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Glucose and trehalose metabolism through the cyclic pentose phosphate pathway shapes pathogen resistance and host protection in Drosophila [1]

['Michalina Kazek', 'Department Of Molecular Biology', 'Genetics', 'Faculty Of Science', 'University Of South Bohemia', 'České Budějovice', 'Czech Republic', 'Lenka Chodáková', 'Katharina Lehr', 'Lukáš Strych']

Date: 2024-05

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 13 C-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.

Immune cells have privileged access to nutrients during immune response [ 17 ]. We hypothesize that hemocytes secure prioritized allocation of carbohydrates using trehalose, i.e., by expressing Tret1-1 along with Treh. To test this hypothesis, we employed 13 C stable isotope tracing [ 18 ] to analyze metabolic changes in hemocytes and by genetically manipulating trehalose metabolism to investigate its role during immune response to parasitoid wasps. We found that hemocytes express several carbohydrate transporters, some of which are dramatically up-regulated upon infection. 13 C tracing experiments showed that activated hemocytes uptake more glucose and trehalose and increase glycolysis and cyclic PPP, all essential for lamellocyte production and larval resistance. Systemic trehalose metabolism is important for an effective immune response, but trehalose itself is only metabolized in fully differentiated lamellocytes, which is not necessary for resistance but instead appears to be important for lamellocyte-mediated host protection.

One of the most important branches from increased glycolysis during immune response is the PPP [ 3 ], generating NADPH and pentoses as precursors for nucleotide and coenzyme synthesis [ 14 ]. NADPH is essential in activated immune cells for lipid biosynthesis, for the ROS production [ 15 ], as well as for the production of antioxidants, such as glutathione, to protect the host from excessive ROS exposure [ 16 ]. Cells can dramatically increase NADPH production through cyclic PPP, which repeatedly oxidizes glucose-6-phosphate (G6P), as recently demonstrated in neutrophils [ 3 ].

Melanization/encapsulation serves the dual purpose of producing and concentrating toxic substances inside the capsule to kill the parasitoid while protecting the host from toxic radicals [ 12 ]. Therefore, there are metabolic requirements associated with lamellocyte differentiation (global changes in gene expression, cytoskeletal, and membrane rearrangements, etc.), production of toxic molecules (e.g., reactive oxygen species, ROS), and host protection mechanisms (e.g., antioxidant production) [ 13 ]. Metabolic reprogramming for the individual tasks involved in killing pathogens and protecting the host is essential for the survival of the infected larva and for fitness of the surviving fly. However, the actual changes in metabolism of larval hemocytes during immune response to wasp infection have not yet been investigated.

We use a model of infection where the parasitoid wasp Leptopilina boulardi injects its egg into developing Drosophila larvae [ 11 ]. Within a few hours, the egg is recognized by circulating hemocytes (plasmatocytes), which induce an immune response. Sessile hemocytes enter the circulation and some attach to the egg while others differentiate into the lamellocytes, large flat cells which later encapsulate the egg. A melanization cascade is initiated in the forming capsule [ 12 ] and within approximately 48 h the parasitoid egg is destroyed. If the immune response is slow or inefficient, the parasitoid larva emerges from the egg and eventually consumes the host fly at the pupal stage [ 4 ].

Trehalose is the primary carbohydrate of insects; when cleaved by trehalase (Treh), it provides a rapid source of glucose [ 5 ]. Unlike glucose, trehalose is a nonreducing sugar and thus the hemolymph of Drosophila larvae contains a tenfold higher concentration of trehalose than glucose [ 4 , 6 ]. This serves as a buffer for glucose homeostasis to ensure robust and stable development [ 7 ]. We previously found that hemocytes strongly up-regulate expression of the trehalose transporter Tret1-1 and Treh during immune response [ 4 ]. This was later confirmed by single cell transcriptomics studies [ 8 , 9 ], which showed lamellocyte-specific expression of Tret1-1 and Treh. These results suggest an important role for trehalose during immune response, which has also been shown in house flies [ 10 ].

When activated by a challenge, immune cells must rapidly undertake new functions, facilitated by the rapid generation of energy and biosynthetic intermediates [ 1 ]. The majority of mammalian immune cells and insect immune cells become more dependent on glucose and have an increased rate of glycolysis during infection [ 1 , 2 ]. This allows for the rapid generation of ATP and branching into other metabolic pathways, for example, the pentose phosphate pathway (PPP) [ 3 ]. We have shown that during infection of Drosophila melanogaster larvae by a parasitoid wasp, immune cells (hemocytes) consume more than double the amount of total systemic glucose than hemocytes in uninfected larvae [ 4 ]. Hemocytes release adenosine to suppress carbohydrate consumption by nonimmune tissues to ensure their own supply, which is critical for an effective immune response [ 4 ].

In summary, we used Treh -/- mitotic clones to test for a cell-autonomous role of trehalose metabolism in hemocytes and found that it is not required for lamellocyte differentiation or host resistance. Elimination of the ability to metabolize trehalose in 40% of lamellocytes enhanced resistance, which was associated with increased hemolymph ROS levels and reduced fitness of survivors. A similar increased in resistance was achieved with 2 independent Treh RNAi lines, but this was associated with higher host lethality. Since hemocytes metabolize trehalose via cyclic PPP, generating NADPH and antioxidants, these results suggest that trehalose may play a role in protecting the host from ROS that serve to kill the pathogen.

We attempted to verify the effect of Treh[cs1] hemocytes mutant clones via another trehalase manipulation. The Treh[cs1] null mutation affects systemic trehalose/glucose metabolism and homozygous cTreh-specific Treh[RAΔG4] loss is compensated by expression of alternative transcripts. Therefore, we used the heterozygous Treh[RAΔG4] allele that (1) partially reduces cTreh expression ( S14 Fig ); and (2) expresses Gal4 in the cTreh pattern, including a strong up-regulation in lamellocytes. To suppress Treh in the cTreh expression pattern, we combined the Gal4 driver within Treh[RAΔG4] with either of 2 UAS-Treh-RNAi constructs, Treh HMC03381 or P{GD5118}v30731 ( S14 Fig ). Both RNAi lines recapitulated the cTreh null mutant phenotype (10% lethality in pupae and lethargic adults compared to 20% lethality in pupae and dying young adults in the null mutant). In both cases, larvae with Treh RNAi showed significantly increased resistance by killing parasitoids more often than control larvae ( S14 Fig ), reproducing the results obtained with Treh -/- mutant clones. However, in the case of RNAi, the increased resistance did not result in improved survival, but was associated with increased host lethality ( S14 Fig ). The effect of RNAi might be more severe as, unlike in Treh -/- hemocyte clones, Treh depletion was not limited to hemocytes but affected all tissues where cTreh is expressed and the Treh[RAΔG4] driver is active. Nevertheless, the increased pathogen killing at the expense of own survival suggests that trehalose may indeed play a role in host protection.

There is a small, statistically insignificant increase in pupal lethality of infected larvae with induced Treh -/- clones ( Fig 6G ), suggesting that the increased ROS levels were not lethal to the host. To explore further, we looked at the lifespan of adult flies that survived the infection. Male lifespan was comparable ( S13 Fig ); however, a greater number of females with induced clones died earlier than controls, with the median survival significantly reduced from 48 to 34 days, although the maximum lifespan was comparable ( S13 Fig ). A more pronounced effect was observed in the production of viable offspring: females with induced clones that survived infection produced less than half of progeny of controls, whereas no such difference was observed in the absence of infection ( Fig 6I ). Thus, induction of Treh[cs1] mutant clones in hemocytes during parasitoid infection of larvae results in reduced fitness of survivors. Whether there is a relationship between increased hemolymph H 2 O 2 and the viability of future offspring of the infected larvae remains to be tested.

