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.

Macrophages play a nutritive role in post-metamorphic maturation in Drosophila.

In the body of multicellular organisms, macrophages play an indispensable role in maintaining tissue homeostasis by removing old, apoptotic and damaged cells. In addition, macrophages allow significant remodeling of body plans during embryonic morphogenesis, regeneration and metamorphosis. Although the huge amount of organic matter that must be removed during these processes represents a potential source of nutrients, their further use by the organism has not yet been addressed. Here, we document that, during metamorphosis, Drosophila larval adipose tissue is infiltrated by macrophages, which remove dying adipocytes by efferocytosis and engulf leaking RNA-protein granules and lipids. Consequently, the infiltrating macrophages transiently adopt the adipocyte-like metabolic profile to convert remnants of dying adipocytes to lipoproteins and storage peptides that nutritionally support post-metamorphic development. This process is fundamental for the full maturation of ovaries and the achievement of early fecundity of individuals. Whether macrophages play an analogous role in other situations of apoptotic cell removal remains to be elucidated.

Inhibition of mevalonate pathway by macrophage-specific delivery of atorvastatin prevents their pro-inflammatory polarisation.

Adjustment of the cellular metabolism of pro-inflammatory macrophages is essential for their bactericidal function; however, it underlies the development of many human diseases if induced chronically. Therefore, intervention of macrophage metabolic polarisation has been recognised as a potent strategy for their treatment. Although many small-molecule inhibitors affecting macrophage metabolism have been identified, their in vivo administration requires a tool for macrophage-specific delivery to limit their potential side effects. Here, we establish Drosophila melanogaster as a simple experimental model for in vivo testing of macrophage-specific delivery tools. We found that yeast-derived glucan particles (GPs) are suitable for macrophage-specific delivery of small-molecule inhibitors. Systemic administration of GPs loaded with atorvastatin, the inhibitor of hydroxy-methyl-glutaryl-CoA reductase (Hmgcr), leads to intervention of mevalonate pathway specifically in macrophages, without affecting HMGCR activity in other tissues. Using this tool, we demonstrate that mevalonate pathway is essential for macrophage pro-inflammatory polarisation and individual's survival of infection.

Innate immunity based cancer immunotherapy: B16-F10 murine melanoma model.

BACKGROUND: Using killed microorganisms or their parts to stimulate immunity for cancer treatment dates back to the end of 19th century. Since then, it undergone considerable development. Our novel approach binds ligands to the tumor cell surface, which stimulates tumor phagocytosis. The therapeutic effect is further amplified by simultaneous application of agonists of Toll-like receptors. We searched for ligands that induce both a strong therapeutic effect and are safe for humans. METHODS: B16-F10 murine melanoma model was used. For the stimulation of phagocytosis, mannan or N-formyl-methionyl-leucyl-phenylalanine, was covalently bound to tumor cells or attached using hydrophobic anchor. The following agonists of Toll-like receptors were studied: monophosphoryl lipid A (MPLA), imiquimod (R-837), resiquimod (R-848), poly(I:C), and heat killed Listeria monocytogenes. RESULTS: R-848 proved to be the most suitable Toll-like receptor agonist for our novel immunotherapeutic approach. In combination with covalently bound mannan, R-848 significantly reduced tumor growth. Adding poly(I:C) and L. monocytogenes resulted in complete recovery in 83% of mice and in their protection from the re-transplantation of melanoma cells. CONCLUSION: An efficient cancer treatment results from the combination of Toll-like receptor agonists and phagocytosis stimulating ligands bound to the tumor cells.

Magnetic Yeast Glucan Particles for Antibody-Free Separation of Viable Macrophages from Drosophila melanogaster.

Currently available methods for cell separation are generally based on fluorescent labeling using either endogenously expressed fluorescent markers or the binding of antibodies or antibody mimetics to surface antigenic epitopes. However, such modification of the target cells represents potential contamination by non-native proteins, which may affect further cell response and be outright undesirable in applications, such as cell expansion for diagnostic or therapeutic applications, including immunotherapy. We present a label- and antibody-free method for separating macrophages from living Drosophila based on their ability to preferentially phagocytose whole yeast glucan particles (GPs). Using a novel deswelling entrapment approach based on spray drying, we have successfully fabricated yeast glucan particles with the previously unachievable content of magnetic iron oxide nanoparticles while retaining their surface features responsible for phagocytosis. We demonstrate that magnetic yeast glucan particles enable macrophage separation at comparable yields to fluorescence-activated cell sorting without compromising their viability or affecting their normal function and gene expression. The use of magnetic yeast glucan particles is broadly applicable to situations where viable macrophages separated from living organisms are subsequently used for analyses, such as gene expression, metabolomics, proteomics, single-cell transcriptomics, or enzymatic activity analysis.

