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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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Related in: MedlinePlus

Purified DENV-2-induced in vitro mTRAIL expression and IFN-α production by purified plasmacytoid dendritic cells.PBMCs from healthy donors were stimulated overnight with DENV-2, mock or none (unstimulated). (A) mTRAIL expression profile on pDCs gated from PBMCs (overlay) and (B) mTRAIL positive pDCs for three donors induced by mock SNT (orange) or DENV-2 SNT (blue) using unstimulated (grey fill) pDCs as negative control. DENV positive C6/36 cells infected for 48 h with supernatant of DENV-2-infected C6/36 cells (DENV-2 SNT) or ultracentrifuged DENV-2 SNT (DENV-2 UC) as described in M&M and figure S1. (C) DENV antigens/AlexaFluor488 (green) and nucleus/DAPI (blue) of C6/36 cells infected with DENV-2 SNT (left) and UC (right) at the same inocula dilution (10−3). (D) DENV positive C6/36 cells by flow cytometry in which cells were infected with SNT (orange) or UC (blue) DENV-2 inocula at different dilutions. PBMCs from healthy donors were stimulated overnight with DENV-2 UC, mock UC or none (unstimulated). (E) mTRAIL expression profile on pDCs gated from PBMCs (overlay) and (F) mTRAIL positive pDCs for four donors induced by mock UC (orange) or DENV-2 UC (blue) using unstimulated (grey fill) pDCs as negative control. Freshly purified pDCs were stimulated overnight with DENV-2 UC, mock UC or not (unstimulated). (G) TRAIL expression induced by different MOIs of DENV-2 UC (blue) using unstimulated cells (grey) as negative control. (H) Purified pDCs positive for mTRAIL expression and (I) IFN-α secretion by unstimulated (grey), mock UC (orange), DENV-2-UC-stimulated pDCs pre-treated (black) or not (blue) with chloroquine, for four donors. Values were submitted to paired t test in which * p<0.05 and ** p<0.005.
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pntd-0002257-g002: Purified DENV-2-induced in vitro mTRAIL expression and IFN-α production by purified plasmacytoid dendritic cells.PBMCs from healthy donors were stimulated overnight with DENV-2, mock or none (unstimulated). (A) mTRAIL expression profile on pDCs gated from PBMCs (overlay) and (B) mTRAIL positive pDCs for three donors induced by mock SNT (orange) or DENV-2 SNT (blue) using unstimulated (grey fill) pDCs as negative control. DENV positive C6/36 cells infected for 48 h with supernatant of DENV-2-infected C6/36 cells (DENV-2 SNT) or ultracentrifuged DENV-2 SNT (DENV-2 UC) as described in M&M and figure S1. (C) DENV antigens/AlexaFluor488 (green) and nucleus/DAPI (blue) of C6/36 cells infected with DENV-2 SNT (left) and UC (right) at the same inocula dilution (10−3). (D) DENV positive C6/36 cells by flow cytometry in which cells were infected with SNT (orange) or UC (blue) DENV-2 inocula at different dilutions. PBMCs from healthy donors were stimulated overnight with DENV-2 UC, mock UC or none (unstimulated). (E) mTRAIL expression profile on pDCs gated from PBMCs (overlay) and (F) mTRAIL positive pDCs for four donors induced by mock UC (orange) or DENV-2 UC (blue) using unstimulated (grey fill) pDCs as negative control. Freshly purified pDCs were stimulated overnight with DENV-2 UC, mock UC or not (unstimulated). (G) TRAIL expression induced by different MOIs of DENV-2 UC (blue) using unstimulated cells (grey) as negative control. (H) Purified pDCs positive for mTRAIL expression and (I) IFN-α secretion by unstimulated (grey), mock UC (orange), DENV-2-UC-stimulated pDCs pre-treated (black) or not (blue) with chloroquine, for four donors. Values were submitted to paired t test in which * p<0.05 and ** p<0.005.

Mentions: PDC activation by DENV-2 was shown to occur by TLR-7 stimulation after endocytosis [31] and this pathway was crucial for IKpDC transformation by HTLV-1 [48]. To assess pDC activation by DENV-2, peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated overnight with virus. Initially, we observed that DENV-2 from mosquito cell line supernatant (SNT) promoted a trend, however not statistically significant, in TRAIL detection on pDC surface after viral stimulation in PBMCs, compared to unstimulated or mock-stimulated pDCs (figure 2A and B). Thereafter, an ultracentrifugation of DENV-2 viral stock was performed in order to concentrate viral particles, increasing MOI (figure S1). The DENV-2 infectivity was assayed for both viral stocks by infecting the mosquito cell line C6/36 and comparing them in serial dilutions. Viral antigens were detected inside cells inoculated with concentrated DENV-2 (UC) as early as 48 hours and at higher frequencies than the non-concentrated supernatant indicating that the concentrated virus had enhanced replication rates and it was intracellularly present as detected by immunofluorescence microscopy and flow cytometry (figure 2C and D). This viral stock (DENV-2 UC) was therefore adopted for assessing DENV-2 induced pDC activation in all experiments described in the present work.


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)

Purified DENV-2-induced in vitro mTRAIL expression and IFN-α production by purified plasmacytoid dendritic cells.PBMCs from healthy donors were stimulated overnight with DENV-2, mock or none (unstimulated). (A) mTRAIL expression profile on pDCs gated from PBMCs (overlay) and (B) mTRAIL positive pDCs for three donors induced by mock SNT (orange) or DENV-2 SNT (blue) using unstimulated (grey fill) pDCs as negative control. DENV positive C6/36 cells infected for 48 h with supernatant of DENV-2-infected C6/36 cells (DENV-2 SNT) or ultracentrifuged DENV-2 SNT (DENV-2 UC) as described in M&M and figure S1. (C) DENV antigens/AlexaFluor488 (green) and nucleus/DAPI (blue) of C6/36 cells infected with DENV-2 SNT (left) and UC (right) at the same inocula dilution (10−3). (D) DENV positive C6/36 cells by flow cytometry in which cells were infected with SNT (orange) or UC (blue) DENV-2 inocula at different dilutions. PBMCs from healthy donors were stimulated overnight with DENV-2 UC, mock UC or none (unstimulated). (E) mTRAIL expression profile on pDCs gated from PBMCs (overlay) and (F) mTRAIL positive pDCs for four donors induced by mock UC (orange) or DENV-2 UC (blue) using unstimulated (grey fill) pDCs as negative control. Freshly purified pDCs were stimulated overnight with DENV-2 UC, mock UC or not (unstimulated). (G) TRAIL expression induced by different MOIs of DENV-2 UC (blue) using unstimulated cells (grey) as negative control. (H) Purified pDCs positive for mTRAIL expression and (I) IFN-α secretion by unstimulated (grey), mock UC (orange), DENV-2-UC-stimulated pDCs pre-treated (black) or not (blue) with chloroquine, for four donors. Values were submitted to paired t test in which * p<0.05 and ** p<0.005.
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Related In: Results  -  Collection

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

pntd-0002257-g002: Purified DENV-2-induced in vitro mTRAIL expression and IFN-α production by purified plasmacytoid dendritic cells.PBMCs from healthy donors were stimulated overnight with DENV-2, mock or none (unstimulated). (A) mTRAIL expression profile on pDCs gated from PBMCs (overlay) and (B) mTRAIL positive pDCs for three donors induced by mock SNT (orange) or DENV-2 SNT (blue) using unstimulated (grey fill) pDCs as negative control. DENV positive C6/36 cells infected for 48 h with supernatant of DENV-2-infected C6/36 cells (DENV-2 SNT) or ultracentrifuged DENV-2 SNT (DENV-2 UC) as described in M&M and figure S1. (C) DENV antigens/AlexaFluor488 (green) and nucleus/DAPI (blue) of C6/36 cells infected with DENV-2 SNT (left) and UC (right) at the same inocula dilution (10−3). (D) DENV positive C6/36 cells by flow cytometry in which cells were infected with SNT (orange) or UC (blue) DENV-2 inocula at different dilutions. PBMCs from healthy donors were stimulated overnight with DENV-2 UC, mock UC or none (unstimulated). (E) mTRAIL expression profile on pDCs gated from PBMCs (overlay) and (F) mTRAIL positive pDCs for four donors induced by mock UC (orange) or DENV-2 UC (blue) using unstimulated (grey fill) pDCs as negative control. Freshly purified pDCs were stimulated overnight with DENV-2 UC, mock UC or not (unstimulated). (G) TRAIL expression induced by different MOIs of DENV-2 UC (blue) using unstimulated cells (grey) as negative control. (H) Purified pDCs positive for mTRAIL expression and (I) IFN-α secretion by unstimulated (grey), mock UC (orange), DENV-2-UC-stimulated pDCs pre-treated (black) or not (blue) with chloroquine, for four donors. Values were submitted to paired t test in which * p<0.05 and ** p<0.005.
Mentions: PDC activation by DENV-2 was shown to occur by TLR-7 stimulation after endocytosis [31] and this pathway was crucial for IKpDC transformation by HTLV-1 [48]. To assess pDC activation by DENV-2, peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated overnight with virus. Initially, we observed that DENV-2 from mosquito cell line supernatant (SNT) promoted a trend, however not statistically significant, in TRAIL detection on pDC surface after viral stimulation in PBMCs, compared to unstimulated or mock-stimulated pDCs (figure 2A and B). Thereafter, an ultracentrifugation of DENV-2 viral stock was performed in order to concentrate viral particles, increasing MOI (figure S1). The DENV-2 infectivity was assayed for both viral stocks by infecting the mosquito cell line C6/36 and comparing them in serial dilutions. Viral antigens were detected inside cells inoculated with concentrated DENV-2 (UC) as early as 48 hours and at higher frequencies than the non-concentrated supernatant indicating that the concentrated virus had enhanced replication rates and it was intracellularly present as detected by immunofluorescence microscopy and flow cytometry (figure 2C and D). This viral stock (DENV-2 UC) was therefore adopted for assessing DENV-2 induced pDC activation in all experiments described in the present work.

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