Replication-Dependent Potent IFN-α Induction in Human Plasmacytoid Dendritic Cells by a Single-Stranded RNA Virus

Veit Hornung, Jörg Schlender, Margit Guenthner-Biller, Simon Rothenfusser, Stefan Endres, Karl-Klaus Conzelmann and Gunther Hartmann
J Immunol November 15, 2004, 173 (10) 5935-5943; DOI: https://doi.org/10.4049/jimmunol.173.10.5935

Abstract

Plasmacytoid dendritic cells sense viral ssRNA or its degradation products via TLR7/8 and CpG motifs within viral DNA via TLR9. Although these two endosomal pathways operate independently of viral replication, little is known about the detection of actively replicating viruses in plasmacytoid dendritic cell (PDC). Replication and transcription of the viral genome of ssRNA viruses as well as many DNA viruses lead to the formation of cytosolic dsRNA absent in noninfected cells. In this study, we used human respiratory syncytial virus (HRSV) encoding a fusion (F) protein for direct cytosolic entry. Both HRSV infection and cytosolic delivery of a 65-nt dsRNA led to potent IFN-α induction in PDC, but not in myeloid dendritic cells. Inactivation of HRSV by UV irradiation abrogated IFN-α induction in PDC. The comparison of two respiratory syncytial virus (RSV) constructs carrying either the HRSV or the bovine RSV F protein revealed that F-mediated cytosolic entry of RSV was absolutely required for IFN-α induction in PDC. HRSV-induced IFN-α production was independent of endosomal acidification and of protein kinase R (PKR) kinase activity, as demonstrated with chloroquine and the PKR inhibitor 2-aminopurine, respectively. In contrast, the induction of IFN-α by the TLR7/8 ligand R848, by the TLR9 ligand CpG-A ODN 2216, and by inactivated influenza virus (TLR7/8 dependent) was completely blocked by 2-aminopurine. IFN-α induction by mouse pathogenic Sendai virus was not affected in PKR- and MyD88-deficient mice, confirming that a ssRNA virus, which is able to directly enter host cells via fusion at the plasma membrane, can be detected by PDC independently of PKR, TLR7/8, and TLR9.

Activation of innate immunity depends on the recognition of molecules that are specific for the pathogen, but absent in the host. Due to their distinct biology, many of such molecules are found in bacteria and protozoa. Examples are peptidoglycans, endotoxin, or flagellin that are detected by TLR2, TLR4, and TLR5, respectively, all members of the TLR family that evolved to detect pathogen-specific molecules (1). The situation is different for viruses. Viruses use the protein synthesis machinery of the host cell for their propagation. As a consequence, specific protein modifications common for viruses (that might serve as recognition pattern) do not exist.

To detect viruses, the vertebrate immune system seems to use an alternative strategy based on specific characteristics of viral nucleic acids. Viral nucleic acids may be detectable due to either a different molecular structure or a distinct subcellular localization. There is recent information that both principles apply. With regard to virus-specific molecular structures, long dsRNA and CpG motifs within viral DNA are involved. Neither is usually present in noninfected cells. For the recognition of dsRNA, two different detection modes are known, the serine threonine kinase protein kinase R (PKR)5 (2, 3, 4) and TLR3 (5). Although PKR is located in the cytosol, TLR3 is present in the endosomal compartment (6). The other molecular structure, CpG motifs in viral DNA, is detected via TLR9 (7, 8). CpG motifs are unmethylated CG dinucleotides with certain flanking bases. The frequency of CpG motifs is suppressed in vertebrates, allowing the vertebrate immune system to detect microbial DNA based on such CpG motifs (9, 10). TLR9 is located in the endosomal/lysosomal compartment, where it directly binds to CpG motifs (11). Due to the effective TLR-mediated detection of dsRNA and CpG DNA, viruses that contain these molecular features have evolved mechanisms that inhibit detection by reducing the accessibility to these structures or that actively antagonize subsequent signaling pathways to escape immune recognition. However, the most divergent group of human pathogenic viruses are positive or negative ssRNA viruses.

This leads to the second principle of viral nucleic acid recognition that has been recently identified, specific subcellular localization of viral nucleic acids. Due to abundant nucleases in the extracellular space, under normal circumstances RNA is absent in the endosome. The results from two recent publications suggest that high concentrations of ssRNA or their degradation products (nucleosides) are detected via TLR7 and TLR8 (12, 13). Guanine analogues have been identified earlier as specific ligands for TLR7 and TLR8 (14, 15, 16). Like TLR9 (receptor for CpG DNA), TLR7 and TLR8 are located in the endosomal membrane (15). Due to abundant ssRNA in the form of mRNA in the cytosol, cytosolic recognition of ssRNA either is not existent or must be sequence specific. However, all ssRNA viruses, once entering the cytosol, depend on the formation of dsRNA intermediates for replication of their genomes. Thus, even if they escape endosomal recognition via TLR7, once they form dsRNA in the cytosol they could become subject to PKR-dependent recognition. These endosomal and cytosolic pathways of viral nucleic acid recognition seem to ensure that most types of viruses can be recognized. Exceptions to this are retroviruses that integrate into the genome and thus do not require the formation of dsRNA for replication.

