1. Keratinocyte-Derived IL-17E Contributes to Inflammation in Psoriasis
Luisa Senra, Romaine Stalder, David Alvarez Martinez, Carlo Chizzolini, Wolf-Henning Boehncke, Nicolò Costantino Brembilla 
Journal of Investigative Dermatology 
Volume 136, Issue 10, Pages (October 2016)
DOI: /j.jid
Copyright © 2016 The Authors Terms and Conditions

2. Figure 1
IL-17E–producing keratinocytes are increased in the epidermis of psoriatic lesions. (a) IL-17A and IL-17E immunohistochemical analysis in the epidermis of a representative skin of 7 NN, 7 PN, and 10 PP. Original magnification ×20; original magnification of insets ×60. Scale bar = 50 μm. Arrows indicate positive cells (brown). (b) IL-17E level (median/interquartile range) in 7 NN, 7 PN, and 10 PP as determined by densitometry analysis of the immunohistochemistry in a. Data relative to the mean of NN are shown. Each symbol represents an individual subject. Significance assessed by Mann-Whitney test. (c) Expression of cytokeratin-10 (K10, green) in combination with IL-17E (red) assessed by immunofluorescence in PP representative of 10 samples. (d) Expression of myeloperoxidase (MPO, green) in combination with IL-17A (pink) and IL-17E (red) assessed by immunofluorescence in PP of 10 samples. Original magnification ×40. Scale bar = 20 μm. (e) IL-17E mRNA levels (blue) in 4 NN, 4 PN, and 4 PP samples as assessed by in situ hybridization. Shown are the irrelevant and no-probe controls. Original magnification ×20. scale bar = 50 μm. Ctr, control subject; K10, cytokeratin-10; MPO, myeloperoxidase; NN, normal skin; PN, nonlesional psoriatic skin; PP, lesional psoriatic skin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

3. Figure 2
Overrepresentation of IL-17E– and IL-17A–positive cells in the dermis of psoriatic lesions. (a) IL-17A and IL-17E immunohistochemical analysis in the dermis of a representative skin of 7 NN, 7 PN, and 10 PP. Original magnification ×20; original magnification of insets ×60. Scale bar = 50 μm. Arrows indicate positive cells (brown). Shown are sections from the same individuals as in Figure 1a. (b) Quantification of IL-17A+ and IL-17E+ cells (median/interquartile range) in papillary and reticular dermis of 7 NN, 7 PN, and 10 PP as determined by histologic examination. Each symbol represents an individual subject. Significance assessed by Mann-Whitney test. (c) Frequency of IL-17A+ or IL-17E+ cells related to the distance from the epidermis in 7 NN, 7 PN, and 10 PP. 0 in the x-axis indicates the epidermal-dermal junction. (d) IL-17E mRNA levels (median/interquartile range), normalized to glyceraldehyde-3-phosphate dehydrogenase in skin, total mRNA extracts of 4 NN, 4 PN, and 4 PP. Shown are data relative to the mean of NN. Each symbol represents an individual subject. (left) Significant difference determined by Mann-Whitney test. (right) Detection of IL-17E in skin total protein extract as assessed by Western blot; shown is one representative experiment of three. (e) IL-17E levels (median/interquartile range) in sera from seven healthy individuals and seven psoriasis patients as assessed by ELISA. Each symbol represents an individual subject. Ctrl, control subject; NN, normal skin; PN, nonlesional psoriatic skin; PP, lesional psoriatic skin; Pso, psoriasis patients.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

4. Figure 3
IL-17E is contained in macrophages in the dermis of psoriatic lesional skin. (a) Expression of IL-17E (red) in combination with CD3, MPO, tryptase, or CD68 (green) assessed by immunofluorescence analysis. (b) Frequency of CD3+, MPO+, tryptase+, and CD68+ cells expressing IL-17E (median/interquartile range) in 6 PP samples. Each symbol represents a distinct individual. Data are expressed as percentage of IL-17E–expressing cells. (c) Expression of CD68 (green) and CD163 (red, left) or CD11c (green) and IL-17E (red, right) assessed by immunofluorescence analysis. (d) Expression of IL-17E (red) in combination with CD1631L1 (green) and CLEC5 (pink) assessed by immunofluorescence. (e) Expression of tryptase (green) in combination with IL-17E (red) and IL-17A (pink) assessed by immunofluorescence. In a, c, d, and e, one representative PP sample of six is shown. Original magnification ×40. Scale bar = 20 μm. Arrows indicate positive cells. MPO, myeloperoxidase; PP, lesional psoriatic skin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

5. Figure 4
IL-17E is internalized by macrophages via receptor-mediated clathrin-dependent endocytosis. Expression of (a) IL-17E (red) in combination with calnexin (endoplasmic reticulum), rab7 (late endosome), or LAMP-1 (lysosome) (green); (b) CD68 (green) and IL-17RB (pink); and (c) clathrin or caveolin-I (green) assessed by immunofluorescence analysis. One representative PP sample of two is shown. Original magnification ×63. Scale bar = 2 μm. (d) IL-17E mRNA levels as detected by quantitative PCR in phorbol 12-myristate 13-acetate/ionomycin-activated Th2 cells and monocyte-derived macrophages from seven healthy individuals in M0 condition or upon polarization toward M1 or M2 phenotype for 24 hours. Shown are data relative to Th2 cells. (e) IL-17E levels in differentiated macrophages exposed to exogenous rhIL-17E for the indicated time points as assessed by Western blot. Shown is one experiment of three. Vertical dashed lines have been inserted to indicate repositioned gel lanes. (f) Expression of IL-17E (red) in combination with IL-17RB (green) in M2-polarized macrophages cultured in presence of IL-17E with or without the selective clathrin inhibitor Pitstop 2. Original magnification ×40. Scale bar = 10 μm. Shown is one experiment of three. min, minutes; Th, T helper.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

6. Figure 5
IL-17E promotes inflammatory responses in M2 macrophages. (a) IL-17RB mRNA levels assessed by quantitative PCR in monocyte-derived macrophages from five healthy donors at resting condition (M0) or upon polarization toward M1 or M2. Data are expressed as relative to M0. Each symbol represents a distinct individual. (b) M2-polarized macrophages were cultured in the presence of increasing doses of IL-17E for 6 hours. Shown are mRNA levels relative to untreated condition as assessed by quantitative PCR. (c) Monocyte-derived macrophages (M0) from six healthy donors were differentiated in M1 or M2 conditions and cultured in the presence of 100 ng/mL of IL-17E for a further 6 hours. Shown are the mean ± standard error of the mean of the mRNA levels relative to the M0 condition as assessed by quantitative PCR. Significant differences of IL-17E treated versus untreated macrophages by Wilcoxon signed-rank test. (d) Correlation between the number of IL-17E+ cells and the number of MPO+ neutrophils (upper panel) or CD3+ T cells (lower panel) as quantified in lesional skin by immunohistological analysis in six PP samples. Significance was determined by Spearman correlation test. Shown is (upper panel) line interpolation according to a linear and (lower panel) a logarithmic regression. MCP-1, monocyte chemotactic protein-1; MPO, myeloperoxidase.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

7. Figure 6
Hypothetical model showing the effects of IL-17E in psoriasis. Keratinocytes in psoriatic lesional skin produce high levels of IL-17E, which might be taken up by dermal macrophages, resulting in their activation (dashed arrow). Activated macrophages produce, among others, TNF-α, IL-8n, and MCP-1, which contribute to enhanced inflammation and recruit further immune cells to the lesion (continuous arrows). MCP-1, monocyte chemotactic protein-1; TNF, tumor necrosis factor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions