Ecdysone Titer Determined by 3DE-3Β-Reductase Enhances The

Ecdysone Titer Determined by 3DE-3β -Reductase Enhances the Immune Response in the Silkworm

This information is current as Wei Sun, Yi-Hong Shen, Liang-Xiao Zhou and Ze Zhang of October 2, 2021. J Immunol 2016; 196:1646-1654; Prepublished online 15 January 2016; doi: 10.4049/jimmunol.1500158 http://www.jimmunol.org/content/196/4/1646 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Ecdysone Titer Determined by 3DE-3b-Reductase Enhances the Immune Response in the Silkworm

Wei Sun,* Yi-Hong Shen,† Liang-Xiao Zhou,† and Ze Zhang*

Although recent studies have demonstrated that 20-hydroxyecdysone (20E), one of the two most important hormones for development, could promote the insect innate immune response, how insects regulate 20E titer to affect the immunity after suffering pathogen attack remains unknown. In this study, to our knowledge, we first found that 20E titer was significantly elevated after bacterial infection in the domesticated silkworm, Bombyx mori. Furthermore, the elevated 20E enhanced the silkworm innate immune system against invading bacteria via ecdysone receptor. During immune response, the expression of the silkworm 3-dehydroecdysone-3b-reductase (3DE-3b-reductase) that converts 3DE released from prothoracic glands into ecdysone was induced. RNA interference experiments suggested that 3DE-3b-reductase is essential to upregulate the 20E titer after bacterial infection. The rescue experiments showed that injection with the recombinant 3DE-3b-reductase protein can significantly elevate the 20E concentration and modulate the expressions of the silkworm immune-related genes. Taken together, 20E titer determined by 3DE-3b-reductase enhances the silkworm Downloaded from defense against the bacterial infection. Thus, our findings reveal an important role of the 20E synthesis pathway from 3DE in enhancing the silkworm immune response and have profound implications for the understanding of interaction mechanisms between insect hormone and immunity. The Journal of Immunology, 2016, 196: 1646–1654.

nsects use a highly effective immune system to defend Indeed, the steroid hormone 20-hydroxyecdysone (20E), one of themselves against foreign microorganisms, and this powerful the two most important regulators of development, metamorphosis, http://www.jimmunol.org/ I system contributes to the successful evolution of insects (1). and reproduction in insects, can also stimulate the insect innate Unlike vertebrates, which have acquired immunity, insects only immune response (7, 8). For example, 20E could promote the contain an innate immune system, including humoral and cellular expressions of immune-related genes, especially AMP genes, in reactions. Pathogen infection rapidly and strongly initiates the infected culture cells and animals (9–14). Furthermore, 20E insect innate immune response. However, more and more studies controls the humoral innate immune response through two dif- showed that several immune-related genes can express without the ferent ways in Drosophila. First, 20E upregulates the transcription stimulation of microorganisms, suggesting that other factors be- of the peptidoglycan recognition protein (PGRP)-LC, and then sides pathogen infection or injury could also regulate the insect induces all AMP genes through the immune deﬁciency (IMD) by guest on October 2, 2021 immune response (2). Indeed, the Drosophila transcription factor pathway; second, 20E regulates the expression of the AMPs, such Forkhead box O (dFOXO) in the insulin/insulin-like growth factor as Diptericin, Metchnikowin, and Drosomycin, by the ecdysteroid- signaling pathway could regulate the expressions of the antimi- related transcription factors independent of PGRP-LC (15). crobial peptides (AMPs) under normal physiological conditions Moreover, 20E also promotes insect cellular immunity. The ex- (3). The target of rapamycin cascade, another nutrient-dependent pressions of the prophenoloxidases could be induced in the pathway in Drosophila, also has the potential impact on the innate Anopheles gambiae cell line 4a-3B (16). Injection with 20E in- immunity and the transcriptions of AMPs (4–6). creases the phagocytic activity of Drosophila plasmatocytes (17), and 20E signaling also enhances hemocyte motility, encapsula- tion, and nodulation (18–21). *Laboratory of Evolutionary and Functional Genomics, School of Life Sciences, Chongqing University, Chongqing 400044, China; and †State Key Laboratory of However, how insects regulate 20E titer to affect immunity after Silkworm Genome Biology, Southwest University, Chongqing 400715, China pathogen attack remains unknown. In insects, the biosynthesis of ORCID: 0000-0002-4787-1626 (L.