1. Volume 48, Issue 4, Pages 787-798.e4 (April 2018)
An Interleukin-25-Mediated Autoregulatory Circuit in Keratinocytes Plays a Pivotal Role in Psoriatic Skin Inflammation 
Miao Xu, Huiping Lu, Young-Hee Lee, Yelin Wu, Kewei Liu, Yuling Shi, Haoran An, Jingren Zhang, Xiaohu Wang, Yuping Lai, Chen Dong 
Immunity 
Volume 48, Issue 4, Pages e4 (April 2018)
DOI: /j.immuni
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2. Immunity 2018 48, 787-798.e4DOI: (10.1016/j.immuni.2018.03.019)
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3. Figure 1 IL-25 Induces Acanthosis and Inflammation in Mouse Skin
(A) IL25 mRNA expression in the skin from healthy controls and psoriasis patients.
(B) Immunofluorescence staining of IL-25 in the skin paraffin sections obtained from healthy control and psoriasis patients.
(C) Il25 mRNA expression in the skin from control mice and IMQ-treated mice.
(D) Immunofluorescence staining of IL-25 in the skin sections of control and IMQ-treated mice.
(E) H&E staining of back skin from the C57BL/6 mice injected with PBS (n = 5) or IL-25 (n = 5).
(F) Ear thickness of the C57BL/6 mice injected with PBS (n = 5) or IL-25 (n = 5).
(G) H&E staining of ear from C57BL/6 mice injected with PBS (n = 5) or IL-25 (n = 5), respectively.
(H) The relative mRNA expression of selected genes regulated by IL-25 in skin from C57BL/6 mice injected with PBS (n = 5) or IL-25 (n = 5).
All the assays were repeated three times with consistent results. Scale bars represent 100 μm in (B) and (D) and 50 μm (zoom in) or 100 μm (zoom out) in (E) and (G). p values were determined by unpaired t test or two-way ANOVA. Data are the means ± SEM. All data are representative of two independent experiments.
See also Figure S1.
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4. Figure 2 IL-25 is Required for the Induction of Psoriasis Model
WT (n = 7) and Il25−/− mice (n = 6) were subjected to IMQ-induced psoriasis-like inflammation, and sacrificed on day 3.5 after disease induction.
(A) H&E staining skin sections (10×). Left: H&E staining data; right: statistical data (means ± SEM).
(B) Infiltration of CD45+ cells in the dermal and epidermal tissues. Top: flow cytometry data. Bottom: statistical data (means ± SEM).
(C) Dermal infiltration of dendritic cells, macrophages, neutrophils, and γδ T cells. Left: flow cytometry data. Right: statistical data (means ± SEM).
All the assays were repeated three times with consistent results. Scale bar represents 50 μm. p values were determined with an unpaired t test. All data are representative of three independent experiments.
See also Figures S2 and S3.
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5. Figure 3
Keratinocyte-Specific Ablation of Il25 Leads to Resistance to IMQ-Induced Psoriasis Model
The Il25f/f and Il25f/f K5Cre mice were subjected to IMQ-induced psoriasis-like inflammation and sacrificed on day 3.5 after disease induction.
(A) Representative H&E staining sections (20×) from the skin tissues of healthy control (n = 5), Il25f/f (n = 5), and Il25f/f K5Cre (n = 5) mice. Right: statistical analysis of skin epidermal and dermal thickness (means ± SEM).
(B) Infiltration of CD45+ cells in the dermal and epidermal tissues.
(C) Dermal infiltration of dendritic cells, macrophages, neutrophils, and γδ T cells. Left: flow-cytometry data. Right: statistical data (means ± SEM).
(D) Il25f/f (n = 5) and Il25f/fK5Cre (n = 5) mice were infected with Staphylococcus aureus for 3 days, and the skin tissues at the infection site were used to measure the size of abscess and calculate the colony-forming unit (CFU).
All the assays were repeated three times with consistent results. The scale bar represents 50 μm. p values were determined by unpaired t test. All data are representative of two independent experiments.
See also Figure S4.
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6. Figure 4
IL17RB Deficiency in Non-Hemapoietic Cells Causes Resistance to IMQ-Induced Psoriasis Model
(A) Expression of Il17rb and Il25 in primary keratinocytes cultured in vitro with or without IL-25 for 24 hr as examined at the mRNA level.
(B) Healthy control (n = 4), WT (n = 5), and Il17rb−/− (n = 5) mice were subjected to IMQ-induced psoriasis-like inflammation and sacrificed on day 3.5 after disease induction. Top: H&E staining of skin lesions (20x). Bottom: statistical analysis of skin epidermal and dermal thickness (means ± SEM).
(C) and (D) Expression of IL-17RB in skin epidermis in WT and Il25−/− mice in IMQ-induced psoriasis model, as determined by flow cytometry (C) or mRNA expression (D).
