1. Immunology and Inflammation
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Drosophila macrophages switch to aerobic glycolysis to mount effective antibacterial defense

  1. Gabriela Krejčová  Is a corresponding author
  2. Adéla Danielová
  3. Pavla Nedbalová
  4. Michalina Kazek
  5. Lukáš Strych
  6. Geetanjali Chawla
  7. Jason M Tennessen
  8. Jaroslava Lieskovská
  9. Marek Jindra
  10. Tomáš Doležal
  11. Adam Bajgar  Is a corresponding author
  1. University of South Bohemia, Czech Republic
  2. Indiana University, United States
  3. Biology Centre CAS, Czech Republic
Research Article
Cite this article as: eLife 2019;8:e50414 doi: 10.7554/eLife.50414
6 figures, 1 table and 1 additional file

Figures

Graphical representation of the experimental approach.

(A) The natural progress of streptococcal infection, with highlighted sampling times during the acute and resolution phases of infection. The Y axis indicates the percentage of surviving adults. (B) The approach used to isolate hemocytes, which are subsequently assayed for gene expression and enzymatic activities. Macrophages sorted from flies at the respective time points post-infection represent acute-phase macrophages (APMФs; 24 hpi) and resolution-phase macrophages (RPMФs; 120 hpi). Control flies were analyzed at the same time points after receiving injection of phosphate-buffered saline (PBS). hpi, hours post-infection; FACS, fluorescence-activated cell sorting; S.p., Streptococcus pneumoniae.

https://doi.org/10.7554/eLife.50414.003
Figure 2 with 1 supplement
Streptococcal infection enhances glycolysis in acute-phase macrophages.

(A–B) Fluorescent images of the dorsal view of the abdomens of infected and control (both Hml >GFP) flies at 24 and 120 hpi, showing NBDG the distribution among the tissues (A) and at a higher magnification (B). Images represent a minimum of ten observations of a similar pattern. (C) Scheme of glycolysis and the TCA cycle, highlighting significant changes in the quantified expression of the indicated genes at 24 and 120 hpi. The expression levels of the mRNA were measured relative to that of the ribosomal protein 49 (rp49), and the statistical significance (p<0.05) was tested using ANOVA (for data see Figure 2—figure supplement 1). Upregulated genes are shown in red, downregulated genes in green; gray indicates no statistically significant difference. (D–F) Enzymatic activities of phosphoglucose isomerase (Pgi) (D) and lactate dehydrogenase (Ldh) (E), as well as the level of NADH (F), at 24 and 120 hpi measured in the homogenate of hemocytes isolated from infected and control flies. The levels of enzymatic activity and NADH concentration were normalized per ten thousand cells per sample. (G) The concentration of circulating lactate measured in the hemolymph of infected and control flies at 24 and 120 hpi. In all plots (D–G), individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001).

https://doi.org/10.7554/eLife.50414.004
Figure 2—source data 1

Metabolic characterization of macrophages post-infection.

https://doi.org/10.7554/eLife.50414.007
Figure 2—figure supplement 1
Gene expression of glycolytic enzymes is increased in acute-phase macrophages.

Gene expression of glycolytic (HexA (A), Pgi (B), Pfk (C), Tpi (D), Gapdh1 (E), Eno (F)) and TCA (Cis (G), Scsα1 (H), CG10219 (I)) genes in the hemocytes of infected and control flies (both Hml >GFP) at 24 and 120 hpi. The mRNA expression levels, normalized against rp49, are given as fold change (F.C.) relative to the expression of noninfected controls. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001).

https://doi.org/10.7554/eLife.50414.005
Figure 2—figure supplement 1—source data 1

Expression of metabolic genes in macrophages post-infection.

https://doi.org/10.7554/eLife.50414.006
Macrophage-specific activities of Hif1α and Ldh increase upon infection.

