Ado Route to Immunity (Tomas Dolezal)

8. 6. 2015 Tomas Dolezal Articles 828x

Extracellular adenosine effects in flies

Tomas Dolezal

(part of habilitation thesis of Tomas Dolezal 2015)



Extracellular adenosine Drosophila e-Ado model established by mutagenesis of ADGF genes Adenosine deaminase deficiency increased e-Ado and caused larval death Genetic screen linked e-Ado to energy metabolism Increased e-Ado led to energy release ADGF-A expression reporter linked e-Ado to immune response Does e-Ado regulate energy during immune response? “Selfish” immune system Ado route to immunity - summary References


Here, I describe the story of characterizing extracellular adenosine role in organism leading to a discovery of the adenosine role in energy regulation during immune response. I refer to this as “Ado Route to Immunity”. Five research papers are presented with this story:

1. Dolezal T, Dolezelova E, Zurovec M, Bryant PJ (2005). A Role for Adenosine Deaminase in Drosophila Larval Development. PLoS Biology 3(7): e201. This is the first broader characterization of phenotype caused by elevated levels of extracellular adenosine due to a deficiency of adenosine deaminase which established a Drosophila model for the following studies.

2. Zuberova M, Fenckova M, Simek P, Janeckova L, Dolezal T (2010). Increased extracellular adenosine in adenosine deaminase deficient flies activates a release of energy stores leading to wasting and death. Dis Model Mech 3(11-12): 773-84. Here we demonstrate the role of extracellular adenosine in regulation of energy metabolism in Drosophila. This study represents the first important part in the story – linking adenosine to energy regulation.

3. Novakova M and Dolezal T (2011). Expression of Drosophila adenosine deaminase in immune cells during inflammatory response. PLoS ONE 6(3): e17741.  This study shows an importance of the regulation of adenosine during immune response; this is the second important part in the story linking adenosine to immune response.

4. Fenckova M, Hobizalova R, Fric Z, Dolezal T (2011). Functional characterization of ecto-5’-nucleotidases and apyrases in Drosophila melanogaster. Insect Biochem Mol Biol 41(12): 956-967.  This study characterizes a mechanism of regulation of extracellular adenosine levels in Drosophila demonstrating a similarity in adenosine system with other organisms including humans.

5. Bajgar A, Kucerova K, Jonatova L, Tomcala A, Schneedorferova I, Okrouhlik J, Dolezal T (2015). Extracellular adenosine mediates a systemic metabolic switch during immune response. PLoS Biology 13(4): e1002135.  The most recent study put together the previous links of adenosine to energy regulation and to immune response leading to a discovery of novel role of extracellular adenosine as a mediator of metabolic switch during immune response. This study has also established a concept of “selfish immune system”.


Extracellular adenosine

Extracellular adenosine (e-Ado) is an important regulatory molecule with a low physiological concentration that can rapidly increase during tissue damage, inflammation, ischemia or hypoxia. During hypoxia or cellular metabolic stress, adenosine can be transported by nucleoside transporters to extracellular space from cells where its concentration raises up due to excessive breakdown of ATP (Figure 1). e-Ado then informs the surrounding tissues about the metabolic state of the cells/tissues via adenosine receptors. This can lead to various responses as vasodilation, increase of blood flow and therefore substrate delivery or it can stimulate glycogenolysis, providing energy substrates from stores to overcome stress. Increased e-Ado may also lead to a suppression of metabolic processes in certain tissues to conserve overall energy (Buck, 2004).

ATP leaks out from damaged tissues and e-Ado is produced from the released ATP by a cascade of ectoenzymes – Apyrases/NTPDases and Ecto-5’-nucleotidases – during inflammatory response; ATP is a strong pro-inflammatory signal, at least in mammals (Figure 1). Both extracellular ATP and e-Ado stimulate purinergic receptors; therefore their release, signaling and progressive decrease regulate the onset of the acute inflammatory response, the fine-tuning of ongoing inflammation and its eventual down-regulation (Bours et al., 2006).

e-Ado binds to adenosine receptors, which are G-protein coupled receptors and their activation may have various consequences. In mammals, four types of adenosine receptors exist – A1, A2A, A2B and A3. A1 and A3 inhibit adenylate cyclase, whereas A2A and A2B stimulate this enzyme. In some cells, the A2B receptors are also coupled to the calcium-mobilizing G-protein subunit - Gαq. The distribution of the receptors on different cells varies and thus e-Ado may have various effects on physiology. In summary, e-Ado is an immunomodulatory molecule with the most prominent role being its anti-inflammatory role but inflammatory actions were also described (Bours et al., 2006). Another important role is regulation of metabolism – e-Ado is able to suppress metabolism and to stimulate release of energy reserves (Buck, 2004; Cortés et al., 2009).

