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: Recombinant human IL-36α (6995-IL; aa 6-158), IL-36γ (6835-IL; aa 18-169), and mouse IL-36α (7059-ML; aa 6-160) were purchased from R&D Systems.

Techniques: Staining, Control, Injection, Western Blot

Journal: Life Science Alliance

Article Title: Gene-selective transcription promotes the inhibition of tissue reparative macrophages by TNF

doi: 10.26508/lsa.202101315

Figure Lengend Snippet: (A, B, C) RNA from wild-type BMDMs left untreated (Ctrl), stimulated with IL4/IL13, IL4/IL13 + TNF (A), IL4/IL13 + IL1b (B) or IL4/IL13 + IL36 (C) was used for qRT-PCR. Data shown are the mean fold-increase compared with the Ctrl group. (D) TnfR1 −/− BMDMs were stimulated with IL4/IL13 or IL4/IL13 + TNF over time. RNA was isolated and analyzed for Retnla expression. Data shown are the mean fold-increase of the Ctrl group. All values are means ± SEM; **** P < 0.0001. Statistically significant differences were determined by one-way ANOVA with Tukey correction (A, B, C) or by two-way ANOVA (D). If not indicated otherwise superscripts show statistical significance compared with the control group. n = 3 biological replicates.

Article Snippet: Where indicated, BMDMs were stimulated with 10 ng/ml IL4 (produced in insect cells), 10 ng/ml IL13 (210-13; Peprotech), 10 ng/ml TNF (315-01A; Peprotech), 10 ng/ml IL1β (401-ML/CF; R&D System), 33.3 ng/ml IL36α (7059-ML/CF; R&D Systems), 33.3 ng/ml IL36β (7060-ML/CF; R&D Systems), 33.3 ng/ml IL36γ (6996-IL/CF; R&D Systems), 5 ng/ml LPS from Escherichia coli O111:B4 (L4391; Sigma-Aldrich), 10 μg/ml Etanercept (Erelzi; Sandoz), 20 μM JNK-IN-8 (SML1246; Sigma-Aldrich), and 10 μM SP600126 (BML-EI305; Enzo).

Techniques: Quantitative RT-PCR, Isolation, Expressing, Control

Journal: The Journal of Clinical Investigation

Article Title: IL-36 γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

doi: 10.1172/JCI128678

Figure Lengend Snippet: ( A ) Gene expression profiling from lesional skin of 5 patients and 5 healthy controls (HC). Heatmap of the top 12 most differentially expressed genes ranked from lowest false discovery rate (FDR) and 12 selected genes are shown. ( B ) Quantitative PCR (qPCR) of mRNA from lesional skin samples of 10 EGFR inhibitor–treated patients with acneiform eruption and 10 healthy control skin biopsies. Data represent mean ± SD. ( C ) Immunohistochemical staining with goat anti–IL-36γ antibody of formalin-fixed, paraffin-embedded skin sections of acneiform eruption patient and normal donors. Scale bars: 100 μm. Pictures are representative of 5 patients and 5 healthy individuals. ( D ) PHKs were exposed to erlotinib (EGFR inhibitor, 1 μM) and C . acnes (MOI of 10) for 6 hours. Total RNA was analyzed by qPCR. Data represent mean ± SEM ( n = 3). ( E ) PHKs were exposed to erlotinib (1 μM) or C . acnes (MOI of 10) or both for 24 hours. Cell lysates were analyzed by Western blotting using specific antibodies against IL-36γ and β-actin. Blots were run contemporaneously with the same protein samples. ( F ) PHKs were exposed to erlotinib (1 μM) and Pam3CSK4 (5 μg/mL). IL-36γ secretion was measured by ELISA in culture supernatants. Data represent mean ± SEM ( n = 3). ( G ) Ex vivo skin explants were exposed to erlotinib (1 μM), Pam3CSK4 (5 μg/mL), and/or human IL-36Ra (1 μg/mL). The skin samples were then analyzed by qPCR. Data represent mean ± SEM ( n = 4). Data were analyzed with 2-tailed unpaired t test ( B ), and 1-way ANOVA followed by Dunnett’s ( D and F ) or Tukey’s multiple-comparisons test ( G ). * P < 0.05; ** P < 0.01; *** P < 0.001. Data are representative of 3 independent experiments.