Detection of viral infection leads to the production of type I IFN (IFN-α and IFN-β). The major producer of type I IFN in humans is the plasmacytoid dendritic cell (PDC; also called IFN-producing cell). The PDC is a highly specialized subset of dendritic cells that is thought to function as a sentinel for viral infection and that is responsible for the vast amount of type I IFN during viral infection (17). TLR expression of human and mouse PDC is limited to TLR7 and TLR9 (18, 19, 20). As a consequence, the PDC is able to detect inactivated herpes virus (DNA virus) (7, 8) and inactivated influenza virus (ssRNA virus) (12) independent of viral replication. In contrast to influenza virus, which enters cells after endocytosis and membrane fusion at low pH, many ssRNA viruses such as human respiratory syncytial virus (HRSV) and other paramyxoviruses are known to directly enter the cytosol via membrane fusion at the cell surface, and thus may circumvent endosomal recognition via TLR7 and TLR9. If TLR-mediated recognition were the only mode of virus detection in PDC, how could the PDC fulfill its proposed function as the major sentinel of viral infection and major producer of type I IFN?

Therefore, one of the questions arising from the literature today is whether PDC are capable to detect ssRNA virus replication via cytosolic dsRNA, or whether other cell types such as myeloid dendritic cells (MDC) take over responsibility for viruses that escape endosomal recognition. In this context, it is interesting to note that TLR3 is absent in PDC (18, 19), and that polyinosinic-polycytidylic acid (poly(I:C)), the model stimulus for PKR, was found to be inactive in PDC (21).

In this study, we used respiratory syncytial virus (RSV) as a model virus to demonstrate that PDC are capable of detecting a ssRNA virus that directly enters the cytosol via a fusion protein. This detection leads to the production of vast amounts of IFN-α independently of endosomal acidification. We demonstrate that recognition of RSV in PDC is independent of PKR activity and that a 65-nt dsRNA delivered into the cytosol, but not the PKR model stimulus poly(I:C), the TLR7/8 ligand R848 or the TLR9 ligand CpG oligodeoxynucleotide (ODN) 2216, mimics RSV-induced IFN-α production in PDC. Cytosolic detection of dsRNA extends the repertoire of PDC for virus detection that is the basis for its function as the major producer of type I IFN in humans. In fact, other than in mice (22), we found that human PDC was the only immune cell subset that produces considerable amounts of IFN-α upon infection with ssRNA virus, while monocytes and MDC were also responsive, but produced other cytokines.

Materials and Methods

Medium and reagents

RPMI 1640 culture medium (Biochrom, Berlin, Germany) supplemented with 10% (v/v) FCS (BioWhittaker, Walkersville, MD), 1.5 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Sigma-Aldrich, Munich, Germany) was used throughout the studies (culture medium). OPTIMEM (Invitrogen Life Technologies, Karlsruhe, Germany) without additional supplements was used for transfection. All compounds were purchased endotoxin tested. ODN 2216 was kindly provided by Coley Pharmaceutical Group (Wellesley, MA) (small letters, phosphorothioate linkage; capital letters, phosphodiester linkage 3′ of the base: 5′-ggGGGACGATCGTCgggggG-3′. R848 was purchased from InvivoGen (Toulouse, France). Poly(I:C) and LPS (Salmonella typhimurium) were from Sigma-Aldrich. The 65-nt dsRNA, derived from the GFP sequence, was kindly provided by Alnylam Pharmaceuticals (Cambridge, MA): sense, 5′-GGGGCACAAG CUGGAGUACA ACUACAACAG CCACAACGUC UAUAUCAUGG CCGACAAGCA GAAGA-3′; antisense, 5′-UCUUCUGCUU GUCGGCCAUG AUAUAGACGU UGUGGCUGUU GUAGUUGUAC UCCAGCUUGU GCCCC-3′.

Mice

MyD88−/− mice (kindly provided by S. Akira, Osaka University, Osake, Japan) (23) were backcrossed six generations into the C57BL/6J strain. Control wild-type (Wt) C57BL/6 mice were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). PKR−/− mice (129S2/SvHsd) were kindly provided by J. Pavlovic (University of Zürich, Zürich, Switzerland) (24). Control Wt 129S2/SvHsd were purchased from Harlan Winkelmann (Borchen, Germany).

Isolation of PDC, MDC, monocytes, and B cells

Human PBMC were prepared from whole blood donated by young healthy donors. PBMC were obtained from whole blood by Ficoll-Hypaque density gradient centrifugation (Biochrom). PDC were isolated by MACS using the blood dendritic cell Ag (BCDA)-4 dendritic cell isolation kit from Miltenyi Biotec (Auburn, CA). Briefly, PDC were labeled with anti-BDCA-4 Ab coupled to colloidal paramagnetic microbeads and passed through a magnetic separation column once (LS column; Miltenyi Biotec). The purity of isolated PDC (lineage-negative, MHC-II-positive, and CD123-positive cells) was between 75 and 100%. Contaminating cells were mainly T cells. Before isolation of MDC, monocytes, and B cells, PDC were depleted by MACS (CS column; Miltenyi Biotec). MDC and B cells were isolated by MACS using the BDCA-1 dendritic cell isolation kit and B cell isolation kit, respectively (both Miltenyi Biotec). Monocytes were isolated via plastic adherence for 2 h. The purity of isolated MDC, B cells, and monocytes was higher than 75, 95, and 95%, respectively. Viability was >95%, as determined by trypan blue exclusion. Murine bone marrow-derived dendritic cells were generated by incubating pooled bone marrow cells in the presence of human recombinant fms-related tyrosine kinase 3 ligand (10 ng/ml; R&D Systems, Minneapolis, MN). After 7 days, these cultures contained ∼60% CD11c+, CD11b−, B220+ PDC.