Due to its numerous actions, e-Ado needs to be tightly regulated. It is converted to inosine by adenosine deaminase and further metabolized to uric acid. There are two types of adenosine deaminases, ADA1 and ADA2 (Maier et al., 2005). ADA1 can be localized both in intracellular and extracellular space while ADA2 is secreted enzyme. e-Ado can also be transported by nucleoside transporters into the cells, converted to AMP by adenosine kinase activating AMPK (da Silva et al., 2006).

Dynamic formation of e-Ado, its secretion, signaling via adenosine receptors, its uptake and degradation all form an intricate network influencing various physiological responses within organism.
We are using Drosophila as a simpler model to study e-Ado roles in vivo. We have shown by several functional studies and characterization of various mutants that the system for e-Ado production, signaling and degradation in flies is remarkably similar to mammalian organisms (Figure 1). e-Ado is produced from AMP by ecto-5’-nucleotidases (Fenckova et al., 2011), signalizes through G-protein coupled receptor AdoR (Dolezal et al., 2005; Dolezelova et al., 2007; Kucerova et al., 2012) and is degraded by adenosine deaminases – ADGFs (Dolezal et al., 2005; Zurovec et al., 2002).

Adenosine generation, signaling and regulation

Figure 1. Schematic representation of generation, regulation and signaling of extracellular adenosine and human and fly enzymes and receptors involved in these processes. There are generally two ways of e-Ado generation – (1) intracellular one when e-Ado is generated upon metabolic stress by action of adenylate kinase and 5’-nucleotidase and released to extracellular space by nucleoside transporters.  (2) Extracellular production from ATP, which leaks out from damaged tissues (or gets released during stress), with subsequent generation of ADP→AMP→e-Ado by ecto-enzymes NTPDase/Apyrase and ecto-5’-nucleotidase. e-Ado can signal through adenosine receptors, can be converted to inosine by extracellular adenosine deaminases, or can be transported into cells and converted either to AMP by adenosine kinase or to inosine by intracellular adenosine deaminase (ADA).


Drosophila e-Ado model established by mutagenesis of ADGF genes

We have started studying the role of e-Ado in Drosophila by mutating genes for enzymes with adenosine deaminase activity. There are seven genes in Drosophila melanogaster encoding proteins with adenosine deaminase domains. One of them, Ada (CG11994), encodes an ortholog of mammalian ADA1 and the remaining six, ADGF-A (CG5992), Msi (CG32178), ADGF-B (CG5998), ADGF-C (CG9345), ADGF-D (CG9621) and ADGF-E (CG10143), encode orthologs of mammalian ADA2/ADGF.

We used an approach of Yikang Rong and Kent Golic (Rong and Golic, 2000) based on ends-in homologous recombination for targeted mutagenesis of ADGFs. Their approach used transgenic flies carrying modified homologous sequences to target regions with desired mutation/modification, sites for nuclease cleavage (SceI and CreI), sites for flipase recombination and miniwhite selection marker. Such transgenic flies were then crossed to other transgenic flies carrying Flipase and SceI-endonuclease under heat shock promoters. Heat shocking the progeny induced homologous recombination within the target region, first by mobilizing the modified construct by flipase to make extrachromosomal circular DNA, then by cutting the circle by SceI endonuclease producing linear fragment carrying modified homologous sequence. Presence of such linear DNA fragment induced homologous recombination with the target sequence and if the recombination occurred in the germline cells, the mutagenized sequence could be transmitted to the next generation.

