhibitor Thor. Toll signaling impairs lipid storage but increases phospholipids that may permit ER expansion to support AMP secretion and consequently bacterial killing. Toll signaling reduces expression and production of the growth hormone Dilp6, thereby reducing animal size. Fat body glycogen stores are depleted during infection, and circulating sugar is increased in a process stimulated by adenosine secreted by plasmatocytes. LDs, lipid droplets; PL, phospholipid; TG, triglyceride.

Fig. 3.

Immune and metabolic interactions in the adult fat body. Toll and Imd signaling in the adult fat body disrupts insulin signaling. The transcription factors CrebA and Mef2 participate in regulation of genes induced by infection, and the Imd pathway transcription factor Relish represses Foxo to inhibit its activation of Thor and brummer during infection. Fat body glycogen stores are depleted during infection, and circulating sugar is increased in a process stimulated by plasmatocyte-derived adenosine. Infection with T. ratisbonensis depletes adult triglyceride stores and leads to fusion of fat body cells to form syncytial xenomas (not shown). Host phosphatidic acid, an intermediate in phospholipid and triglyceride synthesis, is consumed by T. ratisbonensis and drives its growth. LDs, lipid droplets; DNL, de novo lipogenesis; TG, triglyceride.

Hemocytes, the blood cells of Drosophila, carry out the cellular response to infection [101]. Three main hemocyte cell types – plasmatocytes, lamellocytes, and crystal cells – perform distinct roles during infection. Greater than 90% of hemocytes are plasmatocytes, a cell type that is functionally equivalent to mammalian macrophages. Plasmatocytes engulf and consume microbes and dead host cells. Ablation of phagocytic plasmatocytes reduces survival in flies infected with various microbes without altering the fat body humoral immune response [102]. However, interactions between plasmatocytes and fat body cells serve immune function. Plasmatocytes release spatzle during infection to stimulate fat body Toll signaling and AMP production [103], and activating Toll signaling in fat body drives plasmatocyte proliferation and mobilization [104]. Fruit fly larvae that are parasitized by wasp eggs mount a cellular immune response that involves proliferation of lamelloblasts and transdifferentiation of plasmatocytes into lamellocytes, a cell type that encapsulates wasp eggs [105]. Both lamellocytes and crystal cells drive melanization (refer back to Section 2.2). Recently, the use of single-cell transcriptomic techniques has provided deep insight into developmental and physiological regulation of Drosophila blood cell lineages [106,107].

3. Metabolic regulation and the immune response

3.1. Metabolism and metabolic regulation in Drosophila

Drosophila melanogaster is a holometabolous insect with four major life stages – embryo, larva, pupa, and adult – that differ in terms of nutrient intake and whole-animal energy metabolism [108–110]. The embryonic and pupal stages are non-feeding, while the larval stage is characterized by constant food intake that drives high levels of triglyceride and glycogen storage, protein synthesis, and growth. Larvae experience a two hundred-fold increase in body mass over three instar stages that are punctuated by molts driven by the steroid hormone ecdysone, with the final larval size determining adult size [108]. Adult flies consume far less food than larvae and they store lower levels of glycogen and triglyceride, devoting most of their energy toward reproduction. Major shifts in core metabolic pathways accompany transitions from one life stage to another. For example, increased expression of glycolytic enzymes in mid-stage embryos is required for the high rate of glycolysis that drives larval growth [111,112]. The reader is referred to excellent, recent reviews of whole-animal energy metabolism [113], lipid metabolism [114], and carbohydrate metabolism [115] for more details on metabolic regulation in flies and its control by hormones such as the seven Dilps that signal through the conserved insulin receptor to promote growth and nutrient storage.

Host metabolism is altered in response to infection and immune signaling in ways that can be detrimental or beneficial to the host organism. Below, in Sections 3.2–3.5, the effects of infections on lipid, carbohydrate and protein metabolism, and the influence of dietary nutrients on resistance to and tolerance of infection are described. In Sections 4.1–4.3, the effects of infection on insulin signaling, the roles of “metabolic” transcription factors in regulating metabolic responses to infection, and the roles of secreted signaling molecules in coordinating immunometabolic responses among organs are considered. Figures accompanying the text illustrate immunometabolic phenotypes in gut, larval and adult fat body, and plasmatocytes.

3.2. The varied roles of lipid metabolism during infection

Lipid metabolism is regulated by and contributes to immune function. In many cases, activation of innate immune signaling in flies, either genetically or via infection, leads to reduced triglyceride storage. Enteric infection with Vibrio cholerae and septic infection with the intracellular pathogen M. marinum lead to a progressive loss of triglyceride stores in adult flies [116,117]. Similarly, infection of adults with Gram-positive Micrococcus luteus or the entomopathogenic fungus B. bassiana lowers triglycerides [118], and infection of Drosophila larvae with Gram-positive Enterococcus faecalis leads to a transient reduction in triglyceride storage [119]. Impaired triglyceride storage during infection is consistent with wasting that is observed in many systemic infections in humans. However, reducing triglyceride storage also frees fatty acids for other purposes such as fueling the immune response via beta-oxidation or production of other molecules such as membrane phospholipids.

