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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, PI3K and Akt inhibitors block the induction of IFN-α and TNF in human pDCs by heat-inactivated vaccinia virus.Human pDCs (2×105) were infected as indicated with heat-inactivated vaccinia for 1 h. Cells were washed and incubated in fresh medium in the presence or absence of increasing concentrations of chloroquine (A), LY294002 (B), Akt inhibitor VIII (C), or Akt inhibitor X (D). Supernatants were collected at 20 h post treatment and assayed for IFN-α and TNF concentrations. 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-g005: Chloroquine, PI3K and Akt inhibitors block the induction of IFN-α and TNF in human pDCs by heat-inactivated vaccinia virus.Human pDCs (2×105) were infected as indicated with heat-inactivated vaccinia for 1 h. Cells were washed and incubated in fresh medium in the presence or absence of increasing concentrations of chloroquine (A), LY294002 (B), Akt inhibitor VIII (C), or Akt inhibitor X (D). Supernatants were collected at 20 h post treatment and assayed for IFN-α and TNF concentrations. 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: We next asked if Heat-VAC induces an antiviral response in pDCs via a similar pathway triggered by myxoma virus. We addressed this issue with the battery of small molecule inhibitors discussed above. First, we observed that chloroquine reduced IFN-α and TNF production by pDCs infected with heat-inactivated vaccinia in a dose-dependent fashion: 25 µM chloroquine completely blocked the production of IFN-α and reduced TNF level by 52% (Fig. 5A). By comparison, as little as 2 µM chloroquine completely blocked IFN-α production and reduced TNF secretion by 55% in myxoma-infected pDCs (Fig. 2A). Therefore, the induction of IFN-α and TNF by Heat-VAC is at least 10-fold less sensitive to chloroquine inhibition than is induction by myxoma virus infection. 10 µM PI3K inhibitor LY294002 inhibited IFN-α and TNF production by 93% and 33%, respectively in pDCs infected with Heat-VAC (Fig. 5B). 10 µM Akt inhibitor VIII inhibited IFN-α and TNF production by 89% and 71%, respectively (Fig. 5C); and 10 µM of Akt X reduced IFN-α and TNF production by 93% and 64%, respectively (Fig. 5D). These results indicate that Heat-VAC is sensed by pDCs through a pathway that is similar, but not identical, to that for detecting myxoma virus.


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, PI3K and Akt inhibitors block the induction of IFN-α and TNF in human pDCs by heat-inactivated vaccinia virus.Human pDCs (2×105) were infected as indicated with heat-inactivated vaccinia for 1 h. Cells were washed and incubated in fresh medium in the presence or absence of increasing concentrations of chloroquine (A), LY294002 (B), Akt inhibitor VIII (C), or Akt inhibitor X (D). Supernatants were collected at 20 h post treatment and assayed for IFN-α and TNF concentrations. 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-g005: Chloroquine, PI3K and Akt inhibitors block the induction of IFN-α and TNF in human pDCs by heat-inactivated vaccinia virus.Human pDCs (2×105) were infected as indicated with heat-inactivated vaccinia for 1 h. Cells were washed and incubated in fresh medium in the presence or absence of increasing concentrations of chloroquine (A), LY294002 (B), Akt inhibitor VIII (C), or Akt inhibitor X (D). Supernatants were collected at 20 h post treatment and assayed for IFN-α and TNF concentrations. 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: We next asked if Heat-VAC induces an antiviral response in pDCs via a similar pathway triggered by myxoma virus. We addressed this issue with the battery of small molecule inhibitors discussed above. First, we observed that chloroquine reduced IFN-α and TNF production by pDCs infected with heat-inactivated vaccinia in a dose-dependent fashion: 25 µM chloroquine completely blocked the production of IFN-α and reduced TNF level by 52% (Fig. 5A). By comparison, as little as 2 µM chloroquine completely blocked IFN-α production and reduced TNF secretion by 55% in myxoma-infected pDCs (Fig. 2A). Therefore, the induction of IFN-α and TNF by Heat-VAC is at least 10-fold less sensitive to chloroquine inhibition than is induction by myxoma virus infection. 10 µM PI3K inhibitor LY294002 inhibited IFN-α and TNF production by 93% and 33%, respectively in pDCs infected with Heat-VAC (Fig. 5B). 10 µM Akt inhibitor VIII inhibited IFN-α and TNF production by 89% and 71%, respectively (Fig. 5C); and 10 µM of Akt X reduced IFN-α and TNF production by 93% and 64%, respectively (Fig. 5D). These results indicate that Heat-VAC is sensed by pDCs through a pathway that is similar, but not identical, to that for detecting myxoma virus.

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