Steroid hormone signaling is essential to regulate innate immune cells and fight bacterial infection in Drosophila

Abstract

Coupling immunity and development is essential to ensure survival despite changing internal conditions in the organism. Drosophila metamorphosis represents a striking example of drastic and systemic physiological changes that need to be integrated with the innate immune system. However, nothing is known about the mechanisms that coordinate development and immune cell activity in the transition from larva to adult. Here, we reveal that regulation of macrophage-like cells (hemocytes) by the steroid hormone ecdysone is essential for an effective innate immune response over metamorphosis. Although it is generally accepted that steroid hormones impact immunity in mammals, their action on monocytes (e.g. macrophages and neutrophils) is still not well understood. Here in a simpler model system, we used an approach that allows in vivo, cell autonomous analysis of hormonal regulation of innate immune cells, by combining genetic manipulation with flow cytometry, high-resolution time-lapse imaging and tissue-specific transcriptomic analysis. We show that in response to ecdysone, hemocytes rapidly upregulate actin dynamics, motility and phagocytosis of apoptotic corpses, and acquire the ability to chemotax to damaged epithelia. Most importantly, individuals lacking ecdysone-activated hemocytes are defective in bacterial phagocytosis and are fatally susceptible to infection by bacteria ingested at larval stages, despite the normal systemic and local production of antimicrobial peptides. This decrease in survival is comparable to the one observed in pupae lacking immune cells altogether, indicating that ecdysone-regulation is essential for hemocyte immune functions and survival after infection. Microarray analysis of hemocytes revealed a large set of genes regulated at metamorphosis by EcR signaling, among which many are known to function in cell motility, cell shape or phagocytosis. This study demonstrates an important role for steroid hormone regulation of immunity in vivo in Drosophila, and paves the way for genetic dissection of the mechanisms at work behind steroid regulation of innate immune cells.

Figure 1. Ecdysone signaling is required for hemocyte activation at metamorphosis.

(A–D) Analysis of the morphology of control hemocytes (A, C) and hemocytes expressing a DN form of the EcR receptor (EcRB1DN; B, D) at precise time points before and after puparium formation (APF). (A, B) Ex vivo analysis of bleeds; (C, D) in vivo analysis of cells visualized under the dorsal epithelium. Green, endogenous GFP. Blue, DAPI. Red, phalloidin. (E,F) Forward scatter (FSC)–Area/side scatter (SSC)–Area plots reflecting size (x axis) and granularity (y axis) of hemocytes retrieved from L3W, 8 h APF and 18 h APF control (E) and HmlΔ>EcRB1DN (F) animals. (G–K) Hemocytes insensitive to ecdysone do not activate motility nor disperse at metamorphosis (data retrieved from in vivo time-lapse imaging; see typical movies 1 and 2). (G,H) Epithelia-associated (‘sessile patch’) hemocytes are visible as groups of cells at 1 h APF, but have largely dispersed by 4 h APF in control animals (G). EcRB1DN-hemocytes do not undergo dispersal (H). (I,J) Tracks corresponding to the trajectories of twenty cells, for 80 min (starting 2h40 APF) were superimposed at (0;0). Center of mass of all endpoint positions is marked with a red cross (x/y coordinates indicated on figure) and indicates a random migration of control hemocytes (I; center of mass not significantly different from (0;0)). EcRB1DN-hemocytes are largely immotile (J). (K) Cell velocity measured at 1 h APF. Mean and SEM are displayed. Scale bars represent 20 µm in all panels.

Figure 2. Ecdysone directly regulates hemocyte ability to phagocytose dead cells during metamorphosis.

(A) Ex vivo analysis of circulating hemocytes at precise time points APF. Phagosomes containing dead cells (fragmented nuclei) and muscle fragments (striated phalloidin-positive inclusions) are indicated by solid and forked arrowheads, respectively. Green, endogenous GFP. Blue, DAPI. Red, phalloidin. Scale bars represent 20 µm. (B) Quantification of muscle cell engulfment. The graph displays the number of hemocytes with at least one phalloidin-positive vacuole (corresponding to muscle cell). For statistical analysis, significances based on t-test are indicated on the graph (comparison of control and EcRDN-expressing hemocytes at each developmental stage). One way-ANOVA was used to compare EcRDN-expressing hemocytes at different stages (ns) or control hemocytes at different stages (***). For control hemocytes, Tukey-Kramer post-test revealed significant differences between L3W and 8 h (***) or 18 h (***), between 1 h and 8 h (***) or 18 h (***), between 4 h and 8 h (*) or 18 h (***), and between 8 h and 18 h (**).

Figure 3. Pupae with ecdysone-insensitive hemocytes are susceptible to septic injury and oral infection.

