Macrophages are the immune system's first line of defense against infection. These immune cells can be found in all tissues and organs, watching for signs of disease-causing agents and targeting them for destruction. Maintaining macrophages costs energy, so to minimize waste, these cells spend most of their lives in 'low power mode'. When macrophages sense harmful bacteria, they rapidly awaken and trigger a series of immune events that protect the body from infection. However, to perform these protective tasks macrophages need a sudden surge in energy.

In mammals, activated macrophages get their energy from aerobic glycolysis – a series of chemical reactions normally reserved for low oxygen environments. Switching on this metabolic process requires a protein called hypoxia inducible factor 1α (HIF-1 α), which switches on the genes that macrophages need to generate energy as quickly as possible. Macrophages then maintain their energy supply by sending out chemical signals which divert glucose away from the rest of the body.

Fruit flies are regularly used as a model system for studying human disease, as the mechanisms they use to defend themselves from infections are similar to human immune cells. However, it remains unclear whether their macrophages undergo the same metabolic changes during an infection.

To address this question, Krejčová et al. isolated macrophages from fruit flies that had been infected with bacteria. Experiments studying the metabolism of these cells revealed that, just like human macrophages, they responded to bacteria by taking in more glucose and generating energy via aerobic glycolysis. The macrophages of these flies were also found to draw in energy from the rest of the body by raising blood sugar levels and depleting stores of glucose. Similar to human macrophages, these metabolic changes depended on HIF1α, and flies without this protein were unable to secure the level of energy needed to effectively fight off the bacteria.

These findings suggest that this metabolic switch to aerobic glycolysis is a conserved mechanism that both insects and mammals use to fight off infections. This means in the future fruit flies could be used as a model organism for studying diseases associated with macrophage mis-activation, such as chronic inflammation and autoimmune diseases.

Macrophages represent a highly specialized and versatile population of cells that occur in all animals and perform a diversity of functions (Lim et al., 2017). In the absence of an activating stimulus, macrophages reside as quiescent sentinel cells that have minimal metabolic requirements (Davies and Taylor, 2015). In response to extracellular triggers, however, macrophages undergo a dramatic change in behavior that coincides with an enhanced metabolic rate and increased energy demands (Pearce and Pearce, 2013). In this regard, the manner by which macrophages mount a response is dictated by the activating stimuli, which include tissue-damage-, pathogen- or microbe-associated molecular patterns (DAMPs, PAMPs and MAMPs, respectively), as well as signaling molecules that are secreted by other cells, such as cytokines. Each challenge requires the induction of specific metabolic and physiological processes that allow for an adequate immune response (Kawai and Akira, 2011) – cellular changes that are collectively known as a polarization phenotype.

Macrophages polarize into bactericidal (M1) or healing (M2) functional phenotypes characterized mainly by metabolism (Mills et al., 2000). M1 and M2 polarization phenotypes utilize distinct ways of generating ATP (glycolysis vs. oxidative phosphorylation) and metabolizing arginine (NO synthesis vs. the ornithine cycle) (O'Neill and Pearce, 2016). Nowadays, the whole spectrum of polarization phenotypes corresponding to particular functions has been described (Mosser and Edwards, 2008; Martinez and Gordon, 2014). Perhaps the most dramatic change in macrophage metabolism associates with the M1 bactericidal phenotype, in which cells increase both glucose consumption and lactate production independently of oxygen concentration - a phenomenon known as aerobic glycolysis (AG) (Warburg et al., 1927; Warburg, 1956). The resulting metabolic program promotes increased glucose catabolism, thus allowing M1 macrophages to generate enough of the ATP and glycolytic intermediates necessary for elevated phagocytic cell activity (Liberti and Locasale, 2016). This shift in cellular metabolism towards AG appears to be a determining factor in macrophage function and in the development of the pro-inflammatory phenotype (Galván-Peña and O'Neill, 2014).