Cell culture

Isolated cells were cultured in 96-well flat-bottom plates at a concentration of 5 × 104 cells (PDC) or 10 × 104 cells (MDC, monocytes, or B cells) in 200 μl of medium/well at 37°C and 5% CO2. PDC cultures were supplemented with 10 ng/ml IL-3 (R&D Systems). R848 or ODN 2216 was added at a final concentration of 500 ng/ml and 3 μg/ml, respectively. Viruses were added at a multiplicity of infection of 3. Chloroquine or 2-aminopurine (both Sigma-Aldrich) was added 30 min before stimulation at the indicated concentrations. Transfection experiments were conducted with lipofectamine 2000 (Invitrogen Life Technologies). For each transfection, 200 ng of nucleic acid was mixed with 25 μl of OPTIMEM. In a separate tube, 0.5 μl of lipofectamine 2000 was added to 25 μl of OPTIMEM and incubated for 5 min at room temperature. For complex formation, both solutions were mixed, incubated for an additional 20 min at room temperature, and added to cells (100 μl) in a 96-well plate (final volume 150 μl). Cells in transfection solution were incubated at 37°C without additional washing.

Viruses

The HRSV strain for these studies (human RSV strain Long; American Type Culture Collection (ATCC), Manassas, VA) was selected based on its ability to induce IFN-α in PBMC. Bovine RSV (BRSV) Gh/Fh is recombinant and contains the attachment protein G and the fusion protein F from HRSV Long (25). Viruses were grown on Vero cells (ATCC), which are not able to produce type I IFN. For preparation of virus stocks, 80% confluent Vero cell layers were infected at a multiplicity of infection of 0.1 in DMEM in the absence of FCS. The inoculum was removed after 1 h, and cells were incubated in DMEM supplemented with 2.5% FCS at 37°C in a 5% CO2 atmosphere. Upon development of extensive cytopathic effects, virus was released by freezing and thawing, followed by centrifugation at 3500 rpm for 20 min at 4°C. Infectious virus titers were determined on Vero cells by end point dilution and counting of infected cell foci stained for indirect immunofluorescence with RSV F-specific mAb RSV-F (Serotec, Oxford, U.K.). As described elsewhere, influenza virus was heat inactivated to be deficient in replication, while maintaining its IFN-α induction in PDC (12). Sendai virus was obtained from Charles River Laboratories (Wilmington, MA).

Detection of cytokines

Because total IFN-α is comprised of 14 different isoforms, the quantity of IFN-α measured by ELISA depends on the specificity of the detection Ab for these isoforms, and thus is not identical between different ELISA. For this study, the IFN-α module set Bender MedSystems (Graz, Austria) (detection range 8–500 pg/ml) was used. This ELISA detects most of IFN-α isoforms, but not IFN-B and IFN-F. The human IL-12p70 ELISA (detection range 8–500 pg/ml) and the human TNF-α ELISA (detection range 8–500 pg/ml) were from BD Pharmingen (Heidelberg, Germany). All ELISA procedures were performed, according to manufacturer’s recommendations. Murine IFN-α was measured by ELISA using the PBL murine IFN-α ELISA kit (PBL Biomedical Laboratories, Piscataway, NJ; IFN-α concentration in pg/ml) or according to the following protocol: monoclonal rat anti-mouse IFN-α (clone RMMA-1) was used as the capture Ab, and polyclonal rabbit anti-mouse IFN-α serum for detection (both from PBL Biomedical Laboratories) together with HRP-conjugated donkey anti-rabbit IgG as the secondary reagent (Jackson ImmunoResearch Laboratories). Mouse rIFN-αA (PBL Biomedical Laboratories) was used as the standard (IFN-α concentration in IU/ml).

Flow cytometry

Flow cytometric data were acquired on a BD Biosciences (Heidelberg, Germany) FACSCalibur equipped with two lasers (excitation at 488 and 635 nm wavelength). Viral infection was monitored by FACS analysis of ∼104 cells after fixation with 3% paraformaldehyde at room temperature for 5 min. After washing with FACS buffer (PBS containing 0.4% FCS and 0.02% NaN3), cell surface staining of viral envelope proteins for 30 min on ice was performed with RSV-F Ab and an isotype-specific Ab control (Serotec). After washing, cells were incubated for 30 min with FITC-labeled anti-mouse Ab (Dianova, Hamburg, Germany), followed by washing and FACS analysis. For PDC survival, cells were harvested 36 h after stimulation, and the absolute number of viable cells per 50 μl aspirated volume was determined by flow cytometry. Bona fide viable PDC were identified in a TO-PRO-3 iodide (Molecular Probes, Eugene, OR) and morphology-based live gate. Data were analyzed using CellQuest (BD Biosciences) or FlowJo software (version 2.5.1; Tree Star, Stanford, CA).

