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Innate immune response of human plasmacytoid dendritic cells to poxvirus infection is subverted by vaccinia E3 via its Z-DNA/RNA binding domain.

Cao H, Dai P, Wang W, Li H, Yuan J, Wang F, Fang CM, Pitha PM, Liu J, Condit RC, McFadden G, Merghoub T, Houghton AN, Young JW, Shuman S, Deng L - PLoS ONE (2012)

Bottom Line: Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1.These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway.Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists.

View Article: PubMed Central - PubMed

Affiliation: Dermatology Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT
Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus.

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Myxoma virus infection induces IFN-α and TNF production in human pDCs.(A) Freshly isolated pDCs (2×105) were stimulated with CpG2216 (10 μg/ml) or imiquimod (5 μg/ml), or infected with vaccinia or myxoma virus at a multiplicity of 10 (MOI = 10). The concentrations of IFN-α and TNF in the culture supernatants collected at 20 h post treatment were determined by ELISA. The values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors. (B) pDCs were infected with vaccinia followed by addition of CpG2216 (10 μg/ml) or imiquimod (5 μg/ml), or co-infected with vaccinia plus myxoma virus at a MOI of 10 for each virus. Control cells that were treated with CpG or imiquimod, or infected singly with vaccinia or myxoma virus were included. The concentrations of IFN-α and TNF in the culture supernatants collected at 20 h post treatment were determined by ELISA. The values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors (***, p<0.001). (C) Freshly isolated human pDCs were infected with WT vaccinia, GFP-expressing vaccinia or myxoma (GFP-Vaccinia or GFP-Myxoma) alone at a MOI of 10, or co-infected with WT vaccinia plus GFP-Myxoma. GFP expression in infected pDCs (CD123+BDCA2+ cells) was determined by FACS. The experiments were repeated twice. The results of a representative are shown.
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pone.0036823-g001: Myxoma virus infection induces IFN-α and TNF production in human pDCs.(A) Freshly isolated pDCs (2×105) were stimulated with CpG2216 (10 μg/ml) or imiquimod (5 μg/ml), or infected with vaccinia or myxoma virus at a multiplicity of 10 (MOI = 10). The concentrations of IFN-α and TNF in the culture supernatants collected at 20 h post treatment were determined by ELISA. The values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors. (B) pDCs were infected with vaccinia followed by addition of CpG2216 (10 μg/ml) or imiquimod (5 μg/ml), or co-infected with vaccinia plus myxoma virus at a MOI of 10 for each virus. Control cells that were treated with CpG or imiquimod, or infected singly with vaccinia or myxoma virus were included. The concentrations of IFN-α and TNF in the culture supernatants collected at 20 h post treatment were determined by ELISA. The values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors (***, p<0.001). (C) Freshly isolated human pDCs were infected with WT vaccinia, GFP-expressing vaccinia or myxoma (GFP-Vaccinia or GFP-Myxoma) alone at a MOI of 10, or co-infected with WT vaccinia plus GFP-Myxoma. GFP expression in infected pDCs (CD123+BDCA2+ cells) was determined by FACS. The experiments were repeated twice. The results of a representative are shown.

Mentions: To test whether primary human pDCs respond differently to vaccinia (an Orthopoxvirus that is potentially pathogenic in humans) and myxoma virus (a Leporipoxvirus that is non-pathogenic in humans), we purified pDCs from human peripheral blood mononuclear cells using anti-BDCA-4 antibody-coated magnetic beads. The resulting pDC-enriched preparations (CD123+/BDCA2+ cells) had a purity of 60–80% as assessed by flow cytometry (data not shown). Treatment of pDCs with either TLR9 agonist CpG or TLR7 agonist imiquimod co-induced the production and secretion of IFN-α and TNF (Fig. 1A). Infection of pDCs with myxoma virus also induced the production of comparable levels of IFN-α and TNF (Fig. 1A). By contrast, pDCs did not secrete IFN-α or TNF when infected with vaccinia virus (Fig. 1A).


Innate immune response of human plasmacytoid dendritic cells to poxvirus infection is subverted by vaccinia E3 via its Z-DNA/RNA binding domain.

Cao H, Dai P, Wang W, Li H, Yuan J, Wang F, Fang CM, Pitha PM, Liu J, Condit RC, McFadden G, Merghoub T, Houghton AN, Young JW, Shuman S, Deng L - PLoS ONE (2012)

