Genome-wide regulation of innate immunity by juvenile hormone and 20-hydroxyecdysone in the Bombyx fat body
- Ling Tian†1,
- Enen Guo†2,
- Yupu Diao1,
- Shun Zhou1,
- Qin Peng1,
- Yang Cao2,
- Erjun Ling1 and
- Sheng Li1Email author
© Tian et al; licensee BioMed Central Ltd. 2010
Received: 9 April 2010
Accepted: 9 October 2010
Published: 9 October 2010
Insect innate immunity can be affected by juvenile hormone (JH) and 20-hydroxyecdysone (20E), but how innate immunity is developmentally regulated by these two hormones in insects has not yet been elucidated. In the silkworm, Bombyx mori, JH and 20E levels are high during the final larval molt (4 M) but absent during the feeding stage of 5th instar (5 F), while JH level is low and 20E level is high during the prepupal stage (PP). Fat body produces humoral response molecules and hence is considered as the major organ involved in innate immunity.
A genome-wide microarray analysis of Bombyx fat body isolated from 4 M, 5 F and PP uncovered a large number of differentially-expressed genes. Most notably, 6 antimicrobial peptide (AMP) genes were up-regulated at 4 M versus PP suggesting that Bombyx innate immunity is developmentally regulated by the two hormones. First, JH treatment dramatically increased AMP mRNA levels and activities. Furthermore, 20E treatment exhibited inhibitory effects on AMP mRNA levels and activities, and RNA interference of the 20E receptor EcR-USP had the opposite effects to 20E treatment.
Taken together, we demonstrate that JH acts as an immune-activator while 20E inhibits innate immunity in the fat body during Bombyx postembryonic development.
Molting and metamorphosis in insects are coordinately regulated by the molting hormone 20-hydroxyecdysone (20E) and juvenile hormone (JH). Overall, 20E orchestrates the molting process, while JH determines the nature of the molt. In the presence of JH, 20E directs larval molting; while in the absence of JH, 20E directs larval-pupal-adult metamorphosis. In other words, JH antagonizes the 20E-induced physiological and developmental events to assure larval molting and to prevent larval-pupal-adult metamorphosis [1, 2]. At the molecular level, for example, JH prevents 20E-induced programmed cell death (PCD) by suppressing the 20E-triggered transcriptional cascade and modulating mRNA levels of several caspase genes [3, 4].
Insect fat body is the major organ involved in innate immunity, producing antimicrobial peptides (AMP) and other humoral response molecules . AMP plays a central role in fighting against invading pathogens, which in turn up-regulate AMP gene expression via two distinct signaling pathways: the Toll pathway that is largely activated by fungi and Gram-positive bacteria and the Imd pathway that is mainly activated by Gram-negative bacteria . In mammals, sex hormones and their nuclear receptors systematically regulate AMP production and thus innate immunity . Nuclear receptors LXR, RXR, and PPAR also regulate AMP production . However, little is known about hormonal regulation of innate immunity in insects apart from the fruitfly, Drosophila melanogaster. It has been suggested that 20E renders Drosophila mbn-2 cells and flies competent to induce AMP genes, such as diptericin and drosomycin [9–11]. Interestingly, diptericin expression could be induced by infection only after 3rd instar larvae are mature enough to produce sufficient 20E . Recently, it was shown that RNA interference (RNAi) of genes encoding the 20E receptor complex EcR-USP in Drosophila S2 cells prevented 20E-induced immune competence. In addition, JH III and JH agonist (JHA) strongly interfere with this 20E-dependent immune competence. This led to the suggestion that 20E acted as an immune-activator while JH an immune-suppressor . In contrast, the genome-wide microarray study by Beckstead et al (2005) revealed that several AMP genes, including cecropin C, attacin A, drosocin, drosomycin, and defensin, were down-regulated by 20E in EcR-dependent manners. These authors assumed that 20E blocked innate immunity at the onset of metamorphosis . The conflicting reports in Drosophila imply that 20E and JH regulate AMP mRNA expression in a complex manner and it is necessary to clarify this conflict in other insect species.
