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B and IRF-3
1 Department of Viral Diseases and Immunology, National Public Health Institute, Helsinki, Finland
2 Institute of Medical Microbiology and Immunology, University of Aarhus, DK-8000 Aarhus C, Denmark
Correspondence
Jesper Melchjorsen
jesper{at}microbiology.au.dk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, IFN-
and interleukin 28 (IL-28), IL-29, respectively], tumour necrosis factor alpha and the chemokines CCL5 and CXCL10 after herpes simplex virus 1 (HSV-1) infection. The cytokine-inducing activity of HSV-1 was dependent on viability of the virus, because UV-inactivated virus did not induce a cytokine response. Pretreatment of the cells with IFN- or IL-29 strongly enhanced the HSV-1-induced cytokine response. Both IFN- and IL-29 decreased viral immediate-early (IE) gene infected-cell protein 27 (ICP27) transcription, suggesting that IL-29 possesses antiviral activity against HSV-1 comparable to that of IFN-. Macrophage infection with HSV-1 lacking functional ICP27 (d27-1 virus) resulted in strongly enhanced cytokine mRNA expression and protein production. In contrast, viruses lacking functional IE genes ICP0 and ICP4 induced cytokine responses comparable to those of the wild-type viruses. The activation of transcription factors IRF-3 and NF-
B was strongly augmented when macrophages were infected with the ICP27 mutant virus. Altogether, the results demonstrate that HSV-1 both induces and inhibits the antiviral response in human cells and that the type III IFN IL-29, together with IFN-, amplifies the antiviral response against the virus. It is further identified that viral IE-gene expression interferes with the antiviral response in human macrophages and ICP27 is identified as an important viral protein counteracting the early innate immune response.
Present address: Institute of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Romagnani, 1997; Duerst & Morrison, 2003). In the first line of defence, natural killer cells, macrophages and dendritic cells (DCs) have an important role (Biron et al., 1999; Kodukula et al., 1999; Zhao et al., 2003). Virus-infected macrophages and DCs are the major cell types producing chemokines, type I interferons (IFNs) and other cytokines, and regulating the inflammatory response and restricting the spread of virus infection (Sen, 2001; Duerst & Morrison, 2003). Especially, type I IFNs are the key antiviral cytokines (Sen, 2001; Duerst & Morrison, 2003), because they activate hundreds of host genes, including the ones whose gene products have direct antiviral activities (Der et al., 1998). Recently, two novel type I IFN-like genes (type III IFNs), interleukin 28 [IL-28 (IFN-
2/3)] and IL-29 (IFN-1) have been identified and have been shown to be produced after virus infection or by stimulation of cells with double-stranded RNA (dsRNA) (Kotenko et al., 2003; Sheppard et al., 2003; Coccia et al., 2004; Robek et al., 2005; Sirén et al., 2005). Type III IFNs utilize the IL-28R–IL-10R receptor complex, but activate signal transduction in a manner highly similar to that of type I IFNs and possess antiviral activities at least against RNA viruses (Kotenko et al., 2003; Sheppard et al., 2003; Coccia et al., 2004; Robek et al., 2005). At present, there is no information on whether IL-28 and IL-29 have antiviral activity against DNA viruses in primary human cells.
Herpes simplex virus (HSV-1) and HSV-2 are two closely related human DNA viruses causing a number of clinical manifestations, including genital herpes, cold sores and keratitis (Whitley & Roizman, 2001). Like most viruses, HSV has evolved different strategies to inhibit or evade host innate immune-defence mechanisms. For instance, HSV-infected cell protein 34.5 (ICP34.5) and Us11, which are expressed at late stages of infection, inhibit RNA-activated protein kinase R (PKR) activity by reversing the phosphorylation of eIF2 or by binding directly to dsRNA, respectively (He et al., 1997; Poppers et al., 2000). Immediate-early (IE) gene products, such as ICP0, have been shown to inhibit nuclear accumulation of IFN regulatory factor 3 (IRF-3), as well as to interfere with type I IFN signalling (Mossman et al., 2000; Eidson et al., 2002; Härle et al., 2002b; Lin et al., 2004; Melroe et al., 2004). ICP27 represses host-cell gene transcription, reduces the amount of host mRNAs and regulates the shutoff of host protein synthesis during HSV infection (Sacks et al., 1985; McCarthy et al., 1989; Hardwicke & Sandri-Goldin, 1994; Spencer et al., 1997; Song et al., 2001; Mogensen et al., 2004). In addition, ICP4 has been shown to reduce the stability of host-cell mRNAs (Mogensen et al., 2004).