Decreases in steady state levels of triglycerides may be due to decreased de novo lipogenesis, increased lipolysis, or redirection of triglyceride building blocks into other lipid molecules. In the case of M. marinum infection in adult flies, expression of genes encoding lipogenic enzymes is impaired, and infection leads to a significant and prolonged decrease in expression of the diacylglycerol acyltransferase homolog midway that carries out the final step in triglyceride synthesis [116] (Fig. 2). In contrast, in the adult fat body, Relish antagonizes the transcription factor Foxo to repress expression of the adipocyte triglyceride lipase homolog brummer, the rate-limiting enzyme in lipolysis (Fig. 3). Elevated brummer levels in Relish mutants accelerate triglyceride breakdown during starvation in uninfected adult flies, indicating an important basal role of immune pathway signaling in the regulation of lipid metabolism [120]. De novo lipogenesis produces fatty acids not only for storage as triglyceride, but also for synthesis of membrane phospholipids. Toll signaling in the larval fat body regulates both processes. Larval fat bodies expressing constitutively-active Toll^10b receptors exhibit reduced expression of midway and the phosphatidic acid phosphatase Lipin, but forced expression of midway and Lipin in larval fat body only partially rescues reduced triglyceride storage, consistent with redirection of fatty acids to another metabolic pathway [119]. Toll signaling in fat body increases expression of Kennedy pathway enzymes that synthesize membrane phospholipids and leads to increased levels of phosphatidylcholine and phosphatidylethanolamine that may support the endoplasmic reticulum expansion observed in fat body cells with active immune signaling (Fig. 2). Larvae with fat body-specific knockdown of two Kennedy pathway enzymes, eas and Pcyt1, exhibit impaired AMP secretion and reduced bacterial clearance during E. faecalis infection with prolonged induction of Drosomycin compared with wild type larvae [119].

In agreement with findings from infection experiments, genetic activation of Toll or Imd signaling in larval fat body inhibits triglyceride storage [118,119,121,122]. In contrast, genetic activation of Toll or Imd signaling in the adult fly fat body promotes triglyceride storage, and the transcription factor Relish restrains loss of triglyceride during starvation [120,123]. Why genetic activation of fat body Toll or Imd signaling in the absence of microbe infection has opposing effects on triglyceride storage in larvae and adult flies is unknown. However, inherent differences in lipid metabolism between larvae and adults may underlie the differential effects of innate immune signaling on fat accumulation. First, unlike adult flies, larvae are actively growing, and they must devote some resources and energy to this process, even when immune signaling is active, perhaps at the expense of fat storage. Second, elevated phospholipid synthesis in larvae with active immune signaling may divert fatty acids from storage to membrane synthesis [119]; whether this also occurs in adult flies is not known. Third, lipolysis is a greater contributor to whole-animal triglyceride levels in adult flies, especially males, compared with larvae [124]. It is also possible that triglycerides accumulate outside of the fat body, perhaps in gut or muscle, in uninfected adult flies with constitutive innate immune signaling in fat body. Finally, a systematic examination of lipid metabolism in larval and adult flies infected with different microbes may reveal infections that differentially regulate lipid storage across life stages.

3.2.1. Lipid droplets and intracellular infections

Cells store triglycerides and sterol esters in lipid droplets, organelles bounded by a phospholipid monolayer and proteins such as perilipin that regulate lipolysis [125]. Lipid droplets play roles in the innate immune response that go beyond serving as repositories for host cellular energy. In Drosophila, extra-nuclear histones bound to lipid droplets protect precellular blastoderm stage embryos from infection with Gram-positive Staphylococcus epidermidis, Gram-negative Escherichia coli, or the intracellular pathogen L. monocytogenes [126,127], raising the possibility that histones act as a maternally-deposited defense against intracellular pathogens. On the other hand, lipid droplets can promote intracellular pathogen growth. Drosophila hemocytes infected with M. marinum induce the cytokine unpaired 3 (upd3), which acts in an autocrine manner to repress expression of the autophagy-related gene Atg2, leading to accumulation of large lipid droplets (Fig. 4). Disrupting upd3 signaling or inhibiting triglyceride synthesis in hemocytes improves resistance to M. marinum, suggesting that large host lipid droplets are favorable for growth of this intracellular pathogen [128]. In contrast, the microsporidian parasite Tubulinosema ratisbonensis colonizes the adult fat body, generating syncytial xenomas that are progressively depleted of lipid droplets and their triglyceride contents. Genetic screening identified that lipid transport from gut to fat body, lipolysis in fat body, and peroxisomal breakdown of fatty acids promote T. ratisbonensis proliferation in the host. These parasites rely on host fatty acids with phosphatidic acid likely serving as a key driver of growth [129] (Fig. 3).

Fig. 4.

Regulation of lipid and carbohydrate metabolism in plasmatocytes during infection. Lipid droplets accumulate in plasmatocytes infected intracellularly with M. marinum (green) in response to autocrine upd3 signaling that inhibits Atg2 and autophagy-mediated lipid droplet breakdown. S. pneumoniae infection stabilizes HIF1α in plasmatocytes, activating expression of lactate dehydrogenase (LDH) and promoting aerobic glycolysis and host survival. Secretion of adenosine from plasmatocytes stimulates trehalose output from fat body to drive aerobic glycolysis. LDs, lipid droplets; TG, triglyceride.