The silkworm, Bombyx mori, is one of the best models to study insect physiology and biochemistry. In the Bombyx genome, there are 35 AMPs belonging to 6 different types, namely Cecropins, Moricins, Gloverins, Attacins, Enbocins, and Lebocins. Among them, Cecropins, Moricins, Enbocins, and Lebocins have antibacterial activities against both Gram-positive and Gram-negative bacteria, while Gloverins and Attacins only have antibacterial activities against Gram-negative bacteria. Thus, the antibacterial spectrum of Bombyx shows much higher antibacterial activities against Gram-negative bacteria than those against Gram-positive bacteria .
Although some progress related to innate immunity has been made recently in Bombyx, very little is known about how innate immunity is developmentally regulated by insect hormones in this and other insect species. We have used a genome-wide microarray to analyze expression profiles of the fat body from the silkworm, Bombyx mori, during three developmental stages of animals displaying different 20E and JH levels. It revealed a large number of differentially-expressed genes, including 6 AMP genes. Hormone treatment and RNAi experiments demonstrate that JH acts as an immune-activator while 20E inhibits innate immunity in the fat body during Bombyx postembryonic development.
Differentially-expressed genes revealed by microarray analyses
Differentially-expressed genes related to innate immunity
Developmental changes of AMP activities
Increase of AMP mRNA levels and activities by JH
Decrease of AMP mRNA levels and activities by 20E
Increase of AMP mRNA levels and activities by EcR-USP RNAi
A number of studies in Drosophila imply that 20E induces AMP mRNA expression and acts as an immune-activator [9, 10, 22], while JH acts as an immune-suppressor by antagonizing 20E signaling . In contrast, genome-wide microarray studies in Drosophila strongly suggest that 20E-EcR-USP suppress AMP mRNA expression at the onset of metamorphosis . The conflicting reports promoted us to study how 20E and JH coordinately regulate AMP mRNA expression in the Bombyx fat body.
The most important discovery in this paper is that JH is an immune-activator rather than an immune-suppressor in the Bombyx fat body. This conclusion is supported by a series of experiments. First, in our microarray and qPCR validation analyses, 6 AMP genes were up-regulated at 4 M (high JH level) versus PP (low JH level) in the Bombyx fat body (Fig. 2B). Again, developmental changes of AMP activities (Fig. 3) were in consistent with those of AMP mRNA levels (Fig. 2B). Moreover, JHA treatment increased mRNA levels of all the 6 AMP genes and antibacterial activities to both bacteria (Fig. 4).
In the larvae of many insect orders, particularly in Coleoptera, Orthoptera and Lepidoptera, the larval-pupal metamorphosis results from a low titer of JH and a high titer of 20E. In these insects, application of JH or JHA can prevent normal metamorphic events, resulting in a supernumerary larval molt. This is also the case in Bombyx [23, 24]. For this reason, JH is referred to as the status quo hormone [1, 25]. The major physiological function of JH during the larval molts is to antagonize 20E action and to prevent the 20E-induced physiological and developmental events [1, 2]. For example, JH plays an important role by preventing 20E-induced PCD. At the molecular level, JH suppresses the 20E-triggered transcriptional cascade and down-regulates caspase genes [3, 4]. However, because the JH receptor has never been identified, the JH signal transduction pathway remains unclear .
To our surprise, 20E did not down-regulate all the 6 AMP genes (Fig. 5), all of which were up-regulated by JH (Fig. 4) in Bombyx, although the fact that 20E up-regulates some AMP genes but down-regulates others is similar to Drosophila [12, 13]. The biggest question that remains here is whether JH antagonizes the inhibitory effects of 20E to act as an immune-activator during larval molts. As expected, in the Bombyx fat body, JH was able to effectively prevent the 20E-triggered transcriptional cascade at EW (Supplementary Fig. 1). We assume the status quo action of JH is normally conserved in regulating innate immunity in the Bombyx fat body during the larval molts. To identify the JH receptor and to illustrate the JH signal transduction pathway will be necessary to understand the molecular mechanism how JH and 20E coordinately regulate innate immunity in Bombyx and other insect species.
