We wished to determine whether human dM
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 dM
and CD1a by dDC 
. 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 , CD1a+
dDC showed only a limited capacity to interact with DV3 sE protein, whereas CD14+
readily bound the protein. This is corroborated by the distinct expression of CD209 by dM
(), 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. shows that even at high concentrations, there was little binding of DV3 sE protein to dDC, whereas it bound to dM
in a dose-dependent fashion. These findings identify dM
as potential key cellular targets of DV.
To address the question of whether dM
are infected by DV and whether they are permissive for viral production, we established cell culture conditions to generate dermal-type M
from monocytes. We observed on human skin tissue sections that dM
expressing CD14 or CD209, but not the CD1a+
dDC, stained for IL-10 (). 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 (). Similar to DC 
, the addition of GM-CSF increased CD209 levels (), so that a homogeneous CD14+
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 
). The M
expressed coagulation factor XIIIa and CD163, two other cell surface markers of dM 
(). The M
and the DC were both able to bind eGFP-tagged DV3 sE protein, which was inhibited by EDTA (). This distinguishes the monocyte-derived DC from dDC. Upon activation by lipopolysaccharide (LPS), the M
rapidly released IL-10, whereas DC or monocytes produced little of this cytokine (Figure S1C
Figure 2 Generation of dermal-type M with IL-10.
) and monocyte-derived DC (MDDC) were analyzed for DV infection using low-passage DV1 and DV3 strains grown in mosquito cells 
as well as the prototype DV2 strain 16681 
. The cells were exposed to DV1 at a multiplicity of infection (MOI) of 1 for 2 h, washed, and then cultured for 40 h. As shown in , intracellular viral antigen was clearly detected in MDDC by flow cytometry, whereas no specific immuno-labeling was observed in MDdM
. An analysis of DV replication in these cells infected at an MOI of 1 (DV1 and DV3) or 2 (DV2) showed that MDDC were highly permissive to productive infection (~105
FFU/ml or PFU/ml) (); in contrast, progeny virus production was undetectable in DV-infected MDdM
FFU/ml or PFU/ml). Consistent with this finding, no IFN-α was produced by DV-infected MDdM
, even at an MOI of 10, whereas MDDC readily released IFN-α when infected with DV at an MOI of 1 or 10 
(). To verify that MDdM
acquired the virus, both myeloid cell-types were exposed to high DV input (MOI of 100) and electron microscopy analysis was performed after 30 min at 4°C and after 1 h at 37°C (). Cell surface-bound (at 4°C) and endosomal vesicle-associated virus particles (at 37°C) were clearly detected in both cell-types. Thus, internalization of DV can occur in MDdM
but does not result in productive infection.
Figure 3 MDdM are resistant to permissive DV infection.
In an effort to define the molecular basis of the inability of DV to grow in MDdM
. we asked whether internalized DV was sequestered in a manner that hampers productive infection, using DV3 sE-eGFP fusion protein. To monitor DV3 sE protein internalization in MDdM
and MDDC, the cells were incubated with pH-sensitive LysoSensor dye and analyzed by confocal microscopy (). This dye accumulates in acidic organelles, where its fluorescence emission is highest. After 5 min at 37°C, DV3 sE protein was observed in vesicle-like structures in both cell-types. By 30 min and 60 min, DV3 sE protein dispersed to acidified perinuclear lysosomes in MDDC. In marked contrast, when MDdM
were examined at these time-points, a large fraction of internalized DV3 sE protein was excluded from the acidic compartment and remained in non-acidic, large endosomes. Electron microscopy analysis using a colloidal gold-conjugated antibody to GFP demonstrated that DV3 sE protein accumulated in large phagosomes in MDdM
, located close to the plasma membrane (). On the other hand, at 30 min, in MDDC, DV3 sE protein was mostly found in small perinuclear vesicles in the environment of the endoplasmic reticulum. Taken together, these data suggest that the inability of DV to productively infect MDdM
is due to accumulation of virus particles in immature endosomal vesicles whose pH does not allow efficient viral-cell membrane fusion and subsequent virus uncoating.
Figure 4 DV3 sE-eGFP protein is excluded from acidic compartments in MDdM.
Figure 5 DV3 sE-eGFP protein-containing vesicles are distinct in MDDC and MDdM.