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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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TLR7 and MyD88 are required for the induction of type I IFN by Heat-VAC in murine pDCs.Purified murine pDCs were obtained using FACS from Flt3L-BMDCs generated from MyD88−/− (A), TLR7−/− (B), TLR9−/− (C) mice and age-matched WT controls. pDCs (2×105) were stimulated with CpG, or infected with myxoma virus at a MOI of 10, or with an equivalent amount of Heat-VAC. Supernatants were collected at 22 h post infection. The concentrations of IFN-α/β were determined by ELISA. Data are means ± SEM. The combined results of three independently performed experiments are shown.
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pone.0036823-g006: TLR7 and MyD88 are required for the induction of type I IFN by Heat-VAC in murine pDCs.Purified murine pDCs were obtained using FACS from Flt3L-BMDCs generated from MyD88−/− (A), TLR7−/− (B), TLR9−/− (C) mice and age-matched WT controls. pDCs (2×105) were stimulated with CpG, or infected with myxoma virus at a MOI of 10, or with an equivalent amount of Heat-VAC. Supernatants were collected at 22 h post infection. The concentrations of IFN-α/β were determined by ELISA. Data are means ± SEM. The combined results of three independently performed experiments are shown.

Mentions: We took advantage of the murine genetic system to determine the mechanism of induction of type I IFN in pDCs by Heat-VAC. We purified pDCs from Flt3L-BMDCs from MyD88−/−, TLR7−/−, TLR9−/− or age-matched WT control mice by FACS as described 15. The isolated pDCs are CD11c+B220+PDCA-1+, with a purity of greater than 98%. They were treated with CpG, or infected with myxoma virus at a MOI of 10, or with an equivalent amount of Heat-VAC. Supernatants were collected at 22 h post infection. The level of IFN-α/β was determined by ELISA. We found that Heat-VAC-induced production of IFN-α/β was abolished in MyD88−/− or TLR7−/− pDCs, but only modestly reduced in TLR9−/− pDCs (Fig. 6A, B, and C). In contrast, myxoma-induced type I IFN induction was abolished in MyD88−/−, or TLR9−/− pDCs, but modestly reduced in TLR7−/− pDCs as reported previously (Fig. , 15). As a control, CpG induced type I IFN is abolished in TLR9−/− or MyD88−/− pDCs, but not affected in TLR7−/− pDCs (Fig. 6). Taken together, these results indicate that Heat-VAC infection of pDCs leads to the production of RNA species that are detected by the endosomal RNA sensing pathway mediated by TLR7/MyD88.


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)

TLR7 and MyD88 are required for the induction of type I IFN by Heat-VAC in murine pDCs.Purified murine pDCs were obtained using FACS from Flt3L-BMDCs generated from MyD88−/− (A), TLR7−/− (B), TLR9−/− (C) mice and age-matched WT controls. pDCs (2×105) were stimulated with CpG, or infected with myxoma virus at a MOI of 10, or with an equivalent amount of Heat-VAC. Supernatants were collected at 22 h post infection. The concentrations of IFN-α/β were determined by ELISA. Data are means ± SEM. The combined results of three independently performed experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351467&req=5

pone.0036823-g006: TLR7 and MyD88 are required for the induction of type I IFN by Heat-VAC in murine pDCs.Purified murine pDCs were obtained using FACS from Flt3L-BMDCs generated from MyD88−/− (A), TLR7−/− (B), TLR9−/− (C) mice and age-matched WT controls. pDCs (2×105) were stimulated with CpG, or infected with myxoma virus at a MOI of 10, or with an equivalent amount of Heat-VAC. Supernatants were collected at 22 h post infection. The concentrations of IFN-α/β were determined by ELISA. Data are means ± SEM. The combined results of three independently performed experiments are shown.
Mentions: We took advantage of the murine genetic system to determine the mechanism of induction of type I IFN in pDCs by Heat-VAC. We purified pDCs from Flt3L-BMDCs from MyD88−/−, TLR7−/−, TLR9−/− or age-matched WT control mice by FACS as described 15. The isolated pDCs are CD11c+B220+PDCA-1+, with a purity of greater than 98%. They were treated with CpG, or infected with myxoma virus at a MOI of 10, or with an equivalent amount of Heat-VAC. Supernatants were collected at 22 h post infection. The level of IFN-α/β was determined by ELISA. We found that Heat-VAC-induced production of IFN-α/β was abolished in MyD88−/− or TLR7−/− pDCs, but only modestly reduced in TLR9−/− pDCs (Fig. 6A, B, and C). In contrast, myxoma-induced type I IFN induction was abolished in MyD88−/−, or TLR9−/− pDCs, but modestly reduced in TLR7−/− pDCs as reported previously (Fig. , 15). As a control, CpG induced type I IFN is abolished in TLR9−/− or MyD88−/− pDCs, but not affected in TLR7−/− pDCs (Fig. 6). Taken together, these results indicate that Heat-VAC infection of pDCs leads to the production of RNA species that are detected by the endosomal RNA sensing pathway mediated by TLR7/MyD88.

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