Since trehalose metabolism in hemocytes did not appear to be important for resistance, we speculated that it may rather mediate the antioxidant host protection against own immune response. This would be consistent with trehalose being metabolized by cyclic PPP to produce NADPH, which is required for antioxidant production. Removing this activity from larval lamellocytes could decrease their ROS scavenging capacity and thus increase toxicity and resistance towards the pathogen. Therefore, we analyzed the effect of trehalase mutant hemocytes on the level of hydrogen peroxide in the hemolymph. The melanization response is generally associated with strong catalase activity in insect hemolymph, making it difficult to detect increased ROS during infection without catalase inhibitors [ 32 ]. A small increase in H 2 O 2 was detected only at 30 hpi, but not at 20 or 40 hpi during the Drosophila response to parasitoid [ 33 ]. We did not use antioxidant inhibitors because we were interested in differences in antioxidant activities, and this is likely why we failed to detect a difference in hemolymph H 2 O 2 between infected and uninfected larvae ( S13 Fig ). Induction of mitotic Treh[cs1] mutant clones did not alter H 2 O 2 levels in uninfected larvae ( S13 Fig ). However, we detected a consistent, significant increase in H 2 O 2 levels in the hemolymph of larvae with induced clones at 30 hpi ( Fig 6H ). This result supports the notion that trehalose may play a role in mechanisms that protect the host from oxidative stress. Down-regulation of trehalose metabolism in hemocytes leading to increased ROS may explain the observed increased resistance ( Fig 6N ).

To kill the wasp egg, the larval immune cells generate ROS [ 29 ]. However ROS are potentially harmful also to the host, which prevents damage to its own tissues by producing antioxidants [ 13 ]. Flies use the thioredoxin system instead of glutathione reductase to reduce GSSG [ 30 ], and hemocytes highly express thioredoxin reductase Trxr1 and thioredoxin Trx2 with greater expression in lamellocytes ( S12 Fig ). Trx2 is also a substrate for peroxiredoxins [ 31 ], which are highly expressed in hemocytes as well ( S12 Fig ). The thioredoxin system requires NADPH ( S12 Fig ), which is produced by cyclic PPP. We found increased cyclic PPP activity and NADPH levels in hemocytes, including lamellocytes, during infection ( Fig 6J ). We also observed increased levels of the reduced glutathione form (GSH, Fig 6K ) in hemocytes from infected larvae, whereas the oxidized GSSG form remained at comparable levels ( Fig 6L ). Besides GSH, infection elevated another antioxidant, taurine, in hemocytes ( Fig 6M ). Therefore, besides eliminating the pathogen, hemocytes seem to protect the host by mounting an antioxidant response.

To test the effect on resistance, we used the larvae bearing approximately 40% of lamellocytes mutant for Treh. If trehalose metabolism in the lamellocytes is necessary for parasitoid killing, we would expect lower resistance. However, induction of Treh[cs1] mutant clones resulted in the opposite effect, with increased resistance compared to animals with the same genetic background in which clones were not induced ( S11 Fig ). Although a relatively large variability was observed, the average percentage of surviving adult flies in controls was 18% and never exceeded 30%, whereas in flies with induced Treh -/- clones the average was 35% and reached up to 64% ( Fig 6G ). Subsequently, the percentage of surviving parasitoid wasps decreased from 65% in controls to 35% when Treh -/- clones were induced ( Fig 6G ).

(A) Generation of hemocyte clones with null Treh[cs1] mutation by Srp-Gal4 UAS-Flp induced mitotic recombination. Genotypes of parental and daughter cells after mitotic recombination are color marked red: RFP-marked Treh[cs1] mutant cells; green: GFP-marked wild-type sister clone; yellow: RFP/GFP nonrecombinant heterozygous cells. (B) Red-, green-, and yellow-marked hemocytes (both plasmatocytes and lamellocytes) upon mitotic recombination from infected larvae in circulation (left) and attached to a wasp egg (right). (C) Percentage of wild-type recombinant and nonrecombinant hemocytes in uninfected larvae without any mutation. (D) Percentage of Treh[cs1] homozygous, sister wild-type and nonrecombinant heterozygous hemocytes in uninfected larvae. (E) Percentage of Treh[cs1] homozygous, sister wild-type and nonrecombinant heterozygous lamellocytes in infected larvae. (C–E) Bars represent mean percentage, dots represent counts from single larvae, error bars represent ± SEM; samples were compared using unpaired two-tailed t test; asterisks indicate p value (* P < 0.05; ns = not significant). (F–I) Controls without recombination (gray) compared to larvae with Treh[cs1] mutant hemocyte clones (FRT42D GFP / FRT42D Treh[cs1] RFP; Srp-Gal4 / UAS-Flp; purple). (F) Number of lamellocytes 22 hpi. Each dot represents number of lamellocytes per larva, lines represent mean, samples were compared by unpaired t test, no significant difference. (G) Percentages of surviving adult flies, wasp larvae, dead pupae (neither fly nor wasp survived), and adult parasitoid wasps. Bars represent mean percentage, dots represent biological replicates (individual infection experiments), error bars represent ± SEM, samples were compared by ordinary one-way ANOVA with Šídák’s test for multiple comparisons, asterisks indicate p values (* P < 0.05; ** P < 0.01, ***P < 0.001; ns = not significant). (H) H 2 O 2 levels in the hemolymph of infected control larvae (gray) and larvae with Treh[cs1] mutant clones (purple) at 30 hpi. Dots represent paired biological replicates (infection performed and H 2 O 2 measured in parallel for 1 control and 1 mutant sample); data were compared using two-tailed paired t test, asterisks indicate P < 0.01. (I) The average number of progeny produced by an uninfected female and a female surviving the larval infection. Control (gray) and females bearing Treh[cs1] mutant clones (purple) were compared by two-tailed Welch’s t test; asterisk indicates P < 0.05. Bars represent the average numbers of progeny produced per female, each dot represents 1 infection experiment with at least 10 surviving females, error bars represent ± SEM. (J–M) Levels of unlabeled NADPH (J), GSH (K), GSSG (L), and taurine (M) in hemocytes from experiments ex vivo or in vivo. The bars show the mean metabolite amounts expressed by the normalized peak area, samples were obtained from hemocytes from uninfected (Uninf) or infected (INF) larvae, and were compared by two-tailed Welch’s t test; asterisks indicate p values (** P < 0.01, ***P < 0.001, ****P < 0.001; ns = not significant). (N) Model for the role of trehalose in producing antioxidants during response to parasitoid infection. Numerical values for C–M are available in S1 Data . hpi, hours post infection.

