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by Conventional Dendritic Cellsin TLR7/9-Induced Type I IFN Production

αB Kinase κCutting Edge: Critical Role of I

Kikutani and Tsuneyasu KaishoTakahiro Yano, Chihiro Yamazaki, Teruhito Yasui, Hitoshi Katsuaki Hoshino, Izumi Sasaki, Takahiro Sugiyama,

http://www.jimmunol.org/content/184/7/3341doi: 10.4049/jimmunol.0901648March 2010;

2010; 184:3341-3345; Prepublished online 3J Immunol 

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8.DC1http://www.jimmunol.org/content/suppl/2010/03/01/jimmunol.090164

Referenceshttp://www.jimmunol.org/content/184/7/3341.full#ref-list-1

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CuttingEdge:CriticalRoleof IkBKinasea inTLR7/9-InducedType I IFN Production by Conventional Dendritic CellsKatsuaki Hoshino,*,1 Izumi Sasaki,*,†,1 Takahiro Sugiyama,* Takahiro Yano,*Chihiro Yamazaki,*,† Teruhito Yasui,‡ Hitoshi Kikutani,‡ and Tsuneyasu Kaisho*,†,x

A plasmacytoid dendritic cell (DC) can produce largeamounts of type I IFNs after sensing nucleic acids throughTLR7 and TLR9. IkB kinase a (IKKa) is critically in-volved in this type I IFN production through its interac-tion with IFN regulatory factor-7. In response to TLR7/9signaling, conventional DCs can also produce IFN-bbut not IFN-a in a type I IFN-independent manner. Inthis study, we showed that IKKa was required for pro-duction of IFN-b, but not of proinflammatory cytokines,by TLR7/9-stimulated conventional DCs. Importantly,IKKa was dispensable for IFN-b gene upregulation byTLR4 signaling. Biochemical analyses indicated thatIKKa exerted its effects through its interaction withIFNregulatory factor-1. Furthermore, IKKawas involvedinTLR9-induced type I IFN-independent IFN-b produc-tion in vivo. Our results show that IKKa is a unique mol-ecule involved in TLR7/9-MyD88–dependent type I IFNproduction through DC subset-specific mechanisms.The Journal of Immunology, 2010, 184: 3341–3345.

Dendritic cells (DCs) sense nucleic acid immuneadjuvants ssRNA and unmethylated CpG DNA viaTLR7 and TLR9, respectively, and produce proin-

flammatory cytokines or type I IFNs (1–4). Accumulatingevidence indicates that TLR7/9-induced type I IFN inductionis important not only for antiviral defense but also for thepathogenesis of certain autoimmune diseases (5, 6). There-fore, clarifying the underlying mechanisms of this type I IFNproduction should contribute to the development of im-munomanipulation for these diseases.TLR7 and TLR9 associate with the cytoplasmic adaptor,

MyD88, through the homophilic interaction of their re-spective Toll/IL-1R homologous domains (3). MyD88 is es-sential for all TLR7/9-mediated effects. Signaling downstream

of MyD88 bifurcates into two pathways leading to the acti-vation of NF-kB and IFN regulatory factor (IRF)-7 (7). NF-kB activation leads to production of proinflammatory cyto-kines and requires phosphorylation and degradation of IkB.IkB phosphorylation depends mainly on a serine threoninekinase, IkB kinase (IKK)b (8).IRF-7 activation leads to type I IFN production (9). This

pathway functions mainly in a specialized DC subset, theplasmacytoid DC (pDC) (10, 11). The pDC expresses TLR7and TLR9 exclusively among TLRs, has constitutively highlevels of IRF-7, and is notable for its potent ability to producetype I IFNs, including IFN-a and IFN-b, following TLR7/9signaling. IRF-7 activation requires its phosphorylation, andwe have found that IKKa, also known as Chuk, is involved inthis IRF-7 activation (12).In response to TLR7/9 signaling, another DC subset, the

conventional DC (cDC), can also produce IFN-b, but notIFN-a, through the distinct molecularmechanisms frompDCs(13, 14). In this study, we have investigated the involvement ofIKKa in IFN-b production by TLR7/9-stimulated cDCs.

