Tomas Dolezal

Adenosine deaminase-related growth factors stimulate cell proliferation in Drosophila by depleting extracellular adenosine

We describe a protein family in Drosophila containing six adenosine deaminase-related growth factors (ADGFs), which are homologous to a mitogenic growth factor discovered in conditioned medium from cells of a different fly species, Sarcophaga. Closely related proteins have been identified in other animals, and a human homolog is implicated in the genetic disease Cat-Eye Syndrome. The two most abundantly expressed ADGFs in Drosophila larvae are ADGF-A, which is strongly expressed in the gut and lymph glands, and ADGF-D, which is mainly expressed in the fat body and brain. Recombinant ADGF-A and ADGF-D are active adenosine deaminases (ADAs), and they cause polarization and serum-independent proliferation of imaginal disk and embryonic cells in vitro. The enzymatic activity of these proteins is required for their mitogenic function, making them unique among growth factors. A culture medium prepared without adenosine, or depleted of adenosine by using bovine ADA, also stimulates proliferation of imaginal disk cells, and addition of adenosine to this medium inhibits proliferation. Thus ADGFs secreted in vivo may control tissue growth by modulating the level of extracellular adenosine.

A Role for Adenosine Deaminase in Drosophila Larval Development

Adenosine deaminase (ADA) is an enzyme present in all organisms that catalyzes the irreversible deamination of adenosine and deoxyadenosine to inosine and deoxyinosine. Both adenosine and deoxyadenosine are biologically active purines that can have a deep impact on cellular physiology; notably, ADA deficiency in humans causes severe combined immunodeficiency. We have established a Drosophila model to study the effects of altered adenosine levels in vivo by genetic elimination of adenosine deaminase-related growth factor-A (ADGF-A), which has ADA activity and is expressed in the gut and hematopoietic organ. Here we show that the hemocytes (blood cells) are the main regulator of adenosine in the Drosophila larva, as was speculated previously for mammals. The elevated level of adenosine in the hemolymph due to lack of ADGF-A leads to apparently inconsistent phenotypic effects: precocious metamorphic changes including differentiation of macrophage-like cells and fat body disintegration on one hand, and delay of development with block of pupariation on the other. The block of pupariation appears to involve signaling through the adenosine receptor (AdoR), but fat body disintegration, which is promoted by action of the hemocytes, seems to be independent of the AdoR. The existence of such an independent mechanism has also been suggested in mammals.

Extracellular Adenosine Mediates a Systemic Metabolic Switch during Immune Response

Immune defense is energetically costly, and thus an effective response requires metabolic adaptation of the organism to reallocate energy from storage, growth, and development towards the immune system. We employ the natural infection of Drosophila with a parasitoid wasp to study energy regulation during immune response. To combat the invasion, the host must produce specialized immune cells (lamellocytes) that destroy the parasitoid egg. We show that a significant portion of nutrients are allocated to differentiating lamellocytes when they would otherwise be used for development. This systemic metabolic switch is mediated by extracellular adenosine released from immune cells. The switch is crucial for an effective immune response. Preventing adenosine transport from immune cells or blocking adenosine receptor precludes the metabolic switch and the deceleration of development, dramatically reducing host resistance. Adenosine thus serves as a signal that the “selfish” immune cells send during infection to secure more energy at the expense of other tissues.

Expression of Drosophila adenosine deaminase in immune cells during inflammatory response.

Extra-cellular adenosine is an important regulator of inflammatory responses. It is generated from released ATP by a cascade of ectoenzymes and degraded by adenosine deaminase (ADA). There are two types of enzymes with ADA activity: ADA1 and ADGF/ADA2. ADA2 activity originates from macrophages and dendritic cells and is associated with inflammatory responses in humans and rats. Drosophila possesses a family of six ADGF proteins with ADGF-A being the main regulator of extra-cellular adenosine during larval stages. Herein we present the generation of a GFP reporter for ADGF-A expression by a precise replacement of the ADGF-A coding sequence with GFP using homologous recombination. We show that the reporter is specifically expressed in aggregating hemocytes (Drosophila immune cells) forming melanotic capsules; a characteristic of inflammatory response. Our vital reporter thus confirms ADA expression in sites of inflammation in vivo and demonstrates that the requirement for ADA activity during inflammatory response is evolutionary conserved from insects to vertebrates. Our results also suggest that ADA activity is achieved specifically within sites of inflammation by an uncharacterized post-transcriptional regulation based mechanism. Utilizing various mutants that induce melanotic capsule formation and also a real immune challenge provided by parasitic wasps, we show that the acute expression of the ADGF-A protein is not driven by one specific signaling cascade but is rather associated with the behavior of immune cells during the general inflammatory response. Connecting the exclusive expression of ADGF-A within sites of inflammation, as presented here, with the release of energy stores when the ADGF-A activity is absent, suggests that extra-cellular adenosine may function as a signal for energy allocation during immune response and that ADGF-A/ADA2 expression in such sites of inflammation may regulate this role.

