of June 21, 2018.This information is current as

in Dendritic Cellsversus RLR-Induced Type 1 IFN Expression B Signaling Module in Regulating TLR-

κ/NF-βDifferential Requirement for the IKK

BegAdolfo García-Sastre, Siddharth Balachandran and Amer A.Emily L. Hopewell, Randy A. Albrecht, Shoko Nogusa, Xingyu Wang, Junmei Wang, Hong Zheng, Mengyu Xie,

http://www.jimmunol.org/content/193/5/2538doi: 10.4049/jimmunol.1400675July 2014;

2014; 193:2538-2545; Prepublished online 23J Immunol 

MaterialSupplementary

5.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/07/23/jimmunol.140067

Referenceshttp://www.jimmunol.org/content/193/5/2538.full#ref-list-1

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is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Differential Requirement for the IKKb/NF-kB SignalingModule in Regulating TLR- versus RLR-Induced Type 1 IFNExpression in Dendritic Cells

Xingyu Wang,*,1 Junmei Wang,*,1 Hong Zheng,* Mengyu Xie,* Emily L. Hopewell,*,†

Randy A. Albrecht ,‡,x Shoko Nogusa,{ Adolfo Garcıa-Sastre ,‡,x,‖

Siddharth Balachandran ,{ and Amer A. Beg*

Host innate-immune responses are tailored by cell type to control and eradicate specific infectious agents. For example, an acute

RNAvirus infection can result in high-level expression of type 1 IFNs by both conventional dendritic cells (cDCs) and plasmacytoid

dendritic cells (pDCs), but whereas cDCs preferentially use RIG-I–like receptor (RLR) signaling to produce type 1 IFNs, pDCs

predominantly use TLRs to induce these cytokines. We previously found that the IkB kinase b (IKKb)/NF-kB pathway regulates

early IFN-b expression, but not the magnitude of type 1 IFN expression following RLR engagement. In this study, we use IKKb

inhibition and mice deficient in IKKb or canonical NF-kB subunits (p50, RelA/p65, and cRel) to demonstrate that the IKKb/NF-

kB axis is critical for virus-induced type 1 IFN expression in pDCs, but not in cDCs. We also reveal a crucial and more general

requirement for IKKb/NF-kB in TLR- but not RLR-induced expression of type 1 IFNs and inflammatory cytokines. Together,

these findings reveal a previously unappreciated specificity of the IKKb/NF-kB signaling axis in regulation of antimicrobial

responses by different classes of pattern recognition receptors, and therefore by individual cell types reliant on particular pattern

recognition receptors for their innate-immune transcriptional responses. The Journal of Immunology, 2014, 193: 2538–2545.

The mammalian immune system is exquisitely sensitive tothe nature of invading infectious agents and tailors hostresponses that are best suited to eradicate specific agents

(1, 2). Mammalian pattern recognition receptors (PRRs) such asTLRs, RIG-I–like receptors (RLRs), cGAS/STING, and NOD-likereceptors play key roles in protective innate and adaptive immuneresponses against microbial agents (3–8). Because different PRRsrecognize distinct microbial components, and because individualimmune cell types differ in the classes of PRR they express, hostinnate-immune responses to an infectious agent are specified atboth the molecular and the cellular level. For example, infection of

different dendritic cell (DC) subsets with bacterial, viral, or fungalpathogens triggers distinct and specialized transcriptional responsesin each subset (1), the molecular basis for which remains an area ofintense research.DCs are a mobile subset of leukocytes specialized for regulating

diverse host responses against microbes (9–12). They are broadly

characterized as conventional DCs (cDCs) and plasmacytoid DCs

(pDCs). cDCs play a crucial role in T cell activation and express

high levels of costimulatory molecules, proinflammatory cyto-

kines, and type 1 IFNs following direct infection by virus (13–15).

