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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
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
* Corresponding author (e-mail: neal.silverman{at}umassmed.edu)
Accepted 3 June 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: Drosophila, innate immunity, humoral immune response, antimicrobial peptides, juvenile hormone, ecdysone, hormone receptors
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Medzhitov and Janeway, 1998;
Hoffmann and Reichhart, 2002;
Tzou et al., 2002;
Hoffmann, 2003). Insects have
multiple effector mechanisms to combat microbial pathogens. Infection or
wounding stimulates proteolytic cascades in the host, causing blood clotting
and activation of a prophenoloxidase cascade leading to melanization. Cellular
immunity involves hemocytes (blood cells), which mediate phagocytosis,
nodulation and encapsulation of pathogens. Systemic and local infections also
induce a robust antimicrobial peptide (AMP) response. For example, in a
systemic infection, AMPs are rapidly produced in the fat body (the equivalent
of the mammalian liver) and secreted into the hemolymph (bloodstream)
(Gillespie et al., 1997;
Kimbrell and Beutler, 2001;
Hoffmann, 2003).
The molecular events initiating the transcriptional induction of AMP genes
are well characterized (Silverman and
Maniatis, 2001; Tzou et al.,
2002; Hoffmann,
2003; Kaneko and Silverman,
2005). Upon infection, Drosophila recognizes pathogens
using microbial pattern recognition receptors (PRRs), such as peptidoglycan
recognition proteins (PGRPs) and Gram-negative binding proteins (GNBPs).
Binding of pathogen-derived molecules to these receptors activates two
signaling cascades, the Toll pathway and the immune deficiency (IMD) pathway.
While the Toll pathway responds to many Gram-positive bacteria and fungal
pathogens and activates the nuclear factor kappa B (NF-
B) transcription
factors Dorsal and Dif (Dorsal-related immunity factor), the IMD pathway
responds to Gram-negative bacteria, activating the NF-B homolog Relish.
Subsequently, these NF-B factors induce the expression of a broad range
of AMP genes that are effective against Gram-negative and -positive bacteria
(e.g. Attacin, Cecropin, Diptericin) and fungi (e.g. Drosomycin,
Metchnikowin) (Engström,
1999; Lehrer and Ganz,
1999; Silverman and Maniatis,
2001; Tzou et al.,
2002; Hoffmann,
2003). Because the immune system of insects has much in common
with the innate immune response of mammals, Drosophila is an
excellent model for studying the mechanisms of innate immunity
(Silverman and Maniatis, 2001;
Hoffmann and Reichhart,
2002).
Increasing evidence suggests that hormones and nuclear hormone receptors
systemically regulate adaptive and innate immunity in vertebrates
(Rollins-Smith et al., 1993;
Rollins-Smith, 1998;
Webster et al., 2002;
Glass and Ogawa, 2006;
Pascual and Glass, 2006;
Chow et al., 2007). In
mammals, several nuclear hormone receptors have been implicated in regulating
innate immunity and proinflammatory gene expression, including
peroxisome-proliferator-activated receptors (PPARs), liver X receptors (LXRs),
vitamin D receptors (VDRs), estrogen receptors (ERs), and the glucocorticoid
receptor (GR) (Ricote et al.,
1998; Beagley and Gockel,
2003; Joseph et al.,
2003; Smoak and Cidlowski,
2004; Glass and Ogawa,
2006; Ogawa et al.,
2005). For example, GR represses proinflammatory NF-B
targets, and VDR and its ligand 1,25-dihydroxyvitamin D3 induce
expression of the human AMPs cathelicidin (camp) and
defensin β2 (defB2)
(Wang et al., 2004;
Glass and Ogawa, 2006;
Schwab et al., 2007;
Chow et al., 2007).
In contrast, little is known about the hormonal regulation of immunity in
invertebrates such as insects. Several findings suggest that the steroid
hormone 20-hydroxy-ecdysone (20E), an important regulator of development,
metamorphosis, reproduction and aging in insects
(Nijhout, 1994;
Kozlova and Thummel, 2000;
Tu et al., 2006), modulates
cellular and humoral innate immunity. In the mosquito Anopheles
gambiae, 20E induces expression of prophenoloxidase 1
(PPO1), a gene containing ecdysteroid regulatory elements
(Ahmed et al., 1999;
Müller et al., 1999). In
Drosophila melanogaster, 20E causes mbn-2 cells, a tumorous blood
cell line, to differentiate into macrophages and to increase their phagocytic
activity (Dimarcq et al.,
1997), and injection of mid-third instar larvae with 20E increases
the phagocytic activity of plasmatocytes
(Lanot et al., 2001). 20E
signaling is also required for Drosophila lymph gland development and
hematopoiesis, both necessary for pathogen encapsulation
(Sorrentino et al., 2002), and
in flesh fly larvae (Neobelliera bullata), 20E promotes the
nodulation reaction (Franssens et al.,
2006). In terms of humoral immunity, 20E renders D.
melanogaster mbn-2 cells and flies competent to induce AMP genes such as
Diptericin (Dpt) and Drosomycin (Drs)
(Meister and Richards, 1996;
Dimarcq et al., 1997;
Silverman et al., 2000). The
ability to express Dpt in fly larvae depends on the developmental
stage; Dpt expression could be induced by infection only after third
instar larvae were mature enough to produce sufficient 20E
(Meister and Richards, 1996).