Since we could not test the importance of trehalose metabolism in hemocytes with cTreh mutations, we decided to generate mitotic recombination clones [ 28 ] in the hematopoietic lineage with the Treh[cs1] mutation, which we verified as blocking trehalose metabolism ( Fig 5C ). We recombined flippase (Flp) target site FRT42D, Treh[cs1] mutation and RFP marker on the second chromosome and crossed this line to flies with FRT42D and the GFP marker to generate heterozygous FRT Treh[cs1] RFP / FRT GFP flies ( Fig 6A ). To induce mitotic recombination in the hematopoietic lineage of the progeny, the parental flies also carried Srp-Gal4 and UAS-Flp on their third chromosomes. Mitotic recombination resulted in RFP-labeled hemocytes with a Treh[cs1] null mutation and their GFP-labeled wild-type siblings ( Fig 6A and 6B ). When mitotic clones were first induced with only RFP and GFP markers without any mutation, the expected equal ratio of RFP to GFP sibling hemocytes was detected ( Fig 6C ), approximately 40% each, indicating high recombination efficacy (only the remaining 20% were non-recombined GFP/RFP hemocytes). A similar result was obtained in uninfected larvae with the Treh[cs1] mutation, although there was a minor increase in RFP-labeled Treh[cs1] -/- hemocytes compared to the GFP-labeled wild-type hemocytes (49% versus 43%; Fig 6D ). The difference is more likely to result from different genetic backgrounds of the chromosomal arms that become homozygous after recombination than from the lack of trehalase activity. A comparable result was obtained with lamellocytes from infected animals, where we found similar proportions of red and green cells—the small difference was consistent with the difference observed in uninfected larvae ( Fig 6E ). Generating clones with the Treh[cs1] null mutation did not change the total number of lamellocytes compared to larvae without induced recombination ( Fig 6F ). These results indicate that trehalose metabolism is dispensable for lamellocyte differentiation in a cell-autonomous manner, which is consistent with cytoplasmic trehalase not being expressed until in fully differentiated lamellocytes ( Fig 1H ).

Null Treh mutation impairs systemic trehalose metabolism. Since lamellocytes specifically increase the expression of the cytoplasmic Treh, we used the specific mutations in cTreh to test a cell-autonomous role of trehalose metabolism in hemocytes. The Treh[c1] mutation ( S10 Fig ) should block the utilization of trehalose inside the cells but not trehalose-glucose metabolism in the circulation [ 22 ]. However, when we incubated Treh[c1] mutant hemocytes with 13 C-labeled trehalose ex vivo, we observed no effect on trehalose metabolism in hemocytes ( S10 Fig and S2 Table ). The failure of the Treh[c1] mutation to block intracellular trehalose metabolism could be due to a second start codon in the cTreh-RA/RD/RG/RE transcripts ( S10 Fig ). However, when both start codons were removed in our Treh[RAΔG4] mutant (Figs 1 and S10 ), the homozygous mutant hemocytes still metabolized labeled trehalose almost normally ( S10 Fig and S2 Table ), and the mutation did not affect the number of lamellocytes ( S10 Fig ). This is likely due to the compensatory expression occurring at the alternative transcription start sites common to both Treh-RB (cTreh) and Treh-RC (sTreh) transcripts ( Fig 1 ).

Next, we knocked down the Tret1-1 transporter specifically in hemocytes (Srp>Tret1-1-RNAi—P{TRiP.HMS02573}attP2), which resulted in a reduction of Tret1-1 expression upon infection to one-third compared to control ( Fig 5D ). Because the knockdown did not prevent the increase in Tret1-1 expression during infection (it only partially suppressed it), ex vivo metabolism of 13 C-labeled trehalose, indicated by lower incorporation into G6P ( Fig 5E and S2 Table ) as well as the number of lamellocytes ( Fig 5F ) were only slightly reduced. Since Tret1-1 knockdown did not substantially suppress trehalose uptake, we used a null mutant of Tret1-1 together with a null mutation of MFS3. The double mutant MFS3[CRISPR] Tret1-1[XCVI] significantly reduced the number of lamellocytes, albeit again only slightly ( Fig 5G ), suggesting that other transporters ensure carbohydrate supply.

(A) Number of lamellocytes 22 hpi in control (heterozygous Mi{y[+mDint2] = MIC}Tps1[MI03087] / +) and hypomorphic Tps1 mutant (Mi{y[+mDint2] = MIC}Tps1[MI03087] / Tps1[d2]). Each dot represents number of hemocytes per larva, lines represent mean, samples were compared by unpaired t test, asterisks indicate p value (**** P < 0.0001). (B) Number of lamellocytes 22 hpi in control (heterozygous Treh[cs1] / CyO Ubi-GFP) and null Treh[cs1] homozygotes. (C) Heat map of 13 C-labeled fraction of metabolites from control and Treh[cs1] hemocytes in uninfected (Uninf) and infected (INF) conditions incubated ex vivo with α,α−trehalose- 13 C 12 (data in S2 Table ). (D) Efficiency of hemocyte-specific RNAi of Tret1-1 tested by RT-qPCR in hemocytes from uninfected and infected larvae 18 hpi. Box plots (median, 75th and 25th percentiles) show fold change compared to uninfected Tret1-1-RA control samples (expression levels were normalized by RpL32 expression in each sample), each dot represents a biological replicate. Gray boxes represent control (Srp> P{y[+t7.7] = CaryP}attP2) and red boxes Tret1-1 RNAi (Srp>P{TRiP.HMS02573}attP2). Unpaired two-tailed t test was used to compare control with RNAi; asterisks indicate p values (* P < 0.05, ** P < 0.01, *** P < 0.001). (E) Fraction (%) of G6P- 13 C n in hemocytes from uninfected and infected larvae, incubated ex vivo with labeled glucose (left bars) or trehalose (right bars), respectively. Gray bars represent mean in control (Srp> P{y[+t7.7] = CaryP}attP2) and red bars mean in hemocyte-specific Tret1-1 RNAi (Srp>P{TRiP.HMS02573}attP2). Unpaired two-tailed t test was used to compare control with RNAi; asterisks indicate p value (** P < 0.01). (F) Number of lamellocytes 22 hpi in control (Srp> P{y[+t7.7] = CaryP}attP2) and in hemocyte-specific Tret1-1 RNAi (Srp>P{TRiP.HMS02573}attP2). Each dot represents number of lamellocytes per larva, lines represent mean, samples were compared by unpaired t test, asterisks indicate p value (*** P < 0.001). (G) Number of lamellocytes 22 hpi in control (MFS3[CRISPR] Tret1-1[XCVI] / Cyo Ubi-GFP heterozygotes) and in MFS3 Tret1-1 double-null mutants (MFS3[CRISPR] Tret1-1[XCVI]). Each dot represents number of lamellocytes per larva, lines represent mean, samples were compared by unpaired t test, asterisks indicate p value (** P < 0.01). Numerical values for A, B, D, E, F, and G are available in S1 Data . hpi, hours post infection.

Metabolomics with 13 C-labeled trehalose shows that trehalose is metabolized by infection-activated hemocytes. Therefore, we tested the importance of trehalose for the immune response. We first tested the hypomorphic mutation in trehalose-synthesizing gene Tps1 (Mi{y[+mDint2] = MIC}Tps1[MI03087] / Tps1[d2]). Tps1 mutant larvae develop with normal levels of glucose, glycogen, and triglycerides but their trehalose levels are only 20% those of controls [ 7 ]. Infected Tps1 mutants differentiated significantly fewer lamellocytes than control larvae ( Fig 5A ). Similarly, fewer lamellocytes differentiated in null Treh[cs1] mutants ( Fig 5B ) that cannot convert accumulating trehalose to glucose [ 22 ]. We verified that hemocytes from the Treh[cs1] mutant were unable to metabolize 13 C-labeled trehalose supplied ex vivo ( Fig 5C and S2 Table ). These results demonstrate that trehalose is important for efficient lamellocyte differentiation.

