Glucose and trehalose metabolism through the cyclic pentose phosphate pathway shapes pathogen resistance and host protection in Drosophila.

Activation of immune cells requires the remodeling of cell metabolism in order to support immune function. We study these metabolic changes through the infection of Drosophila larvae by parasitoid wasp. The parasitoid egg is neutralized by differentiating lamellocytes, which encapsulate the egg. A melanization cascade is initiated, producing toxic molecules to destroy the egg while the capsule also protects the host from the toxic reaction. We combined transcriptomics and metabolomics, including 13C-labeled glucose and trehalose tracing, as well as genetic manipulation of sugar metabolism to study changes in metabolism, specifically in Drosophila hemocytes. We found that hemocytes increase the expression of several carbohydrate transporters and accordingly uptake more sugar during infection. These carbohydrates are metabolized by increased glycolysis, associated with lactate production, and cyclic pentose phosphate pathway (PPP), in which glucose-6-phosphate is re-oxidized to maximize NADPH yield. Oxidative PPP is required for lamellocyte differentiation and resistance, as is systemic trehalose metabolism. In addition, fully differentiated lamellocytes use a cytoplasmic form of trehalase to cleave trehalose to glucose and fuel cyclic PPP. Intracellular trehalose metabolism is not required for lamellocyte differentiation, but its down-regulation elevates levels of reactive oxygen species, associated with increased resistance and reduced fitness. Our results suggest that sugar metabolism, and specifically cyclic PPP, within immune cells is important not only to fight infection but also to protect the host from its own immune response and for ensuring fitness of the survivor.

Macrophage-derived insulin antagonist ImpL2 induces lipoprotein mobilization upon bacterial infection.

The immune response is an energy-demanding process that must be coordinated with systemic metabolic changes redirecting nutrients from stores to the immune system. Although this interplay is fundamental for the function of the immune system, the underlying mechanisms remain elusive. Our data show that the pro-inflammatory polarization of Drosophila macrophages is coupled to the production of the insulin antagonist ImpL2 through the activity of the transcription factor HIF1α. ImpL2 production, reflecting nutritional demands of activated macrophages, subsequently impairs insulin signaling in the fat body, thereby triggering FOXO-driven mobilization of lipoproteins. This metabolic adaptation is fundamental for the function of the immune system and an individual's resistance to infection. We demonstrated that analogically to Drosophila, mammalian immune-activated macrophages produce ImpL2 homolog IGFBP7 in a HIF1α-dependent manner and that enhanced IGFBP7 production by these cells induces mobilization of lipoproteins from hepatocytes. Hence, the production of ImpL2/IGFBP7 by macrophages represents an evolutionarily conserved mechanism by which macrophages alleviate insulin signaling in the central metabolic organ to secure nutrients necessary for their function upon bacterial infection.

Drosophila macrophages switch to aerobic glycolysis to mount effective antibacterial defense.

Macrophage-mediated phagocytosis and cytokine production represent the front lines of resistance to bacterial invaders. A key feature of this pro-inflammatory response in mammals is the complex remodeling of cellular metabolism towards aerobic glycolysis. Although the function of bactericidal macrophages is highly conserved, the metabolic remodeling of insect macrophages remains poorly understood. Here, we used adults of the fruit fly Drosophila melanogaster to investigate the metabolic changes that occur in macrophages during the acute and resolution phases of Streptococcus-induced sepsis. Our studies revealed that orthologs of Hypoxia inducible factor 1α (HIF1α) and Lactate dehydrogenase (LDH) are required for macrophage activation, their bactericidal function, and resistance to infection, thus documenting the conservation of this cellular response between insects and mammals. Further, we show that macrophages employing aerobic glycolysis induce changes in systemic metabolism that are necessary to meet the biosynthetic and energetic demands of their function and resistance to bacterial infection.

Molecular regulations of metabolism during immune response in insects.