-X.Z.). the 20E is mainly from the cholesterol pathway in the prothoracic Received for publication January 22, 2015. Accepted for publication December 17, glands (8). Nevertheless, ecdysone can also be directly synthe- 2015. sized from 3-dehydroecdysone (3DE) in other tissues (22–24). In This work was supported by National Natural Science Foundation of China Grants immature stages of most lepidopteran species, 3DE is the major 31402014 (to W.S.) and 31272363 (to Z.Z.) and by Fundamental Research Funds for the Central Universities Grant 0903005203271 (to W.S.). product of the prothoracic glands. After release from the glands, 3DE is rapidly reduced to ecdysone by 3DE-3b-reductase in he- Z.Z. and W.S. conceived and designed the experiments; W.S. and L.-X.Z. performed the experiments; W.S. analyzed the data; Y.-H.S. contributed reagents, materials, and molymph, and then the ecdysone is converted into 20E by 20- analysis tools; and W.S. and Z.Z. wrote the paper. hydroxylase in the target tissues (22–26). 3DE-3b-reductase is Address correspondence and reprint requests to Dr. Ze Zhang, Laboratory of Evolutionary considered to be an important enzyme in the biosynthesis of the and Functional Genomics, School of Life Sciences, Chongqing University, 174 Shazheng ecdysone (27). A previous study demonstrated that Trichoplusia ni Road, Shapingba, Chongqing 400044, China. E-mail address: [email protected] 3DE-3b-reductase gene was strongly induced by the pathogen The online version of this article contains supplemental material. infection (28). This means that insects may actively increase Abbreviations used in this article: AMP, antimicrobial peptide; 3DE, 3-dehydroecdysone; b 3DE-3b-reductase, 3-dehydroecdysone-3b-reductase; 20E, 20-hydroxyecdysone; EcR, hormone level through 3DE-3 -reductase after stimulation of ecdysone receptor; EGFP, enhanced GFP; EIA, enzyme immunoassay; IMD, immune the microorganisms. In this study, we functionally characterize deﬁciency; LB, lysogeny broth; PGRP, peptidoglycan recognition protein; RNAi, RNA the 3DE-3b-reductase gene in the silkworm and describe how the interference. silkworm 3DE-3b-reductase regulates ecdysone titer to enhance Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 the immune response. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500158 The Journal of Immunology 1647

Materials and Methods according to the manufacturer’s instruction. The polyclonal Abs against b Insects and cell culture 3DE-3 -reductase were produced by immunizing mice with the purified proteins as described (33). The strain DaZao of the domesticated silkworm was reared on fresh mulberry leaves at 25˚C under a 12/12-h light/dark photoperiod. The day 3 Immunohistochemistry larvae of the fifth instar silkworm were used for all experiments. The fifth Sections of the fat body were dissected from the day 3 larvae of the fifth instar is the last larval stage for the DaZao strain, and the duration of this instar silkworm. The sections were fixed in 4% paraformaldehyde at room ∼ instar is 7 d. Moreover, the fifth instar larvae begin wandering behavior temperature for 3–4 h. They were then embedded in paraffin and were cut late in day 7 and purge their gut contents when the ecdysone titer increases. to 5- to 6-mm-thick sections. For hemocytes, 200 ml hemolymph was About forty-eight hours later, ecdysone reaches the peak. collected and mixed with 2 vol PBS. Two hundred–microliter cell sus- The silkworm ovarian cell line (BmN) was maintained in TC-100 insect pensions were moved on the slides for 20 min and then fixed with 4% cell culture medium (Invitrogen) supplemented with 10% FCS (PAA paraformaldehyde for 20 min at room temperature. Immunohistochemical Laboratories, Pasching, Austria). staining was performed following the previous study (33). Bacteria Eukaryotic expression of the silkworm 3DE-3b-reductase gene a Escherichia coli strain DH5 (Gram-negative bacteria) and Bacillus The cDNA of the coding sequence excluding the signal peptide region of the bombyseptieus (Gram-positive bacteria) were used for infection experi- silkworm 3DE-3b-reductase gene (GenBank ID no. KU233524) was ments. Both bacteria were cultured on petri dishes containing 2.5% ly- cloned into pIZ/V5-His vector (Invitrogen). The constructed plasmid was sogeny broth (LB) medium and 1.0% agar (Sangon Biotech, Shanghai, confirmed by sequencing. Then the recombinant plasmids were transfected China) at 37˚C. Before injection, bacterial colony was grown in LB liquid into a Bombyx mori cell line BmN using X-tremeGENE HP DNA trans- culture at 37˚C overnight. The cells were collected after centrifugation, fection reagent (Roche Diagnostics, Indianapolis, IN). The transfected and the pellets were washed