By Conventional Dendritic Cells in TLR7/9-Induced Type I IFN

Cutting Edge: Critical Role of IκB Kinase α in TLR7/9-Induced Type I IFN Production by Conventional Dendritic Cells

This information is current as Katsuaki Hoshino, Izumi Sasaki, Takahiro Sugiyama, of September 28, 2021. Takahiro Yano, Chihiro Yamazaki, Teruhito Yasui, Hitoshi Kikutani and Tsuneyasu Kaisho J Immunol 2010; 184:3341-3345; Prepublished online 3 March 2010;

doi: 10.4049/jimmunol.0901648 Downloaded from http://www.jimmunol.org/content/184/7/3341

Supplementary http://www.jimmunol.org/content/suppl/2010/03/01/jimmunol.090164 Material 8.DC1 http://www.jimmunol.org/ References This article cites 32 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/184/7/3341.full#ref-list-1

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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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Cutting Edge: Critical Role of IkBKinasea in TLR7/9-Induced Type I IFN Production by Conventional Dendritic Cells ,1 ,†,1 Katsuaki Hoshino,* Izumi Sasaki,* Takahiro Sugiyama,* Takahiro Yano,* x Chihiro Yamazaki,*,† Teruhito Yasui,‡ Hitoshi Kikutani,‡ and Tsuneyasu Kaisho*,†, Aplasmacytoiddendriticcell (DC) can produce large of MyD88 bifurcates into two pathways leading to the acti- amounts of type I IFNs after sensing nucleic acids through vation of NF-kB and IFN regulatory factor (IRF)-7 (7). NF- TLR7 and TLR9. IkBkinasea (IKKa) is critically in- kB activation leads to production of proinﬂammatory cyto- volved in this type I IFN production through its interac- kines and requires phosphorylation and degradation of IkB. tion with IFN regulatory factor-7. In response to TLR7/9 IkB phosphorylation depends mainly on a serine threonine signaling, conventional DCs can also produce IFN-b kinase, IkB kinase (IKK)b (8). but not IFN-a in a type I IFN-independent manner. In IRF-7 activation leads to type I IFN production (9). This Downloaded from this study, we showed that IKKa was required for pro- pathway functions mainly in a specialized DC subset, the duction of IFN-b, but not of proinﬂammatory cytokines, plasmacytoid DC (pDC) (10, 11). The pDC expresses TLR7 by TLR7/9-stimulated conventional DCs. Importantly, and TLR9 exclusively among TLRs, has constitutively high IKKa was dispensable for IFN-b gene upregulation by levels of IRF-7, and is notable for its potent ability to produce type I IFNs, including IFN-a and IFN-b, following TLR7/9

TLR4 signaling. Biochemical analyses indicated that http://www.jimmunol.org/ signaling. IRF-7 activation requires its phosphorylation, and a IKK exerted its effects through its interaction with we have found that IKKa, also known as Chuk, is involved in IFN regulatory factor-1. Furthermore, IKKa was involved this IRF-7 activation (12). in TLR9-induced type I IFN-independent IFN-b produc- In response to TLR7/9 signaling, another DC subset, the tion in vivo. Our results show that IKKa is a unique mol- conventional DC (cDC), can also produce IFN-b, but not ecule involved in TLR7/9-MyD88–dependent type I IFN IFN-a, through the distinct molecular mechanisms from pDCs production through DC subset-specific mechanisms. (13, 14). In this study, we have investigated the involvement of The Journal of Immunology, 2010, 184: 3341–3345. IKKa in IFN-b production by TLR7/9-stimulated cDCs. Materials and Methods by guest on September 28, 2021 endritic cells (DCs) sense nucleic acid immune Mice 2 2 2 2 2 2 adjuvants ssRNA and unmethylated CpG DNA via Ikka / , Myd88 / , and Tlr4 / mice have been described previously (15– 2 2 D TLR7 and TLR9, respectively, and produce proin- 17). TNFR-associated factor 3 (TRAF3)-deficient (Traf3 / ) mice were generated by T. Yasui and H. Kikutani (manuscript in preparation). C57BL/ flammatory cytokines or type I IFNs (1–4). Accumulating 2 2 6, osteopontin (Opn)-deficient (Opn / ), and IFN-a/b R-deficient (Ifna/ evidence indicates that TLR7/9-induced type I IFN induction 2 2 br / ) mice were purchased from CLEA Japan (Shizuoka, Japan), is important not only for antiviral defense but also for the The Jackson Laboratory (Bar Harbor, ME), and B&K Universal (Hull, U.K.), 2 2 pathogenesis of certain autoimmune diseases (5, 6). There- respectively. IL-1R–associated kinase-1 (IRAK-1)–deficient (Irak1 / ) mice fore, clarifying the underlying mechanisms of this type I IFN were provided by Dr. J. A. Thomas (University of Texas Southwestern Medical Center, Dallas, TX) (18). Bone marrow (BM) chimeric mice 2 2 2 2 production should contribute to the development of im- were generated by transferring wild-type, Ikka / ,orTraf3 / fetal 2 2 munomanipulation for these diseases. liver cells into C57BL/6 (CD45.1) mice (19, 20). Ikka+/+Ifna/br / and 2 2 2 2 Ikka / Ifna/br / chimeric mice were generated by transferring fetal TLR7 and TLR9 associate with the cytoplasmic adaptor, 2 2 2 2 2 2 liver cells from Ikka+/+Ifna/br / or Ikka / Ifna/br / mice into irradiated MyD88, through the homophilic interaction of their re- 2 2 Ikka+/+Ifna/br / mice. All mice were maintained under specific pathogen- spective Toll/IL-1R homologous domains (3). MyD88 is es- free conditions, and animal experiments were conducted according to the sential for all TLR7/9-mediated effects. Signaling downstream institutional guidelines.