Oxidative PPP generates NADPH, which is important for lipid synthesis. While lipid metabolism is beyond the scope of our study, we found a rather striking difference in lipid droplets between hemocytes from control larvae and larvae with hemocyte-specific RNAi of Zw during infection ( S9 Fig ). This indicates that properly functioning oxidative PPP in hemocytes is important for their lipid metabolism during infection.

(A) Schematic representation of pentose phosphate pathway with metabolites in black and Drosophila genes encoding enzymes in blue. Genetic manipulations include hemocyte-specific RNAi of Zw induced by Srp-Gal4 (red lightning) and double null mutant in Zw and Pgd (Pgd[n39] pn[ 1 ] Zw[lo2a]; red crosses). (B) Efficiency of hemocyte-specific RNAi of Zw tested by RT-qPCR in hemocytes from uninfected (Uninf) and infected (INF) larvae 18 hpi. Bars represent mean values of fold change compared to uninfected control samples (Zw expression levels normalized by RpL32 expression in each sample); dots show individual samples, error bars represent ± SEM, the control (Srp> P{y[+t7.7] = CaryP}attP2) was compared to RNAi (Srp>P{TRiP.HMC03068}attP2) using unpaired t test, asterisks indicate p values (* P < 0.05, ** P < 0.01). (C) Number of hemocytes 22 hpi in control (Srp> P{y[+t7.7] = CaryP}attP2), Zw Pgd null mutant (Pgd[n39] pn[ 1 ] Zw[lo2a]), and hemocyte-specific RNAi of Zw. Each dot represents number of hemocytes in one larva, lines represent the mean, samples were compared by unpaired t test, asterisks indicate p value (* P < 0.05, **** P < 0.0001). (D) Survival of parasitoid wasp infection in control, null Zw Pgd mutant and hemocyte-specific RNAi of Zw. Bars show percentages of surviving flies (white), developing parasitoids (gray) and dead pupae when neither fly nor parasitoid survived (dark). Bars represent mean values; dots represent biological replicates, error bars represent ± SEM; survival rates were compared using ordinary one-way ANOVA with multiple comparisons; asterisks indicate p values (** P < 0.01, *** P < 0.001; numerical values in S1 Data ). hpi, hours post infection.

Our metabolomics analysis shows that carbohydrate uptake and oxidative PPP increase in hemocytes upon infection. To test the importance of oxidative PPP during immune response, we used a double mutant in G6P dehydrogenase (Zwischenferment or Zw) and 6-phosphogluconate dehydrogenase (Pgd), the first and the third enzymes in oxidative PPP, respectively ( Fig 4A ). The Pgd[n39] pn[ 1 ] Zw[lo2a] mutant flies are fully viable [ 27 ]. We also used a hemocyte-specific RNAi of Zw ( Fig 4A and 4B ) using Srp-Gal4 and the Zw RNAi construct under the UAS promoter (P{TRiP.HMC03068}attP2). The number of plasmatocytes was altered neither in the double mutant nor by hemocyte-specific Zw knockdown, suggesting that basal hematopoiesis was unaffected by these manipulations ( Fig 4C ). However, both the double mutant and knockdown displayed decreased lamellocyte number ( Fig 4C ) and larval survival upon infection ( Fig 4D ), indicating that oxidative PPP is important for effective lamellocyte differentiation and parasitoid killing.

In summary, metabolic changes occurring in hemocytes during infection include increased glucose uptake and, in lamellocytes, glucose production from trehalose. The prevailing fate of glucose during infection appears to be in cyclic PPP, where G6P is oxidized in multiple rounds, as evidenced by elevated partial G6P labeling. Increased glycolysis correlates with higher lactate release from hemocytes. A substantial portion of glucose is used for de novo purine synthesis. While the degree of glucose utilization in mitochondrial metabolism remains unclear, amino acids appear to be the source for the TCA cycle and potentially for lipid synthesis.

Ribose-5P can be used for de novo purine/pyrimidine synthesis. This is indicated by the 13 C labeling of AMP in vivo ( S8 Fig ). While all ribulose-5P/ribose-5P fractions in infected larvae were significantly lower (due to liberation of unlabeled endogenous glucose) than in uninfected larvae, the AMP fractions were similar, indicating increased purine production during infection. This is supported by the overall increase of AMP/ADP/ATP levels during infection ( S8 Fig ). Interestingly, the m+1 fraction of AMP was markedly higher compared to ribulose-5P/ribose-5P. In addition to m+1 ribose-5P as a source of m+1 AMP, labeled glycine (produced from labeled glucose similarly to glutamine; S8 Fig ), added to unlabeled ribose-5P during purine synthesis, could increase the m+1 fraction of AMP. The carboxylation step utilizing labeled CO 2 released during oxidative PPP could be another source for the increased m+1 fraction of AMP [ 26 ].

Since the incorporation of 13 C into tricarboxylic acid (TCA) cycle metabolites was very low (<1%) after 40 min of ex vivo incubation, we compared the incorporation into citrate from the in vivo experiment with feeding 13 C-labeled glucose for 6 h. As we detected only the m+3 fraction of pyruvate ( S7 Fig ), we expected either m+2 (via pyruvate dehydrogenase) or m+3 (via pyruvate carboxylase) fractions of citrate. However, we detected an m+1 fraction as the highest ( S7 Fig ), which we also found in other TCA metabolites ( S7 Fig ). Therefore, we checked the 13 C labeling of glutamine and glutamate as a potential source and found that it matched the fractional pattern of the TCA metabolites ( S7 Fig ). Since we fed larvae with labeled glucose for 6 h, some of the glucose was metabolized to amino acids; the labeled amino acids were released into the hemolymph and taken up by hemocytes as a source for mitochondrial metabolism or, via citrate, potentially for lipid synthesis. Pyruvate derived from glucose could still be used in mitochondria and contribute to the m+2/m+3 fractions, but the amino acids seem to be an important source for mitochondrial metabolism in hemocytes, both without and during infection. Nevertheless, changes in mitochondrial metabolism need to be further investigated.

The m+3 fraction of glyceraldehyde-3P, which is a product of both glycolysis and PPP, increased during infection from both labeled glucose and trehalose ( Fig 3G and 3H ). Pyruvate was more difficult to detect, and the labeled pyruvate reached detectable levels in the ex vivo experiment only after infection ( S2 Table ). We have previously shown that hemocytes release more lactate during infection [ 25 ], which is confirmed here by higher levels in hemolymph during infection ( S4 Fig ). The increased incorporation of 13 C into lactate, from both glucose and trehalose ( Fig 3I ), demonstrates its increased production during infection.

