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

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

Institute of Neuropathology, Faculty of Medicine, Medical Center, 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
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
Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, 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
Hilde-Mangold-Haus, Faculty of Biology, University of Freiburg, Freiburg, Germany
CIBSS-Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany

https://doi.org/10.7554/eLife.86700.2

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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 (C) (n=20) and 15mM Paraquat (P) (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) RT-qPCR 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 (TLC). Left panel: Representative image of a TLC plate, each band represents a sample with ten flies. Right panel: Quantification thereof. n= 3 per group were analyzed. Student’s unpaired t-test: **p<0.01. (G) Representative images of 7 days old HmlΔ4>eGFP flies treated with 5% sucrose solution and 15mM PQ respectively. Scale bars = 500µm. (H) Hemocyte quantification of 7 days 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. (J) TG amounts in crq-Gal80^ts/+ control flies and hemocyte-deficient crq-Gal80^ts>reaper flies on control food or Paraquat food. Left panel: Representative image of a TLC plate, each band represents a sample with ten flies. Right panel: Quantification thereof. n= 3 per group were analyzed. One-way ANOVA: *p<0.05. (K) Glucose and (L) Glycogen levels of whole flies were measured in crq-Gal80^ts/+ control flies and hemocyte-deficient crq-Gal80^ts>reaper flies. Each dot (n= 3) represents a sample with three whole flies. One-way ANOVA: ****p<0.0001.

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 hemocyte 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 top 20 DE genes for each hemocyte 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 hemocyte cluster vs. all 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). Non-regulated genes are shown in grey, not significantly regulated genes are shown in green, significantly regulated genes with a FC<1.5 are shown in blue and significantly regulated genes with a FC>1.5 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).

Oxidative stress induced different transcriptomic states in fat body cells, including cells with a distinct Jak/STAT activation signature
(A) UMAP visualization of fat body cells from Figure 2A by unsupervised clustering. Colors and dashed lines indicate different clusters. Each dot represents one nucleus. n=3150 nuclei are shown. (B) UMAP of fat body cell clusters split by treatment. Nuclei from control flies are labeled in red and nuclei from PQ treated flies are labeled in blue. Dashed lines indicate different clusters. Each dot represents one nucleus. n=3150 nuclei are shown. (C) Heat map of top 20 DE genes for each fat body cluster. Gene names indicated on the left. Fold change of gene expression is color coded as indicated in legend. (F) Volcano plots comparing pseudo bulk gene expression of individual fat body cluster vs. all fat body cells. 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). Non-regulated genes are shown in grey, not significantly regulated genes are shown in green, significantly regulated genes with a FC<1.5 are shown in blue and significantly regulated genes with a FC>1.5 are shown in red.

Loss of 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/+ (B)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 (C) and 15mM Paraquat (P). 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 Hml/+ (n=241), upd3^null;Hml/+ (n=122), Hml>upd3 (n=104), upd3^null;Hml>upd3 (n=57), Hml>mei-41-IR;tefu-IR (n=103) and upd3^null;Hml>mei-41-IR;tefu-IR (n=50) flies 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.

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