Drosophila macrophages control systemic cytokine levels in response to oxidative stress via a non-canonical DNA damage repair signaling cascade

Fabian Hersperger
Tim Meyring
Pia Weber
Chintan Chhatbar
Gianni Monaco
Anne-Kathrin Classen
Marc S. Dionne
Katrin Paeschke
Marco Prinz
Olaf Groß
Katrin Kierdorf author has email address

Institute of Neuropathology, Faculty of Medicine, University of Freiburg, Freiburg, Germany
Faculty of Biology, University of Freiburg, Freiburg, Germany
Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany
Center for Biological Systems Analysis, University of Freiburg, Freiburg, Germany
CIBSS-Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany
MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom
Department of Life Sciences, Imperial College London, London, United Kingdom
Department of Oncology, Haematology and Rheumatology, University Hospital Bonn, Venusberg-Campus 1, Bonn, Germany
Center for Basics in NeuroModulation (NeuroModulBasics), Faculty of Medicine, University of Freiburg, Freiburg, Germany
Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany

https://doi.org/10.7554/eLife.86700.1

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Figures and data

Loss of hemocytes increases the susceptibility of adult Drosophila to oxidative stress by Paraquat feeding.
(A) Survival of Hml/+ flies with control food (n=20) and 15mM Paraquat (n=28) at 29°C. Five independent experiments were performed. Student’s unpaired t-test: ****p<0.0001, each data point represents a sample with 10 flies. (B-C) RTqPCR of whole flies were performed to investigate gene-expressions of the JAK-STAT-signaling pathway (B) and insulin-signaling pathway (C). All transcript levels were normalized to the expression of the loading control Rpl1 and are shown in arbitrary units [au]. Each data point (n=5) represents a sample of three whole flies. Student’s unpaired t-tests were performed for each transcript to compare the gene-expression between control and treated flies: **p<0.01; ***p<0.001; ****p<0.0001. (D) Glucose and (E) glycogen levels of whole flies were measured in control and PQ-treated flies. Each dot (n=3) represents a sample containing three flies. Student’s unpaired t-test: **p<0.01. (F) Triglyceride (TG) amounts were determined via thin-layer chromatography. Student’s unpaired t-test: **p<0.01. (G) Representative images of 7 day old HmlΔ4>eGFP flies treated with 5% sucrose solution and 15mM PQ respectively. Scale bars = 500µm. (H) Hemocyte quantification of 7 day old HmlΔ4>eGFP flies treated with control food (5% sucrose solution) (n=5) or 15mM Paraquat (n=5). Each data point represents one fly. Statistical significance was tested using student’s unpaired t-test: p=0.508. (I) Survival of crq-Gal80^ts/+ control flies (n=13) and hemocyte depleted crq-Gal80^ts>reaper flies (n=7) on control food (n=20) and 15mM Paraquat (n=28) at 29°C. Student’s unpaired t-test: ****p<0.0001, each data point represents a sample with 10 flies.

Unbiased single nuclei transcriptomic profiling identified diverse transcriptomic states of hemocytes associated with oxidative stress response.
(A) Uniform Manifold Approximation and Projection (UMAP) visualization of single nucleus states from flies exposed to 15mM PQ containing or control food. Dashed lines indicate the broad cell types. Male accessory gland main cells (MAG), Malpighian tubule principal cells (MTC), male germline cells (MGC), outer photoreceptor cells (PRC), tracheolar cells (TC), epithelial cells (EC). Colors indicate distinct clusters. n=11839 nuclei are shown. (B) UMAP visualization of nuclei from A colored by treatment. Nuclei from control group are labeled in red and nuclei from PQ group are labeled in blue. Dashed lines indicate the broad cell type as A. (C) UMAP visualization of hemocytes from A after sub-setting and re-clustering. Colors and dashed lines indicate distinct clusters. n=1354 nuclei are shown. (D) UMAP visualization of hemocytes clusters colored by treatment. Nuclei from control group are labeled in red and nuclei from PQ group are labeled in blue. Dashed lines indicate distinct clusters. n=1354 nuclei are shown. (E) Heat map of 20 genes enriched in each hemocytes cluster. Gene names are indicated on the left. Colors in the heat map correspond to normalized scaled expression. (F) Volcano plots comparing pseudo bulk gene expression of individual hemocytes cluster vs all remaining hemocytes. The –log₁₀-transformed adjusted P value (P adjusted, y-axis) is plotted against the log₂-transformed fold change (FC) in expression between the indicated cluster vs remaining hemocytes (x-axis). Genes under log₂ FC and – Log₁₀ P value cut off are shown in grey (NS). Genes above log₂ FC but under P value cut off in green, Genes under log₂ FC and above –Log₁₀ P value cut off are shown in blue and genes above log₂ FC and – Log₁₀ P value cut off are shown in red.