Increased labeling of ribulose-5P/ribose-5P ( Fig 3B and 3E ) and sedoheptulose-7P ( Fig 3C and 3F ), the products of PPP and transketolase/transaldolase activity, respectively, further supports enhanced PPP in hemocytes during infection; we were unable to distinguish between ribulose-5P and ribose-5P ( S2 Table ) and therefore present them together. Ribulose-5P and ribose-5P can arise either from oxidative or nonoxidative PPP. The opposite direction of transketolase/transaldolase activity in the latter generates pentoses without NADPH (scheme in Figs 2 , 3J , and S6 ). While m+3 and m+5 ribulose-5P and m+5 sedoheptulose-7P can be generated by oxidative PPP, m+2 ribulose-5P, and m+4 sedoheptulose-7P are rather markers of nonoxidative PPP (Figs 3B, 3C, 3E, 3F , S4 , S5 and S6 ). The detected mixtures of these forms indicate that hemocytes use both oxidative/cyclic PPP and nonoxidative PPP. Different hemocyte types in our heterogenous cell population likely engage these 2 branches of PPP differently.

(A–K) 13 C labeling of glycolytic and pentose phosphate pathway metabolites in hemocytes from uninfected (light gray/blue bars) and infected (dark gray/blue bars) larvae. Hemocytes were incubated ex vivo for 40 min in media containing 0.5 mM D-glucose- 13 C 6 and 5 mM unlabeled trehalose (gray) or 5 mM α,α−trehalose- 13 C 12 and 0.5 mM unlabeled glucose (blue). m+0 indicates the fraction of the compound with unlabeled molecular mass; m+1 indicates the fraction of the compound with one 13 C-labeled carbon, and so on. Bars represent means of 5 biological replicates with 95% CI, each dot represents 1 biological replicate; asterisks represent significant differences between uninfected and infected fractions tested by unpaired two-tailed Welch’s t test (numerical values in S1 Data ). Note that ribulose-5P and ribose-5P (B, E) and fructose-6P and glucose-1P (G, H) were indistinguishable. For lactate (K), only the m+3 fraction is shown, as m+1 and m+2 were not detected above naturally occurring levels (m+0 and m+3 make up 100%). Schematic representation (L) of the cyclic PPP in blue and nonoxidative PPP-producing pentoses for de novo purine/pyrimidine synthesis in orange. PPP, pentose phosphate pathway.

After 6 h of feeding D-glucose- 13 C 6 , 21% of the glucose in the larval hemolymph was labeled ( S4 Fig ). Some of the labeled glucose was converted to trehalose in the fat body, resulting in 16% of the circulating trehalose being labeled at one of the 2 glucose monomers ( S4 Fig ). Both glucose and trehalose in the hemolymph increased during infection and the increase was mainly due to the unlabeled sugars ( S4 Fig ), suggesting a release from stores consistent with our previous results [ 4 ]. Hemocytes can directly uptake labeled dietary glucose or convert labeled trehalose to glucose. Five-fold increased content of 13 C-labeled and unlabeled glucose in infection-activated relative to control hemocytes indicated that hemocytes increased sugar uptake upon infection ( S4 Fig ), again in agreement with our previous results [ 4 ]. A direct comparison of downstream metabolite labeling between uninfected and infected larvae is complicated by the different fraction of labeled glucose in hemocytes of uninfected and infected larvae—15% versus 7%, respectively ( S4 Fig ), where the increased amount of labeled glucose in infected larvae is diluted by unlabeled glucose, also taken up by the hemocytes. Nevertheless, while the m+6 fraction (with all 6 carbons labeled) of intracellular glucose after infection is half that of the uninfected fraction, the m+6 fractions of glucose-6-phosphate (G6P) are comparable and in addition there are m+2 and m+3 fractions (i.e., with 2 or 3 labeled carbons) in infected samples that are not present in the uninfected samples ( S4 Fig ). Thus, the overall increase in labeled G6P indicates more intense glucose metabolism of hemocytes after infection.

In order to monitor changes in hemocyte metabolism during infection, we took 2 approaches based on tracing of metabolites labeled with the stable 13 C isotope: in vivo and ex vivo. In the former, we fed D-glucose- 13 C 6 (fully labeled, i.e., on all 6 carbon atoms) to larvae at 16 hpi for 6 h before collecting hemocytes at 22 hpi. In addition to following metabolism in activated plasmatocytes, we assessed the last 6 h of differentiation of the first lamellocytes, which appeared complete by the end of the experiment. In the ex vivo approach, we incubated hemocytes in media containing either fully labeled D-glucose- 13 C 6 or α,α−trehalose- 13 C 12 . Hemocytes collected by bleeding larvae 22 hpi were immediately incubated for 40 min with the labeled sugars. About half of the cells have already differentiated into lamellocytes. By examining only 1 time point, we are unable to determine metabolic fluxes, but we could still observe metabolic changes. It is important to note that in both approaches we worked with a heterogeneous population of hemocytes and thus observed either averaged metabolic changes that occurred across different types of hemocytes or a metabolism that occurred only in a specific hemocyte type.

Whereas in our previous study, we detected slightly increased expression (1.5- to 2-fold) of most glycolytic genes after infection using qPCR analysis [ 4 ], here we detected decreased expression of most of these genes using bulk RNAseq analysis ( Fig 2 ). This can be explained by increased overall gene expression in activated hemocytes after infection, where normalization of RNAseq data to one million transcripts (TPM) versus normalization to a selected housekeeping gene may lead to such differences. This difference is evident, for example, when comparing the change in trehalase expression, where RNAseq shows only an 11.5-fold increase (7.2 versus 83 TPM, Fig 1B ), whereas qPCR shows a 25-fold increase ( Fig 1E ).

Diagram showing metabolic pathways; metabolites and enzymes are abbreviated with Drosophila gene names; strongly expressed genes in hemocytes are in large font in bold, moderately expressed genes in large font, and insignificantly expressed genes in small font. Trehalose metabolism is shown in gray, glycolysis in black, pentose phosphate pathway in blue, purine metabolism in orange, and mitochondrial metabolism in green. Tables show glycolytic and PPP gene expression based on bulk RNAseq in TPM in circulating hemocytes at 18 hpi and from the uninfected (18 h Uninf) larvae; the shading highlights the strength of expression—white the weakest, red the strongest—data in S1 Table . hpi, hours post infection; PPP, pentose phosphate pathway; TPM, transcripts per million.

Our bulk transcriptomic analysis showed that all enzymes associated with glycolysis and PPP are strongly expressed in hemocytes in both the uninfected and infected states ( Fig 2 and S2 File and S2 Table ). The levels of specific mRNAs in each pathway correspond well with scRNAseq analyses ( S2 File ; [ 8 , 9 ]). The same glycolytic and PPP genes are similarly expressed in the lymph gland and wing disc with or without infection ( S2 File and S2 Table ). Combining our bulk transcriptomics and scRNAseq data revealed a tendency for a slight decrease in the expression of glycolytic genes in most prohemocytes and plasmatocyte-like cells during infection, while the expression shifted towards lamellocytes and crystal cells ( S2 File ). Expression of PPP genes was generally unchanged during infection. Phosphofructokinase (Pfk) showed the lowest expression of all glycolytic genes and was further reduced in all hemocyte types during infection, suggesting a shift away from glycolysis to PPP and back to glycolysis at the glyceraldehyde-3P level. Overall, the expression analysis does not indicate specific changes in these metabolic pathways in either of the hemocyte types, or during infection.

Our expression analysis showed that hemocytes express multiple carbohydrate transporters, some of which are dramatically induced during infection. This is consistent with our previous results showing enhanced sugar consumption under these conditions. The combination of highly elevated expression of trehalose transporters and of the cytoplasmic form of Treh exclusively in lamellocytes, together with the compensatory expression of cTreh, suggests the importance of trehalose metabolism in these cells.

