1

THE INTERFERON REGULATORY FACTOR, IRF5, IS A CENTRAL MEDIATOR OF

TLR7 SIGNALING.

Annett Schoenemeyer1, Betsy J. Barnes2, Margo. E. Mancl2, Eicke Latz1, Nadege Goutagny1, Paula M. Pitha2, Katherine A. Fitzgerald1* and Douglas T. Golenbock1*

1Division of Infectious Diseases & Immunology, University of Massachusetts Medical School, Worcester, MA 01605, USA. 2Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins

University School of Medicine, Baltimore, MD 21231, USA.

* These authors contributed equally to this work.

Running Title: MyD88 and TRAF6-dependent activation of IRF-5 and IRF-7 in TLR7 signaling.

Correspondence should be addressed to:

Douglas T. Golenbock or Kate A. Fitzgerald, 364 Plantation Street, LRB 308-9, Worcester, MA 01605, USA. Tel: 508 856 (5980), (6518); Fax: 508 856 5463;

e-mail: Douglas.Golenbock@umassmed.edu or Kate.Fitzgerald@umassmed.edu

JBC Papers in Press. Published on January 28, 2005 as Manuscript M412584200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

2

Interferon regulatory factors (IRFs) are critical components of virus-induced immune activation and type I interferon regulation. IRF-3 and IRF-7 are activated in response to a variety of viruses or following engagement of Toll-Like Receptor (TLR) 3 and TLR4 by double stranded RNA (dsRNA) and LPS, respectively. The activation of IRF-5, is much more restricted. Here we show that in contrast to IRF3 and IRF7, IRF5 is not a target of the TLR3 signaling pathway but is activated by TLR7 or TLR8 signaling. We also demonstrate that MyD88, IL-1 receptor associated kinase (IRAK) 1 and TNF-receptor associated factor (TRAF) 6 are required for the activation of IRF-5 and IRF-7 in the TLR7 signaling pathway. Moreover, ectopic expression of IRF-5 enabled type I interferon production in response to TLR7 signaling, while knockdown of IRF-5 by small interfering RNA (siRNA) reduced type I interferon induction in response to the TLR7 ligand, R-848. IRF-5 and IRF-7 therefore emerge from these studies as critical mediators of TLR7 signaling.

Members of the Toll-like receptor family are essential recognition and signaling components of mammalian anti-viral host defense (1). TLR3, TLR7, TLR8 and TLR9 recognize viral nucleic acids and induce type I IFNs. TLR7 and TLR8 are similar in sequence and together with TLR9 form an evolutionarily related subgroup within the TLR superfamily (2, 3). While unmethylated CpG DNA (4), HSV type 1 (5) and HSV type 2 genomic DNA (6) specifically stimulate TLR9 (7, 8), TLR7 is activated by infections with ssRNA viruses, including Influenza virus and Vesicular Stomatitis virus [VSV, (7, 9)]. Consequently, plasmacytoid dendritic cells (pDCs) from TLR7-deficient mice fail to produce type I IFNs upon infection with

Influenza virus or VSV (7, 10). In addition to ssRNA, the synthetic imidazoquinoline, imiquimod, a low molecular weight immune response modifier, activates TLR7 in both humans and mice while its derivative resiquimod (R-848) activates TLR7 and TLR8 in humans, but only TLR7 in mice (10, 11). Both imiquimod and R-848 elicit robust anti-viral and anti-tumor immune responses in vivo, which correlate with a strong induction of type I IFNs (12-14). As a consequence of this activity, imiquimod is used for the treatment of external genital warts caused by human papillomavirus (15). Interferon regulatory factors (IRFs) coordinate the expression of type I IFNs (16-19) as well as chemokines such as IP-10 and RANTES (20-22). Viral infections, dsRNA or LPS signaling can activate IRF-3 and IRF-7 (23-25). In contrast, the activation of IRF-5 another member of the IRF family is much more restricted. Only certain viruses, including Newcastle Disease virus (NDV), VSV and HSV type 1 have been shown to activate IRF-5 (22), while Sendai virus (SeV) and dsRNA (pI:C) which activate IRF-3 and IRF-7, do not activate IRF-5 (22). These observations suggest that IRF5 is activated by distinct signaling mechanisms to those regulating IRF3. In unstimulated cells IRFs reside in the cytoplasm. The activation of these factors requires phosphorylation on the C-terminus, leading to dimerization, nuclear translocation and binding to promoters containing IRF binding elements (IRF-E) (26-28). The IKK-related kinases, IκB kinase ε, IKKε (also called IKKi) (29, 30) and TANK binding kinase 1, TBK1 (also called T2K or NAK) (31-33) can directly phosphorylate IRF-3 and IRF-7 at the C-terminus and control the expression of type I IFNs in response to SeV infection and TLR3 or TLR4 stimulation (34-36). The role of these two non-canonical IKKs in the regulation of IFN gene expression resulting from TLR7, TLR8 or TLR9 ligation,

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

3

or their ability to phosphorylate IRF-5 has not yet been addressed.

TLR3 and TLR4 are known to induce IFNB gene expression. This induction requires IRF-3 and/or IRF-7. TLR3 recruits the TIR domain containing adapter-inducing IFNβ, TRIF (37-39), while TLR4 signaling utilizes TRIF and the adapter molecule, TRIF-related adapter molecule, TRAM (23, 40). The adapter molecules MyD88 and Mal/TIRAP are not involved in type I IFN induction by the TLR3 or TLR4 signaling pathway. In contrast, studies with MyD88-deficient mice revealed that TLR7 and TLR9 signaling to IFNα is dependent on MyD88 (10, 41).

Unlike IRF-3 which is expressed constitutively in all cell types, the expression of IRF-5 and IRF-7 is restricted to B cells and dendritic cells, although their expression is inducible in other cell types by type I IFNs (19, 42). The predominant source of type I IFNs in human blood is the plasmacytoid dendritic cell (pDC). Plasmacytoid DCs release large amounts of type I IFN upon viral infection or stimulation with either R-848 or CpG DNA (43, 44). Consistent with these observations, pDCs express high levels of TLR7 and TLR9, while the expression of other TLRs is either very low or absent (45, 46). The molecular mechanisms responsible for the induction of IFNs by TLR7, TLR8 and TLR9 signaling are unclear at present. Since pDCs express high constitutive levels of IRF-5 as well as IRF-7, we were prompted to investigate the functional importance of these two IRFs in the regulation of type I IFNs by these TLRs. We focused the present study on the TLR7 and TLR8 signaling pathway. We demonstrate that TLR7 and TLR8 activate both IRF-5 and IRF-7 and do not appear to activate IRF-3. We also show using reconstitution experiments and siRNA silencing approaches that IRF-5 is a critical mediator of TLR7 signaling. Both IRF-5 and IRF-7 are regulated in a MyD88, IRAK-1 and TRAF6 dependent manner, in

