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Dermal-type macrophages expressing CD209/DC-SIGN show inherent resistance to dengue virus growth.

Kwan WH, Navarro-Sanchez E, Dumortier H, Decossas M, Vachon H, dos Santos FB, Fridman HW, Rey FA, Harris E, Despres P, Mueller CG - PLoS Negl Trop Dis (2008)

Bottom Line: Here, we showed that dermal macrophages bound recombinant envelope E glycoprotein fused to green fluorescent protein.The macrophages were able to internalize the virus, but progeny virus production was undetectable in the infected cells.In addition, no IFN-alpha was produced in response to the virus.

View Article: PubMed Central - PubMed

Affiliation: CNRS, Laboratory of Therapeutic Immunology and Chemistry, IBMC, Université Louis Pasteur, Strasbourg, France.

ABSTRACT

Background: An important question in dengue pathogenesis is the identity of immune cells involved in the control of dengue virus infection at the site of the mosquito bite. There is evidence that infection of immature myeloid dendritic cells plays a crucial role in dengue pathogenesis and that the interaction of the viral envelope E glycoprotein with CD209/DC-SIGN is a key element for their productive infection. Dermal macrophages express CD209, yet little is known about their role in dengue virus infection.

Methods and findings: Here, we showed that dermal macrophages bound recombinant envelope E glycoprotein fused to green fluorescent protein. Because dermal macrophages stain for IL-10 in situ, we generated dermal-type macrophages from monocytes in the presence of IL-10 to study their infection by dengue virus. The macrophages were able to internalize the virus, but progeny virus production was undetectable in the infected cells. In addition, no IFN-alpha was produced in response to the virus. The inability of dengue virus to grow in the macrophages was attributable to accumulation of internalized virus particles into poorly-acidified phagosomes.

Conclusions: Aborting infection by viral sequestration in early phagosomes would present a novel means to curb infection of enveloped virus and may constitute a prime defense system to prevent dengue virus spread shortly after the bite of the infected mosquito.

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Related in: MedlinePlus

Generation of dermal-type Mφ with IL-10.A. Human CD14+ and CD209+ dMφ stained for IL-10. Formaldehyde-fixed sections of human skin were incubated with anti-IL-10 goat Ab and anti-CD14, anti-CD209 or anti-CD1a mAb. The goat Ab was detected using a biotin-labeled donkey anti-goat Ab, followed by streptavidin-Alexa 488, and the mAbs were revealed by rabbit anti-mouse Ab followed by Cy3-donkey anti-rabbit Ab. The data is representative of 3 donors. The dotted line delimits the dermo-epidermal junction. B. Adult blood monocytes were cultured for 5 days in medium containing fetal calf serum and 10 ng/ml M-CSF without or with a low (10 ng/ml) or a high (30 ng/ml, bold type) concentration of IL-10 in the absence or presence of 30 ng/ml GM-CSF. Non-adherent cells were collected and, after gating for viable cells, were analyzed for expression of CD14 and CD209. Dot plot quadrants were placed according to isotype controls. C. Dermal-type Mφ were generated in M-CSF/IL-10/GM-CSF as in panel B and compared to DC obtained from monocytes in GM-CSF and IL-4. Expression of cell surface markers was measured by FACS. Specific staining is in black and isotype controls are in grey. DV3 sE-eGFP protein binding was measured by FACS after incubating the cells with DV3 sE protein in the absence or presence of EDTA. Shown in black is the fluorescence of cells incubated with DV3 sE-eGFP protein and in grey is fluorescence of cells without the fusion protein.
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pntd-0000311-g002: Generation of dermal-type Mφ with IL-10.A. Human CD14+ and CD209+ dMφ stained for IL-10. Formaldehyde-fixed sections of human skin were incubated with anti-IL-10 goat Ab and anti-CD14, anti-CD209 or anti-CD1a mAb. The goat Ab was detected using a biotin-labeled donkey anti-goat Ab, followed by streptavidin-Alexa 488, and the mAbs were revealed by rabbit anti-mouse Ab followed by Cy3-donkey anti-rabbit Ab. The data is representative of 3 donors. The dotted line delimits the dermo-epidermal junction. B. Adult blood monocytes were cultured for 5 days in medium containing fetal calf serum and 10 ng/ml M-CSF without or with a low (10 ng/ml) or a high (30 ng/ml, bold type) concentration of IL-10 in the absence or presence of 30 ng/ml GM-CSF. Non-adherent cells were collected and, after gating for viable cells, were analyzed for expression of CD14 and CD209. Dot plot quadrants were placed according to isotype controls. C. Dermal-type Mφ were generated in M-CSF/IL-10/GM-CSF as in panel B and compared to DC obtained from monocytes in GM-CSF and IL-4. Expression of cell surface markers was measured by FACS. Specific staining is in black and isotype controls are in grey. DV3 sE-eGFP protein binding was measured by FACS after incubating the cells with DV3 sE protein in the absence or presence of EDTA. Shown in black is the fluorescence of cells incubated with DV3 sE-eGFP protein and in grey is fluorescence of cells without the fusion protein.