On the origin of the functional versatility of macrophages.

Macrophages represent the most functionally versatile cells in the animal body. In addition to recognizing and destroying pathogens, macrophages remove senescent and exhausted cells, promote wound healing, and govern tissue and metabolic homeostasis. In addition, many specialized populations of tissue-resident macrophages exhibit highly specialized functions essential for the function of specific organs. Sometimes, however, macrophages cease to perform their protective function and their seemingly incomprehensible response to certain stimuli leads to pathology. In this study, we address the question of the origin of the functional versatility of macrophages. To this end, we have searched for the evolutionary origin of macrophages themselves and for the emergence of their characteristic properties. We hypothesize that many of the characteristic features of proinflammatory macrophages evolved in the unicellular ancestors of animals, and that the functional repertoire of macrophage-like amoebocytes further expanded with the evolution of multicellularity and the increasing complexity of tissues and organ systems. We suggest that the entire repertoire of macrophage functions evolved by repurposing and diversification of basic functions that evolved early in the evolution of metazoans under conditions barely comparable to that in tissues of multicellular organisms. We believe that by applying this perspective, we may find an explanation for the otherwise counterintuitive behavior of macrophages in many human pathologies.

Polarization of Macrophages in Insects: Opening Gates for Immuno-Metabolic Research.

Insulin resistance and cachexia represent severe metabolic syndromes accompanying a variety of human pathological states, from life-threatening cancer and sepsis to chronic inflammatory states, such as obesity and autoimmune disorders. Although the origin of these metabolic syndromes has not been fully comprehended yet, a growing body of evidence indicates their possible interconnection with the acute and chronic activation of an innate immune response. Current progress in insect immuno-metabolic research reveals that the induction of insulin resistance might represent an adaptive mechanism during the acute phase of bacterial infection. In Drosophila, insulin resistance is induced by signaling factors released by bactericidal macrophages as a reflection of their metabolic polarization toward aerobic glycolysis. Such metabolic adaptation enables them to combat the invading pathogens efficiently but also makes them highly nutritionally demanding. Therefore, systemic metabolism has to be adjusted upon macrophage activation to provide them with nutrients and thus support the immune function. That anticipates the involvement of macrophage-derived systemic factors mediating the inter-organ signaling between macrophages and central energy-storing organs. Although it is crucial to coordinate the macrophage cellular metabolism with systemic metabolic changes during the acute phase of bacterial infection, the action of macrophage-derived factors may become maladaptive if chronic or in case of infection by an intracellular pathogen. We hypothesize that insulin resistance evoked by macrophage-derived signaling factors represents an adaptive mechanism for the mobilization of sources and their preferential delivery toward the activated immune system. We consider here the validity of the presented model for mammals and human medicine. The adoption of aerobic glycolysis by bactericidal macrophages as well as the induction of insulin resistance by macrophage-derived factors are conserved between insects and mammals. Chronic insulin resistance is at the base of many human metabolically conditioned diseases such as non-alcoholic steatohepatitis, atherosclerosis, diabetes, and cachexia. Therefore, revealing the original biological relevance of cytokine-induced insulin resistance may help to develop a suitable strategy for treating these frequent diseases.

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.

Yeast glucan particles enable intracellular protein delivery in Drosophila without compromising the immune system.

Glucan particles derived from yeast have been recently proposed as potential drug delivery carriers. Here, we demonstrate the potential of glucan particles for protein delivery in vivo, using the insect Drosophila melanogaster as a model organism. By employing genetic tools, we demonstrate the capacity of yeast glucan particles to spread efficiently through the Drosophila body, to enter macrophages and to deliver an active transcription factor protein successfully. Moreover, the glucan particles were nontoxic and induced only minimal immune response. The injection of glucan particles did not impair the ability of Drosophila to fight and survive infection by pathogenic bacteria. From this study, Drosophila emerges as an excellent model to test and develop drug delivery systems based on glucan particles, specifically aimed to regulate macrophages.