Previously, we and others have demonstrated that HSV infection triggers a biphasic production of chemokines and cytokines: an early response not requiring replication-competent virus, but dependent on the presence of viral surface and tegument proteins and a later cytokine response dependent on virus replication (Ankel et al., 1998; Paludan, 2001; Paludan & Mogensen, 2001; Melchjorsen et al., 2002; Malmgaard et al., 2004). Presently, the virus replication events regulating the second wave of cytokine-gene expression have not been well characterized.
In this paper, we used monocyte-derived primary human macrophages and DCs to address the following questions: (i) how is the cytokine response regulated during HSV-1 infection of human monocyte-derived macrophages and DCs, (ii) do IFN- and IL-29 have antiviral activity against HSV-1 and do they provide positive-feedback signals for virus-induced cytokine-gene expression, (iii) is the HSV-1-induced cytokine response dependent on virus entry or does it require virus replication, and finally (iv) do specific HSV-1 IE genes regulate the virus-induced host-cell response? We observed that HSV-1-induced cytokine responses were comparable in macrophages and DCs and that the antiviral cytokine response was enhanced strongly by pretreatment of the cells with IFN- or IL-29. We further demonstrated that virus attachment and entry, as such, are not sufficient to activate cytokine-gene expression, but that this response requires virus replication. Finally, we showed that an HSV-1 ICP27-deletion mutant activated elevated levels of the transcription factors IRF-3 and NF-B compared with wild-type (wt) virus, and also induced higher amounts of cytokine-gene expression. This suggests that IE viral gene expression is required both for induction of the cytokine response and for activation of viral counteracting mechanisms.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The isolated cells were determined to be macrophages by their typical morphology and cell-surface CD14 expression (Pirhonen et al., 1999). For DCs, the mononuclear cells were further purified by centrifugation over a Percoll gradient (Amersham Pharmacia Biotech) (Veckman et al., 2004). Percoll gradients of 34, 47·5 and 60 % were made by mixing Percoll with RPMI 1640 medium (Sigma) supplemented with penicillin (0·6 µg ml–1), streptomycin (60 µg ml–1), glutamine (2 mM), HEPES (20 mM) and 10 % fetal calf serum (FCS; Integro BV). Mononuclear cells were suspended in a 34 % Percoll solution and the three Percoll layers were mixed. Cells were centrifuged at 1700 g for 35 min and the top layer containing monocytes was collected. Next, the cells were washed twice with serum-free RPMI 1640 medium with supplements as above, and the remaining T or B cells were depleted by using anti-CD3 and anti-CD19 magnetic beads (Dynal). After this, cells were washed once with RPMI 1640 medium and counted. Monocytes were allowed to adhere to plastic six-well or 24-well plates for 1 h at 37 °C in RPMI 1640 medium without FCS. After incubation, non-adherent cells were removed and the wells were washed once with PBS. Monocytes were allowed to differentiate to immature DCs for 6 days in RPMI 1640 medium with the same supplements as mentioned above plus 10 % FCS, 10 ng GM-CSF ml–1 and 20 ng rhIL-4 ml–1 (R&D Biosystems). Cultured cells were CD1a (semi), CD14–, CD80 (low), CD83– and CD86 (semi), and they showed a typical DC morphology (Veckman et al., 2004). Fresh medium was added every 2 days. Cells from individual blood donors were grown separately, but after stimulation experiments, they were pooled.
Virus preparations.
