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Dendritic Cell Differentiation Induced − Critical Role of AZI2 in GM-CSF

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Dendritic Cell Differentiation Induced − Critical Role of AZI2 in GM-CSF Critical Role of AZI2 in GM-CSF−Induced Dendritic Cell Differentiation Masahiro Fukasaka, Daisuke Ori, Tatsukata Kawagoe, Satoshi Uematsu, Kenta Maruyama, Toshihiko Okazaki, This information is current as Tatsuya Kozaki, Tomoko Imamura, Sarang Tartey, Takashi of September 27, 2021. Mino, Takashi Satoh, Shizuo Akira and Osamu Takeuchi J Immunol published online 22 April 2013 http://www.jimmunol.org/content/early/2013/04/21/jimmun ol.1203155 Downloaded from Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 27, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published April 22, 2013, doi:10.4049/jimmunol.1203155 The Journal of Immunology Critical Role of AZI2 in GM-CSF–Induced Dendritic Cell Differentiation Masahiro Fukasaka,*,† Daisuke Ori,*,‡,x Tatsukata Kawagoe,*,1 Satoshi Uematsu,*,2 Kenta Maruyama,* Toshihiko Okazaki,*,† Tatsuya Kozaki,* Tomoko Imamura,*,‡,x Sarang Tartey,*,‡,x Takashi Mino,‡,x Takashi Satoh,* Shizuo Akira,*,{ and Osamu Takeuchi*,‡,x TNFR-associated factor family member–associated NF-kB activator (TANK)–binding kinase 1 (TBK1) is critical for the activation of IFN regulatory factor 3 and type I IFN production upon virus infection. A set of TBK1-binding proteins, 5-azacytidine–induced gene 2 (AZI2; also known as NAP1), TANK, and TBK1-binding protein 1 (TBKBP1), have also been implicated in the production of type I IFNs. Among them, TANK was found to be dispensable for the responses against virus infection. However, physiological Downloaded from roles of AZI2 and TBKBP1 have yet to be clarified. In this study, we found that none of these TBK1-binding proteins is critical for type I IFN production in mice. In contrast, AZI2, but not TBKBP1, is critical for the differentiation of conventional dendritic cells (cDCs) from bone marrow cells in response to GM-CSF. AZI2 controls GM-CSF–induced cell cycling of bone marrow cells via TBK1. GM-CSF–derived DCs from AZI2-deficient mice show severe defects in cytokine production and T cell activation both in vitro and in vivo. Reciprocally, overexpression of AZI2 results in efficient generation of cDCs, and the cells show enhanced T cell activation in response to Ag stimulation. Taken together, AZI2 expression is critical for the generation of cDCs by GM-CSF and http://www.jimmunol.org/ can potentially be used to increase the efficiency of immunization by cDCs. The Journal of Immunology, 2013, 190: 000–000. endritic cells (DCs) play a central role in instructing DCs and macrophages sense the invasion of pathogens through a T cells to activate acquired immunity upon infection set of pattern-recognition receptors, including TLRs and retinoic D (1–4). They phagocytose microbes such as bacteria and acid inducible gene-I (RIG-I)–like receptors (RLRs) (5, 6). Whereas viruses, process them into Ags, and present the Ags on MHC TLRs are transmembrane proteins that recognize microbial com- molecules. Besides Ag presentation, innate immune cells such as ponents on the cell surface or in endosomes, RLRs sense viral dsRNA in the cytoplasm. The signaling pathways triggered by the receptors lead to the production of proinflammatory cytokines, in- by guest on September 27, 2021 *Laboratory of Host Defense, Immunology Frontier Research Center; †Nitto Denko cluding TNF, IL-6, IL-12p40, and type I IFNs (7). In addition, the Company, Ibaraki, Osaka 567-8680, Japan; ‡Laboratory of Infection and Prevention, x signaling pathways increase the surface expression of costimula- Institute for Virus Research, Kyoto University; Core Research for Evolutional Sci- ence and Technology, Japan Science and Technology Agency, Sakyo-ku, Kyoto 606- tory molecules, such as CD40, CD80, and CD86, on DCs (8). It is 8507, Japan; and {Research Institute for Microbial Diseases, Osaka University, Suita, well documented that costimulation of DCs is critical for mounting Osaka 565-0871, Japan efficient T cell activation and vaccination (9). 