20E also promotes expression of the immunoglobin hemolin in the fat
body of diapausing pupae of the Cecropia moth (Hyalophora
cecropia) (Roxström-Lindquist et
al., 2005). In contrast, 20E may also counteract immune function,
since the Toll ligand dorsal, the Toll effector
spätzle, and several AMPs were downregulated at the onset of
Drosophila metamorphosis in a 20E-dependent manner in gene profiling
studies (Beckstead et al.,
2005). Similarly, 20E downregulates antibacterial activity in
diapausing larvae of the blowfly (Calliphora vicina)
(Chernysh et al., 1995). Thus,
20E might either induce or suppress innate immunity, depending on the
developmental stage and immune response assayed.
While pulses of 20E provide signals for initiating developmental and
physiological transitions (Kozlova and
Thummel, 2000), juvenile hormone (JH) specifically promotes or
inhibits 20E signaling in a stage-specific manner
(Nijhout, 1994;
Riddiford, 1994;
Berger and Dubrovsky, 2005;
Flatt et al., 2005). Recent
results suggest that JH – like 20E – might modulate immunity in
insects. In the tobacco hornworm (Manduca sexta), JH inhibits
granular phenoloxidase (PO) synthesis and thus prevents cuticular melanization
(Hiruma and Riddiford, 1998); likewise, JH reduces PO levels and suppresses
encapsulation in the mealworm beetle (Tenebrio molitor)
(Rolff and Siva-Jothy, 2002;
Rantala et al., 2003). In
honeybees (Apis mellifera), JH-mediated downregulation of the yolk
precursor vitellogenin reduces hemocyte number
(Amdam et al., 2004), and in
flesh fly larvae (Neobelliera bullata) JH suppresses the 20E-induced
nodulation reaction (Franssens et al.,
2006). These findings suggest that 20E is typically a positive
regulator of innate immunity, while JH acts as an immuno-suppressor
(Flatt et al., 2005). Although
JH induces expression of the AMP Ceratotoxin A in female accessory
glands of the medfly (Ceratitis capitata), this peptide is not
induced by bacterial infection (Manetti et
al., 1997). Thus, it remains unclear how JH affects the expression
of pathogen-inducible AMPs in humoral immunity. Furthermore, whether and how
20E and JH interact to regulate AMP expression has not been investigated.
Here we demonstrate that 20E promotes humoral immunity by potentiating AMP induction in D. melanogaster, but that this 20E-induced response is specifically and strongly inhibited by JH and juvenile hormone analogs (JHa). We further show that immune induction by 20E requires ecdysone receptor (EcR)/ultraspiracle (USP), but that immune suppression by JH is independent of the putative JH receptor methoprene-tolerant (MET).
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Riddiford and Ashburner, 1991;
Wilson, 2004;
Zera and Zhao, 2004;
Flatt and Kawecki, 2007). For
details on hormone delivery, see below.
Drosophila stocks and culture
We used the yellow white (y, w) strain for the microarray
experiment and the northern blot on Diptericin mRNA (courtesy of Eric
Rulifson, University of California, San Francisco); for the green fluorescent
protein (GFP) reporter assays, we used the Drosomycin-GFP reporter
strain DD1 [y, w, P(ry+, Dpt-lacZ),
P(w+, Drs-GFP); cn, bw; courtesy of
Dominique Ferrandon, CNRS, Strasbourg]
(Reichhart et al., 1992;
Ferrandon et al., 1998) and
the Diptericin-GFP reporter strain DIG [w; P(Dpt-GFP,
w+)D3-2, P(Dpt-GFP, w+)D3-4; courtesy of Bruno
Lemaitre, EPFL, Lausanne] (Vodovar et al.,
2005). Flies were reared on a standard fly food medium consisting
of cornmeal/sugar/yeast/agar at 25°C, 40% relative humidity, and a 12 h
light–dark cycle.
Microarrays
To examine the transcriptional response of y, w flies to treatment
with exogenous JH, we performed a microarray experiment on uninfected females
treated with JH or solvent (control). Since the physiological effects of JH
are better understood in females than in males, we only used females in this
experiment. Flies were grown on regular yeast diet, switched to no-yeast food
within 1h of eclosion, and yeast-starved for 5days posteclosion to lower their
endogenous JH titer and to synchronize their physiology (see
Tu and Tatar, 2003;
Gershman et al., 2007).