The wt viruses used in this study were the KOS and 17+ strains of HSV-1. The viruses were essentially produced as described previously (Paludan et al., 2002). The ICP0 mutant dl1403 (Stow & Stow, 1986) is on a 17+ genetic background, whereas mutants lacking ICP4 (vi-13; Shepard & DeLuca, 1991) or ICP27 (d27-1; Rice & Knipe, 1990) are on a KOS genetic background. The infectivity of the virus was determined by plaque titration on U2OS cells (17+ and dl1403), Vero cells (KOS) or Vero-derived cell lines (vi-13 and d27-1) (Mogensen et al., 2004). Virus preparations were also examined by electron microscopy. Prior to use, the virus was thawed and used as infectious virus or inactivated by exposure to UV light for 15 min (Malmgaard et al., 2004).
Stimulation experiments.
To minimize inter-individual variation among blood donors, all experiments were carried out by using cells from three to six buffy coats. For priming experiments, cells were pretreated overnight with human IFN- (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland) at a concentration of 100 IU ml–1 or human recombinant IL-29 (kindly provided by Dr Kevin Klucher, ZymoGenetics Inc., Seattle, WA, USA) at a concentration of 20 ng ml–1, or left unprimed (PBS or untreated). Cells were infected with an m.o.i. of 1–2. Supernatants, RNA and cell extracts were stored at –70 °C before analysing the specimens by cytokine-specific ELISA, Northern blotting or DNA oligoprecipitation, respectively.
RNA isolation and Northern blot analysis.
After experiments, cells were washed once with PBS and lysed, and total cellular RNA was recovered by using an RNA purification kit (Qiagen Midi Kit). Samples of total cellular RNA (10 µg) were size-fractionated on 1 % denaturing formaldehyde agarose gels and transferred onto Hybond-N nylon membranes (Amersham Biosciences). Ethidium bromide staining of rRNA bands was used to ensure equal RNA loading. The probes used in Northern blot hybridizations were IFN-, IFN-, IL-28/IFN-2/3, IL-29/IFN-1, tumour necrosis factor alpha (TNF-), RANTES/CCL5, IP-10/CXCL10 (Matikainen et al., 2000; Sirén et al., 2005) and viral IE ICP27. The probe for ICP27 was cloned from total cellular RNA obtained from HSV-1-infected macrophages by RT-PCR using oligonucleotides 5'-AGACCAGACGGATCCCCTGGGAAACCT-3' and 5'-AAACACGAAGGATCCAATGTCCTTAAT-3'. The probes were labelled with [-32P]dCTP [3000 Ci (111 TBq) mol–1; Amersham Biosciences] by using a random-primed DNA labelling kit (Boehringer Mannheim). Hybridizations were performed in conditions of high stringency (50 % formamide, 5x Denhardt's solution, 5x saline sodium phosphate/EDTA and 0·5 % SDS at 42 °C). Filters were washed twice with 1x standard saline citrate/0·1 % SDS at room temperature for 30 min and once at 60 °C for 30 min. Kodak X-Omat AR film was used for autoradiography at –70 °C with intensifying screens.
Cytokine- and chemokine-specific ELISAs.
Cytokine and chemokine levels from cell-culture supernatants were analysed by the sandwich-ELISA method as described previously (Miettinen et al., 1998; Veckman et al., 2003). TNF-, CCL5/RANTES and CXCL10/IP-10 levels were determined with antibody pairs and standards obtained from BD PharMingen.
Oligonucleotide DNA precipitation and Western blotting.