Article Snippet: The goat anti–IL-36γ (catalog AF2320) and anti-mouse KLF4 (catalog AF3158) antibodies were from R&D Systems.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Control, Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded, Western Blot, Enzyme-linked Immunosorbent Assay, Ex Vivo

Journal: The Journal of Clinical Investigation

Article Title: IL-36 γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

doi: 10.1172/JCI128678

Figure Lengend Snippet: ( A ) Luciferase reporter assay of human IL-36γ transcriptional activity in PHKs transfected with IL-36γ-pGL3 (1630 bp) reporter plasmid, followed by exposure to erlotinib and C . acnes for 16 hours. Renilla luciferase activity was measured to determine transfection efficiency. Data represent mean ± SEM ( n = 4). E.V., empty vector. ( B and D ) Schematic of 5′-deletion and mutant constructs of the human IL-36γ promoter. Site-directed mutagenesis was performed to introduce the indicated mutation at the EGFR inhibitor–responsive site (EiRS) and p65 binding site. ( C and E ) 5′-Deletion and mutation study of the human IL-36γ promoter activity. PHKs were transfected with indicated plasmids, followed by exposure to erlotinib and C . acnes for 16 hours. Data represent mean ± SEM ( n = 4). ( F ) Quantitative PCR was performed to evaluate the gene expression of transcription factor candidates binding to the EiRS. PHKs were exposed to erlotinib for 6 hours. Data represent mean ± SEM ( n = 3). ( G ) Gel shift, competition, and supershift EMSA analysis using a Cy5-labeled oligonucleotide probe for the EiRS-containing region and HEK293T cell lysate containing KLF4 protein. ( H ) PHKs were exposed to erlotinib and C . acnes for 24 hours. ( I ) DNA pull-down assay using biotinylated wild-type– or mutant-oligonucleotide probe of the EiRS-containing region. These probes were incubated with extracts from PHKs exposed to erlotinib for 24 hours. DNA-associated proteins were visualized by Western blotting. ( J ) Ex vivo skin explants from healthy controls were exposed to erlotinib for 24 hours and KLF4 expression was assessed by Western blotting. The blot shown is representative of 2 different skin donors. Data were analyzed with 1-way ANOVA followed by Dunnett’s multiple-comparisons test ( A and E ) or with 2-tailed unpaired Mann-Whitney U ( C ) or t test ( F ). * P < 0.05; ** P < 0.01; *** P < 0.001. All blots were run contemporaneously with the same protein samples.

Article Snippet: The goat anti–IL-36γ (catalog AF2320) and anti-mouse KLF4 (catalog AF3158) antibodies were from R&D Systems.

Techniques: Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Construct, Introduce, Binding Assay, Real-time Polymerase Chain Reaction, Gene Expression, Gel Shift, Labeling, Pull Down Assay, Incubation, Western Blot, Ex Vivo, Expressing, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: IL-36 γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

doi: 10.1172/JCI128678

Figure Lengend Snippet: ( A ) PMKs were exposed to erlotinib (1 μM) and murine IL-36γ (100 ng/mL) for 6 hours; isolated RNA was analyzed by quantitative PCR. Data represent mean ± SEM ( n = 3). ( B ) PMKs from wild-type or KLF4-knockout mouse were exposed to murine IL-36γ (100 ng/mL) for 6 hours. Data represent mean ± SEM ( n = 3). PMK cell lysates were analyzed by SDS-PAGE and immunoblotting. Blots were run contemporaneously with the same protein samples. ( C ) Schematic of the human and murine IL-36γ promoter with predicted KLF4 binding site and p65 binding site by JASPAR. ( D ) Gel shift and competition EMSA analysis using a Cy5-labeled oligonucleotide probe for human and mouse KLF4 binding sites and HEK293T cell lysate containing murine KLF4 protein. Sequence-specific binding of human probe to murine KLF4 was demonstrated as a positive control. Gel shift reflecting formation of protein-DNA complexes with the murine probe, and KLF4 was not observed. Data are representative of 3 independent experiments.

Article Snippet: The goat anti–IL-36γ (catalog AF2320) and anti-mouse KLF4 (catalog AF3158) antibodies were from R&D Systems.

Techniques: Isolation, Real-time Polymerase Chain Reaction, Knock-Out, SDS Page, Western Blot, Binding Assay, Gel Shift, Labeling, Sequencing, Positive Control

Journal: The Journal of Clinical Investigation

Article Title: IL-36 γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes

doi: 10.1172/JCI128678

Figure Lengend Snippet: ( A ) Representative images of KLF4 expression (green) in PHKs after erlotinib or control DMSO exposure for 24 hours. Nuclei were stained with DAPI. Scale bars: 10 μm. Data are representative of 3 independent experiments. ( B and C ) Immunofluorescent staining with mouse anti-KLF4 (green) and rabbit anti–IL-36γ (red) antibodies of formalin-fixed, paraffin-embedded skin sections of acneiform eruption patients and healthy controls (HC). Nuclei were stained with DAPI. The white-boxed regions in B were zoomed separately in C . Scale bars: 50 μm ( B ) and 10 μm ( C ). Pictures are representative of 5 patients and 3 healthy individuals.

Article Snippet: The goat anti–IL-36γ (catalog AF2320) and anti-mouse KLF4 (catalog AF3158) antibodies were from R&D Systems.