Subsequently, flies were anesthetized on ice and topically treated with
0.1µl of 187mmoll–1 JH III in acetone or with 0.1µl
100% acetone (control) using a 1µl Hamilton syringe with a repeating
dispenser; 12h after hormone administration, samples were snap-frozen in
liquid nitrogen and stored at –80°C. Total RNA from whole flies was
isolated from samples (two JH samples, two control samples, each with 30
females) by lysis, as previously described
(Gershman et al., 2007). cDNA
products were hybridized at the Brown University Genomics Core Facility to
Affymetrix GeneChip Drosophila_1 Genome Arrays (two replicate chips per
treatment). The dataset consisted of 14,009 probe sets, with 6142 probe sets
annotated. Expression data were analyzed for significant over- or
underrepresentation of gene ontology (GO) terms with the web application
FatiGO (Al-Shahrour et al.,
2004), using a two-fold change criterion. To test whether JH
treatment significantly suppresses expression of AMPs, we used Student's
t-tests implemented in JMP IN 5.1 (SAS Institute, Cary, NC, USA)
(Sall et al., 2004). The
microarray dataset has been deposited in Gene Expression Omnibus (GEO;
http://www.ncbi.nlm.nih.gov/geo/)
with accession number GSE9001. Results of the microarray experiment were
confirmed by analyzing two additional, independent microarray experiments: one
experiment on JH- and solvent-treated y, w females, following the
time course design of Gershman and colleagues
(Gershman et al., 2007); the
other experiment with S2* cells treated with solvent, JHa
(methoprene), 20E or both 20E and JHa (three replicates each; data not
shown).
Fly GFP reporter experiments
To test whether the JHa methoprene suppresses AMP expression in
vivo we used a whole-fly GFP reporter assay of the DD1 (Drs-GFP)
and the DIG (Dpt-GFP) strains, combining hormonal manipulation (JHa
application vs control) with manipulation of infection status
(unjabbed control; sterile, ethanol-jabbed control; and bacteria jabbed). Each
of the 2x3 [(JHa; control)x(unjabbed; ethanol jabbed; bacteria
jabbed)] treatment groups consisted of fifteen 3 day old females (total
N=90 females). Prior to manipulating infection status, flies were
exposed for 24 h in vials to vaporized JHa methoprene (10 µl at 7.9
mmoll–1) or 70% ethanol (control; 10 µl). The next day,
flies were lightly anesthetized with moist CO2 and jabbed at the
abdomen intersegment with a fine (0.2 mm diameter) Minuten pin needle (Fisher
Scientific, Pittsburgh, PA, USA), dipped in live Gram-negative bacteria
(E. coli, strain 1106; bacterial pellets made by centrifugation of a
liquid overnight culture in LB growth medium) or in 70% ethanol (sterile
jabbed control), or left unjabbed. Twenty-four hours after infection, flies
were anesthetized using CO2 and their GFP expression visualized
under fluorescent (FITC) light with a Zeiss Stemi SV11 dissecting scope;
images of individual flies were taken with an AxioCam MRm camera (Carl Zeiss,
Jena, Germany; exposure time, 5 s) and processed with AxioVision LE Rel. 4.3
software. For analysis, images were imported into ImageJ
(http://rsb.info.nih.gov/ij/).
After thresholding images, surface areas of flies were estimated using the
polygon selection tool. Image exposure time and threshold parameters were kept
constant for all images. Data were analyzed with two-way analysis of variance
(ANOVA) implemented in JMP IN 5.1 (Sall et
al., 2004), using infection status and hormone treatment as fixed
factors.
