Several studies indicated that TLR as well as retinoic acid–inducible gene I–like helicase (RLH) signaling contribute to vesicular stomatitis virus (VSV)–mediated triggering of type I IFN (IFN-I) responses. Nevertheless, TLR-deficient MyD88−/−Trif−/− mice and RLH-deficient caspase activation and recruitment domain adaptor inducing IFN-β (Cardif)−/− mice showed only marginally enhanced susceptibility to lethal VSV i.v. infection. Therefore, we addressed whether concomitant TLR and RLH signaling, or some other additional mechanism, played a role. To this end, we generated MyD88−/−Trif−/−Cardif−/− (MyTrCa−/−) mice that succumbed to low-dose i.v. VSV infection with similar kinetics as IFN-I receptor–deficient mice. Three independent approaches (i.e., analysis of IFN-α/β serum levels, experiments with IFN-β reporter mice, and investigation of local IFN-stimulated gene induction) revealed that MyTrCa−/− mice did not mount IFN-I responses following VSV infection. Of note, treatment with rIFN-α protected the animals, qualifying MyTrCa−/− mice as a model to study the contribution of different immune cell subsets to the production of antiviral IFN-I. Upon adoptive transfer of wild-type plasmacytoid dendritic cells and subsequent VSV infection, MyTrCa−/− mice displayed significantly reduced viral loads in peripheral organs and showed prolonged survival. On the contrary, adoptive transfer of wild-type myeloid dendritic cells did not have such effects. Analysis of bone marrow chimeric mice revealed that TLR and RLH signaling of radioresistant and radiosensitive cells was required for efficient protection. Thus, upon VSV infection, plasmacytoid dendritic cell–derived IFN-I primarily protects peripheral organs, whereas concomitant TLR and RLH signaling of radioresistant stroma cells as well as of radiosensitive immune cells is needed to effectively protect against lethal disease.

Host cells are alerted to virus infection by conserved pathogen components or by products of pathogen replication that may trigger pattern recognition receptors (PRR). PRR comprise transmembrane proteins such as TLR and C-type lectin receptors, cytosolic RNA detection systems, such as retinoic acid–inducible gene I (RIG-I)–like helicases (RLH), DNA sensors including DAI, IFI16, cGAS, and AIM2, and presumably other not yet identified signaling platforms (1, 2). Previous studies indicated that viruses, such as the ssRNA-encoded vesicular stomatitis virus (VSV), are recognized by multiple mechanisms. Several TLRs including TLR4 (3, 4), TLR3 (5), TLR7 (6), and recently identified TLR13 (7), LRRFIP1 (8), as well as RIG-I (913), have been implicated in VSV recognition. Additionally, protein kinase R is triggered by VSV RNA and seems to play a role in protection (14). Several studies showed that VSV stimulates plasmacytoid dendritic cells (pDC) in a TLR7-dependent manner, whereas myeloid DC (mDC), macrophages, and murine embryonic fibroblasts are activated by RIG-I stimulation. This suggested that TLR and RLH signaling contribute to antiviral responses in a cell type–specific manner (9, 10, 15), whereas the in vivo significance of these signaling platforms remained unclear.

In the case of VSV infection, the induction of protective type I IFN (IFN-I) is critically required to promote host survival. This was demonstrated by type I IFN receptor (IFNAR)–deficient mice that succumb to VSV challenge within 1 to 2 d, whereas wild-type (WT) mice control the virus without developing signs of disease (16). For VSV-mediated induction of IFN-I responses, TLR7 and RIG-I play a more dominant role than TLR3 (5, 6, 9). As shown in several different studies, upon viral infection, pDC can be one major producer of IFN-I (1719). To study the significance of pDC in viral pathogenesis in greater detail, mice were generated that allowed selective in vivo depletion of pDC. Upon VSV infection of such pDC-depleted mice, at early time points, the induction of IFN-I responses was marginally reduced, whereas the overall survival was normal (20). Thus, this model was not very informative about the pDC function in VSV pathogenesis. Interestingly, mice devoid of MyD88, which is an obligatory adaptor protein of all TLR except TLR3, the latter of which is linked to the adaptor Toll/IL-1R domain–containing adapter inducing IFN-β (Trif), showed only moderately enhanced sensitivity to i.v. VSV infection (21, 22). Mice carrying a nonfunctional caspase activation and recruitment domain adaptor inducing IFN-β (Cardif; also known as mitochondrial antiviral signaling adaptor, virus-induced signaling adaptor, and IFN-β promoter stimulator-1), which is situated in the outer mitochondrial membrane and links activated RIG-I and MDA5 with downstream signaling molecules, were devoid of RLH signaling and showed only marginally impaired survival when compared with WT mice (23). Similar to Cardif-deficient animals, mice devoid of the intracellular DNA sensor stimulator of IFN genes (STING) also showed only slightly enhanced sensitivity to lethal VSV infection (24, 25). Because STING is predominantly an endoplasmic reticulum–resident protein linking RIG-I and DNA-mediated intracellular innate signaling, it was speculated that STING was required to exert effective RIG-I function (26). Collectively, these results support the hypothesis that upon VSV infection, TLR, RLH, and maybe other signaling platforms play a role in promoting protective IFN-I responses. Nevertheless, currently only very limited information is available about crosstalk between different PRR systems in sensing virus infections and whether only TLR and RLH signaling was critically involved in the protection against VSV or whether additional mechanisms played a role (27, 28).

To study whether redundant TLR and RLH signaling conferred protection upon i.v. VSV infection, we intercrossed MyD88−/−Trif−/− (MyTr−/−) and Cardif−/− (Ca−/−) mice to obtain MyTrCa−/− mice devoid of TLR and RLH signaling. Of note, such mice were as sensitive to VSV infection as mice with a complete IFNAR deletion. Based on this observation, we readdressed the in vivo role of DC subsets in promoting survival of VSV infection. Instead of studying mice depleted of pDC, we adoptively transferred fully functional DC subsets to TLR/RLH signaling-ablated mice and monitored the induction of adaptive immunity and survival. We found that pDC were triggered by VSV to mount early IFN-I responses with kinetics that confer antiviral effects in peripheral tissues and that promote the induction of adaptive immunity. Importantly, effective control of VSV infection additionally depended on TLR/RLH competence of radioresistant stroma cells.

All animals were handled in compliance with regulations of the German animal welfare law (Tierschutzgesetz). The protocol was approved by the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit (Oldenburg, Germany, identification numbers 09/1655 and 12/1025).