Myxoma virus infection induces IFN-α and TNF production in human pDCs.(A) Freshly isolated pDCs (2×105) were stimulated with CpG2216 (10 μg/ml) or imiquimod (5 μg/ml), or infected with vaccinia or myxoma virus at a multiplicity of 10 (MOI = 10). The concentrations of IFN-α and TNF in the culture supernatants collected at 20 h post treatment were determined by ELISA. The values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors. (B) pDCs were infected with vaccinia followed by addition of CpG2216 (10 μg/ml) or imiquimod (5 μg/ml), or co-infected with vaccinia plus myxoma virus at a MOI of 10 for each virus. Control cells that were treated with CpG or imiquimod, or infected singly with vaccinia or myxoma virus were included. The concentrations of IFN-α and TNF in the culture supernatants collected at 20 h post treatment were determined by ELISA. The values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors (***, p<0.001). (C) Freshly isolated human pDCs were infected with WT vaccinia, GFP-expressing vaccinia or myxoma (GFP-Vaccinia or GFP-Myxoma) alone at a MOI of 10, or co-infected with WT vaccinia plus GFP-Myxoma. GFP expression in infected pDCs (CD123+BDCA2+ cells) was determined by FACS. The experiments were repeated twice. The results of a representative are shown.
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pone.0036823-g001: Myxoma virus infection induces IFN-α and TNF production in human pDCs.(A) Freshly isolated pDCs (2×105) were stimulated with CpG2216 (10 μg/ml) or imiquimod (5 μg/ml), or infected with vaccinia or myxoma virus at a multiplicity of 10 (MOI = 10). The concentrations of IFN-α and TNF in the culture supernatants collected at 20 h post treatment were determined by ELISA. The values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors. (B) pDCs were infected with vaccinia followed by addition of CpG2216 (10 μg/ml) or imiquimod (5 μg/ml), or co-infected with vaccinia plus myxoma virus at a MOI of 10 for each virus. Control cells that were treated with CpG or imiquimod, or infected singly with vaccinia or myxoma virus were included. The concentrations of IFN-α and TNF in the culture supernatants collected at 20 h post treatment were determined by ELISA. The values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors (***, p<0.001). (C) Freshly isolated human pDCs were infected with WT vaccinia, GFP-expressing vaccinia or myxoma (GFP-Vaccinia or GFP-Myxoma) alone at a MOI of 10, or co-infected with WT vaccinia plus GFP-Myxoma. GFP expression in infected pDCs (CD123+BDCA2+ cells) was determined by FACS. The experiments were repeated twice. The results of a representative are shown.
Mentions: To test whether primary human pDCs respond differently to vaccinia (an Orthopoxvirus that is potentially pathogenic in humans) and myxoma virus (a Leporipoxvirus that is non-pathogenic in humans), we purified pDCs from human peripheral blood mononuclear cells using anti-BDCA-4 antibody-coated magnetic beads. The resulting pDC-enriched preparations (CD123+/BDCA2+ cells) had a purity of 60–80% as assessed by flow cytometry (data not shown). Treatment of pDCs with either TLR9 agonist CpG or TLR7 agonist imiquimod co-induced the production and secretion of IFN-α and TNF (Fig. 1A). Infection of pDCs with myxoma virus also induced the production of comparable levels of IFN-α and TNF (Fig. 1A). By contrast, pDCs did not secrete IFN-α or TNF when infected with vaccinia virus (Fig. 1A).

Bottom Line: Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1.These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway.Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists.

View Article: PubMed Central - PubMed

Affiliation: Dermatology Service, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, United States of America.

ABSTRACT
Plasmacytoid dendritic cells (pDCs) play important roles in antiviral innate immunity by producing type I interferon (IFN). In this study, we assess the immune responses of primary human pDCs to two poxviruses, vaccinia and myxoma virus. Vaccinia, an orthopoxvirus, was used for immunization against smallpox, a contagious human disease with high mortality. Myxoma virus, a Leporipoxvirus, causes lethal disease in rabbits, but is non-pathogenic in humans. We report that myxoma virus infection of human pDCs induces IFN-α and TNF production, whereas vaccinia infection does not. Co-infection of pDCs with myxoma virus plus vaccinia blocks myxoma induction effects. We find that heat-inactivated vaccinia (Heat-VAC; by incubating the virus at 55°C for 1 h) gains the ability to induce IFN-α and TNF in primary human pDCs. Induction of IFN-α in pDCs by myxoma virus or Heat-VAC is blocked by chloroquine, which inhibits endosomal acidification required for TLR7/9 signaling, and by inhibitors of cellular kinases PI3K and Akt. Using purified pDCs from genetic knockout mice, we demonstrate that Heat-VAC-induced type I IFN production in pDCs requires the endosomal RNA sensor TLR7 and its adaptor MyD88, transcription factor IRF7 and the type I IFN feedback loop mediated by IFNAR1. These results indicate that (i) vaccinia virus, but not myxoma virus, expresses inhibitor(s) of the poxvirus sensing pathway(s) in pDCs; and (ii) Heat-VAC infection fails to produce inhibitor(s) but rather produces novel activator(s), likely viral RNA transcripts that are sensed by the TLR7/MyD88 pathway. Using vaccinia gene deletion mutants, we show that the Z-DNA/RNA binding domain at the N-terminus of the vaccinia immunomodulatory E3 protein is an antagonist of the innate immune response of human pDCs to poxvirus infection and TLR agonists. The myxoma virus ortholog of vaccinia E3 (M029) lacks the N-terminal Z-DNA/RNA binding domain, which might contribute to the immunostimulating properties of myxoma virus.

Show MeSH
Related in: MedlinePlus