Mounting an immune response is an energy-consuming process. Activating immune functions requires the synthesis of many new molecules and the undertaking of numerous cellular tasks and it must happen rapidly. Therefore, immune cells undergo a metabolic switch, which enables the rapid production of ATP and new biomolecules. Such metabolism is very nutrient-demanding, especially of glucose and glutamine, and thus the immune response is associated with a systemic metabolic switch, redirecting nutrient flow towards immunity and away from storage and consumption by non-immune processes. The immune system during its activation becomes privileged in terms of using organismal resources and the activated immune cells usurp nutrients by producing signals which reduce the metabolism of non-immune tissues. The insect fat body plays a dual role in which it is both a metabolic organ, storing energy and providing energy to the rest of the organism, but also an organ important for humoral immunity. Therefore, the internal switch from anabolism to the production of antimicrobial peptides occurs in the fat body during infection. The mechanisms regulating metabolism during the immune response ensure adequate energy for an effective response (resistance) but they must be properly regulated because energy is not unlimited and the energy needs of the immune system thus interfere with the needs of other physiological traits. If not properly regulated, the immune response may in the end decrease fitness via decreasing disease tolerance.

Effective cancer immunotherapy based on combination of TLR agonists with stimulation of phagocytosis.

Immunotherapy emerges as a fundamental approach in cancer treatment. Up to date, the efficacy of numerous different immunotherapies has been evaluated. The use of microorganisms or their parts for immune cell activation, referred to as Pathogen-Associated Molecular Patterns (PAMPs), represents highly promising concept. The therapeutic effect of PAMPs can be further amplified by suitable combination of different types of PAMPs such as Toll like receptor (TLR) agonists and phagocytosis activating ligands. Previously, we used the combination of phagocytosis activating ligand (mannan) and mixture of TLR agonists (resiquimod (R-848), poly(I:C), inactivated Listeria monocytogenes) for successful treatment of melanoma in murine B16-F10 model. In the present study, we optimized the composition and timing of previously used mixture. Therapeutic mixture based on well-defined chemical compounds consisted of mannan anchoring to tumor cell surface by biocompatible anchor for membranes (BAM) and TLR agonists resiquimod, poly(I:C), and lipoteichoic acid (LTA). The optimization resulted in (1) eradication of advanced stage progressive melanoma in 83% of mice, (2) acquisition of resistance to tumor re-transplantation, and (3) potential anti-metastatic effect. After further investigation of mechanisms, underlying anti-tumor responses, we concluded that both innate and adaptive immunity are activated and involved in these processes. We tested the efficacy of our treatment in Panc02 murine model of aggressive pancreatic tumor as well. Simultaneous application of agonistic anti-CD40 antibody was necessary to achieve effective therapeutic response (80% recovery) in this model. Our results suggest that herein presented immunotherapeutic approach is a promising cancer treatment strategy with the ability to eradicate not only primary tumors but also metastases.

The use of Zymosan A and bacteria anchored to tumor cells for effective cancer immunotherapy: B16-F10 murine melanoma model.

The idea of using killed microorganisms or their parts for a stimulation of immunity in the cancer immunotherapy is very old, but the question of interactions and binding of these preparations to tumor cells has not been addressed so far. The attachment of Zymosan A and both Gram-positive and Gram-negative bacteria to tumor cells was tested in in vivo experiments. This binding was accomplished by charge interactions, anchoring based on hydrophobic chains and covalent bonds and proved to be crucial for a strong immunotherapeutic effect. The establishment of conditions for simultaneous stimulation of both Toll-like and phagocytic receptors led to very strong synergy. It resulted in tumor shrinkage and its temporary or permanent elimination. The role of neutrophils in cancer immunotherapy was demonstrated and the mechanism of their action (frustrated phagocytosis) was proposed. Finally, therapeutic approaches applicable for safe human cancer immunotherapy are discussed. Heat killed Mycobacterium tuberculosis covalently attached to tumor cells seems to be promising tool for this therapy.