DNA damage signaling in Drosophila macrophages modulates systemic cytokine levels in response to oxidative stress

Environmental factors, infection, or injury can cause oxidative stress in diverse tissues and loss of tissue homeostasis. Effective stress response cascades, conserved from invertebrates to mammals, ensure reestablishment of homeostasis and tissue repair. Hemocytes, the Drosophila blood-like cells, rapidly respond to oxidative stress by immune activation. However, the precise signals how they sense oxidative stress and integrate these signals to modulate and balance the response to oxidative stress in the adult fly are ill-defined. Furthermore, hemocyte diversification was not explored yet on oxidative stress. Here, we employed high throughput single nuclei RNA-sequencing to explore hemocytes and other cell types, such as fat body, during oxidative stress in the adult fly. We identified distinct cellular responder states in plasmatocytes, the Drosophila macrophages, associated with immune response and metabolic activation upon oxidative stress. We further define oxidative stress-induced DNA damage signaling as a key sensor and a rate-limiting step in immune-activated plasmatocytes controlling JNK-mediated release of the pro-inflammatory cytokine unpaired-3. We subsequently tested the role of this specific immune activated cell stage during oxidative stress and found that inhibition of DNA damage signaling in plasmatocytes, as well as JNK or upd3 overactivation, result in a higher susceptibility to oxidative stress. Our findings uncover that a balanced composition and response of hemocyte subclusters is essential for the survival of adult Drosophila on oxidative stress by regulating systemic cytokine levels and cross-talk to other organs, such as the fat body, to control energy mobilization.

Methods
Drosophila melanogaster stocks:

Flies were reared on a high yeast food containing 10% brewer’s yeast, 8% fructose, 2% polenta and 0.8% Agar. Propionic acid and nipagin were added to prevent bacterial or fungal growth. All crosses (except those containing the tub-Gal80ts construct or otherwise noted) were performed at 25°C with a 12 hours dark/light cycle. The crosses with tub-Gal80ts were performed at 18°C to ensure the inhibition of the Gal4-protein during developmental stages of the fly. The experimental male F1 flies were transferred to 29°C as soon as they hatched and were aged for six days. All transgenic lines used in this study are listed below:

List of Drosophila lines used in this study
Source

w1118;Hml∆ -Gal4,UAS-2xeGFP
Gift from Marc S. Dionne

w1118;UAS-tefu-IR
Vienna Drosophila Research Center

w1118;UAS-mei41-IR
Vienna Drosophila Research Center

w1118;UAS-nbs-IR
Vienna Drosophila Research Center

w1118;UAS-mei41-IR;UAS-tefu-IR
made for this study

w1118;UAS-bsk-DN
Vienna Drosophila Research Center

w1118;;HmlΔ-DsRed.nuc
Gift from Katie J. Woodcock

w1118;UAS-upd3-IR
Vienna Drosophila Research Center

w1118;Hml∆ -Gal4,UAS-2xeGFP;tubGal80/tm6csb´
Gift from Katie J. Woodcock

w1118;UAS-hep[act]/CyO; Dr/tm6csb'
Gift from Anne-Kathrin Classen

w1118;UAS-upd3/Sm6a
Gift from Katie J. Woodcock

w[*] upd3[Δ]
Bloomington Drosophila Stock Center

w´upd3Δ; UAS-upd3/Sm6a
made for this study

w1118;tub-Gal80ts/Sm6a;crq-Gal4/TM6 c, Sb'
Gift from Marc S. Dionne

w1118;;UAS-rpr/TM6 c, Sb1
Bloomington Drosophila Stock Center

w1118;UAS-CD8-mCherry
Bloomington Drosophila Stock Center

Lifespan/Survival assays:

Male flies were collected after eclosion and groups of 20 age-matched flies per genotype were housed in a food vial. The survival experiments were performed at 29°C. The vials were placed horizontal to avoid that flies fall into the food and become stuck. Dead flies were counted in a daily manner. The flies were transferred into a fresh food vial twice per week without CO2 anesthesia. 

Paraquat treatment:

Flies were maintained at 29°C for six days prior to treatment. On day six they were starved for 6 hours. A filter paper soaked in 5% sucrose solution with or without 15mM Paraquat (PQ, Methyl viologen hydrate, Acros Organics) was added after starvation. Flies were fed for 18 hours on the PQ containing food in groups of ten. The PQ treatment was performed at 29°C in the dark20. To assess the survival rate the dead flies per vial were counted and the percentage was calculated. The living flies were further analyzed in other assays used in this study.