MyTrCa−/− mice were obtained by intercrossing MyTr−/− (29, 30) and Ca−/− mice (31) under conditions as described previously (32). Additionally, MyTr−/−, Ca−/−, and MyTrCa−/− mice were intercrossed with IFN-β+/∆β-luc mice (33) in a manner that they carried one IFN-β∆β-luc allele. IFN-β+/∆β-luc, My−/−, Tr−/−, and Ca−/− mouse strains were 10-fold backcrossed to the C57BL/6 background before intercrossing. IFNAR 1 chain–deficient mice [IFNAR−/− (16)] were 20-fold backcrossed to the C57BL/6 background (34). Mice were kept under specific pathogen-free conditions in the central mouse facility of the Helmholtz Centre for Infection Research, Braunschweig, and at TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany. C57BL/6 mice, also referred to as WT, were purchased from Harlan-Winkelmann or Janvier Laboratories. Animal experiments were conducted under specific pathogen-free conditions and with the permission of the Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit. For experiments, 8–14-wk-old mice were used. VSV-Indiana (Mudd-Summers isolate), originally obtained from D. Kolakofsky (University of Geneva, Geneva, Switzerland), and VSV-enhanced GFP (eGFP) (35) were grown on BHK-21 cells. Virus was harvested from conditioned culture medium, and titers were determined by plaque formation on Vero cells. The VSV variant M2 (VSV-M2) was shown to be a particularly strong IFN-I–inducing variant of the HR strain of WT VSV (36). The M15R mutant M protein of VSV-M2 is associated with a reduced sequestration of mRNA nuclear export (37).

Mice were warmed up in their cage for 3 min by an infrared lamp. Subsequently, mice with minimal body weight of 20 g were introduced into a restrainer and injected i.v. with 200 μl virus suspension diluted in PBS with titers of 2 × 102, 2 × 104, or 2 × 106 PFU, as indicated, or with 1 × 107 bone marrow (BM) cells or BM-derived DC subsets resuspended in PBS into the lateral tail vein. For s.c. injection, mice were anesthetized with isoflurane, and 50 μl of the indicated reagents was injected under the skin of the neck using an insulin injection syringe.

For VSV neutralization assay, blood samples were drawn at the indicated time points, centrifuged at 20,000 × g for 1.5 min, and serum was frozen at −20°C. For determination of IgG, sera were reduced with 280 mM 2-ME (Sigma-Aldrich) for 1 h at room temperature. For IgM analysis sera not treated with 2-ME were used. Sera were 1:40 prediluted with MEM supplemented with 5% FCS and 1% Glutamax (Life Technologies) and incubated 30 min at 56°C. Serial 2-fold dilutions were mixed with equal volumes of virus diluted to contain 3 × 103 PFU/ml. The serum–virus mixture was incubated for 1.5 h at 37°C and then transferred onto Vero cell monolayers grown in a 96-well plate. After 1 h of incubation at 37°C, the monolayers were overlaid with 100 μl MEM containing 1% methylcellulose and incubated for 24 h at 37°C. Then the overlay was removed, and the monolayer was fixed and stained using 0.5% crystal violet dissolved in 5% formaldehyde, 50% ethanol, and 4.25% NaCl.

For VSV plaque assay, tissues were homogenized in 1 ml MEM supplemented with 5% FCS (Sigma-Aldrich). Serial 10-fold dilutions of homogenates were transferred onto Vero cell monolayers in six-well plates and incubated for 1 h at 37°C. Monolayers were overlaid with 2 ml MEM containing 1% methylcellulose and incubated for 24 h at 37°C. Then the overlay was removed, and the monolayer was fixed and stained using crystal violet.

BM cells were isolated by flushing femur and tibia with RPMI 1640 medium supplemented with 10% FCS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM Glutamax, 100 U/ml penicillin, 100 μg/ml streptomycin (all from Life Technologies), and 0.1 mM 2-ME. Cells were then treated with RBC lysis buffer (Sigma-Aldrich) and washed with PBS. For in vitro differentiation of BM-mDC or BM-pDC, BM cells were seeded at a density of 1 × 106 or 2 × 106 cells/ml and incubated for 8 d in medium supplemented with 100 ng/ml GM-CSF (R&D Systems) or 100 ng/ml Flt3 ligand (Flt3-L; R&D systems), respectively. pDC were purified using the Plasmacytoid Dendritic Cell Isolation Kit II (Miltenyi) following the manufacturer’s instructions. For generation of BM chimeric mice, mice were lethally irradiated with 9 Gy, and the following day, they were i.v. reconstituted with 1 × 107 BM cells of the indicated genotype. BM chimeric mice were used for experiments after at least 8 wk of recovery.

A total of 1 × 106 Flt3-L–producing B16 tumor cells was injected s.c. in one flank of WT mice. Tumor growth was monitored until a maximal diameter of 1.5 cm was reached (38, 39). Tumor-bearing mice containing between 10 and 15% Siglec-H+ pDC in the spleen were sacrificed, and pDC were isolated from the spleen by negative MACS selection using the Plasmacytoid Dendritic Cell Isolation Kit II (Miltenyi Biotec).

Serum was analyzed for IFN-α and IFN-β by ELISA methods following the manufacturer’s instructions (IFN-β ELISA: PBL Biomedical Laboratories; IFN-α ELISA: eBioscience). For determination of IFN-α, the incubation with the biotin labeled anti–IFN-α Ab was performed for 1 h at room temperature followed by overnight incubation at 4°C.

Mice were anesthetized using isoflurane. Immediately following i.v. injection of 100 μl luciferin (30 mg/ml in PBS)/20 g mouse weight, the mice were analyzed in an in vivo imaging instrument (IVIS Spectrum CT; PerkinElmer). The acquired images were analyzed using Living Image 4.3.1 software. For the detection of luciferase activity in different organs and lymph nodes, the respective organs were prepared at the indicated time points after adjuvant application and stored at −80°C until analysis. After tissue homogenizing in Glo Lysis Buffer (Promega), Bright Glo Luciferase Assay System (Promega) was used to determine bioluminescence activity using a plate reader (BioTek).

For FACS analysis, 25 μl blood was stained with a VSV nucleoprotein (NP)–specific pentamer (H-2Kb–RGYVYQGL; ProImmune) for 10 min at room temperature. Then the samples were stained with anti-B220 Pacific Blue (BD Pharmingen), anti-CD3 Alexa Fluor 647 (Caltag Laboratories), anti-CD8 PE-Cy5 (Invitrogen), and anti-CD4 FITC (BioLegend) as indicated. Absolute cell numbers were determined by adding 25 μl AccuCheck Counting-Beads (Invitrogen) to the samples. Data were acquired on an LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).

For quantification of mRNA expression, RNA was extracted from spleen and inguinal lymph nodes (LN) of mice or from 6 × 105 BM-mDC using NucleoSpin RNA Kit (Qiagen) following the manufacturer’s instructions. A total of 100 ng total RNA was used for cDNA synthesis using PrimeScript FirstStrand cDNA Synthesis Kit (TaKaRa) according to the manufacturer’s instructions. Primers and SYBR Green (Bioline) were added to ∼10 ng cDNA, and quantitative real-time PCR (qRT-PCR) was carried out. PCR reactions were run in a LightCycler 480 (Roche). Fold changes of target genes were normalized to housekeeping gene GAPDH (40). The following primers were used in this study: IFN-stimulated gene (ISG) 15 (5′-GAGCTAGAGCCTGCAGCAAT-3′, 5′-TTCTGGGCAATCTGCTTCTT-3′), 2′,5′ oligoadenylate synthetase (2',5' OAS; 5′-GGATGCCTGGGAGAGAATCG-3′, 5′-TCGCCTGCTCTTCGAAACTG-3′), and GAPDH (5′-TGCACCACCAACTGCTTAGC-3′, 5′-GGCATGGACTGTGGTCATGAG-3′).

