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Dengue virus activates membrane TRAIL relocalization and IFN-α production by human plasmacytoid dendritic cells in vitro and in vivo.

Gandini M, Gras C, Azeredo EL, Pinto LM, Smith N, Despres P, da Cunha RV, de Souza LJ, Kubelka CF, Herbeuval JP - PLoS Negl Trop Dis (2013)

Bottom Line: Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA.This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment.Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunologia Viral, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil.

ABSTRACT

Background: Dengue displays a broad spectrum of clinical manifestations that may vary from asymptomatic to severe and even fatal features. Plasma leakage/hemorrhages can be caused by a cytokine storm induced by monocytes and dendritic cells during dengue virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) are innate immune cells and in response to virus exposure secrete IFN-α and express membrane TRAIL (mTRAIL). We aimed to characterize pDC activation in dengue patients and their function under DENV-2 stimulation in vitro. METHODS FINDINGS: Flow cytometry analysis (FCA) revealed that pDCs of mild dengue patients exhibit significantly higher frequencies of mTRAIL compared to severe cases or healthy controls. Plasma levels of IFN-α and soluble TRAIL are increased in mild compared to severe dengue patients, positively correlating with pDC activation. FCA experiments showed that in vitro exposure to DENV-2 induced mTRAIL expression on pDC. Furthermore, three dimension microscopy highlighted that TRAIL was relocalized from intracellular compartment to plasma membrane. Chloroquine treatment inhibited DENV-2-induced mTRAIL relocalization and IFN-α production by pDC. Endosomal viral degradation blockade by chloroquine allowed viral antigens detection inside pDCs. All those data are in favor of endocytosis pathway activation by DENV-2 in pDC. Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA. This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment. Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production.

Conclusions: This investigation characterizes, during DENV-2 infection, activation of pDCs in vivo and their antiviral role in vitro. Thus, we propose TRAIL-expressing pDCs may have an important role in the outcome of disease.

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IFN-α treatment and activated pDC coculture in DENV-2-infected monocytes.Freshly purified monocytes were infected with DENV-2 (MOI 10) for 48 hours, pre-treated or not with IFN-α. (A) Nucleus/DAPI (blue) and virus (green) of monocytes mock- (upper panels) or DENV-2-infected (lower panels). Viral particles were detected in the cytoplasm only in DENV-2-infected monocytes. (B) Nucleus/DAPI (blue), virus (green) and phase contrast (grey) for different stimuli (mock – top, DENV-2 only – middle, IFN-α+DENV-2 – bottom) in monocytes. Green arrows show intracellular detection of DENV particles in DENV-2 infected monocytes, whereas pre-treatment with IFN-α strongly reduced viral antigen detection. Quantification of DENV antigens in mock, DENV-2 and IFN-α pre-treated DENV-2-infected monocytes using microscopy (C) or flow cytometry (D). (E) Freshly purified monocytes were DENV-2-infected then immediately cocultured with DENV-2-stimulated pDCs. DENV antigen detection in CD14-CD11c-CD123+ pDCs or CD14+CD11c+ monocytes after 48 h of culture. Monocytes were DENV-2-infected then immediately cocultured with CpG-stimulated only (CpG), Chroloquine pre-treated CpG-stimulated (CpG+Chloro) or unstimulated (unst) pDCs (figure S2). Cocultures were analysed 48 h later for viral antigens (F) and IFN-α production (G). Data represent independent experiments from two different donors and values were submitted to paired t test in which * p<0.05; ** p<0.005 and *** p<0.0005.
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pntd-0002257-g005: IFN-α treatment and activated pDC coculture in DENV-2-infected monocytes.Freshly purified monocytes were infected with DENV-2 (MOI 10) for 48 hours, pre-treated or not with IFN-α. (A) Nucleus/DAPI (blue) and virus (green) of monocytes mock- (upper panels) or DENV-2-infected (lower panels). Viral particles were detected in the cytoplasm only in DENV-2-infected monocytes. (B) Nucleus/DAPI (blue), virus (green) and phase contrast (grey) for different stimuli (mock – top, DENV-2 only – middle, IFN-α+DENV-2 – bottom) in monocytes. Green arrows show intracellular detection of DENV particles in DENV-2 infected monocytes, whereas pre-treatment with IFN-α strongly reduced viral antigen detection. Quantification of DENV antigens in mock, DENV-2 and IFN-α pre-treated DENV-2-infected monocytes using microscopy (C) or flow cytometry (D). (E) Freshly purified monocytes were DENV-2-infected then immediately cocultured with DENV-2-stimulated pDCs. DENV antigen detection in CD14-CD11c-CD123+ pDCs or CD14+CD11c+ monocytes after 48 h of culture. Monocytes were DENV-2-infected then immediately cocultured with CpG-stimulated only (CpG), Chroloquine pre-treated CpG-stimulated (CpG+Chloro) or unstimulated (unst) pDCs (figure S2). Cocultures were analysed 48 h later for viral antigens (F) and IFN-α production (G). Data represent independent experiments from two different donors and values were submitted to paired t test in which * p<0.05; ** p<0.005 and *** p<0.0005.

Mentions: Because viral load is considered to be an important factor in dengue severity [8], we next studied the role of pDCs in viral replication. For that purpose, we used primary autologous human monocytes that allow efficient DENV-2 replication in order to assess whether pDC could inhibit viral replication or not. Analysis of purified monocytes infected for 48 hours revealed in 2D microscopy a robust intracellular but not nuclear staining of DENV proteins (figure 5A, lower panel), consistent with flavivirus replication cycle [4]. Considering that pDCs produce high levels of IFN-α upon DENV-2 stimulation, we evaluated its antiviral effect. Monocytes that were pre-treated with IFN-α 24 hours before DENV-2 incubation showed a great reduction in viral antigen detection compared to untreated cells (figure 5B). Quantification by microscopy of DENV-2 positive/negative cells showed that IFN-α treatment reduced by 80% (p<0.001) the number of DENV-2 positive cells (figure 5C) and the same reduction was observed by flow cytometry (figure 5D). We also observed a low production of IFN-α by DENV-infected monocytes and confirmed IFN-α on supernatants of monocytes pre-treated with the cytokine (figure S2). These data are supporting that IFN-α has a restricting antiviral role during DENV infection.


Dengue virus activates membrane TRAIL relocalization and IFN-α production by human plasmacytoid dendritic cells in vitro and in vivo.

Gandini M, Gras C, Azeredo EL, Pinto LM, Smith N, Despres P, da Cunha RV, de Souza LJ, Kubelka CF, Herbeuval JP - PLoS Negl Trop Dis (2013)

IFN-α treatment and activated pDC coculture in DENV-2-infected monocytes.Freshly purified monocytes were infected with DENV-2 (MOI 10) for 48 hours, pre-treated or not with IFN-α. (A) Nucleus/DAPI (blue) and virus (green) of monocytes mock- (upper panels) or DENV-2-infected (lower panels). Viral particles were detected in the cytoplasm only in DENV-2-infected monocytes. (B) Nucleus/DAPI (blue), virus (green) and phase contrast (grey) for different stimuli (mock – top, DENV-2 only – middle, IFN-α+DENV-2 – bottom) in monocytes. Green arrows show intracellular detection of DENV particles in DENV-2 infected monocytes, whereas pre-treatment with IFN-α strongly reduced viral antigen detection. Quantification of DENV antigens in mock, DENV-2 and IFN-α pre-treated DENV-2-infected monocytes using microscopy (C) or flow cytometry (D). (E) Freshly purified monocytes were DENV-2-infected then immediately cocultured with DENV-2-stimulated pDCs. DENV antigen detection in CD14-CD11c-CD123+ pDCs or CD14+CD11c+ monocytes after 48 h of culture. Monocytes were DENV-2-infected then immediately cocultured with CpG-stimulated only (CpG), Chroloquine pre-treated CpG-stimulated (CpG+Chloro) or unstimulated (unst) pDCs (figure S2). Cocultures were analysed 48 h later for viral antigens (F) and IFN-α production (G). Data represent independent experiments from two different donors and values were submitted to paired t test in which * p<0.05; ** p<0.005 and *** p<0.0005.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675005&req=5

pntd-0002257-g005: IFN-α treatment and activated pDC coculture in DENV-2-infected monocytes.Freshly purified monocytes were infected with DENV-2 (MOI 10) for 48 hours, pre-treated or not with IFN-α. (A) Nucleus/DAPI (blue) and virus (green) of monocytes mock- (upper panels) or DENV-2-infected (lower panels). Viral particles were detected in the cytoplasm only in DENV-2-infected monocytes. (B) Nucleus/DAPI (blue), virus (green) and phase contrast (grey) for different stimuli (mock – top, DENV-2 only – middle, IFN-α+DENV-2 – bottom) in monocytes. Green arrows show intracellular detection of DENV particles in DENV-2 infected monocytes, whereas pre-treatment with IFN-α strongly reduced viral antigen detection. Quantification of DENV antigens in mock, DENV-2 and IFN-α pre-treated DENV-2-infected monocytes using microscopy (C) or flow cytometry (D). (E) Freshly purified monocytes were DENV-2-infected then immediately cocultured with DENV-2-stimulated pDCs. DENV antigen detection in CD14-CD11c-CD123+ pDCs or CD14+CD11c+ monocytes after 48 h of culture. Monocytes were DENV-2-infected then immediately cocultured with CpG-stimulated only (CpG), Chroloquine pre-treated CpG-stimulated (CpG+Chloro) or unstimulated (unst) pDCs (figure S2). Cocultures were analysed 48 h later for viral antigens (F) and IFN-α production (G). Data represent independent experiments from two different donors and values were submitted to paired t test in which * p<0.05; ** p<0.005 and *** p<0.0005.
Mentions: Because viral load is considered to be an important factor in dengue severity [8], we next studied the role of pDCs in viral replication. For that purpose, we used primary autologous human monocytes that allow efficient DENV-2 replication in order to assess whether pDC could inhibit viral replication or not. Analysis of purified monocytes infected for 48 hours revealed in 2D microscopy a robust intracellular but not nuclear staining of DENV proteins (figure 5A, lower panel), consistent with flavivirus replication cycle [4]. Considering that pDCs produce high levels of IFN-α upon DENV-2 stimulation, we evaluated its antiviral effect. Monocytes that were pre-treated with IFN-α 24 hours before DENV-2 incubation showed a great reduction in viral antigen detection compared to untreated cells (figure 5B). Quantification by microscopy of DENV-2 positive/negative cells showed that IFN-α treatment reduced by 80% (p<0.001) the number of DENV-2 positive cells (figure 5C) and the same reduction was observed by flow cytometry (figure 5D). We also observed a low production of IFN-α by DENV-infected monocytes and confirmed IFN-α on supernatants of monocytes pre-treated with the cytokine (figure S2). These data are supporting that IFN-α has a restricting antiviral role during DENV infection.

Bottom Line: Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA.This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment.Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Imunologia Viral, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, Brazil.

ABSTRACT

Background: Dengue displays a broad spectrum of clinical manifestations that may vary from asymptomatic to severe and even fatal features. Plasma leakage/hemorrhages can be caused by a cytokine storm induced by monocytes and dendritic cells during dengue virus (DENV) replication. Plasmacytoid dendritic cells (pDCs) are innate immune cells and in response to virus exposure secrete IFN-α and express membrane TRAIL (mTRAIL). We aimed to characterize pDC activation in dengue patients and their function under DENV-2 stimulation in vitro. METHODS FINDINGS: Flow cytometry analysis (FCA) revealed that pDCs of mild dengue patients exhibit significantly higher frequencies of mTRAIL compared to severe cases or healthy controls. Plasma levels of IFN-α and soluble TRAIL are increased in mild compared to severe dengue patients, positively correlating with pDC activation. FCA experiments showed that in vitro exposure to DENV-2 induced mTRAIL expression on pDC. Furthermore, three dimension microscopy highlighted that TRAIL was relocalized from intracellular compartment to plasma membrane. Chloroquine treatment inhibited DENV-2-induced mTRAIL relocalization and IFN-α production by pDC. Endosomal viral degradation blockade by chloroquine allowed viral antigens detection inside pDCs. All those data are in favor of endocytosis pathway activation by DENV-2 in pDC. Coculture of pDC/DENV-2-infected monocytes revealed a dramatic decrease of antigen detection by FCA. This viral antigens reduction in monocytes was also observed after exogenous IFN-α treatment. Thus, pDC effect on viral load reduction was mainly dependent on IFN-α production.

Conclusions: This investigation characterizes, during DENV-2 infection, activation of pDCs in vivo and their antiviral role in vitro. Thus, we propose TRAIL-expressing pDCs may have an important role in the outcome of disease.

Show MeSH
Related in: MedlinePlus