S2* cell culture and cell induction
For cell culture experiments we used an embryonic hemocyte- or
macrophage-like Drosophila cell line known as Schneider
S2* cells (Samaklovis et al., 1992). S2* cells were
maintained at 25°C in Schneider S2 Drosophila medium (Gibco,
Gaithersburg, MD, USA; Invitrogen, Carlsbad, CA, USA) or Schneider's insect
media (Sigma), supplemented with 10% fetal bovine serum (FBS, HyClone, Logan,
UT, USA), 1% GlutaMax-1 (Invitrogen), and 0.2% Penicillin–Streptomycin
(Pen-Strep, Invitrogen). The Diptericin-luciferase cell line
(Dpt-luc) was a stable S2* transfectant containing the
reporter plasmid pJM648 (Tauszig et al.,
2000; Kaneko et al.,
2004); at each passage, cells were selected with Geneticin (G418
sulfate, Gibco, Invitrogen, 800µg ml–1). Cell counts were
made with a Fuchs-Rosenthal ultraplane counting chamber (1/16mm2;
2/10mm deep; Hausser Scientific, Horsham, PA, USA). For experiments, cells
were immune stimulated with 1 µg ml–1 E. coli
peptidoglycan (PGN; InvivoGen, San Diego, CA, USA; 1 mg ml–1
stock) for 5–6 h or left untreated (control). In one experiment, we used
crude lipopolysaccharide (LPS) from E. coli (0111:B4; Sigma); the
active Drosophila immune-stimulating component of crude LPS has been
shown to be PGN (Kaneko et al.,
2004). For northern or western blotting without RNA interference
(RNAi), cells were plated at 106 cells ml–1 in
six-well tissue culture plates (3 ml of cells per well); after 24 h, cells
were split to 106 cells ml–1 in six-well plates (3
ml cells per well) and incubated with hormones (no hormone; 20E; JH or JHa; JH
or JHa plus 20E). For each hormone, we added 3 µl of stock solution per
well (see above; 1000x dilution). After 24 h of hormone incubation,
cells were stimulated with PGN for 5–6 h or left unstimulated (control).
For experiments with Dpt-luc cells, procedures were identical, except
that cells were plated at 103 cells µl–1 in
96-well plates (100µl cells per well); hormones were administered as 1
µl of stock per well (1000x dilution). Each cell culture experiment
was replicated at least four times.
RNAi
To study the genetics of the hormonal response we performed RNAi-mediated
silencing of Drosophila ecdysone receptor (EcR),
ultraspiracle (Usp) and methoprene-tolerant
(Met). Double-stranded RNA (dsRNA) was synthesized from a
PCR-amplified template, with T7 promoter sequences flanking a 
500 bp
fragment of the gene of interest, using the Ribomax kit (Promega, Madison, WI,
USA), as previously described (Silverman
et al., 2000). dsRNA was purified by phenol/chloroform extraction
and ethanol precipitation. As RNAi controls, we used dsRNA for E. coli
LacZ (encoding β-galactosidase) or E. coli MalE (encoding
maltose binding protein). Primers used to generate dsRNA are described in
supplementary material Table S1. dsRNA for MalE was generated using
the HiScribe RNAi transcription kit (New England BioLabs, Ipswich, MA, USA);
an 808bp (BglII–EcoRI) fragment of MalE was
inserted into the Litmus 28i vector and amplified using the T7 minimal primer.
For RNAi-mediated silencing, cells were plated at 106 cells
ml–1 (see above) and then soaked with 30 µg of dsRNA in 1
ml FBS-free medium for 30 min, followed by addition of 2 ml of complete
medium. Twenty-four hours later, cells were split to 106 cells
ml–1 in six-well plates (for northern and western blotting)
or plated at 103 cells µl–1 in 96-well plates
(for luciferase assays); subsequently, cells were treated with hormones and
immune stimulated, as described above.
Luciferase reporter assays
To examine how hormones affect Dpt promoter activity, we performed
luciferase assays with Dpt-luc reporter cells in 96-well plates,
using 100 µl cells per well (103 cells µl–1;
see above). Five to six hours after induction with PGN, samples on
experimental plates were transferred to black 96-well assay plates (BD Falcon,
Franklin Lakes, NJ, USA) and lysed for 2 min in Bright-Glo Assay Reagent
(Promega; 100 µl per well). Luciferase activity (in relative luciferase
units) of samples was assayed with a SpectraMax M5 microplate reader and
SoftMax Pro 4.8. software (Molecular Devices, Sunnyvale, CA, USA); samples
were automixed for 5 s and luciferase activity determined using the
luminescence read mode (top read, three points per well, integration time 1000
ms). For each experiment we used a minimum of three replicate wells per
treatment; each experiment was repeated at least four times. Assays combined
with RNAi were analyzed with two-way ANOVA implemented in JMP IN 5.1
(Sall et al., 2004), using
RNAi (RNAi vs control) and hormone (20E vs 20E plus JHa) as
fixed factors.
Northern and western blotting
For northern blotting, dsRNA, DNA or dsRNA plus DNA were transfected into
S2* cells using a standard calcium phosphate method. dsRNA and DNA
were prepared in 2x BBS [BES-buffered saline; 50
mmoll–1
N,N-bis(2-hydroxyethyl)2-aminoethane-sulfonic acid (Sigma), 0.28
moll–1 NaCl, 1.5 mmoll–1
Na2HPO4, at pH 6.95], followed by addition of
CaCl2. Transfection mixtures were vortexed thoroughly and, after 15
min of incubation at room temperature, added dropwise to the S2*
cells. After 24 h, transfected cells were split, treated with hormone and
immune stimulated as described above. As controls we used untransfected and
mock-transfected S2* cells (transfected with the transfection
mixture only, without dsRNA or DNA). Total RNA from cultured cells was
isolated with TRIzol reagent (Invitrogen) and expression of Dpt and
control Rp49 (encoding ribosomal protein RP49) was analyzed by RNA
blotting as previously described
(Silverman et al., 2000).