1 Current address: Department of Ophthalmology, Yokohama City University School DCs are classified into several different subsets, including CD82 of Medicine, Yokohama, Japan. and CD8+ conventional DCs (cDCs) and plasmacytoid DCs (pDCs) 2Current address: International Research and Development Center for Mucosal Vac- cines, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan. (10, 11). pDCs, which are characterized by surface expression of CD11c and B220, are known to produce vast amounts of IFN-a in Received for publication November 14, 2012. Accepted for publication March 26, 2013. response to virus infection via TLR7 and TLR9 (12). On the other This work was supported by Special Coordination Funds of the Japanese Ministry of hand, cDCs recognize pathogen infection via TLRs and RLRs for Education, Culture, Sports, Science and Technology and grants from the Ministry the production of cytokines. Among the cDCs, CD8+ DCs differ- of Health, Labour and Welfare in Japan and the Japan Society for the Promotion of entiate in a Batf3-dependent manner (13) and have a strong capacity Science through the Funding Program for World-Leading Innovative R&D on Sci- ence and Technology (FIRST Program). This work was also supported in part by for cross-presentation of foreign Ags to MHC class I molecules grants from the Takeda Science Foundation, the Naito Foundation, and the Mochida (11). In mice, both cDCs and pDCs can be generated from bone Foundation. marrow (BM) cells by culture with distinct cytokines. FLT3 ligand Address correspondence and reprint requests to Prof. Osamu Takeuchi, Laboratory of (FLT3L) is essential for pDC and a set of cDC differentiation from Infection and Prevention, Institute for Virus Research, Kyoto University, 53 Shogoin Kawara-cho, Sakyo-ku, Kyoto 606-8507, Japan. E-mail address: [email protected]. BM precursor cells (14). In contrast, culture of mouse BM cells or ac.jp human PBMCs in the presence of GM-CSF is widely used to induce Abbreviations used in this article: AZI2, 5-azacytidine–induced gene 2; BM, bone cDCs (15). GM-CSF induces the survival, proliferation, and dif- marrow; BMDC, bone marrow–derived dendritic cell; cDC, conventional dendritic ferentiation of hematopoietic cells including DCs (16). The GM- cell; DC, dendritic cell; DKO, double knockout; EMCV, encephalomyocarditis virus; FLT3L, FLT3 ligand; FLT3L-DC, FLT3L-derived BMDC; Flu, influenza virus; GM- CSF receptor is composed of an a subunit and a common b subunit DC, GM-CSF–derived BMDC; IKK-i,IkB kinase-i; IRF, IFN regulatory factor; KO, shared by IL-3Rs and IL-5Rs (17). After stimulation of GM-CSF knockout; MOI, multiplicity of infection; NDV, Newcastle disease virus; pDC, plas- macytoid DC; poly(I:C), polyinosinic-polycytidylic acid; RLR, retinoic acid–induc- receptors, signaling pathways are activated through the formation of ible gene-I–like receptor; TANK, TNFR-associated factor family member–associated a unique dodecameric receptor complex (18, 19). The transcription NF-kB activator; TBK1, TANK-binding kinase 1; TBKBP1, TBK1-binding protein factors STAT5 and IFN regulatory factor 4 (IRF4) (20, 21) are es- 1; TRAF, TNFR-associated factor; WT, wild-type. sential for GM-CSF–mediated signaling pathways, and MAPK and Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 AKT are also activated by GM-CSF (17). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203155 2 DC DIFFERENTIATION REQUIRES AZI2 The molecular mechanisms for how TLRs and RLRs transactivate deoxynucleotide 1668) were purchased from Invivogen. LPS from Escher- type I IFN genes and proinflammatory cytokines have been ex- ichia coli was purchased from Sigma. GM-CSF and FLT3L were purchased tensively studied. With the exception of TLR3, TLRs trigger an from PeproTech. MyD88-dependent signaling pathway that induces activation of Generation of GM-DCs and FLT3L-derived BMDCs the transcription factor NF-kB, thereby leading to the expression BM cells were harvested from the femurs and tibias of mice by flushing the of proinflammatory cytokine genes. In contrast, TLR3 and TLR4 marrow cavity with HBSS. GM-DC:BM cells were resuspended at the signal through another adaptor molecule, Toll/IL-1R domain– density of 1 3 106 cells/ml in complete RPMI 1640 (supplemented with containing adaptor protein inducing IFN-b, which results in the 10% FCS, 50 mM 2-ME, 1% penicillin [100 U/ml] and 1% streptomycin activation of a set of kinases called IkB kinase-i (IKK-i, also known [100 U/ml]) and 10 ng/ml mouse GM-CSF. The cell culture medium was ε replaced on days 2 and 4, and cells were grown for 6 d. FLT3L-derived as IKK ) and TNFR-associated factor (TRAF) family member–as- BMDC (Flt3L-DC):BM cells were lysed with RBC lysing buffer (Sigma) sociated NF-kB activator (TANK)–binding kinase 1 (TBK1). These and resuspended in complete RPMI 1640 containing 20 ng/ml Flt3L, and kinases phosphorylate IRF3 and IRF7, which induce transactivation cells were grown for 8 d. of type I IFN and IFN-inducible genes (6, 22). RLR signaling also Quantitative real-time PCR and RT-PCR activates IKK-i and TBK1 for the production of type I IFNs. IKK-i and TBK1 are known to associate with several adaptor molecules, Total RNA was extracted using TRIzol (Invitrogen), and cDNA was gen- including TANK, 5-azacytidine–induced gene 2 (AZI2; also known erated by using ReverTraAce (Toyobo) according to the manufacturer’s instructions.
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