Hormonal regulation of the humoral innate immune response in Drosophila melanogaster

Abstract

Juvenile hormone (JH) and 20-hydroxy-ecdysone (20E) are highly versatile hormones, coordinating development, growth, reproduction and aging in insects. Pulses of 20E provide key signals for initiating developmental and physiological transitions, while JH promotes or inhibits these signals in a stage-specific manner. Previous evidence suggests that JH and 20E might modulate innate immunity, but whether and how these hormones interact to regulate the immune response remains unclear. Here we show that JH and 20E have antagonistic effects on the induction of antimicrobial peptide (AMP) genes in Drosophila melanogaster. 20E pretreatment of Schneider S2 cells promoted the robust induction of AMP genes, following immune stimulation. On the other hand, JH III, and its synthetic analogs (JHa) methoprene and pyriproxyfen, strongly interfered with this 20E-dependent immune potentiation, although these hormones did not inhibit other 20E-induced cellular changes. Similarly, in vivo analyses in adult flies confirmed that JH is a hormonal immuno-suppressor. RNA silencing of either partner of the ecdysone receptor heterodimer (EcR or Usp) in S2 cells prevented the 20E-induced immune potentiation. In contrast, silencing methoprene-tolerant (Met), a candidate JH receptor, did not impair immuno-suppression by JH III and JHa, indicating that in this context MET is not a necessary JH receptor. Our results suggest that 20E and JH play major roles in the regulation of gene expression in response to immune challenge.

Fig. 1

JH III suppresses basal AMP expression in whole flies. Shown are log₂-fold change values (JH/control) for 12 AMP transcripts from microarray analyses performed in duplicate. *P<0.05 (t-test). Since the physiological role of JH is not well understood in males, we only used females in this array experiment. We suggest that microarrays might be a particularly useful tool when studying whole organism effects of hormonal signaling: hormones can be topically applied or injected, are taken up into the circulation, act on responsive target tissues, and elicit a systemic, whole organism response (e.g., immune modulation).

Fig. 2

JHa methoprene reduces expression of Drosomycin (Drs) in females of the Drs-GFP reporter strain DD1. (A) uninfected (EtOH jabbed), no JHa, (B) infected (E. coli jabbed), no JHa, (C) uninfected (EtOH jabbed), JHa, (D) infected (E. coli jabbed), JHa, (E) quantification of GFP signals from images; shown are means ± 1 standard error; sample size per group, N = 3. Note the strong autofluorescence in the ovaries (C, E). Qualitatively similar results were obtained with a GFP reporter for Diptericin (Dpt-GFP; DIG) and in a Northern blot on Dpt mRNA in y, w females (data not shown), suggesting that JH/JHa acts as a suppressor of AMP induction in vivo.

Fig. 3

20E and JHa methoprene have antagonistic effects on AMP expression. (A) Northern blot monitoring expression of AMPs Diptericin, Attacin, and Cecropin in S2* cells treated with or without PGN and with different combinations of hormones. (B) Luciferase assay in S2* cells stably transfected with a Dpt-luciferase reporter construct; shown are means ± 1 standard error.

Fig. 4

20E potentiates Dpt induction in a dose-dependent manner (A); JH III (B) and the JHa methoprene (C) and pyriproxyfen (D) suppress the 20-mediated response dose-dependently. Results are from luciferase assays with Dpt-luc cells, immune stimulated with PGN; all JH/JHa treatments were performed in combination with 20E; plotted are means ± 1 standard error.

Fig. 5

Dpt potentiation by 20E requires at least 18 hours of hormone exposure in presence of PGN (A), but suppression by JHa methoprene is rapid and does not depend on preincubation (B). Results in (A) are from a Northern blot, with quantification of Dpt expression normalized to that of a Rp49 control (Northern blot not shown). The x-axis displays the period (in hours) during which cells were exposed to 20E; crude PGN contaminated LPS preparations were used to stimulate the immune response. Results in (B) are from a luciferase assay with Dpt-luc cells, immune stimulated with PGN; all JH/JHa treatments were performed in combination with 20E. The x-axis displays the different hormone treatments: no hormone, 20E only (for 24 hours), or 20E (for 24 hrs) in presence of JHa added to cell culture at hours 4, 6, 8, or 24 prior to the luciferase assay. Shown are means ± 1 standard error.

Fig. 6

20E blocks S2* cell proliferation, but proliferation is unaffected by JHa methoprene; JHa treatment does not prevent the 20E-mediated block in cell proliferation. Cells were left untreated (filled triangles), exposed to 20E (squares), JHa (filled squares), or both hormones (triangles); cell counts were monitored every 24 hours over the next three days. Shown are cell counts (in units of 10⁶ cells/ml cell culture media) over time. The result shown is representative of 3 independent experiments (data not shown).

Fig. 7

(A–B) Northern blotting for Dpt induction shows that EcR and Usp are both required for the potentiation of Dpt induction by 20E, as determined by RNAi-mediated silencing using dsRNA. (C) Western blot with mouse monoclonal antibody AB11 against USP; the Western blot was performed on the same samples used in the Northern blot; RNAi successfully silenced Usp.

Fig. 8

Dpt promoter activity with RNAi-mediated silencing (dsRNA) directed against EcR (A), Usp (B), and Met (C). Silencing EcR and Usp abolishes the 20E response; JHa methoprene seems to suppress the weak Dpt induction that occurs in EcR or Usp knock-down cells; however, this effect is not significant (Table S3). In contrast, silencing Met does not impair the 20E response, and JHa is fully effective in suppressing immune induction by 20E. Results are from luciferase assays with Dpt-luc cells, immune stimulated with PGN; all JH/JHa treatments were performed in combination with 20E; plotted are means ± 1 standard error.

Fig. 9

(A) Northern blotting for Dpt shows that Met is not required for induction of Dpt expression by 20E; notably, JHa methoprene in presence of 20E is able to fully suppress Dpt expression even when Met function is silenced; pMET refers to cells transfected with Met expression vector plasmid. (B) Western blot with rabbit polyclonal antibody against MET; the Western blot was performed on the same samples used in the Northern blot; RNAi successfully silenced Met.