BM-mDC were treated with serial dilutions of recombinant, Chinese hamster ovary cell–expressed, murine IFN-β (stock concentration: 179 μg/ml = 3.58 × 107 U/ml) for 2 h. Subsequently, cells were infected with VSV-eGFP at a multiplicity of infection of 0.01. After 18 h incubation, eGFP expression of BM-mDC was monitored cytofluorometrically. For treatment with human rIFN-αB/D hybrid (rIFN-α) (41), mice were anesthetized using isoflurane, and 1.6 μg (∼105 U) rIFN-α (stock concentration: 165 μg/ml = 1 × 107 U/ml reactive on mouse cells) in 50 μl PBS was administered s.c. into the neck.

To generate IFN-containing serum, C57BL/6 mice were infected with 2 × 107 PFU VSV-M2, and serum was collected 12 h postinfection (hpi) (42). The IFN-α content of the serum was analyzed by an ELISA method. In the described experiment, serum was used containing 2 ng/μL IFN-α.

Mean cytokine levels, viral and Ab titers, and cytotoxic lymphocyte responses were compared by the Mann–Whitney U test. For survival analysis, the Mantel–Cox survival analysis with log-rank statistics was used. A p value ≤0.05 was considered statistically significant. For statistical analysis, the software package GraphPad Prism Version 5.0 (GraphPad) was used.

To study the role of TLR and RLH signaling in the protection against VSV infection, TLR signaling-deficient MyTr−/− mice and Ca−/− mice devoid of RLH signaling were i.v. challenged with 2 × 106 PFU VSV. As observed in previous studies, under such conditions, MyTr−/− and Ca−/− mice showed enhanced sensitivity to VSV infection as indicated by ∼70% of MyTr−/− and 25% of Ca−/− mice succumbing to infection within 15 d, whereas essentially all WT controls survived (Fig. 1A). At infection doses of 2 × 104 or 2 × 102 PFU, MyTr−/− and Ca−/− mice controlled VSV as efficiently as WT mice (Fig. 1B, 1C). As readout for the induction of adaptive immunity, VSV-NP–specific CTL and VSV-neutralizing IgM and IgG responses were analyzed. Of note, previous studies showed that unlike VSV-neutralizing Ab responses, CTL did not critically contribute to the protection against lethal VSV infection (4346). Upon i.v. challenge with 2 × 106 PFU VSV, WT controls, MyTr−/−, and Ca−/− mice showed similar expansion of VSV-specific CTL peaking 1 wk postinfection at ∼20% VSV-NP–specific CTL among CD8+ T cells (Fig. 1D). The analysis of virus-neutralizing Ab responses revealed that similar to WT controls MyTr−/− and Ca−/− mice mounted IgM responses on day 4 that switched to IgG on day 8 (Fig. 1E).

FIGURE 1.

MyTrCa−/− mice are as vulnerable to lethal VSV infection as IFNAR−/− mice. WT controls, IFNAR−/−, MyTr−/−, Ca−/−, and MyTrCa−/− mice were i.v. infected with 2 × 106 (A), 2 × 104 (B), or 2 × 102 (C) PFU VSV, and survival was monitored daily. n ≥ 11. *p < 0.04, ***p < 0.0001 log-rank, Mantel–Cox test. Mice infected with 2 × 106 PFU VSV were bled at the indicated time points, and the expansion of VSV-NP–specific CTL (D) and VSV-neutralizing serum Abs (E) was monitored (n ≥ 6). Horizontal bars indicate means ± SEM of individually evaluated mice (one-tailed Mann–Whitney U test). All data shown are pooled from two to three independently performed experiments.

FIGURE 1.

MyTrCa−/− mice are as vulnerable to lethal VSV infection as IFNAR−/− mice. WT controls, IFNAR−/−, MyTr−/−, Ca−/−, and MyTrCa−/− mice were i.v. infected with 2 × 106 (A), 2 × 104 (B), or 2 × 102 (C) PFU VSV, and survival was monitored daily. n ≥ 11. *p < 0.04, ***p < 0.0001 log-rank, Mantel–Cox test. Mice infected with 2 × 106 PFU VSV were bled at the indicated time points, and the expansion of VSV-NP–specific CTL (D) and VSV-neutralizing serum Abs (E) was monitored (n ≥ 6). Horizontal bars indicate means ± SEM of individually evaluated mice (one-tailed Mann–Whitney U test). All data shown are pooled from two to three independently performed experiments.

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To next address whether redundant TLR and RLH signaling would suffice to protect against VSV infection or whether an additional mechanism was needed, we intercrossed MyTr−/− and Ca−/− mice to obtain MyTrCa−/− mice devoid of TLR and RLH signaling. MyTrCa−/− mice were as sensitive to VSV infection as IFNAR−/− mice (Fig. 1A, 1B), and even at low-dose infection with 2 × 102 PFU, MyTrCa−/− mice died within 2 d with similar kinetics as IFNAR−/− mice (Fig. 1C). One day postinfection with 2 × 106 PFU VSV, neither in WT nor MyTr−/− mice were virus titers detectable in the periphery or in the CNS (Fig. 2A, 2B), whereas Ca−/− mice displayed low viral burden in spleen and liver (Fig. 2A). In contrast, IFNAR−/− and MyTrCa−/− mice showed elevated virus titers in all organs tested (Fig. 2A, 2B). These results demonstrated that in addition to TLR and RLH signaling, no other mechanism was needed to trigger protection against VSV infection.

FIGURE 2.

VSV-infected MyTrCa−/− mice do not produce serum IFN-I and show enhanced virus titers in tissues. WT, IFNAR−/−, MyTr−/−, Ca−/−, and MyTrCa−/− mice were i.v. infected with 2 × 106 PFU VSV, and 24 h later, virus titers were determined in spleen, liver, and lung (A) or in olfactory bulb, cerebrum, cerebellum, brain stem, and spinal cord (B) (n ≥ 3). Blood samples were drawn to determine serum concentrations of IFN-α (n ≥ 7) (C) and IFN-β (n ≥ 5) (D) by ELISA methods. All data presented are pooled from two to three independently performed experiments, or one representative experiment out of three similar ones is shown. Horizontal bars indicate means ± SEM. *p ≤ 0.05, ***p < 0.0001 one-tailed Mann–Whitney U test.

FIGURE 2.

VSV-infected MyTrCa−/− mice do not produce serum IFN-I and show enhanced virus titers in tissues. WT, IFNAR−/−, MyTr−/−, Ca−/−, and MyTrCa−/− mice were i.v. infected with 2 × 106 PFU VSV, and 24 h later, virus titers were determined in spleen, liver, and lung (A) or in olfactory bulb, cerebrum, cerebellum, brain stem, and spinal cord (B) (n ≥ 3). Blood samples were drawn to determine serum concentrations of IFN-α (n ≥ 7) (C) and IFN-β (n ≥ 5) (D) by ELISA methods. All data presented are pooled from two to three independently performed experiments, or one representative experiment out of three similar ones is shown. Horizontal bars indicate means ± SEM. *p ≤ 0.05, ***p < 0.0001 one-tailed Mann–Whitney U test.