Relative quantification of Dpt expression was performed by comparing
the intensities of the experimental bands and the Rp49 control bands.
For the northern blot on y, w flies for Dpt mRNA, we
followed standard procedures, as previously described
(Silverman et al., 2000).
For western blot analysis of USP, cell lysates from S2* cells
transfected with Usp RNAi were prepared and 50 µg of protein per
lane was applied on a 10% SDS-PAGE gel. After electrophoresis, proteins were
transferred onto a PVDF membrane. Non-specific binding was blocked with TBS
(25 mmoll–1 Tris-HCl, 0.5 moll–1 NaCl, pH
7.5), supplemented with 5% non-fat dried milk for 1 h at room temperature.
Blots were incubated for 2 h at room temperature with a 1:100 dilution of the
mouse monoclonal antibody AB11 (courtesy of Carl Thummel, University of Utah
School of Medicine) directed against USP
(Christianson et al., 1992).
After 3x15 min washes in TBST (TBS containing 0.1% Triton X-100), blots
were incubated for 1 h in peroxidase-conjugated anti-mouse IgG (Amersham,
Little Chalfont, Bucks, UK) diluted 1:2500 in TBS, and washed three times for
15 min with TBST. Proteins were visualized with West Pico SuperSignal (Pierce,
Rockford, IL, USA).
MET protein was examined with Western blotting performed on S2*
cells transfected with Met dsRNA, Met plasmid expression
vector, or double transfected with Met dsRNA and Met
expression vector. Transfection with Met expression vector [pAC5.1(C)
MET-V5-6xHis; estimated molecular mass 82.2kDa; courtesy of Thomas G.
Wilson, Ohio State University] was used because endogenous MET levels in
S2* cells were low (data not shown). Transfection was performed
using a standard calcium phosphate method; 24 h after transfection, cells were
split, treated with hormones and immune stimulated as described above. After a
further 24 h, whole-cell extracts were prepared with lysis buffer and 50 µg
of protein extract per lane was applied on an 8% SDS-PAGE gel. After
electrophoresis, proteins were transferred onto a PVDF membrane and
non-specific binding was blocked with TBS containing 0.1% Tween 20 (TBST),
supplemented with 10% non-fat dried milk overnight at room temperature. The
next day, blots were incubated for 3 h at room temperature with rabbit
polyclonal MET antibody (courtesy of Thomas G. Wilson)
(Pursley et al., 2000), at a
dilution of 1:2500 in 10% milk in TBS, followed by 3x15 min washes in
TBST. Blots were incubated for 1 h at room temperature in
peroxidase-conjugated anti-rabbit IgG (BioRad, Hercules, CA, USA) diluted at
1:10,000 in 10% milk in TBS and washed three times for 15 min with TBST.
Proteins were visualized with West Pico SuperSignal.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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To confirm that JH/JHa suppresses AMP expression in vivo, we analyzed Drs-GFP reporter expression in DD1 females (Fig. 2) and Dpt-GFP reporter expression in DIG females (data not shown). For both reporters, we observed substantial variation among individuals in GFP expression intensity, both within and among treatments, as well as among replicate experiments. Therefore, to test whether JH/JHa treatment suppresses GFP induction upon infection, we estimated Drs-GFP expression using quantitative image analysis. Infection with Gram-negative E. coli strongly increased Drs-GFP expression (Fig. 2A,B; two-way ANOVA, F1,7=7.09, P=0.03), while treatment with JHa methoprene significantly reduced expression (Fig. 2C,D; F1,7=6.6, P=0.0375), in both uninfected (Fig. 2C,E) and infected flies (Fig. 2D,E; infectionxhormone interaction effect: F1,7=0.008, P=0.93). Qualitatively similar results were obtained in independent repeats of this experiment and in trials using flies infected with Gram-positive M. luteus (data not shown). To further confirm the JH-mediated suppression of AMP induction in vivo we performed northern blotting on y, w females and found that infection-induced Dpt expression was reduced 2-fold in females treated with JHa (methoprene) vapor relative to controls exposed to solvent only (data not shown). Thus, JH/JHa suppresses the expression of genes involved in innate immunity, including several AMPs (Figs 1 and 2; supplementary material Table S2).
|
;
Dimarcq et al., 1994;
Dimarcq et al., 1997;
Silverman et al., 2000). Since
JH often counteracts 20E-dependent responses
(Riddiford, 1994;
Dubrovsky, 2005) and might
function as an immuno-suppressor [see above (see also
Flatt et al., 2005)], we first
verified that pretreatment with 20E is required for efficient AMP
transcription upon immune challenge and then examined whether JHa could
repress this response. To monitor AMP gene induction, we performed northern
blotting on RNA extracted from S2* cells, using probes for the AMP
genes Dpt, Cecropin and Attacin
(Fig. 3). S2* cells
not exposed to 20E showed little or no AMP gene induction in response to PGN
immune stimulation (Fig. 3A,
left lanes). In contrast, S2* cells pretreated with 20E 24 h prior
to immune challenge robustly and strongly expressed AMPs upon PGN stimulation
(Fig. 3A, middle lanes).