(A) X-gal staining of infected and control flies bearing the HRE-LacZ reporter construct. Images represent a minimum of ten observations of a similar pattern. (B) An uninfected Hml >GFP, Ldh-mCherry adult fly (24 hpi) shows localization of the Ldh reporter activity (red) in many of the immune cells (green). The image is a Z-stack at maximal projection of 25 confocal slices. (C, D) Expression of Ldh (C) and Hif1α (D) mRNAs in hemocytes isolated from infected and control flies (both Hml >GFP; 24 and 120 hpi). (E) Expression of Ldh mRNA in hemocytes of infected and control Hml >GFP flies with and without a hemocyte-specific knockdown of Hif1α at 24 hpi. In all plots (C–E), expression levels, normalized against rp49, are given as fold change (F.C.) relative to levels in PBS-injected Hml >GFP controls (24 hpi), which were arbitrarily set to 1. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001).

https://doi.org/10.7554/eLife.50414.008
Figure 3—source data 1

Expression pattern of Hif1α and Ldh genes.

https://doi.org/10.7554/eLife.50414.009
Figure 4 with 1 supplement
Effects of Hif1α and Ldh hemocyte-specific knockdown on macrophage metabolism.

(A) Dorsal view of the abdomens of S.p.-infected flies (24 hpi) showing the distribution of the fluorescent NBDG probe. Controls (left) are compared to flies subjected to hemocyte-specific knockdown of Hif1α. Images represent a minimum of ten observations of a similar pattern. (B) Schematic representation of the expression of genes encoding metabolic enzymes in the hemocytes of infected control flies (left) and of flies with Hif1α hemocyte-specific knockdown (right) at 24 hpi. The expression levels of the mRNAs were measured relative to that of rp49, and the statistical significance (p<0.05) was tested using ANOVA (for data see Figure 4—figure supplement 1). Upregulated genes are shown in red; gray indicates no statistically significant difference. (C) Dorsal view of the abdomens of S.p.-infected flies (24 hpi) showing the distribution of the fluorescent NBDG probe. Controls (left) are compared to flies subjected to hemocyte-specific knockdown of Ldh. Images represent a minimum of ten observations of a similar pattern. (D–F) Enzymatic activity of Ldh (D), level of NADH (E), and enzymatic activity of Pgi (F) at 24 and 120 hpi measured in lysates of hemocytes isolated from infected and non-infected control flies and from flies with Hif1α hemocyte-specific knockdown. (G–I) Enzymatic activity of Ldh (G), level of NADH (H), and enzymatic activity of Pgi (I) at 24 and 120 hpi measured in lysates of hemocytes isolated from infected and non-infected control flies and from flies with Ldh hemocyte-specific knockdown. In all plots (D–I), the enzyme activities and NADH concentrations were normalized per ten thousand cells per sample. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001).

https://doi.org/10.7554/eLife.50414.010
Figure 4—source data 1

Effect of macrophage-specific Hif1α knockdown on metabolic features of macrophages.

https://doi.org/10.7554/eLife.50414.013
Figure 4—figure supplement 1
Expression of genes encoding glycolytic enzymes is not increased in acute-phase macrophages with Hif1α knock-down.

(A–F) Gene expression of glycolytic genes (HexA (A), Pgi (B), Pfk (C), Tpi (D), Gapdh1 (E) and Eno (F)) in the hemocytes of infected and control Hml >GFP flies and of flies with hemocyte-specific Hif1α knockdown at 24 hpi. (G, H) Gene expression of Hif1α (G) and Ldh (H) at 24 hpi in infected and control Hml >GFP flies and in flies with hemocyte-specific Hif1α knockdown representing the efficiency of RNAi treatment. The mRNA expression levels, normalized against rp49, are given as fold change (F.C.) relative to the expression in noninfected controls. Individual dots represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001).

https://doi.org/10.7554/eLife.50414.011
Figure 4—figure supplement 1—source data 1

Effect of macrophage-specific Hif1α knockdown on expression of metabolic genes.

https://doi.org/10.7554/eLife.50414.012
Systemic effects of Hif1α and Ldh hemocyte-specific knockdown.