Combining the microarray, developmental changes, hormone treatments, and RNAi results together, we conclude that JH is an immune-activator rather than an immune-suppressor, while 20E inhibits innate immunity via its receptor complex EcR-USP. The two hormones dynamically regulate innate immunity in the fat body during Bombyx postembryonic development (Fig. 8).
Bombyx larvae (Nistari) were provided by The Sericultural Research Institute, Chinese Academy of Agricultural Sciences. They were reared with fresh mulberry leaves in the laboratory at 25°C under 14 hour light/10 hour dark cycles .
The gene expression profiles in the Bombyx fat body were analyzed using silkworm genome 70-mer oligonucleotide microarray which covering over 23,000 Bombyx genes. The microarray was originally designed and constructed by Xia et al. (2007) and microarray analysis were performed and analyzed in the CapitalBio Corporation (Beijing, China). Fat body tissues were collected from larvae of three developmental stages: 4 M, 5 F, and PP and quickly frozen in liquid nitrogen. For 5 F, the same amount of fat body tissue was collected from day 1 to 7. Three biological replicates were used in this experiment. Total RNA was extracted from samples using Trizol reagent (Invitrogen, Gaithersburg, MD, USA), according to manufacturer's instructions. cDNA labeling with a fluorescent dye (Cy5 and Cy3-dCTP) was produced by Eberwine's linear RNA amplification method and subsequent enzymatic reaction using the procedure previously described , but with little modification by using CapitalBio cRNA Amplification and Labeling Kit (CapitalBio) for producing higher yields of labeled cDNA. After hybridization, the arrays were scanned with a confocal LuxScanTM scanner and the images obtained were then analyzed using LuxScanTM 3.0 software (Both from CapitalBio). Followed array data extracting, faint signals were removed if the intensities were below 400 units after background subtracted for both channels (Cy3 and Cy5). A space- and intensity-dependent normalization based on a LOWESS program was employed . We identified significant differences in gene expression as those probes showing fold change > 2 and probewise P value < 0.05 . The volcano plot method was used to estimate fold change The P values were calculated for the microarray data using the one sample or two sample t-test. Note that this does not control for false discovery rate and may overlook significant expression differences less than 2-fold, but we verified expression differences in the genes of primary interest using qPCR. For hierarchical analysis, we used average linkage clustering of the gene expression data (Cluster 3.0). Java Treeview (Stanford University, Stanford, Calif) was used for tree visualization. Gene functional categories were analyzed by Molecule Annotation System and KEGG http://www.kegg.com. The microarray data has been submitted to the Gene Expression Omnibus (GEO) under the accession number GSE23424.
After a number of trials, day 2 of 5 F (48 hrs after 4 M) was chosen for 20E injection (Sigma Aldrich, USA) (3 μg/larva) and the controls were injected with the same volume of control solvent. At this stage, hemolymph 20E levels were low and the fat body was sensitive to 20E. Topical application of a JHA (methoprene, Dr. Ehrenstorfer GmbH, Germany, 15 μg/larva), was performed at 12 hrs after the initiation of EW. At this stage, 20E level just began to rise and the fat body was sensitive to JH. At 6 hours after 20E or JHA treatment, hemolymph was collected, and then larvae were sacrificed to dissect fat body tissues. Hemolymph samples were used for measurements of AMP activities and fat body samples were used for qPCR analysis. Ten animals were used for each group and 5 biological replicates were conducted.
