Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A) Coomassie blue stained gel demonstrating the purity of IL-36α preparation. Ten µg IL-36α was loaded on the lane. B) Western immunoblotting of IL-36α protein preparation detects a band around 18 KDa, the predicted molecular weight of mouse IL-36α. C) Cytospin preparations demonstrating neutrophil influx in the BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. D) Total cell counts from BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. E) Differential cell count percentages and F) Differential cell count numbers in the BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. *Indicates significant differences ( P<0.05 ) compared to PBS treated mice. Data represent mean±SEM from 4–5 mice/group. G) Flow cytometry on cells recovered from BAL fluid from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. A majority of cells from the IL-36α instilled lungs were CD11c − CD11b + Ly6G + neutrophils. Depicted flow cytometry plots are representative of 4–5 mice/group. H) Hematoxylin & Eosin stained lung sections isolated from mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Depicted sections are representative of 4–5 mice/group.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Staining, Western Blot, Molecular Weight, Cell Counting, Flow Cytometry, Isolation

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A–H) Transcript expression of early response cytokines (TNFα, IL-1α, IL-1β, IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R and the neutrophil specific chemokines CXCL1 and CXCL2 in the lungs of mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. I–K) Protein expression of TNFα, IL-1α, IL-1β and CXCL1 in the BAL fluid recovered from mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Protein expression was quantified by multiplexed cytometric bead arrays. *Indicates significant differences ( P<0.05 ) compared to PBS treated mice. Data represent mean±SEM from 4–5 mice/group.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A) Total lung resistance and B) lung compliance were not significantly different in the lungs of IL-36α challenged mice compared to PBS controls. Airway responses in mice were measured using invasive plethysmography 24 h following i.t instillation of IL-36α or PBS. Data presented are percentage changes from baseline (0mg/mL) measurements. Data represent mean±SEM from 5–7 mice/group.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques:

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A) Total cell counts from BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. B) Differential cell count percentages and C) Differential cell count numbers in the BAL fluid recovered from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. *Indicates significant differences ( P<0.05 ) compared to PBS treated mice. Data represent mean±SEM from 3–4 mice/group. D) Flow cytometry on cells recovered from BAL fluid from mice 24 h following a single i.t. instillation of PBS or 10 µg IL-36α. A majority of cells from the IL-36α instilled lungs were CD11c − CD11b + Ly6G + neutrophils. Depicted flow cytometry plots are representative of 3–4 mice/group. H) Hematoxylin & Eosin stained lung sections isolated from mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Depicted sections are representative of 3–4 mice/group.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Cell Counting, Flow Cytometry, Staining, Isolation

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A–F) Transcript expression of early response cytokines (TNFα and IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R and the neutrophil specific chemokines CXCL1 and CXCL2 in the lungs of IL-1αβ −/− mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. G–H) Protein expression of TNFα and CXCL1 in the BAL fluid recovered from IL-1αβ −/− mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Protein expression was quantified by multiplexed cytometric bead arrays. *Indicates significant differences ( P<0.05 ) compared to PBS treated mice. Data represent mean±SEM from 3–4 mice/group.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A–H) Transcript expression of early response cytokines (TNFα, IL-1α, IL-1β, IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R as well as the neutrophil specific chemokines CXCL1 and CXCL2 in splenic CD11c + cells 2 h following incubation with increasing concentrations of IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. *Indicates significant differences ( P<0.05 ) compared to 0 µg/mL group. Data represent mean±SD of quadruplicate samples from one of two representative experiments. I) Diff-quik stained cells from cytospun BAL cells from the lungs of naïve mice demonstrates that the majority of lung resident immune cells are alveolar macrophages. J) PCR on cDNA from naïve mouse alveolar macrophages demonstrating the constitutive mRNA expression of an endogenous control (β-actin), IL-1R1, IL-36R and IL-1RAcP. Image of a DNA electrophoresis gel has been color-inverted for clarity. bp – base pairs.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Expressing, Incubation, SYBR Green Assay, Real-time Polymerase Chain Reaction, Diff-Quik, Staining, Control, Nucleic Acid Electrophoresis