Statistical analysis

Data are expressed as means ± SEM. Statistical significance of differences was determined by the paired two-tailed Student‘s t test. Differences were considered statistically significant for p < 0.05. Statistical analyses were performed using StatView 4.51 software (Abacus Concepts, Calabasas, CA).

Results

HRSV Long is a potent inductor of IFN-α production in PDC, but not in MDC, monocytes, or B cells

RSV have been described to induce type I IFN in different cell types, but little is known about the ability of HRSV to activate PDC. In preliminary experiments, IFN-α was found in the supernatants of PBMC that were exposed to HRSV Long (data not shown). To identify the cell type responsible for IFN-α production within PBMC, different subsets of APCs were isolated. Purified monocytes, B cells, MDC, and PDC were tested for their ability to produce IFN-α upon incubation with replication-competent HRSV Long (ATCC; see Materials and Methods). Supernatants were collected at 12, 24, and 36 h after HRSV infection, and IFN-α production was assessed by ELISA. Among all APCs tested, PDC were identified as the major source of IFN-α production (Fig. 1⇓A). The corresponding negative (mock) control of HRSV consisting of culture supernatant from noninfected cells was inactive, indicating that indeed the virus and not cellular contaminants in the virus preparation contained the IFN-α-inducing activity. Compared with IFN-α induction by HRSV in PDC, IFN-α production in monocytes, B cells, and MDC was marginal (Fig. 1⇓A; note different scale). The quantity of IFN-α induction by HRSV in PDC was in the same range as after stimulation with the CpG-A ODN 2216 (Fig. 1⇓B), a well-established TLR9 stimulus that was used as a positive control. A strong correlation of the HRSV and CpG response was observed in PDCs from different donors (Fig. 1⇓B).

FIGURE 1.
FIGURE 1.

PDC, but not MDC, monocytes, or B cells, produce IFN-α upon exposure to RSV. PDC (50,000 cells/well), MDC, B cells, and monocytes isolated from human PBMC (all 100,000 cells/well) were incubated with HRSV, the corresponding mock control, or the CpG-A ODN 2216 (6 μg/ml). A, After 12, 24, and 36 h, supernatants were collected and IFN-α was measured by ELISA. Results are depicted as means ± SEM of independent experiments with cells from individual donors (PDC, n = 8; MDC, n = 6; monocytes, n = 3; B cells, n = 3). B, IFN-α production was analyzed after 36 h of incubation with RSV or CpG-A ODN 2216. The results of 12 independent experiments with cells from different individual donors are plotted (IFN-α in response to RSV, x-axis; IFN-α in response to CpG-A ODN 2216, y-axis). The correlation coefficient was determined using the Spearman calculation.

Infection of MDC may lead to the synthesis of other cytokines than IFN-α. Despite the lack of IFN-α production, MDC indeed were found to produce large amounts of IL-12p70 upon infection with HRSV (Fig. 2⇓), whereas IL-12p70 in PDC was negligible (data not shown). These results suggest that the cytokine response is cell type rather than stimulus specific.

FIGURE 2.
FIGURE 2.

MDC infected by HRSV produce IL-12 p70. MDC were isolated from human PBMC and were incubated with HRSV or the corresponding mock control. MDC (100,000 cells/well) were incubated for 12, 24, and 36 h. IL-12p70 production in response to RSV (▴) and to the mock control (x) was determined in the supernatants by ELISA. Results of four experiments with cells from individual donors are presented as means ± SEM.

HRSV entry via fusion (F) protein is necessary to induce IFN in PDC

In previous studies, we demonstrated that the species-specific permissivity of RSV is determined by the viral F glycoprotein (26). The F protein of RSV mediates species-specific fusion of the virus membrane with the cell membrane. To determine the role of viral entry for IFN-α induction in PDC, we made use of recombinant virus constructs that contained either HRSV or BRSV G and F genes on the otherwise identical genetic background of BRSV (BRSV Gh/Fh: BRSV with HRSV G and F gene; see Materials and Methods). Isolated PDC were incubated with BRSV Gh/Fh and BRSV. After 36 h, PDC were harvested and analyzed by flow cytometry using an Ab that detects both the human and the bovine F protein. Only the BRSV Gh/Fh construct containing the HRSV F protein, but not BRSV, was able to infect PDC, as documented by the expression of F protein on the cell surface (Fig. 3⇓A). Consistent with the ability of BRSV Gh/Fh to enter human cells by membrane fusion only, BRSV Gh/Fh was found to induce IFN-α in PDC (Fig. 3⇓B). These results suggested that cytosolic delivery of the RSV RNA via the viral F protein is required for IFN-α induction in PDC. Exposure of PDC to an intact RSV particle that lacks the ability of direct cytosolic entry (but would still be available for endosomal uptake) seems not sufficient for IFN-α induction in PDC.

FIGURE 3.
FIGURE 3.

Induction of IFN-α in PDC is dependent on the entry of RSV via F protein. PDC isolated from PBMC (50,000 cells/well) were incubated with an BRSV construct that contained the HRSV F protein (BRSV Gh/Fh) or the bovine F protein (BRSV) or the mock control. A, The mean fluorescence intensity (MFI) of surface expression of the F protein on PDC was determined by flow cytometry 36 h after incubation of PDC with either BRSV (upper panel, filled curves) or BRSV Gh/Fh (lower panel, filled curves) or the mock control (open curves). One representative experiment is shown. B, After 36 h, supernatants were collected and IFN-α production was determined by ELISA. Results are depicted as means ± SEM of experiments with cells from six individual donors.