Double-stranded RNA (dsRNA) of Bombyx EcR and USP  were generated using the T7 RiboMAX™ Express RNAi system (Promega, USA) according to the manufacturer's instruction. At the initiation of EW, each individual larva was injected with 5 μl of ddH2O, EcR dsRNA (5 μg), USP dsRNA (5 μg), or EcR (5 μg) and USP (5 μg) dsRNA. At this stage, the Bombyx larvae were sensitive to RNAi treatments. At 24 hrs after RNAi treatment, hemolymph was collected and the larvae were sacrificed for further measurements as described above. Thirty animals were used for each group and 3 biological replicates were conducted.
Quantitative real-time PCR
Total RNA was extracted from larval fat body tissues of different developmental stages and used for quantitative real-time PCR (qPCR) analysis as previously described . Primers used here and somewhere else in this paper are listed in Additional file 3.
Measurements of AMP activities
The AMP activities of the hormone-treated silkworm cell-free plasma were measured using the paper count plates method as previously described . Gram-negative and Gram-positive bacteria used in this study were Escherichia coli and Staphyloccocus aureus, respectively.
Western blot analysis
AB11 USP-specific monoclonal antibody was provided by Dr. K.F. Kafatos (Harvard University). The Tubulin monoclonal antibody was purchased from Invitrogen. Western blot analysis was performed using standard procedures.
This study was supported by 2006CB943902, 2007CB947100, 30870299, 30770271, 2007AA10Z155, 2006AA10A119, KSCX-YW-N-009, and Hundred Talent Project to SL. We are grateful for Drs. William G. Bendena, Wen Wang and Qisheng Song for improving this manuscript.
- Riddiford LM: Cellular and molecular actions of juvenile hormone I. General considerations and premetamorphic actions. Adv Insect Physiol. 1994, 24: 213-274. full_text.View ArticleGoogle Scholar
- Riddiford LM: Juvenile hormone action: a 2007 perspective. J Insect Physiol. 2008, 54: 895-901. 10.1016/j.jinsphys.2008.01.014.PubMedView ArticleGoogle Scholar
- Wu Y, Parthasarathy R, Bai H, Palli SR: Mechanisms of midgut remodeling: juvenile hormone analog methoprene blocks midgut metamorphosis by modulating ecdysone action. Mech Dev. 2006, 123: 530-547. 10.1016/j.mod.2006.05.005.PubMedView ArticleGoogle Scholar
- Liu Y, Sheng Z, Liu H, Wen D, He Q, Wang S, Shao W, Jiang RJ, An S, Sun Y, Bendena WG, Wang J, Gilbert LI, Wilson TG, Song Q, Li S: Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death in Drosophila. Development. 2009, 136: 2015-2025. 10.1242/dev.033712.PubMedView ArticleGoogle Scholar
- Ferrandon D, Imler JL, Hetru C, Hoffmann JA: The Drosophila systemic immune response: sensing and signaling during bacterial and fungal infections. Nature Rev Immunol. 2007, 7: 862-874. 10.1038/nri2194.View ArticleGoogle Scholar
- Lemaitre B, Hoffmann JA: The host defense of Drosophila melanogaster. Annual Rev Immunol. 2007, 25: 697-743. 10.1146/annurev.immunol.25.022106.141615.View ArticleGoogle Scholar
- Beagley KW, Gockel CM: Regulation of innate and adaptive immunity by the female sex hormones oestradiol and progesterone. FEMS Immunol Med Microbiol. 2003, 38: 13-22. 10.1016/S0928-8244(03)00202-5.PubMedView ArticleGoogle Scholar