A specific cluster of plasmatocytes responds to oxidative stress with immune activation by Jak/STAT, DDR and JNK signaling.
(A) Key transcription factors (TFs) regulating hemocytes states. Heat map of scaled TF activity at a single cell level in hemocytes states from Figure 2C. Colors in the heat map correspond to scaled expression. Numbers on top indicate the cluster identity. Labels on the left and right indicate the TFs. (B) Feature plots showing scaled expression of selected genes associated with JAK/STAT signaling (upd3, Socs36E, TotA, TotM, Fas3), JNK and DNA damage signaling (puc, Gadd45), insulin signaling (Thor, InR), TGFβ signaling (dpp, daw) and TNF signaling (egr).

Loss of non-canonical DNA damage signaling activity in hemocytes leads to an increase in systemic upd3 levels and a higher susceptibility to oxidative stress.
(A) Survival of Hml/+ (n= 28), Hml>mei41-IR (n= 23); Hml>tefu-IR (n= 24); Hml>mei41-IR,tefu-IR (n= 16) and Hml>nbs-IR (n= 22) flies on control food and PQ. Each data point represents a sample of 10 flies. Mean ± SEM is shown. Three independent experiments were performed. One-way ANOVA: ***p<0.001; ****p<0.0001. (B) Hemocyte quantification of Hml/+ control flies and DDR-knockdowns on control food (C) and 15mM Paraquat (P). Each data point represents one fly (n=5 for all groups). Mean ± SEM is shown. One way ANOVA: Hml/+ (C) vs Hml>tefu-IR (C), **p=0.0052; Hml>tefu-IR (C) vs Hml>tefu-IR (P), *p=0.03 (C) Comet assay of isolated hemocytes from Hml/+ (C: n=73; P: n=129), Hml>mei41-IR (C: n=119; P: n=132) and Hml>tefu-IR (C: n=175; P: n=125) - flies on control food and PQ. Olive tail moment of comet assays of sorted hemocyte nuclei is shown. One-way ANOVA: *p<0.05; ***p<0.001; ****p<0.0001. (D) Gene expression analysis for Jak/STAT target genes via RT-qPCR in Hml/+ control flies and DDR-knockdowns on control food (C) and 15mM Paraquat (P). All transcript levels were normalized to the expression of the loading control Rpl1 and are shown in arbitrary units [au]. Each data point (n=5) represents a sample of three individual flies. Mean ± SEM is shown. Two-way ANOVA: *p<0.05; **p<0.01; *** p<0.001; ****p<0.0001. (E) Gene expression analysis for insulin signaling target genes and Ilps via RT-qPCR in Hml/+ control flies and DDR-knockdowns on control food (C) and 15mM Paraquat (P). All transcript levels were normalized to the expression of the loading control Rpl1 and are shown in arbitrary units [au]. Each data point (n=5) represents a sample of three individual flies. Mean ± SEM is shown. Two-way ANOVA: *p<0.05; **p<0.01; *** p<0.001; ****p<0.0001. (F) Triglyceride (TG) levels of Hml/+, Hml>mei41-IR, Hml>tefu-IR, Hml>mei41-IR,tefu-IR and Hml>nbs-IR flies on control food and PQ determined via thin-layer chromatography (TLC). One representative TLC is shown (left panel). Quantification thereof is shown on the right. Each data point (n=4 per group) represents a sample of ten individual flies. Mean ± SEM is shown. n.d. = not detectable. One-way ANOVA was performed for both groups (15mM PQ and control) separately: *p=0.0106.