(A) Survival to septic injury with Gram-negative (E. carotovora) and Gram-positive (E. faecalis) bacteria. (B) Stage of lethality after septic injury with E. carotovora. Control or HmlΔ>GFP/EcRB1DN pupae were carefully staged and infected by septic injury with E. carotovora (O.D. 100) at 3 h or 24 h APF. Proportions of individuals dying at different stages over metamorphosis were determined by ‘post-mortem’ examination of the pupae. Pupae were classified into four arbitral categories, based on their appearance at arrested development: “early”, “intermediate 1”, “intermediate 2”, and “late” stage, here represented by a colour gradient (the darker the color, the older the pupa at time of death). The “late” stage corresponds to pupae that look ready to eclose, but finally did not emerge. Statistical analysis displayed on the graph corresponds to Wilcoxon test made without taking into account the stage of death but only the final survival over metamorphosis. An additional analysis with Wilcoxon test taking into account the stage of death indicates significant differences between control and EcRDN at both time point (***), between control at 2 h and 24 h (***) and between EcRDN at 2 h and 24 h (***). (C) Survival to oral infection performed at larval stage with E. carotovora bacteria. Control: HmlΔ>GFP/+ pupae; EcRB1DN: HmlΔ>GFP/EcRB1DN pupae; ‘Phago^less’: HmlΔ>GFP/UAS-Bax. Graphs display mean and SEM.

Figure 4. Acquisition of wound responsiveness at metamorphosis is dependent on ecdysone signaling.

(A–E) Laser wounds were made in the epithelium of white prepupae (A, B). Red and blue dashed lines in (B) delineate central patches and lateral stripes of attached hemocytes, respectively. 200 min after wounding numerous hemocytes are visible at the wound (C); example tracks are shown (see video S3). Tracks of individual cells measured over 80 min, 70 min after wounding (wound performed at 1h30 APF) are superimposed at (0;0) in the tracking plot; all trajectories were rotated to maintain their relative positions towards the wound center (D). The center of mass of all tracks, indicated with red cross, is shifted towards the wound (positive x-axis) and significantly different from (0;0) (p = 0.01). Recruitment rate (cell/min) is significantly higher in pupae wounded at 120 min APF than in pupae wounded 55 min APF (E). Profile of hemocyte recruitment to epithelial wounds in control (brown) or HmlΔ>EcRB1DN pupae (blue) (F). Tracks corresponding to trajectories of EcRB1DN-hemocytes after epithelial wounding were superimposed and rotated as above in tracking plot (G); note that the center of mass (red cross) remains close to (0;0). Rate of recruitment (cell/min) in HmlΔ>EcRB1DN pupae and controls wounded 1h30 APF (H). Mean and SEM are displayed on graphs.

Figure 5. Bacterial phagocytosis is activated by ecdysone signaling at metamorphosis and is required to survive infection.

(A, B) Ecdysone-dependent increase of phagocytic index at metamorphosis. Flow cytometry measurements of ex vivo phagocytosis (pHRodo fluorescence intensity) in hemocytes retrieved from larvae and white prepupae (A); top panel: control hemocytes; bottom panel: EcRB1DN-hemocytes. Phagocytic index (percentage of positive cells x mean intensity) of larval and prepupal control hemocytes or hemocytes expressing EcRB1DN (B). (C–I) Ecdysone signaling is required in vivo for full activation of phagocytosis at metamorphosis. Schematic demonstrating in vivo technique in white prepupa (C). Live imaging of pHRodo particles in control hemocytes (D), or EcRB1DN-expressing hemocytes (E). Flow cytometry measure of phagocytosis (F): pHRodo-fluorescence intensity profile of pHRodo-positive EcRDNB1-hemocytes (corresponding to 38.8% of total EcRDNB1 hemocytes) and pHRodo-positive control hemocytes (88.2% of total control hemocytes). Phagocytic index (flow cytometry experiment) of hemocytes expressing DN forms of all three EcR isoforms or EcR RNAi (G). Striped and full-colour bars correspond to DN forms ^W650A and ^F645A, respectively. EcRDNB1-hemocytes are also impaired in attachment (H) and internalization (I) indexes of injected live E.coli-RFP (in vivo live imaging experiments). Mean and SEM are displayed on graphs.

Figure 6. Transcriptomic analysis of the impact of ecdysone signaling on hemocytes at metamorphosis.

(A) Distribution of the hit genes per fold change and per dependence to EcR. Genes whose expression is significantly (p<0.001) changed in hemocytes between late feeding larval 3^rd instar and early prepupae (1 h–2 h APF) were identified by microarrays and classified based on FC. FC Indicated are linear and only genes which FC>2 are represented on this figure. Up- or down-regulation of a gene was defined to be EcR-dependent when its expression was significantly altered in EcRB1DN-expressing pupal hemocytes (p<0.001; see Table S1 for gene list). (B) qPCR confirmation of the expression of 12 genes from the microarrays. qPCR was performed on RNA extracted from FACS-sorted hemocytes. The results displayed represent the mean and SEM of three biological repeats; samples were independent from samples used for microarrays. Statistical analysis was performed using one-way ANOVA and Tukey-Kramer post-test. For comparison between control larval hemocytes and control pupal hemocytes, as well as between control pupal hemocytes and EcRDN pupal hemocytes, significance are indicated on the graph. There was no significant difference between control larval hemocytes and EcRDN pupal hemocytes except for GstS1 (*), NimC3 (*), Mmp2 (*) and Dscam (***).