and resuspended with saline. Then, the cell method was used according to the manufacturer’s instructions. Forty-eight numbers were measured. hours after transfection, the cells were collected and centrifuged at 6000 3 g Downloaded from Insect infection for 10 min at 4˚C and resuspended by 150 mM phosphate buffer (pH 6.8). Then the cell suspension was sonically treated by an ultrasonic cell crusher E. coli (104 cells/larva) or B. bombyseptieus (103 cells/larva) or saline was (Ningbo Scientz Biotechnology, Ningbo City, China). The sample was injected into the hemocoel of the silkworm. After incubation for different cooled in a bath of an iced water mixture during sonic treatment. The time points, hemocytes and fat body tissues were dissected on ice and supernatant of the cell extracts was collected by centrifugation at 12,000 3 g immediately frozen and stored in liquid nitrogen, respectively. Every tissue for 20 min at 4˚C. Because the recombinant proteins contain the His- sample was collected from five larvae. tag, we used an affinity nickel resin column Ni-NTA superflow (Qiagen, http://www.jimmunol.org/ Hilden, German) to purify the His-tag fusion protein. The purified Injection of 20E and RNA interference of ecdysone receptor protein was dialyzed with 75 mM phosphate buffer (pH 6.8) and then To survey whether 20E may affect the silkworm immune, we first injected quantified using the BCA protein assay and visualized after SDS-PAGE 20E (0.5 mg/larva) (Sigma-Aldrich, St. Louis, MO), and 3 h after treat- by staining with Coomassie blue. ment, the same cell numbers of bacteria were injected as shown above. RNAi and rescued experiments of 3DE-3b-reductase DMSO was used as the control. The survival rate of the silkworm was measured after injection. The tissues were collected 24 h after microor- Based on the cDNA sequence of the silkworm 3DE-3b-reductase gene, ganism infection. Additionally, CFU assays were performed to further test we designed specific primers containing the T7 promoter sequence. The the effect of 20E according to a previous study with modification (29). primers were designed by Primer Premier 5 software and listed in Different concentrations of 20E (50, 500, and 5000 ng/larva) and bacteria Supplemental Table I (34). The methods to generate and inject dsRNA are were coinjected into the silkworm larvae. DMSO was also used as the the same as shown above. by guest on October 2, 2021 control. After 0, 12, and 24 h, 200 ml hemolymph from each treatment was To further confirm 3DE-3b-reductase activity in the silkworm immunity, diluted in series (1021,1022,1023) with PBS. Then the dilutions were we performed rescue experiments by injecting the recombinant 3DE-3b- plated on LB plates and incubated at 37˚C overnight. Then the colonies reductase protein and 20E into the gene knockdown silkworm, respec- were counted. tively. Six hours after the second dsRNA injection, the recombinant protein Because the 20E signal is transduced by the ecdysone receptor (EcR) and (1 mg/larva) was injected. The same concentration of BSA was used as ultraspiracle complex, we can block the 20E action by downregulating the the control. Three hours later, E. coli (104 cells/larva) or B. bombyseptieus expression level of the EcR gene (30, 31). The specific primers containing (103 cells/larva) was injected to infect the silkworm. Insect 3DE-3b- T7 promoter for the silkworm EcR gene were the same as described in Tian reductase can convert 3DE to ecdysone, which is the precursor of the et al. (31). dsRNAs were generated according to our previous paper (32). active 20E. Hence, we could inject 20E to rescue the 3DE-3b-reductase On the second day of the fifth instar, larvae were used to perform an RNA gene knockdown larvae. Similar to the injection of the recombinant pro- interference (RNAi) experiment. Ten-microliter solutions containing 30 mg tein, we injected 20E (0.5 mg/larva) after the second dsRNA injection, EcR dsRNA were injected into each larva. To ensure the effective RNAi, followed by infection with the bacteria. The DMSO was used as the another 30 mg EcR dsRNA was injected into each larva after 24 h. The control. For both rescue experiments, the survival rates of the silkworm same concentration of enhanced GFP (EGFP) dsRNA was used as con- were surveyed, and the fat body tissues were dissected at 24 h after bac- trols. Six hours after the second injection, E. coli (104 cells/larva) or terial infection. B. bombyseptieus (103 cells/larva) was used to infect the treated silkworm. The survival rates of the silkworm were surveyed after injection. The Real-time PCR tissues were collected 24 h after microorganism infection. For each treatment shown above, the stored tissues were grinded in