contrast to IRF-3, which is regulated via TRIF in TLR3 or TLR4 signaling. Experimental Procedures Reagents. pFlag-CMV1-TLR3 was cloned by PCR from a full length cDNA clone. pFlag-CMV1-TLR7 was cloned by PCR from THP-1 genomic DNA. The plasmid pcDNA3.1-TLR8 was a gift from the Eisai Research Institute (Andover, MA). Dominant negative IRF-5 (DN IRF-5) is a DNA-binding-domain deletion mutant of wild type IRF-5 (variant 3, GenBank accession # AY504946 and is missing the first N-terminal 137 amino acids. The pcDNA3-MyD88-TIR-AU1 dominant negative (DN) plasmid was generated as described (47). The Gal4-upstream activation sequence (UAS(GAL)) driven luciferase reporter gene and Gal4-IRF-3 were from T. Fujita [Tokyo, Japan (48)]. Gal4-IRF-5 (19), Gal4-IRF-7 (49), Flag-IRF-5 (19), IRF-7-GFP and IRF-5-GFP (19) were described previously. The IFNA1 and IFNB-secreted alkaline phosphatase (SAP) reporter plasmids were as described (50). TRAF2 and TRAF6 mutant constructs were from H. Wesche [Tularik, CA (51)]. Poly (I) poly (C) (pI:C) was from Amersham Pharmacia (Piscataway, NJ). Resiquimod (R-848) was from GLSynthesis Inc. (Worcester, MA). SeV (Cantrell strain) and NDV (VR-699) were purchased from American Type Culture Collection (ATCC, Manassas, VA). VSV was from Phil Marcus (Connecticut, CT). The IRAK-1 deficient I1A-HEK293 cells were form X. Li [Cleveland, OH, (52)]. Primary human fibroblasts and bovine kidney cells (MDBK) were from Gary Hayward (Baltimore, MD). Reporter gene assays. HEK293 cells or I1A-HEK293 cells (0.2 x 105 cells per well of a 96-well plate) were transfected with 10 ng of pFlag-CMV1-TLR7, pcDNA3.1-TLR8, pFlag-CMV1-TLR3 or empty vector control and co-transfected with 40 ng of Gal4-IRF-5 (variant 3), Gal4-IRF-7 or Gal4-DBD plus 40

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

4

ng of the UAS(GAL)-luciferase reporter gene. Cells were additionally co-transfected with dominant negative constructs for MyD88 (MyD88 TIR), TBK1 (TBK1 K38A), IKKε (IKKε K38A), TRAF2 (DN TRAF2) or TRAF6 (DN TRAF6), as indicated. In all cases, 40 ng per well of the thymidine kinase driven renilla-luciferase reporter gene (Promega, Madison, WI) was co-transfected to normalize for transfection efficiency. After 24h or 36h of transfection, cells were stimulated for 8h or 15h as indicated. Post stimulation, cell lysates were prepared and reporter gene activity was measured using the Dual Luciferase Assay System (Promega, WI). 5 x 105 2fTGH cells were plated in six well plates and transfected for 20h with 1 µg IFNA1 or IFNB SAP reporter, 500 ng pFlag-CMV1-TLR7 and/or 1 µg Flag-IRF-5 and/or 500 ng DN IRF-5 using Superfect transfection reagent (Qiagen, Santa Clarita, CA). Additionally cells were transfected with 50 ng of the beta-galactosidase expression plasmid, to allow for the normalization of the data. 20h after transfection, cells were divided and seeded into 24 well plates and stimulated with 10 µM R-848 or infected with NDV (240 HAU) or VSV (MOI 2) for an additional 16h. The SAP activity was determined as described (50). All data are expressed as the mean relative stimulation + S.D. All of the experiments described were performed a minimum of three occasions and gave similar results. Confocal microscopy. RAW 264.7 cells (1 x 106) were transiently transfected with 2 µg IRF-5-GFP or IRF-7-GFP using GeneJuice (Novagen, Madison, WI), in order to analyze the nuclear translocation of IRF-5 or IRF-7 visually. After 48h of transfection, cells were stimulated for an additional 24h with 15µM of R-848, 100µg/ml of pI:C or PBS. The analysis of nuclear translocation was performed using a Leica TCS SP2 AOBS confocal microscope.

Type I IFN Bioassay. HEK293T, HEK293T/IRF-5, 2fTGH and 2fTGH/IRF-5 cells (0.75x104 cells per well of a 96 well plate) were transiently transfected with 50 ng of pFlag-CMV1-TLR7. 24h later, cells were stimulated with 10 µM R-848 or left untreated. THP-1 cells (1x106) were stimulated with 10 µM R-848 or infected with NDV (240 HAU) or VSV (MOI 2) or left untreated. The levels of biologically active type I IFNs were determined in the cell culture supernatant after 16h of stimulation by the viral cytopathic effect assay (53). Vesicular stomatitis virus was used as the challenging virus and the cytopathic effect was determined in human fibroblasts and/or MDBK cells. RNA interference. The coding region of IRF-5 was targeted with the following IRF-5 siRNA: 5’-ATACACCGAAGGCGTGGAT-3’ (Dharmacon Inc., Lafayette, CO). The conditions for IRF-5 gene silencing were determined by transfecting 2fTGH/Flag-IRF-5 cells with 0, 2, 5, 7 or 10 nM of IRF-5 siRNA, a scrambled siRNA control or a LacZ siRNA control (seq. – 5’-AACGTACGCGGAATACTTCGA-3’) using Mirus TransIT® TKO reagent (Mirus, Madison, WI). The efficiency of IRF-5 silencing was analyzed after 24h of transfection by RT-PCR and/or immunoblot. Amaxa electroporation of suspension cells. THP-1 cells were electroporated using the Amaxa Kit (Gaithersburg, MD) according to manufacturer’s specifications. Briefly, THP-1 cells (1 x 106) were harvested and resuspended in 100 µl nucleofector solution. After the addition of IRF-5 siRNA (7 nM) or LacZ siRNA (10 nM), cells were electroporated using Amaxa program U-01 or V-01. 8h following transfection, cells were exposed to 10 µM R-848 or left untreated. After an additional 16h, cells were subjected to RT-PCR analysis.

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

5

RT-PCR analysis. RNA was isolated from 1x106 THP-1 cells using an RNeasy Mini Kit (Qiagen, Santa Clarita, CA). One microgram of DNase-treated total RNA was reverse-transcribed to cDNA with oligo(dT) primers in a 20 µl reaction. Human IFNα (consensus primers designed to recognize most human IFNα subtypes), IFNβ, IRF-5 and β-actin cDNA were amplified by PCR using primers and PCR conditions as previously described (42, 54). Recombinant proteins. GST fusion proteins and baculovirus proteins were prepared as described (55) . Kinase assays. In vitro kinase assays were performed as described (55) using 10ng of recombinant IKKβ or TBK1 and 1µg of GST-IRF3, IRF5 , IκBα wt and IκBα S32/36A. Results TLR7 and TLR8 activate IRF-5 and IRF-7 but not IRF-3.