- Glass CK, Ogawa S: Combinatorial roles of nuclear receptors in inflammation and immunity. Nature Rev Immunol. 2006, 6: 44-55. 10.1038/nri1748.View ArticleGoogle Scholar
- Meister M, Richards G: Ecdysone and insect immunity: the maturation of the inducibility of the diptericin gene in Drosophila larvae. Insect Biochem Mol Biol. 1996, 26: 155-160. 10.1016/0965-1748(95)00076-3.PubMedView ArticleGoogle Scholar
- Dimarcq JL, Imler JL, Lanot R, Ezekowitz RA, Hoffmann JA, Janeway CA, Lagueux M: Treatment of l(2)mbn Drosophila tumorous blood cells with the steroid hormone ecdysone amplifies the inducibility of antimicrobial peptide gene expression. Insect Biochem Mol Biol. 1997, 27: 877-886. 10.1016/S0965-1748(97)00072-6.PubMedView ArticleGoogle Scholar
- Silverman N, Zhou R, Stöven S, Pandey N, Hultmark D, Maniatis T: A Drosophila IkappaB kinase complex required for Relish cleavage and antibacterial immunity. Gene Dev. 2000, 14: 2461-2471. 10.1101/gad.817800.PubMed CentralPubMedView ArticleGoogle Scholar
- Flatt T, Heyland A, Rus F, Porpiglia E, Sherilock C, Yamamoto R, Garbuzov A, Palli SR, Tatar M, Silverman N: Hormonal regulation of the humoral innate response in Drosophila melanogaster. J exp Biol. 2008, 211: 2712-2724. 10.1242/jeb.014878.PubMed CentralPubMedView ArticleGoogle Scholar
- Beckstead RB, Lam G, Thummel CS: The genomic response to 20-hydroxyecdysone at the onset of Drosophila metamorphosis. Genome Biol. 2005, 6: R99-10.1186/gb-2005-6-12-r99.PubMed CentralPubMedView ArticleGoogle Scholar
- Tanaka H, Ishibashi J, Fujita K, Nakajima Y, Sagisaka A, Tomimoto K, Suzuki N, Yoshiyama M, Kaneko Y, Iwasaki T, Sunagawa T, Yamaji K, Asaoka A, Mita K, Yamakawa M: A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori. Insect Biochem Mol Biol. 2008, 38: 1087-1110. 10.1016/j.ibmb.2008.09.001.PubMedView ArticleGoogle Scholar
- Xia Q, Zhou Z, Lu C, Cheng D, Dai F, Li B, Zhao P, Zha X, Cheng T, Chai C: A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science. 2004, 306: 1937-1940. 10.1126/science.1102210.PubMedView ArticleGoogle Scholar
- International Silkworm Genome Consortium: The genome of a lepidopteran model insect, the silkworm Bombyx mori. Insect Biochem Mol Biol. 2008, 38: 1036-1045. 10.1016/j.ibmb.2008.11.004.View ArticleGoogle Scholar
- Xia Q, Cheng D, Duan J, Wang G, Cheng T, Zha X, Liu C, Zhao P, Dai F, Zhang Z, He N, Zhang L, Xiang Z: Microarray-based gene expression profiles in multiple tissues of the domesticated silkworm, Bombyx mori. Genome Biol. 2007, 8: R162-10.1186/gb-2007-8-8-r162.PubMed CentralPubMedView ArticleGoogle Scholar
- Kinjoh T, Kaneko Y, Itoyama K, Mita K, Hiruma K, Shinoda T: Control of juvenile hormone biosynthesis in Bombyx mori: cloning of the enzymes in the mevalonate pathway and assessment of their developmental expression in the corpora allata. Insect Biochem Mol Biol. 2007, 37: 808-818. 10.1016/j.ibmb.2007.03.008.PubMedView ArticleGoogle Scholar
- Muramatsu D, Kinjoh T, Shinoda T, Hiruma K: The role of 20-hydroxyecdysone and juvenile hormone in pupal commitment of the epidermis of the silkworm, Bombyx mori. Mech Dev. 2008, 125: 411-420. 10.1016/j.mod.2008.02.001.PubMedView ArticleGoogle Scholar
