Toll-like receptors (TLRs) mediate host cell activation by various microbial components. TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 are the receptors that have been associated with virus-induced immune response. We have previously reported that all these TLRs, except TLR9, are expressed at mRNA levels in human monocyte-derived macrophages. Here we have studied TLR2, TLR3, TLR4, and TLR7/8 ligand-induced IFN-α, IFN-β, IL-28, and IL-29 expression in human macrophages. IFN-α pretreatment of macrophages was required for efficient TLR3 and TLR4 agonist-induced activation of IFN-α, IFN-β, IL-28, and IL-29 genes. TLR7/8 agonist weakly activated IFN-α, IFN-β, IL-28, and IL-29 genes, whereas TLR2 agonist was not able to activate these genes. IFN-α enhanced TLR responsiveness in macrophages by up-regulating the expression of TLR3, TLR4, and TLR7. IFN-α also enhanced the expression of TLR signaling molecules MyD88, TIR domain-containing adaptor inducing IFN-β, IκB kinase-ε, receptor interacting protein 1, and IFN regulatory factor 7. Furthermore, the activation of transcription factor IFN regulatory factor 3 by TLR3 and TLR4 agonists was dependent on IFN-α pretreatment. In conclusion, our results suggest that IFN-α sensitizes cells to microbial recognition by up-regulating the expression of several TLRs as well as adapter molecules and kinases involved in TLR signaling.
The mammalian innate immune system detects invading pathogens via TLRs. These pattern recognition receptors are expressed on professional APCs, such as macrophages and dendritic cells. TLRs detect conserved microbial components and activate intracellular signaling cascades leading to the expression of several cytokine and chemokine genes. To date, 13 mammalian TLRs have been characterized (1). The vast majority of TLR research has focused on bacterial recognition by TLRs. Less is known about viruses and TLRs, albeit the fact that during virus-infections the rapid response of innate immune system, particularly production of type I IFNs, is essential for the clearance of viral pathogens. TLRs associated with antiviral immunity include TLR4, which has been reported to recognize the fusion protein of respiratory syncytial virus (2) and TLR2 that has been shown to mediate recognition of measles virus and HSV (3, 4). Similarly, TLR9 mediates recognition of HSV DNA in plasmacytoid dendritic cells (5, 6). In addition, TLR3 is a receptor for dsRNA (7) that is formed during the replication of many RNA viruses. TLR7/8 was initially found to recognize small synthetic antiviral compounds, imidazoquinolines (8, 9). Therefore, it was assumed that TLR7/8 is involved in virus-induced immune responses. Recently TLR7/8 was reported to mediate recognition of viral ssRNA (10, 11, 12). Therefore, TLR3, TLR7, and TLR8 constitute a powerful system to detect viral genetic material.
TLR signaling is mediated by adapter proteins and protein kinases, ultimately leading to the activation of IFN regulatory factor (IRF)3 and NF-κB family of transcription factors and subsequent induction of TLR-regulated genes. The TLR signal transduction pathways are activated when the cytoplasmic Toll/IL-1 receptor (TIR) domain of TLRs recruits cytosolic TIR-domain containing adapter molecules to the receptor complex (13). Most TLRs utilize a common adaptor molecule MyD88 that mediates the activation of NF-κB and AP-1. However, TLR3 and TLR4 use other adaptor molecules as well, since their ligands induce IFN-β expression independently of MyD88 (14, 15). Recently, it was shown that TIR domain-containing adaptor inducing IFN-β (TRIF) mediates signal transduction downstream of TLR3 and TLR4 (16, 17, 18, 19). The hierarchy of TRIF-dependent signaling needs further elucidation, but it is known that TRIF mediates the activation of both IRF3 and NF-κB. TRIF associates with IRF3-activating IκB kinase-ε (IKKε) and TANK-binding kinase-1 (TBK1) (20) and the disruption of these genes in mice results in defective TLR3- and TLR4-mediated IFN responses (16, 21, 22). Furthermore, TLR3/TRIF-induced NF-κB activation is dependent on receptor interacting protein (RIP) kinases (23). Ultimately, the differences in TLR signaling are reflected to differences in TLR-activated gene expression.
Here we have studied TLR2, TLR3, TLR4, TLR7/8 agonists-induced expression of IFN-α, IFN-β, and novel type I IFN homologues, IL-28 (IFN-λ2/3) and IL-29 (IFN-λ1) (24, 25) in human monocyte-derived macrophages. We report that TLR3 and TLR4 ligand-induced IFN-α, IFN-β, IL-28, and IL-29 gene expression in macrophages is dependent on IFN-α. Our results suggest that IFN-α-dependent responsiveness to TLR triggering is achieved by IFN-α-regulated expression of TLRs and TLR-specific signaling components.