pDCs (also known as IFN-producing cells) are a unique subset of

circulating DCs that, upon stimulation by microbial components,

are specialized in the production of very high levels of type 1 IFN

(15–19). Recent studies have revealed critical differences in the

manner in which cDCs and pDCs produce type 1 IFNs following

an acute RNA virus infection. Although cDCs are critically reliant

on the RLR pathway for induction of type 1 IFNs following in-

fection, pDCs instead employ TLR-dependent sensing mecha-

nisms to detect RNA viruses (20–22). In particular, TLR7 and

TLR9 expression in pDCs detects viral ssRNA and DNA, re-

spectively, leading to high type 1 IFN expression (8, 23).Members of the IFN regulatory factor (IRF) and NF-kB tran-

scription factor families are activated following engagement of

multiple PRRs (3, 23–31). Both IRF3 and IRF7 are crucial for

inducing IFN-a/b (32, 33). However, although IRF3 contributes to

IFN expression following RLR engagement in cDCs, IRF7 is

essential for optimal IFN expression in pDCs (32–35). NF-kB

activation by PRRs typically depends on IkB kinase b (IKKb)

(36). Our previous studies indicate that although IKKb/NF-kB is

required for early induction of IFN-b after virus infection, it does

not regulate the magnitude of type 1 IFN expression following

virus triggering of the RLR pathway in cDCs and mouse embry-

onic fibroblasts (MEFs) (37, 38). In the current study, we exam-

ined the role of the NF-kB signaling axis in TLR signaling. We

*Department of Immunology, Moffitt Cancer Center, Tampa, FL 33612; †CancerBiology Ph.D. Program, Department of Oncologic Sciences, University of SouthFlorida, Tampa, FL 33612; ‡Department of Microbiology, Icahn School of Medicineat Mount Sinai, New York, NY 10029; xGlobal Health and Emerging PathogensInstitute, Icahn School of Medicine at Mount Sinai, New York, NY 10029; {FoxChase Cancer Center, Philadelphia, PA 19111; and ‖Department of Medicine, Divi-sion of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY10029

1X.W. and J.W. contributed equally to this work.

Received for publication March 14, 2014. Accepted for publication June 24, 2014.

This work was supported by National Institutes of Health Grants R01 AI059715 andDOD BC011057, and institutional funds from the Moffitt Cancer Center (to A.A.B.).This work was partially supported by National Institute of Allergy and InfectiousDiseases Grants U19AI083025 and U01AI095611 (to A.G.-S.) and by National In-stitutes of Health Grant R21 AI104212 (to S.B.).

Address correspondence and reprint requests to Dr. Amer A. Beg, Moffitt CancerCenter, SRB-2, 12902 Magnolia Drive, Tampa, FL 33612. E-mail address: amer.beg@moffitt.org

The online version of this article contains supplemental material.

Abbreviations used in this article: BM-pDC, bone marrow–derived pDC; cDC, con-ventional DC; DC, dendritic cell; IKKb, IkB kinase b; IRF, IFN regulatory factor;MEF, mouse embryonic fibroblast; NDV, Newcastle disease virus; pDC, plasmacy-toid DC; poly(I:C), polyinosinic-polycytidylic acid; PRR, pattern recognition recep-tor; RLR, RIG-I–like receptor; SeV, Sendai virus; WT, wild-type.

Copyright� 2014 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400675

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report that, in contrast to the RLR pathway, NF-kB is essential forthe induction of type 1 IFNs downstream of TLRs. We show thatinduction of type 1 IFNs by pDCs is severely defective whenIKKb is inhibited, or when components of the IKKb/canonicalNF-kB signaling module are genetically ablated. We also reveala broad requirement for NF-kB activity in TLR-driven expressionof type 1 IFNs and other proinflammatory genes in multiple celltypes. Together with our previous findings, these results demon-strate that the IKKb/NF-kB axis is essential for TLR- but notRLR-mediated induction of type 1 IFNs, indicative of a previouslyunappreciated specificity in transcription factor use by distinct celltypes and classes of PRRs.