RSV-induced IFN-α production in PDC is independent of endosomal maturation

It has been shown that the detection of virus via endosomal TLR7 and TLR9 and subsequent IFN-α induction strictly depends on endosomal maturation and acidification (7, 12, 15). To study whether these endosomal pathways contribute to IFN-α induction by RSV, we used chloroquine as an inhibitor of endosomal acidification. Isolated PDC were pretreated with chloroquine for 30 min before they were exposed to RSV or the CpG-A ODN 2216 that we used as an endosome-dependent positive control stimulus. Inhibition of endosomal acidification by chloroquine resulted in a dose-dependent inhibition of CpG-induced IFN-α. At a chloroquine dose that already inhibited CpG-induced IFN-α to <10%, RSV-induced IFN-α in PDC was not affected (Fig. 4⇓). Higher doses of chloroquine reduced viability of PDC and thus were not useful to study the influence of endosomal maturation on IFN-α production (our unpublished observation). These results indicated that the induction of IFN-α by RSV in PDC, unlike CpG, is independent of endosomal maturation.

FIGURE 4.
FIGURE 4.

IFN-α induction in PDC by RSV is independent of endosomal maturation. PDC (50,000 cells/well) isolated from PBMC were pretreated with medium alone or with increasing concentrations of chloroquine as indicated for 30 min before HRSV or CpG-A ODN 2216 (3 μg/ml) were added. After 36 h, supernatants were collected and IFN-α was measured by ELISA. The relative changes of IFN-α production (IFN-α in the absence of chloroquine was set 100%) in response to increasing concentrations of chloroquine are indicated. Results from three experiments are depicted as means ± SEM.

Inhibition of RSV replication by UV irradiation abrogates the ability of RSV to induce IFN-α production in PDC

It has been reported that the fusion protein of RSV is recognized independent of viral replication via TLR4 present on monocytes (27). Although TLR4 is absent in PDC, and although our results above suggested that cytosolic delivery of the RSV via the species-specific F protein was necessary for RSV to induce IFN-α in PDC, we still wished to exclude the possibility that direct recognition of the F protein is involved in the IFN-α induction. For this purpose, RSV was inactivated by UV cross-linking at a dose that leaves the functional cell-binding activity of the F protein intact (0.1 J/cm2) (28). UV inactivation completely abrogated the ability of RSV to induce IFN-α in PDC (Fig. 5⇓A), while monocytes displayed the same vigorous TNF-α response to both intact RSV and UV-inactivated virus (Fig. 5⇓B). These results clearly indicated that the mechanisms involved in RSV-induced activation of monocytes, such as recognition of the F protein, are not involved in the pathway that leads to RSV-induced IFN-α production in PDC. Furthermore, they exclude the possibility that other preformed components of the virus particle such as the ssRNA are responsible for IFN-α induction in PDC. Thus, in contrast to monocyte activation, induction of IFN-α production in PDC seems to rather depend on the ability of RSV to use their genome for replication and transcription, because both is lost upon UV irradiation.

FIGURE 5.
FIGURE 5.

Blockade of viral replication by UV inactivation abolishes RSV-induced IFN-α production in PDC, but does not affect RSV-induced TNF-α production in monocytes. PDC (50,000 cells/well) and monocytes (100,000 cells/well) isolated from PBMC were incubated with intact or UV-inactivated HRSV or the mock control. In addition, monocytes were stimulated with LPS (100 ng/ml). A, After 36 h, IFN-α in the supernatants of PDC was determined by ELISA. The means ± SEM from six individual donors are depicted. B, After 24 h, supernatants from monocytes were collected and TNF-α production was measured by ELISA. Results from three independent experiments are shown as means ± SEM.

TLR7/8- and TLR9-, but not HRSV- and dsRNA-induced IFN-α production is sensitive to the PKR inhibitor 2-aminopurine

