Dynamic Changes in Resident and Infiltrating Epidermal Dendritic Cells in Active and Resolved Psoriasis Elisa Martini, Maria Wikén, Stanley Cheuk, Irène.

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Dynamic Changes in Resident and Infiltrating Epidermal Dendritic Cells in Active and Resolved Psoriasis Elisa Martini, Maria Wikén, Stanley Cheuk, Irène Gallais Sérézal, Faezzah Baharom, Mona Ståhle, Anna Smed-Sörensen, Liv Eidsmo Journal of Investigative Dermatology Volume 137, Issue 4, Pages 865-873 (April 2017) DOI: 10.1016/j.jid.2016.11.033 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Detection of LCs and DCs in lesional psoriasis epidermis. (a) Confocal images of cryopreserved healthy and lesional psoriasis skin. Langerin (red), CD74 (green), and DAPI (blue) are depicted. Scale bars: 100 μm. (b) Transmission electron microscopy images of lesional psoriasis epidermis. Arrows indicate Birbeck granules. Bars: 5 μm (left), 500 nm (central), 2 μm (right). (c) Representative FACS plots depicting DC populations in healthy skin or lesional psoriasis skin. DC, dendritic cell; LC, Langerhans cell. Journal of Investigative Dermatology 2017 137, 865-873DOI: (10.1016/j.jid.2016.11.033) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Phenotypic profiling and enumeration of eDCs in psoriasis and healthy skin. (a) Number of cells per mm2 skin surface in healthy skin (H), nonlesional psoriasis (NL), and lesional psoriasis (L) determined by FACS. Median is shown. (b) Relative gene expression heat map in cells sorted from healthy (n = 8), nonlesional (n = 7), and lesional psoriasis (n = 9). Columns: individual donors; rows: individual genes. House-keeping gene RPLPO. (c) Representative FACS plots and graphs of frequency of CD14, CD1a, and CD1c expression in LCs, eDCs, and dDCs from lesional psoriasis. Median is shown. (d) Relative gene expression in sorted cells from lesional psoriasis epidermis (n = 9). (e) Representative histogram and graph depicting MFI (geometric mean) of CCR2 expression in cells from lesional psoriasis (n = 6). Mean ± SD is shown. dDC, dermal dendritic cell; eDC, epidermal dendritic cell; LC, Langerhans cell; MFI, mean fluorescence intensity; SD, standard deviation. Journal of Investigative Dermatology 2017 137, 865-873DOI: (10.1016/j.jid.2016.11.033) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Inflammatory gene expression in sorted LCs and eDCs in lesional psoriasis and healthy skin. (a) Relative gene expression heat map of inflammatory genes in sorted LCs from healthy epidermis (n = 9), and LCs and eDCs from lesional psoriasis (n = 9). Columns: individual donors, rows: individual genes. (b) Bar graphs comparing selected gene expression in LCs from healthy skin with LCs and eDCs from lesional psoriasis. House-keeping gene RPLPO. Mean ± SD is shown. eDC, epidermal dendritic cell; LC, Langerhans cell; SD, standard deviation. Journal of Investigative Dermatology 2017 137, 865-873DOI: (10.1016/j.jid.2016.11.033) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Production of intracellular cytokines in LCs and eDCs in response to TLR7/8 or TLR4 stimulation. Cell suspensions were cultured for 6 hours in the presence or absence of R848 or LPS and brefeldin A. Intracellular cytokine production was detected using flow cytometry. (a) Proportion of cytokine producing cells in LCs from healthy epidermis (n = 9–10), or LCs (n = 5), eDCs, and dDCs (n = 7) from lesional psoriasis. Mean ± SD is shown. (b) Representative plots of intracellular cytokines in healthy LCs, lesional psoriasis LCs, eDCs, or dDCs after stimulation with R848 for 6 hours (top) and spice charts showing the median proportion of IL-23p19, IL-1β, and IL-10 production in healthy LCs or LCs, eDCs, and dDCs from lesional psoriasis unstimulated or after R848 stimulation (bottom). dDC, dermal dendritic cell; eDC, epidermal dendritic cell; LC, Langerhans cell; LPS, lipopolysaccharide; R848, resiquimod; SD, standard deviation; TLR, toll-like receptor. Journal of Investigative Dermatology 2017 137, 865-873DOI: (10.1016/j.jid.2016.11.033) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Assessment of eDCs and LCs in psoriasis during treatment and in resolved lesions. (a) Left: photograph of psoriasis during anti-TNF treatment. Right: number of LCs and eDCs per mm2 of skin surface prepared from healthy epidermis (H), during anti-TNF or after UVB treatment assessed by FACS. Median is shown. (b) Gene expression correlation plots of LCs in resolved skin and healthy LCs normalized to house-keeping RPLPO. Dashed lines represent a two-fold change. (c) Relative gene expression in sorted LCs from healthy and resolved skin. Median is shown. (d) Percentage of cytokine producing LCs ± R848 stimulation in healthy anti-TNF-treated skin (n = 5). (c, d) Significance calculated against healthy LCs. Mean ± SD is shown. (e) Confocal image of anti-TNF-treated skin. Langerin (red), CD3 (cyan), Phalloidin (gray). Scale bar: 100 μm. Mean ± SD is shown. eDC, epidermal dendritic cell; LC, Langerhans cell; R848, resiquimod; SD, standard deviation; TNF, tumor necrosis factor. Journal of Investigative Dermatology 2017 137, 865-873DOI: (10.1016/j.jid.2016.11.033) Copyright © 2017 The Authors Terms and Conditions