Materials and MethodsMice

Ikka2/2, Myd882/2, and Tlr42/2 mice have been described previously (15–17). TNFR-associated factor 3 (TRAF3)-deficient (Traf32/2) mice weregenerated by T. Yasui and H. Kikutani (manuscript in preparation). C57BL/6, osteopontin (Opn)-deficient (Opn2/2), and IFN-a/b R-deficient (Ifna/br2/2) mice were purchased from CLEA Japan (Shizuoka, Japan),The Jackson Laboratory (Bar Harbor, ME), and B&K Universal (Hull, U.K.),respectively. IL-1R–associated kinase-1 (IRAK-1)–deficient (Irak12/2) micewere provided by Dr. J. A. Thomas (University of Texas SouthwesternMedical Center, Dallas, TX) (18). Bone marrow (BM) chimeric micewere generated by transferring wild-type, Ikka2/2, or Traf32/2 fetalliver cells into C57BL/6 (CD45.1) mice (19, 20). Ikka+/+Ifna/br2/2 andIkka2/2Ifna/br2/2 chimeric mice were generated by transferring fetalliver cells from Ikka+/+Ifna/br2/2 or Ikka2/2Ifna/br2/2 mice into irradiatedIkka+/+Ifna/br2/2 mice. All mice were maintained under specific pathogen-free conditions, and animal experiments were conducted according to theinstitutional guidelines.

*Laboratory for Host Defense, RIKEN Research Center for Allergy and Immunology;xDepartment of Supramolecular Biology, Graduate School of Nanobioscience, Yokoha-ma City University, Yokohama; and †Department of Allergy and Immunology, Grad-uate School of Medicine and ‡Department of Molecular Immunology, Research Institutefor Microbial Diseases and World Premier International Immunology Frontier ResearchCenter, Osaka University, Osaka, Japan

1K.H. and I.S. contributed equally to this work.

Received for publication May 27, 2009. Accepted for publication January 27, 2010.

This work was supported by grants from the Ministry of Education, Culture, Sports,Science, and Technology and the Japan Science and Technology Corporation, theUehara Memorial Foundation, the Mochida Memorial Foundation for Medical andPharmaceutical Research, and the Japan Intractable Diseases Research Foundation.

Address correspondence and reprint requests to Dr. Tsuneyasu Kaisho, Laboratory forHost Defense, RIKEN Research Center for Allergy and Immunology, Suehiro-cho 1-7-

22, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. E-mail address: tkaisho@rcai.riken.jp

The online version of this article contains supplemental material.

Abbreviations used in this paper: BM, bone marrow; CBB, Coomassie brilliant blue;cDC, conventional dendritic cell; DC, dendritic cell; IKK, IkB kinase; IRAK-1, IL-1R–associated kinase-1; IRF, IFN regulatory factor; Opn, osteopontin; pDC, plasmacytoiddendritic cell; poly(I:C), polyinosinic:polycytidylic acid; RLR, RIG-I–like receptor;TRAF3, TNFR-associated factor 3; TRIF, Toll/IL-1R domain-containing adaptor-inducing IFN-b.

Copyright� 2010 by TheAmerican Association of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901648

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Cells

GM-CSF–induced BM DCs were generated as previously described (21) andused as cDCs.

Reagents

A phosphorothioate oligodeoxynucleotide containing an unmethylated CpGmotif, ODN1668, was used as CpG DNA (22). LPS derived from Salmonellaminnesota Re595, polyinosinic:polycytidylic acid [poly(I:C)], and R848 werepurchased from Sigma-Aldrich (St. Louis, MO), Amersham Biosciences(Piscataway, NJ), and InvivoGen (San Diego, CA), respectively.

Measurement of cytokine production

Cells were treated for 20–24 h with the indicated stimuli, and cytokineproduction was measured by ELISA as described previously (12).

Northern blot analysis

Northern blot analysis was performed as described previously (21).

EMSA

IFN-stimulated response element-binding activities were analyzed as describedpreviously (12).

Nuclear translocation of IRF-1 and NF-kB

Nuclear extracts were subjected to immunoblot analyses with anti–IRF-1 oranti-p65 Abs (Santa Cruz Biotechnology, Santa Cruz, CA) as describedpreviously (12).

Immunoprecipitation assay

The 293T human embryonic kidney cell line was transiently transfected withexpression 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 immunoblottingexperiments were performed as described previously (12).