Double stranded RNA and LPS induce type I IFN gene expression via the adapter molecule TRIF and the transcriptional regulator IRF-3 (37-39). The molecular mechanisms responsible for the induction of type I IFNs in TLR7 or TLR8 signaling is unclear at present. We were interested in elucidating the role of the transcription factors IRF-3, IRF-5 and IRF-7 in signaling by TLR7 and TLR8. To this end we monitor the activation of these factors individually. We employed an in vivo reporter assay that utilizes hybrid proteins consisting of the yeast Gal4 DNA binding domain (DBD) fused either to IRF-3, IRF-5 or IRF-7, lacking its own DNA binding domain (19, 49, 56). In this assay, the UAS(GAL) driven luciferase reporter gene expression requires activation of the corresponding IRF fusion protein (56). The basal level of all three IRF fusion proteins was similar in untreated cells. TLR7 or TLR8-expressing HEK293 cells transfected with

Gal4-IRF-3, Gal4-IRF-5 or Gal4-IRF-7 plasmids were stimulated with R-848. TLR7 or 8 signaling activated IRF-5 and IRF-7, but not IRF-3 (Fig. 1A, upper panel). Stimulation of TLR3-expressing HEK293 with pI:C did not activate IRF-5 (Fig. 1A, bottom left panel), although IRF-3 and IRF-7 were induced in a robust manner. Furthermore, infection of HEK293 cells with SeV, a well-characterized activator of IRF-3 and IRF-7, also failed to activate IRF-5 (Fig. 1A, bottom right panel), in agreement with published reports (19). Thus, TLR7 and TLR8 activate IRF-5 and IRF-7 and do not appear to activate IRF-3. Neither Sendai virus nor TLR3 signaling activated IRF-5.

As a second independent methodology, we examined the nuclear translocation of these three IRFs in RAW264.7 macrophage-like cells transfected with IRF-GFP fusion constructs. The IRF-5-GFP or IRF-7-GFP fusion proteins were expressed in the cytoplasm of unstimulated cells (Fig. 1B, C). Stimulation of these cells with R-848 resulted in the nuclear translocation of both IRF-5-GFP and IRF-7-GFP (Fig. 1B, C, middle panel) but did not induce nuclear translocation of IRF3-GFP (data not shown). In agreement with the Gal4-IRF5 assay, IRF-5-GFP did not translocate to the nucleus in cells stimulated with pI:C (Fig. 1B, lower panel). IRF-7-GFP did however translocate in response to pI:C in agreement with previous reports (Fig. 1C, lower panel). These nuclear translocation data are representative of several fields analysed. We also looked at IRF-3 phosphorylation using phospho specific IRF-3 antibodies in R-848 and LPS stimulated cells. There was no detectable phosphorylation of IRF-3 with R-848 seen (data not shown), in contrast to LPS which did induce IRF3 phosphorylation. Taken together, these observations establish that unlike TLR3, TLR7 and TLR8 can activate IRF-5 but not IRF-3. Consistent with these observations, R-848 did not induce IRF3-DNA binding activity to an ISG-15

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

6

probe (which binds activated IRF3) under conditions where NF-κB was activated (data not shown), further supporting the idea that IRF-3 is not a mediator of the TLR7 signaling pathway. While this manuscript was in preparation, two independent reports from the Akira and Taniguichi laboratories demonstrated that IRF3 was not activated in either the TLR7 or TLR9 signaling pathway (57, 58). TLR7 can induce type I IFNs via IRF-5.

IRF-5 and IRF-7 have been shown to regulate the expression of overlapping as well as distinct IFNα subsets, which are encoded by at least 13 IFNA genes in humans (17, 19, 49, 54). IRF-3 alone is sufficient for the induction of IFNβ (16, 18, 59). Given the specific activation of IRF-5 by the TLR7 pathway, we were prompted to evaluate its contribution to the induction of type I IFNs by R-848. We therefore monitored the activation of IFNB and IFNA1 promoter reporter genes in 2fTGH cells, which transiently expressed TLR7 in addition to Flag-tagged IRF-5. We compared this response to the parental IRF-5-null cell line, which transiently expressed only TLR7. Cells either expressing or lacking IRF-5 were stimulated with R-848 or infected with NDV or VSV. The IFNB reporter was not induced in parental TLR7-expressing 2fTGH cells lacking IRF-5 following R-848 stimulation (Fig. 2A). IFNB reporter gene activity was however induced when IRF-5 was expressed in these cells (Fig. 2A). Similarly, VSV, a type I IFN-inducing virus, known to signal via TLR7 (9) also required IRF-5 to activate the IFNB promoter. In contrast, NDV induced the IFNB reporter via IRF-5 and did not require TLR7 (Fig. 2A). Similar results were obtained in all cases using the IFNA1 reporter (Fig. 2B). Noteworthy, although 2fTGH cells constitutively express IRF-3, there was no induction of the IFNB promoter in response to TLR7 engagement by R-848, further supporting the observation that

TLR7 signaling does not activate IRF-3 (Fig. 2A). To evaluate further the importance of IRF-5 in mediating these responses, we generated a dominant negative mutant of IRF-5, lacking the DNA binding domain. As shown in Fig. 2C overexpression of this IRF-5 mutant inhibited the induction of the IFNA1 promoter following R-848 stimulation. We also measured the synthesis of endogenous biologically active type I IFNs in TLR7-expressing 2fTGH cell lines in the presence or absence of IRF-5. Stimulation of TLR7-expressing 2fTGH cells with R-848 did not result in the induction of endogenous type I IFNs. In contrast, ectopic expression of Flag-tagged IRF-5 conferred on these cells the ability to induce type I IFNs upon R-848 stimulation (Fig. 2D). Similar results were obtained using HEK293T cells expressing TLR7 and IRF-5 (Fig. 2D). In HEK293T cells, the induced type I IFN response consisted primarily of IFNβ, while in 2fTGH cells both IFNβ and IFNα were induced.

IRF-5 siRNA impairs R-848 induced IFNA induction. Having shown that IRF-5 enabled TLR7-expressing cells to produce type I IFNs upon R-848 stimulation, we next analyzed the requirement for IRF-5 in the TLR7 pathway in a more physiologically relevant setting by using siRNA silencing technology to knock down endogenous IRF-5. It is difficult to find an appropriate system to perform these studies since few cell lines express endogenous TLR7 and IRF-5 and respond to R-848 to induce type I IFNs. Plasmacytoid DCs would be the ideal cell type to study, but these cells are not amenable to siRNA silencing. However, the human monocytic cell line, THP-1, fulfilled all the criteria required, including the capability of being transfectable with siRNA. As seen in Figure. 3A, THP-1 cells constitutively expressed IRF-5 (Fig. 3A, lane 1). These cells also express IRF7, although the

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

7

expression of IRF7 is lower than that of IRF5. Stimulation of these cells with R-848 further increased IRF-5 and IRF7 expression (Fig. 3A, lane 4) and strongly induced both IFNB and IFNA mRNA expression (Fig. 3A), which correlated with the synthesis of endogenous biologically active IFNs. R-848 was a much more potent inducer of IFNB and IFNA mRNA and stimulated higher levels of interferon synthesis than either NDV or VSV.

