Date Published: October 1, 2008
Publisher: Public Library of Science
Author(s): Wing-Hong Kwan, Erika Navarro-Sanchez, Hélène Dumortier, Marion Decossas, Hortense Vachon, Flavia Barreto dos Santos, Hervé W. Fridman, Félix A. Rey, Eva Harris, Philippe Despres, Christopher G. Mueller, Ricardo Toshio Fujiwara
Abstract: BackgroundAn 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 FindingsHere, 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-α 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.ConclusionsAborting 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.
Partial Text: Dengue is probably the most important mosquito-transmitted viral disease of humans worldwide. It is caused by dengue virus (DV), which exists as four serotypes (DV1-4) and circulates in an endemic-epidemic mode in most tropical and sub-tropical territories. Transmission of DV to humans occurs when an infected mosquito probes for blood vessels and during a blood meal, through injection of infectious saliva into the human dermis. As a member of the Flaviviridae family, DV infection involves virus uptake into endosomal vesicles that undergo acidification. The low pH induces structural alterations in the envelope (E) protein that lead to membrane fusion and the release of the nucleocapsid into the cytoplasm . After uncoating, the RNA genome is translated to initiate virus replication. It has been proposed that non-neutralizing antibodies raised against one DV serotype may enhance infection by a heterotypic serotype . This may explain why secondary infections are often associated with the more severe forms of dengue fever (hemorrhagic fever with or without shock).
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 –. 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.
In the present study, we demonstrated for the first time the interaction of dMφ with DV3 sE glycoprotein, which correlates with the expression of the DV attachment receptor CD209. Dermal DC displayed only a limited capacity to interact with DV3 sE protein and expressed little CD209. In accordance with these findings, in situ immuno-labeling of human skin section revealed CD209 expression by dMφ but little on DC –. Both cell types carry the MR , which also recognizes DV E protein . Due to the nature of our binding assay, the dermal cells with the highest affinity for DV3 sE protein would acquire the most DV3 sE protein, suggesting that dDC may capture the recombinant envelope protein when physically isolated from dMφ. In the skin, the abundance, the location and the co-expression of CD209, L-SIGN and MR are likely to determine the nature of the DV-capturing immune cell.