Starvation experiments:

10-20 age-matched male flies were kept in a vial containing 1% agar supplemented with 2% 1xPBS. The starvation experiments were performed at 25°C. Dead flies were counted every hour. Several individual experiments were performed and started on different daytimes to exclude diurnal derived artifacts. The data of the individual experiments were pooled and analyzed with GraphPad Prism 9.2.0.

Paraffin sections and stainings:

Anaesthetized flies were washed in 75% ethanol for 5-10 seconds and transferred into 4% PFA for 30 minutes at room temperature. The flies were washed for 1 hour in PBS on a shaker. Subsequently the flies were transferred into tissue cassettes, dehydrated and embedded in paraffin. The flies were cut in 7µm thick sections de-paraffinated and stained on slides with Hematoxylin-Gill (II) for five minutes. Subsequently the sections were blued in running water for ten minutes. It followed a 0.5% Eosin treatment for five minutes and a final ethanol treatment with increasing concentrations until 100%. The sections were transferred into Xylol and finally covered with synthetic resin and a cover slip.

Cryo sections and Oil Red O staining:

Anaesthetized flies were washed in 75% ethanol for 5-10 seconds and transferred into 4% PFA for 30 minutes at room temperature. Subsequently, the flies were dehydrated for 1h at 37°C in 30% sucrose solution. The dehydrated flies were placed in a Tissue-Tek cryo-mold and embedded in Tissue-Tek® O.C.T.™ Compound. The cryo-mold was placed on dry-ice which was embedded in a 100% ethanol bath until the whole block was completely frozen. The flies were cut in 10µm sections and stained on the slide. The slides were put on RT and dried. Subsequently, they were submerged in Oil red O solution for 30 minutes. The slides were rinsed two times in deionized water and subsequently stained for two minutes with hematoxylin to stain the nuclei. Finally, the slides were washed with water for 5 minutes and covered with a cover slip and glycerin.

Semi-quantitative Real-Time PCR:

Three flies were pooled and smashed in 100µl TRIzol to stabilize and isolate the RNA. Chloroform was used to extract the RNA followed by an isopropanol precipitation step. The RNA was cleaned with 70% ethanol and solubilized in water. A DNAse treatment was performed to digest potential genomic DNA contaminations. The purified and DNAse treated RNA was written into cDNA using RevertAid Reverse Transcriptase (Thermo Scientific) at 37°C for one hour. The reaction was stopped by incubating for 10 minutes at 70°C. The subsequent RTqPCR was done in SensiMix SYBR Green no-ROX (Meridian Bioscience) and was performed on a LightCycler 480 (Roche). The qPCR cycling started with a 10-minute 95°C step followed by 50 cycles with the following times and temperatures: 15s at 95°C, 15s at 60°C and 15s at 72°C. The gene expression levels were normalized to the value of the measured loading control gene Rpl1.

Confocal microscopy:

The flies were anesthetized with CO2, glued on a cover slip and imaged immediately. Confocal microscopy was done with a Leica SP8 microscope (Leica) and a 10x/NA0.4 Leica air objective. The images were acquired in a resolution of 512x512 with a scan speed of 600 Hz. Z-Stacks with a step size of 5µm and tile scans (2x2 or 2x3 images per fly) were acquired to quantify the hemocytes in whole flies. The tiled images were merged with the LAS-X software (Leica) and maximum projections were created using Fiji/ImageJ. The hemocytes were counted in the maximum projection images of whole flies.

Smurf Assay:

Smurf assays were performed to check the feeding behavior as well as the gut integrity of 7 days old, male flies upon PQ treatment. Brilliant Blue dye (FCF) was added into 5% sucrose solution in a concentration of 1% (w/v). PQ was added in concentrations of 2mM, 15mM and 30mM. Control food was 5% sucrose and 1% Brilliant Blue without PQ. The flies were fed with blue food according to the PQ-treatment protocol, as described above. Flies were analyzed after 18h of feeding on brilliant-blue food. Three kinds of flies were distinguished. Flies that did not eat at all were excluded. Flies with a blue gut or crop were classified as “non-smurf”. Flies which turned completely blue or showed distribution of blue dye outside the gut were classified as “smurf”.