liquid cDNA cloning and RACE nitrogen to powders. Total RNA was extracted by the Ultrapure RNA kit (Beijing CoWin Biotech, Beijing, China) and treated with DNase I (Takara To obtain the full-length cDNA of the silkworm 3DE-3b-reductase genes, a Bio, Shiga, Japan) to remove the genomic DNA contamination. The RNA GeneRacer kit (Invitrogen, Carlsbad, CA) was used according to the was quantified by the UV spectrophotometer, and then 1 mg RNA was manufacturer’s instruction. The procedure was shown in our previous reverse transcribed to the first strand of cDNA by the EasyScript one-step study (33). All the amplified products were purified with an agarose gel gDNA removal and cDNA synthesis SuperMix kit (TransGen Biotech). purification kit (Omega Bio-tek, Norcross, GA) and then cloned into The specific primers were designed and used in the quantitative real-time pEASY-T1-Clone vectors (TransGen Biotech, Beijing, China). Orientation PCR analysis (Supplemental Table I). The quantitative real-time PCR was and presence of the inserted cDNA were confirmed by sequencing of the performed using a real-time PCR detection system (CFX96, Bio-Rad, recombinant plasmid. Hercules, CA) with a SsoAdvanced SYBR Green supermix kit (Bio- Rad). The PCR was carried out as follows: 30 s at 95˚C, followed by 40 Preparation of antiserum cycles of 5 s at 95˚C and 40 s at 60˚C. The silkworm translation initiation The silkworm 3DE-3b-reductase gene was cloned into the prokaryotic factor 4A gene was used as the reference gene. expression vector pGEX-4T-1 and transformed into E. coli BL21 (DE3) Western blotting analyses strain. The 3DE-3b-reductase gene was induced by isopropyl b-D-1-thio- galactopyranoside, and the recombinant protein with the GST tag was For RNAi and bacterial infection experiments, 3DE-3b-reductases were purified using a GSTrap FF column (GE Healthcare, Uppsala, Sweden) detected at protein level using Western blotting. Total proteins from tissues 1648 REGULATION OF ECDYSONE TITER AFFECTING SILKWORM IMMUNITY or cells were extracted and quantified. SDS-PAGE was used to separate challenged the day 3 larvae of the fifth-instar silkworm with E. coli proteins. The polyclonal Ab against 3DE-3b-reductase (at a dilution of and B. bombyseptieus, respectively. At different time points after 1:8000) shown above was used to perform this assay. The method was also infection, the hemolymph of the treated silkworm was collected described previously (33). and the level of the 20E concentration was measured by the Ecdysteroid measurements competitive EIA assay. Our results showed that the 20E equiva- For ecdysteroid measurements, ecdysteroids were extracted from the lents were significantly elevated after bacterial infection (12 h silkworm hemolymph after bacterial infection at different time points. postinfection: saline, 33.568 6 2.133 ng/ml; E. coli, 45.198 6 Briefly, 100 ml hemolymph was collected from the treated silkworm (three 1.457 ng/ml, p , 0.001; B. bombyseptieus, 47.832 6 1.646 ng/ml, to five individuals) and added with 9 vol methanol. After centrifugation at , 6 3 p 0.001; 24 h postinfection: saline, 33.946 1.779 ng/ml; 12,000 g for 10 min, an aliquot of supernatant was combined and dried 6 , at 70˚C, and then the dried extract was dissolved with 150 ml enzyme E. coli, 54.305 1.646 ng/ml, p 0.001; B. bombyseptieus, immunoassay (EIA) buffer (0.4 M NaCl, 1 mM EDTA, and 0.1% BSA in 56.646 6 1.886 ng/ml, p , 0.001) (Fig. 1A). This suggests that 0.1 M phosphate buffer) (Sangon Biotech). Ecdysteroid levels were the silkworm could increase the 20E titer in the context of path- quantified via competitive EIA (Cayman Chemical, Ann Arbor, MI) using ogen attack. anti-20E rabbit antiserum (Cayman Chemical), 20E acetylcholinesterase tracer (Cayman Chemical) and standard 20E (Sigma-Aldrich). The ace- Effects of 20E on silkworm immunity tylcholinesterase activity was quantified by Ellman’s reagent (Cayman Chemical), and the absorbance at 405 nm was detected with an ELx800 To ascertain whether the elevated 20E may induce or reduce the absorbance microplate reader (BioTek, Winooski, VT). silkworm immunity, we injected 20E into the silkworm before microbial infection. When different pathogens were used to Statistical analysis challenge the silkworm, the survival rates of the silkworm All the experiments shown above were independently repeated three times. injected with 20E were significantly higher than those of the Downloaded from All statistical analyses in this study were performed in the statistical R control (log-rank test: E. coli, 20E versus DMSO, p = 0.0134; package (35). B. bombyseptieus, 20E versus DMSO, p = 0.0237) (Fig. 1B). Then, CFU assays were performed to further examine the effect of Results 20E on the silkworm immunity. From Fig. 1C, we found that 20E Bacterial infection could elevate 20E titer in the silkworm treatment could significantly enhance silkworm to kill both bac-

Previous studies have demonstrated that some stress factors could teria. Note that the low 20E concentration (50 ng/larva) has a http://www.jimmunol.org/ elevate the 20E titer (36–38). Thus, we examined whether patho- similar effect. This indicated that ecdysone could efficiently in- gen stress could increase the 20E titer in the silkworm. We first fluence the immune system of the silkworm. by guest on October 2, 2021

FIGURE 1. 20E could affect silkworm innate immunity. (A) 20E equivalents after bacterial infection; (B) survival rates of the pathogen-infected silkworms after 20E injection; (C) clearance of FITC-labeled bacteria; (D) expression patterns of immune-related genes. For (A), 6, 12, and 24 h after bacterial infection, hemolymph was collected. 20E concentration was measured by competitive EIA. For (B) and (D), every larva was injected with 0.5 mg 20E, followed by injection of different bacteria or saline. The silkworm translation initiation factor 4A gene was used as the reference gene. Each bar reﬂects the average value obtained from three samples. The statistical test for survival rate is log-rank test. For (C), living bacteria and different concentrations of 20E were coinjected into silkworm larvae. Then the hemolymph was collected and plated on LB plates and incubated at 37˚C overnight. Then colonies were counted. *p , 0.05, **p , 0.01 by t test. hpi, hours postinfection. The Journal of Immunology 1649

Similar to other insects, silkworm also contains Toll and IMD nonsecretory protein containing 323 aa (42). However, cotton signaling pathways to stimulate the expressions of different AMP leafworm (Spodoptera littoralis)3DE-3b-reductase protein genes, such as cecropin, gloverin, and moricin, although it is still has a signal peptide (26). Therefore, we used RACE to obtain the unclear which AMPs are specifically regulated by which pathway full length of the silkworm 3DE-3b-reductase gene. As shown in (many AMPs can be upregulated by both positive and negative Supplemental Fig. 1, the complete length of the coding sequence bacteria) (39–41). To assess the effect of 20E on silkworm innate is 1032 bp. The resulting cDNA sequence encodes a protein of immunity, we surveyed the transcriptional responses of several 343 aa with a signal peptide. We further surveyed the expression immune-related genes, including AMP genes (Gloverin and pattern of the gene and found that the 3DE-3b-reductase protein Morcin), adaptor of the Toll receptor (Myd88), peptidoglycan was expressed in the hemocyte and fat body (Supplemental Fig. recognition protein (PGRP-S6), and cellular immune-related gene 2). This is consistent with a previous study (43). The hemocyte (C-type lectin). Under the normal condition (saline), 20E could and fat body are the two important immune tissues or organs. We significantly induce the expressions of all tested genes excepted therefore first investigated the induced expression pattern of the for the Toll signaling adaptor Myd88 gene (Fig. 1D). After injection silkworm 3DE-3b-reductase gene after pathogen infection. In with different bacteria, we obtained similar results, which further hemocyte, both E. coli and B. bombyseptieus did not induce the show that 20E has the potential to upregulate silkworm immunity. expression of the 3DE-3b -reductase gene (Supplemental Fig. 3A). The effect of the ecdysone is thought to be mediated by EcR. In contrast, in fat body, both E. coli and B. bombyseptieus Several studies have proven that RNAi-mediated depletion of EcR stimulated the 3DE-3b-reductase gene expression levels from 6 to gene could prevent 20E signal transduction (13, 15, 31). We then 48 h postinfection (Fig. 3A). Additionally, we also confirmed this knocked down the expression of the silkworm EcR gene using result at the protein level using Western blotting (Fig. 3B). A dsRNA to further investigate the role of ecdysone on the silkworm previous study suggested that T. ni 3DE-3b-reductase gene was Downloaded from immunity. The larvae treated with EcR RNAi exhibited a lower mainly induced in fat body by bacteria (28). This indicates that ability to defend against microorganisms (log-rank test: E. coli, the 3DE-3b-reductase can rapidly take part in the bacterial- ds-EGFP versus ds-EcR, p = 0.0115; B. bombyseptieus, ds-EGFP infected silkworm fat body immune system, and this immune versus ds-EcR, p = 0.0323) (Fig. 2A). As expected, the expres- response may be common at least in the superfamily Bomb- sions of the immune-related genes were reduced in EcR RNAi ycoidea (including both