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.

Liver macrophages regulate systemic metabolism through non-inflammatory factors.

Liver macrophages (LMs) have been proposed to contribute to metabolic disease through secretion of inflammatory cytokines. However, anti-inflammatory drugs lead to only modest improvements in systemic metabolism. Here we show that LMs do not undergo a proinflammatory phenotypic switch in obesity-induced insulin resistance in flies, mice and humans. Instead, we find that LMs produce non-inflammatory factors, such as insulin-like growth factor-binding protein 7 (IGFBP7), that directly regulate liver metabolism. IGFBP7 binds to the insulin receptor and induces lipogenesis and gluconeogenesis via activation of extracellular-signal-regulated kinase (ERK) signalling. We further show that IGFBP7 is subject to RNA editing at a higher frequency in insulin-resistant than in insulin-sensitive obese patients (90% versus 30%, respectively), resulting in an IGFBP7 isoform with potentially higher capacity to bind to the insulin receptor. Our study demonstrates that LMs can contribute to insulin resistance independently of their inflammatory status and indicates that non-inflammatory factors produced by macrophages might represent new drug targets for the treatment of metabolic diseases.

Author Correction: Liver macrophages regulate systemic metabolism through non-inflammatory factors.

In the version of this article initially published, author Volker M. Lauschke had affiliation number 13; the correct affiliation number is 12. The error has been corrected in the HTML and PDF versions of the article.

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.

Cyclosporine A inhibits acetylcholinesterase activity in selected parts of the rat brain.

Cyclosporine A (CsA) is the major immunosuppressive drug used for organ and neural transplantation and the therapy of selected autoimmune diseases. We investigated the effect of CsA on the activity of acetylcholinesterase (AChE) in the frontal cortex, hippocampus, septum, and basal ganglia. AChE was determined spectrophotometrically with acetylthiocholine as substrate and 5,5-bis-2-nitrobenzoic acid as chromogen. CsA was administered in single doses of 20 or 45 mg/kg perorally; in the case of the higher dose we also performed a repeated administration of CsA in three consecutive doses separated by 24 h intervals. Both lower and higher doses of CsA decreased AChE activity in the frontal cortex and hippocampus to practically the same extent. On the contrary, AChE activity was more diminished in the case of the higher dose of CsA used in the septum and basal ganglia. Repeated administration of the higher dose of CsA did not lead, with the exception of the hippocampus, to a further decrease in AChE activity in the brain structures observed. These findings contribute to rare evidence concerning the interaction of CsA and the cholinergic system in the brain.

Anticholinergic drugs--functional antidotes for the treatment of tabun intoxication.

1. To study the influence of antidotes on tabun-induced neurotoxicity, the rats were injected intramuscularly with organophosphate tabun (LD50). The efficacy of choice antidotal treatment consisting of acetylcholinesterase reactivator obidoxime and one of four anticholinergic drugs (atropine, benactyzine, biperiden, scopolamine) was compared. 2. Testing of tabun-induced neurotoxicity progress was carried out using the method Functional observational battery. The experimental animals as well as controls were observed at 24 hours and 7 days following tabun or saline administration. 3. The results were compared to the condition of animals without anticholinergic drug (oxime alone) and control rats that received physiological solution instead of tabun and treatment. Antidotal treatment involving centrally acting anticholinergic drugs (benactyzine, biperiden, scopolamine) showed significantly higher neuroprotective efficacy compared to antidotal treatment containing atropine.

Effect of atropine and the oxime HI-6 on low-level sarin-induced alteration of performance of rats in a T-maze.

1. To study the influence of antidotes on low-level sarin-induced alteration of cognitive functions, male albino Wistar rats were exposed to three various low concentrations of sarin for 60 minutes in the inhalation chamber. One minute following sarin exposure, the rats were i.m. treated with the oxime HI-6 in combination with atropine. Control rats were treated with antidotes as experimental rats but exposed to the pure air instead of sarin. Cognitive functions of the rats were tested using a T-maze where spatial memory and spatial orientation were evaluated. The performance of sarin-exposed and treated rats in the T-maze was tested several times within six weeks (single exposure) or five weeks (repeated exposure) following inhalation exposure to evaluate cognitive impairments. 2. In the case of single exposure to sarin, no statistically significant differencies between the performances of the control and the experimental groups in the alteration of spatial memory and spatial orientation were observed. The repeated exposure of treated rats to clinically asymptomatic dose of sarin (LEVEL 2) did not change the effect of low-level sarin exposure on spatial memory of the experimental rats compared to the single exposure to the same dose of sarin. 3. The decrease in the T-maze performance of the control rats was caused by the impairments of rat's mobility due to the features of a solution of antidotes.