Productive viral infection by a ssRNA virus such as RSV requires transcription of viral mRNAs and replication of the genome, which is believed to involve the formation of dsRNA or at least dsRNA intermediates. Therefore, we hypothesized that recognition of dsRNA in the cytosol of productively infected PDC is the critical event leading to IFN-α production. Indeed, cytosolic delivery of a 65-nt dsRNA molecule by lipofection into PDC was found to induce similar levels of IFN-α as the positive control CpG-A ODN 2216 treated in the same way (Fig. 6⇓A). The dsRNA did not induce IFN-α in MDC (Fig. 6⇓A; note different scale). This suggested that in PDC the dsRNA-binding PKR might be involved in dsRNA recognition and IFN-α induction. However, poly(I:C), a potent model stimulus for PKR, did not induce considerable amounts of IFN-α in PDC (Fig. 6⇓A; note different scale). Furthermore, 2-aminopurine, an inhibitor of PKR activity, did not inhibit, but even increased dsRNA-induced IFN-α in PDC (Fig. 6⇓B) at dosages that did not affect cellular viability (Fig. 6⇓C). The same 2-aminopurine-dependent increase of IFN-α production was found in HRSV-stimulated PDC, suggesting that both dsRNA and RSV induce IFN-α in a similar way independent of PKR activity (Fig. 6⇓D). To our surprise, 2-aminopurine at doses that increased the dsRNA- and RSV-induced IFN-α production strongly inhibited the TLR-mediated IFN-α induction in PDC. This 2-aminopurine-mediated inhibition of IFN-α induction was seen for the TLR7/8 ligand R848 and the TLR9 ligand CpG-A ODN 2216 (Fig. 6⇓D) as well as for influenza virus that was recently shown to be completely TLR7 dependent (12) (Fig. 6⇓E). Unlike IFN-α, the induction of IL-8 (NF-κB-dependent cytokine) by R848 and CpG-A ODN 2216 was not affected by 2-aminopurine (our unpublished observation), indicating that 2-aminopurine is not competing with R848 or CpG-A ODN 2216 for binding to TLR7/8 or TLR9. In addition to 2-aminopurine, chloroquine completely abrogated the influenza-induced IFN-α production of PDC (Fig. 6⇓E), confirming that recognition of influenza virus, unlike HRSV (compare Fig. 4⇑), occurs in the endosomal compartment. Together, these results suggest that RSV- and dsRNA-induced IFN-α in PDC is independent of TLR7/8, TLR9, and PKR activity, and support the concept that RSV, but not influenza virus, is detected by PDC via dsRNA formed in the cytosol during productive viral infection.

FIGURE 6.
FIGURE 6.

PKR- and TLR7-independent IFN-α induction by RSV and cytosolic dsRNA. PDC (50,000 cells/well) or MDC (100,000 cells/well) isolated from PBMC were stimulated with dsRNA (200 ng/ml), poly(I:C) (200 ng/ml), CpG-A ODN (3 μg/ml), R848 (500 ng/ml), or RSV in the presence or absence of 30-min pretreatment with the PKR-specific inhibitor 2-aminopurine. After 36 h, the supernatants were collected and IFN-α was determined in the supernatants by ELISA. A, Lipofectamine was used for comparison of cytosolic delivery of dsRNA, poly(I:C), and CpG-A ODN 2216 in MDC and PDC. Results are depicted as means ± SEM from three individual donors. B, The relative changes of IFN-α production (IFN-α in the absence of 2-aminopurine is set 100%) in response to increasing concentrations of 2-aminopurine are indicated. Results from three independent experiments are depicted as mean values ± SEM. C, Viability of PDC (absolute number of viable cells in 50 μl vol; see Materials and Methods) at 36 h after stimulation with dsRNA or R848 in the presence of increasing concentrations of 2-aminopurine. The mean values ± SEM of two independent donors are depicted. D, Stimulation of PDC with RSV, CpG-A ODN 2216, and R848 is compared. The relative changes of IFN-α production (IFN-α in the absence of 2-aminopurine is set 100%) in response to increasing concentrations of 2-aminopurine are indicated. Data are shown as means ± SEM. E, IFN-α production in response to stimulation of PDC with inactivated influenza virus in the presence or absence of 2-aminopurine or chloroquine. The relative changes of IFN-α production (IFN-α in the absence of 2-aminopurine and chloroquine, respectively, is set 100%) are indicated. The absolute values of influenza-induced IFN-α production in the absence of 2-aminopurine and chloroquine for the three individual donors tested were 2.1, 3.6, and 1.6 ng/ml. Data are shown as means ± SEM.

Sendai virus induces IFN-α in murine PDC independent of MyD88 and PKR

Because F protein-dependent entrance of RSV was species specific, we used the mouse pathogenic Sendai virus to study virus recognition in the absence of MyD88 and PKR in mice. Like RSV, Sendai virus is a negative-strand RNA virus of the family of paramyxoviridae that directly enters host cells via fusion at the plasma membrane. We found that Sendai virus induced similar amounts of IFN-α production in murine PDC as the TLR9 ligand ODN 2216 (Fig. 7⇓A). Although TLR9-mediated IFN-α production was completely dependent on the presence of the adaptor protein MyD88, Sendai virus-induced IFN-α was independent of MyD88. Furthermore, Sendai virus stimulated similar levels of IFN-α production in PKR−/− and Wt mice (Fig. 7⇓B). These results indicated that the detection of Sendai virus by murine PDC is independent of TLR7 and TLR9 (both dependent on MyD88) and PKR, thereby confirming the results obtained for RSV in the human system.

FIGURE 7.
FIGURE 7.

MyD88- and PKR-independent induction of IFN-α by Sendai virus in murine PDC. Bone marrow-derived dendritic cells (200,000 cells/well) containing 50–60% CD11c+ CD11b− B220+ PDC were prepared. A, Cells of MyD88−/− and of Wt mice were incubated in the presence of Sendai virus and ODN 2216. After 36 h, IFN-α was measured in the supernatant. Data are shown as means ± SEM of three independent experiments. B, Cells from PKR−/− mice and of Wt mice were incubated in the presence of Sendai virus. After 36 h, supernatant was collected and IFN-α was analyzed. The results from one of two representative experiments each performed in duplicates are depicted as means ± SEM.