(A–C) The concentration of circulating glucose (A), glycogen stores (B) and circulating lactate (C) in infected and non-infected flies with Hif1α or Ldh hemocyte-specific knockdown and their respective controls at 24 hpi. The concentrations of metabolites were normalized to the amount of proteins in each sample. Individual dots in the plot represent biological replicates. Values are mean ± SD, asterisks mark statistically significant differences (*p<0.05; **p<0.01; ***p<0.001).

https://doi.org/10.7554/eLife.50414.014
Figure 5—source data 1

Effect of macrophage-specific Hif1α and Ldh knockdown on systemic carbohydrate metabolism.

https://doi.org/10.7554/eLife.50414.015
Effects of Hif1α and Ldh hemocyte-specific knockdown on resistance to infection.

(A–B) The survival rate of infected flies of the control genotype and of flies with hemocyte-specific Hif1α (A) and Ldh (B) knockdown. Vertical dotted lines denote medium time to death for each genotype; survival rate during the first 120 hr is shown in detail. Three independent experiments were performed and combined into one survival curve. The average number of individuals per replicate was more than 500 for each genotype. (C, D) Colony forming units (CFUs) obtained from infected flies of control genotype and from flies with hemocyte-specific Hif1α (C) and Ldh (D) knockdown at 0, 24, 48, and 72 hpi. Individual dots in the plot represent the number of bacteria raised from one individual. The data show results merged from three independent biological replicates.

https://doi.org/10.7554/eLife.50414.016
Figure 6—source data 1

Effect of macrophage-specific Hif1α and Ldh knockdown on the resistance to bacterial infection.

