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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

Dermal Mφ bind DV3 sE protein.A. The single-cell dermal suspension was incubated without (None) or with 2 µg of DV3 sE-eGFP fusion protein for 30 min at 37°C, washed, labeled for CD1a and CD14, and analyzed by flow cytometry. Live and large cells were pre-gated using the FSC/SSC channels. To test for CD209 expression, the cell suspension was incubated with anti-CD209 mAb or its isotype control together with anti-CD14 mAb and analyzed by flow cytometry. Arrows point to DV3 sE protein+ cells. The data is representative of 4 donors. B. The cell suspension was incubated with increasing amounts of DV3 sE-eGFP protein before labeling for CD1a, CD14 and HLA-DR. The relative mean fluorescence intensity (MFI) of DV3 sE protein was determined for CD14+HLA-DR+ (dMφ) and CD1a+HLA-DR+ (dDC) cells and shown as a function of DV3 sE-eGFP protein concentration. The analysis was performed in triplicate (mean±SD) for 3 donors.
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pntd-0000311-g001: Dermal Mφ bind DV3 sE protein.A. The single-cell dermal suspension was incubated without (None) or with 2 µg of DV3 sE-eGFP fusion protein for 30 min at 37°C, washed, labeled for CD1a and CD14, and analyzed by flow cytometry. Live and large cells were pre-gated using the FSC/SSC channels. To test for CD209 expression, the cell suspension was incubated with anti-CD209 mAb or its isotype control together with anti-CD14 mAb and analyzed by flow cytometry. Arrows point to DV3 sE protein+ cells. The data is representative of 4 donors. B. The cell suspension was incubated with increasing amounts of DV3 sE-eGFP protein before labeling for CD1a, CD14 and HLA-DR. The relative mean fluorescence intensity (MFI) of DV3 sE protein was determined for CD14+HLA-DR+ (dMφ) and CD1a+HLA-DR+ (dDC) cells and shown as a function of DV3 sE-eGFP protein concentration. The analysis was performed in triplicate (mean±SD) for 3 donors.

Mentions: We wished to determine whether human dMφ are targets of DV infection. To this end, healthy human skin from patients undergoing plastic surgery was processed to obtain a dermal cell suspension. The cells were then cultured without additional cytokines for 48 h to allow re-expression of cell surface markers, such as CD1a and CD209, lost during the collagenase treatment (data not shown). Binding of DV3 E protein to dermal cells was assessed by flow cytometry after staining with CD14 and CD1a-specific antibodies. CD14 is expressed by dMφ and CD1a by dDC [7]–[9]. To detect E protein binding, the soluble form of DV3 E protein (sE) was fused to the reporter protein eGFP and purified from a Drosophila expression system. As shown in Figure 1A, CD1a+ dDC showed only a limited capacity to interact with DV3 sE protein, whereas CD14+ dMφ readily bound the protein. This is corroborated by the distinct expression of CD209 by dMφ (Fig. 1A), whereas dDC expressed little, if any, CD209 (data not shown). Increasing amounts of DV3 sE protein were added to the dermal cell suspension to test if dDC bound the protein at higher concentrations. Figure 1B shows that even at high concentrations, there was little binding of DV3 sE protein to dDC, whereas it bound to dMφ in a dose-dependent fashion. These findings identify dMφ as potential key cellular targets of DV.


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)

Dermal Mφ bind DV3 sE protein.A. The single-cell dermal suspension was incubated without (None) or with 2 µg of DV3 sE-eGFP fusion protein for 30 min at 37°C, washed, labeled for CD1a and CD14, and analyzed by flow cytometry. Live and large cells were pre-gated using the FSC/SSC channels. To test for CD209 expression, the cell suspension was incubated with anti-CD209 mAb or its isotype control together with anti-CD14 mAb and analyzed by flow cytometry. Arrows point to DV3 sE protein+ cells. The data is representative of 4 donors. B. The cell suspension was incubated with increasing amounts of DV3 sE-eGFP protein before labeling for CD1a, CD14 and HLA-DR. The relative mean fluorescence intensity (MFI) of DV3 sE protein was determined for CD14+HLA-DR+ (dMφ) and CD1a+HLA-DR+ (dDC) cells and shown as a function of DV3 sE-eGFP protein concentration. The analysis was performed in triplicate (mean±SD) for 3 donors.
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Related In: Results  -  Collection

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pntd-0000311-g001: Dermal Mφ bind DV3 sE protein.A. The single-cell dermal suspension was incubated without (None) or with 2 µg of DV3 sE-eGFP fusion protein for 30 min at 37°C, washed, labeled for CD1a and CD14, and analyzed by flow cytometry. Live and large cells were pre-gated using the FSC/SSC channels. To test for CD209 expression, the cell suspension was incubated with anti-CD209 mAb or its isotype control together with anti-CD14 mAb and analyzed by flow cytometry. Arrows point to DV3 sE protein+ cells. The data is representative of 4 donors. B. The cell suspension was incubated with increasing amounts of DV3 sE-eGFP protein before labeling for CD1a, CD14 and HLA-DR. The relative mean fluorescence intensity (MFI) of DV3 sE protein was determined for CD14+HLA-DR+ (dMφ) and CD1a+HLA-DR+ (dDC) cells and shown as a function of DV3 sE-eGFP protein concentration. The analysis was performed in triplicate (mean±SD) for 3 donors.
Mentions: We wished to determine whether human dMφ are targets of DV infection. To this end, healthy human skin from patients undergoing plastic surgery was processed to obtain a dermal cell suspension. The cells were then cultured without additional cytokines for 48 h to allow re-expression of cell surface markers, such as CD1a and CD209, lost during the collagenase treatment (data not shown). Binding of DV3 E protein to dermal cells was assessed by flow cytometry after staining with CD14 and CD1a-specific antibodies. CD14 is expressed by dMφ and CD1a by dDC [7]–[9]. To detect E protein binding, the soluble form of DV3 E protein (sE) was fused to the reporter protein eGFP and purified from a Drosophila expression system. As shown in Figure 1A, CD1a+ dDC showed only a limited capacity to interact with DV3 sE protein, whereas CD14+ dMφ readily bound the protein. This is corroborated by the distinct expression of CD209 by dMφ (Fig. 1A), whereas dDC expressed little, if any, CD209 (data not shown). Increasing amounts of DV3 sE protein were added to the dermal cell suspension to test if dDC bound the protein at higher concentrations. Figure 1B shows that even at high concentrations, there was little binding of DV3 sE protein to dDC, whereas it bound to dMφ in a dose-dependent fashion. These findings identify dMφ as potential key cellular targets of DV.

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