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A–C) Transcript expression of the co-stimulatory molecules CD80, CD86 and CD40 in splenic CD11c + cells 2 h following incubation with increasing concentrations of IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. *Indicates significant differences ( P<0.05 ) compared to 0 µg/mL group. Data represent mean±SD from quadruplicate samples from one of two representative experiments. D) Flow cytometric evaluation of splenic CD11c + cells 24 h following incubation with increasing concentrations of IL-36α for 2 h. E) Cell surface expression of co-stimulatory molecules in splenic CD11c + cells 24 h following incubation with increasing concentrations of IL-36α for 2 h. MFI – mean fluorescence intensity. *Indicates significant differences ( P<0.05 ) compared to 0.1 µg/mL group. Data represent mean±SD from triplicate samples from one of two representative experiments.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Expressing, Incubation, SYBR Green Assay, Real-time Polymerase Chain Reaction, Fluorescence

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A) Flow cytometric evaluation of CD4 + T cell proliferation responses induced by IL-36α stimulated splenic CD11c + cells. Splenic CD11c + cells were incubated with increasing doses of IL-36α for 2 h. Following incubation, media containing IL-36α was removed and CFSE-labeled CD4 + T cells were co-cultured with IL-36α stimulated CD11c + cells. CFSE dilution was used to evaluate T cell proliferation responses 96 h following co-culture. CD4 + T cell proliferation was proportional to the concentration of IL-36α used for stimulating CD11c + cells used in the co-culture. Flow cytometry plot presented is representative of quadruplicate samples in one out of two independent experiments. B) Flow cytometric evaluation of antigen-specific CD4 + T cell proliferation responses induced by IL-36α stimulated splenic CD11c + cells. Splenic CD11c + cells were incubated with increasing doses of IL-36α and 100 ng/mL OVA 323-339 for 2 h. Following incubation, media containing IL-36α and the OVA peptide was removed and CFSE-labeled CD4 + T cells from OTII TCR transgenic mice were co-cultured with IL-36α stimulated, OVA peptide pulsed CD11c + cells. CFSE dilution in the culture was used to evaluate T cell proliferation responses 96 h following co-culture. CD4 + T cell proliferation was proportional to the concentration of IL-36α used for stimulating CD11c + cells used in the co-culture. Flow cytometry plot presented is representative of quadruplicate samples in one out of two independent experiments.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Incubation, Labeling, Cell Culture, Co-Culture Assay, Concentration Assay, Flow Cytometry, Transgenic Assay

Journal: PLoS ONE

Article Title: IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

doi: 10.1371/journal.pone.0045784

Figure Lengend Snippet: A) Cells from a mouse macrophage NF-κB reporter cell line (RAW-ELAM cells) were stimulated with increasing concentrations of IL-36α. Green fluorescent protein (GFP) expression, indicative of NF-κB activation, was increased in a dose-dependent manner upon incubation with IL-36α. Flow cytometry plot presented is representative of triplicate samples in one out of two independent experiments.

Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes, probed with rat anti-mouse IL-36α antibody (R&D Systems, Catalog No. MAB2297) and detected with a horseradish peroxidase conjugated goat anti-rat secondary antibody (Abcam) and ECL Plus chemiluminescent detection system (Amersham).

Techniques: Expressing, Activation Assay, Incubation, Flow Cytometry

Journal: Cell reports

Article Title: Consumption of fish oil high-fat diet induces murine hair loss via epidermal fatty acid binding protein in skin macrophages

doi: 10.1016/j.celrep.2022.111804

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For IL-36 intracellular staining, cells were treated with 200 μM PA, DPA or control BSA for 48 h. After surface staining with anti-mouse CD11c and anti-mouse F4/80, cells were permeabilized for intracellular staining of IL-36α (32103-05161, Assaypro) or IL-36γ (PAL621Mu01, Cloud-clone Corp).