*Laboratory for Host Defense, RIKEN Research Center for Allergy and Immunology; 22, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. E-mail address: tkaisho@rcai. x Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokoha- riken.jp ma City University, Yokohama; and †Department of Allergy and Immunology, Grad- ‡ The online version of this article contains supplemental material. uate School of Medicine and Department of Molecular Immunology, Research Institute for Microbial Diseases and World Premier International Immunology Frontier Research Abbreviations used in this paper: BM, bone marrow; CBB, Coomassie brilliant blue; Center, Osaka University, Osaka, Japan cDC, conventional dendritic cell; DC, dendritic cell; IKK, IkB kinase; IRAK-1, IL-1R– associated kinase-1; IRF, IFN regulatory factor; Opn, osteopontin; pDC, plasmacytoid 1K.H. and I.S. contributed equally to this work. dendritic cell; poly(I:C), polyinosinic:polycytidylic acid; RLR, RIG-I–like receptor; Received for publication May 27, 2009. Accepted for publication January 27, 2010. TRAF3, TNFR-associated factor 3; TRIF, Toll/IL-1R domain-containing adaptor- inducing IFN-b. This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology and the Japan Science and Technology Corporation, the Ó Uehara Memorial Foundation, the Mochida Memorial Foundation for Medical and Copyright 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 Pharmaceutical Research, and the Japan Intractable Diseases Research Foundation. Address correspondence and reprint requests to Dr. Tsuneyasu Kaisho, Laboratory for Host Defense, RIKEN Research Center for Allergy and Immunology, Suehiro-cho 1-7- www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901648 3342 CUTTING EDGE: IkB KINASE a IN TLR7/9 SIGNALING

Cells Critical roles of IKKa in IFN-b production by TLR7/9-stimulated GM-CSF–induced BM DCs were generated as previously described (21) and cDCs used as cDCs. We have analyzed involvement of IKKa. TLR9-induced IFN- 2 2 b induction was severely impaired in Ikka / cDCs (Fig. 1A). Reagents 2 2 This was a specific effect, because wild-type and Ikka / A phosphorothioate oligodeoxynucleotide containing an unmethylated CpG cDCs produced comparable amounts of proinflammatory motif, ODN1668, was used as CpG DNA (22). LPS derived from Salmonella minnesota Re595, polyinosinic:polycytidylic acid [poly(I:C)], and R848 were cytokines, such as IL-12p40 and TNF-a, in response to purchased from Sigma-Aldrich (St. Louis, MO), Amersham Biosciences TLR9 stimuli (Fig. 1A). (Piscataway, NJ), and InvivoGen (San Diego, CA), respectively. We next investigated expression of these cytokine genes by Northern blot analysis (Fig. 1B,1C). CpG DNA could up- Measurement of cytokine production regulate expression of IFN-b as well as proinflammatory cy- 2 2 Cells were treated for 20–24 h with the indicated stimuli, and cytokine tokine genes in wild-type but not in Myd88 / cDCs, 2 2 production was measured by ELISA as described previously (12). indicating their dependence on MyD88. Ikka / cDCs Northern blot analysis showed a severe defect in TLR9-induced IFN-b gene expression (Fig. 2B) but normal induction of IL-12p40 and Northern blot analysis was performed as described previously (21). TNF-a gene expression. Similar defects were observed also in 2/2 EMSA TLR7-stimulated Ikka cDCs (Fig. 2C). Thus, as with Downloaded from IFN-stimulated response element-binding activities were analyzed as described previously (12).

Nuclear translocation of IRF-1 and NF-kB Nuclear extracts were subjected to immunoblot analyses with anti–IRF-1 or anti-p65 Abs (Santa Cruz Biotechnology, Santa Cruz, CA) as described previously (12). http://www.jimmunol.org/

Immunoprecipitation assay The 293T human embryonic kidney cell line was transiently transfected with expression vectors for FLAG-tagged mouse IRF-1 (FLAG-IRF-1) and/or Myc- tagged mouse IKKa (Myc-IKKa) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Subsequently, immunoprecipitation and immunoblotting experiments were performed as described previously (12).

In vitro kinase assay by guest on September 28, 2021 One hundred nanograms of puriﬁed recombinant IKKa (Upstate Bio- technology, Lake Placid, NY) was incubated with 1 mg substrates, GST, GST fused to residues 2–329 of mouse IRF-1 (GST-IRF-1), or GST fused to residues 5–55 of mouse IkBa (GST-IkBa) and subjected to an in vitro kinase assay as described previously (12).

Serum IFN-b levels after CpG DNA injection

Each mouse was injected i.p. with 20 nmol CpG DNA and 20 mg D- (+)-galactosamine. At the indicated times, the mice were bled, and serum IFN- b levels were measured by ELISA (PBL InterferonSource, Piscataway, NJ).

Statistical methods A two-tailed, unpaired Student t test was used for assessment of the differences between groups. Prism (Graphpad Software, La Jolla, CA) was used for statistical analysis. Differences were considered to be signiﬁcant when the value of p , 0.05. Results and Discussion Type I IFN production from TLR9-stimulated cDC 2 2 An Ifna/br / pDC has severe defects in TLR9-induced type I IFN production (9), suggesting the involvement of positive feedback loop through IFN-a/b R. We have analyzed whether this feedback loop is also working in cDCs. Wild-type cDCs produced IFN-b, but not IFN-a, in response to the TLR9 agonist CpG DNA, and the IFN-b production increased in 2 2 a dose-dependent manner. Ifna/br / cDCs also produced FIGURE 1. IKKa is required for IFN-b induction by TLR7 or TLR9- comparable amounts of IFN-b (Supplemental Fig. 1). This stimulated cDCs. A, cDCs were stimulated with the indicated concentrations 2/2 of CpG DNA. Cytokine production was measured by ELISA. Data are IFN-b production was dependent on TLR9, because Tlr9 representative of three experiments. B–D, cDCs were stimulated with 0.1 mM cDCs failed to produce any IFN-b upon stimulation with CpG DNA, 100 nM R848, or 100 ng/ml LPS. At the indicated time points, CpG DNA (data not shown). Thus, TLR9-stimulated cDCs total RNA was prepared and subjected to Northern blot analysis using probes can produce IFN-b in an IFN-a/b R-independent manner. for IFN-b, TNF-a, IL-12p40, or b-actin. The Journal of Immunology 3343

FIGURE 2. Role of the MyD88-associated molecules, TRAF3, Opn, and IRAK-1, in TLR9-induced IFN-b gene expression by cDCs. cDCs were stimulated with 0.1 mM CpG DNA. Northern blot analysis was performed as described in Fig. 1B–D.