B. mori and T. ni). larvae (Fig. 2B–F), suggesting that the 20E signaling pathway via http://www.jimmunol.org/ 3DE-3b-reductase gene is required for the silkworm to defend EcR is an important regulator for the silkworm immune response against bacteria after bacterial infection. Taken together, these results suggest that 20E could promote the To ascertain the function of the 3DE-3b-reductase gene in the expressions of several immune-related genes and enhance the re- silkworm immunity, we performed an RNAi experiment to knock sistance of the silkworm against pathogen through the activity of EcR. down the expression of the gene. Twenty-four hours after dsRNA injection, compared with the control, the protein level of 3DE-3b- Bacterial infection could rapidly induce the expression of the reductase significantly decreased (Fig. 3B). Note that dsRNA b silkworm 3DE-3 -reductase gene could not completely reduce the expression of 3DE-3b-reductase A previous study showed that the whole length of the silkworm due to relative low efficiency of RNAi in lepidopteran insects. by guest on October 2, 2021 3DE-3b-reductase gene is 972 bp, and the gene encodes a When E. coli or B. bombyseptieus was used to challenge the

FIGURE 2. EcR RNAi resulted in the repres- sion of the silkworm immune response. (A) Survival rates of the EcR knocked down larvae after bacterial infection; (B–F) expression patterns of immune-related genes. EcR gene was downregulated by dsRNA. After the second injection of the dsRNA, the larvae were infected with bacteria or saline. The relative expression levels are expressed as fold change compared with control (uninfected treatment). The statistical methods are the same as in Fig. 1. *p , 0.05, **p , 0.01 by t test. hpi, hours postinfection. 1650 REGULATION OF ECDYSONE TITER AFFECTING SILKWORM IMMUNITY Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 3. 3DE-3b-reductase plays an important role in silkworm immunity. (A) Expression of the silkworm 3DE-3b-reductase gene after bacterial infection; (B) Western blotting analysis of the silkworm 3DE-3b-reductase protein after bacterial infection (top) and 3DE-3b-reductase RNAi (bottom); (C) survival rate of the RNAi larvae after bacterial infection; (D) change of 20E equivalents in the RNAi larvae after bacterial infection; (E) expression patterns of immune-related genes in 3DE-3b-reductase RNAi larvae. For (A), at different time points after infection with bacteria or use of saline (control), total RNA of fat body was extracted. The relative expression levels of the gene were normalized using the threshold cycle (Ct) values of the reference gene, translation initiation factor 4A (sw22934). For (B), the hemolymphs from different treatments were collected and used for Western blotting analyses. For (C) and (E), RNAi was used to survey the function of the silkworm 3DE-3b-reductase gene. After the second injection of the 3DE-3b-reductase dsRNA, the larvae were injected with bacteria or saline. The survival rates were surveyed. Twenty-four hours after infection, the hemolymph was collected. Then, the 20E concentration was measured. In (E), 24 h postinfection, the fat body was dissected and the total RNA was extracted. Each bar reﬂects the average value obtained from three samples.*p , 0.05, **p , 0.01 by t test. ds-EGFP, larvae injected with the EGFP dsRNA; ds-gene-Bb, gene RNAi larvae were infected with B. bombyseptieus; ds-gene-Ec, gene RNAi larvae were infected with E. coli; ds-3b-red, larvae injected with the 3DE-3b-reductase dsRNA. silkworms, the survival rates of the silkworms treated with 3DE- 0.0248; B. bombyseptieus, ds-3b-red versus ds-EGFP, p = 0.0142) 3b-reductase RNAi were signiﬁcantly lower than those of the (Fig. 3C). Previous studies showed that 3DE-3b-reductase plays control (log-rank test: E. coli, ds-3b-red versus ds-EGFP, p = role in the insect developmental processes (22–24, 27). To exclude The Journal of Immunology 1651 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 4. Rescue experiments promoted RNAi silkworm immunity. (A) Survival rate of the rescued RNAi larvae after bacterial infection; (B) change of 20E equivalents in the rescued RNAi larvae after bacterial infection; (C) expression patterns of immune-related genes in 3DE-3b-reductase RNAi rescued larvae. Six hours after the second injection of the 3DE-3b-reductase dsRNA, the larvae were injected with puriﬁed recombinant 3DE-3b-reductase protein and 20E, respectively, followed by infection with bacteria or saline. BSA and DMSO were used as the controls. Twenty-four hours after infection, the fat body was dissected and total RNA was extracted. Each bar reﬂects the average value obtained from three samples. *p , 0.05, **p , 0.01 by t test.