- Tian L, Guo E, Diao Y, Wang S, Liu S, Cao Y, Jiang R-J, Ling E, Li S: Developmental regulation of glycolysis by 20-hydroxyecdysone and juvenile hormone in fat body tissues of the silkworm, Bombyx mori. J Mol Cell Bio. 2010, 2: 255-263. 10.1093/jmcb/mjq020.View ArticleGoogle Scholar
- Riddiford LM, Cherbas P, Truman JW: Ecdysone receptors and their biological actions. Vitam Horm. 2000, 60: 1-73. full_text.PubMedView ArticleGoogle Scholar
- Roxström-Lindquist K, Assefaw-Redda Y, Rosinska K, Faye I: 20-hydroxyecdysone indirectly regulates Hemolin gene expression in Hyalophora cecropia. Insect Mol Biol. 2005, 14: 645-652. 10.1111/j.1365-2583.2005.00593.x.PubMedView ArticleGoogle Scholar
- Kamimura M, Kiuchi M: Applying fenoxycarb at the penultimate instar triggers an additional ecdysteroid surge and induces perfect extra larval molting in the silkworm. Gen Comp Endocrionol. 2002, 128: 231-237. 10.1016/S0016-6480(02)00507-5.View ArticleGoogle Scholar
- Tan A, Tanaka H, Tamura T, Shiotsuki T: Precocious metamorphosis in transgenic silkworms overexpressing juvenile hormone esterase. PNAS. 2005, 102: 11751-11756. 10.1073/pnas.0500954102.PubMed CentralPubMedView ArticleGoogle Scholar
- Riddiford LM, Hiruma K, Zhou X, Nelson CA: Insights into the molecular basis of the hormonal control of molting and metamorphosis from Manduca sexta and Drosophila melanogaster. Insect Biochem Mol Biol. 2003, 33: 1327-1338. 10.1016/j.ibmb.2003.06.001.PubMedView ArticleGoogle Scholar
- Liu Y, Zhou S, Ma L, Tian L, Wang S, Sheng Z, Jiang R-J, Bendena WG, Li S: Transcriptional regulation of the insulin signaling pathway genes by starvation and 20-hydroxyecdysone in the Bombyx fat body. J Insect Physiol. 2010, 56: 1436-1444. 10.1016/j.jinsphys.2010.02.011.PubMedView ArticleGoogle Scholar
- Guo Y, Guo H, Zhang L, Xie H, Zhao X, Wang F, Li Z, Wang Y, Ma S, Tao J, Wang W, Zhou Y, Yang W, Cheng J: Genomic analysis of anti-hepatitis B virus (HBV) activity by small interfering RNA and lamivudine in stable HBV-producing cells. J Virol. 2005, 79: 14392-14403. 10.1128/JVI.79.22.14392-14403.2005.PubMed CentralPubMedView ArticleGoogle Scholar
- Yang YH, Dudoit S, Luu P, Lin DM, Peng V, Ngai J, Speed TP: Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 2002, 30: e15-10.1093/nar/30.4.e15.PubMed CentralPubMedView ArticleGoogle Scholar
- Patterson TA, Lobenhofer EK, Fulmer-Smentek SB, Collins PJ, Chu TM, Bao W, Fang H, Kawasaki ES, Hager J, Tikhonova IR, Walker SJ, Zhang L, Hurban P, de Longueville F, Fuscoe JC, Tong W, Shi L, Wolfinger RD: Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project. Nat Biotechnol. 2006, 24: 1140-1150. 10.1038/nbt1242.PubMedView ArticleGoogle Scholar
- Cherbas P, Iatrou K: Bombyx EcR (BmEcR) and Bombyx USP (BmCF1) combine to form a functional ecdysone receptor. Insect Biochem Mol Biol. 1996, 26: 217-221. 10.1016/0965-1748(95)00097-6.PubMedView ArticleGoogle Scholar
- Yang W, Wen S, Huang Y, Ye M, Deng X, Han D, Xia Q, Cao Y: Functional divergence of six isoforms of antifungal peptide Drosomycin in Drosophila melanogaster. Gene. 2006, 379: 26-32. 10.1016/j.gene.2006.03.017.PubMedView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.