Techniques: Purification, Recombinant, Activation Assay, SYBR Green Assay, Detection Assay, Enzyme-linked Immunosorbent Assay, Selection, Software

Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: (A) Human primary keratinocytes were treated for 1 hour with 100 ng/mL IL-36α or 200 ng/mL IL-17A and 10 ng/mL TNF-α. The CDK4/6 inhibitor abemaciclib (Abe) or an ethanol vehicle control (Ctrl) were added in parallel. Phosphorylation of RB (pRB) served as a control for CDK4/6 inhibition, and actin as a loading control. Relative mRNA levels of IκBζ (NFKBIZ) were normalized to the reference gene RPL37A. (B) Luciferase assay of IκBζ (NFKBIZ) promoter activity in HaCaT cells that were cytokine-stimulated for 24 hours in the presence or absence of the CDK4/6 inhibitors abemaciclib or palbociclib (Pal). Relative luciferase (luc) activity was normalized to an internal Renilla luciferase control that was transfected in parallel. Endogenous protein levels were analyzed as input controls by immunoblotting (bottom). (C and D) CDK4 and CDK6 were depleted in primary human keratinocytes by lentiviral transduction of shRNA. Ctrl shRNA– or CDK4/6 shRNA–depleted cells were treated with (C) IL-36α or (D) IL-17A/TNF-α, similar as in A. (E and F) Human primary keratinocytes were stimulated with IL-36α as in A. (E) Cytokine gene expression in CDK4/6 inhibitor–treated cells. (F) Relative gene expression levels in IL-36α–treated control or CDK4/6-depleted cells. (G) Transient overexpression of CDK4, CDK6, or CDK9 in HaCaT cells, treated for 1 hour with 100 ng/mL IL-36α. (H) Cytokine gene expression in IL-36α–treated primary keratinocytes overexpressing IκBζ in the presence or absence of abemaciclib. For all analyses, n = 3 ± SD. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: In the IL-36α–mediated psoriasis model, ears of male C57BL/6 mice (8–12 weeks old, Jackson Laboratory) were treated by intradermal injections of 1 μg murine IL-36α (7059-ML, R&D Systems) or PBS control for 5 consecutive days.

Techniques: Inhibition, Luciferase, Activity Assay, Transfection, Western Blot, Transduction, shRNA, Expressing, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: (A) Luciferase assay of the NFKBIZ promoter in HEK293T cells after transient expression of CDK4, CDK6, STAT3, or p65, alone or in combination. The plasmid amounts for STAT3 (200 ng) and p65 (70 ng) were adjusted to achieve similar luciferase activity in the absence of CDK4/6 expression. Overexpression of the HA-tagged CDK4 and CDK6 proteins was detected using a HA-antibody. (B) Primary human keratinocytes with a transient overexpression of hyperactive STAT3 (STAT3C) were treated for 1 hour with 100 ng/mL IL-36α and abemaciclib (Abe). NFKBIZ mRNA levels normalized to RPL37A. Immunoblot analysis of STAT3C overexpression and CDK4/6 inhibition. (C) IκBζ target gene expression in STAT3C-overexpressing primary keratinocytes. Treatment as in B. (D) Luciferase activity assay of the NFKBIZ promoter in HEK293T cells overexpressing STAT3 alone or in combination with WT CDK6 (wt), hyperactive CDK6 (S178P), or a kinase-dead CDK6 mutant (CDK6 DN). For all analyses, n = 3 ± SD. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: In the IL-36α–mediated psoriasis model, ears of male C57BL/6 mice (8–12 weeks old, Jackson Laboratory) were treated by intradermal injections of 1 μg murine IL-36α (7059-ML, R&D Systems) or PBS control for 5 consecutive days.