Since the increased Treh expression during infection is mainly owing to the Treh-RA transcript, we expected that this increase would be abolished in the homozygous Treh[RAΔG4] mutant. The overall expression of Treh was reduced to one-third in uninfected Treh[RAΔG4] homozygotes ( Fig 1E ), but was still up-regulated nine-fold in the infected mutant (compared to a 25-fold increase in the wild type; Fig 1F ). Using transcript-specific qPCR, we found that there was a compensatory expression from the transcription start site common to both Treh-RB (cTreh) and Treh-RC (sTreh) transcripts, which are weakly expressed in wild-type hemocytes ( Fig 1G ). Thus, while there is a strong increase in cTreh expression in lamellocytes during infection from the Treh-RA (or RD, RG, RE) transcripts, loss of this transcript is compensated by the expression of Treh-RB.

We generated a cTreh specific mutant, Treh[RAΔG4], by replacing 47 bp including the first 2 start codons with the Gal4 coding sequence, which can also serve as a cTreh expression reporter ( Fig 1D ). The homozygous Treh[RAΔG4] mutant shows a phenotype similar to the cTreh-specific mutant Treh[c1] [ 22 ], with 20% lethality during pupal development and two-thirds of adult flies becoming lethargic and dying within 3 days of eclosion. Using a heterozygous Treh[RAΔG4] line crossed with flies carrying UAS-GFP, we found lamellocyte-specific expression of cTreh ( S1 Fig ) consistent with scRNAseq data. cTreh was also expressed in other larval tissues, such as imaginal discs ( S1 Fig ) and brain ( S2 Fig ), with and without infection. To verify cTreh expression in lamellocytes, we combined Treh[RAΔG4] driven UAS-mCherry with a lamellocyte-specific marker MSNF9-GFP (Figs 1H and S3 ) and found that cTreh is indeed specifically expressed in fully differentiated lamellocytes. While the expression of MSNF9 is already visible at 18 hpi in not yet fully differentiated lamellocytes ( S3 Fig ), cTreh starts to appear at 24 hpi ( S3 Fig ) and is expressed in most lamellocytes at 40 hpi ( Fig 1H ), including in those encapsulating the parasitoid egg, which is already being melanized ( S3 Fig ).

Notably, in addition to the transporter Tret1-1, expression of the enzyme Treh, which converts trehalose to 2 glucose molecules, is strongly up-regulated in hemocytes upon infection ( Fig 1A and 1B ), primarily in lamellocytes ( S1 File ). Since Treh occurs in 2 forms, cytoplasmic (cTreh) and secreted (sTreh; Fig 1D ) [ 22 ], we used transcript-specific qPCR to distinguish between them. Multiple cTreh transcripts (RA, RD, RG, RE) increased 35-fold in hemocytes upon infection ( Fig 1C ), whereas those encoding sTreh (RC, RF) were induced by infection only slightly above the basal level of cTreh. Therefore, substantially more cTreh than sTreh is expressed in hemocytes during infection.

While sut1 is moderately expressed in the lymph gland, we did not detect increased expression of transporters other than CG1208 at 18 hours post infection (hpi) ( Fig 1A ). MFS3 and sut1 also appear to be the major carbohydrate transporters in the wing discs which, however, display no changes in carbohydrate transporter expression during infection ( Fig 1A ).

(A) Expression heat map (bulk RNAseq) of selected transporters and trehalase in circulating hemocytes, lymph gland and wing disc from uninfected (Uninf) and infected (INF) third-instar larvae collected 9 and 18 hpi (0 h = 72 h after egg laying). Mean values given in each cell are TPM—data in S1 Table . (B) Expression of the trehalase (Treh) gene (bulk RNAseq) in circulating hemocytes and lymph gland. Each dot represents a biological replicate in TPM, bars represent mean ± SEM. Samples were analyzed using DESeq2 in Geneious Prime ( S1 Table ), **** shows adjusted P < 0.0001, ns = not significant. (C) Transcript-specific analysis of Treh expression by RT-qPCR 18 hpi. cTreh, represents cytoplasmic trehalase (primers cTreh-F and Treh-R shown in (D)), increases 35-fold after infection. sTreh represents secreted trehalase (primers sTreh-F and Treh-R shown in (D)). Bars show fold change compared to uninfected cTreh samples (expression levels were normalized by RpL32 expression in each sample), each dot represents a biological replicate. An unpaired two-tailed Welch’s t test was used to compare uninfected and infected samples; ****P < 0.0001 (numerical values in S1 Data ). (D) Map of the Treh gene with individual transcripts (RA-RG). Lines indicate introns, boxes represent exons with coding sequence in orange. Labeled arrows show primers used for RT-qPCR expression analysis. Schematic representation of a Gal4 knock-in into the first exon of Treh-RA, creating a 47-base deletion that removes both cTreh start codons, replaced by a cassette containing the Gal4 coding sequence and an RFP marker, expressed downstream of the P3 regulatory sequence in the fly eye. The resulting fly strain is Treh[RAΔG4]. (E, F) General Treh expression analysis of hemocytes from control and homozygous Treh[RAΔG4] mutant by RT-qPCR 18 hpi using primers common for all transcripts (Treh-F1 and Treh-R1 shown in (D)). (G) Treh-RB and RC transcript specific expression analysis of hemocytes from control and Treh[RAΔG4] mutant by RT-qPCR 18 hpi using primers specific to Treh-RB/RC (Treh-RB-F and Treh-R shown in (D)). (E–G) Bars show fold change compared to uninfected control samples (expression levels were normalized by RpL32 expression in each sample), each dot represents a biological replicate. An unpaired two-tailed Welch’s t test was used to compare samples; *P < 0.05, **P < 0.01, ****P < 0.0001 (numerical values in S1 Data ). (H) Analysis of cTreh expression in hemocytes at 40 hpi. Treh[RAΔG4], expressing Gal4 in the cTreh expression pattern, drives UAS-mCherry expression (red) together with a lamellocyte-specific marker MSNF9-GFP (green) in differentiated lamellocytes but not in plasmatocytes. hpi, hours post infection; PPP, pentose phosphate pathway.

Since Drosophila hemocytes increase their uptake of carbohydrates during parasitoid wasp infection [ 4 ], we analyzed the expression of carbohydrate transporters in the hemocytes. The SLC2 family of hexose sugar transporters in Drosophila comprises 31 genes ( S1 File ; FlyBase ID: FBgg0000691), most of which have not been functionally characterized. Our transcriptomics shows that the expression of 10 of these genes is greater than 3 transcripts per million (TPM) in hemocytes ( S1 File and S1 Table ) and 4 of these (Tret1-1, CG4607, sut1, and CG1208) become expressed in hemocytes during infection ( Fig 1A ). Tret1-1 has been characterized as a glucose and trehalose transporter [ 19 ] and CG4607 appears to be involved in lysosomal glucose metabolism [ 20 ]. Both Tret1-1 and CG4607 are weakly expressed in hemocytes in the uninfected state, but their expression strongly increases upon infection ( Fig 1A and S1 File). Based on scRNAseq [ 9 ], both are expressed exclusively in lamellocytes ( S1 File ). There are 2 mRNA variants of Tret1-1, with Tret1-1-RA increasing 31-fold upon infection, whereas there is no increase in the Tret1-1-RB variant ( S1 File ). sut1 is highly expressed in most hemocyte types in both uninfected and infected states ( Fig 1A and S1 File). MFS3, which belongs to the SLC17 family of organic anion transporters, has also been shown to transport glucose and trehalose [ 21 ] and its high expression in uninfected hemocytes decreases during infection ( Fig 1A and S1 File). The as yet functionally uncharacterized CG1208 shows the strongest increase in expression upon infection, predominantly in lamellocytes ( Fig 1A and S1 File). Thus, MFS3 and sut1 may provide basal carbohydrate transport in most hemocyte types in the uninfected state, whereas Tret1-1 and presumably CG4607 and CG1208 likely enhance carbohydrate transport in lamellocytes during infection.