In vitro kinase assay

One hundred nanograms of purified recombinant IKKa (Upstate Bio-technology, Lake Placid, NY) was incubated with 1 mg substrates, GST, GSTfused to residues 2–329 of mouse IRF-1 (GST-IRF-1), or GST fused toresidues 5–55 of mouse IkBa (GST-IkBa) and subjected to an in vitro kinaseassay 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 differencesbetween groups. Prism (Graphpad Software, La Jolla, CA) was used for sta-tistical analysis. Differences were considered to be significant when the valueof p , 0.05.

Results and DiscussionType I IFN production from TLR9-stimulated cDC

An Ifna/br2/2 pDC has severe defects in TLR9-induced type IIFN production (9), suggesting the involvement of positivefeedback loop through IFN-a/b R. We have analyzed whetherthis feedback loop is also working in cDCs. Wild-type cDCsproduced IFN-b, but not IFN-a, in response to the TLR9agonist CpG DNA, and the IFN-b production increased ina dose-dependent manner. Ifna/br2/2 cDCs also producedcomparable amounts of IFN-b (Supplemental Fig. 1). ThisIFN-b production was dependent on TLR9, because Tlr92/2

cDCs failed to produce any IFN-b upon stimulation withCpG DNA (data not shown). Thus, TLR9-stimulated cDCscan produce IFN-b in an IFN-a/b R-independent manner.

Critical roles of IKKa in IFN-b production by TLR7/9-stimulatedcDCs

We have analyzed involvement of IKKa. TLR9-induced IFN-b induction was severely impaired in Ikka2/2 cDCs (Fig. 1A).This was a specific effect, because wild-type and Ikka2/2

cDCs produced comparable amounts of proinflammatorycytokines, such as IL-12p40 and TNF-a, in response toTLR9 stimuli (Fig. 1A).We next investigated expression of these cytokine genes by

Northern blot analysis (Fig. 1B, 1C). CpG DNA could up-regulate expression of IFN-b as well as proinflammatory cy-tokine genes in wild-type but not in Myd882/2 cDCs,indicating their dependence on MyD88. Ikka2/2 cDCsshowed a severe defect in TLR9-induced IFN-b gene ex-pression (Fig. 2B) but normal induction of IL-12p40 andTNF-a gene expression. Similar defects were observed also inTLR7-stimulated Ikka2/2 cDCs (Fig. 2C). Thus, as with

FIGURE 1. IKKa is required for IFN-b induction by TLR7 or TLR9-

stimulated cDCs. A, cDCs were stimulated with the indicated concentrations

of CpG DNA. Cytokine production was measured by ELISA. Data are

representative of three experiments. B–D, cDCs were stimulated with 0.1 mM

CpG DNA, 100 nM R848, or 100 ng/ml LPS. At the indicated time points,

total RNA was prepared and subjected to Northern blot analysis using probes

for IFN-b, TNF-a, IL-12p40, or b-actin.

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pDCs, IKKa is critical for TLR7/9-provoked induction oftype I IFN.Although a pDC fails to respond to a TLR4 agonist, LPS,

a cDC can upregulate expression of proinflammatory cytokinesand IFN-b upon stimulation with LPS (Fig. 1D). InMyd882/2

cDCs, induction of proinflammatory cytokine genes wasabolished, but IFN-b gene induction was preserved as reportedpreviously (21, 23). LPS-mediated IFN-b induction does notdepend on MyD88 but on another cytoplasmic adaptor, Toll/IL-1R domain-containing adaptor-inducing IFN-b (TRIF)(23, 24). When an Ikka2/2 cDC was stimulated with LPS,expression of IFN-b as well as proinflammatory cytokines wasupregulated (Fig. 1D). These results indicate that IKKa iscritical for TLR7/9/MyD88- but not TLR4/TRIF-mediatedIFN-b gene induction in cDCs.cDCs can also produce type I IFNs in response to the dsRNA

analog poly(I:C). This response depends on a cytosolic RNAsensor, MDA5, which belongs to the RIG-I–like receptor(RLR) family (25, 26). When stimulated with poly(I:C), wild-type and Ikka2/2 cDCs produced comparable amounts ofIFN-a and IFN-b (Supplemental Fig. 2), indicating thatIKKa is dispensable for RLR-induced type I IFN productionfrom cDCs.

