1. Interplay between Keratinocytes and Myeloid Cells Drives Dengue Virus Spread in Human Skin 
Parichat Duangkhae, Geza Erdos, Kate D. Ryman, Simon C. Watkins, Louis D. Falo, Ernesto T.A. Marques, Simon M. Barratt-Boyes 
Journal of Investigative Dermatology 
Volume 138, Issue 3, Pages (March 2018)
DOI: /j.jid
Copyright © 2017 The Authors Terms and Conditions

2. Figure 1
Dengue virus (DENV) replicates primarily in the epidermis and induces transient IFN-α expression. (a) NS3 (green) expression in skin after DENV infection. Scale bar = 100 um. (b) Quantification of infection over time. (c) Immunofluorescence with antibodies (Abs) to AE1 (keratinocytes [KC], red) or CD207 (Langerhans cells, red) and NS3 (green) at 6 hours. Arrows indicate infected KC. Scale bar = 25 um. (d) Proportion of infected KC and Langerhans cells (LC) at 6 hours. Each symbol represents one individual and horizontal lines indicate means. (e) IFN-α (red) expression before and after infection. Scale bar = 100 um. (f) Quantification of IFN-α−expressing cells. (b, f) Data expressed as mean ± standard error of mean from four individuals. Blue staining in images represents nuclei and dotted lines indicate epidermal−dermal junction. ∗P < 0.05 relative to mock infection.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2
Dengue virus (DENV) replicates in a wide range of cells with keratinocytes the major contributor to infection. Staining with antibodies to specific cell markers (red) and NS3 (green) in epidermis (a) and dermis (b) at 48 hours post infection with DENV or K0049 strains. Arrows indicate infected cells. Scale bar = 25 um. (c) Proportion of each cell type infected (left), area of infection attributed to each cell type (middle), and infection as a percent of all infected cells (right). Symbols represent keratinocytes (KC), Langerhans cells (LC), macrophages (MØ), dermal DC (dDC), fibroblasts (FB), and mast cells (MC). Data expressed as mean ± standard error of mean from four individuals. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P <0.001, ∗∗∗∗P < comparing KC with other cell types.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3
Dengue virus (DENV) infection in skin causes influx and infection of myeloid cells. (a) Immunofluorescence with antibodies to individual cell markers (red) at intervals after infection with DENV or mock infection. Scale bar = 100 um. (b) Density of Langerhans cells (LC), macrophages (MØ), dermal DC (dDC), and mast cells (MC) in mock- and DENV-infected skin. Data expressed as mean ± standard error of mean for four individuals. ∗P < 0.05; ∗∗P < 0.01 comparing mock and infected skin. (c) Relationship between cell density and DENV infection for each cell type.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 4
Dengue virus (DENV) infection promotes myeloid cell emigration from skin. (a) Staining with antibodies to CD207 (red) to identify Langerhans cells in dermis at 48 hours post infection with DENV or mock infection. Scale bar = 100 um. (b) Number of migrated cells collected from media per square inch of skin at 24 and 48 hours after DENV or mock infection. (c) Representative flow cytometry plots of migrated cells stained with indicated antibodies to identify Langerhans cells (LC), macrophages (MØ), and dermal DC (dDC) at 24 hours. SSC, side scatter. (d) Quantification of migrated cells. Each symbol represents a different individual. ∗P < 0.05 relative to mock infection.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

6. Figure 5
Expression of cytokines/chemokines by dengue virus (DENV)–infected keratinocytes in skin. (a) Change in expression of genes in skin in the presence or absence of DENV at 48 hours relative to 0 hours. Each line represents one individual. ∗P < (b) Immunofluorescence in skin at 48 hours post infection with DENV (top) or K0049 (bottom) labeled with antibodies to AE1 (keratinocytes, red), NS3 (green), and the specific cytokine or chemokine (white). Arrows indicate expression of cytokine/chemokine in infected-keratinocytes; arrowheads indicate cytokine expression in cells other than infected keratinocytes. Scale bar = 25 um.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

7. Figure 6
Blocking IL-1 or CCL20 prevents recruitment and infection of myeloid cells in skin. (a) (Left) Density of Langerhans cells (LC), macrophages (MØ), and dermal DC (dDC) in uninfected skin (UI) at 0 hours and mock-infected skin at 24 hours and in dengue virus (DENV)−infected skin at 24 hours after exposure to different antibodies. (Right) Number of DENV-infected cells of each cell type after exposure to different antibodies. (b) Total number of DENV-infected cells in epidermis and dermis 24 hours after exposure to isotype control antibodies or antibodies to IL-1β. Each symbol represents a different individual. ∗P < 0.05, ∗∗P < (c) Immunofluorescence with antibodies to individual cell markers (red) and NS3 (green) at 24 hours after DENV infection and exposure to isotype control antibodies or antibodies to IL-1β. Arrows indicate co-localization of NS3 with specific cell marker. Scale bar = 100 um.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions