Journal: The Journal of investigative dermatology

Article Title: IL-17E (IL-25) Enhances Innate Immune Responses during Skin Inflammation.

doi: 10.1016/j.jid.2019.01.021

Figure Lengend Snippet: Figure 1. IL-17E promotes skin inflammation and favors the preferential recruitment of neutrophils in vivo. Balb/c mice were intradermally injected with recombinant mouse IL-17E or saline in the back for 2 consecutive days. Skin samples were harvested on day 3. (a) Representative hematoxylin and eosin staining of skin from mice treated with IL-17E or saline (n ¼ 8). Original magnification 10. Scale bar ¼ 50 mm. (b) Profiling of the cellular infiltrate in the skin of saline- (black bars) and IL-17Eeinjected (red bars) mice by flow cytometry. Cumulative data from three independent experiments (n ¼ 8) are presented as absolute counts per cm2. (c) Representative flow cytometry plots of the experiment shown in b. Full-thickness line gates depict cell populations modified by IL- 17E treatment. Pseudocolor scale indicates absolute counts, and numbers in the gates refer to frequency relative to CD45þ cells. (d) Immunofluorescence analysis of skin sections stained for DAPI (blue), Ki67 (green), and cytokeratin 10 (red). Original magnification 40. Scale bar ¼ 20 mm. One representative result is shown (n ¼ 5). White arrows depict Ki-67 positive cells (left panel). Quantitative analysis of Ki67þ cells in the basal layer (n ¼ 5, right panel). (e) Quantitative analysis of the epidermal thickness based on hematoxylin and eosin-stained sections (n ¼ 5). (f) Gene expression analysis was performed using NanoString (Seattle, WA) technology in the skin of mice injected with IL-17E or saline for 3 hours. Volcano plot (left panel) displays differentially expressed genes upon IL-17E treatment. The y-axis corresponds to the mean expression value of log10 P-value, and the x-axis displays the log2 fold change value. The red dots represent the differentially expressed transcripts (threshold > 2 fold change). Heatmap (right panel) of differentially expressed mRNAs in IL-17Eeinjected versus saline (control) group (n ¼ 5). Expression values are colored based on their z-score after normalization across treatments.

Article Snippet: Mouse antihuman IL-17E mAb (clone 182293) and polyclonal goat anti-human myeloperoxidase antibody were from R&D Systems, rabbit anti-human cytokeratin 10 was from BioSB (Santa Barbara, CA), and rat anti-mouse Ki67 mAb (clone 16A8) and rat anti-mouse Ly6G (clone 1A8) were from Biolegend.

Techniques: In Vivo, Injection, Recombinant, Saline, Staining, Cytometry, Gene Expression, Expressing, Control

Journal: The Journal of investigative dermatology

Article Title: IL-17E (IL-25) Enhances Innate Immune Responses during Skin Inflammation.

doi: 10.1016/j.jid.2019.01.021

Figure Lengend Snippet: Figure 3. IL-17E neutralization through blocking antibodies ameliorates imiquimod-induced skin inflammation. Wild-type balb/c mice were treated with imiquimod for 3 consecutive days on the shaved back skin and one ear. Next, 100 mg of neutralizing IL-17E antibody or isotype control were injected intraperitoneally1 day before the first application and 1 hour before every application. Skin and ear tissue samples were harvested on day 4. (a) Daily body weight variation and percentage of increase in ear swelling expressed as mean standard error of the mean (n ¼ 5). (b) Representative hematoxylin and eosin staining of the back skin and quantitative analysis of the epidermal thickness of imiquimod-treated mice injected with anti-IL-17E (n ¼ 11) or isotype control

Article Snippet: Mouse antihuman IL-17E mAb (clone 182293) and polyclonal goat anti-human myeloperoxidase antibody were from R&D Systems, rabbit anti-human cytokeratin 10 was from BioSB (Santa Barbara, CA), and rat anti-mouse Ki67 mAb (clone 16A8) and rat anti-mouse Ly6G (clone 1A8) were from Biolegend.

Techniques: Neutralization, Blocking Assay, Control, Injection, Staining

Journal: The Journal of investigative dermatology

Article Title: IL-17E (IL-25) Enhances Innate Immune Responses during Skin Inflammation.

doi: 10.1016/j.jid.2019.01.021

Figure Lengend Snippet: Figure 4. IL-17E neutralization affects innate immune cell infiltration during imiquimod-induced skin inflammation. Experimental condition, as in Figure 4. (a) Visualized t-SNE map of cells isolated from the skin of mice treated with imiquimod. Clusters (cell populations) were identified by manual gating. One representative result of each group is shown (n ¼ 10 for isotype control group; n ¼ 11 for a-IL-17E-treated group). (b) Based on clusters identified in a, median intensities for each marker were calculated and plotted as a heatmap. (c, d) Histogram showing the mean fluorescence intensity of the indicated markers in each cell cluster (c) belonging to the NK population (clusters 1e3) or (d) belonging to the neutrophil population (clusters 10 to 13) in the anti-IL-17E treated group. max, maximum; NK, natural killer; t-SNE, t-distributed stochastic neighbor embedding.