Materials and MethodsMice and materials

RelA2/2, p502/2, and cRel2/2 mice have been described previously (39).IKKb+/2 mice (40) were kindly provided by Dr. Zhi-Wei Li (MoffittCancer Center). E14 wild-type (WT) and RelA2/2 fetal liver hematopoi-etic precursors (CD45.2) were adoptively transferred into lethally irra-diated CD45.1 recipient mice, as previously described (39). Recipientmice were typically used after 6–8 wk. All experiments with mice werecarried out in accordance with institutional guidelines. E. coli LPS 0127:B8 (Sigma-Aldrich) was used at 100 ng/ml, and R837 (imiquimod; Invi-trogen) was used at 5 mg/ml. The IKKb inhibitor PS-1145 was used at10 mM and was obtained from Sigma-Aldrich. PS-1145 does not preventphosphorylation (activation) of IKKb but inhibits the ability to phos-phorylate substrates (41).

Cells

Bone marrow–derived cDCs were cultured from mouse bone marrowprecursors, as described (38). To obtain Flt-3 ligand–induced bone mar-row–derived pDCs (BM-pDCs), bone marrow cells (2 3 106 cells permilliliter) were cultured in 24-well plates in DC medium (RPMI 1640, 5%FBS, 5 mM 2-ME, antibiotics) in the presence of 200 ng/ml Flt-3 ligand.On day 7, pDCs were purified using anti-B220 MicroBeads (MiltenyiBiotec) and resuspended in fresh DC medium. Mouse splenic pDCs werepurified using anti–mPDCA-1 magnetic beads or FACS sorted (B220+

CD11cint population) and cultured in DC medium. MEFs were preparedfrom day 12.5 embryos.

Sendai virus and Newcastle disease virus infections

Unless otherwise indicated, Sendai virus (SeV) was used at 200 hemag-glutinin units/ml, and Newcastle disease virus (NDV) was used at a mul-tiplicity of infection of 2 in DCs and 10 in MEFs, as described previously(38). For infection with NDV in vivo, mice received i.v. injections of virus(1 3 107 PFU per mouse) or saline control. At 8 h after injection, splenicpDCs were purified using anti–mPDCA-1 MicroBeads (Miltenyi Biotec).

Real-time PCR, RNase protection assay, immunoblotting, andtransfection

Real-time PCR methods and primers were used as previously described(38). Whole-cell extracts were prepared by lysing cells in RIPA buffer (25mM Tris-HCl, pH 7.9, 150 mM NaCl, 2 mM EDTA, 0.5% Nonidet P-40)supplemented with protease inhibitor mixture (Roche) and phosphataseinhibitors (NaF and Na3VO4). The extracts were subjected to immunoblotanalysis using Abs against phospho-IKKa/b and total IKKb (Cell Sig-naling). IRF3 dimerization was detected by native-gel immunoblot anal-ysis, as described (42). Western blotting for IRF3 was performed usinganti-IRF3 Ab (Zymed). The RNase protection assay was performedas previously described (43). Polyinosinic-polycytidylic acid [poly(I:C)](Invivogen) was used as dsRNA for transfection, using electroporation aspreviously described (44). Briefly, 4 3 106 cDCs were resuspended in 300ml RPMI medium with or without 3 mg poly(I:C) and pulsed once at 300 V,150 mF (Bio-Rad Gene Pulser II). Cell suspensions were washed with 1.7ml DC media once, then seeded to low-attachment 24-well plates for 6 h,after which cells were collected for RT-PCR.

ELISA and EMSA

After different treatments, cells were cultured for the indicated time peri-ods, and supernatants were collected. Expression of IFN-a and IFN-b wasdetermined by ELISA kits from PBL Biomedical Laboratories followingthe manufacturer’s recommendations. Readings below the first standard

curve sample were considered undetected. For detection of NF-kB acti-vation, nuclear extracts were prepared and EMSA was performed as pre-viously described (45).