Mutagenesis of ADGF genes by homologous recombination

Figure 2. Scheme of ADGF genes targeting by homologous recombination. First, we produced a transgenic fly carrying 8.5-kb long homologous sequence with stop codons introduced into all three target genes (top). Crossing this fly to hs>SceI hs>Flp fly induced mobilization of this sequence by Flp action and linearization by SceI cutting. Such fragment induced ends-in homologous recombination inserting the construct and thus producing a duplication within the target region with miniwhite marker for eye color (middle). adgf-akarel mutant was produced already during this step due to a gene conversion resulting in both adgf-a mutant alleles within the duplication. Second step, induced by CreI cutting, resulted in homologous recombination between the duplicated arms of the same chromosome and thus in reduction of the duplication with different possible mutant combinations (bottom).

At the time of designing our experiment, no one had repeated Rong and Golic’s approach yet. In addition, we used this approach to target multiple genes at once. The scheme (Figure 2) for targeting three genes at once and producing various possible mutant combinations by unverified approach seemed quite unrealistic to many people. However, the mutagenesis not only worked (Figure 3) but thanks to the gene conversion, it produced all possible mutant combinations of the three target genes including single, double and triple mutants (Dolezal et al. 2003). The resulted adgf-a mutant (also known as karel) became the basis for most of our following work described in this thesis.

homologous recombination mutant screening

Figure 3. Detection of ADGF gene targeting by eye color. The targeting construct carried a miniwhite marker which produced an orange/red pigment in white eyes of white mutant. The mosaic eyes (with orange regions within the eye) marked the ongoing process of recombination; some cells lost the original construct – white, some cells underwent the recombination marked by the presence of miniwhite. Progeny with stable recombination event had full orange eyes – that is how the mutant was recognized.


Adenosine deaminase deficiency increased e-Ado and caused larval death

The adgf-a mutation, produced by homologous recombination, caused a loss of function of adenosine deaminase.  Deficiency of the adenosine deaminase activity led to more than a magnitude-higher level of e-Ado in larval hemolymph and caused death at the end of the larval development in 70% of cases or during metamorphosis in the rest; rarely the adgf-a null adult flies emerged (Dolezal et al., 2005). Rescue of the phenotype by expressing transgenically-provided wild-type ADGF-A but not by ADGF-A lacking the adenosine deaminase activity (catalytically-dead version) demonstrated that the lethality is caused by the failure to regulate e-Ado by deamination to inosine. In addition, the adgf-a mutants were rescued by blocking AdoR signaling (adoR null mutation) further indicating that the phenotype was mainly caused by an excessive e-Ado signaling and not by the toxicity of increased e-Ado or the lack of inosine, the product of deamination.

The adgf-a mutant larvae were delayed in their development and late third-instar larvae were almost translucent compared to wild-type control (Figure 4). This was caused by a disintegration of fat body and its almost complete disappearance in cases of largely-delayed development. Such larvae never pupated and eventually died. Fat body is the major metabolizing organ in larvae, accumulating energy reserves in form of triglycerides and glycogen, ensuring metabolic homeostasis and nutrient utilization, biosynthesis etc. Fat body normally disintegrates during metamorphosis (Aguila et al., 2007). Fat body disintegration in adgf-a thus suggested metabolic problems or precocious metamorphic changes.

adgf-a mutant with disintegrated fat body

Figure 4. Fat body disintegration in the adgf-a mutant. The dark mass filling up the whole wild-type larva is fat body which is almost missing in the adgf-a mutant (small remaining chunks are still visible).

Another phenotypic feature of the adgf-a mutant was a melanotic capsule formation, which is caused by abnormal differentiation of hematopoietic cells (hemocytes). We found that adgf-a had largely increased number of hemocytes, including specialized immune cells called lamellocytes. Lamellocytes do not appear during normal development; their differentiation is for example induced by parasitoid wasp infection when lamellocytes are produced to encapsulate parasitoid egg (Keebaugh and Schlenke, 2014). Lamellocytes are often detected in mutants with melanotic capsule formation. This was an interesting observation since hematopoietic lineage together with brain and gut expressed strongly ADGF-A. In addition, expression of transgenically-provided ADGF-A only in hematopoietic lineage was sufficient to fully rescue the adgf-a mutant. Somehow, the expression of ADGF-A in hemocytes was important for normal development.