3.2.2. Enteric infection and innate immune signaling regulate intestinal lipid metabolism

Gut enterocytes are the entry points for dietary lipids into host metabolism. Enterocytes also defend the host against ingested pathogenic microbes via Duox-dependent production of ROS from the substrates NADPH and O₂ in response to bacterial-derived uracil in the gut lumen. Uracil stimulates TRAF3, AMPK and the serine/threonine kinase warts to inhibit mTORC1 in enterocytes, leading to a lipid metabolic switch that supports Duox activity by decreasing the NADPH-consuming process of fatty acid synthesis (Fig. 1). In response to enteric infection with Erwinia carotovorum spp. carotovorum strain 15 (Ecc15), enterocytes reduce expression of lipogenic enzymes, increase expression of lipolytic enzymes, and activate lipophagy; blocking these pathways reduces ROS production and enhances susceptibility to Ecc15 [130]. In contrast, oral infection of adult flies with V. cholerae leads to lipid accumulation in enterocytes. V. cholerae consume commensal-derived acetate, thereby depriving the host of acetate. This leads to intestinal steatosis and depletion of fat body lipids, suggesting impaired transport of dietary lipids to fat body. Supplementing the diet of V. cholerae-infected flies with acetate improves intestinal steatosis [117].

Infection and innate immune signaling alter local paracrine signaling that regulates lipid accumulation in enterocytes. In Drosophila, tachykinins secreted from gut enteroendocrine cells suppress lipogenesis and lipid accumulation in neighboring enterocytes [131]. Commensal-derived acetate activates Imd signaling in enteroendocrine cells to stimulate tachykinin production, and loss of Imd signaling in enteroendocrine cells induces intestinal lipid accumulation due to reduced tachykinin levels (Fig 1). Activation of Imd signaling in tachykinin-producing enteroendocrine cells is sufficient to rescue reduced growth, delayed larval development, and triglyceride accumulation in germ-free flies that lack commensal-derived acetate. In this way, innate immune signaling communicates health of the microbiota to the host to regulate growth and metabolism [132].

3.2.3. Lipid transport that facilitates infection and resolves inflammation

Lipid transport processes move lipids between organs, and lipid transporters play diverse roles during infection. The brain is relatively impervious to microbial infiltration due to the blood brain barrier, but Gram-positive Streptococcus agalactiae can enter the Drosophila larval brain through interactions between a bacterial surface lipoprotein and a host lipoprotein receptor, LpR2, that permits transcellular crossing of the blood brain barrier and infection of the central nervous system [133]. Another lipid transport process, lipid clearance, is a key component of the successful resolution of an immune response. The actions of Duox in the gut, NADPH oxidase in hemolymph, and phenoloxidases at the sites of infection or wounding help to kill and contain microbes and initiate wound healing. However, ROS produced by these enzymes threaten the host because they can cause lipid peroxidation that can ultimately damage DNA and protein via reactive aldehyde production. Infected flies remove lipids from hemolymph through the kidney-like Malpighian tubules via the stress-induced lipid-binding protein Materazzi. The action of Materazzi reduces lipid peroxidation in hemolymph of infected flies and promotes survival of infections, sterile injury and oxidative stress [134].

3.3. Carbohydrate metabolism in infected flies and phagocytic hemocytes

Compared with the wide-ranging effects of infection and innate immune signaling on lipid metabolism, less is known about how carbohydrate metabolism is regulated by infection. Infections with M. marinum, V. cholerae, and L. monocytogenes lead to progressive loss of stored glycogen [116,117,135], and wasp egg infection in Drosophila larvae leads to reduced glycogen levels in body wall musculature [136]. In fruit fly larvae parasitized by wasp eggs, glycogen accumulation in fat body is suppressed and glycogenolysis and glucose output are stimulated; these processes provide energy for lamellocyte proliferation that is required for wasp egg encapsulation and melanization [137]. Similarly, glycogen breakdown in fat body during Streptococcus pneumoniae infection promotes pathogen resistance [138].

Glucose metabolism regulates cellular immunity in adult flies infected with S. pneumoniae. During the acute phase of septic S. pneumoniae infection, phagocytic plasmatocytes increase glucose uptake, induce expression and activity of glycolytic enzymes such as phosphoglucose isomerase and lactate dehydrogenase, and increase lactate production and NADH levels, all hallmarks of aerobic glycolysis (Fig. 4). Aerobic glycolysis is terminated during the resolution stage of infection, but loss of lactate dehydrogenase in phagocytic plasmatocytes strongly reduces survival of infected flies, with increased bacterial load indicative of reduced pathogen resistance [139]. What plasmatocyte function(s) the shift to aerobic glycolysis serves is not yet known; however, this work opens new avenues for investigation of distinct metabolic phenotypes in Drosophila cellular immunity [140].

