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. 2004 Aug 3;101(31):11416-21.
doi: 10.1073/pnas.0403555101. Epub 2004 Jul 22.

Herpes simplex virus type-1 induces IFN-alpha production via Toll-like receptor 9-dependent and -independent pathways

Hubertus Hochrein  1 , Beatrix Schlatter, Meredith O'Keeffe, Cornelia Wagner, Frank Schmitz, Matthias Schiemann, Stefan Bauer, Mark Suter, Hermann Wagner
Affiliations

Herpes simplex virus type-1 induces IFN-alpha production via Toll-like receptor 9-dependent and -independent pathways

Hubertus Hochrein et al. Proc Natl Acad Sci U S A. .

Abstract

Type I IFN production in response to the DNA virus herpes simplex virus type-1 (HSV-1) is essential in controlling viral replication. We investigated whether plasmacytoid dendritic cells (pDC) were the major tissue source of IFN-alpha, and whether the production of IFN-alpha in response to HSV-1 depended on Toll-like receptor 9 (TLR9). Total spleen cells or bone marrow (BM) cells, or fractions thereof, including highly purified pDC, from WT, TLR9, and MyD88 knockout mice were stimulated with known ligands for TLR9 or active HSV-1. pDC freshly isolated from both spleen and BM were the major source of IFN-alpha in response to oligodeoxynucleotides containing CpG motifs, but in response to HSV-1 the majority of IFN-alpha was produced by other cell types. Moreover, IFN-alpha production by non-pDC was independent of TLR9. The tissue source determined whether pDC responded to HSV-1 in a strictly TLR9-dependent fashion. Freshly isolated BM pDC or pDC derived from culture of BM precursors with FMS-like tyrosine kinase-3 ligand, produced IFN-alpha in the absence of functional TLR9, whereas spleen pDC did not. Heat treatment of HSV-1 abolished maturation and IFN-alpha production from all TLR9-deficient DC but not WT DC. Thus pDC and non-pDC produce IFN-alpha in response to HSV-1 via both TLR9-independent and -dependent pathways.

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Figures

Fig. 2.
Fig. 2.
Total, enriched, or highly purified CD11c+ and CD11c– cells from BM are activated in response to HSV-1 to produce IFN-α, even in the absence of TLR9. WT total BM cells [2.5 × 106 per ml, in the presence of IL-3 and GM-CSF (A)] or CD45RA-enriched [2.5 × 106 per ml (B)], B220-enriched [2.5 × 106 per ml (C)], or CD19-enriched [1 × 106 per ml (D)] WT BM cells were stimulated with HSV-1or CpG-2216. B220-enriched (E) or CD11c-enriched (F) BM cells (2.5 × 106 per ml) from WT or TLR9 KO mice were stimulated with HSV-1 or CpG-2216 in the presence of IL-3 and GM-CSF. Sorted CD11c+B220highCD45RAhigh pDC (G) or CD11c+B220–CD45RA– non-pDC (H) cells (2.5 × 105 per ml) were stimulated with either 107 pfu/ml [multiplicity of infection (MOI) 40] or 106 pfu/ml (MOI 4) of HSV-1 or CpG-2216, in the presence of IL-3 and GM-CSF. All supernatants were tested by ELISA for IFN-α production. One representative experiment of at least three is shown. Error bars represent the range of duplicate samples; asterisks indicate that production of IFN-α was below the detection limit of the ELISA.
Fig. 3.
Fig. 3.
GM-CSF-generated DC are activated in response to HSV-1 in the absence of TLR9. GM-DC cultures (2.5 × 106 cells per ml) from WT or TLR9 KO mice were stimulated with HSV-1 or CpG-2216 and the supernatants were tested by ELISA for the production of IFN-α, IL-6, or TNF-α. Error bars represent the range of duplicate samples; asterisks indicate that cytokines were not detectable by ELISA. Data are representative of one of three replicate experiments.
Fig. 4.
Fig. 4.
M-CSF-generated macrophages are activated in response to HSV-1 in the absence of TLR9. M-CSF macrophage cultures (2.5 × 106 cells per ml) from WT or TLR9 KO mice were stimulated with HSV-1 or CpG-2216 and the supernatants were tested by ELISA for the production of IFN-α. Error bars represent the range of duplicate samples. Data are representative of one of three replicate experiments.
Fig. 5.
Fig. 5.
FL-DC and CD11c+ BM cells are not activated by heat-treated HSV-1 in the absence of TLR9. (A) FL-DC (2.5 × 106 per ml) generated from WT or TLR9 KO mice were stimulated with HSV-1 (107 pfu/ml) that was either left untreated or heated to 56°C for 30 min (HSV-1-heat). Cells were stained with antibodies directed to CD40 or CD62-L. Open black histograms represent HSV-1-stimulated cells, shaded histograms represent HSV-1-heat stimulated cells, and open gray histograms represent cells in media only. (B) Supernatants were tested by ELISA for the production of IFN-α or IL-6. (C) Magnetic cell-sorting-enriched CD11c+ cells (2 × 105 per ml) purified directly from BM were stimulated with HSV-1, HSV-1-heat, or CpG-2216. Supernatants were tested by ELISA for production of IFN-α or TNF-α. Error bars represent the range of duplicate samples; asterisks indicate that cytokine production was below the detection limits of the ELISA. Data are representative of one of at least three replicate experiments.
Fig. 1.
Fig. 1.
FL-DC do not require TLR9 or MyD88 for activation in response to HSV-1. Total FL-DC (2.5 × 106 per ml) generated from WT, TLR9 KO, or MyD88 KO mice were stimulated with HSV-1 (107 pfu/ml), CpG-2216 (1 μM) or R848 (1 μg/ml). (A) The supernatants were removed and tested for IFN-α or IL-6 production by ELISA. (B) Cells were stained with antibodies directed to CD86 or CD40. (C) Total FL-DC (1 × 106 per ml) from WT or TLR9 KO mice were then stimulated for 22 h with a titration of HSV-1 from 1 × 107 – 0.16 × 106 pfu/ml [10 – 0.16 multiplicity of infection (MOI)], and the supernatants were tested by ELISA for IFN-α, TNF-α, or IL-6 production. (D) Sorted pDC and cDC (2.5 × 105 per ml) purified from FL-DC of WT or TLR9 KO mice were stimulated with HSV-1 or CpG-2216. Supernatants were tested by ELISA for IFN-α, TNF-α, or IL-6 production. (E) Sorted pDC or cDC (5 × 105 per ml) from FL-DC of WT mice were stimulated with HSV-1 in the presence or absence of IL-3 and GM-CSF and the supernatants were tested by ELISA for IFN-α. One representative experiment of at least three is shown. Error bars represent the range of duplicate samples; asterisks indicate that cytokines were not detectable by ELISA.
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