We next monitored the effect of IRF-5 siRNA silencing on the TLR7-mediated IFN response in THP-1 cells. Transfection of THP-1 cells with IRF-5 siRNA decreased endogenous IRF-5 mRNA, whereas the LacZ siRNA control had no effect on its expression (Fig. 3B). The analysis of IFNA expression monitored by RT-PCR revealed that the induction of IFNA by R-848 was strongly reduced in cells transfected with IRF-5 siRNA but not in untransfected THP-1 cells or cells transfected with the LacZ control siRNA (Fig. 3B). Taken together, these data provide strong evidence that IRF-5 is an essential transducer of the TLR7-dependent induction of type I IFNs. MyD88, IRAK-1 and TRAF6 activate IRF-5 in the TLR7 signaling pathway. Neither LPS nor dsRNA require the adaptor MyD88 to activate the type I IFN signaling pathway. In contrast, TLR7 signaling is completely dependent on the adapter molecule MyD88. R-848 or Influenza virus or VSV infection fail to induce IFN-α in cells deficient in TLR7 or MyD88 (7, 10). Since the induction of type I IFNs by the TLR7 and TLR9 subgroup is dependent on MyD88, this pathway clearly differs from that induced by the TLR3 and TLR4 pathways.

We next addressed the question of whether MyD88 could couple to IRF-5 and/or IRF-7 activation. Consistent with this idea, the activation of the Gal4-IRF-5 and Gal4-IRF-7 reporter gene by R-848 in TLR7 or TLR8-expressing HEK293 cells was inhibited in a

dose-dependent manner by a dominant negative mutant of MyD88 (MyD88-TIR) suggesting that MyD88 acts upstream of IRF-5 and IRF-7 (Fig. 4A, data not shown). In contrast, activation of the Gal4-IRF-3 and Gal4-IRF-7 reporter constructs in TLR3-expressing cells by pI:C was unaffected by expression of the dominant negative MyD88 (Fig. 4A, data not shown).

The association of MyD88 with the TIR domains of IL-1R/TLR family members recruits the serine/threonine kinase IRAK-1 (60), a critical event leading to the activation of NF-κB. Neither IRAK-1 (61, 62) nor the related serine/threonine kinase, IRAK-4 (63), appear to be required for the activation of IRF-3 by MyD88 independent signaling pathways. We examined if IRAK-1 was also important for TLR7 signaling to IRF-5 using a mutant I1A-HEK293 cell line, which is deficient in IRAK-1 and is defective in IL-1 signaling to NF-κB (52). Activation of IRF-3 and NF-κB in TLR3 signaling has previously been shown to be intact in these cells (61). We transfected wild type or mutant I1A-HEK293 cells with either TLR7 or TLR8 and monitored the effect of R-848 on the activation of IRF-5. In contrast to wild-type HEK293 parental cells, TLR7 or TLR8-expressing I1A-HEK293 cells did not activate IRF-5 (Fig. 4B). Similar results were obtained for IRF-7 (data not shown). While activation of the NF-κB reporter gene was also abrogated in these cells, TNFα induced the NF-κB reporter in both parental HEK293 and I1A-HEK293 cells (data not shown).

TRAF6 is critical for NF-κB activation by IL-1R/TLR family members, however, activation of IRF-3 in TLR3 and TLR4 signaling is independent of TRAF6 (61, 64). Since IRAK-1 was required for IRF-5 and IRF-7 activation, we wondered if TRAF6 might also function downstream of IRAK-1 in this pathway. Activation of the Gal4-IRF-5 reporter gene by TLR7 and TLR8 following R-848 stimulation was inhibited in a dose-

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

8

dependent manner by a dominant negative mutant of TRAF6 (Fig. 4C, right panels). Similar results were obtained with IRF-7 (data not shown). In contrast, a dominant negative mutant of TRAF2 did not inhibit the activation of IRF-5 (Fig. 4C, left panels). These novel observations demonstrate that TRAF6 is a critical transducer in the IRF-5 activation pathway and broaden our understanding of TRAF6 as a protein that participates in signaling pathways other than those leading to the activation of NF-κB and AP-1. TBK1 and IKKε phosphorylate IRF-5 in vitro. The IKK-related kinases, IKKε and TBK1 are essential regulators of the IFN response (31-33). They both phosphorylate and activate IRF-3 and IRF-7 (34, 36). Their role in IRF-5 activation has not yet been addressed. We therefore investigated if IRF-5 was also a target of TBK1 kinase activity in vitro. As seen in Fig. 5A, TBK1, but not the related kinase IKKβ, efficiently phosphorylated an IRF-5-GST fusion protein in vitro. Similar results were obtained with a GST-IRF3 substrate, while a GST alone construct was not phosphorylated by either kinase. Recombinant IKKε also efficiently phosphorylated GST-IRF3 and IRF5. While IKKβ failed to phosphorylate IRF-5 and IRF-3 it efficiently phophorylated IκBα. Consistent with a role for IKKε and TBK1 in the regulation of the IRF-5 signaling pathway, TLR7-induced activation of Gal4-IRF-5 was inhibited in a dose-dependent manner by the TBK1K38A and IKKεK38A kinase inactive mutants (Fig. 5 B). Similar results were seen in TLR8-expressing cells (not shown). R-848 signaling via TLR7 and TLR8 also induced NF-κB activation, however, this response was unaffected by expressing TBK1K38A or IKKεK38A (data not shown). To further analyze the role of TBK1 in the TLR7 signaling pathway we attempted to monitor IFNA induction by ELISA and quantitative

PCR analysis in embryonic fibroblasts derived from TBK1-deficient mice. Wild type embryonic fibroblasts did not induce type I IFN following R-848 stimulation, eliminating their usefulness for this approach. Indeed, embryonic fibroblasts were found to lack IRF-5 and IRF-7 expression, which may explain these observations (Barnes and Pitha, unpublished). Discussion

The innate immune system has

evolved several distinct viral recognition systems, which integrate complex networks of signaling pathways, leading to the activation of pathway-specific transcription factors and the induction of immune response genes. TLRs, together with their associated downstream signaling molecules, constitute key viral recognition systems in the innate immune response. Considerable work over the last few years has revealed that a subset of TLRs (TLR3, TLR7, TLR8 and TLR9) recognize viral nucleic acids and induce type I IFNs. It is clear that the signaling mechanisms involved in the induction of IFNs differ depending on the receptor system activated. TLR3 and TLR4 signaling have been most extensively characterized, while much less is known about how the TLR7, TLR8 and TLR9 subfamily regulate these responses. Understanding the molecular mechanisms regulating the induction of type I IFNs by these TLRs is likely to reveal novel therapeutic and immune-modulation strategies, aimed at eliminating acute and chronic viral infections. We report here for the first time the novel finding that engagement of TLR7 and TLR8 by R-848 activates IRF-5 as well as IRF-7 and does not appear to activate IRF-3. These observations led us to focus the present study on IRF-5, since its role in TLR signaling had not been addressed previously. We discovered that IRF-5 is a central mediator of

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

9

TLR7 and TLR8 signaling. IRF5 contributes to IFN induction in human cells. We were surprised to find that IRF-5 was important not only for IFNα but also for IFNβ induction. However, in agreement with these observations, we have recently shown that NDV-activated IRF-5 induced high levels of endogenous IFNβ in B cells (65). Small interfering RNA silencing of IRF-5 attenuated IFN induction in response to R-848. Although significantly reduced, this response was not completely impaired. While there are several possibilities to explain this observation, we propose that IRF-7 mediates this residual IFN response, since IRF7 is also activated in the TLR7 and TLR8 pathway in a similar manner as IRF-5 (17, 66). IRF-5 has previously been shown to form both homodimers as well as heterodimers with IRF-3 or IRF-7 in response to virus infection (67). Under these conditions, the formation of IRF-5/IRF-7 heterodimers can modulate the assembly of the IFNA enhanceosome and alter the profile of IFNA subtypes induced. In the case of TLR7 signaling, where IRF-3 is not activated, IRF-5 and IRF-7 may function cooperatively to regulate IFNA gene transcription. The relative roles of IRF-5 and IRF-7 in TLR7 signaling can best be studied when IRF-5 and IRF-7-deficient mice become available.

