Dengue virus inoculation to human skin explants: an effective approach to assess in situ the early infection and the effects on cutaneous dendritic cells - PubMed (original) (raw)
doi: 10.1111/j.0959-9673.2005.00445.x.
Mayra Perez-Tapia, Iris Estrada-Garcia, Gilberto Vaughan, Alejandro Escobar-Gutierrez, Juana Calderon-Amador, Sara Elisa Herrera-Rodriguez, Adriana Brizuela-Garcia, Monica Heras-Chavarria, Adriana Flores-Langarica, Leticia Cedillo-Barron, Leopoldo Flores-Romo
Affiliations
- PMID: 16191104
- PMCID: PMC2517443
- DOI: 10.1111/j.0959-9673.2005.00445.x
Dengue virus inoculation to human skin explants: an effective approach to assess in situ the early infection and the effects on cutaneous dendritic cells
Alberto Yairh Limon-Flores et al. Int J Exp Pathol. 2005 Oct.
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.
Figures
Figure 1
Cytopathic effects of the new dengue virus 2 (DV2) clinical isolate on target insect cells and RT-PCR from conventional target mosquito cells and from cultured explants of human skin. The classical mosquito cell-line C6/36 (Aedes albopictus) was used to test whether this new viral isolate from a patient would infect a conventional target cell. 107 C6/36 cells were infected using a multiplicity of infection of 0.1 PFU/cell in tissue-culture flasks of 25 cm2 (Corning, NY, USA) and humid atmosphere with 5% CO2 at 37 °C for 2 days. DV2 was added, since the beginning of culture, and 24 h later, the typical cytopathic effects were observed (b), compared with control non-infected cultures (a). To detect the negative-strand DV-RNA indicative of active viral replication, RT-PCR was done with RNA obtained from control-cultured non-inoculated and DV-inoculated skin explants, as well as conventional target (C6/36) mosquito cells. Lane 1: molecular weight markers, lane 2: cultured non-inoculated skin explants, lane 3: DV-inoculated explants cultured for 48 h, lane 4: cultured non-inoculated explants (72 h), lane 5: DV-inoculated explants cultured for 72 h, lane 6: mosquito cells infected with DV2 isolate and lane 7: PCR negative control. Arrowhead indicates the negative-strand viral amplicon (119 bp size). Results are representative of five different experiments from skin samples obtained from five individuals.
Figure 4
Non-structural protein-1 (NS1) can be identified only in dengue virus (DV)-inoculated explants. Tissue sections obtained from the skin explants under different treatments were all incubated with a murine monoclonal antibody to recombinant NS1 from DV. Positivity was developed with peroxidase to obtain blue dark colour, while counterstaining was performed with nuclear fast red, providing a reddish staining. (a) illustrates sections from freshly prepared skin, (b) skin sections from controlcultured explants, (c) skin from mock-infected explants and (d) the blue dark labelling obtained in explants inoculated with DV. Pictures are representative of three different subjects assessed.
Figure 2
Effects of dengue virus 2 (DV2) infection upon epidermal dendritic Langerhans cells (LC). Monoclonal antibodies to MHC-CII (DR), CD1a and Langerin were used to assess epidermal LC. (a) Far left panel (top to bottom pictures) shows epidermal sheets from freshly obtained skin; middle left panel (top to bottom) represents non-inoculated cultured explants; middle right panel (top to bottom) represents mock-infected explants; far right panel (top to bottom) illustrates DV-inoculated skin explants. Pictures were taken at 5 days of culture and are representative examples of five different skin samples evaluated. (b) MHC-CII, CD1a and Langerin were used as markers to quantify LC frequency per area in each of these different experimental conditions. Closed bars indicate LC frequency in epidermal sheets from freshly (non-cultured, non-infected) obtained skin; open bars indicate LC in cultured non-infected skin; grey bars illustrate LC in mock-infected explants; squared bars indicate LC in DV-infected skin explants. Results are expressed as the mean ± SD of five different experiments from skin samples obtained from five individuals. Statistically significant differences are indicated by asterisks (***P < 0.001).
Figure 3
Dengue virus 2 positive (DV2+) cells can be identified in the basal layer of the epidermis. Conventional sections of skin explants cultured for 5 days after DV2 inoculation clearly revealed DV positivity in many cells of the basal epidermis (b, square). Inset (c) shows the intense blue dark labelling in basal cells of epidermis, while very few DV+ cells were identified in the suprabasal layer (b, small arrows), presumably Langerhans cells. In contrast, no DV+ blue cells were observed in the dermis (b, large open arrow). Lack of staining with an isotype-matched control antibody is shown (a). Inset in (c) represents a magnification from (b) to illustrate the typical DV (dark blue) labelling representative of skin samples from five different individuals.
Figure 5
Infection with new dengue virus 2 (DV2) induces the appearance of (activation) CD80 and (maturation) CD83 molecules in epidermis. Conventional skin sections of DV2-infected explants (b–d, f–h) revealed the emergence of CD83+-mature dendritic cell (DC) (blue labelling in b-d, g and arrows in b) and of CD80+-activated DC (blue labelling in f) confined to the epidermis, but without apparent correspondence to the scant and weak DV positivity found (faint blue labelling in h, arrows) in skin serial sections of DV-inoculated explants. (c) represents a magnification of b, while d is a magnification of another skin explant. No CD83+-mature DCs were found in the dermis of DV2-infected explants (b, c, d, g). Absence of mature CD83+ DC and of CD80 is also observed in control-cultured non-infected explants (a and e, respectively). Pictures were taken at 5 days after culture initiation and are representatives of five skin samples from different individuals. Dotted line (b) indicates the dermo-epidermal junction, and brownish colour is due to melanin+ cells in these skin types.
Figure 6
Cutaneous dengue virus 2 (DV2) infection induces apoptosis (TUNEL positivity) mainly in the epidermis. The TUNEL technique was used to assess apoptotic cell death following DV2 infection. (a) shows TUNEL-negative control-skin explants (no Dnase), whereas (b) illustrates TUNEL-positive control sample treated with Dnase, indicated by the green fluorescence. (c) shows TUNEL results in skin from control-cultured non-infected explants, whereas (d) shows skin from DV2-infected explants. (e) represents a magnification of (c), while (f) is a magnification of (d). Arrows in (d) indicate the outer part of epidermis where TUNEL positivity is more apparent in DV-infected skin, whereas arrows in (c) illustrate the lack of apoptosis in the corresponding areas of control skin. Results are representative of three different experiments in samples from three different subjects.
References
- Avirutnan P, Malasit P, Seliger B, Bhakdi S, Husmann M. Dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis. J Immunol. 1998;161:6338–6346. - PubMed
- Bachmann MF, Zinkernagel RM, Oxenius A. Immune responses in the absence of costimulation: viruses know the trick. J Immunol. 1998;161:5791–5794. - PubMed
- Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature. 1998;392:245–252. - PubMed
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