Concluding the work published in (Dolezal et al., 2005), increased e-Ado due to the ADGF-A deficiency caused suppression of development through AdoR signaling, leading to larval/pupal death. However, the reason for the lethality remained unclear as well as the role of e-Ado during normal development.

ADGF-A deficiency resulted in lack of e-Ado regulation and thus in its accumulation. However, what was the source of e-Ado causing an accumulation of e-Ado in the adgf-a mutant? One of the possibilities, which I decided to test, was that environmental stress including interaction with microorganisms stimulated the release of e-Ado. While this would not be a problem in wild type flies, the lack of e-Ado regulation in the adgf-a mutant could lead to e-Ado accumulation and its excessive signaling. Therefore I tried to rescue the adgf-a mutant by raising it in axenic conditions. I used autoclaved yeast media (yeast and agar) and I sterilized embryos by washing them in 70% ethanol before placing on the axenic media. To my surprise, while the control line developed normally under these conditions, all adgf-a mutants died before reaching mid or late 3rd instar stage. Later, this result became very important for understanding the phenotype but at the time of the experiments, I was not able to explain them and thus I abandoned them.


Genetic screen linked e-Ado to energy metabolism

Trying to find the reason for lethality caused by the elevated e-Ado, I decided to use the most powerful tool available in Drosophila model – large genetic screen. I was hoping that identifying the genes, genetically interacting with the adgf-a phenotype, might uncover the reason for lethality.

I used an advantage of deficiency kit available from Bloomington Stock Centre in 2005. The kit covered most of the Drosophila genome by limited number of larger overlapping deficiencies (Figure 5).

Bloomington deficiency kit

Figure 5. Example of overlapping deficiencies covering the left arm of Chromome II from the Bloomington Deficiency Kit. Image of the left arm of the polytene chromosome II with marked cytological bands is shown on top; the corresponding map of overlapping deficiencies is at the bottom. Each deficiency included couple to several tens of genes with boundaries more or less precisely  defined at the molecular level.

I could cross each deficiency into the adgf-a null mutant background and test if the heterozygosity of certain genomic regions suppressed or enhanced the adgf-a phenotype (Figure 6). Since the adgf-a mutation was located on chromosome III, I used deficiencies only on chromosome X (total 76 deficiencies) and on chromosome II (total 99 deficiencies; some of them shown in Figure 5).

First, I used low resolution screen with larger deficiencies (from the kit) and identified 11 regions increasing survival of the adgf-a mutant. My doctorate student Monika Zuberova took up the project in 2006 with the goal to identify the individual genes responsible for the suppressing effects within the regions. She was successful in 6 regions linking the suppression effects either to adenosine metabolism (which was logical) but interestingly also to energy metabolism (Žuberová, 2011). The most interesting region for us turned out to contain a gamma subunit of phosphorylase kinase (PhKγ), a gene involved in regulation of glycogen metabolism.

Genetic cross used for deficiency screen

Figure 6. Genetic cross for identification of deletions suppressing the adgf-a mutant phenotype. Balanced deletion on chromosome X or II with TM6B Tb balancer (marked by Tb causing tubby phenotype) on III was crossed to the adgf-a mutant balanced by TM3 Ser. Females with unbalanced deletion and adgf-a mutation balanced by TM6B Tb (recognized by presence of Tb and absence of Ser markers) were crossed to males with adgf-a balanced by TM6B Tb. Half of the progeny homozygous for the adgf-a mutation (recognized by the absence of Tb marker) carried also the deletion. If the deletion suppressed the phenotype it was recognized by an increased presence of non-Tb pupae without melanotic capsules in vial.