Similarly, treatment of immune-stimulated Dpt-luc cells with 20E
caused a dramatic increase in Dpt promoter activity (80-fold
increase), whereas PGN treatment in the absence of 20E had a markedly smaller
effect on activation of the Dpt promoter (five-fold increase;
Fig. 3B). Thus, the effects on
Dpt mRNA transcript levels, as monitored by northern blotting
(Fig. 3A), are also reflected
at the level of Dpt promoter activity, as monitored in luciferase
assays.
|
We found that cells treated with both JHa (methoprene) and 20E did not gain the immune capacity of cells treated with 20E alone (Fig. 3A, right lanes; and Fig. 3B, 17-fold decrease compared with 20E alone), confirming our in vivo observation that JH functions as an immuno-suppressor. JHa in the absence of 20E, on the other hand, only weakly suppressed the immune capacity of PGN-stimulated cells (3.4-fold suppression by JHa compared with no hormone control; Fig. 3B). This suggests that JHa is an antagonist of 20E. Dose–response experiments with 20E alone and with JH (or its synthetic analogs methoprene and pyriproxyfen) in the presence of 20E confirmed that JH and JHa antagonize the 20E response (Fig. 4). Increasing concentrations of 20E upon immune stimulation strongly increased Dpt reporter activity (Fig. 4A), but JH and its synthetic analogs decreased this response in a dose-dependent manner (Fig. 4B,C,D).
|
|
). However, the
JHa methoprene did not affect these 20E-mediated developmental phenotypes.
Cells treated with both 20E and JHa stopped proliferating and morphologically
differentiated, just like cells treated with 20E alone
(Fig. 6; and data not shown).
Thus, JHa appears to be a rapid and specific inhibitor of the ability of 20E
to increase immune capacity.
|
;
Thomas et al., 1993;
Yao et al., 1993;
Hall and Thummel, 1998). We
therefore tested whether 20E potentiation of Dpt requires
EcR and Usp (Figs
7 and
8; supplementary material Table
S3). RNAi directed against EcR completely prevented 20E-mediated
potentiation of Dpt mRNA expression and reporter activity
(Fig. 7A and
Fig. 8A; supplementary material
Table S3). Similarly, RNAi directed against Usp abolished 20E
potentiation (Fig. 7B and
Fig. 8B; supplementary material
Table S3); western blot analysis confirmed the effectiveness of Usp
RNAi (Fig. 7C). Although
20E-mediated potentiation of Dpt reporter activity was markedly
decreased by RNAi targeting of EcR and Usp, the residual
level of reporter activity was still inhibited by JHa
(Fig. 8A,B), but this
suppression was not statistically significant (supplementary material TableS3;
interaction contrasts analysis). Thus, it is difficult to firmly conclude
whether or not USP is required for the JHa-mediated suppression of immune
inducibility. Similar results were obtained when using JH III (data not
shown). However, it is very clear that 20E regulates Dpt expression
and promoter activity by signaling through EcR/USP.
|
|
JH repression of AMPs is independent of MET
In contrast to 20E, the mechanisms underlying signal transduction
downstream of JH remain unknown (Wilson,
2004; Berger and Dubrovsky,
2005; Dubrovsky,
2005; Flatt et al.,
2005). Therefore, to understand how JH down-modulates immune
function, we asked whether repression of the 20E response by JH and JHa
depends on Met, a candidate receptor for JH
(Wilson and Fabian, 1986;
Shemshedini and Wilson, 1990;
Shemshedini et al., 1990;
Wilson and Ashok, 1998;
Pursley et al., 2000).
RNAi-mediated silencing of Met did not affect 20E potentiation of
Dpt promoter activity and mRNA expression
(Fig. 8C and
Fig. 9A; supplementary material
Table S3), suggesting that Met is not involved in 20E signaling. To
confirm the effectiveness of Met RNAi, we performed western blot
analysis with rabbit polyclonal antibody against MET. Since endogenous MET
levels were low (data not shown), we overexpressed Met with a plasmid
expression vector (pMET) and found that RNAi successfully silenced
Met (Fig. 9B).
