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The Journal of Immunology, 2007, 178, 6770 -6776
Copyright © 2007 by The American Association of Immunologists, Inc.
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Lack of Essential Role of NF-{kappa}B p50, RelA, and cRel Subunits in Virus-Induced Type 1 IFN Expression1

Xingyu Wang*, Sofia Hussain*, Emilie-Jeanne Wang{dagger}, Xiuyan Wang{ddagger}, Ming O. Li{ddagger}, Adolfo García-Sastre§ and Amer A. Beg2,*

* Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center & Research Institute, University of South Florida, Tampa, FL 33612; {dagger} Department of Microbiology, Columbia University, New York, NY 10032; {ddagger} Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520; and § Department of Microbiology, and Emerging Pathogens Institute, Mount Sinai School of Medicine, New York, NY 10028


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Type 1 IFNs (IFN-{alpha}beta) play pivotal roles in the host antiviral response and in TLR-induced signaling. IFN regulatory factor (IRF) and NF-{kappa}B transcription factors are thought to be crucial for virus-induced mRNA expression of IFN-beta. Although recent studies have demonstrated essential roles for IRF3 and IRF7, the definitive role of NF-{kappa}B factors in IFN-beta (or IFN-{alpha}) expression remains unknown. Using mice deficient in distinct members of the NF-{kappa}B family, we investigated NF-{kappa}B function in regulating type 1 IFN expression in response to Sendai virus and Newcastle disease virus infection. Surprisingly, IFN-beta and IFN-{alpha} expression was strongly induced following virus infection of mouse embryonic fibroblasts (MEFs) from p50–/–, RelA/p65–/–, cRel–/–, p50–/–cRel–/–, and p50–/–RelA–/– mice. Compared with wild-type MEFs, only RelA–/– and p50–/–RelA–/– MEFs showed a modest reduction in IFN-beta expression. To overcome functional redundancy between different NF-{kappa}B subunits, we expressed a dominant-negative I{kappa}B{alpha} protein in p50–/–RelA–/– MEFs to inhibit activation of remaining NF-{kappa}B subunits. Although viral infection of these cells failed to induce detectable NF-{kappa}B activity, both Sendai virus and Newcastle disease virus infection led to robust IFN-beta expression. Virus infection of dendritic cells or TLR9-ligand CpG-D19 treatment of plasmacytoid dendritic cells from RelA–/– or p50–/–cRel–/– mice also induced robust type 1 IFN expression. Our findings therefore indicate that NF-{kappa}B subunits p50, RelA, and cRel play a relatively minor role in virus-induced type 1 IFN expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Type 1 IFNs (IFN-{alpha}beta) play a central role in the host response to viral infection (1). The two main subtypes of type 1 IFNs are IFN-{alpha} and IFN-beta. The IFN-{alpha} gene family is comprised of more than a dozen members, whereas IFN-beta is encoded by a single gene. Recent studies have identified RIG-I and Mda5 in viral detection and initiating signaling via the IFN-beta promoter stimulater/mitochondrial antivirus signaling protein adapter to TANK-binding kinase-1/1{kappa}B kinase (IKK)3-{epsilon} kinases, resulting in activation of key transcription factors (2, 3, 4, 5). The mechanisms governing virus- and TLR-induced signaling leading to expression of type 1 IFN transcription have been a topic of intense interest for several years (6). In particular, numerous studies have investigated regulation of the IFN-beta gene (6). Four cis elements are thought to be crucial for virus-induced IFN-beta expression (7). These elements, designated positive regulatory domain (PRD)-I, II, III, and IV, bind key transcription factor that mediates virus-induced expression. The PRD-II element binds to NF-{kappa}B transcription factors, and is thought to play an especially important role in virus-induced IFN-beta enhanceosome assembly (8, 9). Elements PRD-I and PRD-III associate with members of the IFN regulatory factor (IRF) transcription factor family (6). Using gene-targeted mice, recent studies have defined an essential role for IRF3 and IRF7 in regulation of type 1 IFN expression (10, 11). Absence of IRF7 or combined absence of IRF3 and IRF7 prevents induction of IFN-{alpha} and IFN-beta following viral infection or TLR stimulation of dendritic cells (DCs) (10, 11). In contrast, no definitive study has addressed the function of members of the NF-{kappa}B family in virus-induced type 1 IFN expression.

Regulation of type 1 IFN expression is subject to a critically important positive feedback loop (6, 12). Two type 1 IFN gene products, IFN-{alpha}4 and IFN-beta, are rapidly induced following viral infection. Newly synthesized IFN-{alpha}4 and IFN-beta bind to the type 1 IFN receptor, resulting in induction of the IFN-stimulated gene factor-3 transcription factor (6, 12). IFN-stimulated gene factor-3 is a complex of STAT1, STAT2, and IRF9, and is critical for expression of non-{alpha}4 subtypes of IFN-{alpha} (6, 12). This most likely occurs indirectly through transcriptional induction of IRF7 expression (13). Indeed, high-level induction of IFN-{alpha}4/IFN-beta and other IFN-{alpha} genes is dependent on IRF7 (11).