Discussion

Two recent publications provide evidence that PDC detect replication-deficient herpes virus via TLR9, resulting in an instant type I IFN response that is independent of viral replication (7, 8). Considering the crucial importance of type I IFN in the control of local and systemic HSV infection (29) and the proposed central role of PDC in this context (30), it was surprising that TLR9−/− mice showed no diminished ability to control herpes viral infection (8). This observation is in conflict with the hypothesis that PDC are the major type I IFN-producing cells, except if PDC would feature additional TLR9-independent mechanisms of virus detection.

It is long known that dsRNA is formed in the cytosol during herpes viral replication as the result of overlapping convergent transcription (31). However, by using herpes virus, it is difficult to separate TLR9 (CpG DNA) mediated from cytosolic dsRNA-mediated activation of human PDC. To selectively study a possible contribution of cytosolic dsRNA, we used the ssRNA virus HRSV (negative single-stranded virus) as a model. The appearance of HRSV F protein on the surface of cells demonstrates successful infection, including RNA replication and transcription in the cytosol.

In this study, HRSV strain Long induced the production of similar levels of IFN-α in PDC as CpG-A ODN 2216, a well-established positive control (32). UV irradiation of HRSV abolished its IFN-α-inducing activity. Furthermore, IFN-α induction by RSV was dependent on the presence of the corresponding species-specific fusion protein for cytosolic entry, because only a mutant of BRSV virus that contained the HRSV F protein (BSRV Gh/Fh) (but not the authentic BRSV) was able to enter human PDC and to induce IFN-α by replication of the BRSV RNA. These results suggested that cytosolic entry of a replication-competent virus rather than ssRNA or other preformed components of the virus particle was required for IFN-α induction in PDC.

There are four lines of evidence that HRSV particles are not detected via TLR in the endosomal compartment of PDC: first, RSV-induced IFN-α production was independent of endosomal acidification that is critical for activation via endosomal TLRs (7, 15). Second, 2-aminopurine potently inhibited TLR7-mediated (R848, influenza virus)- and TLR9-mediated (CpG)- but not HRSV-induced IFN-α production, while IL-8 production was not affected by any of these stimuli. Third, IFN-α induction by HRSV was mimicked by cytosolic delivery of dsRNA, and both were equally insensitive to 2-aminopurine. Fourth, replication-deficient RSV particles (HRSV-UV or BRSV) did not induce IFN-α in PDC, although monocytes were still activated.

Further evidence comes from our studies with MyD88-deficient mice. MyD88-deficient and Wt mice showed similar levels of IFN-α production in bone marrow-derived PDC stimulated with the mouse pathogenic Sendai virus. Sendai virus is a member of the paramyxoviridae family like RSV and was used because entry of RSV was species specific (F protein). MyD88-independent IFN-α induction by Sendai virus in PDC confirmed that ssRNA viruses indeed can be detected by PDC without the usage of endosomal TLR7, TLR8, and TLR9, because these TLRs depend on the adaptor molecule MyD88 (33, 34).

The finding that UV irradiation abolished the activity of HRSV to induce IFN-α in PDC was surprising, because inactivated influenza virus (a ssRNA virus like HRSV) is known in the literature as a stimulus for IFN-α induction in PDC (12, 35). In our study, UV inactivation was performed at a dose that leaves the fusion protein of RSV functionally intact (28). In addition, we observed that, similar to HRSV, other human paramyxoviruses lost their activity to induce IFN-α in PDC upon UV inactivation (J. Schlender, V. Hornung, S. Finke, S. Marozin, K. Brzozka, G. Hartmann, and K.-K. Conzelman, manuscript in preparation). The distinct routes of viral entry might explain these differences. Influenza virus is internalized into endosomal compartments, where a low pH triggers a conformational change in hemagglutinin, allowing fusion and release of the nucleocapsid into the cytosol (36). Influenza virus suppresses dsRNA recognition of the infected host cell by using the viral protein NS1 to sequester dsRNA (22). Conversely, productive entry of paramyxoviruses like RSV into host cells is by fusion of the viral envelope with the host cell plasma membrane in a pH-independent manner (37). Lund et al. (38) used chloroquine at 0.1 mM to demonstrate that recognition of vesicular stomatitis virus-RSV-F in murine bone marrow cells is dependent on endosomal acidification. It is important to note that in our hands, 0.1 mM (32 μg/ml) chloroquine leads to massive cell death of human PDC, highlighting the importance of dose-finding studies for the specific cell subset to be examined.

The different mode of viral entry for RSV and influenza virus and the inhibition of dsRNA recognition by NS1 of influenza virus are consistent with replication-dependent recognition of cytosolic dsRNA for RSV in this study and the recently described endosomal TLR7-mediated recognition of ssRNA or its degradation products for inactivated influenza virus (12). Of note, like for influenza virus, RSV nonstructural proteins interfere with IFN induction in different cell lines (39, 40). Although such inhibitory mechanisms are virtually absent in HRSV strain Long used as a model stimulus in this study, it will be interesting to assay clinical HRSV isolates for their capacity to prevent IFN induction in PDCs.