Statistical analysis

All statistical analysis was performed using the Student t test, with a p ,0.05 considered significant. The p values in the figure legends are specifiedas *p , 0.05, **p , 0.01, ***p , 0.001.

ResultsCrucial role of IKKb /NF-kB in virus-induced type 1 IFNexpression in pDCs

We previously found that the overall magnitude of type I IFNexpression after virus infection of cDCs is not altered in the absenceof NF-kB RelA, p50, or cRel subunits (38). However, RelA isuniquely required for basal and early virus-induced IFN-b ex-pression in cDCs and MEFs (37, 38, 46). Type 1 IFN expressionby pDCs is believed to be especially important for the host anti-virus response. To determine the potential role of the IKKb/NF-kB pathway in virus-induced type 1 IFN expression in pDCs, weused PS-1145, a highly specific IKKb inhibitor (41, 47). Of in-terest, SeV-induced type 1 IFN production in WT splenic pDCs aswell as BM-pDCs was significantly reduced in the presence of PS-1145 (Fig. 1A). Similar results were obtained following NDVinfection of splenic and BM-pDCs (Supplemental Fig. 1). Incontrast, and consistent with our previous results indicating lackof essential requirement for IKKb/NF-kB (37, 38), PS-1145 didnot inhibit SeV-induced expression of IFN-a or IFN-b in cDCs(Fig. 1B). Furthermore, no significant effect of PS-1145 was no-

FIGURE 1. Differential effect of IKKb inhibition on virus-induced type

1 IFN expression in cDCs and pDCs. (A) Effect of IKKb inhibitor PS-1145

on SeV infection–induced type 1 IFN production by splenic pDCs and

BM-pDCs determined by ELISA. (B) Effect of PS-1145 on SeV infection–

induced IFN-b and IFN-a protein expression in cDCs determined by

ELISA. All virus infections were for 6 h. pDCs. Representative results

from three independent experiments are shown.**p , 0.01, ***p , 0.001.

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ticed after 24-h SeV infection, whereas NDV-induced IFN-bwas only slightly reduced (Supplemental Fig. 2). Together, theseresults suggest that virus-induced type 1 IFN is selectively de-pendent on IKKb in pDCs, but not in cDCs.After SeV infection, IFN-b expression was slightly but signif-

icantly reduced by ∼30% in RelA2/2 cDCs, whereas no reduc-tion was noticed in p502/2, cRel2/2, and p502/2cRel2/2 cDCs(Fig. 2A). In contrast, IFN-b and IFN-a production was sub-

stantially impaired in pDCs derived from RelA2/2, p502/2,cRel2/2, and p502/2cRel2/2 knockout mice, with the most re-duction in RelA2/2 and p502/2cRel2/2 pDCs (Fig. 2B). Al-though absence of RelA in cDCs reduced IFN-b expression by∼30%, an ∼75% reduction in RelA absence was seen in pDCs(Fig. 2A, 2B). The p50 + cRel absence did not reduce IFN-bexpression in cDCs but significantly reduced it in pDCs (Fig. 2A,2B). Therefore, these NF-kB subunits are substantially more

FIGURE 2. NF-kB deficiency severely impairs in vitro and in vivo virus-induced type 1 IFN expression in pDCs. (A) ELISA analysis of IFN-b pro-

duction induced by SeV infection in WTand NF-kB knockout cDCs. (B) ELISA analysis of type 1 IFN production induced by SeV infection in WT and NF-

kB knockout splenic pDCs. (C) WT and p502/2cRel2/2 cDCs were transfected with poly(I:C) by electroporation. Poly(I:C) administration and transfection

are indicated. Real-time PCR analysis of IFN-b mRNA expression is shown. (D) Real-time PCR analysis of IFN-b mRNA expression in splenic pDCs from

WT and p502/2cRel2/2 mice infected with NDV. All virus infections were for 6 h. Representative results from two independent experiments are shown.