3.4. Targeting protein synthesis for host resistance and pathogen virulence

An effective humoral immune response requires a massive induction of AMP synthesis and high functional capacity of the protein secretory pathway. During infection, AMPs reach concentrations of ~1–100 μM in hemolymph, concentrations that match and, for the most part, greatly exceed their measured LD₅₀ values for neutralizing bacteria and fungi [80,141]. Many AMPs are induced simultaneously during infection, meaning that many millions of individual proteins are produced throughout an immune response. Protein synthesis is energetically costly, requiring hydrolysis of four high-energy phosphate bonds per peptide bond formed. Consequently, cells regulate protein synthesis tightly, driving it at high rates to promote growth, for example, when nutrients are plentiful and anabolic signaling pathways prevail [142]. It is possible that cellular energy normally used for growth is diverted toward immune processes such as AMP synthesis in response to infection. Although this hypothesis remains to be formally tested, it is supported by the finding that activation of Toll signaling in individual larval fat body cells dominantly reduces cell size even in the presence of constitutive PI3K signaling [122] (Fig. 2).

As in mammals, protein synthesis in flies is regulated by the protein kinase complex mTORC1, which activates S6 kinase and inhibits eukaryotic initiation factor 4E binding protein (4E-BP, encoded by Thor in Drosophila) to promote 5’-cap-dependent mRNA translation [143, 144]. Paradoxically, Thor is induced upon infection, and Thor promotes survival during infection with various microbes [145]. In Thor mutants, AMP gene transcription following infection is normal, but AMP translation is impaired. Close examination of AMP mRNAs revealed that they enable cap-independent translation, likely via internal ribosome entry sequences identified in their 5’-untranslated regions. Induction of Thor during infection imposes a bias toward cap-independent translation and thus high-level synthesis of AMPs [146] (Fig. 2). In agreement with these findings, rapamycin treatment to inhibit mTORC1 enhances immune function and AMP expression in adult flies infected with Gram-negative Pseudomonas aeruginosa [147]. Interestingly, the Imd pathway transcription factor Relish is required to repress expression of Thor in midgut enterocytes when dietary protein is low. This may limit AMP production to favor associations between the host and its microbiome to promote nutrient homeostasis [148].

While a selective translational block can be beneficial during infection, some pathogens disrupt host defense by blocking host protein synthesis [149]. For example, in adult flies infected orally with P. entomophila, Duox-dependent ROS production in the gut activates the kinases Gcn2 and AMPK, leading to translation inhibition. Blocking translation inhibits production of host factors that promote tissue repair following infection, thus enhancing P. entomophila pathogenicity [150] (Fig. 1). Oral infection of Drosophila larvae with virulent Ecc15 indirectly disrupts host protein synthesis and consequently whole-animal growth by impeding digestion of dietary protein. The presence of Ecc15 in the gut leads to induction of immune genes while repressing expression of digestive peptidases, a class of genes that is positively regulated by the microbiota in uninfected animals [151].

3.5. Influence of diet on innate immune function

Dietary nutrients influence the outcome of infection, often in a microbe-specific manner. Flies infected with either of two intracellular pathogens, L. monocytogenes or S. typhimurium, voluntarily reduce food intake. Experimentally decreasing caloric intake via dietary restriction from the larval stage onward differentially affects outcomes of these infections. Dietary restriction of flies infected with L. monocytogenes decreases microbial resistance, characterized by increased bacterial load and impaired antimicrobial peptide expression and melanization, thereby reducing survival. However, the same calorie-restricted diet leads to increased median survival characterized by increased microbial tolerance in flies infected with S. typhimurium, although the mechanism for increased tolerance induced by diet restriction is unknown [92].

Titration of the two major components of the lab fruit fly diet, sucrose and yeast, differentially affects nutrient storage, lifespan, and fecundity, with optimal effects on each parameter occurring at different ratios of sugar to protein [152]. The balance of dietary carbohydrate and protein shapes the immune response. For example, in cotton leafworm (Spodoptera littoralis) larvae, lysozyme responses to infection peak with high dietary protein, while phenoloxidase responses are highest with increased dietary sugar [153]. In adult fruit flies infected with P. aeruginosa or Staphylococcus aureus, restriction of dietary yeast improves median survival time. This occurs via stabilization of the transcription factor myc, downstream of mTORC1 inhibition, leading to enhanced humoral immunity and increased pathogen resistance [147].

Manipulating dietary sugar also modulates immune function. Systemic infection with P. rettgeri results in higher pathogen loads in flies reared for several generations on a 10% glucose diet compared with flies reared on 2.5% glucose [154]. Similarly, adult flies fed a 1 M sucrose diet exhibit reduced AMP gene expression and greater lethality in response to septic infection with P. aeruginosa compared with flies fed 0.15 M sucrose [155]. In contrast, adult female flies fed a holidic diet supplemented with glucose (~0.6 M) exhibited increased median survival time during enteric infection with V. cholerae compared with flies fed a control diet (~0.05 M glucose) [156]. This high glucose diet increases basal AMP expression and induces expression of cell junction components that form the epithelial barrier of the intestine [157]. Interestingly, adaptation of Drosophila larvae to low nutrient diets during experimental evolution leads to susceptibility to P. entomophila without defects in AMP gene expression or ROS production but instead with clear defects in intestinal barrier function [158], suggesting a trade-off between these two critical gut functions.