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As in VSV-infected MyTrCa−/− mice survival kinetics and viral loads were reminiscent of IFNAR−/− mice, we hypothesized that in the absence of TLR and RLH signaling, VSV infection would not trigger protective IFN-I responses. To study this, IFN-α and IFN-β serum protein levels were determined 24 h following i.v. infection of mice with 2 × 106 PFU VSV. Although WT, MyTr−/− and Ca−/− mice produced significant levels of IFN-α, in the serum of IFNAR−/− mice reduced IFN-α and in MyTrCa−/− mice, no IFN-α was detected (Fig. 2C). Interestingly, no IFN-β was measurable in the serum of tested mice, except for IFNAR−/− mice, which showed moderately enhanced IFN-β levels (Fig. 2D). To address whether minimal IFN-β induction played a role, we next studied the particularly sensitive IFN-β reporter mouse model expressing a luciferase gene under the control of the IFN-β promoter (IFN-β+/∆β-luc) (33). These mice were bred on the MyTr−/−, Ca−/−, or MyTrCa−/− backgrounds, and 24 and 48 h after i.v. infection with 2 × 104 PFU, VSV in vivo imaging was pursued. Although IFN-β+/∆β-luc, IFN-β+/∆β-lucMyTr−/−, and IFN-β+/∆β-lucCa−/− mice displayed an overall similar IFN-β induction, IFN-β+/∆β-lucMyTrCa−/− mice showed no detectable luciferase expression (Fig. 3A, 3B). For measuring the luciferase activity directly in the organs of all genotypes, tissues were prepared following treatment with an enhanced infection dose of 2 × 106 PFU. The analysis revealed that IFN-β+/∆β-luc and IFN-β+/∆β-lucMyTr−/− mice showed similarly induced luciferase activity in spleen, cervical LN (Fig. 3C), inguinal LN (Supplemental Fig. 1A), and lumbar LN (Supplemental Fig. 1B), whereas IFN-β+/∆β-lucCa−/− mice showed even slightly enhanced luciferase activity in these tissues (Fig. 3C, Supplemental Fig. 1A, 1B). Most importantly, also under conditions of enhanced virus infection dose, VSV-infected IFN-β+/∆β-lucMyTrCa−/− mice did not show luciferase activity in the tested tissues (Fig. 3C, Supplemental Fig. 1A, 1B).

FIGURE 3.

VSV-infected MyTrCa−/− mice do not show local IFN-β induction. (A) WT, MyTr−/−, Ca−/−, and MyTrCa−/− mice carrying an IFN-β∆β-luc allele were infected with 2 × 104 PFU VSV, and 24 and 48 h later, luciferin was i.v. injected, and luciferase activity was determined by in vivo imaging. The ventral and lateral views of three mice per group of one experiment out of three similar ones are shown. (B) Spleen and cervical LN from (A) were marked as regions of interest, and light signals within the respective areas were quantified (n = 3; *p ≤ 0.05; one-tailed Mann–Whitney U test). (C) WT, MyTr−/−, Ca−/−, and MyTrCa−/− mice carrying an IFN-β∆β-luc allele were infected with 2 × 106 PFU VSV, spleen and cervical LN were prepared 24 hpi, and luciferin activity was determined in vitro from tissue homogenates. n ≥ 4. **p ≤ 0.007 one-tailed Mann–Whitney U test. (D) Mice treated as in (C) were sacrificed 24 h postinfection, cervical LN and spleen were prepared, and mRNA induction of ISG 15 and 2’,5′ OAS was measured by qRT-PCR n ≥ 3. *p ≤ 0.0286, **p ≤ 0.0021, ***p ≤ 0.0008 one-tailed Mann–Whitney U test.

FIGURE 3.

VSV-infected MyTrCa−/− mice do not show local IFN-β induction. (A) WT, MyTr−/−, Ca−/−, and MyTrCa−/− mice carrying an IFN-β∆β-luc allele were infected with 2 × 104 PFU VSV, and 24 and 48 h later, luciferin was i.v. injected, and luciferase activity was determined by in vivo imaging. The ventral and lateral views of three mice per group of one experiment out of three similar ones are shown. (B) Spleen and cervical LN from (A) were marked as regions of interest, and light signals within the respective areas were quantified (n = 3; *p ≤ 0.05; one-tailed Mann–Whitney U test). (C) WT, MyTr−/−, Ca−/−, and MyTrCa−/− mice carrying an IFN-β∆β-luc allele were infected with 2 × 106 PFU VSV, spleen and cervical LN were prepared 24 hpi, and luciferin activity was determined in vitro from tissue homogenates. n ≥ 4. **p ≤ 0.007 one-tailed Mann–Whitney U test. (D) Mice treated as in (C) were sacrificed 24 h postinfection, cervical LN and spleen were prepared, and mRNA induction of ISG 15 and 2’,5′ OAS was measured by qRT-PCR n ≥ 3. *p ≤ 0.0286, **p ≤ 0.0021, ***p ≤ 0.0008 one-tailed Mann–Whitney U test.

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Because VSV-infected MyTrCa−/− mice did not mount IFN-α serum responses and also did not show local IFN-β induction, we next studied local IFNAR triggering by qRT-PCR analysis of ISG 24 h after VSV infection with 2 × 106 PFU. Although spleen and LN of WT, MyTr−/−, and Ca−/− mice showed induction of ISG 15 and 2′,5′ OAS, in MyTrCa−/− mice as well as in IFNAR−/− mice, no such induction was detected (Fig. 3D, Supplemental Fig. 1C, 1D). Collectively, we interpreted the absence of serum IFN-α, lack of IFN-β induction, and deficiency of ISG stimulation that upon VSV challenge of MyTrCa−/− mice, no IFN-I responses were induced.

To verify that TLR/RLH ablation did not affect IFNAR signaling, BM-mDC generated from WT, IFNAR−/−, MyTr−/−, Ca−/−, and MyTrCa−/− mice were treated with recombinant murine IFN-β (rIFN-β), and the induction of ISG was determined. Except for IFNAR−/− BM-mDC, BM-mDC of the other genotypes tested showed similar ISG 15 mRNA induction (Fig. 4A). To address whether a comparable ISG induction also conferred an equivalent antiviral state, BM-mDC generated from WT, MyTr−/−, Ca−/−, and MyTrCa−/− mice were treated with serially diluted rIFN-β, then infected with VSV-eGFP, and following incubation for 18 h, GFP expression of infected cells was measured by FACS analysis. Although IFNAR−/− BM-mDC did not show antiviral effects, following incubation with ≥4 U concentrations of rIFN-β, cells from all other genotypes tested were protected against VSV infection (Fig. 4B). These results indicated that MyTrCa−/− BM-mDC retained a similar sensitivity for IFNAR triggering as WT BM-mDC.