Monocyte-derived macrophages or DCs were stimulated with infectious HSV-1, UV-inactivated HSV-1 or HSV-1 ICP27-deletion mutant as indicated in the figures and figure legends. Equal amounts of cells were harvested (1x107 per sample), washed and lysed in a buffer containing 10 mM HEPES, 400 mM KCl, 10 % glycerol, 2 mM EDTA, 1 mM EGTA, 0·01 % Triton X-100, 0·5 mM dithiothreitol (DTT), 1 mM Na3VO4 and protease inhibitors (Complete; Roche). Cleared cell lysates were incubated with streptavidin–agarose beads (Neutravidin; Pierce) coupled to 5'-biotinylated, 5'-6 bp-extended oligonucleotides (DNA Technology). The oligonucleotides used were: IFN- PRDI–III, 5'-GGATCCGAAAACTGAAAGGGAGAAGTGAAAGTG-3' (upstream) and 5'-GGATCCCACTTTCACTTCTCCCTTCTTTCAGTTTTC-3' (downstream) for IRF-3 and -7 precipitation; and IFN- PRDII, 5'-GGATCCGGAATTTCCCGGAATTTCCC-3' (upstream) and 5'-GGATCCGGGAAATTCCGGGAAATTCC-3' (downstream) for NF-B precipitation (Sirén et al., 2005). The upstream oligonucleotide sequence was 5'-biotinylated. The binding reactions were performed for 2 h at 4 °C in binding buffer containing 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 protease inhibitors. After washing, the oligonucleotide-bound proteins were released in SDS sample buffer by boiling for 5 min, separated by SDS-PAGE (10 % gel) and transferred onto Immobilon-P membranes (Millipore). Rabbit anti-IRF3, anti-p50 and anti-p65 antibodies (Abs) were purchased from Santa Cruz Biotechnology. Abs were allowed to bind for 1 h at room temperature in PBS containing 5 % non-fat milk. Peroxidase-conjugated goat anti-rabbit IgG (DakoCytomation) was allowed to bind for 1 h at room temperature and the proteins on membranes were visualized on Amersham Hyper-Max film by the enhanced chemiluminescence system (Amersham Biosciences).
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RESULTS |
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and IL-29 enhance the cytokine response in HSV-1-infected monocyte-derived human macrophages and DCs/ provides positive-feedback signals for RNA virus-induced type I IFN-gene expression (Marié et al., 1998; Sirén et al., 2005). To study whether similar mechanisms are involved in DNA virus-induced cytokine-gene expression, macrophages were left untreated or pretreated with IFN- or IL-29 followed by infection with HSV-1 (17+) virus for 6 or 9 h. At 6 and 9 h after infection, IFN-, IFN-, IL-28 (IFN-2/3), IL-29 (IFN-1), TNF-, CXCL10 and CCL5 mRNA expression was induced in macrophages (Fig. 1). Pretreatment of macrophages with IFN- or IL-29 strongly enhanced HSV-1-induced IFN-, IFN-, IL-28 and IL-29 mRNA expression (Fig. 1). IFN- or IL-29 priming had only a modest enhancing effect on TNF- and CXCL10 mRNA expression. IFN- priming also augmented HSV-induced expression of CCL5, with the strongest effect observed 9 h p.i. A similar cytokine mRNA expression pattern was seen in HSV-1-infected human monocyte-derived DCs (data not shown). The ability of IFN-/ to reduce HSV replication is well established (Oberman & Panet, 1988; Klotzbücher et al., 1990; Härle et al., 2002a). IL-28 and IL-29 have been shown to possess antiviral activity against several RNA viruses (Kotenko et al., 2003; Sheppard et al., 2003; Robek et al., 2005), whereas no information is available on their ability to inhibit HSV. As shown in Fig. 1, preteatment of the cells with IL-29, as well as with IFN-, reduced viral IE-gene expression as measured by ICP27 mRNA accumulation, thus suggesting that IL-29 has clear antiviral activity against HSV infection in human macrophages.
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pretreatment (18 h). Supernatants were harvested and the levels of TNF-, CCL5 and CXCL10 were measured by ELISA. We used two different strains of HSV-1 (KOS and 17+) to examine whether our observations were strain-specific. As seen in Fig. 2, TNF- was induced at modest levels in unprimed macrophages and more strongly in DCs, and IFN priming had a varying effect, depending on the cell type and virus strain. CCL5 was barely induced in unprimed cells, whereas IFN priming greatly enhanced the response. Finally, we observed that CXCL10 was clearly induced in the unprimed cells and that IFN pretreatment augmented the response in macrophages, but not in DCs. Whilst the two HSV-1 strains induced similar levels of cytokines in unprimed macrophages and DCs, we observed that 17+-induced cytokine production was generally enhanced more by IFN- pretreatment than cytokine production that was induced by KOS.