Materials and Methods
Macrophages and NK-92 cells
Human macrophages were obtained from leukocyte-rich buffy coats from healthy blood donors (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland). PBMCs were isolated from buffy coats by density gradient centrifugation using Ficoll-Paque (Amersham Pharmacia Biotech). Monocytes were allowed to adhere to plastic six-well plates (Falcon Multiwell; BD Biosciences) and differentiated into macrophages for 1 wk in macrophage serum-free medium (Invitrogen Life Technologies) supplemented with GM-CSF (10 ng/ml; Nordic Biosite), 0.6 μg/ml penicillin and 60 μg/ml streptomycin. In all experiments, cells from four different blood donors were used. Human NK-92 cell line was obtained from American Type Culture Collection (ATCC CRL-2407) and maintained in continuous culture in MEM α medium supplemented with 12% horse serum (Invitrogen Life Technologies), 12% FCS (Integro), 0.2 mM i-inositol, 20 mM folic acid, 40 mM 2-ME, 2 mM l-glutamine, antibiotics, and 100 IU/ml IL-2 (Chiron). Before stimulation, NK-92 cells were starved in IL-2-free RPMI 1640 media for 18 h. All experiments were performed three times with similar results.
Cytokines and TLR ligands
Human leukocyte IFN-α was provided by the Finnish Red Cross Blood Transfusion Service and was used at 100 IU/ml. IFN-β (Schering-Plough) was used at 100 IU/ml. TLR3 ligand poly(I:C) (Sigma-Aldrich), TLR2 ligand Pam3Cys (EMC Microcollections), TLR4 ligand LPS (Escherichia coli 0111:B4; Sigma-Aldrich) and TLR7/8 ligand R848 (InVivoGen) were used at 30 μg/ml, 100 ng/ml, 1 μg/ml and 1 μg/ml, respectively. TLR stimulations were performed in RPMI 1640 medium containing 10% FCS.
RNA isolation and Northern blot analysis
Total cellular RNA was isolated with the RNeasy kit (Qiagen) according to the manufacturer’s instructions. Samples containing equal amounts of RNA (10 μg) were size fractioned on 1% formaldehyde-agarose gel, transferred to a nylon membrane (Hybond; Amersham Biosciences) and hybridized with IFN-α2 (26), IFN-β (27), IL-28, IL-29, MyD88 (28), TRIF, TRIF-related adaptor molecule (TRAM), IKKε, TBK1, RIP1, IRF1 (26), IRF7, IFN-γ (29), TLR3, TLR4, TLR7, or TLR8, (30) probes. Probes for IL-28, IL-29, TRIF, TRAM, IKKε, TBK1, IRF7, and RIP1 were cloned from total cellular RNA obtained from Sendai virus-infected macrophages by RT-PCR using oligonucleotides GTCTCCACAGGATCCGCAGGCCTT and CAGCCAGGGGGATCCTTTTTTGGG (IL-28), GAAGGCCAGGGATCCCTTGGAAGA and GTGTCAGGTGGATCCAGGGTGGGT (IL-29), GAACAGGGATCCTATAACTTTGTGATCCTC and GTGGGGGGATCCGGTGTTGGCCACCTTCCT (TRIF), GGGCAAAGGGGATCCTTTCTCGGGGAA and GCACAGTGTGGATCCAAGTCCAGGATA (TRAM), TCAGACGGATCCAGGTGCTGGGACTCTAT and TACATGGGATCCGAAGCATCCAGCAGA (IKKε), ATTGCAGGATCCGATTTACTATCAGTTC and CTAAAGGGATCCAACGTTGCGAAGGCCAC (TBK1), ATGATGGTCGGATCCAGCGCCCCTGGG and GCTCAGGCGGGATCCGTGCTGGCGAGA (IRF7), and AAAGAAAGAGGATCCAAACGAAAA and GCTGTTCCTGGATCCAGTGGTCTT (RIP1). To ensure equal RNA loading, β-actin mRNA expression was analyzed. The probes were labeled with [α-32P]dATP (3000 Ci/mmol; Amersham Biosciences) using a random primed DNA labeling kit (Boehringer Mannheim). The membranes were hybridized (Ultrahyb; Ambion) and washed in 1× SSC/0.1% SDS and exposed to Kodak AR X-omat films at −70°C using intensifying screens.