FIGURE 4.

IFN-I treatment confers antiviral effects in MyTrCa−/− BM-mDC and protects VSV-infected MyTrCa−/− mice. (A) BM-mDC were generated from WT, IFNAR−/−, MyTr−/−, Ca−/−, and MyTrCa−/− mice and treated with 400 U murine rIFN-β. After the indicated time, cells were harvested, and RNA was purified and examined by qRT-PCR for the induction of ISG 15 (n = 3). (B) BM-mDC of the indicated genotypes were treated for 2 h with the specified concentrations of murine rIFN-β and then infected with VSV-eGFP at a multiplicity of infection of 0.01. After 18 h incubation, eGFP expression of BM-mDC was monitored by flow cytometry. n = 6. *p ≤ 0.05 one-tailed Mann–Whitney U test. The data shown are pooled from two independently performed experiments, or one representative experiment out of three similar ones is shown. Horizontal bars indicate means ± SEM. (C) WT controls (circles) and MyTrCa−/− mice (diamonds) were s.c. treated at the indicated time points with PBS (white), 2 ng IFN-containing serum (red), or 1 × 105 U of rIFN-α (red with cross) and i.v. infected with 2 × 104 PFU VSV. Survival was monitored daily.

FIGURE 4.

IFN-I treatment confers antiviral effects in MyTrCa−/− BM-mDC and protects VSV-infected MyTrCa−/− mice. (A) BM-mDC were generated from WT, IFNAR−/−, MyTr−/−, Ca−/−, and MyTrCa−/− mice and treated with 400 U murine rIFN-β. After the indicated time, cells were harvested, and RNA was purified and examined by qRT-PCR for the induction of ISG 15 (n = 3). (B) BM-mDC of the indicated genotypes were treated for 2 h with the specified concentrations of murine rIFN-β and then infected with VSV-eGFP at a multiplicity of infection of 0.01. After 18 h incubation, eGFP expression of BM-mDC was monitored by flow cytometry. n = 6. *p ≤ 0.05 one-tailed Mann–Whitney U test. The data shown are pooled from two independently performed experiments, or one representative experiment out of three similar ones is shown. Horizontal bars indicate means ± SEM. (C) WT controls (circles) and MyTrCa−/− mice (diamonds) were s.c. treated at the indicated time points with PBS (white), 2 ng IFN-containing serum (red), or 1 × 105 U of rIFN-α (red with cross) and i.v. infected with 2 × 104 PFU VSV. Survival was monitored daily.

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To next address the IFNAR responsiveness of MyTrCa−/− mice in vivo, WT controls and MyTrCa−/− mice were i.v. challenged with 2 × 104 PFU VSV, and 4 and 8 h later, the animals were s.c. treated with 50 μl serum that was collected from VSV-M2–challenged WT mice and contained ∼2 ng IFN-I. Serum treatment prolonged survival by 1 d when compared with VSV-infected MyTrCa−/− mice treated with PBS (Fig. 4C). Because previous studies showed that recombinant human IFN-αB/D (rIFN-α) exhibited strong reactivity on mouse cells (41), we next s.c. applied 1 × 105 U of rIFN-α 4 and 8 h postinfection. Interestingly, under such conditions, MyTrCa−/− mice were fully protected (Fig. 4C).

Besides the fact that these experiments verified that not only MyTrCa−/− cells but also MyTrCa−/− mice retained IFN-I sensitivity, it also showed that high-dose treatment with rIFN-α conferred protection against VSV infection. These characteristics qualified MyTrCa−/− mice as a model for adoptive transfer studies to test the contribution of selected immune cell subsets to promote protection against lethal VSV infection.

To study whether DC subsets contribute to protection against lethal VSV infection, BM cells isolated from WT mice were differentiated in medium supplemented with either Flt3-L or GM-CSF to obtain cultures that contained ∼30% Siglec-H+B220+ pDC or ∼96% CD11c+ mDC, respectively. Upon adoptive transfer of 1 × 107 Siglec-H+ pDC purified from WT BM-pDC cultures and subsequent VSV challenge, MyTrCa−/− mice showed improved survival and died 4–6 d later than controls that did not receive pDC (Fig. 5A). On the contrary, adoptive transfer of BM-mDC cultures was not effective (Fig. 5A). In addition, MyTrCa−/− mice carrying adoptively transferred pDC mounted increased neutralizing Ab responses when compared with pDC-treated WT mice (Fig. 5B). At the time of death, VSV-infected MyTrCa−/− mice carrying WT pDC showed no or very low viral titers in peripheral organs, whereas high viral loads were detected within the CNS (Fig. 5C). In contrast, MyTrCa−/− mice treated with BM-mDC showed high virus burden in all tissues tested (Fig. 5C). To study the site where upon VSV infection of MyTrCa−/− mice carrying adoptively transferred pDC IFN-I responses were induced, BM-pDC were generated from IFN-β+/∆β-luc mice, and 1 × 107 purified IFN-β+/∆β-luc BM-pDC were adoptively transferred to MyTrCa−/− mice. After VSV infection, such mice showed induction of luciferase activity at similar sites as in the studies with IFN-β+/∆β-luc mice (compare Figs. 3A and 5D). No IFN-β induction was measured upon VSV infection of WT and MyTrCa−/− mice adoptively transferred with IFN-β+/∆β-luc BM-derived mDC (Fig. 5D). These results indicated that in MyTrCa−/− mice carrying TLR/RLH-competent pDC VSV infection triggered IFN-β induction at similar sites as observed in TLR/RLH-competent animals, whereas mDC did not.

FIGURE 5.

Upon VSV infection, MyTrCa−/− mice adoptively transferred with WT pDC show prolonged survival. (A) Total of 1 × 107 purified Siglec-H+ BM-pDC or 1 × 107 CD11c+ BM-mDC was adoptively transferred to WT controls or MyTrCa−/− mice, and the following day, the mice were i.v. challenged with 2 × 104 PFU VSV. Survival of the mice was monitored twice daily (n = 5). Data shown are representative of three independent experiments. (B) Neutralizing IgM/IgG or IgG serum Ab responses were analyzed from VSV-infected WT and MyTrCa−/− mice treated with BM-pDC (n = 6). (C) At the time of death, organs of mice treated with BM-mDC (blue) or BM-pDC (red) were isolated, and viral loads were determined in peripheral organs (spleen [Sp], liver [Li], and lung [Lu]) and in the CNS (olfactory bulb [OB], cerebrum [CR], cerebellum [CB], brain stem [BS], and spinal cord [SC]). Data were analyzed with one-tailed Mann–Whitney U test. n = 5 mice per group. **p ≤ 0.006. (D) Total of 1 × 107 purified BM-pDC or BM-mDC generated from IFN-β+/∆β-luc mice were adoptively transferred to WT and MyTrCa−/− mice. The following day, mice were infected with 2 × 104 PFU VSV, and IFN-β induction was analyzed by in vivo imaging at the indicated time points. One representative mouse out of six similar ones is shown. Bars indicate mean ± SEM.

FIGURE 5.