https://doi.org/10.7554/eLife.50414.017

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource
reference
IdentifierAdditional
information
Strain, strain background (Streptococcus pneumoniae)EJ1 strainProvided by David SchneiderDilution 20,000 units
Chemical compound, drugTRIzol ReagentInvitrogenCat# 15-596-018
Chemical compound, drugSuperscript III Reverse TranscriptaseInvitrogenCat# 18080044
Chemical compound, drug2x SYBR Master MixTop-BioCat# T607
Chemical compound, drug2-NBDGThermo Fisher
Scientific
Cat# N13195
Chemical compound, drugX-galSigmaCat# B4252
Commercial assay, kitGlucose (GO) Assay KitSigmaCat# GAGO20-1KT
Commercial assay, kitBicinchoninic Acid Assay KitSigmaCat# BCA1
Commercial assay, kitLactate Assay KitSigmaCat# MAK064
Commercial assay, kitLactate Dehydrogenase Activity Assay KitSigmaCat# MAK066
Commercial assay, kitPhosphoglucose Isomerase Colorimetric Assay KitSigmaCat# MAK103
Genetic reagent (Drosophila
melanogaster)
HmlG4G80: w*; HmlΔ-Gal4*; P{tubPGal80ts}*Cross made in our laboratory by Tomas Dolezal
Genetic reagent (D. melanogaster)Hml > GFP: w; HmlΔ-Gal4 UAS-eGFPProvided by Bruno Lemaitre
Genetic reagent
(D. melanogaster)
Hif1α[RNAi]: P{KK110834}VIE-260BVienna Drosophila Resource CenterVDRC: v106504FBst0478328
Genetic reagent (D. melanogaster)TRiP control: y(1) v(1); P{y[+t7.7]=CaryP}attP2Bloomington
Drosophila Stock Center
BDSC: 36303FBst0036303
Genetic reagent (D. melanogaster)KK control: y, w[1118];P{attP,y[+],w[3`]}Bloomington Drosophila Stock CenterBDSC: 60100FBst0060100
Genetic reagent (D. melanogaster)
Ldh[RNAi]: y(1) v(1); P{y[+t7.7] v[+t1.8]=TRiP.HMS00039}attP2Bloomington
Drosophila Stock Center
BDSC: 33640FBst0033640
Genetic reagent (D. melanogaster)HRE-LacZ: HRE-HRE-CRE-LacZProvided by Pablo Wappner (Lavista-Llanos et al., 2002)
Genetic reagent (D. melanogaster)Ldh-mCherryProvided by Jason Tennessen
Genetic reagent (D. melanogaster)w: w1118Genetic background based on CantonS
Sequence-based reagentCis forward: 5′TTCGATTGACTCCAGCCTGG3′KRDCG14740FBgn0037988
Sequence-
based reagent
Cis reverse: 5′AGCCGGGAACCACCTGTCC3′KRDCG14740FBgn0037988
Sequence-based reagentLdh forward:
5′CAGAGAAGTGGAACGAGCTG3′
KRDCG10160FBgn0001258
Sequence-
based reagent
Ldh reverse:
5′CATGTTCGCCCAAAACGGAG3′
KRDCG10160FBgn0001258
Sequence-based reagentEno forward: 5′CAACATCCAGTCCAACAAGG3′KRDCG17654FBgn0000579
Sequence-based reagentEno reverse: 5′GTTCTTGAAGTCCAGATCGT3′KRDCG17654FBgn0000579
Sequence-based reagentGapdh1 forward:
5′TTG TGG ATC TTA CCG TCC GC3′
KRDCG12055FBgn0001091
Sequence-based reagentGapdh1 reverse: 5′CTCGAACACAGACGAATGGG3′KRDCG12055FBgn0001091
Sequence-based reagentHexA forward: 5′ATATCGGGCATGTATATGGG3′KRDCG3001FBgn0001186
Sequence-based reagentHexA reverse:
5′CAATTTCGCTCACATACTTGG3′
KRDCG3001FBgn0001186
Sequence-based reagentPfk forward:
5′AGCTCACATTTCCAAACATCG3′
KRDCG4001FBgn0003071
Sequence-based reagentPfk reverse: 5′TTTGATCACCAGAATCACTGC3′KRDCG4001FBgn0003071
Sequence-based reagentPgi forward: 5′ACTGTCAATCTGTCTGTCCA3′KRDCG8251FBgn0003074
Sequence-based reagentPgi reverse: 5′GATAACAGGAGCATTCTTCTCG3′KRDCG8251FBgn0003074
Sequence-based reagentRp49 forward: 5′AAGCTGTCGCACAAATGGCG3′KRDCG7939FBgn0002626
Sequence-based reagentRp49 reverse: 5′GCACGTTGTGCACCAGGAAC3′KRDCG7939FBgn0002626
Sequence-based reagentHif1α forward: 5′CCAAAGGAGAAAAGAAGGAAC3′KRDCG45051FBgn0266411
Sequence-based reagentHif1α reverse: 5′GAATCTTGAGGAAAGCGATG3′KRDCG45051FBgn0266411
Sequence-based reagentCG10219 forward: 5′GAGATCTCCGTGAGTGCGC3′KRDCG10219FBgn0039112
Sequence-based reagentCG10219 reverse: 5′CTCCACGCCCCAATGGG3′KRDCG10219FBgn0039112
Sequence-based reagentScsα1 forward: 5′TCACAAGCGCGGCAAGATC3′KRDCG1065FBgn0004888
Sequence-based reagentScsα1 reverse: 5′TTGATGCCCGAATTGTACTCG3′KRDCG1065FBgn0004888
Sequence-based reagentTpi forward: 5′AGATCAAGGACTGGAAGAACG3′KRDCG2171FBgn0086355
Sequence-based reagentTpi reverse: 5′ACCTCCTTGGAGATGTTGTC3′KRDCG2171FBgn0086355
Software, algorithmGraphpad Prismhttps://www.graphpad.com/Graphpad PrismRRID:SCR_002798
Software, algorithmMicrosoft Excelhttps://www.microsoft.com/Microsoft Excel
Software, algorithmFijiImageJ - https://fiji.scImageJRRID:SCR_002285
OtherS3e Cell SorterBioRad -http://www.bio-rad.com/BioRad
OtherOlympus FluoView 1000Olympus -https://www.olympus-global.com/OlympusRRID:SCR_017015
RRID:SCR_014215
OtherOlympus SZX12Olympus -https://www.olympus-global.com/Olympus
OtherOlympus IX71Olympus -
https://www.olympus-global.com/
Olympus

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data have been provided for Figures 2 and 4.

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