Antiacetylcholinesterase activity of cyclosporine--a comparison of single and repeated administration and effect of 7-methoxytacrine.

The aim of this work is a comparison of single and repeated peroral administration of cyclosporine (CsA) and the interaction of repeated administration of CsA and 7-methoxytacrine (MEOTA) on the activity of acetylcholinesterase (AChE) in the frontal cortex, hippocampus, septum, and basal ganglia in rats. Both single and repeated administration of CsA diminished the activity of AChE in the frontal cortex, septum and basal ganglia, while the enzyme activity in the hippocampus was diminished only in the case of repeated CsA, as well as repeated CsA + MEOTA administration. Repeated administration of CsA led to a further augmentation of anticholinesterase activity only in the frontal cortex and--in a lesser extent--in the basal ganglia. No augmentation of AChE activity was observed in the hippocampus and septum.

The impairment of spatial memory following low-level sarin inhalation exposure and antidotal treatment in rats.

1. To study the influence of antidotes on low-level sarin-induced impairment of cognitive functions, the rats were exposed to three various low concentrations of sarin (LEVEL 1-3) for 60 minutes in the inhalation chamber. In addition, one group of rats was exposed to LEVEL 2 of sarin repeatedly. 2. Testing of cognitive functions was carried out using the Y-maze evaluating learning and spatial memory. The correct averse behavior of sarin-exposed rats in the Y-maze was tested several times within four weeks following sarin inhalation exposure and antidotal treatment to look for any cognitive impairments. 3. The results were compared to the Y-maze performance of sarin-exposed rats without antidotal treatment and control rats exposed to pure air instead of sarin with or without antidotal treatment. While antidotal treatment was able to eliminate a short-term deficiency in the Y-maze performance in rats exposed to the LEVEL 1 of sarin, a significant decrease in the Y-maze performance in rats exposed to sarin at the LEVEL 2 and 3 was only shortened. Sarin-induced spatial memory impairments in rats exposed repeatedly to sarin at the LEVEL 2 was also shortened when rats were treated following each sarin inhalation exposure. 4. The findings confirm that antidotes currently used for nerve agent poisonings are beneficial for the treatment of rats singly or repeatedly exposed to non-convulsive symptomatic or even clinically asymptomatic concentrations of sarin.

A comparison of the neuroprotective efficacy of pharmacological pretreatment and antidotal treatment in soman-poisoned rats.

1. To study the influence of pharmacological pretreatment (PANPAL or pyridostigmine combined with biperiden) and antidotal treatment (the oxime HI-6 plus atropine) on soman-induced neurotoxicity, male albino rats were poisoned with a lethal dose of soman (54 (g/kg i.m.; 100% of LD50 value) and observed at 24 hours and 7 days following soman challenge. The neurotoxicity of soman was evaluated using a Functional observational battery and an automatic measurement of motor activity. 2. Pharmacological pretreatment as well as antidotal treatment were able to eliminate some of soman-induced neurotoxic effects observed at 24 hours following soman poisoning. The combination of pharmacological pretreatment (PANPAL or pyridostigmine combined with biperiden) and antidotal treatment was found to be more effective in the elimination of soman-induced neurotoxicity in rats at 24 hours following soman challenge in comparison with the administration of pharmacological pretreatment or antidotal treatment alone. To compare both pharmacological pretreatments, the combination of pyridostigmine with biperiden seems to be more efficacious to eliminate soman-induced signs of neurotoxicity than PANPAL. 3. At 7 days following soman poisoning, the combination of pharmacological pretreatment involving pyridostigmine and biperiden with antidotal treatment was only able to completely eliminate soman-induced neurotoxic signs. 4. Thus, our findings confirm that the combination of pharmacological pretreatment and antidotal treatment is able not only to protect the experimental animals from the lethal effects of soman but also to eliminate most soman-induced signs of neurotoxicity in poisoned rats. The pharmacological pretreatment containing pyridostigmine and biperiden appears to be more efficacious to eliminate soman-induced neurotoxic sings than PANPAL.