Upon VSV infection, MyTrCa−/− mice adoptively transferred with WT pDC show prolonged survival. (A) Total of 1 × 107 purified Siglec-H+ BM-pDC or 1 × 107 CD11c+ BM-mDC was adoptively transferred to WT controls or MyTrCa−/− mice, and the following day, the mice were i.v. challenged with 2 × 104 PFU VSV. Survival of the mice was monitored twice daily (n = 5). Data shown are representative of three independent experiments. (B) Neutralizing IgM/IgG or IgG serum Ab responses were analyzed from VSV-infected WT and MyTrCa−/− mice treated with BM-pDC (n = 6). (C) At the time of death, organs of mice treated with BM-mDC (blue) or BM-pDC (red) were isolated, and viral loads were determined in peripheral organs (spleen [Sp], liver [Li], and lung [Lu]) and in the CNS (olfactory bulb [OB], cerebrum [CR], cerebellum [CB], brain stem [BS], and spinal cord [SC]). Data were analyzed with one-tailed Mann–Whitney U test. n = 5 mice per group. **p ≤ 0.006. (D) Total of 1 × 107 purified BM-pDC or BM-mDC generated from IFN-β+/∆β-luc mice were adoptively transferred to WT and MyTrCa−/− mice. The following day, mice were infected with 2 × 104 PFU VSV, and IFN-β induction was analyzed by in vivo imaging at the indicated time points. One representative mouse out of six similar ones is shown. Bars indicate mean ± SEM.

Close modal

To quantify the number of pDC needed to confer protective effects, 1 × 107, 1 × 106, or 1 × 105 pDC purified from WT BM-pDC were adoptively transferred to MyTrCa−/− mice. Similar to the experiments above, upon i.v. infection with 2 × 104 PFU VSV, mice treated with 1 × 107 purified pDC showed prolonged survival of 3–5 d, whereas adoptive transfer of 1 × 106 pDC conferred 1–3 d prolonged survival, and adoptive transfer of 1 × 105 pDC was not effective (Fig. 6A). To address whether in vivo–differentiated pDC similarly conferred protection, WT mice were s.c. treated with 1 × 106 Flt3-L–expressing B16 melanoma cells that promoted enhanced development of endogenous pDC (38, 39). After 10–14 d, mice developed tumors with a diameter of ∼1.5 cm and carried between 10 and 15% Siglec-H+B220+ pDC in the spleen compared with <0.1% pDC in untreated mice. Upon adoptive transfer of 1 × 107 of such ex vivo–isolated and purified pDC, VSV-infected MyTrCa−/− mice showed a similarly improved survival as observed in experiments with purified pDC derived from in vitro–differentiated BM-pDC cultures (Fig. 6B). To assure that adoptively transferred pDC would be triggered by VSV in a TLR7-dependent manner, pDC were generated from BM of TLR7−/− mice. Upon in vitro stimulation with CpG, such cells showed induction of CD86 and CD69, whereas upon treatment with R848 as a direct trigger of TLR7, no upregulation of activation markers was observed (Supplemental Fig. 2). Following adoptive transfer of 1 × 107 TLR7−/− or WT pDC purified from BM-pDC cultures, only WT pDC but not TLR7−/− pDC conferred prolonged survival of VSV-infected MyTrCa−/− mice (Fig. 6C). Collectively, these results indicated that upon VSV infection, pDC were triggered in a TLR7-dependent manner to mount rapid IFN-I responses in secondary lymphoid tissues that protected peripheral organs, but which did not suffice to control virus replication within the CNS.

FIGURE 6.

Increasing numbers of adoptively transferred TLR7-competent pDC enhance protection of MyTrCa−/− mice. (A) Upon adoptive transfer of 1 × 107, 1 × 106, or 1 × 105 purified WT BM-pDC, MyTrCa−/− mice were challenged with 2 × 104 PFU VSV, and survival was monitored (n ≥ 5). (B) pDC were purified from spleen of WT mice treated with Flt3-L–expressing melanoma cells, and 1 × 107 of such pDC was adoptively transferred to MyTrCa−/− mice and WT controls (n ≥ 6). The following day, mice were infected with 2 × 104 PFU VSV, and survival was monitored. (C) Total of 1 × 107 purified BM-pDC generated from TLR7−/− mice (n ≥ 5) was adoptively transferred to WT and MyTrCa−/− mice. The following day, mice were infected with 2 × 104 PFU VSV, and survival was monitored. **p ≤ 0.0082, ***p < 0.0001 log-rank, Mantel–Cox test.

FIGURE 6.

Increasing numbers of adoptively transferred TLR7-competent pDC enhance protection of MyTrCa−/− mice. (A) Upon adoptive transfer of 1 × 107, 1 × 106, or 1 × 105 purified WT BM-pDC, MyTrCa−/− mice were challenged with 2 × 104 PFU VSV, and survival was monitored (n ≥ 5). (B) pDC were purified from spleen of WT mice treated with Flt3-L–expressing melanoma cells, and 1 × 107 of such pDC was adoptively transferred to MyTrCa−/− mice and WT controls (n ≥ 6). The following day, mice were infected with 2 × 104 PFU VSV, and survival was monitored. (C) Total of 1 × 107 purified BM-pDC generated from TLR7−/− mice (n ≥ 5) was adoptively transferred to WT and MyTrCa−/− mice. The following day, mice were infected with 2 × 104 PFU VSV, and survival was monitored. **p ≤ 0.0082, ***p < 0.0001 log-rank, Mantel–Cox test.

Close modal

To address whether also other immune cells or some resident tissue cell subsets were involved in mounting protective IFN-I responses, BM chimeric mice were generated in which either radiosensitive immune cells (MyTrCa−/− > WT) or radioresistant stroma cells (WT > MyTrCa−/−) were refractory to VSV-mediated induction of IFN-I responses. Upon VSV challenge, MyTrCa−/− >WT and WT > MyTrCa−/− showed intermediate sensitivity, whereas WT > WT mice showed only minor lethality and MyTrCa−/− >MyTrCa−/− mice were as sensitive to infection as the original MyTrCa−/− mice (Fig. 7A). WT > WT controls, MyTrCa−/− >WT and WT > MyTrCa−/− mice showed a similar induction of VSV-specific CTL responses after 6 d (Fig. 7B), as well as high IgM- and IgG-neutralizing Ab responses after 8 d of infection (Fig. 7C), indicating that adaptive immunity was normally induced. Moreover, MyTrCa−/− >WT mice showed even elevated CTL responses and enhanced IgG serum levels (Fig. 7B, 7C). Interestingly, adoptive transfer of WT pDC into MyTrCa−/− >WT mice did not significantly prolong survival upon VSV infection (data not shown). These experiments revealed that for complete protection against VSV infection, IFN-I responses of pDC and presumably other immune cells as well as of radioresistant stroma cells were needed.

FIGURE 7.