The genetic screen suggested that the adgf-a mutant larvae might have problems with energy storage and suppressing glycogenolysis by phKγ mutation helped the adgf-a mutant to survive (Zuberova et al., 2010). At that time I attended the 20th European Drosophila Research Conference (EDRC 2007) in Vienna and visited a presentation of Susanne Buch from Michael Pankratz laboratory about dietary effects on Insulin-like peptide signaling (Buch et al., 2008). She showed a result, which was just one of many for most of the people in the audience but was staggering for me – flies reared on a pure-yeast diet had one-quarter glycogen stores compared to flies reared on “regular” diet. The “regular” diets usually contain much more carbohydrates than the pure-yeast diet. I immediately recollected my results with axenic pure-yeast media killing all the adgf-a mutant larvae. All of a sudden, the old results perfectly clicked with the results of the genetic screen. The adgf-a mutant had troubles to accumulate glycogen stores during larval development. Rearing the mutant on yeast diet, which decreased glycogen even in the wild-type flies, worsened the phenotype of the adgf-a mutant, while suppressing the glycogenolysis by phKγ mutation increased its chance to survive. It was clear that elevated e-Ado affected the energy metabolism.

Increased e-Ado led to energy release

Right after we returned from the conference, Monika performed a simple experiment: she put the adgf-a larvae on yeast diet supplemented with different amount of carbohydrates and found a striking dependence of the phenotype on the diet (Figure 7).

adgf-a mutant sensitivity to sugar content

Figure 7. Survival of adgf-a mutant is dependent on the diet. adgf-a mutant and the heterozygous adgf-a/+ siblings as control were raised on pure yeast diet (0%) or yeast diet supplemented with either 5% or 10% sucrose. Number of pupae (grey bars) and adults (black bars) clearly demonstrates that increasing amount of carbohydrates in the diet increases the chance of survival for the adgf-a mutant while the control is not affected by the dietary differences.

We also found that elevated e-Ado caused a hyperglycemia; we detected doubled levels of circulating glucose in the hemolymph of adgf-a larvae. e-Ado thus caused a release of glucose from energy stores or prevented its incorporation into stores leaving more free glucose in circulation. Larvae with elevated levels of e-Ado had problems to accumulate energy stores which are required in further development and this anti-storage effect was either enhanced by low-carbohydrate diet or prevented by high-carbohydrate diet. The hyperglycemia caused by elevated e-Ado was suppressed by adoR mutation indicating that the AdoR signaling was mediating the hyperglycemia.

These results demonstrated that the e-Ado-mediated effects on energy metabolism affected larval development and caused even death when larvae were kept on carbohydrate-poor diet. e-Ado thus turned out to be an important regulator of energy metabolism in flies. This e-Ado role resembled the role of e-Ado in other organisms including mammals. For example, hypoxic tissues release high quantities of adenosine, which stimulate, through adenosine receptor, a release of glucose from liver glycogen, thus promoting hyperglycemia (Cortés et al., 2009).


ADGF-A expression reporter linked e-Ado to immune response

Genetic screen helped us to link e-Ado to energy metabolism. Another approach uncovering the role of e-Ado that we decided to take was to monitor the regulation of e-Ado by ADGF-A. This became a project for another doctorate student, Milena Novakova. She used the same approach that I had used for generating ADGF mutants based on homologous recombination. Instead of incorporation of stop codons, she replaced the whole coding sequence of the ADGF-A gene by a sequence encoding GFP to produce an expression reporter (Figure 8; (Novakova and Dolezal, 2011)).

Generation of the adgf-a expression reporter

Figure 8. Generation of ADGF-A expression reporter by homologous recombination. Complete coding sequence of ADGF-A from start to stop codon was precisely replaced by coding sequence of destabilized version of GFP (dGFP). The 5’and 3’UTR of ADGF-A as well as all surrounding regulatory sequences stayed intact.

GFP-mRNA was now expressed in the same pattern and at the same quantity as ADGF-A-mRNA since all the regulatory sequences stayed intact. This was analyzed in heterozygous animals carrying one copy of intact ADGF-A on one chromosome and one copy of the GFP reporter on the other homologous chromosome. However, we barely detected any GFP protein in the reporter line. This could be caused by a posttranscriptional regulation (both 5’-UTR and 3’-UTR were preserved from the ADGF-A mRNA) or by destabilized GFP version with short half-life which was used in hope to detect dynamic changes in the ADGF-A expression.