Hypoxia inducible factor 1α (HIF1α) is a key regulator of AG within macrophages. Although this transcription factor is normally degraded in the presence of oxygen, the triggering of either Toll-like receptor (TLR) or Tumor necrosis factor receptor (TNFR) signaling within macrophages activates Nuclear factor kappa-B (NFĸB) and stabilizes HIF1α independently of oxygen availability (Siegert et al., 2015; Jung et al., 2003). This normoxic HIF1α stabilization promotes the expression of genes that are under the control of hypoxia response elements (HREs), many of which are involved in cellular metabolism, cell survival, proliferation, and cytokine signaling (Dengler et al., 2014). In this regard, two of the key HIF1α target genes encode the enzymes pyruvate dehydrogenase kinase (PDK) and lactate dehydrogenase (LDH), which together shunt pyruvate away from the mitochondria and maintain NAD⁺/NADH redox balance independently of oxidative phosphorylation. Inhibition of both HIF1α and LDH represents an efficient experimental strategy to direct cellular metabolism from AG to oxidative phosphorylation in both mice and Drosophila (Allison et al., 2014; Geeraerts et al., 2017), demonstrating the crucial role of these enzymes in this metabolic switch.

Although pyruvate metabolism within the tricarboxylic acid (TCA) cycle is limited during AG, the TCA intermediates are essential for many cellular processes. Therefore, cells under AG rely on feeding the TCA cycle with glutamine, causing a TCA cycle to be ‘broken’ (Langston et al., 2017). Such a dramatic change in mitochondrial metabolism leads to significant imbalances in the cytosolic accumulation of TCA metabolites (such as NO, succinate, fumarate, L-2-hydroxyglutarate) that further contribute to HIF1α stabilization (Bailey and Nathan, 2018). While this feedback maintains AG, it simultaneously makes it dependent on a sufficient supply of nutrients from the environment (Iommarini et al., 2017).

Macrophages employing AG must consume sufficient carbohydrates to support biosynthesis and growth. In order to ensure an adequate supply of sugar and other nutrients, these cells produce signaling molecules that affect systemic metabolism in order to secure enough energy for themselves – a concept recently defined as selfish immune theory (Jeong et al., 2003; Straub, 2014). According to this theory, signaling molecules released by immune cells induce systemic metabolic changes such as hyperglycemia and systemic insulin resistance to increase the titer of nutrients that are available for the immune response and to limit their consumption by other tissues and processes (Dolezal, 2015). As many of these signaling molecules are direct HIF1α transcriptional targets, HIF1α stabilization directs the cellular metabolism while it simultaneously induces the expression of genes that have an impact on the whole systemic metabolism (Peyssonnaux et al., 2007; Imtiyaz and Simon, 2010). Thus, macrophages not only phagocytose cells but they also regulate the systemic metabolism of an organism.

Drosophila macrophages, like those of mammals, serve an essential role in the immune system and are capable of responding to a wide array of stimuli, ranging from pathogenic bacteria and fungi to the corpses of apoptotic cells (Wood and Martin, 2017; Sears et al., 2003; Govind, 2008). The mechanism of the bactericidal function itself is highly conserved at the molecular level between Drosophila and mammalian macrophages. In both Drosophila and mammals, two central signaling pathways, Toll and Imd (TLR and TNFR functional homologs) are triggered in response to pathogenic stimuli (Valanne et al., 2011; Buchon et al., 2014; Lemaitre et al., 1996). The Toll and Imd pathways induce the NFĸB signaling in Drosophila, so we can assume that the phagocytic role of macrophages could be accompanied by stabilization of the HIF1α ortholog, Similar (Sima), hereafter referred to as Hif1α (van Uden et al., 2011). Indeed, normoxic stabilization of Hif1α followed by its nuclear localization and increased expression of HRE-controlled genes can induce metabolic changes that are typical of AG (Romero et al., 2008; Li et al., 2013; Liu et al., 2006; Herranz and Cohen, 2017; Eichenlaub et al., 2018). Even though the HRE-controlled genes frequently appear in transcriptomic data for activated insect macrophages (Irving et al., 2005; Johansson et al., 2005), the direct role of Hif1α in the macrophages has not yet been tested.

Considering that the molecular mechanisms that control macrophage activation are similar in both Drosophila and humans, it seems logical that the metabolic changes that occur within these cells would also be comparable, but the metabolism of insect macrophages remains poorly understood. Here, we address this question by analyzing in vivo metabolic and transcriptional changes in adult Drosophila phagocytic macrophages by employing a model of Streptococcus-pneumoniae-induced sepsis. The well-defined progress of this infection allowed us to distinguish three phases of the immune response according to the changing dynamics of bacterial growth (acute, plateau, and resolution phase) (Figure 1A). The acute phase lasts for the first 24 hr, during which the streptococcal population is rapidly growing and its abundance must be limited by phagocytosis to avert early death (Pham et al., 2007; Bajgar and Dolezal, 2018). The established equilibrium between continuous bacterial growth and host bacterial killing results in the plateau phase lasting for the next four days. At the end of this period, the immune system of the host surmounts the infection and clears the majority of the pathogens. The following resolution phase (120 hr post-infection (hpi) and later) is essential for macrophage-mediated clearance of bacterial residues and for the reestablishment of homeostasis (Bajgar and Dolezal, 2018; Chambers et al., 2012).