TLR/RLH signaling of radioresistant and radiosensitive cells is needed to protect against lethal VSV infection. (A) WT and MyTrCa−/− mice were irradiated with 9 Gy, and 1 d later, mice were injected with 1 × 107 BM cells of WT or MyTrCa−/− mice. Eight weeks after the reconstitution, WT > WT, MyTrCa−/− >MyTrCa−/−, WT > MyTrCa−/−, and MyTrCa−/− > WT mice were i.v. challenged with 2 × 104 PFU VSV, and survival was monitored twice daily. n ≥ 11. **p ≤ 0.0069 log-rank, Mantel–Cox test. (B) On day 6 postinfection, blood samples were drawn, and NP-specific CD8+ T cells were determined by pentamer staining (n ≥ 5). (C) Neutralizing Ab responses were analyzed from serum 8 days postinfection (n ≥ 7). Horizontal bars indicate means ± SEM. **p ≤ 0.007 one-tailed Mann–Whitney U test. Data shown are pooled from two to three independently performed experiments.

FIGURE 7.

TLR/RLH signaling of radioresistant and radiosensitive cells is needed to protect against lethal VSV infection. (A) WT and MyTrCa−/− mice were irradiated with 9 Gy, and 1 d later, mice were injected with 1 × 107 BM cells of WT or MyTrCa−/− mice. Eight weeks after the reconstitution, WT > WT, MyTrCa−/− >MyTrCa−/−, WT > MyTrCa−/−, and MyTrCa−/− > WT mice were i.v. challenged with 2 × 104 PFU VSV, and survival was monitored twice daily. n ≥ 11. **p ≤ 0.0069 log-rank, Mantel–Cox test. (B) On day 6 postinfection, blood samples were drawn, and NP-specific CD8+ T cells were determined by pentamer staining (n ≥ 5). (C) Neutralizing Ab responses were analyzed from serum 8 days postinfection (n ≥ 7). Horizontal bars indicate means ± SEM. **p ≤ 0.007 one-tailed Mann–Whitney U test. Data shown are pooled from two to three independently performed experiments.

Close modal

In this study, we addressed the cooperation of TLR and RLH signaling in the induction of VSV-triggered IFN-I responses. To this end, we intercrossed MyTr−/− and Ca−/− mice to obtain MyTrCa−/− mice. Such mice were as sensitive to lethal VSV infection as IFNAR−/− mice and did not mount protective IFN-I. This observation was surprising because MyTr−/− and Ca−/− mice were as resistant to intermediate- and low-dose VSV infection as WT mice. These data indicated that concomitant TLR and RLH signaling was necessary for efficient protection against lethal VSV infection. This mouse model allowed addressing the biological role of different immune cell subsets in virus recognition and their capacity in mounting protective IFN-I responses. Transferred pDC significantly prolonged survival of VSV infection and induced an antiviral state in peripheral organs, whereas virus still entered the CNS and finally killed the host. BM chimeric mice in which either radiosensitive immune cells or radio-resistant stroma cells were TLR/RLH deficient showed an intermediate sensitivity to lethal VSV infection. Collectively, these data showed that pDC alone conferred swift IFN-I responses, which controlled virus replication in peripheral tissues, whereas full protection depended on IFN-I production also of radioresistant cells and presumably of other immune cells.

Although in serum of VSV-infected WT mice high levels of IFN-α were induced, in VSV-infected MyTrCa−/− mice, no IFN-α was detected. Interestingly, IFN-β was neither discovered in the serum of VSV-infected WT nor MyTrCa−/− mice, whereas IFNAR−/− mice mounted slightly enhanced IFN-β responses. The latter observation can be explained by continued PRR triggering in absence of IFNAR feedback or by the lack of IFN-β uptake, as previously suggested in the context of Listeria infection (47). Nevertheless, local IFN-β induction was found in cervical LN and spleen of VSV-infected IFN-β+/∆β-luc reporter mice, which was absent in IFN-β+/∆β-lucMyTrCa−/− animals. To study local IFNAR triggering, ISG induction within secondary lymphoid organs was examined. Whereas spleen, cervical LN, inguinal LN, and lumbar LN of VSV-infected WT mice showed significant ISG 15 and 2′,5′ OAS mRNA induction, no such effects were detected in VSV-infected MyTrCa−/− mice. Because IFN-β+/∆β-luc reporter mice and the induction of ISG were sensitive readouts for IFN-I responses, even when elicited only locally without IFN-I appearing in the serum, we concluded that MyTrCa−/− mice did not mount IFN-I responses upon VSV challenge. Thus, concomitant TLR and RLH triggering was indispensable to promote survival of VSV infection of mice, whereas other additional mechanisms did not seem to be critically involved.

To verify that in MyTrCa−/− mice IFN-I–mediated IFNAR signaling was not perturbed, we generated MyTrCa−/− BM-mDC, treated them with rIFN-β, and determined the induction of ISG 15 and of antiviral effects. Indeed, in these assays, WT and MyTrCa−/− BM-mDC showed very similar antiviral effects, whereas ISG 15 induction was even elevated in MyTrCa−/− BM-mDC. These data indicated that IFN-β–mediated IFNAR signaling was not impaired by MyTrCa ablation. Various studies showed before that during activation of multiple PRR pathways, antagonism between single signaling pathways may shape the overall response (27, 48). Thus, it can be speculated that also basal TLR and RLH signaling modulated ISG mRNA, which would explain enhanced ISG 15 mRNA induction upon IFN-β treatment of TLR- and RLH-deficient cells. To study in vivo whether MyTrCa−/− mice retained IFN-I sensitivity, mice were treated with IFN-I–containing serum or recombinant human IFN-α, which in previous studies was shown to be active in the murine system (41). To recapitulate kinetics of endogenous IFN-I responses, treatment with IFN-I–containing serum or rIFN-α was initiated 4 hpi and repeated at 8 hpi. Although treatment with serum containing ∼2 ng IFN-α prolonged survival of VSV-infected MyTrCa−/− mice by 1 d, treatment with 1 × 105 U rIFN-α conferred quantitative protection of MyTrCa−/− mice. These results indicated that: 1) the short t1/2 of serum IFN-I was not sufficient to mediate significant protective effects; 2) comparably high-level IFN-I responses were needed to protect MyTrCa−/− mice; and 3) in MyTrCa−/− mice, IFNAR signaling was preserved in all relevant cell subsets. The observation that IFN-I sensitivity of MyTrCa−/− mice was retained qualified MyTrCa−/− mice as a suitable model to study the in vivo function of WT immune cell subsets, such as DC, in an environment that is completely blinded for the innate recognition of VSV infection.