Increased extracellular adenosine in Drosophila that are deficient in adenosine deaminase activates a release of energy stores leading to wasting and death

SUMMARY Extracellular adenosine is an important signaling molecule in neuromodulation, immunomodulation and hypoxia. Adenosine dysregulation can cause various pathologies, exemplified by a deficiency in adenosine deaminase in severe combined immunodeficiency. We have established a Drosophila model to study the effects of increased adenosine in vivo by mutating the main Drosophila adenosine deaminase-related growth factor (ADGF-A). Using a genetic screen, we show here that the increased extracellular adenosine in the adgf-a mutant is associated with hyperglycemia and impairment in energy storage. The adenosine works in this regard through the adenosine receptor as an anti-insulin hormone in parallel to adipokinetic hormone, a glucagon counterpart in flies. If not regulated properly, this action can lead to a loss of energy reserves (wasting) and death of the organism. Because adenosine signaling is associated with the immune response and the response to stress in general, our results mark extracellular adenosine as a good candidate signal involved in the wasting syndrome that accompanies various human pathologies.

Casein kinase I epsilon somatic mutations found in breast cancer cause overgrowth in Drosophila.

We are using a candidate gene approach to identify genes contributing to cancer through somatic mutation. Somatic mutations were found in breast cancer samples in the human casein kinase I epsilon (CKIepsilon) gene, a homolog of the Drosophila gene dco in which certain point mutations lead to imaginal disc overgrowth. We therefore created fly genotypes in which the dco gene carried point mutations homologous to those discovered in CKIepsilon, and tested them in vivo. The results show that the most frequent mutation discovered in breast cancer, L39Q, causes a striking overgrowth phenotype in flies. Further experiments show that this mutation affects the newly recognized Fat/Warts signaling pathway, which controls organ size and shape in both flies and mammals. Another mutation, S101R, modifies the mutant phenotype so that the affected tissue disintegrates, mimicking more aggressive forms of breast cancer. Our results thus strongly support the conclusion that CKIepsilon mutations play important roles in breast carcinogenesis.

Adenosine: a selfish-immunity signal?