Western blot analysis
Equal amounts of proteins were separated on SDS-PAGE with the Laemmli buffer system and transferred onto Immobilon-P membranes (Millipore). The membranes were blocked with PBS containing 5% nonfat milk and were stained with anti-TBK1 (SC-9085; Santa Cruz Biotechnology), anti-IKKε (SC-9913), or anti-RIP1 (SC-7881) Abs followed by staining with peroxidase conjugated anti-rabbit or anti-goat IgG (DakoCytomation) and visualization with the ECL system (Amersham Biosciences).
Oligonucleotide DNA precipitation
Equal amounts of cells (1 × 107/sample) were harvested and nuclear extracts were prepared by lysing the cells in buffer containing 10 mM HEPES-KOH, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, 1 mM Na3VO4, and a protease inhibitor mixture (Complete; Roche). The remaining nuclei were lysed in 10 mM HEPES, 400 mM KCl, 10% glycerol, 2 mM EDTA, 1 mM EGTA, 0.01% Triton X-100, 0.5 mM DTT, 1 mM Na3VO4, and a protease inhibitor mixture. DNA binding proteins were incubated for 2 h with streptavidin-agarose beads (Pierce) coupled to 5′-biotinylated oligonucleotides: IFN-α14 positive regulatory domain (PRD)-like (GGATCCGGAAAGCCAAAAGAGAAGTAGAAAAAAA), IFN-β PRDI-III (GGATCCCACTTTCACTTCTCCCTTTCAGTTTTC), and IFN-β PRDII (GGATCCGGAATTTCCCGGAATTTCCC) (31, 32) (DNA Technology). The binding reactions were performed for 2 h at +4°C in binding buffer (10 mM HEPES, 133 mM KCl, 10% glycerol, 2 mM EDTA, 1 mM EGTA, 0.01% Triton X-100, 0.5 mM DTT, 1 mM Na3VO4, and a protease inhibitor mixture) followed by washing the unbound proteins with binding buffer. The oligonucleotide-bound proteins were released in SDS sample buffer, separated on SDS-PAGE, and transferred onto Immobilon-P membranes (Millipore). The proteins were visualized with the ECL system by using guinea pig anti-IRF-3 (IFN-β PRDI-III precipitated), anti-IRF1, anti-IRF7 (IFN-α14 PRD-like precipitated), or rabbit anti-p50 (SC-7178; Santa Cruz Biotechnology), anti-p65 (SC-372), anti-c-rel (SC-70), anti-RelB (SC-226) (IFN-β PRDII precipitated) Abs and peroxidase conjugated goat anti-rabbit or anti-guinea pig IgG (DakoCytomation). Guinea pig anti-IRF-1 Abs have been previously described (33). Anti-IRF3 and anti-IRF7 were prepared in guinea pigs by immunizing the animals four times at 4-wk intervals with preparative SDS-PAGE (BioRad) purified baculovirus-expressed proteins (20 μg per immunization).
Biological IFN-αβ assay
Cell culture supernatants were treated at pH 2, and IFN-αβ titer was measured in HEp2 cells by vesicular stomatitis virus plaque reduction assay as previously described (34). The results are shown as international units per milliliter, using IFN-α preparation as a standard.
IFN-α pretreatment enhances TLR- and virus-induced IFN gene expression
Type I IFNs are an essential factor in the activation of multiple genes during viral infections. TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 are associated with virus-induced immune response and antiviral immunity (35). Hence, we determined IFN-α, IFN-β, IL-28, and IL-29 mRNA expression in macrophages stimulated with TLR2, TLR3, TLR4, or TLR7/8 ligands Pam3Cys, poly(I:C), LPS, or R848, respectively. TLR9 ligand was not included in our studies since TLR9 gene was not found to be expressed in macrophages (data not shown). As shown in Fig. 1, IFN-α pretreatment of macrophages was required for efficient TLR3 and TLR4 ligand-induced IFN-α, IFN-β, IL-28, and IL-29 mRNA expression. TLR7/8 stimulation resulted in a weak induction of IFN genes in IFN-α-pretreated macrophages, whereas TLR2 ligand was not able to induce IFN mRNA expression, regardless of IFN-α pretreatment. These results demonstrate that TLR2, TLR3, TLR4, and TLR7/8 differ in their ability to induce IFN-α, IFN-β, IL-28, and IL-29 gene expression in human macrophages. Furthermore, IFN-α pretreatment was required for the TLR-actived expression of these genes.