pDC have been described as main IFN-I producers in many different virus infections (1719). To study their role in a gain-of-function setting, WT pDC isolated from BM-pDC cultures were adoptively transferred to MyTrCa−/− mice. Upon VSV infection pDC carrying MyTrCa−/− mice showed prolonged survival by 4–6 d, whereas transfer of WT BM-mDC did not have such effect. Notably, a similarly prolonged survival of VSV-infected MyTrCa−/− mice carrying pDC isolated from BM-pDC cultures and MyTrCa−/− mice carrying pDC directly isolated from mice was observed. Although the ex vivo–isolated pDC were derived from mice carrying an Flt3-L–expressing tumor and that therefore showed a significantly enhanced prevalence of pDC in the spleen, such pDC developed in an overall normal environment. Thus, the capacity of pDC to mount life-extending IFN responses in VSV-infected MyTrCa−/− mice was not associated with the in vitro differentiation protocol of pDC but instead with the identity of the transferred DC subset. Additionally, adoptive transfer of reporter transgenic IFN-β+/∆β-luc pDC revealed that 24 h after VSV infection, IFN-β–expressing pDC primarily located to spleen as well as axial, inguinal, and cervical LN. Interestingly, MyTrCa−/− mice carrying IFN-β+/∆β-luc pDC showed even elevated IFN-β induction when compared with IFN-β+/∆β-luc pDC carrying WT controls, presumably due to the enhanced virus burden in MyTrCa−/− mice. This is explained by the fact that in MyTrCa−/− mice, only IFN-β+/∆β-luc pDC were able to produce protective IFN-I, whereas in WT controls, endogenous pDC and other cells also contributed to early IFN-I. Additionally, pDC carrying MyTrCa−/− mice mounted higher Ab responses compared with WT controls. Early after VSV challenge, subcapsular sinus macrophages located in secondary lymphoid organs are among initially infected cells (4951). In MyTrCa−/− mice, these infected cells are not able to restrict VSV infection. This would lead to higher viral titers early after VSV challenge of MyTrCa−/− mice. As a consequence, enhanced Ag concentrations would be present inducing more vigorous Ab responses. In conclusion, pDC-derived IFN-I conferred an antiviral state in peripheral organs and high levels of neutralizing Abs were induced, whereas virus still entered the CNS and eventually killed the host. As expected, VSV-mediated triggering of protective IFN-I responses mounted by adoptively transferred pDC was TLR7 dependent. Although previous studies showed that mice in which pDC were selectively depleted showed enhanced susceptibility to HSV infection (52), pathogenesis of VSV infection was not affected by pDC depletion, except for slightly decreased IFN-I serum levels at early time points (20). These data were interpreted that after VSV infection in addition to pDC other cells mounted protective IFN-I responses which compensated for the loss of pDC. Instead of relying on a loss of function approach, we applied a gain of function model (i.e., adoptive transfer of WT pDC into TLR/RLH-deficient mice), which revealed important functions of pDC in anti-VSV defense.

Our experiments with BM chimeric mice indicated that stroma cells critically contributed to VSV induced IFN-I responses. Already former studies implied that similar to immune cells also stroma might be triggered to express cytokines, when appropriately stimulated (5355). Different stroma cell subsets and LN-resident macrophages, which before have been discussed to be comparatively radioresistant (56), may express different PRR such as TLR3 and TLR4 (53). Nevertheless, little is known about the role of radioresistant cells in acute virus infection. Our data with BM chimeric mice, in which either radiosensitive immune cells or radioresistant stroma cells were TLR/RLH ablated, revealed that both immune cells as well as radioresistant stroma were needed to confer efficient protection against VSV infection. Interestingly, adoptive transfer of WT pDC into chimeric mice, in which immune cells were TLR/RLH deficient, whereas radioresistant cells were TLR/RLH competent, did not show prolonged survival upon VSV infection, suggesting that in addition to pDC other immune cells significantly contributed to protective IFN-I responses.

Because our data clearly demonstrated that interaction between TLR and RLH signaling was critically needed to confer protection against VSV infection, the question arose of how mechanistically this interaction could be envisaged. As published previously, pDC seem to be preferentially triggered by endosomal TLR, whereas mDC and many other cell subsets are primarily triggered by cytosolic RLH to mount IFN-I responses (15). Thus, one explanation for redundant TLR and RLH triggering needed for protection against VSV infection is that depending on the involved cell subset, either TLR or RLH plays a critical role for the induction of protective IFN-I. This may not only apply for immune cell subsets but also for radioresistant cells. A recent report showed that upon TLR triggering of pDC RIG-I expression was upregulated independent of IFNAR stimulation (57). This example indicated another level of TLR and RLH interaction (i.e., that in a first phase, cells are TLR-dependently triggered to become sensitive for RLH triggering). Currently, it is difficult to estimate the in vivo relevance of this mechanism for protection against VSV. Nevertheless, it is certainly possible that in particular on the level of radioresistant stroma cells, this mechanism might be important.

In conclusion, we found that MyTrCa−/− mice were not triggered upon VSV infection to mount protective IFN-I and that such mice succumbed to infection with similar kinetics as IFNAR−/− mice. These data indicated that besides TLR and RLH signaling, no other signaling platforms were critically involved in VSV recognition. Because MyTrCa−/− mice retained IFNAR sensitivity, these mice served as an ideal model to more precisely dissect the role of protective IFN-I responses in viral pathogenesis. We found that rapid IFN-I responses mounted by pDC induced an antiviral state in peripheral organs, whereas IFN-I production of radiosensitive immune cells and of radioresistant stroma was needed to fully protect mice from virus entering the CNS.

We thank Dr. E. Grabski and Dr. M. Bahgat for critically reading the manuscript and Dr. T. Frenz for support and discussion. Flt3-L–producing B16 tumor cells were a gift from Prof. M. Brinkmann.

This work was supported by the German Research Council Grant SFB 854 B15 (to U.K.) and Topic 2 of the Programme Infection Research of the Helmholtz Centre for Infection Research. J.S. holds a stipend from the Lichtenberg Ph.D. program “Dynamics of Host Pathogen Interactions” and was supported by the Center for Infection Biology, which is part of the Hannover Biomedical Research School of the Hannover Medical School.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • BM

    bone marrow

  •  
  • Cardif

    caspase activation and recruitment domain adaptor inducing IFN-β

  •  
  • eGFP

    enhanced GFP

  •  
  • Flt3-L

    Flt3 ligand

  •  
  • hpi

    h postinfection

  •  
  • IFN-I

    type I IFN

  •  
  • IFNAR

    type I IFN receptor

  •  
  • ISG

    IFN-stimulated gene

  •  
  • LN

    lymph node

  •  
  • mDC

    myeloid dendritic cell

  •  
  • My

    MyD88

  •  
  • MyTrCa−/−

    MyD88−/−Trif−/−Cardif−/−

  •  
  • NP

    nucleoprotein

  •  
  • 2′,5′ OAS

    2′,5′ oligoadenylate synthetase

  •  
  • pDC

    plasmacytoid dendritic cell

  •  
  • PRR

    pattern recognition receptor

  •  
  • qRT-PCR

    quantitative real-time PCR

  •  
  • rIFN-α

    recombinant human IFN-αB/D

  •  
  • rIFN-β

    recombinant murine IFN-β

  •  
  • RIG-I

    retinoic acid–inducible gene I

  •  
  • RLH

    retinoic acid–inducible gene I–like helicase

  •  
  • STING

    stimulator of IFN gene

  •  
  • Trif

    Toll/IL-1R domain–containing adapter inducing IFN-β

  •  
  • VSV

    vesicular stomatitis virus

  •  
  • WT

    wild-type.

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The authors have no financial conflicts of interest.

Supplementary data