Mentions: To address the question of whether dMφ are infected by DV and whether they are permissive for viral production, we established cell culture conditions to generate dermal-type Mφ from monocytes. We observed on human skin tissue sections that dMφ expressing CD14 or CD209, but not the CD1a+ dDC, stained for IL-10 (Fig. 2A). When purified human monocytes were cultured in M-CSF and increasing concentrations of IL-10, the cells expressed CD14 and CD209 in an IL-10 dose-dependent manner (Fig. 2B). Similar to DC [12], the addition of GM-CSF increased CD209 levels (Fig. 2B), so that a homogeneous CD14+CD209+ cell population could be obtained with CD209 expression nearly identical to that of DC derived from monocytes in the presence of GM-CSF and IL-4 (Figure S1A). Western blotting of cell lysates confirmed the presence of CD209 as a major band of 49 kDa in both cell-types [13] (Figure S1B). The Mφ expressed coagulation factor XIIIa and CD163, two other cell surface markers of dMφ [14] (Fig. 2C). The Mφ and the DC were both able to bind eGFP-tagged DV3 sE protein, which was inhibited by EDTA (Fig. 2C). This distinguishes the monocyte-derived DC from dDC. Upon activation by lipopolysaccharide (LPS), the Mφ rapidly released IL-10, whereas DC or monocytes produced little of this cytokine (Figure S1C).


Dermal-type macrophages expressing CD209/DC-SIGN show inherent resistance to dengue virus growth.

Kwan WH, Navarro-Sanchez E, Dumortier H, Decossas M, Vachon H, dos Santos FB, Fridman HW, Rey FA, Harris E, Despres P, Mueller CG - PLoS Negl Trop Dis (2008)

Generation of dermal-type Mφ with IL-10.A. Human CD14+ and CD209+ dMφ stained for IL-10. Formaldehyde-fixed sections of human skin were incubated with anti-IL-10 goat Ab and anti-CD14, anti-CD209 or anti-CD1a mAb. The goat Ab was detected using a biotin-labeled donkey anti-goat Ab, followed by streptavidin-Alexa 488, and the mAbs were revealed by rabbit anti-mouse Ab followed by Cy3-donkey anti-rabbit Ab. The data is representative of 3 donors. The dotted line delimits the dermo-epidermal junction. B. Adult blood monocytes were cultured for 5 days in medium containing fetal calf serum and 10 ng/ml M-CSF without or with a low (10 ng/ml) or a high (30 ng/ml, bold type) concentration of IL-10 in the absence or presence of 30 ng/ml GM-CSF. Non-adherent cells were collected and, after gating for viable cells, were analyzed for expression of CD14 and CD209. Dot plot quadrants were placed according to isotype controls. C. Dermal-type Mφ were generated in M-CSF/IL-10/GM-CSF as in panel B and compared to DC obtained from monocytes in GM-CSF and IL-4. Expression of cell surface markers was measured by FACS. Specific staining is in black and isotype controls are in grey. DV3 sE-eGFP protein binding was measured by FACS after incubating the cells with DV3 sE protein in the absence or presence of EDTA. Shown in black is the fluorescence of cells incubated with DV3 sE-eGFP protein and in grey is fluorescence of cells without the fusion protein.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2553280&req=5