Animals homozygous for the GFP reporter were in fact null adgf-a mutants. They resembled perfectly the adgf-a mutant phenotype, verifying that we indeed replaced the ADGF-A coding sequence. The adgf-a mutants differentiated specialized immune cells called lamellocytes and their aggregation led to formation of melanotic capsules (Dolezal et al., 2005). We noticed that the cells aggregated in the melanotic capsules in ADGF-A-GFP reporter line strongly expressed GFP.

Melanotic capsules are formed in various mutants with affected hematopoiesis as, for example, in cactus and hopTum mutants (Harrison et al., 1995; Qiu et al., 1998). The ADGF-A-GFP reporter was also expressed in the aggregated cells of cactus and hopTum mutants (Novakova and Dolezal, 2011). However, the natural role of lamellocytes production and melanization is a defense against parasitoid wasp infection when lamellocytes encapsulate the parasitoid egg, which is destroyed by subsequent melanization (Keebaugh and Schlenke, 2014). Therefore we also tested if ADGF-A was expressed in the immune cells during parasitoid wasp attack and found that it indeed was (Figure 9). These results suggested that the regulation of e-Ado was especially important during immune response.

ADGF-A:GFP expression in lamellocytes encapsulating parasitoid egg

Figure 9. ADGF-A-GFP reporter expression in lamellocytes encapsulating parasitoid wasp egg. LEFT: melanized parasitoid egg is encapsulated in multilayer of lamellocytes (DIC image). RIGHT: corresponding fluorescent image showing strong GFP fluorescence in encapsulating cells.


The results obtained with ADGF-GFP reporter were later confirmed by doctoral student Michaela Fenckova who used a different approach to produce the ADGF-A reporter (Fenckova, 2011). Michaela used a FlyFos system developed by group of Pavel Tomancak in Max Planck Institute, Dresden (Ejsmont et al., 2009). They used a fosmid library of Drosophila genome cut to ~30-kb pieces. These pieces often contained a sequence of the gene with full regulatory sequences. Such sequences could be modified by recombineering to introduce fluorescent tags into resulting fusion proteins. Michaela made a FlyFos-ADGF-A:GFP fly carrying this construct on chromosome II. The FlyFos-ADGF-A:GFP not only reproduced the expression pattern of ADGF-A including lamellocyte expression during wasp attack but also fully rescued the adgf-a null mutant showing that FlyFos-ADGF-A:GFP is in fact a fully functional ADGF-A protein marked by the GFP tag.


Does e-Ado regulate energy during immune response?

Summarizing previous results, we showed that (1) increased e-Ado regulated energy metabolism. We also showed that (2) the regulation of e-Ado was especially important during immune response suggesting that e-Ado was released in higher quantities during infection. Taken together, it was tempting to speculate that e-Ado may regulate energy during immune response.

Regulation of energy during immune response is critical – full response requires significant amount of energy and inability to provide it with nutrients can lead to immune system suppression and reduced resistance (Calder, 2013; Matarese et al., 2002; Rauw, 2012). Immune cells must respond rapidly to the activating signals and thus they change their metabolism, which involves increased aerobic glycolysis, known as the Warburg effect (Cheng et al., 2014; Delmastro-Greenwood and Piganelli, 2013; Wolowczuk et al., 2008). The increased demand for energy and nutrients by the immune system often requires adaptation of the whole organism, which is associated with an overall metabolic suppression and a systemic insulin resistance in all tissues except the immune cells (Chambers et al., 2012; Rauw, 2012; Straub et al., 2010).

The importance of the systemic regulation of energy is demonstrated by examples of certain infections leading to depletion of energy reserves (wasting) and eventually death of the organism (Dionne et al., 2006; Tracey and Cerami, 1992).  The capacity of e-Ado to regulate energy metabolism, to “measure” the level of tissue/organismal stress, and to adapt the energy use to the actual situation all made e-Ado a perfect candidate for the energy regulator during immune response.

We decided to use the parasitoid wasp infection of Drosophila larvae to test the putative role of e-Ado in the energy regulation during immune response. Immune response to parasitoid egg involves a massive and fast production of specialized immune cells (Figure 10) and thus represents energetically demanding process. The wasp infection is therefore an ideal model to study the energy regulation during immune response.