a k a FIGURE 3. Role of IKK in IRF-1 activation. A, IRF-1 and NF- B ac- pDCs, IKK is critical for TLR7/9-provoked induction of tivation in TLR4- or TLR9-stimulated cDCs. cDCs were stimulated with 100 type I IFN. ng/ml LPS or 0.1 mM CpG DNA for the indicated time periods and sub- Although a pDC fails to respond to a TLR4 agonist, LPS, jected to Western blot analysis using Abs against IRF-1 or p65. An anti-lamin a cDC can upregulate expression of proinflammatory cytokines B Ab was used as a control Ab to ensure equal loading. B, Interaction of IKKa 2 2 and IFN-b upon stimulation with LPS (Fig. 1D). In Myd88 / with IRF-1. 293T cells were transfected with Myc-IKKa and/or FLAG-IRF- cDCs, induction of proinflammatory cytokine genes was 1. Immunoprecipitates were prepared and subjected to Western blot analysis with the indicated Abs. C, Recombinant IKKa was subjected to an in vitro abolished, but IFN-b gene induction was preserved as reported Downloaded from kinase assay. GST, GST-IRF-1, or GST-IkBa was used as substrates. The previously (21, 23). LPS-mediated IFN-b induction does not proteins were fractionated by SDS-PAGE and visualized by Coomassie bril- depend on MyD88 but on another cytoplasmic adaptor, Toll/ liant blue (CBB) staining or autoradiography. IL-1R domain-containing adaptor-inducing IFN-b (TRIF) 2/2 (23, 24). When an Ikka cDC was stimulated with LPS, dimer formation is required for LPS-induced IFN-b gene expression of IFN-b as well as proinflammatory cytokines was expression (32). However, CpG DNA fails to induce IRF-3 upregulated (Fig. 1D). These results indicate that IKKa is dimer formation (Supplemental Fig. 3). IRF-7 activation was http://www.jimmunol.org/ 2 2 critical for TLR7/9/MyD88- but not TLR4/TRIF-mediated severely impaired in Ikka / pDCs (12). However, amounts IFN-b gene induction in cDCs. of IFN-stimulated response element-binding complexes con- 2 2 cDCs can also produce type I IFNs in response to the dsRNA taining IRF-7 were increased in TLR9-stimulated Ikka / analog poly(I:C). This response depends on a cytosolic RNA cDCs (Supplemental Fig. 4). Thus, it is unlikely that IKKa is sensor, MDA5, which belongs to the RIG-I–like receptor critically involved in IFN-b gene upregulation in cDCs (RLR) family (25, 26). When stimulated with poly(I:C), wild- 2/2 through IRF-3 or IRF-7. type and Ikka cDCs produced comparable amounts of A cDC does not require IRF-7 or IRF-3 but instead requires IFN-a and IFN-b (Supplemental Fig. 2), indicating that IRF-1 for TLR9-induced IFN-b production (13, 14). We have by guest on September 28, 2021 IKKa is dispensable for RLR-induced type I IFN production investigated the nuclear translocation of IRF-1 and the NF-kB from cDCs. subunit, p65, in LPS or CpG DNA-stimulated cDCs (Fig. Roles of MyD88-associated molecules in IFN-b gene induction in 3A). Upon stimulation with LPS, wild-type cDCs increased nuclear IRF-1 levels, and this increase was not impaired in TLR9-stimulated cDCs 2 2 Ikka / cDCs. When stimulated with CpG DNA, wild-type Several molecules have been reported to be involved in TLR7/9- mediated type I IFN production by pDCs. TRAF3 associates with the TLR adaptors MyD88 and TRIF as well as IKK family members, such as TANK-binding kinase 1, and is critically involved in TLR- and RLR-induced type I IFN production. TRAF3 deficiency leads to defective induction of type I IFNs by RLRs and TLRs, including TLR4, TLR7, and TLR9 (27, 28). Opn functions as an intracellular signaling molecule and, by associating with MyD88, plays critical roles in IRF-7 activation and type I IFN production in pDCs (29). Furthermore, a serine threonine kinase, IRAK-1, also associates with MyD88 and IRF-7 and is critical for TLR7/9-induced type I IFN production (30). 2 2 2 2 2 2 As with wild-type cDCs, Traf3 / , Opn / , and Irak1 / cDCs upregulated IFN-b and IL-12p40 gene expression after TLR9 stimuli (Fig. 2). Thus, IKKa is distinguished from these three molecules in terms of the involvement in TLR9- induced type I IFN production by both pDCs and cDCs.

