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 Supplementary http://www.jimmunol.org/content/suppl/2016/01/15/jimmunol.150015 Material 8.DCSupplemental References This article cites 55 articles, 13 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/196/4/1646.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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 expres- sions 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,