the possibility that RNAi itself may affect larval mortality, the (Fig. 3C). In total, the higher mortality of the 3DE-3b-reductase survival rate of the RNAi knockdown silkworm was examined RNAi larvae indicates that this gene is essential for the silkworm under normal circumstance. In the feeding stage, the 3DE-3b- immune response. reductase RNAi had no effect on the normal development of the We then wanted to know how 3DE-3b-reductase takes part in silkworm (log-rank test: ds-3b-red versus ds-EGFP, p = 0.901) the silkworm immunity. According to previous reports, 3DE-3b- 1652 REGULATION OF ECDYSONE TITER AFFECTING SILKWORM IMMUNITY reductase can convert the 3-dehydro-ecdysteroid to ecdysteroid which may elevate the 20E titer in the hemolymph and thus en- (22–26). If the expression level of the 3DE-3b-reductase gene was hance the silkworm immune response after bacterial infection. downregulated, its enzymatic products might also decrease. There- fore, we collected the hemolymph from the 3DE-3b-reductase and Discussion EGFP RNAi larvae at 24 h after bacterial infection and measured the Hormones are important regulators of many biological processes, 20E titer as shown above. Compared with the control (ds-EGFP), the including metabolism, development, and reproduction. Increasing 20E equivalents significantly decreased in the 3DE-3b-reductase evidence showed that hormones can also systemically regulate RNAi silkworms (E. coli, 50.05 6 2.53 versus 41.81 6 2.02 ng/ml, immunity in vertebrates (44). In mammals, glucocorticoids are the p = 0.013; B. bombyseptieus, 49.37 6 2.58 versus 40.99 6 3.19 ng/ml, main neuroendocrine to regulate the immune system (45). Several p = 0.026) (Fig. 3D). As 20E could regulate the expressions of the studies showed that stressors can rapidly result in the systemic immune-related genes, we further surveyed their transcription release of glucocorticoids, and thereby alter the mammalian innate levels in 3DE-3b-reductase RNAi larvae. The injection with sa- immune and inflammatory response through the glucocorticoid line into the 3DE-3b-reductase or EGFP RNAi larvae could not receptor (44, 46, 47). In insects, molting hormone 20E titers can induce the expressions of the tested genes. In contrast, the silk- also increase when they are exposed to different stressors, such as worms pretreated with 3DE-3b-reductase RNAi prior to bacterial starvation, heat treatment, and sleep deprivation (36–38). How- challenge showed a significant decrease in the expression levels of ever, whether pathogen stress can change the insect 20E titer is the immune-related genes (Fig. 3E). still unclear. Our results showed an almost 40% increase in the Additionally, we performed a rescue experiment to further in- 20E titer after bacterial infection. Therefore, similar to its role in vestigate the function of the 3DE-3b-reductase. First, we expressed starvation and sleep deprivation, elevated 20E might have a Downloaded from the recombinant 3DE-3b-reductase protein with His-tag in the function as a stress hormone in pathogen attack. silkworm cell line BmN. We purified the protein by nickel affinity Ecdysone was thought to be an essential regulator in insect chromatography column. The SDS-PAGE and Western blotting development, metamorphosis, and reproduction. Several previous analyses showed the purified protein (Supplemental Fig. 3B). studies suggested that 20E could promote both cellular and humoral We then did the rescue experiment by injecting the purified innate immunity, and this enhancement requires EcR in Drosophila recombinant protein into the gene knockdown larvae. Moreover, (13–15, 20). However, knowledge about the effects of 20E on the enzymatic product of the 3DE-3b-reductase is ecdysteroid, immune response is very limited in lepidopteran insects. A recent http://www.jimmunol.org/ and therefore we also injected 20E to rescue the RNAi silkworm. study showed that the transcription levels of most Helicoverpa Fig. 4A shows that injection with either 3DE-3b-reductase protein armigera immune-related genes, including pattern recognition or 20E significantly reduced the mortality of RNAi silkworm after receptors and AMPs, significantly increased during the wandering bacterial infection compared with the control (BSA or DMSO). stage (48). Furthermore, injection with 20E, even at very low Injection with the active 3DE-3b-reductase protein could elevate concentrations, could strongly induce the expressions of these the 20E titer after 12 h of treatment (E. coli,36.966 2.24 versus genes (49). Interestingly, for B. mori, two previous studies ob- 47.51 6 1.92 ng/ml, p = 0.0037; B. bombyseptieus,37.986 1.72 tained opposite results. One study suggested that 20E suppressed 6 versus 48.47 2.62 ng/ml, p = 0.0069) (Fig. 4B). Consistent the expressions of the silkworm AMP genes (31). In contrast, by guest on October 2, 2021 with the increase of the 20E titer, we found that the transcription another study suggested that 20E treatment significantly upregu- level of EcR, which is the first factor to pass the 20E signal, is lated the expressions of AMP genes as shown in H. armigera (49). significantly upregulated in the RNAi larvae injected with the 3DE- Therefore, we further surveyed the effect of the elevated 20E titer 3b-reductase protein. Meanwhile, the expressions of the immune- after pathogen attack. related genes were strongly promoted (Fig. 4C). In this study, we used the larvae treated with exogenous 20E and Taken together, our results suggested that the silkworm could bacterial infection to survey the effect of the ecdysone. All tested actively increase the expression level of the 3DE-3b-reductase, immune-related genes except for the Myd88 gene were induced in