As IFN-α positively regulated TLR-mediated IFN production in human macrophages, we determined whether virus-induced IFN production was also enhanced by IFN-α pretreatment (Fig. 2). Both influenza A and Sendai virus-infected human macrophages are known to produce type I IFNs (36, 37). Sendai virus activated high IFN-α, IFN-β, IL-28, and IL-29 mRNA expression in macrophages, which was further enhanced by IFN-α pretreatment. Densitometric scanning with image analysis software (Kodak Digital Science) showed that IFN-α enhances Sendai virus-induced IFN gene expression in macrophages by ∼2-fold. Similar to TLR3 ligand, influenza A virus-induced IFN-α, IFN-β, IL-28, and IL-29 gene expression was strongly enhanced by IFN-α pretreatment (Fig. 2).
IFN-α enhances expression of TLRs and TLR signaling molecules
TLR signaling leads to the activation of NF-κB and IRF families of transcription factors. To date, five potential TIR domain-containing TLR adaptor molecules have been identified (38). Since IFN-α enhanced TLR-activated IFN production in macrophages, we studied whether IFN-α affected the expression of TLRs and TLR signaling molecules. TLR3, TLR4, and TLR7 mRNA expression was clearly enhanced by IFN-α (Fig. 3). This confirms our previous observations that IFN-α regulates TLR gene expression in macrophages (30). IFN-α also up-regulated mRNA expression of MyD88, a common adaptor molecule associated with TLR signaling (Fig. 4 A). However, TLR3 and TLR4 signal MyD88-independently via another adaptor molecule TRIF, leading to the activation of IRF3 and NF-κB (16, 17, 19). TRIF mRNA expression was up-regulated in macrophages by IFN-α. TRIF mediates TLR4-specific, MyD88-independent gene expression with TRAM (18, 39). TRAM gene expression was not affected by IFN-α.
TRIF is reported to associate with IRF3 and IRF7 activating kinases, TBK1 and IKKε (20, 40). Since the activation of IRF family of transcription factors is essential for IFN-α and IFN-β production, we analyzed the expression of IRFs, TBK1 and IKKε in macrophages. TBK1 and IKKε are basally expressed in macrophages (Fig. 4, A and B). IKKε mRNA and protein expression was clearly up-regulated in response to IFN-α stimulation, whereas only a modest increase in TBK1 mRNA and protein expression was observed. IFN-α does not affect IRF3 gene expression (41), whereas IRF1 and IRF7 gene expression was activated by IFN-α treatment (Fig. 4,A). RIP1 is a TLR3/TRIF-specific mediator of NF-κB activation (23). RIP1 mRNA and protein expression was also strongly enhanced by IFN-α (Fig. 4, A and B). We conclude that besides stimulating the expression of TLR3 and TLR4, IFN-α regulates TLR-mediated gene activation in macrophages by enhancing the expression of TLR3- and TLR4-specific signaling molecules.
TLR ligand-induced IRF and NF-κB activation
To gain more insight into TLR3-, TLR4-, TLR7-, and TLR8-regulated IFN induction in human macrophages, we studied the activation of the IRF family of transcription factors in TLR3, TLR4, and TLR7/8 ligand-stimulated macrophages. Nuclear extracts from TLR ligand-stimulated cells were precipitated with oligonucleotides containing an IRF binding site from IFN-α or IFN-β genes. Oligonucleotide-bound transcription factors were analyzed by Western blotting (Fig. 5 A). Basal IRF3 DNA binding to IFN-β promoter PRDI-PRDIII region was detected in untreated macrophages. Both TLR3 and TLR4 ligands induced the phosphorylation of IRF3, and this phosphorylation was clearly higher in TLR3/4-stimulated cells that were pretreated with IFN-α. No IRF7 DNA binding to IFN-α14 PRD-like element was observed in untreated macrophages. However, enhanced IRF7 DNA binding was detected in IFN-α pretreated cells, and this binding was clearly increased by TLR3 and TLR4 stimulation. IRF1 DNA binding to IFN-α14 PRD-like element was also activated by TLR3 and TLR4 ligands. As in the case of IRF7, clearly stronger binding of IRF1 was observed in cells pretreated with IFN-α. Interestingly, TLR7/8 ligand R848 activated IRF1 DNA binding to IFN-α14 PRD-like element, but no significant IRF3 phosphorylation or IRF7 DNA binding was seen. In contrast, R848 weakly inhibited IRF3 and IFN-α-induced IRF7 DNA binding.