*p , 0.05, **p , 0.01, ***p , 0.001.

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important for IFN-b expression after virus infection of pDCsversus cDCs.Because absence of p50 + cRel does not affect mouse viability,

unlike RelA absence, we used p502/2cRel2/2 mice for severaladditional studies. First, we sought to more directly test the role ofthese NF-kB subunits in IFN-b expression after triggering of theRLR pathway. Transfection of dsRNA in cDCs is a powerfulstimulus for IFN-b expression through activation of the RLRpathway (44). Importantly, p502/2cRel2/2 cDCs induced robustand similar expression of IFN-b compared with WT cDCs afterdsRNA transfection (Fig. 2C). Second, unlike p502/2cRel2/2

pDCs (Fig. 2B), cDCs from p502/2cRel2/2 spleens inducednormal levels of IFN-b (Supplemental Fig. 3). Third, we deter-mined the in vivo role of p50 + cRel in virus-induced type 1 IFNexpression in pDCs. WT and p502/2cRel2/2 mice were infectedwith NDV (107 PFU per mouse). At 8 h post infection, mRNAlevels of IFN-b in splenic pDCs were determined. As shown inFig. 2D, IFN-b mRNA in pDCs from NDV-infected WT mice wasrobustly induced compared with the PBS control. In strikingcontrast, induction of IFN-b mRNA in pDCs from p502/2cRel2/2

mice was greatly reduced (Fig. 2D). These findings indicate acritical role for NF-kB p50 and cRel subunits in regulating virus-induced IFN-b expression in pDCs in vivo. In contrast, our resultsindicate that these NF-kB subunits are not essential for RLR-induced IFN-b expression triggered by dsRNA transfection (Fig.2C) or by virus infection of cDCs.

IKKb inhibition suppresses TLR-induced type 1 IFNexpression in cDCs

Asmentioned above, pDCs specifically require TLR, whereas cDCsrequire the RLR pathway for virus-induced type 1 IFN expression.An interesting possibility is that IKKb/NF-kB dependence on virus-induced type 1 IFN expression in pDCs is due to a unique re-quirement for this signaling module in TLR signaling. To test thispossibility, we determined whether TLR-induced type 1 IFN ex-pression in cDCs was dependent on IKKb/NF-kB. Importantly,unlike lack of effect after virus infection (Fig. 1B), TLR4 ligandLPS-induced IFN-b mRNA (not shown) and protein expressionwere substantially inhibited by PS-1145 in cDCs (Fig. 3A). TLR7ligand R837–induced IFN-b expression in cDCs was also signifi-cantly inhibited by PS-1145 (Fig. 3A). As expected, PS-1145significantly inhibited LPS-induced NF-kB activation (Fig. 3B)but had no appreciable effect on IRF3 activation (as determined byactivation-induced dimerization) in cDCs (Fig. 3C). These resultstherefore indicate that TLR-induced IKKb/NF-kB activation inboth pDCs (by virus infection) and cDCs (by LPS, R837) is re-quired for type 1 IFN expression.

IKKb is essential for TLR- but not RLR-induced type 1 IFNand inflammatory cytokine expression in MEFs

We wished to extend analysis of the role for IKKb using IKKb2/2

cDCs. Because IKKb deficiency results in embryonic day 12lethality (40), we used IKKb2/2 fetal liver cells to generateradiation chimeras for obtaining IKKb2/2 cDCs. However, aswe previously observed with p502/2RelA2/2 mice (39), spleencDCs, pDCs, or BM-derived cDCs were not obtained in the ab-sence of IKKb (data not shown). Similar to cDCs, virus-inducedtype 1 IFN expression is known to depend on RLR signaling inMEFs (20). Therefore, to further understand the role of IKKb inTLR- versus RLR-induced gene expression, we used early passageIKKb2/2 MEFs (40). Importantly, LPS-induced IFN-b expressionwas severely impaired in IKKb2/2 MEFs (Fig. 4A). In contrast,SeV or NDV infection induced slightly elevated expression ofIFN-b in IKKb2/2 MEFs compared with WT MEFs (Fig. 4A).

