Much research on DV relies on relevant human cell culture models due to the difficulty of establishing appropriate animal models. Progress has been made by showing that DV E protein recognizes the C-type lectin CD209 and its homologue L-SIGN and that expression of either of these lectins is sufficient to render cells permissive to DV grown in mosquito cells [3],[4]. Recently, the mannose receptor (MR) has also been shown to mediate DV binding and infection [5]. Dendritic cells (DC), generated from monocytes in the presence of GM-CSF and IL-4, express CD209, L-SIGN and the MR and are highly susceptible to DV infection [3],[4],[6]. These monocyte-derived DC are thought to be representative of dermal DC (dDC), yet there is increasing evidence that CD209 is not expressed by dDC but primarily by dermal macrophages (dM) [7]–[9]. This underscores the importance of dM in early infection events and raises the question of whether dM are permissive for productive DV infection. Studies of these cells have been hampered by the lack of suitable isolation techniques from human skin and culture methods to generate the cells from monocytic precursors.

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 (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 M expressed coagulation factor XIIIa and CD163, two other cell surface markers of dM [14] (Fig. 2C). The M 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 M rapidly released IL-10, whereas DC or monocytes produced little of this cytokine (Figure S1C).

Monocyte-derived dM (MDdM) and monocyte-derived DC (MDDC) were analyzed for DV infection using low-passage DV1 and DV3 strains grown in mosquito cells [3] as well as the prototype DV2 strain 16681 [15]. 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 Figure 3A, 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) (Fig. 3B); in contrast, progeny virus production was undetectable in DV-infected MDdM (<10³ 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 [16] (Fig. 3C). 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 (Fig. 3D). 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.

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 (Fig. 4). 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 (Fig. 5). 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.

Based on the observations that dM stained for intracellular IL-10 in situ and that IL-10 is produced by dM ex vivo [17],[18], we tested the effect of IL-10 on the formation of dM from monocytes. By combining IL-10, M-CSF and GM-CSF, a homogenous cell population was obtained which carried CD209 and other markers characteristic of dM, rapidly produced IL-10 in response to LPS or other toll-like receptor ligands (data not shown), and bound DV3 sE protein. Like MDDC, the MDdM were capable of internalizing live DV but, distinct from MDDC, they displayed an inherent resistance to viral growth. In contrast to DV3 sE protein found in acidified compartments in MDDC, we observed that DV3 sE protein accumulated in non-acidified phagosomes in MDdM. The DC vesicles containing DV3 sE protein or live virus were bell-shaped or tubular, whereas they were round, larger and close to the plasma membrane in the M. To our knowledge, this identifies MDdM as the first innate immune cell capable of protecting the human host from DV infection and virus propagation. From this data, we propose that dM can act to trap infecting virions in a fusion-incompetent endosomal environment and thus to prevent DV spread to dDC at the anatomical site of the mosquito bite. We cannot formally exclude the possibility that downstream delays in the viral life cycle contribute to the inability of DV to replicate in MDdM, but the finding that West Nile virus productively infects these cells (data not shown) indicates that they are not generally refractory to flavivirus growth.

IL-10, required for CD209 expression and blockage of endosome acidification, is likely to be produced by the dM themselves, constitutively, or in response to stimuli such as UV-light [17]. In this context, a key question is whether mosquito salivary proteins, co-injected with the infectious virus, would also trigger IL-10 production by dM or, on the contrary, provoke an inflammatory response. Inflammatory cytokines of the Th2 T-helper cell type, IL-4 and IL-13, may be responsible for the formation of CD209⁺MR⁺ DC, which are permissive for DV infection and viral progeny production [3]–[6]. Alternatively, the presence of anti-DV non-neutralizing antibodies raised against a heterotypic DV serotype may render dDC susceptible to DV infection at the site of the mosquito bite.

Figure S1

Alternative Language Abstract S1

Alternative Language Abstract S2

Alternative Language Abstract S3