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, 79 (16), 10376-85

Early Alpha/Beta Interferon Production by Myeloid Dendritic Cells in Response to UV-inactivated Virus Requires Viral Entry and Interferon Regulatory Factor 3 but Not MyD88

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Early Alpha/Beta Interferon Production by Myeloid Dendritic Cells in Response to UV-inactivated Virus Requires Viral Entry and Interferon Regulatory Factor 3 but Not MyD88

Asa S Hidmark et al. J Virol.

Abstract

Alpha/beta interferons (IFN-alpha/beta) are key mediators of innate immunity and important modulators of adaptive immunity. The mechanisms by which IFN-alpha/beta are induced are becoming increasingly well understood. Recent studies showed that Toll-like receptors 7 and 8 expressed by plasmacytoid dendritic cells (pDCs) mediate the endosomal recognition of incoming viral RNA genomes, a process which requires myeloid differentiation factor 88 (MyD88). Here we investigate the requirements for virus-induced IFN-alpha/beta production in cultures of bone marrow-derived murine myeloid DCs (mDCs). Using recombinant Semliki Forest virus blocked at different steps in the viral life cycle, we show that replication-defective virus induced IFN-alpha/beta in mDCs while fusion-defective virus did not induce IFN-alpha/beta. The response to replication-defective virus was largely intact in MyD88-/- mDC cultures but was severely reduced in mDC cultures from mice lacking IFN regulatory factor 3. Our observations suggest that mDCs respond to incoming virus via a pathway that differs from the fusion-independent, MyD88-mediated endosomal pathway described for the induction of IFN-alpha/beta in pDCs. We propose that events during or downstream of viral fusion, but prior to replication, can activate IFN-alpha/beta in mDCs. Thus, mDCs may contribute to the antiviral response activated by the immune system at early time points after infection.

Figures

FIG. 1.
FIG. 1.
UV treatment renders rSFV replication incompetent but leaves the virus fusion competent. (A) Schematic representation of the recombinant viruses used for this study. (B) Infectious titers of nrSFV, rSFV, and UV-rSFV determined on BHK-21 cells using the equivalent of 4 × 107 IU/ml (corresponding to 100 IU/cell) for all three viruses and 10-fold dilutions of rSFV (100 to 0.01 IU/cell). EGFP expression (% infection) was measured after 24 h of incubation with virus. (C) Analysis of E1 trimer formation. MEFs were incubated with [35S]methionine-labeled rSFV or UV-rSFV for 1 hour at 0°C and were then incubated at either 0°C or 37°C for a further 20 min. The cells were washed in PBS and lysed in a buffer containing 1% NP-40. The lysates were analyzed by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. (D) MEFs were infected with equivalent amounts of nrSFV, rSFV, and UV-rSFV (corresponding to 1 IU/cell) and assayed for EGFP expression and IRF-3 localization by immunofluorescence using an anti-IRF-3 antibody. Bar, 50 μm. (E) Bioassay for IFN-α/β produced by MEFs treated with equivalent amounts of rSFV, nrSFV, and UV-rSFV (corresponding to 100 IU/cell) or with a 1:10 dilution of rSFV. *, no IFN-α/β could be detected in the sample. A sample from the rSFV-stimulated cells was preincubated with a specific anti-IFN-α/β antibody (raw data from the bioassay are shown in the inset) to show the specificity of the assay.
FIG. 2.
FIG. 2.
mDCs produce IFN-α/β and up-regulate costimulatory markers in response to rSFV and UV-rSFV. (A) Phenotype (left and center panels) and EGFP expression 24 h after the addition of rSFV (10 IU/cell) (right panel) of representative mDC cultures. (B) IFN-α/β production by mDCs 24 h after incubation with equal amounts of nrSFV, rSFV, and UV-rSFV (corresponding to 10 IU/cell of rSFV) and with rSFV diluted 10-fold and 100-fold. Control cultures were incubated with AM (see Materials and Methods). *, no IFN-α/β could be detected in the sample. (C) NH4Cl was added to the mDC cultures either 2 h before (−2 h) or 1 h after (+1 h) the addition of rSFV (10 IU/cell), and IFN-α/β was measured in the supernatant 24 h after the addition of the virus. (D) Up-regulation of CD40 and CD86 24 h after the addition of rSFV (10 IU/cell) and equivalent amounts of nrSFV and UV-SFV or a 1:1,000 dilution of rSFV. The results shown are representative of at least three experiments using independent preparations of mDCs.
FIG. 3.
FIG. 3.
rSFV and UV-rSFV induce an early systemic IFN-α/β response. IFN-α/β was measured in the sera of individual wt mice 4 h after injection with rSFV (108 IU), an equivalent amount of nrSFV or UV-SFV, or a 1:10 or 1:100 dilution of rSFV. Control mice were injected with the supernatant from mock-packaged recombinant SFV RNA (Sup). *, no IFN-α/β could be detected in the sample. Data from one representative experiment of two are shown. The UV-inactivated virus induced significantly more IFN-α/β than nrSFV or a 100-fold dilution of rSFV (P < 0.05).
FIG. 4.
FIG. 4.
IFN-α/β production and activation of mDCs in response to rSFV and UV-rSFV are intact in the absence of MyD88. (A) Phenotypes of representative mDC cultures generated from wt and MyD88−/− mice. (B) IFN-α/β produced by mDCs generated from wt and MyD88−/− mice. mDCs were incubated with rSFV (25 IU/cell), a 1:10 dilution of rSFV (2.5 IU/cell), UV-rSFV (a volume equivalent to 25 IU/cell of rSFV), CpG (1 μM), or pI-C (50 μg/ml) for 6 or 24 h. *, no IFN-α/β could be detected in the sample. (C) Expression of CD86 on wt and MyD88−/− mDC surfaces 24 h after the addition of rSFV. The results shown are representative of at least three independent preparations of mDCs.
FIG. 5.
FIG. 5.
rSFV induces early systemic IFN-α/β production in mice lacking MyD88. (A) IFN-α/β levels in sera from individual wt and MyD88−/− mice 4 h after injection with UV-rSFV (equivalent of 108 IU). (B) IFN-α/β levels in sera from individual wt and MyD88−/− mice 6 h after injection with rSFV (107 IU). Data from one representative experiment of two experiments using at least four mice per group are shown.
FIG. 6.
FIG. 6.
IRF3−/− mDCs are defective in early IFN-α/β production in response to rSFV and UV-rSFV. (A) Phenotypes of representative mDC cultures generated from wt and IRF-3−/− mice. (B) mDCs were incubated with rSFV (25 IU/cell), a 1:10 dilution of rSFV (2.5 IU/cell), UV-rSFV (a volume equivalent to 25 IU/cell of rSFV), CpG (1 μM), or pI-C (50 μg/ml) for 6 or 24 h. *, no IFN-α/β could be detected in the sample. The results shown are representative of at least three independent preparations of mDCs. (C) IFN-α/β levels in sera from individual wt and IRF-3−/− mice 4 h after injection with UV-rSFV (equivalent of 108 IU).

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