Techniques: Expressing, Control, Staining, Formalin-fixed Paraffin-Embedded

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: IL-36 Induces Bisphosphonate-Related Osteonecrosis of the Jaw-Like Lesions in Mice by Inhibiting TGF-β-Mediated Collagen Expression

doi: 10.1002/jbmr.2985

Figure Lengend Snippet: Microarray gene profiling identifies overexpressed IL-36 family members in ONJ-like lesions in mice. (A) A graphic representation of timeline used in this study. (B) Clinical illustration of the osteomucosal tissues before and after removal at the tooth-extracted areas photographed 2 weeks after tooth extraction. (C) A graphic representation of the microarray profiling that are differentially expressed more than fivefold were represented (n = 3 per group). (D) The ingenuity pathway analysis (IPA) of functional pathways that are associated with ONJ lesions. (E) Lists of genes involved in biosynthesis of steroids. (F) List of genes involved in role of osteoblasts, osteoclasts, and chondrocytes in rheumatoid arthritis. The unhealed osteomucosal tissues at the tooth-extracted sites were harvested and subjected mRNA isolation, cDNA synthesis, and qRT-PCR for (G) IL-18, (H) IL-36α, (I) IL-36β, (J) IL-36γ, and (K) FDPS. The results are from two independent non-BRONJ and BRONJ lesions. (L) Staining for H&E and IHC staining for IL-36α at the tooth-extracted sites in vehicle (Veh)- or Zometa (ZOL)-treated mice. Scale bar = 100 µm. ELISA for (M) IL-18 and (N) IL-36α from the serum obtained from Veh- or ZOL-treated mice 2 weeks after tooth extraction. *p<0.05; **p<0.01; ***p<0.001. Results represent the means ± SD performed in triplicate unless otherwise indicated.

Article Snippet: Recombinant IL-36α (7059-ML), IL-36β (7060-ML), IL-36γ (6996-ML), and TGF-β (7666-MB) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Microarray, Extraction, Functional Assay, Isolation, cDNA Synthesis, Quantitative RT-PCR, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: IL-36 Induces Bisphosphonate-Related Osteonecrosis of the Jaw-Like Lesions in Mice by Inhibiting TGF-β-Mediated Collagen Expression

doi: 10.1002/jbmr.2985

Figure Lengend Snippet: IL-36α inhibits expression of collagen and α-Sma in GMSCs. (A) List of genes associated with connective tissues (eg, collagenous and non-collagenous proteins) from the microarray data. (B) qRT-PCR of Il-6 in gingival mesenchymal stem cells (GMSCs) treated with recombinant IL-36α, IL-36β, and IL-36γ for 4 days. (C) qRT-PCR of Col1a1, Col1a2, and Col3a1 in GMSCs treated with recombinant IL-36α, IL-36β, and IL-36γ for 4 days. (D) Western blotting of IL-1Rrp2 after knocking down with control siRNA (CTLsi) or IL-1Rrp2 siRNA (IL-1Rrp2si) in GMSCs. (E) qRT-PCR of Col1a1, Col1a2, and Col3a1 in GMSCs with IL-1Rrp2 knockdown and IL-36a treatment. (F) Western blotting of Col1a1 and α-Sma in GMSCs treated with IL-36α and TGF-β1 for 2 days. (G) ELISA for collagen type I from supernatants obtained from GMSCs treated with IL-36α and TGF-β1. (H) qRT-PCR of Col1a1, Col1a2, and α-Sma in GMSCs treated with recombinant IL-36α and TGF-β1 for 2 days. *p<0.05; **p<0.01; ***p<0.001. Results represent the means ± SD performed in triplicate.

Article Snippet: Recombinant IL-36α (7059-ML), IL-36β (7060-ML), IL-36γ (6996-ML), and TGF-β (7666-MB) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Expressing, Microarray, Quantitative RT-PCR, Recombinant, Western Blot, Control, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: IL-36 Induces Bisphosphonate-Related Osteonecrosis of the Jaw-Like Lesions in Mice by Inhibiting TGF-β-Mediated Collagen Expression

doi: 10.1002/jbmr.2985

Figure Lengend Snippet: Proposed model. TGF-β1 binds TGF-β receptor and causes phosphorylation of Smad2/3, which in turns translocates into the nucleus and activates the target genes such as Col1a1 or α-Sma. IL-36α binds to IL-36 receptor complex and activates the ERK signaling pathway. Activated Erk inhibits translocation of TGF-β1-activated Smad complex, thereby suppressing the Smad-mediated activation of gene expression.

Article Snippet: Recombinant IL-36α (7059-ML), IL-36β (7060-ML), IL-36γ (6996-ML), and TGF-β (7666-MB) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Phospho-proteomics, Translocation Assay, Activation Assay, Gene Expression