The adapter molecule TRIF was discovered based on its role in TLR3 signaling to IRF-3 (37, 38). A TRAM-TRIF module functions in the TLR4 pathway, since TRIF is not directly recruited to the TLR4-TIR domain (23, 68). Here, we report that TLR7 and TLR8 rely on MyD88, IRAK-1 and TRAF6 to activate IRF-5 (and IRF-7), suggesting that the IRF and NF-κB pathways in TLR7 and TLR8 signaling bifurcate downstream of TRAF6 (Fig. 6). TLRs have thus evolved both MyD88/IRAK-1/TRAF6 dependent and independent signaling pathways to activate IRF proteins and induce type I IFNs. While this manuscript was in submission, two independent groups demonstrated that

MyD88, IRAK1 and TRAF6 form a complex with IRF-7. MyD88 interacts directly with IRF-7 via its N-terminal death domain. IRF-3 was not detected in this complex, consistent with the data presented here in our study that IRF-3 is unlikely to mediate these signaling pathways. Interestingly, Akira and colleagues suggest that TRAF6 may function as an E3 ubiquitin ligase in the MyD88-dependent activation of IRF7. The target of this ubiquitin-dependent pathway however remains to be clearly identified. We believe that our studies show that IRF5 is also involved in TLR7 signaling. Together these studies suggest that the activation of IRF-5 and IRF-7 are functionally important in mediating the MyD88-dependent IFN response. Although we have focused this study on the role of IRF-5 in IFN regulation, a key question that arises from these studies is whether IRF-5 might also contribute to the regulation of additional responses, since the MyD88-TRAF6 module is critical for pro-inflammatory cytokine expression.

The IKK-related kinases, IKKε [IKKi, (29, 30)] and TBK1 [T2K/NAK, (31-33)] phosphorylate IRF-3 and IRF-7 in response to certain viruses, dsRNA or LPS signaling (34-36). In TLR7 signaling however, it is IRF-5 and IRF-7 rather than IRF-3 that are important for mediating the IFN response. Dominant negative versions of both TBK1 and IKKε efficiently inhibit IRF5 activation and IRF5 is an efficient substrate for these two kinases in vitro. However, mice deficient in both of these kinases will be essential to decipher the role of these kinases in TLR7 signaling, since these kinases appear to function in a redundant manner when co-expressed, as might be the case in pDCs. How the MyD88-IRAK1-TRAF6 pathway would activate TBK1 and/or IKKε is unclear, since TRIF a known upstream activator does not participate in TLR7 signaling. The TRAF family member associated NF-κB activator (TANK), also known as I-TRAF, and the related proteins

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

10

NAP1 are bona fide candidates which may recruit TBK1 to this TRAF-6-IRF complex, since TANK has previously been shown to interact with both TBK1 and TRAF proteins (31, 69-72). Acknowledgements This work was supported by grants from the German Academy of Natural Scientists

Leopoldina (BMBF-LPD 9901/8-57; A. S.), by the Wellcome Trust (K. A. F) and the NIH (GM54060, AI52455, AI49309: D. T. G; RO1A1/CA19737-19A1: P.M.P). We would like to thank N. Silverman and E. Lien for critical reading of the manuscript and helpful discussions.

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

11

References 1. Janeway, C.A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu Rev

Immunol 20:197-216. 2. Chuang, T.H., and R.J. Ulevitch. 2000. Cloning and characterization of a sub-family of

human toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur Cytokine Netw 11:372-378. 3. Du, X., A. Poltorak, Y. Wei, and B. Beutler. 2000. Three novel mammalian toll-like

receptors: gene structure, expression, and evolution. Eur Cytokine Netw 11:362-371. 4. Hemmi, H., O. Takeuchi, T. Kawai, T. Kaisho, S. Sato, H. Sanjo, M. Matsumoto, K.

Hoshino, H. Wagner, K. Takeda, and S. Akira. 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740-745.

5. Krug, A., G.D. Luker, W. Barchet, D.A. Leib, S. Akira, and M. Colonna. 2004. Herpes simplex virus type 1 activates murine natural interferon-producing cells through toll-like receptor 9. Blood 103:1433-1437.

6. Lund, J., A. Sato, S. Akira, R. Medzhitov, and A. Iwasaki. 2003. Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells. J Exp Med 198:513-520.

7. Diebold, S.S., T. Kaisho, H. Hemmi, S. Akira, and E.S.C. Reis. 2004. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303:1529-1531.

8. Heil, F., H. Hemmi, H. Hochrein, F. Ampenberger, C. Kirschning, S. Akira, G. Lipford, H. Wagner, and S. Bauer. 2004. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 303:1526-1529.

9. Lund, J.M., L. Alexopoulou, A. Sato, M. Karow, N.C. Adams, N.W. Gale, A. Iwasaki, and R.A. Flavell. 2004. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci U S A 101:5598-5603.

10. Hemmi, H., T. Kaisho, O. Takeuchi, S. Sato, H. Sanjo, K. Hoshino, T. Horiuchi, H. Tomizawa, K. Takeda, and S. Akira. 2002. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 3:196-200.

11. Jurk, M., F. Heil, J. Vollmer, C. Schetter, A.M. Krieg, H. Wagner, G. Lipford, and S. Bauer. 2002. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol 3:499.

12. Testerman, T.L., J.F. Gerster, L.M. Imbertson, M.J. Reiter, R.L. Miller, S.J. Gibson, T.L. Wagner, and M.A. Tomai. 1995. Cytokine induction by the immunomodulators imiquimod and S-27609. J Leukoc Biol 58:365-372.

13. Stanley, M.A. 2002. Imiquimod and the imidazoquinolones: mechanism of action and therapeutic potential. Clin Exp Dermatol 27:571-577.

14. Megyeri, K., W.C. Au, I. Rosztoczy, N.B. Raj, R.L. Miller, M.A. Tomai, and P.M. Pitha. 1995. Stimulation of interferon and cytokine gene expression by imiquimod and stimulation by Sendai virus utilize similar signal transduction pathways. Mol Cell Biol 15:2207-2218.

15. von Krogh, G. 2001. Management of anogenital warts (condylomata acuminata). Eur J Dermatol 11:598-603; quiz 604.