Figure 1

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To analyze processes that are characteristic of highly active phagocytic macrophages in Drosophila, we compared the attributes of acute phase macrophages (APMФs) with those of macrophages from uninfected individuals and resolution-phase macrophages (RPMФs). Using a previously described hemolectin-driven GFP (HmlGal4 >UAS eGFP) (Jung et al., 2005), we isolated Drosophila adult macrophages (approximately 15,000 cells/replicate) and analyzed the metabolic and transcriptional responses that are induced within these cells upon infection (Figure 1B). Our approach revealed that Drosophila macrophages respond to the acute phase of bacterial infection by increasing glucose uptake, elevating glycolytic flux, and producing lactate. Moreover, as in mammals, the activation and maintenance of AG within Drosophila macrophages depend on Hif1α, and require elevated Ldh activity. We also demonstrate that the induction of AG within Drosophila macrophages leads to a change in systemic carbohydrate metabolism. Overall, our findings demonstrate that Drosophila macrophages must induce both autonomous and systemic changes in carbohydrate metabolism to mount a proper bactericidal function and to resist infection.

Mammalian macrophages stimulated by bacteria have been shown to rewire their metabolism temporarily towards AG in order to develop an adequate bactericidal response (Olenchock et al., 2017; Nonnenmacher and Hiller, 2018; Browne et al., 2013). Although well established in mammals, such metabolic adaptation has not been experimentally tested in insect macrophages to date. We show here that Drosophila macrophages that are activated by bacterial infection undergo a dramatic remodeling of cellular metabolism. We demonstrate that acute-phase macrophages exhibit hallmarks of AG, such as elevated uptake of glucose, increased expression and activity of glycolytic genes, elevation of NADH levels, and preferential LDH-mediated conversion of pyruvate to lactate. Through macrophage-specific gene knockdown, we identified Hif1α to be essential for the induction of increased glycolytic flux as well as for the increased activity of LDH. Both Hif1α and Ldh are necessary for the full development of infection-induced changes in systemic carbohydrate metabolism and for resistance to bacterial infection.

A major takeaway of our work is that the cellular response to bacterial infection is an energetically challenging process that imposes significant metabolic demands upon the host. Our findings demonstrate that Drosophila macrophages meet these metabolic demands by inducing AG during the acute phase of S. pneumoniae infection, as evidenced by the increased expression of glycolytic enzyme genes and elevated NADH levels. This increase in LDH enzyme activity in the absence of elevated TCA cycle activity suggests that macrophages preferentially convert pyruvate to lactate and is consistent with the elevated concentration of lactate observed in hemolymph. However, we find that this metabolic adaptation is temporary, as AG is terminated during the resolution phase of infection. This latter observation is important because it reveals that macrophages temporally regulate metabolic flux throughout an infection and because it establishes Drosophila as a powerful model for exploring the molecular mechanisms that control immune cell metabolism.

Our findings also extend the similarities between fly and mammalian models of macrophage polarization, as we identified Hif1α and Ldh as being crucial for the establishment and maintenance of AG in acute-phase macrophages. The importance of these factors is demonstrated by the macrophage-specific Hif1α knockdown experiment described above, in which many of the hallmark characteristics of AG, including expression of the Ldh gene, were abolished. This finding highlights the conserved and ancient role for Hif1α in regulating the switch between glycolytic and oxidative metabolism (Webster, 2003), and suggests that this function evolved as a means of allowing cells to adapt quickly to changing physiological conditions and cell-specific metabolic needs. The role of Hif1α in regulating this switch is of significant interest because, although this transcription factor is classically associated with the response to hypoxia, our study adds to the growing list of examples in which Hif1α remodels cellular metabolism in the context of cell proliferation, activation, and competition, even under normoxic conditions (Miyazawa and Aulehla, 2018). Moreover, our finding is particularly intriguing in light of the fact that Hif1α also serves a key role in promoting AG in neoplastic tumor cells (Herranz and Cohen, 2017; Eichenlaub et al., 2018; Wang et al., 2016). Therefore, our studies of fly macrophages provide a new in vivo system in which we can study how Hif1α promotes cell activity by modulating central carbon metabolism.