Discussion

We previously demonstrated a systemic metabolic switch during infection of Drosophila larvae by parasitoid wasps, when sugar consumption in nonimmune tissues is reduced to provide nutrients for the immune system [4]. We and others have also found a strong increase in the expression of the trehalose transporter Tret1-1 and Treh in hemocytes during infection [4,8,9]. This suggests that trehalose is likely an important carbohydrate source for privileged immune cells.

Analysis of transcriptional changes at single-cell resolution suggested that larval hemocytes use lipids as the primary source to fuel the TCA cycle in the uninfected state [8]. However, half of the plasmatocytes in all clusters express an established glucose/trehalose transporter MFS3 [21] as well as a putative monosaccharide transporter sut1 [9], suggesting that plasmatocytes also utilize glucose. While plasmatocytes may consume lipids during infection, as deduced from gene expression, lamellocytes strongly up-regulate expression of several carbohydrate transporters and appear to mainly rely on saccharides as a source [8]. Our bulk transcriptomic data agree with these findings, and 13C-labeled carbohydrate tracing clearly supports these gene expression-based conclusions, showing that plasmatocytes from uninfected larvae do indeed metabolize glucose with a significant fraction metabolized via PPP.

The infection-induced carbohydrate consumption by hemocytes is mediated by a marked increase in the expression of 3 additional carbohydrate transporters besides MFS3 and sut1. Like MFS3, Tret1-1 is an established glucose/trehalose transporter [19]. The mild reduction in lamellocyte abundance in the Tret1-1 MFS3 double-null mutant indicates that at least one of the other, yet uncharacterized carbohydrate transporters (sut1, CG4607, or CG1208) contributes to glucose transport in hemocytes. Arguably, increased carbohydrate supply during immune challenge may be so critical as to require multiple, functionally redundant, transporters (Fig 7). This redundancy in carbohydrate transporters implies that loss of 1 or 2 transporter genes may not have a serious impact on immune response. However, the importance of carbohydrate supply to hemocytes is evident as hemocyte-specific silencing of oxidative PPP by Zw RNAi leads to loss of resistance. We have previously described the significance of a switch in systemic carbohydrate metabolism during immune response. Here, we show that hemocytes indeed require carbohydrates for an effective response in order to fuel PPP.

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TIFF original image Download: Fig 7. Hemocyte metabolism during parasitoid infection and its links to immune processes. A schematic model of hemocyte metabolism during parasitoid wasp infection. Metabolites, metabolic reactions/pathways, and processes marked in black have been examined in this study. Possible links to other processes and pathways (discussed in the main text) appear in gray. cTreh, cytoplasmic trehalase; GSH, reduced glutathione; GSSG, oxidized glutathione; PPP, pentose phosphate pathway; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; sTreh, secreted trehalase. https://doi.org/10.1371/journal.pbio.3002299.g007

The different ways in which glucose is metabolized depend on the actual needs of cells (Fig 7 and [14]). If a cell primarily requires ATP, glucose is converted via glycolysis to pyruvate, which can further be metabolized in the mitochondria to produce multiple ATPs. If a cell mainly requires nucleotides (typically a proliferating cell), glucose is metabolized by nonoxidative PPP to form ribose-5P (bypassing NADPH production by oxidative PPP), which is further utilized in de novo synthesis of purines/pyrimidines. If a cell primarily needs NADPH, glucose is metabolized in cyclic PPP, where the ribose-5P produced by oxidative PPP is recycled back to G6P, which can enter further rounds of oxidation in PPP. The cell can combine these pathways to obtain the optimal ratios of ATP, NADPH, pyruvate, and pentoses.

Our 13C tracing shows that hemocytes from uninfected larvae predominantly metabolize glucose and very little trehalose. When infected, hemocytes increase glucose metabolism and additionally metabolize trehalose. Expression of cTreh indicates that only differentiated lamellocytes cleave trehalose and likely also metabolize glucose, as MFS3 and Tret1-1 transport both trehalose and glucose [19,21]. Thus, by tracing 13C-labeled trehalose, we can specifically monitor lamellocyte metabolism. It is important to note that we observe metabolism of a heterogeneous pool of hemocyte types, and some hemocytes may use only some of the pathways in which we see 13C labeling.

As summarized in the overall model (Fig 7), hemocytes, including lamellocytes, increase their rate of glycolysis and lactate production during infection. A significant amount of glucose is shunted into the PPP, both in the absence of and during infection. G6P becomes partially labeled by 13C, indicating that a significant fraction of hemocytes, including lamellocytes, use cyclic PPP to re-oxidize G6P and maximize the yield of NADPH per glucose molecule [3]. A portion of G6P is returned to glycolysis at the glyceraldehyde-3P step, ultimately producing lactate. Some of the ribose-5P is diverted to de novo nucleotide synthesis. Thus, hemocytes, especially lamellocytes, couple NADPH production via cyclic PPP with de novo nucleotide synthesis and ATP production via downstream glycolysis. In addition, the specific pattern of partial labeling of pentoses suggests that some hemocytes generate pentoses via nonoxidative PPP regardless of infection. We could not determine how much carbohydrates were used in mitochondrial metabolism; we observed that hemocytes use amino acids as a source for the TCA cycle, or possibly for cytoplasmic citrate and lipid synthesis similarly in uninfected and infected larvae.

13C tracing and impaired resistance in Zw-deficient larvae show the importance of cyclic PPP in immune cells. Although the significance of NADPH production in activated immune cells, particularly in conjunction with oxidative burst, is well known [34], we are aware of only one study showing the importance of cyclic PPP in immunity, specifically in mammalian neutrophils [3]. By providing evidence for the key role of cyclic PPP in invertebrate immune cells, our findings highlight its evolutionary significance. NADPH generated by cyclic PPP may serve a variety of purposes in immunity [34]. Zw null and RNAi animals show that oxidative/cyclic PPP is important for lamellocyte differentiation and for resistance (Fig 7). It is likely that NADPH is used during lamellocyte differentiation for reductive biosynthesis. For example, lamellocytes undergo significant changes in size, morphology, and adhesion capacity that distinguish them from their precursors [35]. Therefore, the use of NADPH in reductive biosynthesis of fatty acids can be hypothesized to play a key role in the remodeling of cell membranes during lamellocyte differentiation. In addition, there is a global shift in gene expression [8,9] and thus a requirement for RNA synthesis. Consequently, ribose-5P generated by PPP enters de novo nucleotide synthesis, as we observed by 13C labeling of AMP/ADP/ATP. De novo synthesized ATP is also required for S-adenosylmethionine production and increased methylation in activated immune cells [36]. Methylation of newly synthesized molecules (e.g., nascent proteins) may be important for lamellocyte differentiation but homocysteine, a product of methylation, is also used in the trans-sulfuration pathway [37] to produce the antioxidants glutathione and taurine (see below).