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

12

16. Juang, Y.T., W. Lowther, M. Kellum, W.C. Au, R. Lin, J. Hiscott, and P.M. Pitha. 1998. Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3. Proc Natl Acad Sci U S A 95:9837-9842.

17. Marie, I., J.E. Durbin, and D.E. Levy. 1998. Differential viral induction of distinct interferon-alpha genes by positive feedback through interferon regulatory factor-7. Embo J 17:6660-6669.

18. Sato, M., H. Suemori, N. Hata, M. Asagiri, K. Ogasawara, K. Nakao, T. Nakaya, M. Katsuki, S. Noguchi, N. Tanaka, and T. Taniguchi. 2000. Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-alpha/beta gene induction. Immunity 13:539-548.

19. Barnes, B.J., P.A. Moore, and P.M. Pitha. 2001. Virus-specific activation of a novel interferon regulatory factor, IRF-5, results in the induction of distinct interferon alpha genes. J Biol Chem 276:23382-23390.

20. Lin, R., C. Heylbroeck, P. Genin, P.M. Pitha, and J. Hiscott. 1999. Essential role of interferon regulatory factor 3 in direct activation of RANTES chemokine transcription. Mol Cell Biol 19:959-966.

21. Genin, P., M. Algarte, P. Roof, R. Lin, and J. Hiscott. 2000. Regulation of RANTES chemokine gene expression requires cooperativity between NF-kappa B and IFN-regulatory factor transcription factors. J Immunol 164:5352-5361.

22. Barnes, B.J., M.J. Kellum, A.E. Field, and P.M. Pitha. 2002. Multiple regulatory domains of IRF-5 control activation, cellular localization, and induction of chemokines that mediate recruitment of T lymphocytes. Mol Cell Biol 22:5721-5740.

23. Fitzgerald, K.A., D.C. Rowe, B.J. Barnes, D.R. Caffrey, A. Visintin, E. Latz, B. Monks, P.M. Pitha, and D.T. Golenbock. 2003. LPS-TLR4 signaling to IRF-3/7 and NF-kappaB involves the toll adapters TRAM and TRIF. J Exp Med 198:1043-1055.

24. Kawai, T., O. Takeuchi, T. Fujita, J. Inoue, P.F. Muhlradt, S. Sato, K. Hoshino, and S. Akira. 2001. Lipopolysaccharide stimulates the MyD88-independent pathway and results in activation of IFN-regulatory factor 3 and the expression of a subset of lipopolysaccharide-inducible genes. J Immunol 167:5887-5894.

25. Doyle, S., S. Vaidya, R. O'Connell, H. Dadgostar, P. Dempsey, T. Wu, G. Rao, R. Sun, M. Haberland, R. Modlin, and G. Cheng. 2002. IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity 17:251-263.

26. Barnes, B., B. Lubyova, and P.M. Pitha. 2002. On the role of IRF in host defense. J Interferon Cytokine Res 22:59-71.

27. Yoneyama, M., W. Suhara, and T. Fujita. 2002. Control of IRF-3 activation by phosphorylation. J Interferon Cytokine Res 22:73-76.

28. Reich, N.C. 2002. Nuclear/cytoplasmic localization of IRFs in response to viral infection or interferon stimulation. J Interferon Cytokine Res 22:103-109.

29. Peters, R.T., S.M. Liao, and T. Maniatis. 2000. IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex. Mol Cell 5:513-522.

30. Shimada, T., T. Kawai, K. Takeda, M. Matsumoto, J. Inoue, Y. Tatsumi, A. Kanamaru, and S. Akira. 1999. IKK-i, a novel lipopolysaccharide-inducible kinase that is related to IkappaB kinases. Int Immunol 11:1357-1362.

31. Pomerantz, J.L., and D. Baltimore. 1999. NF-kappaB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase. Embo J 18:6694-6704.

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

13

32. Bonnard, M., C. Mirtsos, S. Suzuki, K. Graham, J. Huang, M. Ng, A. Itie, A. Wakeham, A. Shahinian, W.J. Henzel, A.J. Elia, W. Shillinglaw, T.W. Mak, Z. Cao, and W.C. Yeh. 2000. Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription. Embo J 19:4976-4985.

33. Tojima, Y., A. Fujimoto, M. Delhase, Y. Chen, S. Hatakeyama, K. Nakayama, Y. Kaneko, Y. Nimura, N. Motoyama, K. Ikeda, M. Karin, and M. Nakanishi. 2000. NAK is an IkappaB kinase-activating kinase. Nature 404:778-782.

34. Sharma, S., B.R. tenOever, N. Grandvaux, G.P. Zhou, R. Lin, and J. Hiscott. 2003. Triggering the interferon antiviral response through an IKK-related pathway. Science 300:1148-1151.

35. Fitzgerald, K.A., S.M. McWhirter, K.L. Faia, D.C. Rowe, E. Latz, D.T. Golenbock, A.J. Coyle, S.M. Liao, and T. Maniatis. 2003. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat Immunol 4:491-496.

36. McWhirter, S.M., K.A. Fitzgerald, J. Rosains, D.C. Rowe, D.T. Golenbock, and T. Maniatis. 2004. IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts. Proc Natl Acad Sci U S A 101:233-238.

37. Yamamoto, M., S. Sato, K. Mori, K. Hoshino, O. Takeuchi, K. Takeda, and S. Akira. 2002. Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling. J Immunol 169:6668-6672.

38. Oshiumi, H., M. Matsumoto, K. Funami, T. Akazawa, and T. Seya. 2003. TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-beta induction. Nat Immunol 4:161-167.

39. Yamamoto, M., S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, O. Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, and S. Akira. 2003. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 301:640-643.

40. Yamamoto, M., S. Sato, H. Hemmi, S. Uematsu, K. Hoshino, T. Kaisho, O. Takeuchi, K. Takeda, and S. Akira. 2003. TRAM is specifically involved in the Toll-like receptor 4-mediated MyD88-independent signaling pathway. Nat Immunol.

41. Krug, A., A.R. French, W. Barchet, J.A. Fischer, A. Dzionek, J.T. Pingel, M.M. Orihuela, S. Akira, W.M. Yokoyama, and M. Colonna. 2004. TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function. Immunity 21:107-119.

42. Izaguirre, A., B.J. Barnes, S. Amrute, W.S. Yeow, N. Megjugorac, J. Dai, D. Feng, E. Chung, P.M. Pitha, and P. Fitzgerald-Bocarsly. 2003. Comparative analysis of IRF and IFN-alpha expression in human plasmacytoid and monocyte-derived dendritic cells. J Leukoc Biol 74:1125-1138.

43. Gibson, S.J., J.M. Lindh, T.R. Riter, R.M. Gleason, L.M. Rogers, A.E. Fuller, J.L. Oesterich, K.B. Gorden, X. Qiu, S.W. McKane, R.J. Noelle, R.L. Miller, R.M. Kedl, P. Fitzgerald-Bocarsly, M.A. Tomai, and J.P. Vasilakos. 2002. Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7 agonists, imiquimod and resiquimod. Cell Immunol 218:74-86.

44. Siegal, F.P., N. Kadowaki, M. Shodell, P.A. Fitzgerald-Bocarsly, K. Shah, S. Ho, S. Antonenko, and Y.J. Liu. 1999. The nature of the principal type 1 interferon-producing cells in human blood. Science 284:1835-1837.

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

14

45. Jarrossay, D., G. Napolitani, M. Colonna, F. Sallusto, and A. Lanzavecchia. 2001. Specialization and complementarity in microbial molecule recognition by human myeloid and plasmacytoid dendritic cells. Eur J Immunol 31:3388-3393.

46. Kadowaki, N., S. Ho, S. Antonenko, R.W. Malefyt, R.A. Kastelein, F. Bazan, and Y.J. Liu. 2001. Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens. J Exp Med 194:863-869.

47. Muzio, M., J. Ni, P. Feng, and V.M. Dixit. 1997. IRAK (Pelle) family member IRAK-2 and MyD88 as proximal mediators of IL-1 signaling. Science 278:1612-1615.

48. Yoneyama, M., W. Suhara, Y. Fukuhara, M. Fukuda, E. Nishida, and T. Fujita. 1998. Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300. Embo J 17:1087-1095.

49. Au, W.C., P.A. Moore, D.W. LaFleur, B. Tombal, and P.M. Pitha. 1998. Characterization of the interferon regulatory factor-7 and its potential role in the transcription activation of interferon A genes. J Biol Chem 273:29210-29217.

50. Dent, C.L., and D.R. Gewert. 1996. A regulatory domain within the virus-response element of the interferon alpha 1 gene acts as a transcriptional repressor sequence and determinant of cell-specific gene expression. Eur J Biochem 236:895-903.

51. Cao, Z., J. Xiong, M. Takeuchi, T. Kurama, and D.V. Goeddel. 1996. TRAF6 is a signal transducer for interleukin-1. Nature 383:443-446.

52. Li, X., M. Commane, C. Burns, K. Vithalani, Z. Cao, and G.R. Stark. 1999. Mutant cells that do not respond to interleukin-1 (IL-1) reveal a novel role for IL-1 receptor-associated kinase. Mol Cell Biol 19:4643-4652.

53. Yeh, T.J., P.T. McBride, J.C. Overall, Jr., and J.A. Green. 1982. Automated, quantitative cytopathic effect reduction assay for interferon. J Clin Microbiol 16:413-415.

54. Yeow, W.S., W.C. Au, Y.T. Juang, C.D. Fields, C.L. Dent, D.R. Gewert, and P.M. Pitha. 2000. Reconstitution of virus-mediated expression of interferon alpha genes in human fibroblast cells by ectopic interferon regulatory factor-7. J Biol Chem 275:6313-6320.

55. Lee, F.S., R.T. Peters, L.C. Dang, and T. Maniatis. 1998. MEKK1 activates both IkappaB kinase alpha and IkappaB kinase beta. Proc Natl Acad Sci U S A 95:9319-9324.

56. Wathelet, M.G., C.H. Lin, B.S. Parekh, L.V. Ronco, P.M. Howley, and T. Maniatis. 1998. Virus infection induces the assembly of coordinately activated transcription factors on the IFN-beta enhancer in vivo. Mol Cell 1:507-518.

57. Kawai, T., S. Sato, K.J. Ishii, C. Coban, H. Hemmi, M. Yamamoto, K. Terai, M. Matsuda, J. Inoue, S. Uematsu, O. Takeuchi, and S. Akira. 2004. Interferon-alpha induction through Toll-like receptors involves a direct interaction of IRF7 with MyD88 and TRAF6. Nat Immunol 5:1061-1068.

58. Honda, K., H. Yanai, T. Mizutani, H. Negishi, N. Shimada, N. Suzuki, Y. Ohba, A. Takaoka, W.C. Yeh, and T. Taniguchi. 2004. Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling. Proc Natl Acad Sci U S A 101:15416-15421.

59. Maniatis, T. 1986. Mechanisms of human beta-interferon gene regulation. Harvey Lect 82:71-104.

60. Cao, Z., W.J. Henzel, and X. Gao. 1996. IRAK: a kinase associated with the interleukin-1 receptor. Science 271:1128-1131.

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

15

61. Jiang, Z., T.W. Mak, G. Sen, and X. Li. 2004. Toll-like receptor 3-mediated activation of NF-{kappa}B and IRF3 diverges at Toll-IL-1 receptor domain-containing adapter inducing IFN-{beta}. Proc Natl Acad Sci U S A.

62. Jiang, Z., M. Zamanian-Daryoush, H. Nie, A.M. Silva, B.R. Williams, and X. Li. 2003. Poly(I-C)-induced Toll-like receptor 3 (TLR3)-mediated activation of NFkappa B and MAP kinase is through an interleukin-1 receptor-associated kinase (IRAK)-independent pathway employing the signaling components TLR3-TRAF6-TAK1-TAB2-PKR. J Biol Chem 278:16713-16719.

63. Suzuki, N., S. Suzuki, U. Eriksson, H. Hara, C. Mirtosis, N.J. Chen, T. Wada, D. Bouchard, I. Hwang, K. Takeda, T. Fujita, S. Der, J.M. Penninger, S. Akira, T. Saito, and W.C. Yeh. 2003. IL-1R-associated kinase 4 is required for lipopolysaccharide-induced activation of APC. J Immunol 171:6065-6071.

64. Sato, S., M. Sugiyama, M. Yamamoto, Y. Watanabe, T. Kawai, K. Takeda, and S. Akira. 2003. Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF) associates with TNF receptor-associated factor 6 and TANK-binding kinase 1, and activates two distinct transcription factors, NF-kappaB and IFN-regulatory factor-3, in the Toll-like receptor signaling. J Immunol 171:4304-4310.

65. Barnes, B.J., J. Richards, M. Mancl, S. Hanash, L. Beretta, and P.M. Pitha. 2004. Global and Distinct Targets of IRF-5 and IRF-7 during Innate Response to Viral Infection. J Biol Chem 279:45194-45207.

66. Au, W.C., P.A. Moore, W. Lowther, Y.T. Juang, and P.M. Pitha. 1995. Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. Proc Natl Acad Sci U S A 92:11657-11661.

67. Barnes, B.J., A.E. Field, and P.M. Pitha-Rowe. 2003. Virus-induced heterodimer formation between IRF-5 and IRF-7 modulates assembly of the IFNA enhanceosome in vivo and transcriptional activity of IFNA genes. J Biol Chem 278:16630-16641.

68. Oshiumi, H., M. Sasai, K. Shida, T. Fujita, M. Matsumoto, and T. Seya. 2003. TICACM-2: a bridging adapter recruiting to Toll-like receptor 4 TICAM-1 that induces interferon-beta. J Biol Chem.

69. Chariot, A., A. Leonardi, J. Muller, M. Bonif, K. Brown, and U. Siebenlist. 2002. Association of the adaptor TANK with the I kappa B kinase (IKK) regulator NEMO connects IKK complexes with IKK epsilon and TBK1 kinases. J Biol Chem 277:37029-37036.

70. Nomura, F., T. Kawai, K. Nakanishi, and S. Akira. 2000. NF-kappaB activation through IKK-i-dependent I-TRAF/TANK phosphorylation. Genes Cells 5:191-202.