In addition to IRFs, NF-κB transcription factors regulate type I IFN gene expression. We studied the effect of TLR stimulation to NF-κB DNA binding activity with oligonucleotide immunoprecipitation method. TLR3, TLR4, and TLR7/8 ligands enhanced p50, p65, c-rel, and RelB DNA binding to IFN-β promoter PRDII element (Fig. 5 B). IFN-α pretreatment as such enhanced the DNA binding of NF-κB p65 protein. TLR3 ligand-induced p65 DNA binding was clearly enhanced in IFN-α-pretreated cells. In conclusion, our results show that especially TLR3 ligand-induced IRF3 and NF-κB activation and TLR4 ligand-induced IRF3 activation were enhanced in IFN-α-pretreated cells.
Cell culture supernatants from TLR3, TLR4, and TLR7/8 ligand-stimulated macrophages have antiviral and IFN-γ-inducing activity
IFN-α has been shown to induce IFN-γ production from NK and T cells (37, 42, 43). We determined the IFN-γ-inducing activity of cell culture supernatants from TLR ligand-stimulated macrophages. Macrophages were stimulated with TLR2, TLR3, TLR4, and TLR7/8 ligands for 20 h, after which the macrophage cell culture supernatants were collected and subjected onto NK-92 cells. After 3 h of stimulation with macrophage supernatants, NK-92 cells were harvested and their IFN-γ mRNA expression was analyzed by Northern blotting (Fig. 6 A). Supernatants from TLR3 and TLR4 ligand-stimulated macrophages induced IFN-γ gene expression in NK-92 cells only when macrophages had been pretreated with IFN-α. This demonstrates the importance of IFN-α in sensitizing the macrophages for TLR3 and TLR4 signaling.
Macrophage supernatants (from Fig. 6,A) were also analyzed for their antiviral activity. This was done with a biological type I IFN assay that is based on vesicular stomatitis virus plaque reduction. IFN-α pretreatment was absolutely required for TLR3 and TLR4 ligand-induced antiviral activity (Fig. 6,B). Supernatants from TLR2 ligand-treated macrophages had neither antiviral nor IFN-γ inducing activity. These results correlate well with IFN-α, IFN-β, IL-28, and IL-29 mRNA expression patterns presented in Fig. 1. Cell culture supernatants from TLR7/8 ligand-stimulated macrophages had both antiviral and IFN-γ-inducing activity (Fig. 6, A and B). Thus, despite hardly detectable IFN-α, IFN-β, IL-28, and IL-29 mRNA expression by Northern blotting (Fig. 1), TLR7/8 triggering as such enhances the production of antiviral cytokines in macrophages.
During viral infections rapid production of IFNs is needed to prevent the spread of the viruses within the organism. In addition to their direct antiviral effects IFNs regulate both innate and adaptive immunity. Recently, a novel family of type I IFN-like cytokines was described (24, 25). IL-28A/B and IL-29 (also known as IFN-λ2/3 and IFN-λ1, respectively), posses antiviral activity and their production is induced by viral infection in several cell types (24, 25, 44). There is evidence that virus infection-induced cytokine production is mediated throught TLRs (45). In this report we have examined the involvement of TLRs in IFN gene expression, by using agonists for those TLRs, that have been associated with RNA virus infections. We show that these TLRs selectively mediate IFN gene expression in human monocyte-derived primary macrophages. In addition, we provide evidence that IFN-α sensitizes the cells to microbial stimulation by up-regulating the expression of TLRs and various adaptor molecules and kinases involved in TLR signaling.
It has been well established that several IFN-α genes are regulated by a positive feedback mechanism by type I IFN-induced IRF7 during virus infection (46). In addition, type I IFNs are essential for TLR3- and TLR4-induced gene activation (14, 47, 48, 49, 50). In our study, TLR3 and TLR4 ligand-induced IFN-α and IFN-β gene expression was strongly enhanced by IFN-α pretreatment of macrophages. Similarly, IL-28 and IL-29 expression was induced by TLR3 and TLR4 stimulation and their expression, too, was dramatically enhanced when macrophages were pretreated with IFN-α. Thus, the positive feedback loop associated with IFN-α and IFN-β induction (51, 52, 53) is also involved in the regulation of IL-28 and IL-29 gene expression. TLR7/8 ligand induced a weak expression of IFN-α, IFN-β, IL-28, and IL-29 genes in IFN-α-pretreated macrophages whereas TLR2 ligand was completely unable to activate these genes. In conclusion, our results suggest that IL-28 and IL-29 genes are regulated in similar fashion as IFN-α and IFN-β genes.