The greatest levels of type 1 IFN are produced by members of the DC lineage (14). Thus, virus infection of conventional DCs (cDCs) and especially plasmacytoid DCs (pDCs) results in very high levels of type 1 IFN expression (3, 14, 15). TLR ligand stimulation of both cDCs and pDCs also results in significant induction of type 1 IFN expression (3, 16). Depending on the specific TLR ligand, IFN expression is controlled by TLR-associated adaptor molecules MyD88 or Toll-IL1 domain-containing adaptor-inducing IFN-beta (3, 16). An especially potent stimulus for type 1 expression is the TLR9 ligand CpG DNA D19 (14, 17). D19 is specifically retained in endosomal vesicles of pDCs, resulting in continuous high-level stimulation that allows potent type 1 IFN expression (17). Additional adapter molecules have also been shown to be essential for virus-induced type 1 IFN expression through both TLR-dependent and -independent pathways (18, 19).

NF-{kappa}B transcription factors are activated by virus infection and by all known TLR ligands (3, 16). NF-{kappa}B activation is mediated by proteasomal degradation of the I{kappa}B proteins, an event triggered by I{kappa}B phosphorylation by IKK-beta (20). Five different members of the NF-{kappa}B family have been identified in mammals: p50, p52, RelA (p65), cRel, and RelB (21). A potential role for NF-{kappa}B in IFN-beta expression was implicated nearly two decades ago, with subsequent studies indicating an important role for NF-{kappa}B factors in virus-induced IFN-beta expression (8, 9, 22, 23). However, no definitive study has addressed the actual requirement for NF-{kappa}B in IFN-beta (or IFN-{alpha}) regulation. Using NF-{kappa}B-deficient mouse embryonic fibroblasts (MEFs), cDCs, and pDCs, we therefore examined the role of NF-{kappa}B factors in virus- and TLR-induced expression of type 1 IFNs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and materials

C57BL/6 CD45.1 and C57BL/6 CD45.2 mice were purchased from Jackson Laboratories and bred in the animal facility of H. Lee Moffitt Cancer Center and Research Institute. RelA–/–, p50–/–, and cRel–/– mice have been described previously (24). All mice used were backcrossed into a C57BL/6 background for 10 generations. E14 wild-type (WT) and RelA–/– fetal liver hemopoietic precursors (CD45.2) were adoptively transferred into lethally irradiated CD45.1 recipient mice, as previously described (24). Recipient mice were typically used after 6–8 wk. All experiments with mice were conducted in accordance with institutional guidelines. D19 CpG DNA was synthesized by Sigma-Aldrich.

Cells

MEFs were cultured in fibroblast medium (DMEM, 10% of donor bovine serum, antibiotics). p50–/–RelA–/– fibroblasts were immortalized by continued culture. A total of 1 x 106 cells was plated in 6-cm tissue culture dishes in 5 ml of medium and cultured overnight before being used in experiments.

Bone marrow-derived DCs (BMDCs) were cultured from mouse bone marrow precursors following a modified procedure (24). Briefly, bone marrow cells were cultured in 24-well plates in DC medium (RPMI 1640, 5% FBS, 100 µM 2-ME and antibiotics) in the presence of 3% supernatant from J558L cells transduced with mouse GM-CSF at 1 x 106 cells/ml. Medium was changed every 2 days. On day 6, DC clusters were collected and resuspended in fresh DC medium containing GM-CSF. Mouse spleen pDCs were purified using Miltenyi Biotec anti-mouse plasmacytoid DC Ag-1 magnetic beads and cultured in DC medium. For all DC experiments, cells were plated at a density of 1 x 106 cells/ml.

Sendai virus (SV) and Newcastle disease virus (NDV) infections

MEFs or BMDCs were washed twice in culture medium without serum. The 200HA U/ml SV (Cantell strain; Charles River Laboratories) or NDV (strain B1 grown in embryonated chicken eggs) at a multiplicity of infection of 5 (for MEFs) or 2 (for DCs) was then added to cells without serum for ~1 h. MEFs were then washed twice in medium without serum and incubated in culture medium for time periods indicated in the text. After a 1-h infection, serum and GM-CSF supernatant were added to DCs, which were then cultured further for time periods indicated in the text.

Retroviral infection of immortalized MEFs

The I{kappa}B{alpha} Ser32/36 S->A mutant gene was cloned into murine stem cell virus internal ribosome entry site GFP (MIG) retroviral expression vector. Retroviruses were produced by transfecting 293T cells with retroviral plasmid DNA and the pCL-Eco packaging plasmid. p50–/–RelA–/– fibroblasts were spin infected at 2600 rpm for 1 h at room temperature. GFP-positive cells were isolated by cell sorting and then cultured in fibroblast medium.