4. Signaling that regulates metabolism and immune function

4.1. Immune responses impair insulin signaling

In Drosophila, seven Dilps can bind to a single known insulin receptor to activate a conserved PI3K-Akt signaling pathway that regulates growth and metabolism. Flies lacking individual Dilps or components of the intracellular insulin signaling pathway exhibit reduced growth and altered carbohydrate, lipid, and protein metabolism [159,160]. Infection and signaling through the Toll or Imd pathways negatively regulate insulin signaling. Infection of adult flies with M. marinum leads to reduced whole-animal Akt phosphorylation [116]. Adult flies infected with M. luteus or B. bassiana exhibit reduced Akt phosphorylation in the fat body [118]. Similarly, enteric infection with V. cholerae disrupts PI3K activation in fat body and strongly inhibits whole-animal Akt phosphorylation [117] (Fig. 2). Genetic activation of Toll signaling in larval fat body via tissue-specific expression of constitutively-active Toll^10b receptors disrupts insulin signaling and impairs whole-animal growth [118]. Similarly, expression of constitutively-active Imd in larval fat body impairs whole-animal growth and leads to reduced Akt phosphorylation on the mTORC2-regulated hydrophobic motif site [121]. Impaired fat body insulin signaling caused by Toll pathway activation is due to intracellular insulin resistance (Fig. 3). Toll acts cell-autonomously to block insulin signaling at or downstream of the serine/threonine kinase Pdk1, which phosphorylates Akt on its activation loop. Expressing Akt with a phospho-mimicking mutation at this site is sufficient to rescue cell and animal growth as well as triglyceride storage when Toll^10b is expressed in larval fat body [122].

The relationship between innate immune signaling and insulin signaling is reciprocal; manipulating insulin signaling affects immune responses to infection. Diminishing insulin signaling via a loss-of-function mutation in chico, the Drosophila insulin receptor substrate homolog, improves survival during infection with P. aeruginosa or E. faecalis in adult flies [161]. AMP expression is basally elevated in chico mutant larvae [162] and in larval fat body with insulin receptor knockdown [155], perhaps due to de-inhibition of Thor that would be predicted with loss of insulin signaling (refer back to Section 3.4). Elevated AMP expression may contribute to improved survival in response to P. aeruginosa observed in flies with GAL4-mediated knockdown of the insulin receptor in fat body from the larval stage onward [155].

Hemocytes contribute to the regulation of insulin signaling in Drosophila. Ablation of Hemolectin-positive (Hml+) plasmatocytes in Drosophila larvae reduces whole-animal growth and triglyceride storage with impaired PI3K activity and increased Foxo nuclear translocation observed in fat body [163,164]. Loss of hemocytes leads to retention of Dilp2 and Dilp5 in brain insulin producing cells and induces expression of AMPs in fat body. However, whether active Toll and/or Imd signaling in fat body contributes to impaired insulin signaling in larvae lacking hemocytes is not known [163]. Hemocytes that remain after Hml+ plasmatocyte ablation secrete the cytokine upd3, which activates JAK-STAT signaling in fat body. Loss of upd3 on this background increases numbers of remaining hemocytes and decreases nuclear Foxo in fat body, suggesting that upd3 signaling either directly or indirectly disrupts insulin signaling in fat body [164].

The innate immune system responds not only to infection and wounding, but also to tissue damage caused by UV irradiation. The latter leads to melanization, hemocyte proliferation, induction of the cytokines upd2 and upd3, and activation of hemocyte JAK-STAT signaling. UV irradiation inhibits insulin signaling in fat body in a hemocyte-dependent manner, and reduced whole-animal insulin signaling in chico heterozygotes improves survival of UV-mediated DNA damage, reminiscent of phenotypes described above. Interestingly, subsequent induction of Relish expression in fat bodies from UV-irradiated larvae mediates repression of Foxo and leads to increased Dilp seceretion from brain insulin-producing cells, thereby restoring animal growth [165].

The impaired growth in animals with active innate immune signaling in fat body is due in part to disrupted production of insulin receptor ligands. Although circulating levels of Dilp2, secreted by brain insulin-producing cells, are not altered by fat body Toll signaling [122], infection with E. faecalis or expression of constitutively-active Toll^10b receptors in fat body decreases transcript levels of the insulin-like growth factor 1 homolog Dilp6 (Fig. 2). Toll signaling in fat body reduces circulating Dilp6 levels by 75%, and restoring Dilp6 expression in fat body rescues whole-animal growth, but not triglyceride storage, in flies with active fat body Toll signaling [166].