FIGURE 5. Mechanism to explain the change of ecdysone titer after bacterial attack. After bacterial infection, the 3-dehydroecdysone (3DE) is released from prothoracic glands to other tissues, such as fat body. Bacterial infection also can increase 3DE-3b- reductase activity, which further converts 3DE into ecdysone. Finally, the elevated ecdysone titer could enhance the silkworm humoral and cellular defense. E, ecdysone. The Journal of Immunology 1653 the silkworm larvae coinjected with 20E and bacteria. Myd88, an was induced in the fat body. A previous study showed that 3DE- adaptor of the Toll receptor, is involved in the Toll-mediated in- 3b-reductase with enzymatic activity was mainly present in the nate immune responses (50). These results are consistent with a silkworm hemolymph (43). Indeed, we also detected the induced previous study in which most of the genes in the Toll signaling protein in the silkworm hemolymph after bacterial infection. pathway cannot be up- or downregulated during the silkworm Therefore, it is likely that 3DE-3b-reductase protein with signal wandering stage (49). Therefore, 20E may have no effect on the peptide formed in fat body and then was transferred to hemo- silkworm Toll pathway. However, 20E depresses the transcription lymph. Then the active 3DE-3b-reductase can convert 3DE into levels of several Drosophila Toll-related genes, such as Toll ligand ecdysone, which, in turn, is rapidly hydroxylated to the active spa¨tzle and Toll transcription factor dorsal (51). The mechanism 20E. Finally, the high 20E concentration mediated by EcR func- to explain this difference between the silkworm and fruit fly re- tion will elevate the expressions of several AMP genes, which mains unclear. 20E signaling exerts its effects through the EcR could enhance the silkworm humoral immunity. Meanwhile, 20E and ultraspiracle complex, which binds to the promoters of target can also induce the cellular immune response through C-type genes (52, 53). EcR is the primitive receptor to transduce the 20E lectin (Fig. 5). Although the elevated 20E titer is limited, it can signal. When the EcR gene was downregulated by RNAi, the efficiently enhance the silkworm immune system. Note that this survival rate of the silkworm and the transcription levels of limited increase of the 20E titer cannot affect the normal devel- immune-related genes significantly decreased (Fig. 2). Taken to- opment of the silkworm. Therefore, we thought that the silkworm gether, bacterial attack could increase 20E titer level, which could evolved a very smart strategy to strengthen its immunity by in- enhance the silkworm immunity mediated by EcR. Note that 20E creasing 20E titer, which is not enough to effect its normal de- concentration increased to be ,50 ng/ml after infection, which is velopment. Collectively, our findings reveal an important role of still too low to affect the silkworm normal development (54). Al- the 20E synthesis pathway from 3DE in enhancing immune re- Downloaded from though the concentration is not very high, it is enough to modulate sponse and represent a first step, to our knowledge, toward the the immune system of the silkworm (Fig. 1C). Additionally, a re- understanding of interaction mechanisms between insect hormone cent work also showed that the injection of a low concentration of and immunity. 20E could enhance the expressions of several immune-related genes in another lepidopteran insect (48). Thus, we speculate Acknowledgments http://www.jimmunol.org/ that insects may use some mechanisms to balance immune and We thank Deng-Wei Qi for help in preparing the polyclonal Abs against developmental processes. Additionally, our results also demon- 3DE-3b-reductase and Dr. Shi-Hong Gu for constructive suggestions on strated that the elevated 20E titer could significantly increase the our experiments. ability of the silkworm to defend against the bacterial infection (Figs. 1, 2), supporting the results of a previous study (49). Disclosures As shown above, pathogen attack, starvation, sleep deprivation, The authors have no financial conflicts of interest. and heat treatment could increase the insect 20E titer. However, the mechanism to regulate the 20E titer under environmental stress remains unclear. In this study, we showed, to our knowledge for

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