Techniques: Luciferase, Expressing, Plasmid Preparation, Activity Assay, Over Expression, Western Blot, Inhibition, Targeted Gene Expression, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: (A) STAT3 activity was detected by analyzing the phosphorylation state at tyrosine 705 (Y705) and threonine 727 (T727) of STAT3 in primary human keratinocytes. After overnight starvation, cells were stimulated for 1 hour with IL-36α or IL-17A/TNF-α in the presence or absence of abemaciclib (Abe). (B) STAT3 activity in CDK4- and CDK6-depleted keratinocytes. Stimulation as in A. (C) Immunoblot detection of phosphorylated STAT3 (Y705) in IL-36α–stimulated keratinocytes, in which EZH2 function was suppressed by the EZH2 inhibitor EPZ6438 (EPZ, 10 μM) or shRNA-mediated knockdown. Detection of H3K27me3 controlled effective EZH2 inhibition or depletion. (D) Immunoblot detection of phosphorylated EZH2 at threonine 345 (T345) and threonine 487 (T487) in abemaciclib-treated or CDK4/6-depleted keratinocytes following stimulation with IL-36α. (E) Coimmunoprecipitation of EZH2 and STAT3 in HaCaT cells treated for 30 minutes with IL-36α in the presence or absence of abemaciclib. An EZH2-specific antibody or IgG was used for pull down of protein complexes. STAT3 and pEZH2 (T345) were detected by immunoblotting. (F) Luciferase activity assay of the NFKBIZ promoter in HEK293T cells, which transiently overexpress CDK6, WT EZH2 (wt), mutant EZH2 (T345A), or STAT3, alone or in combination. Equal protein expression was detected by immunoblotting. n = 3 ± SD. (G) Gene expression in IL-36α– and abemaciclib-treated, primary keratinocytes following transient expression of a phospho-mimicking EZH2 (T345D) mutant. Input controls (left). mRNA levels of NFKBIZ and its target genes were normalized to RPL37A (right). n = 3 ± SD. (H) Overexpression of IκBζ overrides the inhibitory effects of EPZ6438 (EPZ) on IL-36α–stimulated gene expression in primary keratinocytes. n = 3 ± SD. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: In the IL-36α–mediated psoriasis model, ears of male C57BL/6 mice (8–12 weeks old, Jackson Laboratory) were treated by intradermal injections of 1 μg murine IL-36α (7059-ML, R&D Systems) or PBS control for 5 consecutive days.

Techniques: Activity Assay, Western Blot, shRNA, Inhibition, Luciferase, Mutagenesis, Expressing, Over Expression

Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: (A and B) Detection of methylated STAT3 by coimmunoprecipitation. EZH2 and STAT3 (A) or CDK6 and STAT3 (B) were transiently expressed in HEK293T cells. After 1 hour of treatment with (A) abemaciclib (Abe) or (B) EPZ6438 (EPZ), cell lysates were prepared and subjected to immunoprecipitation using a STAT3-specific antibody or control IgG. (C) NFKBIZ promoter-driven luciferase activity in HEK293T cells, transiently expressing CDK6 and EZH2, alone or in combination with WT (wt) STAT3 or methylation-defective STAT3 mutant (K180R). n = 3 ± SD. (D) Analysis of IκBζ and IκBζ target gene expression in STAT3 wt or STAT3 K180R-expressing HaCaT cells. STAT3 wt or STAT3 K180R constructs were transiently expressed in STAT3-KO HaCaT cells, followed by stimulation for 1 hour with IL-36α. n = 3 ± SD. (E) Chromatin immunoprecipitation (ChIP) of STAT3, EZH2, or IgG control in STAT3-KO HaCaT cells reconstituted with either STAT3 wt or STAT3 K180R after 30 minutes of stimulation with IL-36α. Fold enrichment at the NFKBIZ promoter or at the myoglobin genomic region (MB; as negative control) was calculated relative to the IgG control. n = 3 ± SD. (F) ChIP of STAT3, EZH2, CDK4, and CDK6 in IL-36α–stimulated HaCaT cells stimulated for 30 minutes with IL-36α. Shown is the fold enrichment over IgG control. n = 3 ± SD. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: In the IL-36α–mediated psoriasis model, ears of male C57BL/6 mice (8–12 weeks old, Jackson Laboratory) were treated by intradermal injections of 1 μg murine IL-36α (7059-ML, R&D Systems) or PBS control for 5 consecutive days.

Techniques: Methylation, Immunoprecipitation, Luciferase, Activity Assay, Expressing, Mutagenesis, Targeted Gene Expression, Construct, Chromatin Immunoprecipitation, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: (A) Expression data from skin biopsies of 64 healthy individuals and 58 patients with psoriasis were analyzed from the GEO profile data set GDS4602. Shown are normalized expression values for CCND1, CCND2, and CCND3. EZH2 mRNA (B) and protein levels (C) in human skin samples from healthy individuals and patients with psoriasis; retrieved from the same data set as in A and B. Significance was calculated with a 1-way ANOVA test: *P < 0.05; **P < 0.01; ***P < 0.001. Scale bars: 100 μm. (D) Analysis of Ccnd2, Ccnd3, and Ezh2 mRNA levels in IMQ-treated mice ears at day 6. Values were normalized to Actin. n = 6 per group ± SEM. (E) Analysis of Ccnd2, Ccnd3, and Ezh2 mRNA levels in IL-36α–treated mice ears at day 5. n = 6 per group ± SEM. Significance was calculated using a 2-tailed Student’s t test: *P < 0.05; **P < 0.01; ***P < 0.001. (F) IHC staining of EZH2, cyclin D2, and cyclin D3 in untreated (Ctrl) and IMQ-treated mouse ears at day 6. Scale bars: 40 μm.