While Hif1α drives AG in Drosophila macrophages via transcriptional regulation of target genes, the role of Ldh in these cells is more complicated. Although acute-phase macrophages still consume more glucose upon Ldh knockdown, these cells exhibit significantly lower Pgi activity and NADH levels, and the titer of circulating lactate also drops. Our results suggest that even though Ldh acts only at the last step of AG, its role is essential for full metabolic reprograming and efficient function of immune cells. Drosophila Ldh, like its mammalian ortholog, is responsible for the reduction of pyruvate to lactate, which is linked with the regeneration of NAD⁺ from NADH. However, this single reaction has an immense impact on cellular metabolism. Both the accumulation of pyruvate and the lack of NAD⁺ can become limiting in cells with high glycolytic flux (Olenchock et al., 2017). In addition, Ldh-dependent removal of cytosolic pyruvate was recently found to be essential to prevent pyruvate entry into mitochondria and a subsequent change of TCA cycle course (Eichenlaub et al., 2018; Wang et al., 2016).

Although not addressed in our study, changes in mitochondrial metabolism are also closely associated with AG and should be the focus of future studies of activated Drosophila macrophages. The interconnection between the transcriptional activity of Hif1α and the change of mitochondrial metabolism in Drosophila has recently been elucidated. The activation of several direct targets of Hif1α transcriptional activity leads to an inhibition of the classical course of the TCA cycle (Wang et al., 2016; Eichenlaub et al., 2018). One of the well-understood mechanisms is the prevention of pyruvate entry into the TCA cycle, which is caused by increased kinase activity of pyruvate dehydrogenase kinase 1 (PDK1). PDK1-mediated phosphorylation of pyruvate dehydrogenase (PDH) directly inhibits its enzymatic function, which is essential for pyruvate conversion to acetyl-CoA (Wang et al., 2016). This event causes a cytoplasmic accumulation of TCA cycle intermediates and thus promotes a secondary wave of Hif1α stabilization through inhibition of prolyl hydroxylase dehydrogenase (PHD) under normoxic conditions (Koivunen et al., 2007; Freije et al., 2012). The change in the TCA cycle is further needed for mitochondrial production of ROS that are transferred to the phagolysosome for bacterial killing (Forrester et al., 2018; Williams and O'Neill, 2018).

We further demonstrate that both Hif1α and Ldh are crucial not only for full macrophage activation, but also for the bactericidal function of the immune cells, with the rearrangement of macrophage metabolism towards AG being essential for resistance to infection and host survival. An important aspect of AG is the functional dependence of macrophages on sufficient supply of external energy resources, as demonstrated in both mammalian and insect phagocytes (Bajgar and Dolezal, 2018; Anderson et al., 1973a; Anderson et al., 1973b; Langston et al., 2017; Newsholme et al., 1986) and documented here by increased consumption of glucose. Immune cells therefore generate systemic factors to secure sufficient supply of nutrients by altering the function of other organs and by regulating systemic metabolism (Bajgar et al., 2015; Bajgar and Dolezal, 2018). While the identification of specific signaling factors is beyond the scope of this work, there are several candidate molecules in Drosophila that are known to be produced by activated macrophages as a reaction to the metabolic state of the cell. Although it is likely that multiple factors will be involved in this process, we can presume that these factors will reflect the metabolic state of the cells (e.g., extracellular adenosine), or that they will be linked to the transcriptional program that causes the switch towards AG (e.g., Imaginal morphogenesis protein late 2 (ImpL2)). In our previous work, we showed that the systemic metabolic switch upon infection depends on extracellular adenosine, which is produced by the activated immune cells (Bajgar and Dolezal, 2018; Bajgar et al., 2015). The production of adenosine directly reflects a metabolic state of the cell, such as increased consumption of ATP (Worku and Newby, 1983), as well as the accelerated occurrence of methylation events (German et al., 1983; Wu et al., 2005). Expression of ImpL2 was shown to be regulated by Hif1α (Li et al., 2013), and as ImpL2 was previously identified as a mediator of cancer-induced loss of energy reserves in flies due to its anti-insulin role (Kwon et al., 2015; Figueroa-Clarevega and Bilder, 2015), it could represent another link between AG in macrophages and changes in systemic metabolism that ensure sufficient supply of energy resources.