Roles of MyD88-associated molecules in IFN-b gene induction inTLR9-stimulated cDCs

Severalmolecules have been reported to be involved inTLR7/9-mediated type I IFN production by pDCs. TRAF3 associateswith the TLR adaptorsMyD88 and TRIF as well as IKK familymembers, such as TANK-binding kinase 1, and is criticallyinvolved in TLR- and RLR-induced type I IFN production.TRAF3 deficiency leads to defective induction of type I IFNs byRLRs and TLRs, including TLR4, TLR7, and TLR9 (27, 28).Opn functions as an intracellular signaling molecule and, byassociating withMyD88, plays critical roles in IRF-7 activationand type I IFN production in pDCs (29). Furthermore, a ser-ine threonine kinase, IRAK-1, also associates with MyD88 andIRF-7 and is critical for TLR7/9-induced type I IFN pro-duction (30).As with wild-type cDCs, Traf32/2, Opn2/2, and Irak12/2

cDCs upregulated IFN-b and IL-12p40 gene expression afterTLR9 stimuli (Fig. 2). Thus, IKKa is distinguished fromthese three molecules in terms of the involvement in TLR9-induced type I IFN production by both pDCs and cDCs.

Impaired activation of IRF-1 and NF-kB in TLR9-stimulated Ikka2/2

cDCs

IFN-b gene expression is regulated by several IRF familymembers or NF-kB (31). Which signaling molecules are ac-tivated depends on the stimuli or stimulated cell types. IRF-3

dimer formation is required for LPS-induced IFN-b geneexpression (32). However, CpG DNA fails to induce IRF-3dimer formation (Supplemental Fig. 3). IRF-7 activation wasseverely impaired in Ikka2/2 pDCs (12). However, amountsof IFN-stimulated response element-binding complexes con-taining IRF-7 were increased in TLR9-stimulated Ikka2/2

cDCs (Supplemental Fig. 4). Thus, it is unlikely that IKKa iscritically involved in IFN-b gene upregulation in cDCsthrough IRF-3 or IRF-7.A cDC does not require IRF-7 or IRF-3 but instead requires

IRF-1 for TLR9-induced IFN-b production (13, 14). We haveinvestigated the nuclear translocation of IRF-1 and the NF-kBsubunit, p65, in LPS or CpG DNA-stimulated cDCs (Fig.3A). Upon stimulation with LPS, wild-type cDCs increasednuclear IRF-1 levels, and this increase was not impaired inIkka2/2 cDCs. When stimulated with CpG DNA, wild-type

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.

FIGURE 3. Role of IKKa in IRF-1 activation. A, IRF-1 and NF-kB ac-

tivation in TLR4- or TLR9-stimulated cDCs. cDCs were stimulated with 100

ng/ml LPS or 0.1 mM CpG DNA for the indicated time periods and sub-

jected to Western blot analysis using Abs against IRF-1 or p65. An anti-lamin

B Ab was used as a control Ab to ensure equal loading. B, Interaction of IKKawith IRF-1. 293T cells were transfected with Myc-IKKa and/or FLAG-IRF-

1. Immunoprecipitates were prepared and subjected to Western blot analysis

with the indicated Abs. C, Recombinant IKKa was subjected to an in vitro

kinase assay. GST, GST-IRF-1, or GST-IkBa was used as substrates. The

proteins were fractionated by SDS-PAGE and visualized by Coomassie bril-

liant blue (CBB) staining or autoradiography.

FIGURE 4. Involvement of IKKa in serum IFN-b elevation after injection

of CpG DNA. Wild-type and Ifna/br2/2 mice (A) or BM chimeric mice (B)were injected i.p. with CpG DNA and D-(+)-galactosamine and bled at the

indicated times. Then serum IFN-b levels were measured by ELISA. Hori-

zontal bars indicate means. Student t test was used to determine statistical

significance between groups (*p , 0.05).

