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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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Chloroquine and PI3K inhibitor block the induction of IFN-α and TNF in human pDCs by myxoma virus.pDCs (2×105) were stimulated with CpG2216 (10 μg/ml), or infected with myxoma virus (MOI = 10), and were then treated with or without inhibitors including chloroquine (A), and LY294002 (B) 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-g002: Chloroquine and PI3K inhibitor block the induction of IFN-α and TNF in human pDCs by myxoma virus.pDCs (2×105) were stimulated with CpG2216 (10 μg/ml), or infected with myxoma virus (MOI = 10), and were then treated with or without inhibitors including chloroquine (A), and LY294002 (B) 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: pDCs utilize TLR7 and TLR9 to detect viral nucleic acids and initiate an antiviral response. TLR9 has been implicated in recognizing viral DNA, as demonstrated for herpes simplex virus 18-20. pDCs rely on TLR7 in sensing RNA virus infection 21,22. Myxoma virus is sensed by TLR9/MyD88 in murine pDCs 15, whereas myxoma virus infection induces both type I IFN and TNF in primary human macrophages by a RIG-I-dependent sensing mechanism 23. Here we tested whether chloroquine, an inhibitor of endosomal acidification and maturation 21, would affect the innate responses of human pDCs to myxoma virus infection. Treatment of pDCs at 1 h post-inoculation with 2 µM and 5 µM chloroquine blocked IFN-αproduction, while reducing TNF production by 57% and 99%, respectively (Fig. 2A). Induction of IFN-α secretion by TLR9 agonist CpG was also blocked by 2 µM and 5 µM chloroquine, while CpG-induced TNF production was reduced by 33% and 96%, respectively (Fig. 2A). Imiquimod-induced IFN-α and TNF production was also similarly inhibited in the presence of chloroquine (data not shown). The greater sensitivity of IFN-α versus TNF induction to chloroquine inhibition could be related to the spatial and temporal regulation of IFN-α and TNF in early and late endosomes, respectively 24. These data implicate that endosomal acidification, such as that required for TLR9 signaling, is important for myxoma virus sensing by human pDCs.


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)

Chloroquine and PI3K inhibitor block the induction of IFN-α and TNF in human pDCs by myxoma virus.pDCs (2×105) were stimulated with CpG2216 (10 μg/ml), or infected with myxoma virus (MOI = 10), and were then treated with or without inhibitors including chloroquine (A), and LY294002 (B) 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-g002: Chloroquine and PI3K inhibitor block the induction of IFN-α and TNF in human pDCs by myxoma virus.pDCs (2×105) were stimulated with CpG2216 (10 μg/ml), or infected with myxoma virus (MOI = 10), and were then treated with or without inhibitors including chloroquine (A), and LY294002 (B) 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: pDCs utilize TLR7 and TLR9 to detect viral nucleic acids and initiate an antiviral response. TLR9 has been implicated in recognizing viral DNA, as demonstrated for herpes simplex virus 18-20. pDCs rely on TLR7 in sensing RNA virus infection 21,22. Myxoma virus is sensed by TLR9/MyD88 in murine pDCs 15, whereas myxoma virus infection induces both type I IFN and TNF in primary human macrophages by a RIG-I-dependent sensing mechanism 23. Here we tested whether chloroquine, an inhibitor of endosomal acidification and maturation 21, would affect the innate responses of human pDCs to myxoma virus infection. Treatment of pDCs at 1 h post-inoculation with 2 µM and 5 µM chloroquine blocked IFN-αproduction, while reducing TNF production by 57% and 99%, respectively (Fig. 2A). Induction of IFN-α secretion by TLR9 agonist CpG was also blocked by 2 µM and 5 µM chloroquine, while CpG-induced TNF production was reduced by 33% and 96%, respectively (Fig. 2A). Imiquimod-induced IFN-α and TNF production was also similarly inhibited in the presence of chloroquine (data not shown). The greater sensitivity of IFN-α versus TNF induction to chloroquine inhibition could be related to the spatial and temporal regulation of IFN-α and TNF in early and late endosomes, respectively 24. These data implicate that endosomal acidification, such as that required for TLR9 signaling, is important for myxoma virus sensing by human pDCs.

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