RNase protection assay (RPA) and real-time PCR

Total RNA was prepared by TRIzol reagent for RPA (Ambion) for detection of IFN-beta and IL-12 expression. RPA was performed according to manufacturer’s recommendations (Ambion). For real-time PCR, RNA was further purified using Qiagen RNeasy kit. Purified RNA samples were then subjected to real-time PCR analysis by a standard curve method for relative quantitation from Applied Biosystems. Briefly, standards were prepared by serial dilution of a pool of all samples. RNA samples and standards were reverse transcribed into cDNA using TaqMan reverse-transcription reagents from Applied Biosystems. cDNAs were then subjected to real-time PCR analysis by standard curve method in an Applied Biosystems 7900HT Sequence Detection System using SYBR Green I Dye. All samples were run in triplicate, with SDs indicated in figures. Results for target genes are presented after normalizing to beta-actin. PCR primers used were: 5'-AGCTCCAAGAAAGGACGAACAT-3' (IFN-beta-F), 5'-GCCCTGTAGGTGAGGTTGATCT-3' (FIN-beta-R); 5'-GTTCCTGCACTTGGCAATCATCCA-3' (I{kappa}B{alpha}-F), 5'-TCAGGATCACAGCCAGCTTTCAGA-3' (I{kappa}B{alpha}-R); 5'-ATGGAATCCTGTGGCATCCAT-3' (beta-actin-F), 5'-CCACCAGACAACACTGTGTTGG-3' (beta-actin-R); 5'-ARSYTGTSTGATGCARCAGGT-3' (IFN-{alpha} (non-{alpha}4)-F), 5'-GGWACACAGTGATCCTGTGG-3' (IFN-{alpha} (non-{alpha}4)-R) (sequence provided by D. Levy, New York, NY); 5'-CCTGTGTGATGCAGGAACC-3' (IFN-{alpha}4-F), 5'-TCACCTCCCAGGCACTGA-3' (IFN-{alpha}4-R); 5'-CTGGAGCCATGGGTATGCA-3' (IRF-7-F), 5'-AAGCACAAGCCGAGACTGCT-3' (IRF-7-R).

EMSA and ELISA

For detection of NF-{kappa}B activation, nuclear extracts were prepared and EMSA was performed, as described (24). After virus infection or D19 treatment, cells were cultured for indicated time periods and supernatants were collected. Production of mouse IFN-{alpha} and IFN-beta was determined by ELISA kits from PBL Biomedical Laboratories following the manufacturer’s recommendations. Readings below the first standard curve sample were considered undetected.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Regulation of type 1 IFN expression in NF-{kappa}B-deficient embryonic fibroblasts following SV infection

Using MEFs, we investigated the individual and combined roles of NF-{kappa}B p50, RelA, and cRel in regulating IFN-beta expression. To this end, MEFs were cultured for different time periods after infection with SV, following which IFN-beta mRNA expression was determined by RPA. In WT MEFs, IFN-beta expression was detected at 4, 6, or 12 h after SV infection, with maximal expression at 6 h (Fig. 1A). IFN-beta mRNA expression was indistinguishable among WT, p50–/–, and cRel–/– MEFs at all three time points (Fig. 1A). Compared with other genotypes, IFN-beta expression was slightly decreased at 4- and 6-h time points in RelA–/– MEFs (Fig. 1A; also see below). IFN-beta expression may be controlled by redundant function of p50, RelA, and cRel. To address this possibility, we first used p50–/–cRel–/– MEFs. Compared with WT MEFs, p50–/–cRel–/– MEFs showed similar induction of IFN-beta at the different time points tested (Fig. 1A). To quantify changes in IFN-beta expression, we performed real-time PCR on mRNA from SV-infected MEFs. Consistent with RPA results, only RelA–/– MEFs showed a modest reduction in IFN-beta expression at 4- and 6-h, but not 12-h time points (Fig. 1A).


Figure 1
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  		FIGURE 1. Regulation of type 1 IFN expression in NF-{kappa}B-deficient embryonic fibroblasts following SV infection. A, Expression of IFN-beta mRNA determined by both RPA (left panel) and real-time PCR (right panel). B–E, Expression of IFN-{alpha}4 (B), IFN-{alpha} (non-4) (C), IRF-7 (D), and I{kappa}B{alpha} mRNA (E) was determined by real-time PCR. SDs from triplicate samples are also indicated. F, IFN-beta protein expression determined by ELISA. UI, Uninfected; IFN-{alpha} (non-{alpha}4), pan IFN-{alpha}, except for IFN-{alpha}4; U.D., undetected.

 
In addition to IFN-beta, the {alpha}4 subtype of IFN-{alpha} can be directly induced by virus infection, i.e., in the absence of IFN autocrine signaling. Notably, the role of NF-{kappa}B p50, RelA, or cRel in regulating virus-induced IFN-{alpha}4 or IFN-{alpha} (non-{alpha}4) has not been determined. Using real-time PCR, we first determined the roles of the above NF-{kappa}B subunits in IFN-{alpha}4 mRNA expression. As shown in Fig. 1B, no substantial difference in IFN-{alpha}4 expression was noticed between WT and the different knockout MEFs. IFN-{alpha} (non-{alpha}4) requires IFN-beta- and IFN-{alpha}4-induced autocrine signaling for induction of mRNA expression. RelA–/– MEFs showed substantially reduced expression of IFN-{alpha} (non-{alpha}4) at 4 and 6 h, but elevated expression at 12 h (Fig. 1C). This is most likely due to reduced expression of IFN-beta and IFN-{alpha}4 at early time points, resulting in decreased IFN-{alpha} (non-{alpha}4) induction by autocrine signaling. Other knockout MEFs failed to show appreciable reduction in IFN-{alpha} (non-{alpha}4) expression (Fig. 1C). Another crucial target of autocrine signaling is the gene encoding for the IRF7 transcription factor. As with IFN-{alpha} (non-{alpha}4), RelA–/– MEFs showed slightly reduced IRF7 expression, whereas, interestingly, p50–/–cRel–/– MEFs showed slightly elevated expression (Fig. 1D). Because only RelA–/– and p50–/–cRel–/– MEFs showed any appreciable change in virus-induced gene expression, we specifically tested these cells in two additional experiments. First, we tested how SV-induced I{kappa}B{alpha} mRNA expression, a bona fide NF-{kappa}B target gene, was affected in RelA–/– and p50–/–cRel–/– MEFs. Unlike modest reductions in IFN gene expression, I{kappa}B{alpha} expression was dramatically reduced in RelA–/– MEFs, and to a lesser extent in p50–/–cRel–/– MEFs (Fig. 1E). Thus, whereas RelA absence does not have a major impact on IFN-beta, it is critical for induction of a bona fide NF-{kappa}B target gene. Second, we determined SV-induced IFN-beta protein expression in RelA–/– and p50–/–cRel–/– MEF supernatants. Consistent with mRNA results, RelA–/– MEFs, but not p50–/–cRel–/– MEFs, showed a 30% decrease in IFN-beta protein levels compared with WT MEFs.

Type 1 IFN expression in p50–/–RelA–/– MEFs expressing a dominant-negative variant of I{kappa}B{alpha}

Hetreodimers of p50 + RelA comprise the major NF-{kappa}B activity in MEFs. We next used immortalized p50–/–RelA–/– MEFs to determine virus-induced NF-{kappa}B activity. SV infection of WT immortalized MEFs led to highly significant increase in NF-{kappa}B levels that bound to the MHC class I (H-2Kb) and IFN-beta {kappa}B sites (Fig. 2A). Consistent with predominance of p50 and RelA in MEFs, overall SV-induced NF-{kappa}B activity was dramatically reduced in p50–/–RelA–/– MEFs (Fig. 2A). However, a low-level activity was detected, which was partly inhibited by Abs against the three remaining NF-{kappa}B subunits (p52, cRel, and RelB) (Fig. 2A). To inhibit virus-induced activation of these subunits, we transduced p50–/–RelA–/– MEFs with a retrovirus expressing a dominant-negative variant of I{kappa}B{alpha}, which is refractory to signal-induced phosphorylation and degradation (I{kappa}B{alpha}SA). Control retrovirus (MIG)- and I{kappa}B{alpha}SA retrovirus (MIG-I{kappa}B{alpha})-infected p50–/–RelA–/– MEFs were FACS sorted based on GFP expression and then subjected to SV infection. Importantly, MIG-I{kappa}B{alpha}-transduced cells showed virtually no inducible NF-{kappa}B activity that bound to the MHC class I (H-2Kb) and IFN-beta {kappa}B sites after SV infection (Fig. 2A). Thus, I{kappa}B{alpha}SA expression in p50–/–RelA–/– MEFs can inhibit the three remaining NF-{kappa}B subunits (p52, cRel, and RelB), thereby providing a cell type virtually devoid of detectable virus-induced NF-{kappa}B activity.


Figure 2
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  		FIGURE 2. Type 1 IFN expression in p50–/–RelA–/– MEFs expressing a dominant-negative variant of I{kappa}B{alpha}. A, EMSA analysis of NF-{kappa}B activity in MEFs. Left panels, Show binding to the MHC class I (H-2Kb) and IFN-beta {kappa}B sites. Right panels, Show effect of control Ab (IgG) or Abs to cRel, RelB, or p100/p52 on virus-induced complexes (C1-C4) in p50–/–RelA–/– MEFs (longer exposure time was used compared with left panels to show presence of low-level binding complexes). B–E, Expression of IFN-beta (B), IFN-{alpha}4 (C), IFN-{alpha} (non-{alpha}4) (D), and IRF-7 mRNA (E) determined by real-time PCR. p50–/–RelA–/–MIG: p50–/–RelA–/– cells infected with control retrovirus. p50–/–RelA–/– I{kappa}B{alpha}SA: p50–/–RelA–/– cells infected with retrovirus expressing I{kappa}B{alpha}SA.

 
We then used WT, p50–/–RelA–/–, MIG p50–/–RelA–/–, and MIG-I{kappa}B{alpha} p50–/–RelA–/– MEFs to investigate IFN-beta expression after SV infection. The lowest expression of IFN-beta was seen in MIG-I{kappa}B{alpha} p50–/–RelA–/– MEFs (Fig. 2B). However, this was <2-fold reduced compared with WT MEFs at 4 and 6 h, whereas MIG-I{kappa}B{alpha} p50–/–RelA–/– MEFs showed higher expression than WT MEFs at the 9-h time point (Fig. 2B). Furthermore, IFN-{alpha}4, IFN-{alpha} (non-{alpha}4), or IRF7 mRNA levels were also strongly induced in the different cell types (Fig. 2, C–E). These results therefore indicate that SV induces reduced, yet robust expression of type 1 IFNs in the absence of detectable NF-{kappa}B activity.