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, CCL5 and CXCL10 production was detectable starting at 9–13 h after infection (Fig. 3). In accordance with the results presented in Fig. 1
, IFN- pretreatment of macrophages clearly enhanced CCL5 production in macrophages (Fig. 3). Similarly, IFN- pretreatment strongly enhanced HSV-1-induced CCL5 production in DCs at all time points studied (Fig. 3). Thus, HSV-1 infection of primary human macrophages and DCs induces low levels of IFNs and inflammatory cytokines, which are strongly enhanced if the cells have been primed with IFN- or IL-29.
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) suggest that virus replication may be required for the HSV-1-induced cytokine response. To address this question, we used UV-inactivated HSV-1 to stimulate macrophages. UV-inactivated HSV-1 is incapable of transcribing its early genes, leading to an inability of the virus to replicate (Malmgaard et al., 2004). As shown in Fig. 4(a), the UV-inactivated preparations of HSV-1 were incapable of inducing IFN-, IFN-, IL-28 or IL29 mRNA expression in macrophages. Similarly, in DCs, HSV-1-induced IL-28, IL-29, TNF-, CCL5 and CXCL10 mRNA expression and TNF- and CCL5 protein production were severely impaired by UV inactivation of the virus (Fig. 4b). Collectively, the data support the conclusion that HSV-1 gene expression triggers the induction of IFN-, TNF-- and chemokine-gene expression.
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and chemokines after infection with HSV-1 mutant d27-1, IFN-, IL-28, IL-29, TNF-, CCL5 and CXCL10 mRNA expression equally as well as wt viruses (Fig. 5). In contrast, the virus mutant lacking a functional ICP27 gene resulted in highly elevated expression of IFN-, IFN-, IL-28, IL-29, TNF-, CXCL10 and CCL5 mRNAs, especially in cells that had been pretreated with IFN- (Fig. 5). In particular, we noted that, in unprimed cells, the d27-1 mutant was the only virus tested that was able to induce expression of IFN-, which is highly IRF-3-dependent. To examine whether the higher mRNA levels also led to enhanced protein secretion, we determined cell-culture supernatant TNF-, CXCL10 and CCL5 concentrations by ELISA. Significantly higher levels of TNF-, CXCL10 and CCL5 were secreted after infection with d27-1 virus than for wt HSV-1 (Fig. 6). Collectively, the data suggest that HSV-1 interferes with virus-induced cytokine-gene expression through a mechanism dependent on the ICP27 gene product.
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B transcription factorsB and IRF transcription-factor families (Mogensen & Paludan, 2001; Sen, 2001; Melchjorsen et al., 2003). During virus infection, NF-B and IRF-3 are activated and transported into the nucleus, where they regulate transcription of IFNs and other cytokine genes (Mogensen & Paludan, 2001; Sen, 2001; Melchjorsen et al., 2003). Figs 5 and 6 show that virus lacking the ICP27 gene induces elevated levels of cytokine expression. To look into the explanation for this observation, we analysed activation of IRF-3 and NF-B in macrophages after infection with wt or ICP27-deficient virus. As seen in Fig. 7, the wt virus induced activation of NF-B (p65 and p50) and IRF-3 only weakly, whereas the ICP27-deficient mutant virus activated these transcription factors very potently. These data therefore suggest that HSV-1 counteracts activation of IRF-3 and NF-B through a mechanism dependent on ICP27 and that this constitutes a viral mechanism to inhibit expression of genes with antiviral activities.
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DISCUSSION |
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). Macrophages, DCs and their produced cytokines contribute to the innate immune response against HSV infection and further constitute an important link between innate and adaptive immunity (Kodukula et al., 1999; Sen, 2001; Duerst & Morrison, 2003; Zhao et al., 2003). In the present study, we characterize the initial antiviral responses in human monocyte-derived macrophages and DCs after challenge with HSV-1. We report that HSV-1 infection activates IFN-, IFN-, IL-28, IL-29, TNF-, CCL5 and CXCL10 gene expression in these cell types. The identified cytokine response was dependent on virus replication. Furthermore, we show that both IFN- and the type III IFN IL-29 strongly enhance HSV-1-induced cytokine-gene expression and -protein production. These results suggest that, like in RNA virus infections, IFN-/- and IL-29-mediated positive-feedback signals are also operating in DNA virus infections, such as HSV, leading to a greatly enhanced antiviral response.
