Dengue virus inoculation to human skin explants: an effective approach to assess in situ the early infection and the effects on cutaneous dendritic cells

Abstract

Although dengue virus (DV) enters through skin while mosquitoes feed, early contacts remain unexplored regarding the cutaneous viral fate and in situ immune responses. We addressed this by exposing healthy, non-cadaveric, freshly obtained human skin explants to a human DV2 isolate. We demonstrated negative-strand DV-RNA and non-structural protein-1, both suggestive of viral replication in skin. Although control, mock-infected and DV-infected explants showed less (MHC-CII(+)/CD1a(+)/Langerin+) Langerhans cells, deranged morphology and decreased frequency were more apparent in DV-infected explants. Whereas DV+ cells were infrequent in epidermis and completely absent in dermis, some areas of basal epidermis were clearly DV+, presumably keratinocytes, cells where TUNEL positivity revealed apoptosis. Unlike fresh, control and mock-infected skin, DV-infected explants expressed CD80 and CD83, indicative of dendritic cell (DC) activation and maturation, respectively. However, sequential sections indicated that these cells were not DV+, suggesting that activated/mature DCs capable of priming T cells, probably, were not infected. Alternatively, the occasionally infected epidermal DC might not have reached maturation. Interestingly, skin DV infection apparently uncouples the DC activation/maturation process from another crucial DC function, the subsequent migration into dermis. This was suggested, because upon cutaneous DV infection, the few emerging CD83+ (mature) DCs remained within the outer epidermis, while no dermal CD83+ DCs were observed. These paradoxical effects might represent unknown DV subversion strategies. This approach is relatively easy, quick (results in 48 h), economical for developing countries where dengue is re-emerging and advantageous to evaluate in situ viral biology, immunity and immunopathology and potential antiviral strategies.

Gamma Interferon (IFN-γ) Receptor Restricts Systemic Dengue Virus Replication and Prevents Paralysis in IFN-α/β Receptor-Deficient Mice

“…Levels of DENV RNA in most tissues of the A129 mice did not significantly change between 2 and 4 days after infection, with the exception of the skin, in which the levels of virus increased greatly. This observation is interesting considering the skin is an important site of early DENV replication in humans (27,47). Overall, these data demonstrate that IFN-␥R activity causes decreased systemic levels of virus, which is likely a result of reducing early viral replication in the spleen and bone marrow and then maintaining an antiviral state to limit DENV dissemination and further systemic replication.…”

West Nile Virus Capsid Degradation of Claudin Proteins Disrupts Epithelial Barrier Function

“…It is likely that epithelial cells will be infected soon after the bite of a carrier mosquito (i.e., keratinocytes [29]). In infected mice, replication of the virus in kidney tubules has been demonstrated before the virus can be detected in the brain (20), and in humans and hamsters, virus can be detected in the epithelial cells of the kidney and other tissues for weeks p.i.…”

The pathogenesis of dengue

“…After the bite of an infected mosquito it is thought that initially immature Langerhan's cells in the dermis are infected first. 124 These infected cells migrate to the lymph nodes, resulting in infection of cells of the macrophage-monocyte lineage. This amplifies the infection, which is then disseminated via the lymphatic and vascular system.…”