Thin Layer Chromatography (TLC):

Each sample in the TLC analysis contains 10 flies of the respective genotype. The flies were starved for one hour before they were anesthetized with CO2 and transferred into 100µl chloroform:methanol (3:1) on ice. The flies were centrifuged for 5 minutes at 15,000g at 4°C and subsequently homogenized with a pestle. The sample was centrifuged again with the same settings. Lard was dissolved in chloroform:methanol (3:1) and served as triglyceride control (standard 1). Standard 1 was used to produce a standard curve with decreasing triglyceride concentrations. The samples and the standard curve were loaded on a glass silica gel plate (Millipore). The mobile phase in the TLC chamber was made with hexane:diethylether (4:1). The plate was placed in the chamber until the solvent front reached the upper end of the plate. The plate was taken out, air dried and stained with ceric ammonium heptamolybdate (CAM). Subsequently the plate was incubated at 80°C for 2 hours to visualize the stained triglyceride bands. Images were taken with a gel documentation system (gelONE). Triglyceride density was measured and the concentrations were calculated according the pre-determined concentrations of the standard curve. Image analysis and calculations were done using ImageJ.

Glucose Assay:

Seven days old male flies were used for the analysis. They were treated for 18h with PQ or sucrose respectively. Subsequently the flies were starved for one hour to bring all flies into the same metabolic state. Three flies were pooled as one sample and manually homogenized in 75µl TE + 0.1% Triton X-100 (Sigma Aldrich). The samples were incubated for 20min at 75°C to inactivate any intrinsic enzymatic activity. 5µl of the samples were loaded into a flat-bottom 96-well plate. Each sample was measured four times to measure glucose, trehalose, glycogen and the fly background respectively. The fly background was determined by diluting the fly sample with water. Glucose reagent (Sentinel Diagnostics) was added to the fly sample to measure the glucose levels. Trehalase (Sigma Aldrich) or amyloglucosidase (Sigma Aldrich) was added to the glucose reagent to measure trehalose or glycogen respectively. Plates were incubated for 24h at 37°C before the colorimetric measurement was performed at a wavelength of 492nm on a Tecan® Spark plate reader. The concentrations were calculated according to standards loaded on the same plate.

S2 Cell Culture, CellROX staining and flow cytometry:

Drosophila S2 cells (Thermo Scientific, R96007) were cultured in Schneider’s Drosophila medium (Gibco™) at 26°C and atmospheric oxygen and carbon dioxide conditions. The medium contained 10% FCS and 1% penicillin/streptomycin. PQ was added into the medium in concentrations of 15mM or 30mM to investigate the influence on the production of reactive oxygen species (ROS). Control cells remained untreated. To visualize ROS and oxidative stress, the cells were stained with CellROX™ Deep Red Reagent (Invitrogen) for 30 minutes directly in the medium in a concentration of 1:500 at 26°C. The cells were washed twice with 1xPBS containing 2mM EDTA. In order to stain dead cells DAPI was added to the samples in a concentration of 1:1000. Samples were acquired using a BD LSRFortessa™ (BD Biosciences) and analyzed with FlowJo analysis software.

COMET assays:

Comet assays were performed to analyze the amount of DNA damage in S2 cells treated with PQ. S2 cells were treated with 15mM or 30mM PQ for 24h. The cells were brought into a concentration of 1.8x105 cells/ml. Low melting agarose (LMA) was boiled at 90°C and subsequently cooled down to 37°C. The cells were diluted in low melting agarose (LMA) at a ratio of 1:10 (cells:LMA) and transferred onto a CometSlide™ (Trevigen). To ensure proper attachment of the LMA, the slides were cooled for 30 minutes at 4°C. The lysis was performed for one hour at 4°C. Therefore, the slides were submerged in CometAssay® Lysis Solution (Trevigen). Subsequently, the cells were submerged in alkaline unwinding solution (200mM NaOH, 1mM EDTA, pH 13) for 1h at 4°C. The electrophoresis was performed under alkaline conditions (pH13) for 30 minutes with a current of 20V (1V/cm). The slides were neutralized with ddH2O, dehydrated with 37% ethanol and dried properly at 37°C before they were stained with SYBR™Gold (Invitrogen) for 30 minutes. Comets were imaged with an Olympus BX61 fluorescence microscope. Comet data was analyzed via TriTrek CometScore 2.0.0.38 software.