Similar findings were obtained when IFN-a4 and IFN-a (non4)mRNA expression was analyzed in IKKb2/2 MEFs after virus in-fection (Fig. 4A), whereas LPS did not induce detectable expressionof IFN-a4 and IFN-a (non4) mRNA in MEFs or cDCs (data notshown). Overall, these findings indicate a key role for IKKb in TLR-induced but not RLR-induced type 1 IFN expression in MEFs.RNA virus infection also induces inflammatory cytokine ex-

pression in an RLR/RIG-I–dependent manner (20). Next, we de-termined the role of IKKb in RLR-induced expression of TNF-aand IL-6. LPS-induced TNF-a expression was abolished inIKKb2/2 MEFs, whereas TNF-a was still induced after virusinfection of IKKb2/2 MEFs, but at a reduced level (Fig. 4B).Therefore, IKKb presence is more crucial for TLR- versus RLR-induced TNF-a expression. Furthermore, LPS-induced IL-6 wassubstantially reduced in IKKb2/2 MEFs, but not in virus-infectedMEFs, which showed enhanced IL-6 expression in IKKb2/2

MEFs (Fig. 4C). These results therefore indicate that IKKb isessential for TLR-induced but not RLR-induced inflammatorycytokine gene expression.

Differential IKKb/NF-kB and IRF3 activation by TLR versusRLR engagement in cDCs

Overexpression of components of both TLR and RLR pathwayshave been shown to strongly activate NF-kB (48, 49). However, itis possible that natural triggering of these pathways does notequivalently impact NF-kB activation. We hypothesized that thedifference in IKKb/NF-kB requirement between cDCs and pDCscould be linked to a differential activation of this signaling modulein TLR versus RLR pathways. To test this, we first determined the

FIGURE 3. IKKb inhibition suppresses TLR-induced type 1 IFN ex-

pression in cDCs. (A) Effect of PS-1145 on LPS- or R837-induced IFN-b

protein expression (6 h) determined by ELISA in cDCs. (B) Effect of PS-

1145 on LPS-induced NF-kB activation (6 h) in cDCs determined by EMSA.

(C) Effect of PS-1145 on LPS-induced IRF3 activation (dimerization) (6 h)

in cDCs determined by native gel Western blotting. Representative results

from two or three independent experiments are shown. **p , 0.01.

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level of IKKb activation after engagement of the TLR and RLRpathway in cDCs. LPS induced robust activation-specific phos-phorylation of IKKb (Fig. 5A), consistent with strong activationof NF-kB in cDCs (Supplemental Fig. 4). In striking contrast,RLR engagement in cDCs by SeV infection showed little or noactivation of IKKb (Fig. 5A), which corresponded to weaker andless sustained NF-kB activation (Supplemental Fig. 4). However,both SeV and NDV infection led to substantial increase in IRF3activation, whereas LPS induced significantly less IRF3 activation(Fig. 5B). Thus, TLR engagement specifically leads to high IKKbactivation in cDCs. We next determined whether a similar re-sponse is triggered in pDCs following TLR activation by virusinfection. Importantly, a substantial increase in phosphorylatedIKKb was observed in response to SeV infection of pDCs (Fig.5C). Thus, strong IKKb activation specifically occurs in responseto TLR engagement by distinct agonists in both cDCs (i.e., LPS)and pDCs (i.e., virus infection).As RLR engagement results in weak, likely suboptimal acti-

vation of NF-kB in cDCs, we asked if TLR stimulation by virtueof its capacity to robustly activate NF-kB could enhance virus-

induced IFN expression in cDCs. Indeed, LPS treatment of cDCstogether with SeV infection synergistically increased levels ofIFN-b mRNA compared with LPS or SeV alone (Fig. 5D) or LPSand NDV (data not shown). Furthermore, PS-1145 decreased SeV +LPS–induced IFN-b expression to levels comparable to SeV alone(Fig. 5D). Therefore, strong TLR-induced IKKb/NF-kB activationcan enhance RLR-induced IFN-b expression in cDCs, indicatingthat lack of a requirement for IKKb/NF-kB signaling in type 1 IFNgene expression downstream of RLRs is likely due to weak acti-vation of this pathway in cDCs.