Type I IFNs are able to inhibit the replication of many animal viruses (Sen, 2001), including that of HSV (Oberman & Panet, 1988; Klotzbücher et al., 1990; Härle et al., 2002a). The recently identified IFN-like cytokines IL-28 and IL-29 have been shown to have antiviral activity against several RNA viruses, as well as against Hepatitis B virus and poxviruses (Kotenko et al., 2003; Sheppard et al., 2003; Coccia et al., 2004; Bartlett et al., 2005; Robek et al., 2005). Here, we provide the first evidence that IL-29, like IFN-/, possesses potential antiviral activity against HSV-1 infection in human macrophages and DCs. Both of these cytokines repressed HSV-1 IE-gene transcription, thus showing clearly that HSV-1 is also sensitive to the antiviral effects of IL-29. It will be of great interest to see whether IL-28 and IL-29 will have a role in the treatment of HSV-1 infections.
Like most pathogenic viruses, HSV-1 has evolved several mechanisms to block or circumvent the activation of host immune responses (Duerst & Morrison, 2003; Melroe et al., 2004). In HSV-1 infection, both IE genes ICP0, ICP4 and ICP27 and late genes ICP34.5 and Us11 are involved in the evasion of host-cell immune responses (Hardy & Sandri-Goldin, 1994; He et al., 1997; Poppers et al., 2000; Eidson et al., 2002; Melroe et al., 2004; Mogensen et al., 2004). ICP0 inhibits IRF-3- and IFN-stimulated gene activation in certain permissive cell types and renders the cells more resistant to IFN-/ action (Mossman et al., 2000; Eidson et al., 2002; Härle et al., 2002b; Lin et al., 2004; Melroe et al., 2004). In the present study, however, we show that HSV-1 blocks IFN production in the non-permissive human macrophages independently of ICP0, because the ICP0-deficient virus dl1403 is a slightly weaker inducer of IFNs and cytokines than the wt viruses, thus rather suggesting a positive regulatory role of ICP0. This is in accordance with previous studies showing ICP0-dependent production of CCL5 in the weakly permissive murine macrophage cell line RAW264.7 (Melchjorsen et al., 2002). Thus, the role of ICP0 in induction and suppression of IFNs and cytokines seems to depend on the target cell.
ICP4 has been shown to trigger a mechanism that inhibits production of proinflammatory cytokines in a murine macrophage cell line (Mogensen et al., 2004). However, this function of ICP4 was not observed to the same extent in the examined human primary macrophages. However, we did observe elevated expression of IFN- and CXCL10 after infection with the ICP4-deletion mutant compared with wt virus. Therefore, the potential role of ICP4 in inhibition of cytokine expression might be cell type-specific and requires further investigation.
ICP27 is a multifunctional regulatory protein essential for virus replication, mediating the successful expression of a number of viral genes (McCarthy et al., 1989; Rice & Knipe, 1990; Uprichard & Knipe, 1996; Jean et al., 2001). Reports from several laboratories have identified ICP27 as a mediator of HSV-induced immune evasion. ICP27 is involved in host protein shutdown, host mRNA splicing inhibition and repression of host-gene transcription and mRNA stability (Sacks et al., 1985; McCarthy et al., 1989; Hardwicke & Sandri-Goldin, 1994; Spencer et al., 1997; Song et al., 2001; Mogensen et al., 2004). In human macrophages, the ICP27-deletion mutant virus, d27-1, induced clearly higher expression of IFNs and chemokine genes than the wt HSV-1. Because ICP27 inhibits splicing in vitro (Hardwicke & Sandri-Goldin, 1994; Hardy & Sandri-Goldin, 1994), it is tempting to speculate that this phenomenon is a result of inhibition of host-cell mRNA splicing. However, it is worth noting that, although the IFN- and IFN- genes are intron-less, their expression was clearly enhanced in cells infected by ICP27 mutant virus. Concordantly, enhanced expression of cellular transcripts has also been seen in HeLa cells in response to ICP27 mutant virus and, especially, transcripts for IFN-/ were increased significantly (Stingley et al., 2000). This suggests that ICP27, in addition to regulating host mRNA splicing, may also affect other post-transcriptional events in mRNA processing or transport, rendering host-cell transcripts to rapid turnover. Such a mechanism has been described in Influenza A virus (Nemeroff et al., 1998). Alternatively, ICP27 may induce expression of viral IE or late genes involved in