Remarkably, when directing RNAi against Met, the JHa methoprene was
still able to fully suppress Dpt activity, suggesting that
Met does not function in the JH modulation of immunity in
Drosophila (Fig. 8C
and Fig. 9A; supplementary
material Table S3); experiments using JH III yielded similar results (data not
shown).
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|
DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
;
Riddiford, 1994;
Kozlova and Thummel, 2000;
Dubrovsky, 2004; Berger and Dubrovsky,
2005; Flatt et al.,
2005; Flatt et al.,
2006; Tu et al.,
2006). Previous evidence indicates that both hormones can
individually modulate immunity (Meister
and Richards, 1996; Dimarcq et
al., 1997; Rolff and
Siva-Jothy, 2002; Beckstead et
al., 2005). Here, through experiments in Drosophila
S2* cells and whole flies, we have shown that 20E and JH exert
antagonistic effects on mRNA expression and promoter activity of AMP genes.
This is similar to 20E and JH action during midgut remodeling where the JHa
methoprene suppresses the expression of genes involved in 20E signaling and
20E-mediated programmed cell death
(Parthasarathy and Palli,
2007; Parthasarathy et al.,
2008a).
We found that 20E potentiates expression of several AMPs, as previously
observed (Meister and Richards,
1996; Dimarcq et al.,
1994; Dimarcq et al.,
1997; Silverman et al.,
2000). We extend previous findings by showing that 20E is a
specific hormonal potentiator of AMP induction: upon immune stimulation, 20E
enables, in a dose- and time-dependent manner, Dpt induction
following immune stimulation. Interestingly, immune potentiation by 20E
required at least 18 h of hormone exposure. 20E is known to transcriptionally
regulate target genes through enhancers that contain EcR response
elements (EcREs). Since this level of transcriptional activation occurs
rapidly and since many EcR targets are transcription factors
(Thummel, 2002;
Yin and Thummel, 2005), it
seems likely that 20E mediates the increase in immune responsiveness through
secondary or tertiary targets of 20E/EcR signaling.
Although JH has previously been implicated in modulating immunity
(Manetti et al., 1997; Hiruma
and Riddiford, 1998; Rolff and Siva-Jothy,
2002; Rantala et al.,
2003), JH effects on AMP expression have not been investigated. We
found that potentiation of Dpt activity by 20E was strongly
suppressed by compounds with JH activity (juvenoids). Inhibitory effects were
obtained by using not only the JHa methoprene and pyriproxyfen but,
importantly, also the natural hormone JH III (methyl epoxy farnesoate). In
addition, another product of the larval ring gland and adult corpus allatum
(tissues producing JH), the JH precursor methyl farnesoate
(Jones et al., 2006;
Jones and Jones, 2007), also
strongly suppresses 20E induction of Dpt activity (A.G. and T.F.,
unpublished data). While we consistently observed robust JH- or JHa-mediated
suppression of AMP induction in S2* cells, quantitative levels of
suppression were quite variable among experiments, presumably due to slight
variations in the physiological state of the cells or in luciferase assay
conditions. Our dose–response experiments with JH III, MF and JHa
suggest that these inhibitory effects are specific hormonal effects; all
compounds caused strong suppression of the 20E response at concentrations
below 10–10 moll–1. The specificity of these
effects is further suggested by our observation that JHa did not block
S2* cells from attaining 20E-induced growth arrest and
morphological differentiation (see also
Wyss, 1976;
Cherbas et al., 1989;
Echalier, 1997). In contrast
to induction by 20E, immune suppression by juvenoids was rapid and did not
require preincubation, suggesting that the repression is the result of a
primary hormone response. Moreover, inhibitory effects of juvenoids seen in
S2* cells were faithfully mirrored in the fly: in microarrays, GFP
reporter assays and northern blot experiments performed on adult flies, JH/JHa
acted as powerful immuno-suppressors of AMP expression in vivo. Both
JH and 20E act on many target tissues in the fly, including brain, gonads and
fat body, the equivalent of the mammalian liver and a major endocrine target
tissue (Nijhout, 1994;
Riddiford, 1994;
Flatt et al., 2005). Since
upon systemic infection AMPs are mainly produced in the fat body, it is likely
that JH/20E modulation of AMP induction normally takes place in this tissue.
Together, our findings suggest that 20E and JH interact antagonistically to
regulate immunity in Drosophila.