Suppression of oxidative/cyclic PPP affects resistance. NADPH is needed to produce ROS and these are needed to kill the parasitoid. NADPH oxidase reduces oxygen to superoxide anions, which are subsequently dismutated to hydrogen peroxide [38]. Interestingly, Nappi and Vass detected H 2 O 2 in plasmatocytes, eventually attached to the egg, but not in lamellocytes [33]. This suggests that plasmatocytes use cyclic PPP to generate NADPH and H 2 O 2 , although our work does not exclude other sources of NADPH, such as isocitrate dehydrogenase and malic enzyme. Hydrogen peroxide can serve as a signal for further immune stimulation [39,40] or react with nitric oxide to form the hydroxyl radical, a very potent ROS [38]. Lamellocytes expressing the prophenoloxidase PPO3 and crystal cells expressing PPO2 [41] produce additional toxic molecules associated with melanization, which again requires NADPH [12]. Lamellocyte-mediated encapsulation and melanization concentrate the toxic reaction within the capsule, which is crucial for resistance. Thus, NADPH produced by oxidative PPP may be required for resistance due to reductive biosynthesis during lamellocyte differentiation and encapsulation and due to the production of toxic molecules inside the capsule. To kill the parasitoid, the metabolic activities of different hemocytes need to be coordinated. Resolving these roles requires time-controlled genetic manipulation of cyclic PPP, ROS production, and melanization cascade specific to the cell type in combination with, for example, genetically encoded metabolic sensors. Such manipulations would make the Drosophila-parasitoid model an excellent tool for investigating the role of cyclic PPP in immunity.

Trehalose is specifically metabolized by lamellocytes as they express cytoplasmic trehalase. The compensatory expression of alternative transcripts in a mutant lacking cTreh raises the question of why the capacity to metabolize trehalose is so important in lamellocytes. While systemic metabolism of trehalose is necessary for efficient lamellocyte differentiation, metabolism of trehalose within differentiating hemocytes is not. Thus, trehalose in the circulation appears to be important for maintaining adequate glucose levels for differentiating hemocytes [7]. Based on the expression of cTreh, trehalose is only metabolized by fully differentiated lamellocytes via cyclic PPP, generating NADPH. Survival rate of individuals with almost half of their hemocytes mutant for Treh is rather increased compared to controls. Thus, trehalose metabolism in hemocytes does not appear to be important for resistance. Encapsulation and melanization are thought to play a role not only in killing the pathogen but also in protecting the host from its own toxic immune reaction [12,13]. Based on our data, we propose that trehalose metabolism in lamellocytes may play a specific role in protecting the host: (1) Trehalose is metabolized by cyclic PPP, which generates NADPH. NADPH is required for the reduction of GSSG to GSH, which we found increased in hemocytes competent to respond at 22 hpi. Most hemocytes highly express genes of the thioredoxin system, but based on scRNAseq studies [8,9], lamellocytes show an even stronger expression. The thioredoxin system produces antioxidants including GSH. Depletion of trehalase activity from half of the lamellocytes led to increased hemolymph H 2 O 2 levels at 30 hpi, strongly suggesting that trehalose metabolism is involved in ROS scavenging; down-regulation of this role may lead to a more toxic reaction for both the pathogen and the host. (2) Larvae with half of their hemocytes deficient in Treh are more resistant than controls but surviving adults are less fit. Molina-Cruz and colleagues showed that mosquito strains with higher ROS levels survived bacterial and Plasmodium infections at a higher rate, whereas dietary antioxidant supplementation reduced resistance [42]. Interestingly, the same antioxidants also significantly improved age-related loss of fecundity in mosquitoes [43]. It is important to emphasize that our study does not provide a direct causal link between increased ROS during the larval immune response and the reduced adult fitness. (3) Treh knockdown induced in the cTreh expression pattern increased pathogen killing, albeit at the expense of host survival as more flies died as pupae. Here, all tissues expressing cTreh were affected, including those that may normally contribute to host protection, resulting in more severe effects. In conclusion, both the induction of Treh-/- mutant hemocyte clones and Treh RNAi increased resistance but impaired host survival and/or fitness, strongly suggesting that trehalose metabolism is needed for protecting the host from its own toxic immune response.

Although the intracellular metabolism of trehalose is restricted to lamellocytes and appears to be associated with an antioxidant response, this does not mean that these cells exclusively play a role in host protection, they may still mediate resistance. By encapsulating the parasitoid, lamellocytes are likely essential for containing the toxic reaction near the parasitoid, but they may also produce both ROS and antioxidants, which is not uncommon in immune cells [44]. Further studies should focus on lamellocytes to address the relationship between trehalose metabolism in cyclic PPP and the concurrent response to the parasitoid and its redox neutralization by the host.

In summary, an effective immune response to parasitoid wasp infection requires rapid and coordinated hemocyte activity, which includes lamellocyte differentiation. Additionally, capsule formation around the parasitoid egg is required, associated with a melanization cascade, and thus production of toxic molecules within the capsule. Lastly, protection of the host cavity from this toxic reaction is required for host survival. All these actions require changes in carbohydrate metabolism in hemocytes (Fig 7). Here, we show that systemic trehalose metabolism, including synthesis by Tps1 and conversion to glucose by Treh, is essential for adequate carbohydrate supply to hemocytes during infection, for lamellocyte differentiation and for host resistance. Nutrient supply to hemocytes is ensured by the expression of several carbohydrate transporters. While glucose is generally metabolized by hemocytes, trehalose is specifically metabolized only within lamellocytes by cytoplasmic trehalase. We demonstrate that both glucose and trehalose are metabolized via PPP, cyclic PPP in particular, which oxidizes G6P in multiple rounds to maximize NADPH production. PPP also connects to downstream glycolysis, which produces ATP and ends with the release of lactate. PPP and its links to several metabolic pathways support various activities required in the response to parasitoids. (1) We have shown that oxidative PPP is required for lamellocyte differentiation, implicating a role for NADPH in reductive biosynthesis, for example, in membrane remodeling. Differentiation could also be promoted by ribose-5P associated with nucleotide synthesis, which is required for broad changes in gene expression (new RNA and methylation). (2) We also demonstrated that oxidative PPP is required for resistance, which likely involves NADPH in the melanization cascade and in ROS production, both required for pathogen killing. (3) We observed increased production of the antioxidants glutathione and taurine, which requires NADPH/Trxr1-mediated reduction of GSSG to GSH and could also be promoted by coupling PPP-produced ribose-5P to the ATP-SAM-homocysteine-trans-sulfuration pathway. Antioxidants could explain the observed effects on the reduced fitness of trehalase knockout in hemocyte clones. Nevertheless, the potential link between sugars metabolized in PPP and host protective mechanisms requires further work, such as manipulating the thioredoxin system specifically in fully differentiated lamellocytes and studying the effects on larvae as well as development and physiology of surviving animals. It is difficult to separate resistance mechanisms from host protection when, for example, NADPH produced by cyclic PPP appears to be required for both. We were initially surprised that although trehalose metabolism seems to be important in hemocytes, we did not observe any effect on resistance. However, from an evolutionary perspective, protecting the host from its own immune response is no less important and probably no less energetically demanding than fighting the pathogen. The trade-off between survival and reproductive fitness may be of great evolutionary importance.

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