2 2 Impaired activation of IRF-1 and NF-kB in TLR9-stimulated Ikka / FIGURE 4. Involvement of IKKa in serum IFN-b elevation after injection 2 2 cDCs of CpG DNA. Wild-type and Ifna/br / mice (A) or BM chimeric mice (B) were injected i.p. with CpG DNA and D-(+)-galactosamine and bled at the IFN-b gene expression is regulated by several IRF family indicated times. Then serum IFN-b levels were measured by ELISA. Hori- members or NF-kB (31). Which signaling molecules are ac- zontal bars indicate means. Student t test was used to determine statistical tivated depends on the stimuli or stimulated cell types. IRF-3 significance between groups (*p , 0.05). 3344 CUTTING EDGE: IkB KINASE a IN TLR7/9 SIGNALING cDCs also had elevated levels of IRF-1 in the nucleus, but this We have also examined other types of cDCs including 2 2 response was defective in Ikka / cDCs. Nuclear p65 protein splenic or Flt3 ligand-induced BM cDCs (11). IKKa was levels were also increased in wild-type cDCs after stimulation required for these cDCs to produce IFN-b in response to with LPS or CpG DNA. This increase, however, was signifi- TLR9 signaling (Supplemental Fig. 5). In these cDCs, IKKa 2 2 cantly impaired in CpG DNA- but not LPS-stimulated Ikka / was, although partially, involved also in other cytokine gene cDCs. These results indicate that IKKa is required for IRF-1 induction. IKKa should be considered as a unique molecular and p65 activation in TLR9-stimulated cDCs. target for manipulating type I IFN induction by TLR7/9 We then analyzed whether IKKa can interact with IRF-1 independently of the DC subsets. (Fig. 3B). When FLAG-tagged IRF-1 was expressed with Myc- tagged IKKa in 293T cells, IKKa was coimmunoprecipitated with IRF-1. Moreover, an in vitro kinase assay revealed that Acknowledgments IKKa could phosphorylate IRF-1 as well as IkBa (Fig. 3C). We thank N. Iwami, Y. Fukuda, and E. Haga for technical assistance and S. In cDCs, IRF-1 interacts and colocalizes with MyD88. After Haraguchi for secretarial assistance. We also thank P. Burrows for critical read- TLR9 stimuli, IRF-1 is licensed and migrates into the nucleus. ing of the manuscript. IRF-1 is phosphorylated in a MyD88-dependent manner (13), although it remains unclear whether IRF-1 phosphorylation is critical for its activation or nuclear translocation. A critical Disclosures 2/2 involvement of IRF-1 was shown by the finding that Irf1 The authors have no financial conflicts of interest. Downloaded from cDCs fail to produce IFN-b in response to TLR9 (13, 14). 2 2 2 2 Similar to Ikka / cDCs, Irf1 / cDCs retained the ability to produce TNF-a or IL-12p40 in response to CpG DNA. Im- References portantly, we have found that nuclear translocation of IRF-1 1. Akira, S., S. Uematsu, and O. Takeuchi. 2006. Pathogen recognition and innate 2 2 was decreased in TLR9-stimulated Ikka / cDCs, indicating immunity. Cell 124: 783–801. 2. Medzhitov, R. 2007. 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SUPPLEMENTAL FIGURES

Supplemental Figure 1. IFN-α/β R-independent IFN-β production from TLR9- stimulated cDC. Cells were stimulated with the indicated concentrations of CpG DNA. Cytokine production was measured by ELISA. Data are representative of four experiments.

1 Supplemental Data

Supplemental Figure 2. IKKα is dispensable for RLR-induced type I IFN production. cDCs were stimulated with the indicated concentrations of poly(I:C). Cytokine production was measured by ELISA. Data are representative of three experiments.

2 Supplemental Data

Supplemental Figure 3. IRF-3 dimer formation. Wildtype cDCs were stimulated for indicated time periods with 100ng/ml LPS or 0.1μM CpG DNA. Nuclear extracts (10μg protein per lane) were electrophoresed with the running buffer (25mM Tris, 192mM glycine, in the presence and absence of 0.2% deoxycholate in the cathode and anode buffer, respectively) on 7.5% polyacrylamide gel. IRF-3 dimer was detected by immunoblot analysis with anti-IRF-3 Ab (United States Biological, Swampscott, MA, USA). Data are representative of three experiments.

3 Supplemental Data

Supplemental Figure 4. IRF-7 activation in TLR9-stimulated cDC. cDCs were stimulated with 0.1μM CpG DNA for the indicated time periods. Nuclear cell lysates were prepared and subjected to EMSA for ISRE-binding activity. Anti-IRF-7 Ab and normal rabbit IgG (control

Ab) were used for supershift analysis. The arrow indicates the supershifted bands.

4 Supplemental Data

Supplemental Figure 5. Cytokine gene induction in TLR9-stimulated splenic or

Flt3L-induced BM cDC. Single cell suspension was prepared from spleens of Ikka+/- (control) or Ikka-/- chimeric mice (20). Cells were stained with FITC-mPDCA-1 (Miltenyi Biotec),

PE-B220 (eBioscience), APC-CD11c (eBioscience), biotinylated anti-CD45.1 (BD

Bioscience), and streptavidin-PE-Cy7 (eBioscience). CD11c+B220-PDCA-1-CD45.1- cells were sorted as donor-derived splenic cDCs with BD FACSAria II cell sorter (BD Bioscience).

Flt3L-induced BM DCs were generated as described previously (22) and

CD11c+B220-PDCA-1-CD45.1- cells were sorted as Flt3L-induced BM cDCs. Cells were stimulated with 0.1μM CpG DNA for indicated periods. Quantitative real-time RT-PCR analysis was performed using a 7000 Sequence detector (Applied Biosystems). Reactions were performed with the following primers: 18S rRNA (internal control), Ifnb1, Tnf, and

Il12b (Taqman Gene Expression Assays, Applied Biosystems). Data are representative of two

(splenic cDC) and three (Flt3L-induced BM cDC) experiments.