To understand the mode of JH/20E signaling action in immunity we used RNA
interference in S2* cells. We focused on three key genes involved
in 20E and JH signaling: ecdysone receptor (EcR),
ultraspiracle (Usp) and methoprene-tolerant
(Met). 20E signaling requires 20E binding to a heterodimer between
EcR and USP (Koelle et al.,
1991; Thomas et al.,
1993; Yao et al.,
1993; Hall and Thummel,
1998). However, 20E signals can also be mediated by EcR homodimers
[in vitro (see Lezzi et al.,
1999; Lezzi et al.,
2002; Grebe et al.,
2003)], heterodimers between hormone-receptor 38 (DHR38) and USP
(Baker et al., 2003), or
non-genomic actions (Wehling,
1997; Elmogy et al.,
2004; Srivastava et al.,
2005). Confirming the classical model of 20E signal transduction,
we found that 20E potentiation of Dpt induction requires both EcR and
USP function. When EcR and Usp were silenced with RNAi, JHa
still appeared to be able to suppress the residual Dpt induction (see
Fig. 8A,B); however, this
effect was not statistically significant (supplementary material Table S3).
Thus, it is possible that the EcR/USP heterodimer is not involved in the
JH-mediated immune suppression. On the other hand, we cannot exclude the
possibility that EcR/USP integrate both 20E and JH signaling
(Fang et al., 2005); under
such a model, the EcR/USP heterodimer would be required for both Dpt
activation by 20E and its suppression by JH/JHa.
Indeed, the USP part of EcR/USP might be an important mediator of JH
signaling since JH can act as a USP ligand and suppress or potentiate
20E-dependent EcR signaling responses
(Jones and Sharp, 1997; Jones
et al., 2002; Xu et al.,
2002; Henrich et al.,
2003; Maki et al.,
2004; Wozniak et al.,
2004; Fang et al.,
2005; Jones et al.,
2006). For example, JH and 20E can synergistically activate a
JH esterase reporter gene (Fang
et al., 2005). While these two hormones can activate transcription
independently, activation is greater than additive if both hormones are
present. In the absence of 20E, activation by JH is through the USP homodimer,
whereas activation by 20E in the absence of JH is mediated by EcR/USP
(Fang et al., 2005). Notably,
when both hormones are present, EcR/USP mediates integration of JH and 20E
signaling, with JH signaling being mediated by the USP part of the
20E-liganded heterodimer (Fang et al.,
2005). Thus, our results suggest that EcR/USP is required for 20E
signaling in Drosophila immunity, but we cannot rule out the
interesting possibility that JH exerts its inhibitory effects by signaling
through the USP part of EcR/USP. Future work will be needed to test the
requirement of EcR/USP for fat body-specific induction of AMPs in
vivo, using dominant negative or RNAi constructs.
Another candidate for the elusive JH receptor is encoded by Met, a
basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) transcription factor
(Wilson and Fabian, 1986;
Shemshedini and Wilson, 1990;
Shemshedini et al., 1990;
Wilson and Ashok, 1998;
Pursley et al., 2000;
Wilson et al., 2003). While
MET is not a nuclear hormone receptor like EcR or USP, MET binds JH with
higher affinity than USP and functions as a JH-dependent transcription factor
(Miura et al., 2005).
Moreover, Met genetically interacts with the 20E-regulated
transcription factor Broad-Complex (BR-C)
(Wilson et al., 2006), an
important mediator of 20E signaling downstream of EcR, and MET
protein interacts with both EcR and USP in GST pull-down assays
(Li et al., 2007). However,
while Met controls entry into metamorphosis in the beetle
Tribolium castaneum, as one would expect if Met encodes a JH
receptor (Konopova and Jindra,
2007), Drosophila Met null mutants show normal
development (Wilson and Fabian,
1986; Wilson and Ashok,
1998; Flatt and Kawecki,
2004).
To further examine the role of MET in JH signal transduction we directed
RNAi against Met in Dpt-luc S2* cells and found
that silencing Met does not impair 20E induction of Dpt.
Remarkably, we also found that RNAi against Met does not abolish the
immunosuppressive action of JH/JHa, despite the involvement of MET in certain
JH responses (Miura et al.,
2005; Konopova and Jindra,
2007). Similarly, despite its involvement in JH signaling, MET
does not seem to be required for JH suppression of 20E action in
Tribolium (Parthasarathy and
Palli, 2008; Parthasarathy et
al., 2008b). We conclude that MET does not function in the JH
regulation of immunity in Drosophila. Thus, it appears that JH
suppression of 20E action may be mediated by USP (as part of the ECR/USP
heterodimer or as a monomer/homodimer) or by another, as yet unidentified
mechanism. While the identity of the JH receptor remains unresolved, the
endocrine regulation of Drosophila immunity might provide a powerful
model system for studying regulatory cross-talk between JH/20E and for
dissecting the elusive JH signaling pathway. Moreover, given the common
endocrine-based trade-off between reproduction and immunity in mammals, birds
and invertebrates (Muehlenbein and
Bribiescas, 2005; Harshman and
Zera, 2007; Lawniczak et al.,
2007; Miyata et al.,
2008), it will be of major interest to study how the reproductive
insect hormones JH and 20E interact to co-regulate reproduction and immune
function (Flatt et al.,
2005).
LIST OF ABBREVIATIONS
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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