It has been described that monocytes are able to detect the F protein of RSV via TLR4 and CD14 (27). In our study, UV irradiation of RSV abolished IFN-α induction in PDC, while stimulation of TNF-α production in monocytes was still maintained, indicating that detection of the F protein via TLR4 is not involved in the IFN-α induction in PDC. Furthermore, these results show that the dose of UV irradiation leaves the structure of the F protein intact for recognition by monocytes. Although it cannot be excluded that other candidate proteins of the virus possibly involved in PDC activation are more sensitive to UV irradiation than the F protein, it is more likely that RNA is involved. It is well-established that UV irradiation leads to RNA damage, thereby inhibiting the formation of dsRNA required for the transcription of the negative-strand viral genome. In support of dsRNA responsible for IFN-α induction in PDC, our results show that transfection of dsRNA, like RSV, induces similar quantities of IFN-α in PDC and is equally insensitive to inhibition by 2-aminopurine. However, although indicative, our studies provide no definite proof that dsRNA is critical for RSV-induced PDC activation.

In general, recognition of cytosolic dsRNA is believed to be mediated via PKR, a serine threonine kinase (2, 3, 4). Synthetic poly(I:C) is a well-established model stimulus to activate PKR. In addition to PKR-mediated recognition, poly(I:C) is detected via TLR3 (5). In our hands and in those of other investigators (18, 21), poly(I:C) alone did not activate PDC, consistent with the lack of TLR3 in PDC (19). However, to our surprise, even cytosolic delivery of poly(I:C) via transfection was inactive, suggesting that in PDC activation of PKR via long dsRNA is not linked to IFN-α induction. Unlike poly(I:C), potent IFN-α induction was found in response to cytosolic delivery of a 65-nt dsRNA. A PKR-independent mechanism of IFN-α induction by this 65-mer dsRNA as well as of RSV was suggested by the insensitivity of both stimuli to 2-aminopurine, a well-established inhibitor of PKR activity (41, 42). We confirmed PKR-independent recognition of ssRNA viruses by PDC in PKR-deficient mice. Our data are in agreement with results by another group that used PKR-independent and TLR3 knockout mice to show that the induction of IFN-α and IFN-β by another member of the family of paramyxoviridae, Newcastle disease virus, was independent of PKR and TLR3 (43).

The ability to detect HRSV is not limited to PDC. RSV infection strongly stimulated MDC to produce high amounts of IL-12p70. However, only PDC were found to produce high levels of IFN-α in response to RSV infection, while very little IFN-α was produced in MDC. Furthermore, cytosolic delivery of poly(I:C) or a 65-nt dsRNA molecule did not induce IFN-α in MDC. These results further support the concept that in humans, the PDC is the only cell type that is capable of producing high amounts of IFN-α upon viral infection. The situation might be different in mice. It has been reported that upon viral stimulation, murine MDC produce considerable levels of IFN-α (22). In that study, MDC and PDC produced similar levels of IFN-α upon stimulation with cytosolic poly(I:C). However, the absolute values of IFN-α produced by MDC and PDC were very low and only 10-fold higher than the amount of IFN-α produced by B cells. In that paper, the ability of murine MDC to produce IFN-α might have been overestimated by selecting poly(I:C) as a stimulus. Our data show that, at least for the human system, IFN-α induction by poly(I:C) in PDC is marginal. This is supported by the literature (21, 44).

In conclusion, our study provides evidence that PDC have the ability to detect certain ssRNA viruses such as RSV in the cytosol. Based on our results, we propose a concept in which viruses that manage to pass early TLR7/8- and TLR9-mediated recognition in the endosome of PDC may still be detected as soon as they start to form dsRNA in the cytosol for transcription of their genome. Cytosolic recognition of virus adds another mode of virus detection that allows PDC to properly fulfill their proposed duty: to function as the major producer of type I IFN upon viral infection, thereby eliminating viral burden from the host.

Acknowledgments

We thank Jennifer Wang for sharing bone marrow cultures. Furthermore, we thank S. Akira and C. Weissmann for the opportunity to work with MyD88 and PKR knockout mice, respectively. This work is part of the dissertation of M. Guenthner-Biller at the Ludwig-Maximilians-University.

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 study was supported by grants from the Deutsche Forschungsgemeinschaft HA 2780/4-1 and SFB 571, the Dr. Mildred Scheel Stiftung 10-2074, the Friedrich-Baur-Stiftung, and the Human Science Foundation of Japan (to G.H.). S.R. was supported by a grant from the Deutsche Forschungsgemeinschaft RO 2525/1-1; K.-K.C. was supported by SFB 455, SPP1089 Co260/1, and the European Commission 5FP QLK2-CT-1999-00443.

  • ↵2 V.H. and J.S. contributed equally to this work.

  • ↵5 Current address: Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA 01655.

  • ↵3 Address correspondence and reprint requests to Dr. Gunther Hartmann, Department of Internal Medicine, Division of Clinical Pharmacology, Ludwig-Maximilians-University, Ziemssenstrasse 1, 80336 Munich, Germany. E-mail address: ghartmann{at}lrz.uni-muenchen.de

  • 4 Abbreviations used in this paper: PKR, protein kinase R; F, fusion; HRSV, human respiratory syncytial virus; MDC, myeloid dendritic cell; ODN, oligdeoxynucleotide; PDC, plasmacytoid dendritic cell; RSV, respiratory syncytial virus; Wt, wild type; BCDA, blood dendritic cell Ag.

  • Received June 8, 2004.
  • Accepted August 11, 2004.

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