4.2. Metabolic transcription factors that shape the response to infection

Transcription factors that regulate growth and metabolism modulate these processes during infection and in response to innate immune signaling. A clear example is forkhead box, sub-group O (Foxo), a driver of catabolic metabolism that is negatively regulated by insulin signaling. Loss-of-function mutations in Foxo rescue impaired glycogen storage and reduced lipogenic gene expression, although not impaired lipid storage, in M. marinum-infected flies [116]. In the adult fat body, Relish represses Foxo at the promoter of the lipolytic gene brummer during starvation and at the promoter of the translational repressor Thor in adult flies fed a high sugar, low yeast diet [120,148] (Fig. 3). A number of AMP genes contain putative insulin response elements and Foxo binding sites in their promoters, in addition to canonical NF-κB response elements. Starvation of flies or expression of constitutively-active Foxo induces AMP expression in fat body and barrier epithelia in the absence of infection and independently of the Toll and Imd pathways (Fig. 1). This may serve the role of strengthening defenses in stressed animals [162]. Oral infection with Gram-negative Serratia marcescens activates AMP expression in gut in a Foxo-dependent manner, and loss of Foxo reduces survival in infected flies [167]. In the aging gut, Foxo represses expression of PGRP-SC2, a secreted pattern recognition receptor that functions as a negative regulator of Imd signaling, due to its amidase domain that destroys Dap-type peptidoglycan. Loss of PGRP-SC2 leads to commensal overgrowth that shortens lifespan [168].

The transcription factor Myocyte enhancer factor 2 (Mef2) acts in the fat body to regulate nutrient storage and the response to infection. Knocking down Mef2 in fat body reduces transcript levels of genes encoding lipogenic and glycogenic enzymes; flies with loss of Mef2 in fat body store very little triglyceride or glycogen but are euglycemic. Mef2 binding sites are also found in promoters of AMP genes, and loss of Mef2 in fat body reduces expression of multiple AMPs in flies infected simultaneously with M. luteus and E. coli. Loss of Mef2 in fat body renders flies susceptible to M. marinum, L. monocytogenes, E. cloacae and Candida albicans infections, with increased bacterial numbers observed in E. cloacae and M. marinum infections compared with wild type flies. Whether Mef2 activates anabolic or immune genes depends on phosphorylation in its DNA binding domain, with phospho-Mef2 directed to anabolic promoters, perhaps in a S6K dependent manner [169] (Fig. 3). Whether the decreased resistance to infection in flies with knockdown of Mef2 in fat body is exacerbated by their metabolic defects is unknown.

A key component of the humoral immune response to systemic infection is the secretion of large quantities of AMPs from the fat body. This secretory response is transcriptionally regulated downstream of the Toll and Imd pathways by the transcription factors Xbp1 and CrebA. The unfolded protein response mediator Xbp1 is activated by Ire1-dependent splicing, and infection with E. faecalis induces Xbp1 splicing in fat body. Xbp1 contributes to the acute induction of Kennedy pathway enzymes such as Pcyt1 and eas in response to Toll pathway activation [119]. Infections with a variety of bacteria induce expression of CrebA. Loss of CrebA in fat body strongly reduces survival of Gram-positive and Gram-negative bacterial infections, with a smaller bacterial load upon death observed compared with wild type flies, indicating reduced tolerance of infection. Genes encoding components of the secretory pathway are decreased upon CrebA knockdown, indicating that an important role of this transcription factor is preventing ER stress during infection [170].

Hypoxia inducible factor 1α (HIF1α) is a key transcriptional regulator of glycolytic metabolism. HIF1α is stabilized and activated in response to hypoxia or, in normoxic conditions, in response to immune signaling and metabolites such as succinate [171]. In Drosophila, the HIF1α homolog Sima is required for plasmatocytes to induce aerobic glycolysis during S. pneumoniae infection, and expression of Sima in plasmatocytes increases resistance of flies to S. pneumoniae [139] (Fig. 4). In Drosophila larvae parasitized by wasp eggs, succinate accumulation in lymph gland hemocyte progenitors stabilizes Sima. Sima drives lamellocyte differentiation, and loss of Sima expression in hemocyte progenitors reduces lamellocyte population size in parasitized animals [140].

4.3. Secreted regulators of immune function and metabolism

For many infections, a robust immune response requires contributions from multiple cell types: fat body cells, hemocytes, and gut enterocytes. Coordination of immune and metabolic functions among organs in the fly is critical during infection, and a number of peptide hormones, cytokines, and secreted small molecule mediators participate in both the immune response and regulation of nutrient homeostasis. However, whether immune functions of these factors rely on their control of metabolism is largely unexplored. Here, the immune and metabolic roles of adenosine secreted from plasmatocytes and the fat body-derived cytokines Eiger and Dawdle are described.

Circulating levels of the purine adenosine rise in response to cell stress, and binding of extracellular adenosine to G protein-coupled receptors in target organs mediates a number of cytoprotective effects [172]. Drosophila larvae with chronically elevated adenosine levels exhibit hyperglycemia, impaired nutrient storage, precocious hemocyte differentiation, fat body disintegration, and a delay in pupation [173,174]. However, regulated release of adenosine from plasmatocytes in fruit fly larvae parasitized by wasp eggs and in adult flies infected with S. pneumoniae mediates a metabolic shift from fat body glycogen storage to glycogen breakdown and trehalose output (Figs. 2 and 3). This metabolic switch fuels larval lamellocyte proliferation, enabling wasp egg melanization and encapsulation, and promotes resistance to S. pneumoniae in adult flies. This process is mediated by secretion of adenosine from hemocytes and their progenitors in the lymph gland (Fig. 4). Increased trehalose output in response to wasp infection requires expression of the G_s-coupled adenosine receptor (AdoR) in fat body [137,175]. AdoR mutant flies are susceptible to sublethal doses of S. pneumoniae with reduced glycogen mobilization and reduced hemolymph sugar levels at early stages of infection [138].

