Dermal CD14+ Dendritic Cell and Macrophage Infection by Dengue Virus Is Stimulated by Interleukin-4 Evelyne Schaeffer, Vincent Flacher, Vasiliki Papageorgiou,

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Dermal CD14+ Dendritic Cell and Macrophage Infection by Dengue Virus Is Stimulated by Interleukin-4 Evelyne Schaeffer, Vincent Flacher, Vasiliki Papageorgiou, Marion Decossas, Jean-Daniel Fauny, Melanie Krämer, Christopher G. Mueller Journal of Investigative Dermatology Volume 135, Issue 7, Pages 1743-1751 (July 2015) DOI: 10.1038/jid.2014.525 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Increased Dendritic Cell–Specific Intercellular adhesion molecule-3–Grabbing Nonintegrin (DC-SIGN) expression in dermatitis. (a) Images of normal and hypereosinophilic syndrome skin stained for CD163 and DC-SIGN with DAPI (4′,6-diamidino-2-phenylindole) nuclear counterstain. The dotted line marks the dermo–epidermal junction. Scale bars are shown. (b) Graph depicts the number of CD163+ (red), DC-SIGN+ (green), and CD163+/ DC-SIGN+ (brown) cells per field for each condition. The data present the mean value±SD for five donors. The Wilcoxon test was used for statistical analysis; **P<0.01. Journal of Investigative Dermatology 2015 135, 1743-1751DOI: (10.1038/jid.2014.525) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Dermal dendritic cells (DCs) are infected by Dengue virus (DENV). (a) DCs and T cells emigrating from skin explants were identified by FACS. CD14 and CD1c labeling was determined on the gated DC-containing population. (b) HLA-DR, MR, and Dendritic Cell–Specific Intercellular adhesion molecule-3–Grabbing Nonintegrin (DC-SIGN) expression was measured on gated CD14+ dermal DCs (dDCs) (green) and CD1c+ dDCs (red). Isotype staining is in black. Data are representative of at least five donors. (c) DENV infection of CD1a/c+ DCs (HLA-DRhiCD14-) and CD14+ dDCs (HLA-DRloCD14+) was measured by FACS as intracellular staining of DENV-E protein. The mean percentage±SD (n=7) of cells positive for DENV-E protein in comparison to mock-infection is indicated. (d) Confocal microscopic visualization of DENV-E protein within a CD14+ dDC. Scale bar represents 10μm. The image is representative of two donors. Journal of Investigative Dermatology 2015 135, 1743-1751DOI: (10.1038/jid.2014.525) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 IL-4 stimulates Dengue Virus (DENV) infection of CD14+ dermal dendritic cells (dDCs). (a) Viral titers from mock- or DENV-infected DCs stimulated or not by IL-4 were determined on Vero cells as FACS infectious units (FIU) per ml. Each point represents one donor and the horizontal bar the mean value. (b) DENV infection of untreated or IL-4-stimulated CD1a/c+ DCs and CD14+ dDCs was measured by intracellular presence of DENV-E protein. The percentage of DENV-E+ cells is indicated. The graph depicts the collective data for each donor, represented by a symbol. Horizontal bars are the mean values. (c) Microscopic detection of DENV-E protein in a CD14+ dDC but lacking from a neighboring CD1a/c+ DC. Scale bar=10μm. The image is representative of two donors. (d) Expression of DC-SIGN by CD1c+ and CD14+ dDCs emigrating from skin explants with or without IL-4. The graphs depict the mean fluorescence intensity of DC-SIGN and mannose receptor expression for both subsets for each donor. The mean value is shown as horizontal bars. Statistical significance was determined by unpaired Student’s t-test. *P<0.05; ***P<0.001. Journal of Investigative Dermatology 2015 135, 1743-1751DOI: (10.1038/jid.2014.525) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 IL-4-activated dermal macrophages (dMs) are permissive to Dengue virus (DENV) infection. (a) Transmission electron microscopy of FACS-purified dMs cultured without cytokines, with IL-4 or with IL-4/GM-CSF. In comparison is shown a CD1c+ dermal dendritic cell (dDC). Bar=2μm. The data are representative of three donors. (b) Phenotypic characterization of dMs cultured in the absence or presence of the indicated cytokines. The mean fluorescence intensity of the cell surface markers for each skin donor is shown. Horizontal bars represent mean values. (c) FACS analysis of DENV infection of dMs obtained from the indicated conditions. The percentage of DENV-E+ dMs is indicated. The graph depicts this percentage for each donor. The mean value is shown by a horizontal bar. Statistical significance was determined by unpaired Student’s t-test. *P<0.05; **P<0.01; ***P<0.001. Journal of Investigative Dermatology 2015 135, 1743-1751DOI: (10.1038/jid.2014.525) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Immunostimulatory impact of IL-4-activated dermal dendritic cells (dDCs) and dermal macrophages (dMs). (a) Measure of tumor necrosis factor-α (TNF-α) release by mock or Dengue virus (DENV)-infected dDCs stimulated or not by IL-4. The graph depicts the collective data for each donor, and horizontal bars are the mean values. (b) TNF-α release by mock or DENV-infected dMs cultured in the indicated conditions. Data points of individual donors are shown, and the mean values are horizontal bars. (c) Proliferation of naive CD4+ T cells by allogeneic DCs obtained from the skin with or without IL-4. T-cell proliferation was measured as the loss of carboxyfluorescein succinimidyl ester (CFSE) fluorescent dye. The data are representative of three donors. (d) Proliferation of naive CD4+ T cells by allogeneic DCs obtained from the skin with or without IL-4 and exposed to DENV. T-cell proliferation was measured with CFSE, and the data are representative of three donors. (e) Proliferation of CD4+ T cells by allogeneic dMs cultured in the indicated conditions. T-cell proliferation was measured as the loss of CFSE. The data are representative of three donors. Statistical significance was determined by unpaired Student’s t-test. *P<0.05; **P<0.01; ***P<0.001; NS, nonsignificant. Journal of Investigative Dermatology 2015 135, 1743-1751DOI: (10.1038/jid.2014.525) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions