IFN Regulatory Factor Family Members Differentially Regulate the Expression of Type III IFN (IFN-λ) Genes

Cell culture

Monocyte-derived DCs (moDCs) were obtained as previously described (30). Briefly, monocytes were isolated from leukocyte-rich buffy coats of healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland) by consecutive centrifugations in Ficoll-Paque and Percoll gradients (Amersham Biosciences) followed by depletion of the remaining T or B cells with anti-CD3 and anti-CD19 magnetic beads (Dynal Biotech). Monocytes were suspended in RPMI 1640 medium (Sigma-Aldrich) in the absence of serum, and 2.5 × 10⁶ cells/well were allowed to adhere to plastic 6-well plates (Falcon; BD Biosciences). After 1 h of incubation at 37°C, nonadherent cells were removed, and the cells were washed with PBS. Monocytes were differentiated into immature DCs for 6 days in 2 ml of RPMI 1640 medium supplemented with 0.6 mg/ml penicillin, 60 mg/ml streptomycin, 2 mM l-glutamine, 20 mM HEPES, 10% FCS (Integro), 10 ng/ml recombinant human GM-CSF (BioSource International), and 20 ng/ml recombinant human IL-4 (R&D Systems). Fresh medium (1 ml/well) was added every 2 days.

Human embryonic kidney (HEK) 293 cell line (American Type Culture Collection; CLR1573) was maintained in Eagle’s MEM (Sigma-Aldrich) with antibiotics, l-glutamine, HEPES, and 10% FCS.

Virus infections

Murine SV (strain Cantell) originates from the National Public Health Institute (Helsinki, Finland) and it was cultured in embryonated chicken eggs and stored at −70°C (31). The hemagglutination titer of SV was 4096 and the infectivity of the virus stock in DCs was 6 × 10⁹ PFU/ml (4). Cells were infected with a multiplicity of infection (MOI) of 5 (1/500 dilution of virus stock) to reach maximal infectivity without significant cytopathic effects in 18 h of infection. For RNA analysis, DCs were infected with SV for 1, 2, 4, 6, 9, and 12 h, and total cellular RNA was isolated with the RNeasy Midi kit (Qiagen). For DNA-binding experiments, virus-infected DCs were harvested at 2, 4, and 8 h after infection and nuclear protein extracts were prepared (4). The experiments were performed with cells obtained from three to four different blood donors. In transfection assays, HEK293 cells were infected with SV, since IRF3 and IRF7 molecules need to be C-terminally phosphorylated by virus-activated IKKε/TBK-1 kinase complexes to induce their nuclear translocation and transcriptional activity.

Plasmids

The pGL3-luciferase (luc) reporter constructs for IFN-β and IFN-α4 promoters were previously described (32). Promoter sequences for IFN-α1, IFN-λ1, and IFN-λ3 were cloned from pEF2-luc reporter plasmids (33) into MluI and XhoI restriction sites of the pGL3-basic vector (Promega). Expression vectors for IRF1, IRF5 (variant 2, 498 aa) (34), IRF3 (35), IRF7 (36), p50, p65 (37), RIG-I, ΔRIG-I (18), Cardif (33), IKKε, and TBK1 (17) were described elsewhere. IRF8 cDNA was provided by Dr. B.-Z. Levi (Department of Biotechnology and Food Engineering, Technion, Haifa, Israel). The sequence of IRF8 was amplified and cloned into the BamHI site of pcDNA3.1⁺-FLAG-tagged expression vector. The expression constructs for TRIF and MyD88 were gifts from Dr. K. Fitzgerald (University of Massachusetts Medical School, Worcester, MA). The cDNA encoding Pin1 was amplified from total cellular RNA isolated from human macrophages and cloned into the BamHI site of the pcDNA3.1⁺-FLAG-tagged expression vector using primers (upstream) 5′-GCACCTGGATCCACCATGGCGGACGAGGAGAAGCTG and (downstream) 5′-CTGGGCGGATCCTTATCACTCAGTGCGGAGGATGA (BamHI site underlined, initiation codon as bold).

Northern blot analysis

For mRNA expression analyses, total cellular RNA was isolated by a RNeasy Midi RNA isolation kit (Qiagen). RNA was quantified photometrically, and samples containing equal amounts (10 μg) of total cellular RNA were size-fractionated on 1% formaldehyde-agarose gels and transferred onto Hybond-N nylon membranes (Amersham Biosciences). The membranes were hybridized with human IFN-α (α1), IFN-β (provided by Dr. C. Weissman, Zurich, Switzerland), IFN-λ1, IFN-λ2/3, IRF7 (38), and RIG-I (20) cDNA probes. The probes for Northern blot analysis were labeled with [α-³²P]dATP (3000 Ci/mmol; Amersham Biosciences) using a Klenow random primed DNA labeling kit (Boehringer Mannheim). Membranes were hybridized (Ultrahyb buffer; Ambion), washed in 1× SSC/0.01% SDS and exposed to Kodak X-Omat AR films (Eastman Kodak) at −70°C with intensifying screens.

Quantitative RT-PCR

Before cDNA synthesis, 2 μg of purified total cellular RNA was treated with DNase I (Roche Diagnostics) to remove any contaminating DNA. Generation of cDNA was done by using a TaqMan Reverse Transcriptase kit (Applied Biosystems) with random hexamers as primers. The cDNA was amplified by PCR using a TaqMan Universal PCR Mastermix and the commercial Gene Expression Assay with primers and probes for IFN-α1, IFN-β, IFN-λ1, IFN-λ2/3, IRF3, and IRF7 from Applied Biosystems. The data were normalized to 18S rRNA (TaqMan Endogenous Control kit) and presented as the relative gene expression in relation to 18S rRNA.

DNA affinity-binding assay and Western blot analysis

Virus-induced activation of transcription factors was studied using the DNA affinity-binding method as previously described (4). The used putative IFN-stimulated response element (ISRE), positive-regulatory domain I (PRDI), and NF-κB-binding site oligonucleotide sequences from the IFN-λ1 and IFN-λ3 promoters were 5′-GATTTCAGTTTCTCTTTCCTTCTTGA (IFN-λ1 ISRE site), 5′-TTTCCAATTACCTTTTCACTTTACACAC (IFN-λ1 PRDI site), 5′-CAAAGTGGAAATTCTCATTTCC (IFN-λ1 NF-κB1 site), 5′-TCCGTCAGGGACTCCCTGAGGCC (IFN-λ1 NF-κB2 site), 5′-CTCCATCAGTTTCTCTTTCCCTCCAGC (IFN-λ3 ISRE site) 5′-TCCGACACAAAAGTGAAAAGGATGTAG (IFN-λ3 PRDI site), and 5′-GCAGCCCCTGCCCTCAGTGGGCAGCCTCTGC (IFN-λ3 NF-κB site) (transcription factor binding site underlined) (DNA Technology). The bound proteins were separated on SDS-PAGE and visualized by using specific Abs against IRF1 (39), IRF3, IRF7 (4), p-IRF3 S396 (40), IRF8 (Santa Cruz Biotechnology), p65, p50, RelB (Santa Cruz Biotechnology), and p52 (Upstate Biotechnology).

Transfections

HEK293 cells were seeded into 24-well plates at 75,000 cells/well or into 96-well plates at 20,000 cells/well and the cells were allowed to attach overnight. For luc assays, the cells were transfected with 100 ng for a 24-well format and 20 ng for a 96-well format of indicated expression plasmids, unless otherwise stated, along with 15 ng for a 24-well format and 3 ng for a 96-well format of pRL-RSV plasmid (Renilla luc) and 100 ng for a 24-well format and 20 ng for a 96-well format of firefly luc reporter under the control of different IFN promoters using TransIT-LT1 transfection reagent (Mirus Bio). The DNA:TransIT ratio was 1:4. Total amount of DNA was adjusted with the empty expression vector. At 4 h after transfection, the cells were infected with SV for 18 h when indicated. The luc activities were detected by using the Dual-Luciferase Reporter Assay System (Promega) and Victor multilabel reader (Wallac). The firefly luc activities were normalized to Renilla luc activities and the results are presented as the fold induction in relation to the unstimulated promoter and Renilla transfections in the absence of SV infection.

SV-induced expression of IFN genes in human moDCs

Because human DCs produce large quantities of IFNs and they can readily be infected with RNA viruses such as influenza A and SV (4), they form a good system to study the regulation of type I and type III IFN gene expression. To reveal the kinetics and magnitude of IFN gene expression, we infected moDCs with SV and isolated total cellular RNA at different time points after infection. The expression of IFN-α, IFN-β, IFN-λ1, and IFN-λ2/3 genes was analyzed with Northern blotting. For a more quantitative approach, we used RT-PCR. Because human IFN-λ2 and IFN-λ3 genes are almost identical (96% amino acid identity) (2), the probe used in Northern blot analysis and the primers used in RT-PCR could not distinguish between the latter two IFN-λ types. RIG-I and IRF7 mRNAs were also analyzed in Northern blots to verify the expression kinetics of these two IFN-stimulated genes. Using RT-PCR, we quantified the expression levels of IRF3 and IRF7 during virus infection. We observed that the gene expression of IFN-λ1 and IFN-β was induced already 2 h after infection of moDCs with SV, whereas the expression of IFN-α and IFN-λ2/3 genes appeared to take place somewhat later (Fig. 1⇓A). Based on RT-PCR analysis, the kinetics of IFN-β and IFN-λ1 mRNA expression was also a few hours faster as compared with IFN-α and IFN-λ2/3 genes. Quantitative analysis revealed that all studied IFNs showed ∼1000-fold induction after SV infection (Fig. 1⇓B). In Northern blot analysis, the expression of RIG-I and IRF7 became visible at the 4-h time point, showing an expression kinetic that coincided or preceded those of IFN-α and IFN-λ2/3 genes (Fig. 1⇓A). Quantification of IRF3 and IRF7 mRNA showed that IRF3 is expressed constantly whereas the maximal expression level of IRF7 observed at 6 h after SV infection (100-fold increase, Fig. 1⇓B).

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FIGURE 1.

The expression of type I and type III IFN genes in SV-infected human moDCs. moDCs from three different blood donors were infected with SV (MOI = 5). Cells were collected at different times after infection, pooled, and total cellular RNA was isolated and processed for Northern blotting (A) or quantitative RT-PCR (B). A, Total cellular RNA (10 μg/lane) was separated on 1% agarose gels, transferred to nylon filters, and Northern blot analysis with IFN-α- (α1), IFN-β-, IFN-λ1-, IFN-λ2/3-, RIG-I-, IRF7-, and β-actin-specific probes was conducted. A representative experiment of two is shown. B, Virus-induced expression of different IFNs, IRF3, and IRF7 was quantified with RT-PCR. The values were normalized to 18S rRNA and presented as relative gene expression. Error bars, SD of the mean of determinations from two experiments.

Structure of human IFN-λ1 and IFN-λ3 promoters

To characterize the transcription factors regulating the expression of IFN-λ genes, we performed a computer sequence analysis of the human IFN-λ1 and IFN-λ3 promoter regions with the MatInspector (Genomatix software) and TESS (Transcription Element Search software (http://www.cbil.upenn.edu/tess), programs. Because the promoter sequences of IFN-λ2 and IFN-λ3 genes are identical, except for five nucleotides (Fig. 2⇓B) and a 10-nt TATA repeat within the first 1000 nt upstream of the transcription start site, we concentrated on analyzing the IFN-λ3 gene promoter in more detail. The sequences of IFN-λ1 and IFN-λ3 promoters are shown in Fig. 2⇓. In the IFN-λ1 promoter, we found a cluster of IRF-binding ISRE sites between nt −216 to −192 relative to the transcription start site (41). In addition, we identified a PRDI site at −65 to −79 which, according to the computer programs, was expected to be an IRF1 binding site. The promoter of IFN-λ1 had two putative NF-κB-binding sites, one between nt −98 to −89 referred to as NF-κB1 and another one between nt −264 to −253 referred to as NF-κB2. The IFN-λ3 promoter contained a putative ISRE site at −288 to −301 (relative to the transcription start site stated by GenBank) and a PRDI site at −182 to −195. In addition, a putative NF-κB binding site was located inside the transcribed sequence between nt +115 to +125 (Fig. 2⇓).

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FIGURE 2.

Human IFN-λ1 and IFN-λ3 promoter structures. Computer analysis was performed to elucidate the putative transcription factor binding sites on the promoter sequences of IFN-λ1 (A) and IFN-λ3 (B) genes. The putative binding site for IRFs (ISRE and PRDI) and NF-κB are underlined. Sequences for oligonucleotides used in DNA affinity-binding assays are shaded. There are five nucleotides that differ between the IFN-λ2 and IFN-λ3 promoters and they are circled. C, Alignment of transcription factor binding sites of IFN-β, IFN-λ1, and IFN-λ3 promoters compared with the consensus sequence.

Functionality of ISRE, PRDI, and NF-κB elements on IFN-λ1 and IFN-λ3 gene promoters

To analyze the functionality of the putative IFN-λ1 and IFN-λ3 promoter transcription factor binding sites identified by the computer search, we conducted DNA-binding experiments. At 2, 4, and 8 h after SV infection, moDCs were collected and nuclear protein extracts were prepared. An oligonucleotide precipitation assay using ISRE, PRDI, or NF-κB elements was conducted and factors binding to these elements were identified by Western blotting. Both IFN-λ1 and IFN-λ3 promoter elements were able to bind IRF and NF-κB transcription factors (Fig. 3⇓). Virus infection increased the binding of IRF1, IRF3, and IRF7 on ISRE and PRDI elements of these promoters, and moreover the binding of phosphorylated IRF3 was seen already at the 2- and 4-h time points. The binding of IRF1 and IRF7 was observed to take place in cell extracts collected at later time points. The binding of IRF8 on ISRE and PRDI elements was constitutive and not dependent on virus infection. The components of the classical NF-κB pathway, p65 and p50, bound to the NF-κB elements on both IFN-λ1 and IFN-λ3 promoters in response to SV infection. In addition, the binding of the component of the alternative NF-κB pathway RelB, but not p52, was also weakly increased in virus-infected cell extracts.

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FIGURE 3.

SV-induced IRF and NF-κB DNA binding to the ISRE, PRDI, and NF-κB sites of IFN-λ1 and IFN-λ3 promoters. moDCs from three different blood donors were infected with SV (MOI = 5). Cells were collected at 2, 4, or 8 h after infection and nuclear protein extracts were prepared. Nuclear proteins were allowed to bind to agarose-bound oligonucleotides representing ISRE, PRDI, and NF-κB elements of the IFN-λ1 and IFN-λ3 promoters. Oligonucleotide-bound proteins were boiled, separated on SDS-PAGE, transferred to nylon filters, and visualized with IRF- and NF-κB-specific Abs. Promoter elements and proteins are indicated. This experiment was performed three times with similar results. CTRL, Control.

Role of IRF and NF-κB transcription factors in type I and type III IFN gene expression

Next, we wanted to analyze which of the IRF and NF-κB family members activate type I and type III IFN promoters. HEK293 cells were transfected with IFN-α1, IFN-α4, IFN-β, IFN-λ1, and IFN-λ3 promoter-reporter constructs in combination with expression constructs for IRF1, IRF3, IRF5, IRF7, IRF8, and p50 plus p65. Because IRF3 and IRF7 appeared to differentially regulate these promoters, they can be classified as IFN-α type (IFN-α1, IFN-α4, and IFN-λ3) and IFN-β type (IFN-β and IFN-λ1) promoters (Fig. 4⇓A). SV infection increased IFN-β and IFN-λ1 promoter activation significantly and in IRF1, IRF3, or IRF7 gene-transfected cells even more (Fig. 4⇓A). IRF7 induced the activation of all tested IFN promoters already without SV infection (Fig. 4⇓A). IFN-α and IFN-λ3 promoters were not significantly induced by SV infection. Ectopic expression of IRF1 and IRF3 followed by SV infection led to increased IFN-α1, IFN-α4, and IFN-λ3 promoter activation. However, this activation was much greater in IRF7 gene-transfected cells, indicating that IFN-α and IFN-λ3 promoters were predominantly regulated by IRF7 (Fig. 4⇓A).

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FIGURE 4.

Multiple IRF and NF-κB transcription factors activate IFN promoters. Different IRF (A) and NF-κB p50 + p65 (B) expression constructs were cotransfected into HEK293 cells along with IFN-α1, IFN-α4, IFN-λ3, IFN-λ1, and IFN-β promoter luc reporter constructs for 4 h followed by SV infection for 18 h as indicated. A, Expression of IRF1, IRF3, and IRF7 activated IFN-λ1 and IFN-β promoters, whereas only the expression of IRF7 was able to activate IFN-α and IFN-λ3 promoters to a greater extent. B, HEK293 were transfected with IFN promoter constructs for 4 h followed by infection with SV for 18 h. Alternatively, promoter constructs were cotransfected with NF-κB p50 and p65 expression constructs for 22 h followed by analysis of luc activity. The luc activities were normalized with Renilla activities and data are presented as fold induction from basal promoter activation without transfected expression constructs or virus infection. Bars, The means ± SD of triplicate determinations and the results are from one of three experiments.

In another set of experiments, we conducted transfection with p50- and p65-protein-expressing constructs to analyze the role of NF-κB in type I and type III IFN gene expression. Because IκBα is not present, p50 plus p65 heterodimer is transcriptionally active. Although IFN-α1 and IFN-α4 promoters were very weakly activated by p50 plus p65, both IFN-λ promoters as well as the IFN-β promoter were readily induced by transfected NF-κB components (Fig. 4⇑B). It was of great interest that IFN-λ3 promoter activation was induced by p50 plus p65 heterodimers to the level similar to or even greater than that induced by SV infection (Fig. 4⇑B).

IRF5 has been reported to play a crucial role in the expression of type I IFNs (42) and IRF8 has been suggested to be involved in the late-phase type I IFN gene expression in the mouse (11). In our HEK293 cell transfection/SV infection system, IRF5 induced only a minimum increase in the IFN promoter activations, except IFN-β promoter that was clearly activated by IRF5 (Fig. 4⇑A). This observation is consistent with some recent publications (11, 43). We did not see any increasing effect on IFN promoter activation by IRF8, rather there was an inhibitory effect (Fig. 4⇑A). To further characterize the role of IRF8 in IFN gene expression, we cotransfected the IRF8 expression plasmid with IRF3 or IRF7 and infected the cells with SV. There was a remarkable IRF8 dose-dependent decrease in IRF7-mediated IFN promoter activation (Fig. 5⇓). This phenomenon was not so clear with IRF3, although some decrease in IRF3-mediated activation of IFN-λ1 and IFN-β promoters was seen. Since IRF8 was found to bind to the ISRE element on IFN promoters (Fig. 3⇑), it is possible that IRF8 could compete with other IRFs in their DNA-binding activity. Although the expression and stability of IRF3 was not altered by overexpression of IRF8, IRF7 stability seemed to be impaired, because the amount of full-length IRF7 was reduced and correspondingly another form of IRF7 protein appeared (Fig. 5⇓B). This may indicate that IRF8 interferes with IRF7 DNA binding, rendering it unstable and susceptible for proteolytic degradation.

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FIGURE 5.

IRF8 down-regulates IRF3- or IRF7-induced type I and type III IFN promoter activation. HEK293 cells were transfected with IFN promoter constructs along with increasing doses of IRF8 and constant doses of IRF3 or IRF7 expression constructs (in ratios of 0.3:1, 1:1, and 3:1). At 4 h after transfection, cells were infected with SV for 18 h followed by the luc assay and Western blotting. A, Increasing expression of IRF8 inhibits IRF3- or IRF7-dependent activation of all studied IFN promoters. The luc activities from a luc assay were normalized with Renilla activities and data are presented as fold induction from basal promoter activation without transfected expression constructs or virus infection. Bars, The means ± SD of triplicate determinations from one representative experiment of three. B, Cellular proteins were separated on 10% SDS-PAGE, transferred to nylon filters, and visualized with IRF3-, IRF7-, and IRF8-specific Abs as indicated.

