Ecdysone triggered PGRP-LC expression controls Drosophila innate immunity

Abstract

Throughout the animal kingdom, steroid hormones have been implicated in the defense against microbial infection, but how these systemic signals control immunity is unclear. Here, we show that the steroid hormone ecdysone controls the expression of the pattern recognition receptor PGRP-LC in Drosophila, thereby tightly regulating innate immune recognition and defense against bacterial infection. We identify a group of steroid-regulated transcription factors as well as two GATA transcription factors that act as repressors and activators of the immune response and are required for the proper hormonal control of PGRP-LC expression. Together, our results demonstrate that Drosophila use complex mechanisms to modulate innate immune responses, and identify a transcriptional hierarchy that integrates steroid signalling and immunity in animals.

Figure 1

20E controls PGRP-LC expression. (A) Microarray expression profiles for IMD pathway components. Profiles were generated from triplicate samples of S2* cells before and after 24 h of treatment with 20E. The asterisks represent statistical significance (*P value <0.05; **P<0.01) calculated by unpaired t-test. (B) Real-time qRT–PCR analysis of PGRP-LC and Dpt transcripts from S2* cells that were exposed to 20E for various times, as indicated, and treated with PGN for 6 h or left untreated prior to harvest for RNA isolation. The mean of three independent biological replicates is shown, and error bars represent standard deviation. PGRP-LC levels at 12 h of hormonal treatment with or without PGN stimulation, are significantly increased compared with the untreated samples (0h), while Diptericin levels are significantly increased beginning at 18 h after hormonal treatment, only after PGN stimulation. *P<0.05, **P<0.01, ***P<0.001, as determined by one-way ANOVA with Tukey’s multiple comparison test.

Figure 2

20E-independent IMD signalling in PGRP-LCx-FLAG expressing cells. Analysis of whole-cell lysates from parental S2* or PGRP-LCx-FLAG cells with or without 24 h pretreatment with 20E, followed by 10 min stimulation with PGN, as indicated. IMD cleavage was analyzed by immublotting (IB) while IMD ubiquitination was monitored by immunoprecipitation (IP)-IB (upper two panels); Relish cleavage and phosphorylation were analyzed by IB (lower two panels). The percent Relish cleavage was quantified by measuring the intensity of the relevant bands from three different experiments and the mean from three experiments (with s.d.) are shown.

Figure 3

20E-dependent and -independent expression of AMP genes in PGRP-LCx-FLAG cells. qRT–PCR analysis of Dpt, Mtk, Drs, CecA1, AttA and Def expression from parental S2* (grey bars) and PGRP-LCx-FLAG cells (black bars). Cells, with or without 24 h of 20E pretreatment, were stimulated with PGN for 6 h (or not), and then harvested for RNA isolation, as indicated. AMP gene expression was normalized to Rp49 levels, and then normalized between biological replicates and represented as a percentage of the expression level relative to the 20E treated and PGN-stimulated samples from each cell type. For each treatment, the values shown represent the mean of three independent experiments. Error bars represent standard deviations and *P<0.05, **P<0.01, ****P<0.0001 were calculated using unpaired t-test for comparing the corresponding samples with or without hormonal treatment.

Figure 4

Classic ecdysone signalling pathway components regulate PGRP-LC expression and AMP gene induction. (A and B) qRT–PCR analysis of Dpt (A) or Cecropin A1 (B) induction in S2* cells transfected with RNAi targeting for EcR, br-c, Eip78C, Eip93F, Eip 74EF, Eip75B, Hr46, pnr, srp or mock transfected. Cells with or without exposure to 20E for 24 h were then stimulated (or not) with PGN for an additional 6 h, as indicated. (C) The same RNA was analyzed for the expression of PGRP-LC by qRT–PCR. The mean and s.d. of three independent experiments is shown. P values were calculated by one-way ANOVA with Tukey’s multiple comparison test, ***P<0.001.

Figure 5

EcR, br-c, srp and pnr are critical for the PGRP-LC-independent hormonal control of AMP gene induction. qRT–PCR analysis of Dpt, Drs and Mtk expression in parental S2* cells (A–C) and PGRP-LCx-FLAG expressing cells (D–F) treated with RNAi targeting for EcR, br-c, Eip78C, Eip93F, Eip74EF, Eip75B, Hr46, pnr, srp or mock transfected. Cells were exposed or not to 20E treatment for 24 h and then stimulated (or not) with PGN for an additional 6 h, as indicated. The results shown represent the mean of three independent experiments and error bars are s.d. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test, *P<0.05, **P<0.01, ***P<0.001.

Figure 6

EcR, br-c, Eip93F, Eip78C, Eip74EF, Hr46, pnr, and srp knockdown causes immunodeficiency in adult flies. Real-time RT–PCR was used to analyze the expression of Dpt (A), CecA1 (B) and PGRP-LC (C) in EcR, br-c, Eip78C, Eip93F, Eip74EF, Eip75B, Hr46, pnr or srp RNAi expressing flies before or 24 h after infection with E. coli. For all experiments, the yolk protein 1 (Yp1)-GAL4 driver was used to express inverted-repeat RNAs specifically in the adult female fat body, and the Yp1-GAL4 strain is presented as a control. P-values were calculated in comparison to Yp1-GAL4 driver strain by one-way ANOVA with Tukey’s multiple comparison test. *P<0.05, **P<0.01, ***P<0.001, (D) Kaplan–Meier plot showing survival of these same lines after infection with Erwinia carotovora carotovora 15. Survival curves of uninfected animals of all genotypes, overlap and are shown as dashed lines. Statistical significance between the survival of infected RNAi flies and the control Yp1-GAL4 strain were determined by a log-rank test and is equal or less then 0.005 for all comparisons, except for Eip75B RNAi, which survives better with a P=0.03. n=60 for all genotypes, and results are typical of at least three independent experiments.

Figure 7

Model for 20E regulation of IMD innate immune signalling. 20E controls the IMD innate immune signalling by at least two distinct mechanisms. First, 20E regulates the expression of the peptidoglycan receptor PGRP-LC. This hormonal control involves several ecdysone-inducible transcription factors, including BR-C, Eip78C, Eip93F, Eip74EF, Eip75B, HR46, PNR and SRP. Through this steroid-mediated regulation of the key microbial sensor, immune induction of all AMP genes through the IMD pathway is tightly controlled by prior exposure to this hormone. Through a second mechanism, 20E further regulates the expression of a subset of AMP genes (i.e., Dipt, Mtk and Drs), independent of its control of the receptor PGRP-LC. BR-C, SRP and PNR transcription factors are absolutely required for this PGRP-LC independent hormonal effect. On the other hand, the 20E-inducible nuclear hormone receptor Eip75B negatively regulates the IMD pathway, at least in part, by interfering with PGRP-LC expression.