Parasitoid wasp Leptopilina boulardi infection of Drosophila larva

Figure 10. Parasitoid wasp infection of Drosophila larva and subsequent immune response of the host. Wasp Leptopilina boulardi injects its egg into early third-instar Drosophila larva. The parasitoid egg, usually hiding within the gut folds, is soon recognized by circulating immune cells (plasmatocytes marked by Hml>GFP). Plasmatocytes send unknown signal, which activates proliferation and differentiation of specialized immune cells – lamellocytes – in lymph gland. Lamellocytes (marked by Msn>GFP) must appear in the circulation within 24 hours, starting to encapsulate the parasitoid egg. Within 48 hours, the encapsulated egg must be destroyed by melanization otherwise a parasitoid larva will hatch and will consume the host.

First, we tested if the infection-induced immune response led to measurable changes in the energy metabolism. Infection led to hyperglycemia and suppression of energy storage, both glycogen and triglycerides. This was similar to the effect of adgf-a mutation that increased adenosine but this time, the energy effects were caused by natural physiological response to infection, not by genetic mutation. Then we tested if adenosine played a role in the observed energy changes. The easiest way was to block the adenosine signaling by the adoR mutation.  We found with a big excitement that the adoR mutation completely suppressed the hyperglycemia induced by the parasitoid attack implying that the adenosine signaling indeed played a role in the energy regulation during immune response.

In addition, adoR also dramatically decreased the number of lamellocytes and thus the resistance to wasp infection. Although this fit nicely to the idea that blocking the adenosine signaling suppressed hyperglycemia and thus the available energy for the rapid lamellocyte differentiation, adoR mutation could also just simply block the differentiation of lamellocytes. This other possible explanation was supported by results of (Mondal et al., 2011) who showed that adenosine played a role in regulation of hemocyte differentiation during normal development. However, our results demonstrated that adoR mutant was capable of lamellocyte differentiation, it was just less effective. Feeding infected larvae a diet with increased content of glucose, which increased the level of glucose in the hemolymph of such fed animals, significantly increased the number of lamellocytes in adoR. Later, we also showed that changes in Jak-Stat signaling, crucial for induction of lamellocyte differentiation (Makki et al., 2010), were intact in adoR. These results together demonstrated that the adenosine signaling was crucial for proper energy regulation ensuring enough energy for rapid lamellocyte differentiation and thus for effective response of flies to parasitoid wasp attack.

Postdoctoral fellow Adam Bajgar, who joined our team in 2012, started to dig deeper into the effects of parasitoid attack on metabolism and the role of adenosine in these effects. One of the most elegant procedures, which he used, was tracing 14C-labeled glucose during infection within different tissues. Adam demonstrated a substantial shift of glucose usage from non-immune tissues and developing processes towards immune cells during infection; consumption of glucose by immune cells increased from less than 10% during normal conditions to almost one-third of the overall consumption by the organism during infection. Importantly, adoR mutation significantly suppressed this shift. In agreement with the glucose tracing, the growth of developing tissues was suppressed during infection and thus the development was delayed; these effects were again under AdoR control. Adenosine thus turned out to mediate a systemic metabolic switch ensuring that the energy is not consumed by developmental processes and is left for the needs of immune system during infection (Bajgar et al., 2015).

Important aspect of this work was the connection of the observed molecular effects with the impact on organismal physiology, development and immune functions. We did not find only the regulation of energy metabolism by adenosine but the real biological role of this regulation. We were able to see a tradeoff between development and immune response, something which is occurring in nature all the time. In our model, Drosophila larva invests the energy preferentially either into development or into the immune response. In case of serious attack by parasitoid wasp, larva must use the energy for rapid immune response; if larva does not shift the energy as in case of adoR mutant, it develops with the same speed as uninfected larvae but it loses the resistance against the parasitoid. To find these types of connections, it is important to have a good model in vivo; the inter-organ communications cannot be studied in vitro. Drosophila offered to us a genetically well-tractable model and parasitoid wasp infection turned out to be an excellent experimental system in vivo to uncover systemic communications for proper energy regulation during immune response.