Hemocyte-derived upd3 controls susceptibility to oxidative stress in adult Drosophila.
(A) Survival of Hml/+ (n=28), Hml>upd3-IR (n=27) and Hml>upd3 (n=26) flies on 15mM PQ food. Each data point represents a sample of 10-15 flies. Mean ± SEM is shown. Three independent experiments were performed. One-way ANOVA: ****p<0.0001. (B) Gene expression analysis for upd3 via RT-qPCR in Hml/+, Hml>upd3-IR and Hml>upd3 flies on control food (-) and 15mM Paraquat (+). Each data point (n=5-6) represents a sample of three individual flies. Mean ± SEM is shown. Two-way ANOVA: ****p<0.0001. (C) Hemocyte quantifications in Hml/+, Hml>upd3-IR and Hml>upd3 flies on control food and 15mM PQ food. Each data point represents one fly (n= 5-6). Mean ± SEM is shown. One-way ANOVA: **p<0.01. (D) Survival of w¹¹¹⁸ (n=17), Hml/+ (n=16), upd3^null (n=11) and upd3^null;Hml>upd3 flies (n=16) on 15mM PQ food at 29°C. Each data point represents a sample with 10-15 flies. Mean ± SEM is shown. One-way ANOVA: ****p<0.0001. (E) Survival of Hml/+ (n=78), Hml>hep[act] (n=38) and Hml>bsk-DN (n=80) flies on 15mM PQ food. Each data point represents a sample with 10 flies. Mean ± SEM is shown. One-way ANOVA: **p<0.01; ****p<0.0001. (F) Gene expression analysis for upd3 via RT-qPCR of Hml/+, Hml>hep[act] and Hml>bsk-DN flies on control food (-) and 15mM Paraquat (+). Each data point (n=5-6) represents a sample of three individual flies. Mean ± SEM is shown. Two-way ANOVA: *p<0.05.

Paraquat-induced oxidative stress does not cause gut leakage, but detrimental changes in triglyceride storage in the fat body.
(A) Survival of Hml/+ flies treated with control food (n=8), 2mM PQ (n=8), 15mM PQ (n=9) and 30mM PQ (n=8). Each data point represents a sample of 10 flies. Mean ± SEM is shown. One-way ANOVA: **p<0.01; ****p<0.0001. (B) Smurf assay to test the gut integrity of 7 day old Hml/+ flies treated with 2mM PQ (n=79), 15mM PQ (n=90), 30mM PQ (n=80) and controls (n=80). Data was pooled from two independent experiments. (C) Gene expression analysis for AMP genes via RT-qPCR in whole flies on control food (C) and 15mM Paraquat (P). All transcript levels were normalized to the expression of the loading control Rpl1 and are shown in arbitrary units [au]. Each data point (n=5) represents a sample of three individual flies. Mean ± SEM is shown. (D) Gene expression analysis for dpp, daw and eiger via RT-qPCR in whole flies on control food (C) and 15mM Paraquat (P). All transcript levels were normalized to the expression of the loading control Rpl1 and are shown in arbitrary units [au]. Each data point (n=5) represents a sample of three individual flies. Mean ± SEM is shown. (E) Representative light microscopic images of HE stained control flies (left panels) and flies treated with 15mM PQ (right panels). Inserts indicate location of higher magnification images below. Scale bar (overview) = 500 µm, scale bar (inserts) = 100 µm.