TLR signal transduction is regulated by adaptor proteins acting downstream of TLRs, resulting in the activation of NF-κB and IRF family of transcription factors (13). TLR3 and TLR4 are unique among TLRs since they are capable of mediating MyD88-independent NF-κB and IRF3 activation via TRIF (16, 17, 18). Both MyD88 and TRIF expression in human macrophages was enhanced by IFN-α (Fig. 4). In addition to our previously described activation of TLR3 gene expression (30), IFN-α up-regulated expression of TLR4 in macrophages. Therefore, the essential components involved in TLR3- and TLR4-mediated intracellular signaling pathways are up-regulated by IFN-α, giving a putative molecular explanation for the enhanced responsiveness of macrophages to TLR3 and TLR4 triggering after IFN-α stimulation.
Type I IFN gene expression is tightly regulated by transcription factors belonging to the NF-κB and IRF families. Upon appropriate signals, IRF3 and NF-κB are activated and transported into the nucleus where they regulate IFN-α and IFN-β gene expression. The first wave of IFN-α and/or IFN-β produced stimulates IRF7 expression, which activates the expression of most IFN-α subtype genes (51, 52, 53). Thus, IRF7 is considered to be one of the key regulators of type I IFN production. Our knowledge of molecular mechanisms that regulate virus infection-induced or TLR-mediated activation of IRF3 and IRF7 was greatly improved when TBK1 and IKKε were identified to be IRF3- and IRF7-activating kinases (20, 40). In addition, it was recently shown that a serine-threonine kinase, RIP1, mediates TLR3/TRIF-specific NF-κB activation (23). In macrophages IKKε and especially RIP1 mRNA and protein expressions were clearly enhanced by IFN-α stimulation. Up-regulation of RIP1 and IKKε expression is likely to contribute to enhanced NF-κB and IRF3 activation, respectively, which we observed after TLR3 stimulation in IFN-α-pretreated macrophages. RIP1 is specifically involved in TLR3/TRIF signaling pathway and, therefore, it is conceivable that the observed up-regulation of RIP1 gene expression by IFN-α does not affect TLR4 ligand-induced NF-κB activation. However, IFN-α pretreatment strongly enhanced TLR4 ligand-induced IRF3 activation and this likely explains the increased IFN-β gene expression in these cells.
TLR7 and TLR8 recognize viral ssRNA (10, 11, 12) and, therefore, they can be considered as crucial receptors recognizing the genetic material of RNA viruses. TLR7/8-specific ligand, R848 induces IFN-α or IL-12 production in human myeloid or plasmacytoid DCs, respectively (54). This demonstrates that TLR7 and TLR8 mediate their effects in a cell type-specific manner. Although R848 did not stimulate significant IFN-α1, IFN-α2, IFN-α4, IFN-β, IFN-ω, IL-28, or IL-29 mRNA expression in macrophages (Fig. 1 and data not shown), it was able to induce type I IFN-like antiviral activity especially in cells that were pretreated with IFN-α. This suggests that TLR7/8 ligand-induced gene expression in human macrophages should be by DNA microarray analysis.
In addition to IFNs, TLR triggering induces the production of a variety of chemotactic and immunomodulatory cytokines that regulate the activation of immune cells. IFN-γ is essential for the development of Th1 type immune response. Cell culture supernatants from TLR3 or TLR4 ligand-stimulated macrophages induced IFN-γ gene expression in NK cells only when macrophages were pretreated with IFN-α (Fig. 6). These results suggest that IFN-α may significantly contribute to TLR-induced development of Th1 immune response.
In this report we demonstrated that TLRs involved in the recognition of different viral components differentially activate antiviral response in human monocyte-derived macrophages. In addition, our results demonstrate the crucial role of IFN-α in enhancing the innate immune sensing.
We thank Hanna Valtonen, Mari Aaltonen, and Teija Westerlund for expert technical assistance.
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.
This work was supported by the Medical Research Council of the Academy of Finland, The Sigrid Juselius foundation, and the Finnish Cancer Foundation.
Abbreviations used in this paper: IRF, IFN regulatory factor; IKKε, IκB kinase-ε; RIP, receptor interacting protein; TBK1, TANK-binding kinase-1; TRAM, TRIF-related adaptor molecule; TRIF, TIR domain-containing adaptor inducing IFN-β; TIR, Toll/IL-1 receptor; PRD, positive regulatory domain.