Zebrafish NIK Mediates IFN Induction by Regulating Activation of IRF3 and NF- κB Bo Chen, Chen Li, Jian Yao, Lin Shi, Wanmeng Liu, Fang Wang, Shitian Huo, Yongan Zhang, Yuanan Lu, Usama This information is current as Ashraf, Jing Ye and Xueqin Liu of October 1, 2021. J Immunol published online 17 February 2020 http://www.jimmunol.org/content/early/2020/02/14/jimmun ol.1900561 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2020/02/16/jimmunol.190056 Material 1.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published February 17, 2020, doi:10.4049/jimmunol.1900561 The Journal of Immunology Zebrafish NIK Mediates IFN Induction by Regulating Activation of IRF3 and NF-kB Bo Chen,*,†,1 Chen Li,*,†,1 Jian Yao,*,† Lin Shi,*,† Wanmeng Liu,*,† Fang Wang,*,† Shitian Huo,*,† Yongan Zhang,*,† Yuanan Lu,‡ Usama Ashraf,x Jing Ye,x and Xueqin Liu*,† Type I IFN mediates the innate immune system to provide defense against viral infections. NF-kB–inducing kinase (NIK) potentiates the basal activation of endogenous STING, which facilitates the recruitment of TBK1 with the ectopically expressed IRF3 to induce IFN production. Moreover, NIK phosphorylates IKKa and confers its ability to phosphorylate p100 (also known as NF-kB2) in mammals. Our study demonstrated that NIK plays a critical role in IFN production in teleost fish. It was found that NIK interacts with IKKa in the cytoplasm and that IKKa phosphorylates the NIK at the residue Thr432, which is different from k the mammals. Overexpression of NIK caused the activation of IRF3 and NF- B, which in turn led to the production of IFN and Downloaded from IFN-stimulated genes (ISGs). Furthermore, the ectopic expression of NIK was observed to be associated with a reduced replication of the fish virus, whereas silencing of endogenous NIK had an opposite effect in vitro. Furthermore, NIK knockdown significantly reduced the expression of IFN and key ISGs in zebrafish larvae after spring viremia of carp virus infection. Additionally, the replication of spring viremia of carp virus was enhanced in NIK knockdown zebrafish larvae, leading to a lower survival rate. In summary, our findings revealed a previously undescribed function of NIK in activating IFN and ISGs as a host antiviral response. These findings may facilitate the establishment of antiviral therapy to combat fish viruses. The Journal of Immunology, 2020, http://www.jimmunol.org/ 204: 000–000. nnate immunity provides the first step of host defense against including the response to pathogens. Among the IRFs, IRF3 and microbial pathogens. In response to viral infection, the in- IRF7 are critical for the induction of type I IFN signaling cascades I duction of IFNs, especially the type I IFN, plays a decisive in response to the sensing of viral DNA and/or RNA motifs by the role in the host defense processes (1). IFN is imperative for the pattern recognition receptors. Upon activation, both IRF3 and potent activation to IFN-stimulated genes (ISGs), which rapidly IRF7 are capable of translocating from cytoplasm to the nucleus culminate in the inhibition of viral replication and spread (2, 3). of the cell, where they trigger the expression of type I IFNs by guest on October 1, 2021 IFN regulatory factors (IRFs) are transcriptional factors that per- (4, 5). Albeit the expression levels of IRF3 and IRF7 are diverse form crucial functions in several aspects of the immune response, in different cells, they are consistent in terms of virus-induced phosphorylation, dimerization, and nuclear translocation (5). NF-kB activation occurs through two pathways: the canonical *College of Fisheries, Huazhong Agricultural University, Wuhan 430070, Hubei, and noncanonical pathways. Both pathways are vital in regulating † China; Hubei Engineering Technology Research Center for Aquatic Animal Dis- the transcription of an array of genes involved in the several eases Control and Prevention, Wuhan 430070, Hubei, China; ‡Department of Public Health Sciences, University of Hawaii at Manoa, Honolulu, Hawaii 96822; and xState processes of immune and inflammatory responses, such as cyto- Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, kine receptor adhesion molecules, acute phase protein gene tran- Wuhan 430070, Hubei, China scription factors, and some regulatory factors (6, 7). NF-kB is also 1 B.C. and C.L. contributed equally to this work. reported to exert antiviral function by inducing the expression of ORCIDs: 0000-0002-3258-6224 (J.Y.); 0000-0002-0012-4084 (X.L.). IFN (1, 6). A better understanding of the mechanism of NF-kB Received for publication May 20, 2019. Accepted for publication January 9, 2020. activation is of great