Finally, our findings raise an interesting question regarding the links between AG and the ability of immune cells to respond quickly to infection. Recent studies of mammalian macrophage metabolism revealed that AG is essential for the development of innate immune memory - called trained immunity (Netea et al., 2016). The mechanism of trained immunity relies on chromatin remodeling by epigenetic factors that enable cells to react with higher efficiency in response to re-infection by a particular pathogen (Kim et al., 2019). As many chromatin remodeling enzymes need cofactors (such as acetyl-CoA, NAD⁺, α-KG) for the remodeling of the epigenetic landscape, their function can be influenced by the metabolic state of the cell. Induction of AG leads to the accumulation of many cofactors that are essential for a proper function of these enzymes (Kim et al., 2019; Baardman et al., 2015). The concept of trained immunity not only is valid for mammals, but is rather present in many invertebrate clades (where it is called immune priming; Milutinović and Kurtz, 2016; Pham et al., 2007). Our observation of AG as a characteristic feature of activated Drosophila macrophages thus raises a question of its importance for the development of trained immunity in insects and other invertebrates. Taken together, our findings demonstrate how the molecular mechanisms that control AG induction in Drosophila macrophages exhibit a surprisingly high level of evolutionary conservation between mammals and insects, thus emphasizing that this metabolic switch is essential for survival of infection and hinting at the potential role for AG in the development of immune memory.

In conclusion, we have shown that infection-induced systemic changes in carbohydrate metabolism are associated with changes of macrophage cellular metabolism, and that both can be affected by macrophage-specific Hif1α and Ldh knockdown. Our data thus link the metabolic state of macrophages with the systemic metabolic changes. On the basis of our previous research on the selfish nature of the immune system under challenge (Straub, 2014), we envision that the shift in the cellular metabolism of macrophages leads to the production of signals that alter the systemic metabolism, thereby securing the sufficient energy supply necessary to allow the macrophages to fight the infection. By linking the induction of macrophage polarization with systemic metabolism and systemic outcomes in vivo, our experimental system can aid future research towards better understanding of the immune system and of diseases related to its malfunction.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data have been provided for Figures 2 and 4.

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Author details

1. Gabriela Krejčová

   Department of Molecular Biology and Genetics, University of South Bohemia, Ceske Budejovice, Czech Republic

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   krejcovagabriela@seznam.cz

   Competing interests

   No competing interests declared

2. Adéla Danielová

   Department of Molecular Biology and Genetics, University of South Bohemia, Ceske Budejovice, Czech Republic

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

3. Pavla Nedbalová

   Department of Molecular Biology and Genetics, University of South Bohemia, Ceske Budejovice, Czech Republic

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

4. Michalina Kazek

   Department of Molecular Biology and Genetics, University of South Bohemia, Ceske Budejovice, Czech Republic

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

5. Lukáš Strych

   Department of Molecular Biology and Genetics, University of South Bohemia, Ceske Budejovice, Czech Republic

   Contribution

   Data curation, Formal analysis

   Competing interests

   No competing interests declared

6. Geetanjali Chawla

   Department of Biology, Indiana University, Bloomington, United States

   Present address

   Regional Centre for Biotechnology, Faridabad, India

   Contribution

   Data curation

   Competing interests

   No competing interests declared

7. Jason M Tennessen

   Department of Biology, Indiana University, Bloomington, United States

   Contribution

   Resources, Data curation, Funding acquisition, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3527-5683

8. Jaroslava Lieskovská

   1. Department of Medical Biology, University of South Bohemia, Ceske Budejovice, Czech Republic
   2. Institute of Parasitology, Biology Centre CAS, Ceske Budejovice, Czech Republic

   Contribution

   Data curation

   Competing interests

   No competing interests declared

9. Marek Jindra

   1. Department of Molecular Biology and Genetics, University of South Bohemia, Ceske Budejovice, Czech Republic
   2. Institute of Entomology, Biology Centre CAS, Ceske Budejovice, Czech Republic

   Contribution

   Validation, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2196-9924

10. Tomáš Doležal

    Department of Molecular Biology and Genetics, University of South Bohemia, Ceske Budejovice, Czech Republic

    Contribution

    Resources, Supervision, Funding acquisition, Validation, Writing—original draft, Writing—review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-5217-4465

11. Adam Bajgar

    1. Department of Molecular Biology and Genetics, University of South Bohemia, Ceske Budejovice, Czech Republic
    2. Institute of Entomology, Biology Centre CAS, Ceske Budejovice, Czech Republic

    Contribution

    Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    bajgaa00@prf.jcu.cz

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9721-7534

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