71. Rothe, M., J. Xiong, H.B. Shu, K. Williamson, A. Goddard, and D.V. Goeddel. 1996. I-TRAF is a novel TRAF-interacting protein that regulates TRAF-mediated signal transduction. Proc Natl Acad Sci U S A 93:8241-8246.

72. Cheng, G., and D. Baltimore. 1996. TANK, a co-inducer with TRAF2 of TNF- and CD 40L-mediated NF-kappaB activation. Genes Dev 10:963-973.

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

16

Figure Legends Figure 1. TLR7 and TLR8 activate IRF-5 and IRF-7 but not IRF-3. (A) HEK293 cells were transfected with TLR7, TLR8 or TLR3, the UAS(GAL)-reporter plasmid and with either Gal4-IRF-3, Gal4-IRF-5 or Gal4-IRF-7. 36h after transfection, cells were stimulated with 10 µM R-848, 100 µg/ml pI:C, 100 HAU SeV or left untreated. 15h after stimulation, luciferase activity was determined. (B, C) Nuclear translocation of IRF-5-GFP (C) and IRF-7-GFP (D) upon R-848 stimulation. RAW264.7 cells were transiently transfected with IRF-5-GFP or IRF-7-GFP and stimulated with 15 µM R-848 or 100 µg/ml pI:C for 24h or left untreated. Nuclear translocation was monitored by confocal microscopy and the fields shown are representative of several fields examined. Figure 2. TLR7 signaling requires IRF-5 to activate type I IFNs. (A, B) 2fTGH cells were transfected with either IFNB (A) or IFNA1-SAP reporter plasmid (B) and if indicated with pFlag-CMV1-TLR7 and/or Flag-IRF-5. After 20h of transfection cells were stimulated with 10 µM R-848 or infected with NDV (240 HAU) or VSV (MOI 2) for 16h. The SAP activity was determined in the cell lysates. (C) 2fTGH cells were transfected for 20h with the IFNA1-SAP reporter, Flag-IRF-5 and where indicated with pFlag-CMV1-TLR7 and DN IRF-5. Following 16h of stimulation with 10 µM R-848, SAP activity was determined in the cell lysates. (D) HEK293T, HEK293T/IRF-5, 2fTGH and 2fTGH/IRF-5 cells were transiently transfected with pFlag-CMV-TLR7. Following stimulation with 10 µM R-848 for 16h, endogenous type I IFN or IFNα were analyzed in the cell culture supernatants by the viral cytopathic effect assay. Figure 3. The TLR7/8-mediated activation of IFNA in THP-1 cells requires IRF-5. (A) Following 16h of stimulation, as indicated, total RNA was isolated from THP-1 cells, reverse-transcribed into cDNA. IFNA, IFNB, IRF-5, IRF7 and ß-actin cDNA was amplified by PCR. Endogenous type I IFNs were analyzed in the cell culture supernatants by the viral cytopathic effect assay. (B) IRF-5 siRNA or LacZ siRNA was transiently transfected to THP-1 cells by electroporation. After 8h of transfection, cells were stimulated with 10 µM R-848 or left untreated. 16h later, RNA was isolated, reverse-transcribed and the cDNA was subjected to semi-quantitative PCR analysis of IRF-5 and IFNA expression. Figure 4. TLR7 and TLR8 signaling to IRF-5 requires MyD88, IRAK-1 and TRAF6. (A) HEK293 cells were transfected with the indicated TLR plasmids, with the UAS(GAL)-reporter plasmid, with Gal4-IRF-5 and different concentrations of the DN construct, MyD88-TIR. After 36h, cells were exposed to R-848 (10 µM), pI:C (100 µg/ml) or PBS. After 15h of stimulation, luciferase activity was determined. (B) IRAK-1 deficient I1A-HEK293 cells and control HEK293 cells were transfected with TLR7 or TLR8, the UAS(GAL)-reporter plasmid and Gal4-IRF-5. After 36h, cells were stimulated with 10 µM R-848 or left untreated. The production of luciferase was determined after an additional 15h. (C) HEK293 cells were transfected with the indicated TLR constructs, UAS(GAL)-reporter plasmid, with Gal4-IRF-5 and different concentrations of DN TRAF6 or DN TRAF2. After 36h of transfection cells were stimulated with 10 µM R-848 or left untreated for an additional15h before luciferase activity was determined. Figure 5. Purified TBK1 but not IKKβ phosphorylates IRF3 and IRF5. (A) Wild-type TBK1 or IKKβ was expressed in insect cells using baculovirus vectors. Kinase activity of the purified

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

17

proteins was assayed using GST-IRF5, GST-IRF3 or GST alone (not shown) substrates as indicated. (B) HEK293 cells were transfected with the indicated TLR constructs, UAS(GAL)-reporter plasmid, with Gal4-IRF-5 and different concentrations of DN TBK1 (TBK1K38A) or DN IKKε (IKKεK38A). 36h after transfection, cells were stimulated with 10 µM R-848 or left untreated. Luciferase activity was determined after an additional 15h. Figure 6. Regulation of the IRF proteins in TLR7 and TLR3 signaling. In response to dsRNA signaling, the adapter molecule TRIF is recruited to the intracytoplasmic domain of TLR3. TRIF relays signals from activated TLR3 and via the IKK related kinase, TBK1, phosphorylates and activates IRF-3. IRF-3 subsequently binds and activates the IFNβ promoter. In contrast to TLR3, the TLR7 pathway requires MyD88, IRAK-1 and TRAF6 and activates IRF5 and not IRF3. IRF5 contributes to the IFN response in TLR7 signaling. The MyD88-IRAK1-TRAF6-IRF5 pathway may also control additional genes.

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

IRF-5

IFNA

B-actin

IRF5 siRNA lacz siRNA con. R848 con. R848 con. R848

0 1 2 3 4

IFNA

B-actin

IRF-5

IFNB

Type I IFNs U/ml <1/5 30 114 1654

IRF-70 – water control1 – untreated2 – NDV3 – VSV4 – R848

A

B

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

Gal4-IRF5

Rela

tive

Stim

ulat

ion

Rela

tive

Stim

ulat

ion

0

2

4

6

8

0 10 25 50

R-848

PBS

0

2

4

6

8

0 10 25 50

R-848

PBS

TBK1 K38A IKKe K38A

TBK1TBK1

IKKb IKKbIKKb

IKKbKinase

IRF5 IRF3 IkBa IkBa S32/S36A

Figure 5

A. B. by guest on June 27, 2018

http://ww

w.jbc.org/

Dow

nloaded from

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from

Paula M. Pitha, Katherine A. Fitzgerald and Douglas T. GolenbockAnnett Schoenemeyer, Betsy J. Barnes, Margo E. Mancl, Eicke Latz, Nadege Goutagny,

The interferon regulatory factor, IRF5, is a central mediator of TLR7 signaling

published online January 28, 2005J. Biol. Chem. 

  10.1074/jbc.M412584200Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

by guest on June 27, 2018http://w

ww

.jbc.org/D

ownloaded from