TLR and RLR pathways induce distinct gene expressionpatterns in cDCs

We hypothesized that differential IKKb/NF-kB activation by TLRversus RLR pathways may also have an impact on gene expres-sion patterns induced by these PRRs. Both NF-kB target inflam-matory cytokines (IL-1a, IL-1b, TNF-a, and IL-6) and Th1cytokines (IL-12 p40, IL-12 p35, and IL-18) (43) were stronglyinduced by LPS in cDCs (Fig. 6). In contrast, mRNA induction ofthese genes following SeV or NDV infection was substantially

FIGURE 4. IKKb is essential for TLR- but not RLR-induced type 1 IFN and inflammatory cytokine expression in MEFs. (A) IFN-b, IFN-a4, and IFN-a

(non4) mRNA expression in IKKb+/+ and IKKb2/2 MEFs induced by LPS or by SeV or NDV infection, as determined by real-time PCR. (B) TNF-a

mRNA expression in IKKb+/+ and IKKb2/2 MEFs induced by LPS or by SeV or NDV infection, as determined by real-time PCR. (C) IL-6 mRNA

expression in IKKb+/+ and IKKb2/2 MEFs induced by LPS or by SeVor NDV infection, as determined by real-time PCR. Representative results from three

independent MEF isolates are shown. *p, 0.05.

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lower in comparison with LPS (Fig. 6). In contrast, IFN-b wasstrongly induced by virus/RLR but only weakly by LPS/TLR(Fig. 6). These results therefore indicate that differential activa-tion of IKKb/NF-kB by TLR versus RLR pathways in cDCs canalso lead to induction of distinct gene expression responses(Fig. 7).

DiscussionAlthough IKKb/NF-kB is activated by all known microbial PRRs(3), the role of this signaling module in mediating transcriptional

responses to different classes of PRR is not well understood. We

demonstrate in this article that IKKb/NF-kB is selectively required

for TLR- but not RLR- induced expression of type 1 IFN and in-

flammatory cytokines. Consequently, cell types (such as cDCs and

MEFs) that rely on RLR signaling for RNA virus–triggered in-

duction of type 1 IFNs do not require NF-kB signaling, whereas cell

types (e.g., pDCs) that respond to RNA viruses via TLR-mediated

signaling pathways are dependent on IKKb/NF-kB signaling for

optimal induction of type 1 IFNs. Mechanistically, we find that

difference in NF-kB “signal strength” may underlie this differential

requirement for the IKKb/NF-kB module in transcriptional

responses downstream of RLRs versus TLRs in DC subsets: RLR

stimulation in cDCs results in weaker activation of IKKb/NF-kB

than does TLR stimulation in pDCs by the same RNA virus trigger.This selective use of IKKb/NF-kB for induction of type 1 IFNs

by the TLR pathway may explain, at least in part, why pDCs

produce such copious amounts of type I IFNs. In pDCs, endo-

somal vesicle TLR signaling leads to high and sustained IRF7

activation via assembly of a unique MyD88-IRF7 transcriptional–

transductional signaling complex (35, 50). The additional activa-

tion of NF-kB by TLR signaling in pDCs may then allow syner-

gistic interactions between NF-kB and IRF7, leading to the

characteristic high-level IFN expression that is a defining feature

of this cell type. The requirement for NF-kB in high-level IFN

production by pDCs may result from CBP/p300 histone acetyl

transferase transcriptional coactivator recruitment to the ifnb lo-

cus, and perhaps to other type 1 IFN gene loci.In contrast to NF-kB, IRF3 is activated significantly more strongly