Induction of IFN-beta expression in NDV-infected NF-{kappa}B-deficient fibroblasts

It is possible that lack of a requirement for NF-{kappa}B in type 1 IFN expression reflects a unique property of SV. We therefore used select MEF subsets to determine IFN-beta expression following infection with NDV. NDV-induced IFN-beta expression in WT MEFs was strongest at 12 h after infection (Fig. 3A). Thus, SV and NDV induce IFN-beta expression with different kinetics, whereas SV shows maximal expression at 6 h postinfection and NDV shows maximal expression at 12 h postinfection. Notably, high levels of IFN-beta expression were induced in WT, RelA–/–, and p50–/–cRel–/–MEFs (Fig. 3A), although, as with SV infection, NDV-induced IFN-beta was also reduced in RelA–/– MEFs. In addition, IFN-beta expression was strongly induced in WT and p50–/–RelA–/–, MIG p50–/–RelA–/–, or MIG-I{kappa}B{alpha} p50–/–RelA–/– MEFs (Fig. 3B). Taken together, our results thus indicate that both SV and NDV can induce IFN-beta expression in the virtual absence of detectable NF-{kappa}B activity.


Figure 3
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  		FIGURE 3. Induction of IFN-beta expression in NDV-infected NF-{kappa}B-deficient fibroblasts. A, RPA analysis of IFN-beta mRNA expression in RelA–/– or p50–/–cRel–/– MEFs. B, RPA analysis of IFN-beta mRNA expression in indicated fibroblast lines.

 
Regulation of virus- and TLR-induced IFN-beta expression in p50-, cRel-, and RelA-deficient DCs

DCs are critically important for type 1 IFN expression following virus infection or TLR stimulation (14, 25). NF-{kappa}B proteins play a crucial role in DC development (24). Thus, combined absence of p50 and RelA severely impairs DC generation (24). In contrast, DC development can still take place in the combined absence of p50 and cRel (24). To determine how individual absence of RelA or combined absence of p50 and cRel impacts virus-induced IFN-beta expression, we generated DCs from bone marrow of RelA–/– and p50–/–cRel–/– mice. NDV infection of DCs resulted in robust induction of IFN-beta at 6 h postinfection (Fig. 4A), whereas MEFs showed high expression at 12 h (Fig. 3). These distinct kinetics are most likely due to differences in NDV infection kinetics of these two cell types. NDV or SV infection of RelA–/– BMDCs showed a modest reduction in IFN-beta mRNA expression (Fig. 4A), a result similar to virus infection of RelA–/– MEFs. In contrast, p50–/–cRel–/– BMDC infection with SV or NDV showed higher induction of IFN-beta compared with WT DCs (Fig. 4B). Similar to MEFs, we therefore conclude that neither RelA nor p50 + cRel function is essential for virus induction of IFN-beta expression. Virus infection of DCs also leads to production of inflammatory and T cell-stimulatory cytokines such as TNF-{alpha}, IL-6, and IL-12 (26). Importantly, IFN responsiveness is crucial for this because DCs lacking the type 1 IFN receptor exhibit reduced cytokine expression (26). Previously, NF-{kappa}B has been shown to regulate LPS-induced expression of the p40 subunit of IL-12 in DCs and macrophages (24, 27). We therefore examined whether, in addition to the type 1 IFN pathway, virus-induced IL-12 p40 expression also requires NF-{kappa}B proteins. NDV, and to a significantly lesser extent SV, induced expression of IL-12 p40 in WT DCs (Fig. 4). NDV-induced IL-12 p40 expression was reduced in RelA–/– DCs, but completely abolished in p50–/–cRel–/– DCs (Fig. 4). Thus, despite robust IFN-beta expression, p50–/–cRel–/– DCs are unable to induce IL-12 p40 expression. These results therefore indicate that p50 + cRel are independently involved in virus-induced IL-12 p40 expression.


Figure 4
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  		FIGURE 4. Regulation of virus-induced IFN-beta expression in p50-, cRel-, and RelA-deficient DCs. A, RPA analysis of IFN-beta mRNA expression in WT and RelA–/– BMDCs. B, RPA analysis of IFN-beta mRNA expression in WT and p50–/–cRel–/– BMDCs.

 
pDCs are a critically important type 1 IFN-producing cell type. One of the most potent signals for pDC-induced type 1 IFN expression is D19-CpG, which signals though TLR9. To determine the roles of RelA, p50, and cRel in TLR9-induced type 1 IFN expression, we purified pDCs from RelA–/– and p50–/–cRel–/– mouse spleens. After a 20-h stimulation with D19, pDC culture supernatants were collected to determine IFN-{alpha} and IFN-beta expression by ELISA. Remarkably similar to MEFs and BMDCs, a modest reduction in IFN-beta (and IFN-{alpha}) expression was noticed in RelA–/– pDCs (Figs. 5A and 1F). In contrast, p50–/–cRel–/– pDCs showed moderately elevated expression of both IFN-{alpha} and IFN-beta (Fig. 5B), as well as elevated constitutive expression of IFN-beta (Fig. 5B). We therefore conclude that RelA, p50, or cRel is not essential for TLR9-induced type 1 IFN expression in pDCs.


