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First published online August 8, 2008
Journal of Experimental Biology 211, 2712-2724 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.014878
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Hormonal regulation of the humoral innate immune response in Drosophila melanogaster

Thomas Flatt1, Andreas Heyland2, Florentina Rus3, Ermelinda Porpiglia3, Chris Sherlock3,{dagger}, Rochele Yamamoto1, Alina Garbuzov1, Subba R. Palli4, Marc Tatar1 and Neal Silverman3,*

1 Division of Biology and Medicine, Department of Ecology and Evolutionary Biology, Brown University, Providence, RI 02912, USA
2 Department of Integrative Biology, University of Guelph, Ontario, Canada, N1G 2W1
3 Department of Medicine, Division of Infectious Disease, University of Massachusetts Medical School, Worcester, MA 01655, USA
4 Department of Entomology, Agricultural Science Center, College of Agriculture, University of Kentucky, Lexington, KY 40546-0091, USA


Figure 1
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  		Fig. 1. Juvenile hormone III (JH III) suppresses basal antimicrobial peptide (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 (Student's 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).

 

Figure 2
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  		Fig. 2. Juvenile hormone analog (JHa) methoprene reduces expression of Drosomycin (Drs) in females of the Drs-GFP reporter strain DD1. (A) Uninfected (ethanol jabbed), no JHa; (B) infected (E. coli jabbed), no JHa; (C) uninfected (ethanol jabbed), JHa; (D) infected (E. coli jabbed), JHa; (E) quantification of GFP signals from images (means ± 1 s.e.m.; sample size per group, N=3). Note the strong autofluorescence in the ovaries (e.g. in D). 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.

 

Figure 3
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  		Fig. 3. 20-hydroxy-ecdysone (20E) and JHa methoprene have antagonistic effects on AMP expression. (A) Northern blot monitoring expression of AMPs Dpt, Attacin and Cecropin in S2* cells treated with peptidoglycan (PGN) or untreated, and with different combinations of hormones. Rp49, control (encoding ribosomal protein RP49). (B) Luciferase assay in S2* cells stably transfected with a Dpt-luciferase reporter construct (means ± 1 s.e.m.).

 

Figure 4
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  		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 20E-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 (means ± 1 s.e.m.).

 

Figure 5
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  		Fig. 5. Dpt potentiation by 20E requires at least 18 h of hormone exposure in the 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 an Rp49 control (northern blot not shown). The x-axis displays the period (in hours) during which cells were exposed to 20E; crude PGN-contaminated lipopolysaccharide (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 h), or 20E (for 24 h) in the presence of JHa added to cell culture at 4, 6, 8 or 24 h prior to the luciferase assay. Means ± 1 s.e.m.

 

Figure 6
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  		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), or were exposed to 20E (open squares), JHa (filled squares) or both hormones (open triangles); cell counts were monitored every 24 h over the next 3 days. Shown are cell counts (in units of 106 cells ml–1 cell culture media) over time. The result shown is representative of three independent experiments (data not shown).

 

Figure 7
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  		Fig. 7. (A and B) Northern blotting for Dpt induction shows that ecdysone receptor (EcR) and ultraspiracle (Usp) are both required for the potentiation of Dpt induction by 20E, as determined by RNA interference (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.

 

Figure 8
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  		Fig. 8. Dpt promoter activity with RNAi-mediated silencing (dsRNA) directed against EcR (A), Usp (B) and Met (C). E. coli MalE, control (encoding maltose binding protein). 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 (supplementary material 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 (means ± 1 s.e.m.).

 

Figure 9
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  		Fig. 9. (A) Northern blotting for Dpt shows that Met is not required for induction of Dpt expression by 20E; notably, JHa methoprene in the 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.

 

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© The Company of Biologists Ltd 2008