Gating strategy for the FACS isolation of nuclei from control and PQ-treated flies.
(A) Flow cytometric sorting strategy of nuclei used for the snRNA-seq. Doublet exclusion was performed via gating on DRQ7 single positive nuclei. DRQ7⁺DAPI⁺ double positive events were considered to be nuclei and were sorted for the snRNA-seq. (B) Violin plot indicating number of genes identified in analyzed nuclei of the sorted samples. Color code is indicated in legend. (C) Violin plot indicating RNA counts of the analyzed nuclei of each sorted sample. Color code is indicated in legend. (D) Violin plot indicating percentage of mitochondrial genes detected in the nuclei of each sorted samples. Color code is indicated in legend.

Specific signature genes identify plasmatocyte and crystal cell clusters within the eight identified hemocyte clusters.
(A) Dot plot of hemocyte signature gene-expression according to Cattenoz et al²⁵. Color code is indicated in legend. (B)-(L) Feature plots for expression levels of respective hemocyte signature genes across all hemocyte clusters. Color-code, indicating the expression levels of the respective genes, is shown on the right side of each graph. Gene names are indicated above plots shown. (M) Bar plot representing distribution of nuclei derived from flies under control condition (red) and flies treated with 15mM PQ (turquoise) across all eight identified hemocyte clusters. Each cluster is shown as one bar. Total number of hemocytes in the respective cluster is indicated below.