pntd-0000311-g002: Generation of dermal-type Mφ with IL-10.A. Human CD14+ and CD209+ dMφ stained for IL-10. Formaldehyde-fixed sections of human skin were incubated with anti-IL-10 goat Ab and anti-CD14, anti-CD209 or anti-CD1a mAb. The goat Ab was detected using a biotin-labeled donkey anti-goat Ab, followed by streptavidin-Alexa 488, and the mAbs were revealed by rabbit anti-mouse Ab followed by Cy3-donkey anti-rabbit Ab. The data is representative of 3 donors. The dotted line delimits the dermo-epidermal junction. B. Adult blood monocytes were cultured for 5 days in medium containing fetal calf serum and 10 ng/ml M-CSF without or with a low (10 ng/ml) or a high (30 ng/ml, bold type) concentration of IL-10 in the absence or presence of 30 ng/ml GM-CSF. Non-adherent cells were collected and, after gating for viable cells, were analyzed for expression of CD14 and CD209. Dot plot quadrants were placed according to isotype controls. C. Dermal-type Mφ were generated in M-CSF/IL-10/GM-CSF as in panel B and compared to DC obtained from monocytes in GM-CSF and IL-4. Expression of cell surface markers was measured by FACS. Specific staining is in black and isotype controls are in grey. DV3 sE-eGFP protein binding was measured by FACS after incubating the cells with DV3 sE protein in the absence or presence of EDTA. Shown in black is the fluorescence of cells incubated with DV3 sE-eGFP protein and in grey is fluorescence of cells without the fusion protein.
Mentions: To address the question of whether dMφ are infected by DV and whether they are permissive for viral production, we established cell culture conditions to generate dermal-type Mφ from monocytes. We observed on human skin tissue sections that dMφ expressing CD14 or CD209, but not the CD1a+ dDC, stained for IL-10 (Fig. 2A). When purified human monocytes were cultured in M-CSF and increasing concentrations of IL-10, the cells expressed CD14 and CD209 in an IL-10 dose-dependent manner (Fig. 2B). Similar to DC [12], the addition of GM-CSF increased CD209 levels (Fig. 2B), so that a homogeneous CD14+CD209+ cell population could be obtained with CD209 expression nearly identical to that of DC derived from monocytes in the presence of GM-CSF and IL-4 (Figure S1A). Western blotting of cell lysates confirmed the presence of CD209 as a major band of 49 kDa in both cell-types [13] (Figure S1B). The Mφ expressed coagulation factor XIIIa and CD163, two other cell surface markers of dMφ [14] (Fig. 2C). The Mφ and the DC were both able to bind eGFP-tagged DV3 sE protein, which was inhibited by EDTA (Fig. 2C). This distinguishes the monocyte-derived DC from dDC. Upon activation by lipopolysaccharide (LPS), the Mφ rapidly released IL-10, whereas DC or monocytes produced little of this cytokine (Figure S1C).

Bottom Line: Here, we showed that dermal macrophages bound recombinant envelope E glycoprotein fused to green fluorescent protein.The macrophages were able to internalize the virus, but progeny virus production was undetectable in the infected cells.In addition, no IFN-alpha was produced in response to the virus.

View Article: PubMed Central - PubMed

Affiliation: CNRS, Laboratory of Therapeutic Immunology and Chemistry, IBMC, Université Louis Pasteur, Strasbourg, France.

ABSTRACT

Background: An important question in dengue pathogenesis is the identity of immune cells involved in the control of dengue virus infection at the site of the mosquito bite. There is evidence that infection of immature myeloid dendritic cells plays a crucial role in dengue pathogenesis and that the interaction of the viral envelope E glycoprotein with CD209/DC-SIGN is a key element for their productive infection. Dermal macrophages express CD209, yet little is known about their role in dengue virus infection.

Methods and findings: Here, we showed that dermal macrophages bound recombinant envelope E glycoprotein fused to green fluorescent protein. Because dermal macrophages stain for IL-10 in situ, we generated dermal-type macrophages from monocytes in the presence of IL-10 to study their infection by dengue virus. The macrophages were able to internalize the virus, but progeny virus production was undetectable in the infected cells. In addition, no IFN-alpha was produced in response to the virus. The inability of dengue virus to grow in the macrophages was attributable to accumulation of internalized virus particles into poorly-acidified phagosomes.

Conclusions: Aborting infection by viral sequestration in early phagosomes would present a novel means to curb infection of enveloped virus and may constitute a prime defense system to prevent dengue virus spread shortly after the bite of the infected mosquito.

Show MeSH
Related in: MedlinePlus