Figure 5
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  		FIGURE 5. Regulation of TLR-induced IFN-{alpha} and IFN-beta expression in p50-, cRel-, and RelA-deficient pDCs. A, Production of IFN-{alpha} (upper panel) and IFN-beta (lower panel) protein determined by ELISA in WT and RelA–/– pDC. B, Production of IFN-{alpha} (upper panel) and IFN-beta (lower panel) protein determined by ELISA in WT and p50–/–cRel–/– pDCs.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Many studies in the past have proposed critically important roles for NF-{kappa}B in virus-induced IFN-beta expression (8, 9, 22, 23). In the first definitive study of its kind, we have found that NF-{kappa}B/Rel transcription factors play only a modest role in virus-induced IFN-beta expression. Only the individual absence of RelA, but not p50 or cRel, resulted in reduction of IFN-beta mRNA expression. Our most definitive studies were performed on p50–/–RelA–/– fibroblasts. By expressing I{kappa}B{alpha}SA in p50–/–RelA–/– MEFs, we generated a fibroblast cell line that exhibited virtually no detectable virus-induced NF-{kappa}B activity. Even in these cells, albeit reduced compared with WT cells, SV or NDV induced robust IFN-beta expression. Nonetheless, we cannot exclude the possibility that low-level complexes of p52 and RelB, which appeared to be inhibited following I{kappa}B{alpha}SA expression, can still play a role in modulating IFN-beta expression. Based on studies of knockout cells, we can conclude that whereas NF-{kappa}B subunits p50, RelA, and cRel play a role, they are apparently not essential for virus-induced IFN-beta expression.

Three families of transcription factors have been implicated in IFN-beta expression, as follows: IRF, NF-{kappa}B, and AP-1. It has been shown that IRF3/7 factors play an essential role in transcriptional induction of IFN-beta (6). In contrast, our results indicate that NF-{kappa}B factors are largely dispensable for this induction. However, NF-{kappa}B factors may still play an important regulatory role in fine-tuning the kinetics and robustness of IFN-beta induction in response to viral infection or other stimuli. It is also possible that NF-{kappa}B factors are more important for IFN-beta expression in subset of cells or cell types with limiting amounts of other transcription factors, such as IRF3/7 or AP-1. In any case, our results indicate that NF-{kappa}B plays a more subtle role in IFN-beta expression than previously thought. Virus infection leads to formation of the IFN-beta enhanceosome (8), a structure that has been used as a paradigm for regulation of inducible transcription. Furthermore, NF-{kappa}B has been proposed to play a crucial role in assembly of the IFN-beta enhanceosome (8, 9). Our results, however, indicate that either NF-{kappa}B factors are not required for enhanceosome assembly or virus-induced IFN-beta expression can take place in the absence of the enhanceosome. An additional means for regulating mRNA levels is through control of stability. Indeed, mRNA levels of many cytokines, including IFN-beta, are subject to regulation through control of stability. Although we have not examined this possibility in the present study, it is plausible that NF-{kappa}B proteins can positively or negatively modulate IFN-beta (or IFN-{alpha}; see below) mRNA levels posttranscriptionally. Additional studies will be required to test this interesting possibility.

We have also determined the roles of NF-{kappa}B RelA, p50, and cRel subunits in virus- and TLR-induced IFN-beta expression in DCs. Although our findings with DCs also demonstrate that RelA, p50, and cRel are not essential for IFN-beta expression, interesting differences were noticed in IFN-beta expression between WT and knockout DCs. As in fibroblasts, a modest reduction in IFN-beta mRNA and protein expression was noticed in RelA–/– BMDCs and pDCs. Interestingly, virus infection or D19 treatment of p50–/–cRel–/– BMDCs and pDCs, respectively, showed elevated IFN-beta mRNA and protein expression compared with WT DCs. Thus, although not essential for virus-induced IFN-beta expression, NF-{kappa}B proteins may still influence aspects of IFN-beta expression. Previously, the NF-{kappa}B-activating kinase IKK-beta was reported to control type 1 IFN expression, whereas more recently, IKK-{alpha} was shown to participate in IRF7 activation (28, 29). In light of our findings, it is therefore plausible that both IKK-{alpha} and IKK-beta control type 1 IFN expression by regulating the IRF3/IRF7 activation pathway. Importantly, we have found that the combined functions of p50 + cRel are crucial for virus-induced IL-12 p40 expression. Thus, unlike their role in virus-induced IFN-beta expression, NF-{kappa}B proteins play critically important roles in mediating specific host responses to viral infection.

We have also performed the first detailed analysis of the role of NF-{kappa}B proteins in IFN-{alpha} expression. Similar to IFN-beta, we found that virus infection leads to robust IFN-{alpha} expression in NF-{kappa}B-deficient MEFs. In addition, RelA–/–- or p50–/–cRel–/– CpG D19-treated pDCs also strongly induced IFN-{alpha} expression, consistent with a recent report (30). Virus-induced transcriptional induction of IRF7 requires type 1 IFN autocrine signaling. We also examined virus-induced expression of IRF7 to determine a potential role for NF-{kappa}B proteins in regulating IFN autocrine signaling. However, no highly significant differences were seen in IRF7 expression between WT and the different knockout cells, indicating that neither type 1 IFN expression nor downstream signaling events are absolutely dependent on NF-{kappa}B proteins. Interesting differences, especially between WT and p50–/–RelA–/– cells, were nonetheless noted. Thus, although IRF7 regulates IFN{alpha} (non-{alpha}4), IRF-7 levels were decreased, whereas IFN{alpha} (non-{alpha}4) levels were increased in p50–/–RelA–/– cells in comparison with WT cells. Additionally, the main difference in IFN-{alpha} (non-{alpha}4) mRNA levels was observed at 9 and 12 h postinfection, when levels in WT cells were rapidly decreasing. We believe that a possible explanation for these results may be that decay of IFN-{alpha} (non-{alpha}4) mRNA levels is slower in p50–/–RelA–/– cells, suggesting possible control of mRNA stability by NF-{kappa}B proteins, as mentioned above.

Members of the NF-{kappa}B and IRF families of transcription factors are among the most crucial for mediating host responses to microbial agents. Recent studies using IRF3–/– and IRF7–/– fibroblasts and DCs have demonstrated an essential role for these transcription factors in virus-induced type 1 IFN expression (6). In contrast to these findings, our results indicate that robust virus-induced type 1 IFN expression can take place in the absence of key NF-{kappa}B subunits and following dominant-negative I{kappa}B{alpha} expression. Together with previous studies, the results shown in this study indicate discrete functions for these two transcription factor families in mediating host responses to virus infection: IRF3/7 being crucial for induction of type 1 IFN responses and NF-{kappa}B factors playing essential roles in inducing key cytokine genes.


   Acknowledgments
 
We thank Dr. Christian Schindler (Columbia University, New York, NY) for helpful comments on this manuscript. We also acknowledge help provided by Flow Cytometry and Molecular Biology core facilities at the Moffitt Cancer Center.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant R01 AI059715 and institutional funds from Moffitt Cancer Center (to A.A.B.) and National Institutes of Health U19AI62623 funded Center to Investigate Viral Immune Antagonism (to A.G.-S.). Back

2 Address correspondence and reprint requests to Dr. Amer A. Beg, Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center & Research Institute, University of South Florida, 12902 Magnolia Drive, Mail Stop: SRB-2, Tampa, FL 33612. E-mail address: amer.beg{at}moffitt.org Back

3 Abbreviations used in this paper: IKK, I{kappa}B kinase; BMDC, bone marrow dendritic cell; cDC, conventional dendritic cell; DC, dendritic cell; IRF, IFN regulatory factor; MEF, mouse embryonic fibroblast; MIG, murine stem cell virus internal ribosome entry site; NDV, Newcastle disease virus; pDC, plasmacytoid DC; PRD, positive regulatory domain; RPA, RNase protection assay; SV, Sendai virus; WT, wild type. Back

Received for publication December 27, 2006. Accepted for publication March 16, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Stetson, D. B., R. Medzhitov. 2006. Type I interferons in host defense. Immunity 25: 373-381. [Medline]
  2. Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi, M. Miyagishi, K. Taira, S. Akira, T. Fujita. 2004. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5: 730-737. [Medline]
  3. Kawai, T., S. Akira. 2006. Innate immune recognition of viral infection. Nat. Immunol. 7: 131-137. [Medline]
  4. Kato, H., O. Takeuchi, S. Sato, M. Yoneyama, M. Yamamoto, K. Matsui, S. Uematsu, A. Jung, T. Kawai, K. J. Ishii, et al 2006. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441: 101-105. [Medline]
  5. Seth, R. B., L. Sun, C. K. Ea, Z. J. Chen. 2005. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-{kappa}B and IRF 3. Cell 122: 669-682. [Medline]
  6. Honda, K., A. Takaoka, T. Taniguchi. 2006. Type I interferon gene induction by the interferon regulatory factor family of transcription factors. Immunity 25: 349-360. [Medline]
  7. Du, W., D. Thanos, T. Maniatis. 1993. Mechanisms of transcriptional synergism between distinct virus-inducible enhancer elements. Cell 74: 887-898. [Medline]
  8. Thanos, D., T. Maniatis. 1995. Virus induction of human IFN beta gene expression requires the assembly of an enhanceosome. Cell 83: 1091-1100. [Medline]
  9. Merika, M., A. J. Williams, G. Chen, T. Collins, D. Thanos. 1998. Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription. Mol. Cell 1: 277-287. [Medline]
  10. Sato, M., H. Suemori, N. Hata, M. Asagiri, K. Ogasawara, K. Nakao, T. Nakaya, M. Katsuki, S. Noguchi, N. Tanaka, 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. [Medline]
  11. Honda, K., H. Yanai, H. Negishi, M. Asagiri, M. Sato, T. Mizutani, N. Shimada, Y. Ohba, A. Takaoka, N. Yoshida, T. Taniguchi. 2005. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature 434: 772-777. [Medline]
  12. Levy, D. E., I. Marie, A. Prakash. 2003. Ringing the interferon alarm: differential regulation of gene expression at the interface between innate and adaptive immunity. Curr. Opin. Immunol. 15: 52-58. [Medline]
  13. Lu, R., W. C. Au, W. S. Yeow, N. Hageman, P. M. Pitha. 2000. Regulation of the promoter activity of interferon regulatory factor-7 gene: activation by interferon and silencing by hypermethylation. J. Biol. Chem. 275: 31805-31812. [Abstract/Free Full Text]
  14. Liu, Y. J.. 2005. IPC: professional type 1 interferon-producing cells and plasmacytoid dendritic cell precursors. Annu. Rev. Immunol. 23: 275-306. [Medline]
  15. Diebold, S. S., M. Montoya, H. Unger, L. Alexopoulou, P. Roy, L. E. Haswell, A. Al-Shamkhani, R. Flavell, P. Borrow, C. Reis e Sousa. 2003. Viral infection switches non-plasmacytoid dendritic cells into high interferon producers. Nature 424: 324-328. [Medline]
  16. Takeda, K., T. Kaisho, S. Akira. 2003. Toll-like receptors. Annu. Rev. Immunol. 21: 335-376. [Medline]
  17. Honda, K., Y. Ohba, H. Yanai, H. Negishi, T. Mizutani, A. Takaoka, C. Taya, T. Taniguchi. 2005. Spatiotemporal regulation of MyD88-IRF-7 signalling for robust type-I interferon induction. Nature 434: 1035-1040. [Medline]
  18. Balachandran, S., E. Thomas, G. N. Barber. 2004. A FADD-dependent innate immune mechanism in mammalian cells. Nature 432: 401-405. [Medline]
  19. Oganesyan, G., S. K. Saha, B. Guo, J. Q. He, A. Shahangian, B. Zarnegar, A. Perry, G. Cheng. 2006. Critical role of TRAF3 in the Toll-like receptor-dependent and -independent antiviral response. Nature 439: 208-211. [Medline]
  20. Karin, M., Y. Ben-Neriah. 2000. Phosphorylation meets ubiquitination: the control of NF-{kappa}B activity. Annu. Rev. Immunol. 18: 621-663. [Medline]
  21. Ghosh, S., M. J. May, E. B. Kopp. 1998. NF-{kappa}B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16: 225-260. [Medline]
  22. Thanos, D., T. Maniatis. 1995. Identification of the rel family members required for virus induction of the human beta interferon gene. Mol. Cell. Biol. 15: 152-164. [Abstract]
  23. Garoufalis, E., I. Kwan, R. Lin, A. Mustafa, N. Pepin, A. Roulston, J. Lacoste, J. Hiscott. 1994. Viral induction of the human beta interferon promoter: modulation of transcription by NF-{kappa}B/rel proteins and interferon regulatory factors. J. Virol. 68: 4707-4715. [Abstract/Free Full Text]
  24. Ouaaz, F., J. Arron, Y. Zheng, Y. Choi, A. A. Beg. 2002. Dendritic cell development and survival require distinct NF-{kappa}B subunits. Immunity 16: 257-270. [Medline]
  25. Akira, S., K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4: 499-511. [Medline]
  26. Honda, K., S. Sakaguchi, C. Nakajima, A. Watanabe, H. Yanai, M. Matsumoto, T. Ohteki, T. Kaisho, A. Takaoka, S. Akira, et al 2003. Selective contribution of IFN-{alpha}/beta signaling to the maturation of dendritic cells induced by double-stranded RNA or viral infection. Proc. Natl. Acad. Sci. USA 100: 10872-10877. [Abstract/Free Full Text]
  27. Sanjabi, S., A. Hoffmann, H. C. Liou, D. Baltimore, S. T. Smale. 2000. Selective requirement for c-Rel during IL-12 P40 gene induction in macrophages. Proc. Natl. Acad. Sci. USA 97: 12705-12710. [Abstract/Free Full Text]
  28. Chu, W. M., D. Ostertag, Z. W. Li, L. Chang, Y. Chen, Y. Hu, B. Williams, J. Perrault, M. Karin. 1999. JNK2 and IKKbeta are required for activating the innate response to viral infection. Immunity 11: 721-731. [Medline]
  29. Hoshino, K., T. Sugiyama, M. Matsumoto, T. Tanaka, M. Saito, H. Hemmi, O. Ohara, S. Akira, T. Kaisho. 2006. I{kappa}B kinase-{alpha} is critical for interferon-{alpha} production induced by Toll-like receptors 7 and 9. Nature 440: 949-953. [Medline]
  30. O’Keeffe, M., R. J. Grumont, H. Hochrein, M. Fuchsberger, R. Gugasyan, D. Vremec, K. Shortman, S. Gerondakis. 2005. Distinct roles for the NF-{kappa}B1 and c-Rel transcription factors in the differentiation and survival of plasmacytoid and conventional dendritic cells activated by TLR-9 signals. Blood 106: 3457-3464. [Abstract/Free Full Text]



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