It is long known that immune response is energydemanding. It has become clear in recent years [1] that pretty much any immune cell in our body undergoes, upon its activation, a metabolic shift resembling the Warburg effect originally described for cancer cells. Immune cells increase glucose consumption and produce a significant portion of ATP by glycolysis ending with lactate even under oxygenated conditions; increased glycolysis is required for the generation of intermediate metabolites associated with the activation of the immune cell. Increased energy consumption by immune cells requires a metabolic adaptation of the whole organism. During trauma or infection, the organism vitally depends on the immune system, which is therefore privileged in energy/nutrient allocation. According to Rainer Straub [2], insulin resistance caused by pro-inflammatory cytokines is a physiological way of the immune system to usurp energy/nutrients during acute stress from the rest of the organism because immune cells themselves do not become insulin resistant. Such selfish behavior of the immune system may be crucial for an effective immune response. We have recently demonstrated a selfish behavior of the immune system during defense of Drosophila larva against parasitoid wasp infection [3]. The wasp injects its egg into the larva and that activates a production of specialized immune cells called lamellocytes, which encapsulate and destroy the parasitoid egg. Production of lamellocytes is associated with increased glycolysis and glucose consumption by precursors of these cells. We demonstrated that a systemic metabolic switch, which included a suppression of development and energy storage, was required for the rapid production of lamellocytes and thus for the effective immune response. We further showed that lamellocytes precursors released adenosine that suppressed the consumption of glucose by non-immune tissues and thus slowed down the host development. When we blocked adenosine signaling or its release from immune cells, the development proceeded with normal speed but the resistance against parasitoid dropped, demonstrating a trade-off between development and the immune response. In our experimental system, immune cells use adenosine as a selfish signal to usurp energy from the rest of the organism, which is a vital strategy during infection. Extracellular adenosine can be produced in extracellular space from ATP, which for example leaks out from damaged tissues. Alternatively, when demand for ATP exceeds supply in a cell, the decreasing ATP level results in an increased level of AMP that can either activate AMPK and thus can suppress energy consuming processes within the cell or AMP can be converted to adenosine by cytosolic 5′-nucleotidase [4]; adenosine is then released to extracellular space by equilibrative nucleoside transporters where it can inform other tissues about the metabolic stress. The conversion of AMP to adenosine, instead of activating AMPK, would make more sense for activated immune cells, which need to obtain more energy; it remains to be tested if this was the origin of adenosine whose effects on systemic metabolism were observed in our work [3]. Extracellular adenosine could thus represent another type of selfish immune system signal - unlike proinflammatory cytokines, which would rather measure the robustness of the immune system activation (e.g. how many immune cells have been activated), adenosine would measure the actual energy needs of the immune cells and the actual tissue damage (ATP leakage). Can adenosine play a similar role in higher organisms including humans? Adenosine is produced, for example, by activated neutrophils and its systemic level is increased during sepsis. Adenosine generally suppresses energy-consuming processes; this can be observed both at the cellular level, e.g. inhibiting cell growth, and at the systemic level. The systemic suppression effects of adenosine are observed in torpor/hibernation and are important for anoxia-tolerant organisms. Adenosine is known to suppress neuronal firing and to induce sleep; caffeine is the most famous adenosine receptor antagonist. Increased plasma levels of adenosine were associated with chronic fatigue syndrome and adenosine was shown to mediate an exercise-induced fatigue [5]. Fatigue is a hallmark of sickness and thus it is tempting to speculate that adenosine may cause fatigue in proportion to tissue damage and the energy needs of immune cells. Fatigue and suppressing the overall activity of the organism could form, together with insulin resistance, a complex program to conserve energy for the immune system. How would this role of adenosine go together with its well-established anti-inflammatory role in the mammalian immune system [6]? The key might be to distinguish local and systemic effects, effects of different levels of adenosine and timing (Figure ​(Figure1).1). Low circulating levels of adenosine (though increased above the basal level) might have little effect on immune cells (or rather a stimulatory effect) but may have systemic suppressive effects influencing energy distribution within the organism. High levels of adenosine, generated by damaged tissues in sites with excessive inflammation, have anti-inflammatory effects on immune cells at that site; adenosine leaking into circulation from inflamed sites may further contribute to energy regulation at the systemic level. Figure 1 Speculative role of adenosine in metabolic regulation during immune response It would be interesting to test if adenosine, produced during immune response, influences the metabolism of non-immune tissues, activity and wakefulness, regulating thus the energy allocation in higher organisms. How important would such metabolic regulation be for the effectivity of immune response? It might be difficult to study these complex roles of adenosine but purinergic signaling is an important target for new drugs, for example to treat inflammatory diseases [6], and thus it is important to understand the various roles of adenosine.

Extracellular adenosine modulates host-pathogen interactions through regulation of systemic metabolism during immune response in Drosophila

Phagocytosis by hemocytes, Drosophila macrophages, is essential for resistance to Streptococcus pneumoniae in adult flies. Activated macrophages require an increased supply of energy and we show here that a systemic metabolic switch, involving the release of glucose from glycogen, is required for effective resistance to S. pneumoniae. This metabolic switch is mediated by extracellular adenosine, as evidenced by the fact that blocking adenosine signaling in the adoR mutant suppresses the systemic metabolic switch and decreases resistance to infection, while enhancing adenosine effects by lowering adenosine deaminase ADGF-A increases resistance to S. pneumoniae. Further, that ADGF-A is later expressed by immune cells during infection to regulate these effects of adenosine on the systemic metabolism and immune response. Such regulation proved to be important during chronic infection caused by Listeria monocytogenes. Lowering ADGF-A specifically in immune cells prolonged the systemic metabolic effects, leading to lower glycogen stores, and increased the intracellular load of L. monocytogenes, possibly by feeding the bacteria. An adenosine-mediated systemic metabolic switch is thus essential for effective resistance but must be regulated by ADGF-A expression from immune cells to prevent the loss of energy reserves and possibly to avoid the exploitation of energy by the pathogen.