Eiger is the sole Drosophila tumor necrosis factor (TNF) homolog. In flies, Eiger is produced as a transmembrane protein that is cleaved by the metalloprotease TNF-α converting enzyme to yield a secreted ligand that binds the membrane receptors Wengen and Grindelwald to activate c-Jun N-terminal kinase signaling [176–181]. Cell types that secrete Eiger include plasmatocytes and the fat body [176,182,183]. Canonical roles for Eiger include induction of apoptosis [178,180] (but see [184]), but this cytokine also participates in the immune response against microbial pathogens. However, whether it plays a protective or detrimental role depends on the type of infection. For extracellular pathogens, such as B. bassiana, E. faecalis, S. aureus, S. pneumoniae, and Burkholderia cepacia, Eiger-dependent mechanisms contribute to immune function, and eiger mutants succumb to these infections rapidly. In contrast, loss of eiger increases median survival time during infection with intracellular pathogens such as L. monocytogenes and S. typhimurium, suggesting that Eiger can drive pathology when pathogens are protected from classical humoral immune signaling [89,185]. Eiger is induced in fat body during infection, and fat body-specific knockdown of Eiger increases mean survival time during S. typhimurium infection, with a strong reduction in feeding rate that may drive tolerance (refer back to Section 3.5) [182]. Eiger also regulates growth and metabolism independently of infection. In fruit fly larvae, Eiger is secreted from the fat body in response to low protein diets and inhibits secretion of Dilps from brain insulin-producing cells thereby reducing whole-animal growth, and in adult male flies, secretion of Eiger from fat body promotes resistance to starvation stress [176]. Genetic screening for suppressors of Eiger identified a number of glycolytic and citric acid cycle enzymes as well as components of the electron transport chain that when knocked down rescue Eiger-induced cell death, possibly by reducing production of ROS, indicating that metabolism contributes to Eiger-dependent phenotypes [186]. Whether the metabolic functions of Eiger contribute to its regulation of immune function is unknown.

Like Eiger, the cytokine Dawdle regulates metabolic functions and plays a role in the immune response. Dawdle is a homolog of mammalian transforming growth factor-β that signals through the type I receptor Baboon and the type II receptor Punt to activate the Smad2/3 homolog Smox [187–189]. Dawdle is expressed widely, including in fat body and muscle [190,191], and it acts in an endocrine manner to regulate metabolism. In particular, Dawdle promotes secretion of Dilps from brain insulin producing cells, and, accordingly, dawdle mutant larvae are hyperglycemic. These larvae also have increased triglyceride and glycogen levels compared with wild type animals [192]. Independently of its effect on Dilp secretion, Dawdle regulates hemolymph pH, perhaps by negatively regulating expression of components of the citric acid cycle and the electron transport chain that produce metabolic acids such as citrate and succinate [192]. Glucose-rich diets induce expression of Dawdle in larval and adult fat body, and secreted Dawdle mediates glucose repression of amylases that digest dietary sugars [193,194]. Dawdle affects basal immune function and has pathogen-dependent effects on survival of infection. Infection of adult flies with M. luteus or L. monocytogenes activates expression of Dawdle. Flies with ubiquitous knockdown of Dawdle exhibit melanotic tumors in the absence of infection, and elevated expression of Dawdle reduces survival in flies infected with L. monocytogenes [195]. In adult flies, infection with the parasitic nematodes Heterorhabditis gerrardi or Heterorhabditis bacteriophora and their respective symbiotic bacteria, Photorhabdus asymbiotica and Photorhabdus luminescens, activates Dawdle expression. Adult flies homozygous for a transposon insertion in the dawdle locus exhibit decreased survival of nematode infections with elevated nematode numbers compared with control flies [196]. Uninfected dawdle mutant larvae exhibit reduced hemocyte numbers and increased basal prophenoloxidase activity compared with control animals. Infection with the parasitic nematode H. gerrardi leads to elevated Duox expression in dawdle mutants but no significant effects on survival of infection [197]. Whole-animal trehalose levels are elevated in dawdle mutants [198], in keeping with hyperglycemia observed in previous work [192]. Infection with the nematode H. bacteriophora lowers trehalose levels in dawdle mutant larvae only when the nematode is associated with its symbiotic bacteria P. luminescens, a Drosophila pathogen [198]. As with Eiger, whether the metabolic processes regulated by Dawdle influence the outcome of infections has not been determined.