(E) Bone marrow isolated from WT or Il17rb−/− mice was individually transferred into lethally irradiated WT or Il17rb−/− mice; the recipient mice were then subjected to IMQ-induced psoriasis-like skin inflammation and sacrificed for analysis. Top: H&E staining (20×) of skin sections from the chimeric mice. Bottom: statistical analysis of skin epidermal and dermal thickness.
The scale bar represents 50 μm. p values were determined by unpaired t test. All data are representative of two independent experiments.
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7. Figure 5
IL-25 Promotes Keratinocyte Proliferation and Regulates Genes Associated with Inflammation and Proliferation
The primary keratinocytes from multiple mice were pooled and stimulated with IL-25 in duplicates for 8 hr in vitro and then subjected to RNA-seq analysis. The medium-only culture was used as a negative control for RNA-seq or real-time qPCR analysis.
(A) RNA-seq pathway analysis of IL-25-regulated genes.
(B) Scattered map of IL-25 regulated genes.
(C) IL-25 dose-dependently (ng/ml) increased the cell numbers of primary murine keratinocytes in in vitro cultures. The viable cells were determined by Guawa-counting.
(D) Effect of IL-25 on cellularity of primary murine keratinocytes at different culture time points.
(E) Ki67 staining in primary murine keratinocytes stimulated by IL-25 for 48 hr in in vitro cultures.
(F) Cell-cycle analysis of primary murine keratinocytes cultured for 24 hr with or without IL-25 as determined by DAPI staining.
(G) Immuno-histochemical staining of Ki67 staining (10×) in the skin of IMQ-treated WT and Il25−/− mice. The scale bar represents 50 μm.
p values were determined by an unpaired t test or two-way ANOVA. All data are representative of two independent experiments.
See also Figure S5.
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8. Figure 6
IL-25 Induces Keratinocytes Proliferation Dependent on Act1 and STAT3 Signaling
(A) Validation of mRNA expression of selective IL-25-regulated genes by real-time qPCR in both WT and Act1−/− primary murine keratinocyte stimulated with or without IL-25 for 8 hr.
(B) Ki67 staining (left) and cell-cycle analysis (right) of primary keratinocytes isolated from WT and Act1−/− mice stimulated with IL-25 or mock for 24 hr in in vitro cultures.
(C) Phosphorylation of STAT3 (pSTAT3) in primary keratinocytes cultured for 24 hr with or without IL-25 as determined by phophoflow cytometry.
(D) Immuno-histochemical staining (20×) of pSTAT3 in the skin after PBS or IL-25 injection. The dark arrow showed the representative positive staining of pSTAT3.
(E) Ki67 staining (up) and cell-cycle analysis (down) of primary keratinocytes stimulated with IL-25 for 24 hr in the presence or absence of Stattic or anti-IL-6 blocking antibody in in vitro cultures.
(F) Expression of IL-25-regulated genes in primary keratinocytes stimulated by IL-25 in the presence or absence of STAT3 inhibitor Stattic for 8 hr in in vitro cultures.
The scale bar represents 50 μm (20× magnification). p values were determined by unpaired t test or one-way ANOVA. All data are representative of two independent experiments.
See also Figure S6.
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9. Figure 7 IL-25 Activates STAT3 via Act1 and JAK
(A) Primary keratinocytes isolated from both WT and Act1−/− mice were stimulated with IL-25 and collected at indicated time points for immunoblotting of STAT3 or pSTAT3.
(B) Primary keratinocytes isolated from WT mice stimulated by IL-25 in the presence or absence of anti-IL-6 antibody or JAK1&2 inhibitor collected at indicated time points for immunoblotting of STAT3 or pSTAT3. Actin was used as a loading control.
(C) HEK293 cells were transfected with HA-tagged IL-17RB-expressing plasmid, stimulated with IL-25 for different lengths of time, and then collected, lysed, and immunoprecipitated (IPed) with anti-HA antibody. The IPed proteins were analyzed by immunoblotting with anti-Tyr or anti-HA antibodies. The whole-cell lysate (WCL) was used as a loading control.
(D) HEK293 cells were co-transfected with the STAT3 vector and either the empty vector or the HA-tagged IL-17RB vector, and the cells were collected, lysed, and immunoprecipitated with anti-HA antibody, followed by immunoblotting of STAT3 and HA. Top: immunoprecipitated products. Bottom: WCL. Results shown are representative data of three independent experiments.
(E) Keratinocytes were cultured and stimulated with IL-25 for 1 hr and then used in an immunoprecipitation assay with anti-IL-17RA antibody or control IgG antibody, and the immunoprecipitated proteins were blotted with anti-IL-17RA and anti-STAT3 antibodies.
All data are representative of two independent experiments.
See also Figure S7.
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