transcriptional repression. However, previously described evidence speaks against this hypothesis. We have found previously that CCL5 mRNA expression in murine macrophages coincides with the expression of HSV-1 IE genes, but precedes early and late HSV gene transcription (Melchjorsen et al., 2002), implying that the early cytokine response precedes IE- and late-gene expression. Additionally, we have shown that inhibition of viral DNA replication, and thus late-gene expression, does not affect the production of IL-6 (Mogensen et al., 2004). Collectively, the data suggest that ICP27-mediated inhibition of IFN-/ or IL-28/29 gene transcription is independent of DNA replication and late-gene expression. This would argue for a direct effect of ICP27 at host-cell transcription. In accordance with this hypothesis, the d27-1 mutant virus was a hyperpotent activator of IRF-3, suggesting that ICP27 regulates IRF3 activation negatively during HSV infection. Also, our observation that IFN- expression was elevated strongly in cells treated with d27-1 compared with the parental wt virus, together with the fact that the observed IRF-3 activation by d27-1 in unprimed versus IFN--primed cells does not correlate fully with the gene expression observed in Fig. 5, could indicate that IRF-7 could also be regulated negatively by ICP27.
Although we have gained further insight into the mechanisms of HSV-regulated cytokine-gene expression, the mechanism and molecular events governing the production of cytokines during HSV infection still remain to be described. Previous reports suggest that HSV-induced cytokine-gene expression is induced after direct recognition of virus structures or viral genetic material (Ankel et al., 1998; Paludan, 2001; Paludan & Mogensen, 2001; Melchjorsen et al., 2002; Malmgaard et al., 2004). In murine cells, recent findings suggest that recognition of HSV virions proceeds through Toll-like receptor 2 (TLR2) and genomic DNA via TLR9 (Lund et al., 2003; Krug et al., 2004; Kurt-Jones et al., 2004). However, cellular pattern-recognition receptors and viral components responsible for the second wave of cytokine-gene expression need to be further identified. We are currently investigating the mechanism involved in recognition of HSV in human primary cells.
Identifying HSV factors involved in the activation of the host-cell proinflammatory response, as well as the factors that can counteract this activation, is of considerable interest. Previous reports on mouse cultures, together with the data presented here, suggest that HSV-1 IE-gene expression is essential for the transcription of proinflammatory cytokines (Paludan, 2001; Paludan & Mogensen, 2001; Melchjorsen et al., 2002; Malmgaard et al., 2004; Fig. 5). IE-gene products may, however, also counteract the antiviral response. Our present findings show that ICP27 negatively regulates the activation of IRF-3 and NF-B, which is required for the transcription of IFN, cytokine and chemokine genes.
Taken together, we have characterized the proinflammatory response elicited by HSV-1-infected human monocyte-derived macrophages and DCs. We show that type I IFNs, as well as the type III IFN IL-29, provide a strong positive-feedback signal for HSV-1-induced cytokine-gene expression in human macrophages and DCs. Furthermore, the data suggest a novel role for IL-29 in the antiviral response against HSV-1, suggesting a potential therapeutic role for type III IFNs against HSV infections. Finally, we demonstrate that HSV-1 must be replication-competent to induce the cytokine response and that the viral IE gene ICP27 interferes with HSV-1-induced host-cell cytokine-gene expression. In summary, the outcome of HSV infection relies on a delicate balance between HSV-1 propagation, viral interference with host-cell factors and the activated innate antiviral response.
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ACKNOWLEDGEMENTS |
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Received 19 September 2005;
accepted 16 January 2006.
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) or IFN-
) (100 IU ml–1, 18 h) macrophages and DCs were infected with HSV-1 (17+, m.o.i.=1) and cell-culture supernatants were harvested at indicated time points. Cytokine production was analysed by ELISA. The results are the means (±SD) of three individual blood donors. The results are representative of two independent experiments.