Article Snippet: Mouse antihuman IL-17E mAb (clone 182293) and polyclonal goat anti-human myeloperoxidase antibody were from R&D Systems, rabbit anti-human cytokeratin 10 was from BioSB (Santa Barbara, CA), and rat anti-mouse Ki67 mAb (clone 16A8) and rat anti-mouse Ly6G (clone 1A8) were from Biolegend.

Techniques: Neutralization, Isolation, Control, Marker

Journal: The Journal of investigative dermatology

Article Title: IL-17E (IL-25) Enhances Innate Immune Responses during Skin Inflammation.

doi: 10.1016/j.jid.2019.01.021

Figure Lengend Snippet: Figure 5. IL-17E promotes the recruitment of human neutrophils via activation of macrophages in a p38-dependent mechanism. (a) mRNA level of human IL-8 (left panel) and murine Cxcl1 and Cxcl2 (right panel) in IL-17Eetreated M2 macrophages from humans and mice, respectively. (b) IL-8 levels as assessed by ELISA in supernatants of primary keratinocytes (n ¼ 4) cultured in the presence of IL-17E or TNF (right panel). (c) mRNA levels assessed by quantitative PCR in M2 polarized macrophages pretreated for 30 minutes with a p38 MAPK (SB202190) or NF-kB inhibitor (JSH 23) and then stimulated with IL-17E for a further 6 hours. mRNA levels relative to the untreated condition are shown as mean standard error of the mean (n 5). (d) Immunoblotting analysis of M2 polarized macrophages stimulated with 100 ng/ml of IL-17E for the indicated time points (n ¼ 5, one representative result is shown). (e) Neutrophil migration induced by IL-8 (50 ng/ml) or IL-17E (100 ng/ml) in a classic Transwell assay (Nunc, Roskilde, Denmark) (left panel). M2 macrophages were left unstimulated (unstim.) or treated with IL-17E in the presence or not of SB202190 for 24 hours. Supernatants were retrieved and used to assess neutrophil migration. IL-8 neutralizing antibodies were added to the conditioned media of macrophages stimulated with IL-17E 30 minutes before assessing chemotaxis. Data from one representative experiment are expressed as relative to control media and presented as the mean standard error of the mean (n ¼ 3). (f) IL-8 protein levels assessed by ELISA in supernatants of M2 polarized macrophages. The protein levels relative to untreated condition are shown as mean standard error of the mean (n 5). Significant differences were assessed by Wilcoxon signed rank test. *P ¼ 0.01 to 0.05. Ctrl, control; min, minute; p-, phosphorylated.

Article Snippet: Mouse antihuman IL-17E mAb (clone 182293) and polyclonal goat anti-human myeloperoxidase antibody were from R&D Systems, rabbit anti-human cytokeratin 10 was from BioSB (Santa Barbara, CA), and rat anti-mouse Ki67 mAb (clone 16A8) and rat anti-mouse Ly6G (clone 1A8) were from Biolegend.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Migration, Transwell Assay, Chemotaxis Assay, Control

Journal: The Journal of investigative dermatology

Article Title: IL-17E (IL-25) Enhances Innate Immune Responses during Skin Inflammation.

doi: 10.1016/j.jid.2019.01.021

Figure Lengend Snippet: Figure 6. An increase in IL-17ED cells may indicate a neutrophil infiltration in human skin inflammatory conditions. Expression of myeloperoxidase (green) in combination with IL-17E (red) and DAPI (blue) assessed by immunofluorescence in the skin of healthy volunteers (n ¼ 4) and patients with acute generalized exanthematous pustulosis (n ¼ 3) and pyoderma gangrenosum (n ¼ 4). Original magnification 20. Scale bar ¼ 50 mm. (b) Quantification of dermal IL-17Eþ and MPOþ cells in the samples, as in a. (c) Quantification of the expression of IL-17E in the epidermis of the samples shown in a. AGEP, acute generalized exanthematous pustulosis; MPO, myeloperoxidase; PG, pyoderma gangrenosum.

Article Snippet: Mouse antihuman IL-17E mAb (clone 182293) and polyclonal goat anti-human myeloperoxidase antibody were from R&D Systems, rabbit anti-human cytokeratin 10 was from BioSB (Santa Barbara, CA), and rat anti-mouse Ki67 mAb (clone 16A8) and rat anti-mouse Ly6G (clone 1A8) were from Biolegend.

Techniques: Expressing