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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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TRAIL localization in DENV-2-activated pDCs by 3D microscopy analysis.Freshly purified pDCs stimulated with DENV-2 pre-treated or not with chloroquine (Chloro), or mock infected. TRAIL expression was analyzed by flow cytometry or by a 3D microscope. (A) Membrane TRAIL flow cytometry profiles (left column) on pDC stimulated by different stimuli -mock, DENV-2 or DENV-2+Chloro overlaid by unstimulated (grey). Microscopic images from pDC cultured with the mock, DENV-2 or DENV-2+Chloro showing DAPI-colored nucleus, TRAIL staining (green) and overlay. (B) 3D interactive surface plots analysis of 3D microscopic image. Overlay of nucleus (blue), TRAIL (green) and phase contrast (grey) as seen in (C) for different stimuli: DENV-2-stimulated pDC (DENV-2) exhibits membrane TRAIL localization in contrast to DENV-2+Chloro or unstimulated, where TRAIL is detected only intracellularly. (D) Percentage of pDCs expressing intracellular TRAIL only (Intra) or on the membrane (Mb) is shown as percentage of total analyzed cells. Values were submitted to paired t test in which * p<0.05 and *** p<0.0005.
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pntd-0002257-g003: TRAIL localization in DENV-2-activated pDCs by 3D microscopy analysis.Freshly purified pDCs stimulated with DENV-2 pre-treated or not with chloroquine (Chloro), or mock infected. TRAIL expression was analyzed by flow cytometry or by a 3D microscope. (A) Membrane TRAIL flow cytometry profiles (left column) on pDC stimulated by different stimuli -mock, DENV-2 or DENV-2+Chloro overlaid by unstimulated (grey). Microscopic images from pDC cultured with the mock, DENV-2 or DENV-2+Chloro showing DAPI-colored nucleus, TRAIL staining (green) and overlay. (B) 3D interactive surface plots analysis of 3D microscopic image. Overlay of nucleus (blue), TRAIL (green) and phase contrast (grey) as seen in (C) for different stimuli: DENV-2-stimulated pDC (DENV-2) exhibits membrane TRAIL localization in contrast to DENV-2+Chloro or unstimulated, where TRAIL is detected only intracellularly. (D) Percentage of pDCs expressing intracellular TRAIL only (Intra) or on the membrane (Mb) is shown as percentage of total analyzed cells. Values were submitted to paired t test in which * p<0.05 and *** p<0.0005.

Mentions: To better characterize DENV-2-activated pDCs, we analyzed them by 3-dimension (3D) microscopy. Focal plane analysis revealed the presence of intracellular TRAIL expression in unstimulated pDC (figure 3A, upper panels), confirming our cytometry data and our previous study [48]. Images also revealed some ‘peripheral’ TRAIL expression that did not seem to be localized in the cytoplasm but rather on the membrane (figure 3A, middle panels). TRAIL expression profile in DENV-2-stimulated pDC did not seem to differ from unstimulated cells, even if TRAIL appeared to be decreased in the cytoplasm at the expense of “peripheral” TRAIL (figure 3A, middle panels). However, it remained hard to distinguish between intracellular and membrane TRAIL profile expression in both conditions without the use of a membrane marker. The blocking of endosomal acidification by chloroquine use revealed the same profile as mock-stimulated pDCs.


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)

TRAIL localization in DENV-2-activated pDCs by 3D microscopy analysis.Freshly purified pDCs stimulated with DENV-2 pre-treated or not with chloroquine (Chloro), or mock infected. TRAIL expression was analyzed by flow cytometry or by a 3D microscope. (A) Membrane TRAIL flow cytometry profiles (left column) on pDC stimulated by different stimuli -mock, DENV-2 or DENV-2+Chloro overlaid by unstimulated (grey). Microscopic images from pDC cultured with the mock, DENV-2 or DENV-2+Chloro showing DAPI-colored nucleus, TRAIL staining (green) and overlay. (B) 3D interactive surface plots analysis of 3D microscopic image. Overlay of nucleus (blue), TRAIL (green) and phase contrast (grey) as seen in (C) for different stimuli: DENV-2-stimulated pDC (DENV-2) exhibits membrane TRAIL localization in contrast to DENV-2+Chloro or unstimulated, where TRAIL is detected only intracellularly. (D) Percentage of pDCs expressing intracellular TRAIL only (Intra) or on the membrane (Mb) is shown as percentage of total analyzed cells. Values were submitted to paired t test in which * p<0.05 and *** p<0.0005.
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Related In: Results  -  Collection

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pntd-0002257-g003: TRAIL localization in DENV-2-activated pDCs by 3D microscopy analysis.Freshly purified pDCs stimulated with DENV-2 pre-treated or not with chloroquine (Chloro), or mock infected. TRAIL expression was analyzed by flow cytometry or by a 3D microscope. (A) Membrane TRAIL flow cytometry profiles (left column) on pDC stimulated by different stimuli -mock, DENV-2 or DENV-2+Chloro overlaid by unstimulated (grey). Microscopic images from pDC cultured with the mock, DENV-2 or DENV-2+Chloro showing DAPI-colored nucleus, TRAIL staining (green) and overlay. (B) 3D interactive surface plots analysis of 3D microscopic image. Overlay of nucleus (blue), TRAIL (green) and phase contrast (grey) as seen in (C) for different stimuli: DENV-2-stimulated pDC (DENV-2) exhibits membrane TRAIL localization in contrast to DENV-2+Chloro or unstimulated, where TRAIL is detected only intracellularly. (D) Percentage of pDCs expressing intracellular TRAIL only (Intra) or on the membrane (Mb) is shown as percentage of total analyzed cells. Values were submitted to paired t test in which * p<0.05 and *** p<0.0005.
Mentions: To better characterize DENV-2-activated pDCs, we analyzed them by 3-dimension (3D) microscopy. Focal plane analysis revealed the presence of intracellular TRAIL expression in unstimulated pDC (figure 3A, upper panels), confirming our cytometry data and our previous study [48]. Images also revealed some ‘peripheral’ TRAIL expression that did not seem to be localized in the cytoplasm but rather on the membrane (figure 3A, middle panels). TRAIL expression profile in DENV-2-stimulated pDC did not seem to differ from unstimulated cells, even if TRAIL appeared to be decreased in the cytoplasm at the expense of “peripheral” TRAIL (figure 3A, middle panels). However, it remained hard to distinguish between intracellular and membrane TRAIL profile expression in both conditions without the use of a membrane marker. The blocking of endosomal acidification by chloroquine use revealed the same profile as mock-stimulated pDCs.

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