RIG-I- and TLR-dependent signaling pathways regulate type III IFN promoters

Based on current knowledge, RNA virus infection can trigger three separate signal transduction pathways (TLR7, TLR3, and RIG-I), leading to the activation of transcription factors that regulate the expression of antiviral genes. To identify the signaling pathways regulating type III IFN gene expression, we overexpressed RIG-I, the constitutively active form of RIG-I (ΔRIG-I), Cardif, IKKε, TBK1, TRIF, and MyD88 alone or in combination with IRF1, IRF5, or IRF7 and analyzed IFN promoter activation in HEK293 cells. The data revealed that overexpression of upstream signaling molecules ΔRIG-I, Cardif, IKKε, TBK1, and TRIF induced IFN promoter activation independently of SV infection (Fig. 6⇓). Full-length RIG-I, instead, required SV infection for full transcriptional activation. Also in this experiment, there were similarities between IFN-β and IFN-λ1 as well as between IFN-α and IFN-λ3 promoters. All tested signaling molecules activated IFN-β and IFN-λ1 promoters very well, whereas the activation of IFN-α1 and IFN-λ3 promoters remained at a moderate level (Fig. 6⇓).

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FIGURE 6.

Activation of type I and type III IFN promoters by signaling components of the TLR or RIG-I pathways. HEK293 cells were transfected with expression constructs for the components of the RIG-I (A), TLR3, or TLR7 (B) signal transduction pathways along with the IFN promoter luc reporter constructs. Four hours after transfection, cells were infected with SV for 18 h and luc activity was measured. The luc activities were normalized with Renilla activities and data are presented as fold induction from basal promoter activation without transfected expression constructs or virus infection. Bars, The means ± SD of triplicate determinations. One representative experiment of three is shown.

Interestingly, activating the MyD88-dependent signaling cascade (mimicking TLR7 signaling) and, most importantly, MyD88 plus IRF7 combination led to a significant activation of IFN-α and IFN-λ3 promoters (Fig. 6⇑B). The activation induced by the combination of MyD88 and IRF7 appeared to be independent of SV infection. Expression of MyD88 alone induced a moderate activation of IFN-α1 and IFN-α4 promoters. However, the IFN-α4 promoter was only weakly activated with the other signaling cascades. In addition, the IFN-α4 promoter was activated with the combination of MyD88 and IRF5, while IFN-α1 and IFN-λ promoters were not responding significantly to this activation stimulus. The expression of MyD88 with IRF1 enhanced virus-dependent activation of IFN promoters, especially those of IFN-β and IFN-λ1.

Inhibition of signaling cascades by Pin1

Recently, it was demonstrated that a prolyl isomerase Pin1 has suppressive effects on IRF3-mediated activation of IFN-β gene expression (29). This prompted us to analyze whether Pin1 plays a negative regulatory role in virus-induced signaling cascades analyzed herein. HEK293 cells were transfected with plasmids encoding Pin1 and signaling molecules upstream of each signaling cascade in combination with the five IFN promoter constructs. As a control, we transfected IRF3 and Pin1 genes into HEK293 cells and infected them with SV. TRIF- and ΔRIG-I-mediated IFN promoter activation was clearly inhibited by coexpression of Pin1 (Fig. 7⇓A). Interestingly, also the MyD88-dependent activation of IFN promoters was down-regulated by Pin1. The decreased promoter activation induced by Pin1 was seen in samples activated by MyD88 combined with IRF1 or IRF7. Transfected Pin1 had no remarkable effect on the expression of transfected IRFs (Fig. 7⇓B). inhibitory effect of Pin1 was likely IRF specific, since NF-κB-induced promoter activation was not reduced by ectopic expression of Pin1 (data not shown).

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FIGURE 7.

Prolyl isomerase Pin1 inhibits type I and type III IFN gene expression induced by signaling components of the TLR and RIG-I pathways. HEK293 cells were transfected with increasing concentrations of the Pin1 expression construct along with the IFN promoter luc reporter constructs and components of the TLR7 (MyD88, IRF1, and IRF7), TLR3 (TRIF), or RIG-I (ΔRIG-I) pathways for 22 h. As a control, HEK293 cells were transfected with the IRF3 expression construct for 4 h followed by infection with SV for 18 h. The ratios of inhibiting and activating constructs were 0.3:1, 1:1, and 3:1. A, After transfection, luc activity was measured. The values were normalized with Renilla activities and data are presented as fold induction from basal promoter activation without transfected expression constructs or virus infection. Bars, The means ± SD of triplicate determinations. B, Cellular proteins were separated on 10% SDS-PAGE, transferred to nylon filters, and visualized with IRF1-, IRF3-, and IRF7-specific Abs as indicated.