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cDCs also had elevated levels of IRF-1 in the nucleus, but thisresponse was defective in Ikka2/2 cDCs. Nuclear p65 proteinlevels were also increased in wild-type cDCs after stimulationwith LPS or CpG DNA. This increase, however, was signifi-cantly impaired in CpGDNA- but not LPS-stimulated Ikka2/2

cDCs. These results indicate that IKKa is required for IRF-1and p65 activation in TLR9-stimulated cDCs.We then analyzed whether IKKa can interact with IRF-1

(Fig. 3B). When FLAG-tagged IRF-1 was expressed with Myc-tagged IKKa in 293T cells, IKKa was coimmunoprecipitatedwith IRF-1. Moreover, an in vitro kinase assay revealed thatIKKa could phosphorylate IRF-1 as well as IkBa (Fig. 3C).In cDCs, IRF-1 interacts and colocalizes with MyD88. After

TLR9 stimuli, IRF-1 is licensed and migrates into the nucleus.IRF-1 is phosphorylated in a MyD88-dependent manner (13),although it remains unclear whether IRF-1 phosphorylation iscritical for its activation or nuclear translocation. A criticalinvolvement of IRF-1 was shown by the finding that Irf12/2

cDCs fail to produce IFN-b in response to TLR9 (13, 14).Similar to Ikka2/2 cDCs, Irf12/2 cDCs retained the ability toproduce TNF-a or IL-12p40 in response to CpG DNA. Im-portantly, we have found that nuclear translocation of IRF-1was decreased in TLR9-stimulated Ikka2/2 cDCs, indicatingthat IKKa is required for IRF-1 activation by TLR9 signaling.Our present results also demonstrated that IKKa could asso-ciate with IRF-1 and phosphorylate IRF-1. Thus, it can beassumed that IKKa is involved in TLR7/9-mediated IFN-binduction through its interaction with IRF-1. Nuclear trans-location of the NF-kB p65 subunit was also decreased inTLR9-stimulated Ikka2/2 cDCs. However, this decrease didnot lead to defective production of proinflammatory cytokines.

In vivo roles of IKKa in TLR9-induced type I IFN-independentIFN-b gene induction

We have further investigated whether and how the mechanismsdefined in the in vitro cDCs contribute to in vivo responses. Forthis purpose, we have analyzed the role of IKKa in TLR9-induced IFN-b production in the absence of IFN-a/b R sig-naling. As with wild-type mice, Ifna/br2/2 mice injected withCpG DNA showed significant or even exaggerated elevation ofserum IFN-b (Fig. 4A). The results indicate that CpG DNAcan induce IFN-b in vivo in a type I IFN-independent manner.We have further examined the importance of IKKa in Ifna/

br2/2 genetic backgrounds. Compared with Ikka+/+Ifna/br2/2

chimeric mice, the Ikka2/2Ifna/br2/2 mice had a severe de-fect in the elevation of serum IFN-b levels after the injectionof CpG DNA (Fig. 4B). Thus, IKKa is required for type IIFN-independent IFN-b induction in vivo by TLR9.IFN-b production from cDCs is less than that from pDCs.

However, the cDChas a potent ability to activate T cells becauseof a higher expression of costimulatory molecules than thepDC and, moreover, is numerically much more plentiful thanthe pDC. Therefore, type I IFN produced by cDCs shouldplay critical roles in shaping immune responses. Notably, un-like pDCs, cDCs can produce type I IFN in an IFN-a/b R-independent manner, and Ifna/br2/2 mice had a significant in-crease in serum IFN-b levels after injection of a TLR9 agonist.Although it is currently unclear why the increase is more evidentin Ifna/br2/2 mice than in wild-type mice, the results clearlyindicate that TLR9-mediated IFN-a/b R-independent IFN-binduction also functions in an IKKa-dependent manner in vivo.

We have also examined other types of cDCs includingsplenic or Flt3 ligand-induced BM cDCs (11). IKKa wasrequired for these cDCs to produce IFN-b in response toTLR9 signaling (Supplemental Fig. 5). In these cDCs, IKKawas, although partially, involved also in other cytokine geneinduction. IKKa should be considered as a unique moleculartarget for manipulating type I IFN induction by TLR7/9independently of the DC subsets.

AcknowledgmentsWe thank N. Iwami, Y. Fukuda, and E. Haga for technical assistance and S.

Haraguchi for secretarial assistance. We also thank P. Burrows for critical read-

ing of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

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