5. Conclusions

Work in flies defined the key molecular components of conserved innate immune signaling pathways, and it has since extended our understanding of how host metabolism is altered by infection and immune signaling [199]. Many key questions about immunometabolic regulation remain unanswered: How does metabolism change over the course of an infection, from detection of a pathogen to resolution of the immune response? How do biological variables of the host shape immune function? What are the mechanisms employed by immune signaling pathways to regulate metabolism? How do endocrine signals coordinate metabolic regulation with the needs of the immune system? And, ultimately, how do shifts in metabolism contribute to or impair pathogen resistance and tolerance?

Drosophila is well suited to answering these questions. Flies can be infected with a variety of microbes, and their innate immune signaling pathways are well characterized. Measurements of bacterial load, bacterial load upon death, hemocyte cell numbers and phagocytic capacity, wound healing and clotting, and AMP expression allow precise determination of immune function. Metabolic pathways are highly-conserved from flies to man and are increasingly well characterized in Drosophila. Finally, Drosophila is a powerful genetic model organism, with a host of tools that allows tissue-and temporally-specific gene manipulation. These features are key to determining how host metabolism shapes immune function and the response to infection.

How does metabolism change over the course of an infection, from detection of a pathogen to resolution of the immune response? Infections are dynamic. The load of microbes in the host changes over time, as does the host immune response [200]. Examining multiple time points over the course of an infection reveals how metabolism changes to support immune function [119,139,170]. Ultimately and ideally, infections should be cleared, inflammation should resolve, and tissue damage should be repaired [201]. In particular, how metabolism is regulated during the resolution phase of the immune response is not yet well understood. Additionally, whether metabolic changes in each stage of infection – pathogen detection, production of immune effectors and microbe killing, and resolution – serve immediate immune functions or prepare the host for later stages of the immune response is largely unknown. Another aspect of the response to infection that can be studied at high resolution using Drosophila is how distinct pathogens shape immunometabolic phenotypes. Adult flies do not respond in a uniform way to all Gram-positive or all Gram-negative bacteria, nor does pathogen virulence correlate in a predictable manner with the number of genes that change with infection [170]. The type of microbe and its characteristics [203], the infectious dose used, the delivery route (enteric infection versus septic injury), and the diet of flies in a particular lab are factors that can influence metabolic outcomes during the immune response.

How do biological variables of the host shape immune function? Two key biological variables that determine metabolic responses to infection are sex and life stage. Male and female flies differ in their immune responses to infection, particularly after mating [202]. Flies exhibit sex differences in metabolism, with elevated expression of the lipolytic enzyme brummer in males partially accounting for their low triglyceride stores compared with females [124]. Male and female flies differ in their sensitivity to insulin-like peptides [204,205], and in tissue repair and proliferation in gut [27]. Whether inherent sex-differences in metabolism influence observed sex-differences in immune function [84,93,146–149] or vice versa remains to be determined. Inherent differences in metabolism related to growth and nutrient storage in Drosophila larvae and adults may alter responses to infections and immune signaling. For example, genetic activation of Toll or Imd signaling in the larval fat body reduces triglyceride storage [83,85], while activating these pathways in adult fat body increases triglyceride levels [120,123]. The mechanism for this difference is not known.

What are the mechanisms employed by immune signaling pathways to regulate metabolism? Many metabolic genes change in expression during infection, but in most cases, how they are regulated at the transcriptional level is unknown. A key question to answer is whether core transcription factors in the Toll and Imd pathways, Dif, dorsal and Relish, directly bind to metabolic gene promoters to activate or repress expression or whether changes in gene expression are secondary to changes in other transcriptional regulators. Post-transcriptional regulation of metabolism is largely unexplored, but it is likely to be a key component of immunometabolic responses. The use of genetic approaches to tag endogenous proteins will enable studies of protein levels, subcellular localization and enzyme activity and should help to address this component of metabolic regulation during infection. Going forward, the use of advanced metabolic approaches such as metabolomics and metabolic flux experiments will enhance our understanding of metabolism during immune signaling [206]. Linking innate immune signaling to mechanisms of metabolic regulation may well become another contribution of Drosophila to our understanding of host-pathogen interactions.

How do endocrine signals coordinate metabolic regulation with the needs of the immune system? A large network of hormones and neuropeptides regulates many aspects of physiology, including metabolism, in Drosophila larvae and adults [207]. Some of these hormones, such as Dawdle and Eiger, regulate both immune function and metabolism. However, whether the immune-regulatory functions of a hormone rely on its control of metabolism is largely unexplored. Similarly, whether effects of diet on immune responses are direct or depend on changes on endocrine or neural signaling is unknown.

Finally, how do shifts in metabolism contribute to or impair pathogen resistance and tolerance? As described throughout this review, changes in triglyceride and glycogen storage and mobilization, phospholipid metabolism, protein synthesis and alterations in circulating sugars accompany infection and activation of innate immune signaling. Metabolic gene expression is also regulated during the immune response. What these changes do for the animal, and whether they impact resistance to or tolerance of pathogens is the ultimate question. Using the powerful genetic tools of Drosophila to manipulate gene function and metabolism in time and space, we should be able to determine how animals harness metabolism to fight pathogens and how metabolic processes might become detrimental if they persist after an infection is cleared.

Abbreviations:

AMP

antimicrobial peptide

Dilp

Drosophila insulin-like peptide

ROS

reactive oxygen species