by RLRs than by TLRs. High-level IRF3 activation in cell types

(e.g., cDCs, MEFs) in which RLRs are the primary sensors and

producers of type 1 IFNs after RNA virus infection may therefore

minimize a requirement for strong IKKb-induced NF-kB activation

for type 1 IFN gene expression (51). Nonetheless, our earlier work

has demonstrated an important role for NF-kB very early in IFN-b

induction by RLRs (37), consistent with the idea that an initial,

relatively weak NF-kB signal serves to recruit essential coactivators

to type I IFN gene loci immediately after RLR activation, after

which strong IRF3 activity can drive IFN-b gene expression (51).

Although both TLR or RLR pathway components may potently

activate NF-kB in overexpression studies (48, 49), our results in-

dicate that the natural activation of these two pathways leads to

significantly different activation levels of IKKb/NF-kB, with con-

sequently distinct effects on the induction of type 1 IFN genes.Genome-wide gene expression studies have previously revealed

that transcriptional mechanisms play a key role in determining

specificity of host responses against different infectious agents (1,

2). For example, studies in DCs have shown that proinflammatory

cytokines (e.g., TNF-a, IL-1a/b, and IL-6) are strongly induced

by bacteria but weakly by viruses, whereas, conversely, type 1 IFN

genes are strongly induced by viruses but only weakly by bacteria

(1). As the molecular basis for this specificity is unclear, we

explored whether differential activation of NF-kB versus IRF3

pathways by TLRs and RLRs leads to the induction of distinct

gene expression patterns, besides type 1 IFNs. A side-by-side

comparison of LPS stimulation versus SeV and NDV infection

of cDCs showed a dramatic difference in induction of inflamma-

tory cytokines versus IFN-b expression by these stimuli. These

results therefore indicate that 1) strong activation and critical re-

quirement for IKKb/NF-kB by TLR signaling is crucial for high-

level expression of inflammatory cytokines (e.g., TNF-a, IL-1a/b,

FIGURE 5. Differential IKKb/NF-kB and IRF3 activation by TLR

versus RLR engagement in cDCs. (A) Phosphorylation of IKKb induced

by LPS and SeV in cDCs determined by Western blotting. (B) Activation

of IRF3 induced by LPS and SeV in cDCs determined by native gel

Western blotting. (C) Phosphorylation of IKKb induced by SeV infection

in splenic pDCs determined by Western blotting. (D) Effect of PS-1145 on

synergistic induction of IFN-b by LPS plus SeV infection in cDCs de-

termined by real-time PCR. Representative results from two or three in-

dependent experiments are shown. **p ,0.01, ***p ,0.001.

FIGURE 6. TLR and RLR pathways induce distinct gene expression

patterns in cDCs. Total RNA was prepared by TRIzol reagent for RNase

protection assay for determining expression of indicated genes. LPS: 100

ng/uL, SeV: 200 hemagglutinin units/ml, NDV: multiplicity of infection 2.

L.E., long exposure to allow visualization of IL-12 p35.

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and IL-6), whereas, conversely, 2) low activation and lack ofrequirement of IKKb/NF-kB by RLR is likely responsible forlow expression of inflammatory cytokines following RNA virusinfection. More generally, differential activation of IKKb/NF-kB and IRF3 axes may allow induction of distinct gene ex-pression responses by infectious agents activating differentPRRs in distinct cell types. These findings as they relate to ourdiscoveries in cDCs versus pDCs are schematically summa-rized in Fig. 7.

AcknowledgmentsWe thank the Flow Cytometry andMolecular Genomics core facilities at the

Moffitt Cancer Center for help and Dr. Zhi-Wei Li (Moffitt Cancer Center)

for kindly providing IKKb+/2 mice.

DisclosuresThe authors have no financial conflicts of interest.

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FIGURE 7. Regulation of RLR- and TLR-

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