PQ treatment of S2 cells in vitro induces reactive oxygen species, immune activation and DNA damage.
(A) Gating strategy of flow cytometric analysis of control or PQ-treated S2-cells stained for reactive oxygen species (ROS) with CellROX. Control cells, 15mM PQ treated and 30mM PQ treated cells are shown in the first, second and third row respectively. After doublet exclusion, dead cells were excluded via DAPI staining. The amount of ROS was determined in the last gate were the CellROX staining is shown. (B) Representative FACS histograms of cells treated with PQ and analyzed via CellROX staining. Gates indicate the cut-off for determining CellROX⁺ cells. (C) Mean fluorescence intensity (MFI) of FACS analyzed, CellROX⁺ cells. Data were generated from three independent experiments (n=3). One-way ANOVA: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (D) Comet assay of S2 cells treated without PQ (n=351), 5mM PQ (n=208), 10mM PQ (n=129) and 15mM PQ (n=71). Assay was performed in two independent experiments. Data of one representative assay are shown as violin plots with median (red), second and third quartile. One-way ANOVA: ***p<0.001; ****p>0.0001. (E-F) Gene expression analysis of genes associated with JAK-STAT signaling, JNK signaling, insulin signaling is shown in (E) and TGFβ signaling and AMPs is shown in (F). S2-cells treated with 15mM PQ for six or 24 hours. Controls were untreated. Each dot represents a sample containing RNA of 50.000 cells. One-way ANOVA: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Loss of DNA damage, upd3 or JNK signaling in hemocytes alters susceptibility to oxidative stress but not overall lifespan.
(A) Representative images of Hml/+ flies and DDR-Knockdown flies on control food (left panels) and treated with 15mM PQ (right panels). Five individual flies per group were analyzed. (B) Lifespan analysis of Hml/+ (n=251), Hml>mei-41-IR (n=231), Hml>tefu-IR (n=219), Hml>mei-41-IR;tefu-IR (n=144) and Hml>nbs-IR flies (n=299). Hml/+ vs. Hml>mei-41-IR: Log-rank test ****p<0.0001, Χ²=172.2; Wilcoxon test ****p<0.0001, Χ²=155.9. Hml/+ vs. Hml>tefu-IR: Log-rank test ****p<0.0001, Χ²=56.09; Wilcoxon test ****p<0.0001, Χ²=50.95. Hml/+ vs. Hml>mei-41-IR;tefu-IR: Log-rank test ns, Χ²=3.1; Wilcoxon test ***p=0.0005, Χ²=12.11. Hml/+ vs Hml>nbs-IR: Log-rank test ****p<0.0001, Χ²=60.90; Wilcoxon test ****p<0.0001, Χ²=50.21. Data was obtained from three independent experiments. (C) Starvation survival of Hml/+ (n=607), Hml>mei-41-IR (n=421), Hml>tefu-IR (n=480), Hml>mei-41-IR;tefu-IR (n=266) and Hml>nbs-IR (n=341) flies. Hml/+ vs. Hml>mei-41-IR: Log-rank test **p=0.0052, Χ²=7.81; Wilcoxon test ns, Χ²=0.02. Hml/+ vs. Hml>tefu-IR: Log-rank test ns, Χ²=0.03; Wilcoxon test *p=0.0153, Χ²=5.88. Hml/+ vs. Hml>mei-41-IR;tefu-IR: Log-rank test ns, Χ²=0.21; Wilcoxon test *p=0.019, Χ²=5.51. Hml/+ vs Hml>nbs-IR: Log-rank test ***p=0.0003, Χ²=13.30; Wilcoxon test ns, Χ²=1.24. Data was obtained from two independent experiments. (D) Lifespan analysis of Hml-Gal80^ts/+ (n=127), Hml-Gal80^ts>mei-41-IR (n=140), Hml-Gal80^ts>tefu-IR (n=164), Hml-Gal80^ts>mei-41-IR;tefu-IR (n=101) and Hml-Gal80^ts>nbs-IR flies (n=157). Hml-Gal80^ts/+ vs. Hml-Gal80^ts>mei-41-IR: Log-rank test ****p<0.0001, chi²=21.80; Wilcoxon test ****p<0.0001, Χ²=18.48. Hml-Gal80^ts/+ vs. Hml-Gal80^ts>tefu-IR: Log-rank test ***p=0.0003, Χ²=13.20; Wilcoxon test *p=0.033, Χ²=4.56. Hml-Gal80^ts/+ vs. Hml-Gal80^ts>mei-41-IR;tefu-IR: Log-rank test *p=0.0429, Χ²=4.10; Wilcoxon test ***p=0.0001, Χ²=14.95. Hml-Gal80^ts/+ vs Hml-Gal80^ts>nbs-IR: Log-rank test **p=0.0047, Χ²=7.99; Wilcoxon test ns, Χ²=1.68. Data was obtained from five independent experiments. (E) Survival of Hml-Gal80^ts/+ (n=23), Hml-Gal80^ts>mei-41-IR (n=28), Hml-Gal80^ts>tefu-IR (n=18), Hml-Gal80^ts>mei-41-IR;tefu-IR (n=13) and Hml-Gal80^ts>nbs-IR flies (n=31) on PQ food. Each dot represents a sample with ten flies. Mean ± SEM is shown. One-way ANOVA *p<0.05; ****p<0.0001. Data was obtained from five independent experiments. (F) Lifespan analysis of Hml/+ (n=266), Hml>upd3-IR (n=269), and Hml>upd3 flies (n=193). Hml/+ vs. Hml>upd3-IR: Log-rank test ****p<0.0001, Χ²=96.03; Wilcoxon test ****p<0.0001, Χ²=81.16. Hml/+ vs. Hml>upd3: Log-rank test ****p<0.0001, Χ²=15.33; Wilcoxon test *p=0.0124, Χ²=6.26. Results are shown from two independent experiments. (G) Representative images of Hml/+, Hml>upd3-IR and Hml>upd3 flies on control food (left panels) and treated with 15mM PQ (right panels). Six individual flies per group were analyzed. (H) Lifespan analysis of Hml/+ (n=281), Hml>hep[act] (n=117), and Hml>bsk-DN (n=273) flies. Hml/+ vs. Hml>hep[act]; Log-rank test: n.s.; p=0.69, Χ²=0.16; Wilcoxon test: n.s. p=0.18, Χ²=1.77. Hml/+ vs. Hml>bsk-DN: Log-rank test: ****p<0.0001, Χ²=25.31; Wilcoxon test: ****p<0.0001, Χ²=23.61. Data was obtained from two independent experiments.

Differentially expressed genes in hemocyte clusters C1-C8.

Differentially expressed genes in hemocyte clusters C1-C8.

Transgenic Drosophila lines and primers used in the study.

Transgenic Drosophila lines and primers used in the study.

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