“Selfish” immune system

One of many interesting questions was the source of adenosine as the signal for metabolic switch? To answer this question, we used a simple readout characterizing the robustness of the immune response – the number of lamellocytes. The number can be easily checked and thus we could test various genetic manipulations to find which one affected the robustness of the response. We used various Gal4 drivers expressed in different tissues to knockdown selected genes by inducing RNAi (under UAS promoter) in tissues expressing the Gal4 drivers (Brand and Perrimon, 1993). Using this system, we found that suppressing adenosine transport from immune cells, by knocking down the equilibrative nucleoside transporter ENT2, had similar effects as blocking the adenosine signaling by adoR (Bajgar et al., 2015). Immune cells dramatically change their metabolism upon activation, leading to increased aerobic glycolysis akin to the Warburg effect (Cheng et al., 2014; Delmastro-Greenwood and Piganelli, 2013). Our expression analysis of glycolytic genes, glucose and trehalose transporters, and 14C uptake by immune cells suggested a similar behavior for the differentiating immune cells upon wasp attack. Taken together, this suggested that the immune cells could autonomously regulate the systemic metabolic switch based on their acute energy needs.

Adenosine-mediated systemic metabolic switch may resemble a selfish brain theory (Peters et al., 2004) in a way that the immune system, like the brain, is a privileged part of the organism, capable of suppressing energy consumption by other tissues in its own interest. The theoretical concept of selfish immune system was recently proposed to explain various metabolic problems in humans (Straub, 2014). We show that such a selfish immune system would use e-Ado as a signal to appropriate extra energy resources during immune challenge. Brain is hierarchically the highest organ and the most sensitive to energy depletion; immune system becomes indispensable during fight with pathogen and tissue damage and is also energetically demanding. Therefore their privileged positions make sense. Their energy state needs to be constantly monitored and fast response to energy depletion is crucial. Both task may be perfectly ensured by action of adenosine – it is instantly produced when ATP falls down and is released to extracellular space to inform about the energy status of the cell/tissue.

The view of immune system behaving selfishly in terms of energy distribution needs to be tested by additional experimental systems and the role of adenosine as a mediator must be further studied. This phenomenon may underlie various biomedical problems such as wasting, sepsis and metabolic syndrome (Straub, 2014). It is known that during systemic immune response, most of the tissues but immune cells become insulin resistant and this is associated with hyperglycemia (Straub et al., 2010).  Although this might be normal response ensuring enough energy for immune cells as in our parasitoid/fruit fly model and as the selfish immune system theory would suggest, it often becomes a medical complication; and the regulation behind such state is not clear. Our work thus may help to find path to various problems at the border of immunity and energy regulation.


Ado route to immunity - summary

The story started with generation of Drosophila adgf-akarel mutant with elevated levels of extracellular adenosine due to the deficiency of adenosine deaminase. This mutant brought us to the energy regulation by e-Ado in flies, which was a known common role of e-Ado in other organisms. However in flies, the energy regulation seemed to be the most prominent role of e-Ado while in higher organisms, as mammals, situation is complicated by various immunomodulatory roles of e-Ado. Using an expression reporter for the fly adenosine deaminase, we also found a link of e-Ado to immunity. Due to its important energy role, we started to focus on connection of immune response with energy regulation. We used a model of parasitoid wasp infection and discovered that e-Ado was used as a signal for systemic metabolic switch. The switch has been observed in various organisms including humans but its molecular mechanism remained unknown. Uncovering the role of e-Ado in this switch also supported the concept of selfish immune system, in which immune system belongs to privileged organs that can usurp energy from the rest of the organism during stress (Figure 11).

Adenosine role in selfish immune system

Figure 11. Selfish immune system uses e-Ado to mediate systemic metabolic switch.  Parasitoid wasp egg injected into Drosophila larva is recognized by circulating hemocytes which activate differentiation of specialized immune cells – lamellocytes – from pro-hemocytes. Activated pro-hemocytes proliferate and differentiate into lamellocytes and this is associated with increased glycolysis and consumption of glucose. Activated pro-hemocytes usurp energy from the rest of the organism, including developing tissues, by releasing adenosine. e-Ado suppresses metabolism of other tissue by AdoR signaling. Differentiated lamellocytes eventually encapsulate the parasitoid egg and destroy it by melanization.



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