importance for harnessing the uncontrolled This work was supported by the National Key Research and Development Program of activation of immune and inflammatory responses. China (2018YFD0900505), the Natural Science Foundation of China (31972834), In mammals, the role of NIK (also known as MAP3K14) in and Fundamental Research Funds for the Central Universities (2662018YJ022). immunity is well established. According to previous studies, NIK Address correspondence and reprint requests to Assoc. Prof. Jing Ye or Prof. Xueqin k Liu, Huazhong Agricultural University, Wuhan, Hubei, China (X.L.). E-mail ad- activates the noncanonical NF- B pathway, which responds to dresses: [email protected] (J.Y.) or [email protected] (X.L.) selective receptor signals that mediate adaptive immune functions, The online version of this article contains supplemental material. such as lymphoid organ development and B cell survival (7, 8). Abbreviations used in this article: ATCC, American Type Culture Collection; CIP, calf Furthermore, the activation of the nonclassical pathway can also intestinal phosphatase; Co-IP, coimmunoprecipitation; EPC, epithelioma papulosum induce the activation of the classical pathway because the stimu- cyprini; FHM, fathead minnow; GCRV, grass carp reovirus; GSIV, giant salamander lation of LTa1b2 leads to the accumulation of intracellular NIK iridovirus; HEK, human embryonic kidney; IFNw1pro, IFNw1 promoter; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; ISRE, IFN-stimulated response element; and the upregulation of proinflammatory cytokine expression (9). MO, morpholino; MOI, multiplicity of infection; mutNIK, mutant of zebrafish NIK; NIK phosphorylates IKKa at Ser176 and Ser180 residues, and mutation NC, negative control; NIK-MO, MO targeting NIK; poly(I:C), polyinosinic-polycytidylic acid; qRT-PCR, quantitative real-time PCR; S1, sequence 1; S2, sequence 2; S3, sequence 3; in these two residues to glutamate causes the constitutive activation siNIK, siRNA against NIK; siRNA, small interfering RNA; STD-MO, MO targeting of NF-kB (10). Furthermore, IKKa (S176E) constitutively ac- STD; STING, stimulator of IFN geneS; SVCV, spring viremia of carp virus; wtNIK, tivates IRF3/7 and is involved in IFN-a production through wild-type NIK. TLR 7/9 signaling cascades (10). NIK interacts with the DNA Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 pathway adapter stimulator of IFN genes (STING; also known as www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900561 2 ZEBRAFISH NIK REGULATES IFN BY ACTIVATION OF IRF3 AND NF-kB TMEM173/MITA) to enhance the induction of IFN (11). IFN can IKKa (NM_200317.1) was cloned into 33Flag pCMV-14 or pEGFP-N1. induce NIK expression in return (12). Although extensive studies The pGL3–basic firefly luciferase reporter vector harboring the IFNw1 w have been conducted in mammals, the role of NIK in fish has not promoter (IFN 1pro)–Luc or NF-kBpro-Luc and the Renilla luciferase internal control vector (pRL-TK) were provided by professor Yongan yet been investigated. Zhang (College of Fisheries, Huazhong Agricultural University) (22). The Aquatic diseases are the main factors in restricting the develop- IFN-stimulated response element (ISRE)–Luc was received from Professor ment of the aquatic industry and threatening food safety. Among the Shaobo Xiao (College of Veterinary Medicine, Huazhong Agricultural aquatic diseases caused by different pathogens, viral diseases are the University) (23). Wild-type NIK (wtNIK) plasmid was mutated using FastPfu PCR (94˚C most difficult to prevent and cure (13, 14). Spring viremia of carp for 5 min, followed by 25 cycles of 98˚C for 10 s, 55˚C for 5 s, 72˚C for virus (SVCV; ssRNA) (15), giant salamander iridovirus (GSIV; 6 min, and 72˚C for 10 min) (TransGen). Recovered PCR product was dsDNA) (16), and grass carp reovirus (GCRV; dsRNA) (17) are fish treated with Dpn I (TAKARA) at 37˚C for 30 min. Subsequently, the pathogens that cause severe diseases and significant mortalities in plasmid was extracted and stored at 220˚C. the affected fish, which ultimately leads to huge economic losses to Plasmid transfection and virus infection the aquaculture industry. However, there is still no effective method presently available to prevent and control these viral diseases. The recombinant plasmids were transfected using FuGENE HD (Promega) In this study, we demonstrated a previously undescribed following the manufacturer’s protocol. For virus infection assays, cells were infected with SVCV (0.05 multiplicity of infection [MOI]) at 28˚C or function of zebrafish NIK in inducing the immune response GSIV (1.0 MOI) or GCRV (1.0 MOI) at 25˚C.