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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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Akt inhibitors VIII and X block the induction of IFN-α and TNF in human pDCs by myxoma virus.(A, B) Human pDCs were cultured with CpG2216 (10 μg/ml), or infected with myxoma virus (MOI = 10), and were then treated with or without LY294002 (10 μm) or Akt inhibitor X (10 μm) for 90 min (CpG) or 8 h (Myxoma). Cells were stained with Alexa Fluor 647 anti-human AKT antibody that recognizes phospho-S473, and analyzed by flow cytometry. The results shown are representative of three separate experiments. (C, D) pDCs (2×105)were stimulated with CpG2216 (10 μg/ml), or infected with myxoma virus (MOI = 10), and were then treated with or without Akt inhibitors VIII (C), or X (D) at indicated concentrations. Supernatants were collected at 20 h post treatment and measured for IFN-α and TNF concentrations by ELISA. The values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors (*, p<0.05; **, p<0.01; ***, p<0.001).
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pone.0036823-g003: Akt inhibitors VIII and X block the induction of IFN-α and TNF in human pDCs by myxoma virus.(A, B) Human pDCs were cultured with CpG2216 (10 μg/ml), or infected with myxoma virus (MOI = 10), and were then treated with or without LY294002 (10 μm) or Akt inhibitor X (10 μm) for 90 min (CpG) or 8 h (Myxoma). Cells were stained with Alexa Fluor 647 anti-human AKT antibody that recognizes phospho-S473, and analyzed by flow cytometry. The results shown are representative of three separate experiments. (C, D) pDCs (2×105)were stimulated with CpG2216 (10 μg/ml), or infected with myxoma virus (MOI = 10), and were then treated with or without Akt inhibitors VIII (C), or X (D) at indicated concentrations. Supernatants were collected at 20 h post treatment and measured for IFN-α and TNF concentrations by ELISA. The values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors (*, p<0.05; **, p<0.01; ***, p<0.001).

Mentions: Akt (protein kinase B), a serine/threonine kinase and a downstream target of PI3K, is a regulator of cell metabolism, survival, and proliferation 28. PI3K generates PtdIns(3,4,5)P3, which recruits inactive Akt in the cytosol to the plasma membrane. The binding of PtdIns(3,4,5)P3 to the N-terminal pleckstrin homology (PH) domain of Akt allows phosphorylation of threonine-308 at the activation loop of the AKT kinase domain by 3-phosphoinositide-dependent protein kinase-1 (PDK-1). The activity of PDK-1 is also dependent on the binding of PtdIns(3,4,5)P3. Subsequent phosphorylation occurs at serine-473 in the hydrophobic regulatory domain by the mTORC2 complex, which is required for the activation of Akt 29. Guiducci et al. 27 showed that CpG treatment or infection with influenza virus induces Akt phosphorylation at Ser473 in pDCs. This induction can be inhibited by PI3K inhibitor LY. We observed that myxoma virus induction of Akt phosphorylation (p-AKT) at Ser473 occurs at 8 h post infection, as determined by intracellular staining with anti-p-AKT antibody against phospho-Ser473 followed by FACS analysis (Fig. 3A). LY inhibited both CpG- and myxoma-induced Akt phosphorylation in human pDCs (Fig. 3A).


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)

Akt inhibitors VIII and X block the induction of IFN-α and TNF in human pDCs by myxoma virus.(A, B) Human pDCs were cultured with CpG2216 (10 μg/ml), or infected with myxoma virus (MOI = 10), and were then treated with or without LY294002 (10 μm) or Akt inhibitor X (10 μm) for 90 min (CpG) or 8 h (Myxoma). Cells were stained with Alexa Fluor 647 anti-human AKT antibody that recognizes phospho-S473, and analyzed by flow cytometry. The results shown are representative of three separate experiments. (C, D) pDCs (2×105)were stimulated with CpG2216 (10 μg/ml), or infected with myxoma virus (MOI = 10), and were then treated with or without Akt inhibitors VIII (C), or X (D) at indicated concentrations. Supernatants were collected at 20 h post treatment and measured for IFN-α and TNF concentrations by ELISA. The values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors (*, p<0.05; **, p<0.01; ***, p<0.001).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351467&req=5

pone.0036823-g003: Akt inhibitors VIII and X block the induction of IFN-α and TNF in human pDCs by myxoma virus.(A, B) Human pDCs were cultured with CpG2216 (10 μg/ml), or infected with myxoma virus (MOI = 10), and were then treated with or without LY294002 (10 μm) or Akt inhibitor X (10 μm) for 90 min (CpG) or 8 h (Myxoma). Cells were stained with Alexa Fluor 647 anti-human AKT antibody that recognizes phospho-S473, and analyzed by flow cytometry. The results shown are representative of three separate experiments. (C, D) pDCs (2×105)were stimulated with CpG2216 (10 μg/ml), or infected with myxoma virus (MOI = 10), and were then treated with or without Akt inhibitors VIII (C), or X (D) at indicated concentrations. Supernatants were collected at 20 h post treatment and measured for IFN-α and TNF concentrations by ELISA. The values shown are averages of triplicate means (± SEM) of three independent experiments using human pDCs isolated from three different donors (*, p<0.05; **, p<0.01; ***, p<0.001).
Mentions: Akt (protein kinase B), a serine/threonine kinase and a downstream target of PI3K, is a regulator of cell metabolism, survival, and proliferation 28. PI3K generates PtdIns(3,4,5)P3, which recruits inactive Akt in the cytosol to the plasma membrane. The binding of PtdIns(3,4,5)P3 to the N-terminal pleckstrin homology (PH) domain of Akt allows phosphorylation of threonine-308 at the activation loop of the AKT kinase domain by 3-phosphoinositide-dependent protein kinase-1 (PDK-1). The activity of PDK-1 is also dependent on the binding of PtdIns(3,4,5)P3. Subsequent phosphorylation occurs at serine-473 in the hydrophobic regulatory domain by the mTORC2 complex, which is required for the activation of Akt 29. Guiducci et al. 27 showed that CpG treatment or infection with influenza virus induces Akt phosphorylation at Ser473 in pDCs. This induction can be inhibited by PI3K inhibitor LY. We observed that myxoma virus induction of Akt phosphorylation (p-AKT) at Ser473 occurs at 8 h post infection, as determined by intracellular staining with anti-p-AKT antibody against phospho-Ser473 followed by FACS analysis (Fig. 3A). LY inhibited both CpG- and myxoma-induced Akt phosphorylation in human pDCs (Fig. 3A).

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