Article Snippet: In the IL-36α–mediated psoriasis model, ears of male C57BL/6 mice (8–12 weeks old, Jackson Laboratory) were treated by intradermal injections of 1 μg murine IL-36α (7059-ML, R&D Systems) or PBS control for 5 consecutive days.

Techniques: Expressing, Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: (A) Ear thickness measurements during topical treatment of mice with IMQ with or without abemaciclib (Abe; 10 μL of a 2% solution) or the EZH2 inhibitor CPI-169 (CPI, 10 μL of a 5% solution). n = 6 mice per group ± SEM. (B) H&E staining of untreated (Ctrl), IMQ-, IMQ and Abe–, or IMQ and CPI–treated ears. Scale bars: 100 μm. (C) Phospho-RB (pRB) and H3K27me3 staining after 6 days of treatment validated effective CDK4/6 and EZH2 inhibition, respectively. Scale bars: 40 μm. (D) Infiltrating immune cells in mouse ears at day 6 of treatment were quantified as follows: Neutrophils: CD45+, CD11b+, Ly6G+; macrophages: CD45+, CD11b+, F4/80+; T cells: CD45+, CD3+, and αβ-TCR+ or γδ-TCR+. n = 3 mice per group ± SEM. (E) Flow cytometry analysis of IMQ-treated or IMQ and CPI-169–treated mouse ears at day 6. (F) Protein levels in untreated (Ctrl) and treated mouse skin tissue at day 6. (G) Ear thickness of IL-36α–treated mice at day 5. Ears of mice were daily treated by intradermal injections with 1 μg IL-36α. Control mice received injections with PBS. Additionally, mice received topical treatment with ethanol as control (Veh), 2% abemaciclib (Abe), or 5% CPI-169 (CPI). n = 6 mice per group ± SEM. (H) H&E staining of PBS- or IL-36α–treated ears at day 5. Scale bars: 100 μm. (I) Immunoblot analysis of IκBζ, EZH2 phosphorylation (pEZH2 T345) and STAT3 activation (pSTAT3 Y705) in treated mouse skin tissue at day 5. pRB and H3K27me3 were analyzed as positive controls for drug action. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: In the IL-36α–mediated psoriasis model, ears of male C57BL/6 mice (8–12 weeks old, Jackson Laboratory) were treated by intradermal injections of 1 μg murine IL-36α (7059-ML, R&D Systems) or PBS control for 5 consecutive days.

Techniques: Staining, Inhibition, Flow Cytometry, Western Blot, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: The CDK4/6-EZH2 pathway is a potential therapeutic target for psoriasis

doi: 10.1172/JCI134217

Figure Lengend Snippet: All analyses were performed with n = 6 mice per group ± SEM. (A) Treatment scheme for the therapy using the IMQ mouse model. To explore whether CDK4/6 and EZH2 inhibitors suppress already-established psoriasis-like skin inflammation, mice were first treated with IMQ, followed by the application of 2% abemaciclib or 5% CPI-169 solution starting at the third IMQ application. (B) Ear thickness measurements during treatment. (C) H&E staining of untreated (Ctrl), IMQ-, IMQ and Abe–, or IMQ and CPI–treated ears. H&E staining shows the prevalence of psoriasis-like symptoms at IMQ day 2 when the inhibitors were applied for the first time. Scale bars: 100 μm. (D) Quantification of infiltrating immune cells in mouse ears at day 6. Immune cell subpopulations were quantified as in Figure 6D. n = 3 mice per group ± SEM. (E) Protein levels in untreated (Ctrl) and IMQ-treated mouse skin tissue in the presence or absence of abemaciclib or CPI-169 at day 6. Mice were treated as in A. FOXM1 and H3K27me3 were analyzed as positive controls for drug action. (F) Treatment scheme in the IL-36–induced psoriasis mouse model. IL-36–mediated psoriasis-like dermatitis was induced by administration of 1 μg IL-36α at every second day. Control mice received PBS. Starting from day 4 of IL-36α injection, ethanol as Vehicle (Veh), 2% abemaciclib, or 5% CPI-169 were daily applied by topical administration. (G) Ear thickness measurements during IL-36α treatment. (H) H&E staining of PBS- or IL-36α–treated ears at day 9. Scale bars: 100 μm. (I) Immunoblot analysis in IL-36α–treated mouse skin tissue at day 9. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: In the IL-36α–mediated psoriasis model, ears of male C57BL/6 mice (8–12 weeks old, Jackson Laboratory) were treated by intradermal injections of 1 μg murine IL-36α (7059-ML, R&D Systems) or PBS control for 5 consecutive days.

