Ecdysone mediates the development of immunity in the Drosophila embryo

Abstract

Beyond their role in cell metabolism, development, and reproduction, hormones are also important modulators of the immune system. In the context of inflammatory disorders, systemic administration of pharmacological doses of synthetic glucocorticoids (GCs) is widely used as an anti-inflammatory treatment [1, 2]. However, not all actions of GCs are immunosuppressive, and many studies have suggested that physiological concentrations of GCs can have immunoenhancing effects [3-7]. For a more comprehensive understanding of how steroid hormones regulate immunity and inflammation, a simple in vivo system is required. The Drosophila embryo has recently emerged as a powerful model system to study the recruitment of immune cells to sterile wounds [8] and host-pathogen dynamics [9]. Here we investigate the immune response of the fly embryo to bacterial infections and find that the steroid hormone 20-hydroxyecdysone (20-HE) can regulate the quality of the immune response and influence the resolution of infection in Drosophila embryos.

Figure 1

Stage 15 Drosophila Embryos Are Able to Mount Immune Responses to Bacterial Challenge (A–E) A stage 15 embryo expressing Drosocin-GFP stained with anti-GFP (Ai) and the tracheal-specific antibody 2A12 (Aii). The merge (Aiii) shows clear Drc expression throughout the tracheal network 3 hpi with Ecc15. A live, untreated, Drc-GFP-expressing embryo (B) and a Drc-GFP-expressing embryo (C) 6 hpi with PBS show no Drc expression, whereas injection with either E. coli (D) or Ecc15 (E) leads to robust expression in the embryonic epithelium (arrowheads). Arrows show autofluorescence in the yolk. Scale bars represent 20 μm (A) and 50 μm (B–E). (F–K) Real-time qPCR analysis of Drosocin (F), Cecropin A1 (G), Defensin (H), Diptericin (I), Metchnikowin (J), and Drosomycin (K) in stage 15 embryos injected with endotoxin-free PBS or live bacterial cells of E. coli, M. luteus, and E. carotovora (Ecc15) for 2 hr. The expression of antimicrobial peptide genes was normalized to the reference gene rp49 and then standardized to the expression level of nontreated samples. The mean of three independent biological replicates is shown, and error bars represent the SD. ^∗p < 0.05, ^∗∗p < 0.01, and p^∗∗∗ < 0.001 as determined by one-way ANOVA with an ad hoc Tukey’s multiple comparison test. n = 200 embryos.

Figure 2

Stage 15 Drosophila Embryos Are Able to Effectively Distinguish between Different Types of Infection (A) Real-time qPCR analysis of Diptericin expression in stage 15 embryos 2 hr after Ecc15 infection in the wild-type and Relish (RelA^E20) and modular serine protease (modSP¹) mutants shows a clear requirement for Imd signaling in the response to Ecc15. (B) Real-time qPCR showing that the expression of Drosomycin in stage 15 embryos infected with M. luteus depends on the Toll signaling component modSP¹. (C) Percentage survival 24 hpi of RelA^E20, modSP¹, and psh;modSP¹ embryos infected with the Gram-positive bacteria M. luteus, the Gram-negative bacteria Ecc15 and E. coli, and an Aspergillus fumigatus protease cocktail compared with PBS-injected wild-type embryos. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗∗p < 0.001 as determined by two-way ANOVA followed by an ad hoc Tukey’s multiple comparison test. n = 100 embryos for all genotypes. (D) Bacterial load in infected stage 15 embryos. Bacterial load is controlled in wild-type embryos, but not in RelA^E20 or modSP¹ embryos. Infections were performed in groups of 25 embryos and reproduced in at least six independent experiments. ^∗∗∗∗p < 0.001 as determined by determined by two-way ANOVA followed by an ad hoc Tukey’s multiple comparison test.

Figure 3

Stage 11 Embryos Show Reduced Immune Competency in Response to Bacterial Invasion (A) Stage 11 embryos expressing Drosocin-GFP fail to switch on Drosocin upon superficial injection with Ecc15 (compare to stage 15). This development of immune competence coincides with a pulse of ecdysone in the embryo that peaks at approximately 8 hr after egg laying. (B) Survival analysis upon septic injection with E. coli, Ecc15, and M. luteus in stage 15 and stage 11 wild-type embryos clearly shows that early embryos are compromised in their survival after infection with all bacteria tested. Statistical significance was determined by multiple unpaired t tests (^∗p < 0.05, ^∗∗p < 0.01). n = 100 embryos for all genotypes. (C) Stage 11 and stage 15 embryos were injected with Ecc15, E. coli, or M. luteus, and colony-forming units were determined at 8 hpi. Bacterial load is significantly higher in infected stage 11 embryos. The significance was assessed by multiple unpaired t tests (p < 0.01). The infections were performed in groups of 25 embryos and reproduced in six independent experiments. (D) Effect of bacterial infection upon survival of stage 15 ecdysone receptor mutant embryos shows that mutants have compromised survival at 24 hpi with Ecc15. The survival of embryos expressing dominant-negative EcR-B1 receptor in hemocytes (srp>EcR-B1 DN) was not significantly different from that of wild-type embryos, whereas expression of dominant-negative EcR-B1 receptor in the trachea using btl-Gal4 leads to a reduction in survival to levels observed in EcR mutants. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.005). n = 100 embryos for all genotypes. (E) Bacterial load is higher in infected stage 15 btl>EcR-B1 DN embryos than in control embryos at 8 hpi. Statistical significance was determined by multiple unpaired t tests (^∗p = 0.004).

Figure 4

Ecdysone Regulates Embryonic Immune Responses (A) Real-time qPCR analysis of Cecropin A1, Defensin, and Metchnikowin expression in stage 15 wild-type and transheterozygous EcRQ^50st/EcR^M55fs mutant embryos at 2 hpi with Ecc15. Graphs show a reduced expression of all three AMPs in the mutant after infection. Gene expression levels were normalized to rp49 levels and were then standardized to nontreated samples and presented as fold change. For each treatment, the values shown represent the mean of three independent experiments. Error bars represent the SD. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001 as determined by one-way ANOVA with an ad hoc Tukey’s multiple comparison test. (B) Representative images of Drosocin-GFP-expressing stage 11 and stage 15 embryos 12 and 6 hpi, respectively, with Ecc15. Images show an apparent lack of Drc-GFP expression in young embryos upon bacterial infection (Ecc15), which can be rescued upon coinjection with 25 μM ecdysone (Ecc15+20HE). Treatment with ecdysone in the absence of bacteria did not cause an upregulation of Drosocin (20-HE). Scale bars represent 50 μm. (C) Real-time qPCR analysis of Drosocin expression in stage 11 embryos after treatment with 25 μM 20-HE. The graph shows that, consistent with Drc-GFP data in (B), addition of ecdysone is able to rescue Drosocin expression in early embryos upon Ecc15 infection. Error bars represent the SD. ^∗p < 0.05 as determined by one-way ANOVA with an ad hoc Holm-Sidak’s multiple comparison test.