Techniques: Staining, Injection, Western Blot

Journal: The Journal of allergy and clinical immunology

Article Title: IL-1 and IL-36 are dominant cytokines in generalized pustular psoriasis

doi: 10.1016/j.jaci.2016.08.056

Figure Lengend Snippet: IL-36α was not expressed by healthy skin (a), found in the cytoplasm of the uppermost layers of viable KC in PV lesions (b) and expressed at a higher intensity by a wider band of KC in GPP lesions (c). IL-36α expression was strongest in the LC proximal to neutrophilic pustules, but not the neutrophils themselves (d). Faint expression of IL-36γ could be detected in the cytoplasm, but was most apparent in the nuclei of healthy skin KC (e). IL-36γ was intensified in PV lesions (f) yet this was more intense in GPP lesions (g), most pronounced in the KC adjacent to pustules (h). Representative images from 6 GPP, PV and NN donors used (Supplemental Figure 1). DAB with hematoxylin counterstain. Scale bar 100µm.

Article Snippet: Immunohistochemistry was performed on formalin fixed and paraffin embedded human skin biopsies sectioned at 5 μm, and then immunohistochemically stained after antigen retrieval using polyclonal goat anti-IL-36α (1μg/ml, AF1078, R&D Systems), IL-36γ (2.5μg/ml, rat, Y-12, Santa Cruz Biotech), polyclonal goat anti-CXCL8 (5μg/ml, AF2008, R&D Systems), polyclonal rabbit anti-cathepsin G (5μg/ml, NBP233498, Novus Biologicals), neutrophil elastase (1μg/ml, mouse, NP57, Dako), proteinase 3 (1μg/ml, EPR6277, LSBio), and matched isotype control antibodies.

Techniques: Expressing

Journal: The Journal of allergy and clinical immunology

Article Title: IL-1 and IL-36 are dominant cytokines in generalized pustular psoriasis

doi: 10.1016/j.jaci.2016.08.056

Figure Lengend Snippet: Exposure to NETs for 1 hour (a–d) significantly increased the ability of full-length (FL)-IL-36γ to induce CXCL1 and IL8 expression by KC more than 3-fold compared with untreated FL-IL-36γ (a and c), suggesting that enzyme activity within the NETs caused FL-IL-36γ activation. Truncated (T)-IL-36 as positive control for IL-36 activity. Purified neutrophil elastase (NE, e–h) and cathepsin G (CG, i–l) activated FL-IL-36α and FL-IL-36γ respectively which was inhibited by the addition of specific inhibitors of elastase (serpin A1) or cathepsin G (serpin A3). Statistical significance determined with Student’s t-test and indicated *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n.s. non-significant.

Article Snippet: Immunohistochemistry was performed on formalin fixed and paraffin embedded human skin biopsies sectioned at 5 μm, and then immunohistochemically stained after antigen retrieval using polyclonal goat anti-IL-36α (1μg/ml, AF1078, R&D Systems), IL-36γ (2.5μg/ml, rat, Y-12, Santa Cruz Biotech), polyclonal goat anti-CXCL8 (5μg/ml, AF2008, R&D Systems), polyclonal rabbit anti-cathepsin G (5μg/ml, NBP233498, Novus Biologicals), neutrophil elastase (1μg/ml, mouse, NP57, Dako), proteinase 3 (1μg/ml, EPR6277, LSBio), and matched isotype control antibodies.

Techniques: Expressing, Activity Assay, Activation Assay, Positive Control, Purification