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

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: IL-36α inhibits melanoma by inducing pro-inflammatory polarization of macrophages

doi: 10.1007/s00262-023-03477-5

Figure Lengend Snippet: IL-36α inhibits melanoma growth by promoting MHC IIhigh macrophage infiltration in the TME. a Flow cytometry analysis of MHC IIhigh macrophages, M-MDSC and Treg cells in mouse B16-vec and B16-IL-36α melanoma tissues. b Representative flow cytometric plots and percentages of CD45+ lymphocytes in melanoma tissues. c–h Representative flow cytometric plots and percentages of CD11b+F4/80+ TAMs (c), F4/80+MHCII+ MHC IIhigh macrophages (d), CD11b+Ly6C+ M-MDSCs (e), CD4+ T cells (f), CD8+ T cells (g) and CD4+Foxp3+ Tregs (h) within the gated CD45+ population in melanoma tissues. Data are shown as mean ± SEM. Five mice were in each group. *p < 0.05; **p < 0.01; ***p < 0.001, determined by two-tailed unpaired Student’s t test. B16-IL-36α was compared with B16-vec

Article Snippet: Protein expression levels of IL-36α, IL-36β, and IL-36γ were determined by western blotting using anti-IL-36α (AF2297-SP, R&D Systems), anti-IL-36β (PA5-81415, Invitrogen) and anti-IL-36γ (A10165, ABclonal) antibodies.

Techniques: Flow Cytometry, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: IL-36α inhibits melanoma by inducing pro-inflammatory polarization of macrophages

doi: 10.1007/s00262-023-03477-5

Figure Lengend Snippet: IL-36α may have an anti-tumor function in both human and mice. a Relationship between high and low expression of IL-36A, IL-36B and IL-36G and patient survival in patients with thymoma, kidney renal clear cell carcinoma and melanoma. Data were from the Kaplan–Meier plotter database and the cBioPortal database. b Schematic protocol of the mice tumor model. Mice were challenged with 5 × 105 B16-vec or B16-IL-36α s.c. On day 14, melanoma tissue was removed and analyzed. c Mice weights were measured every 2 days from the fourth day after tumor implantation. (n = 5). Data are shown as mean ± SEM. Five mice were in each group. *p < 0.05; **p < 0.01; ***p < 0.001, determined by Mann–Whitney test. B16-IL-36α, B16-IL-36β and B16-IL-36γ was compared with B16-vec separately. d, f On day 14 after implantation, the mice tumors were removed and weighed. (n = 5). Data are shown as mean ± SEM. Five mice were in each group. *p < 0.05; **p < 0.01; ***p < 0.001, determined by one-way ANOVA with Dunn’s test. B16-IL-36α, B16-IL-36β and B16-IL-36γ were compared with B16-vec separately. e Tumor volume in mice was measured daily from day 8 after tumor implantation. (n = 5). Data are shown as mean ± SEM. Five mice were in each group. *p < 0.05; **p < 0.01; ***p < 0.001, determined by Mann–Whitney test. B16-IL-36α, B16-IL-36β and B16-IL-36γ were compared with B16-vec separately. g The survival of mice was monitored. Ten mice were in each group. The p value was based on a log-rank test. B16-IL-36α, B16-IL-36β and B16-IL-36γ were compared with B16-vec separately

Article Snippet: Protein expression levels of IL-36α, IL-36β, and IL-36γ were determined by western blotting using anti-IL-36α (AF2297-SP, R&D Systems), anti-IL-36β (PA5-81415, Invitrogen) and anti-IL-36γ (A10165, ABclonal) antibodies.

Techniques: Expressing, Tumor Implantation, MANN-WHITNEY

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: IL-36α inhibits melanoma by inducing pro-inflammatory polarization of macrophages

doi: 10.1007/s00262-023-03477-5

Figure Lengend Snippet: IL-36α reverses the M2-type polarization and promotes the M1-type polarization of macrophages. a, b Changes in JAK1/STAT6 pathways of BMDMs stimulated with tumor condition medium collected from 1 × 106 B16-vec cells or B16-IL-36α cells were detected by immunoblotting assay at different time points. c, d Changes in JAK1/STAT6 pathways of BMDMs stimulated with TCM collected from 1 × 106 B16-vec cells with or without 100 ng/mL IL-36α were detected by immunoblotting assay at different time points. Data (mean ± SEM) are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, determined by two-way ANOVA with Tukey test. e Heatmap showing the log10 fold change of downregulated and upregulated genes in wild-type BMDMs stimulated with PBS or 100 ng/ml of IL-36α for 6 h by RT-qPCR analysis. (n = 3). f, g. Changes in JAK1/STAT6 pathways of BMDMs stimulated with 20 ng/mL IL-4 or 20 ng/mL IL-4 and 100 ng/ml of IL-36α were detected by immunoblotting assay at different time points. Data (mean ± SEM) are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, determined by two-way ANOVA with Tukey test. h, i. Changes in NF-κB and MAPK molecular pathways of BMDMs stimulated with 100 ng/ml of IL-36α were detected by immunoblotting assay at different time points. Data (mean ± SEM) are representative of at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, determined by one-way ANOVA with Dunn’s post hoc analysis

Article Snippet: Protein expression levels of IL-36α, IL-36β, and IL-36γ were determined by western blotting using anti-IL-36α (AF2297-SP, R&D Systems), anti-IL-36β (PA5-81415, Invitrogen) and anti-IL-36γ (A10165, ABclonal) antibodies.

Techniques: Western Blot, Quantitative RT-PCR

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: IL-36α inhibits melanoma by inducing pro-inflammatory polarization of macrophages

doi: 10.1007/s00262-023-03477-5

Figure Lengend Snippet: IL-36α inhibits melanoma lung metastasis by elevating MHC IIhigh macrophage proportion. a Schematic protocol of the mice lung metastasis model. Mice were challenged with 5 × 105 B16-vec or B16-IL-36α i.v., respectively. On day 20, mice lung tissues were removed and analyzed. b, c Metastatic tumor nodules in the lung were quantified 20 days after B16-vec and B16-IL-36α tumor inoculation. d Lung tissue weights were analyzed after B16-vec and B16-IL-36α metastasis. e Representative flow cytometric plots and percentages of CD45+ lymphocytes in lung tissues. f–k Representative flow cytometric plots and percentages of CD11c+F4/80+ TAMs (f), F4/80+MHCII+ MHC IIhigh macrophages (g), CD11b+Ly6C+ M-MDSCs (h), CD4+ T cells (i), CD8+ T cells (j) and CD4+Foxp3+ Tregs (k) within the gated CD45+ population in lung tissues. Data are shown as mean ± SEM. Five mice were in each group. *p < 0.05; **p < 0.01; ***p < 0.001, determined by two-tailed unpaired Student’s t test. B16-vec was compared with B16-IL-36α

Article Snippet: Protein expression levels of IL-36α, IL-36β, and IL-36γ were determined by western blotting using anti-IL-36α (AF2297-SP, R&D Systems), anti-IL-36β (PA5-81415, Invitrogen) and anti-IL-36γ (A10165, ABclonal) antibodies.

Techniques: Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: IL-36α inhibits melanoma by inducing pro-inflammatory polarization of macrophages

doi: 10.1007/s00262-023-03477-5

Figure Lengend Snippet: IL-36α reverses the M2-type polarization of macrophages. a Changes in cytokines expression of BMDMs stimulated with TCM collected from 1 × 106 B16-vec cells or B16-IL-36α cells for 24 h with or without 100 ng/mL IL-36α using RT-qPCR assays. Data (mean ± SEM) are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, determined by One-way ANOVA with Dunn’s post hoc analysis. b Heatmap showing the downregulated and upregulated genes in B16-vec or B16-IL-36α tumor tissues by RT-qPCR analysis. (n = 5). c Log2 fold changes of cytokine gene expression of the B16-vec tumor tissues compared with B16-IL-36α tumor tissues. (n = 5). d Heatmap showing the downregulated and upregulated genes in B16-vec or B16-IL-36α tumor cells metastatic lung tissues by RT-qPCR analysis. (n = 5). e Log2 fold changes of cytokine gene expression of the B16-vec metastatic lung tissues compared with B16-IL-36α metastatic lung tissues. (n = 5). f Changes in cytokines expression of BMDMs stimulated with PBS, 20 ng/mL IL-4 or 20 ng/mL IL-4 and 100 ng/mL of IL-36α by RT-qPCR after 6 h. Data (mean ± SEM) are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, determined by one-way ANOVA with Dunn’s post hoc analysis

Article Snippet: Protein expression levels of IL-36α, IL-36β, and IL-36γ were determined by western blotting using anti-IL-36α (AF2297-SP, R&D Systems), anti-IL-36β (PA5-81415, Invitrogen) and anti-IL-36γ (A10165, ABclonal) antibodies.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: IL-36α inhibits melanoma by inducing pro-inflammatory polarization of macrophages

doi: 10.1007/s00262-023-03477-5

Figure Lengend Snippet: IL-36α has a significant therapeutic effect on the growth of melanoma. a Schematic protocol of the mice melanoma treatment model. Mice were challenged with 5 × 105 B16-vec cells s. c. and then treated with 10 μg/kg IL-36α i.p. every other day. On day 14, mice melanomas were removed and analyzed. b, c Mice melanoma tissues with or without IL-36α treatment were removed on day 14, and their weights were measured and counted. d Representative flow cytometric plots and percentages of CD45+ lymphocytes in melanoma tissues. e–j Representative flow cytometric plots and percentages of CD11b+F4/80+ TAMs (e), F4/80+MHCII+ MHC IIhigh macrophages (f), CD11b+Ly6C+ M-MDSCs (g), CD4+ T cells (h), CD8+ T cells (i) and CD4+Foxp3+ Tregs (j) within the gated CD45+ population in melanoma tissues. Data are shown as mean ± SEM. Five mice were in each group. *p < 0.05; **p < 0.01; ***p < 0.001, determined by two-tailed unpaired Student’s t test. B16-vec with IL-36α treatment was compared with the group without IL-36α treatment. k The survival of mice was monitored. Ten mice were in each group. The p value was based on a log-rank test. B16-vec with IL-36α treatment was compared with the group without IL-36α treatment

Article Snippet: Protein expression levels of IL-36α, IL-36β, and IL-36γ were determined by western blotting using anti-IL-36α (AF2297-SP, R&D Systems), anti-IL-36β (PA5-81415, Invitrogen) and anti-IL-36γ (A10165, ABclonal) antibodies.

Techniques: Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: IL-36α inhibits melanoma by inducing pro-inflammatory polarization of macrophages

doi: 10.1007/s00262-023-03477-5

Figure Lengend Snippet: IL-36α increases the anti-tumor effect of PD-L1 antibody. a Schematic protocol of the mice PD-L1 antibody in combination with IL-36α model. Mice were challenged with 5 × 105 B16-vec cells s. c. and then treated with 10 μg/kg IL-36α, 10 mg/kg PD-L1 antibody, or PD-L1 antibody in combination with IL-36α i.p. from the eighth day. On day 14, mice melanoma tissues were removed and analyzed. b, c Mice melanoma tissues with or without IL-36α treatment were removed on day 14, and their weights were measured. Data are shown as mean ± SEM. Five mice were in each group. *p < 0.05; **p < 0.01; ***p < 0.001, determined by one-way ANOVA with Dunn’s post hoc analysis. B16-vec group was compared with B16-vec + Anti-PD-L1 group. B16-vec + IL-36α group was compared with B16-vec + Anti-PD-L1 group. B16-vec + Anti-PD-L1 + IL-36α group was compared with B16-vec + Anti-PD-L1. d Mice weights were measured every 2 days from the 4th day after implantation of tumors. e Tumor volumes of mice were measured daily from day 8 after tumor implantation. Data are shown as mean ± SEM. Five mice were in each group. *p < 0.05; **p < 0.01; ***p < 0.001, determined by Mann–Whitney test. B16-vec group was compared with B16-vec + Anti-PD-L1 group. B16-vec + IL-36α group was compared with B16-vec + Anti-PD-L1 group. B16-vec + Anti-PD-L1 + IL-36α group was compared with B16-vec + Anti-PD-L1. f Representative flow cytometric plots and percentages of CD45+ lymphocytes in melanoma tissues. g–l Representative flow cytometric plots and percentages of CD11b+F4/80+ TAMs (g), F4/80+MHCII+ MHC IIhigh macrophages (h), CD11b+Ly6C+ M-MDSCs (i), CD4+ T cells (j), CD8+ T cells (k) and CD4+Foxp3+ Tregs (l) within the gated CD45.+ population in melanoma tissues. Data are shown as mean ± SEM. Five mice were in each group. *p < 0.05; **p < 0.01; ***p < 0.001, determined by one-way ANOVA with Dunn’s post hoc analysis. B16-vec group was compared with B16-vec + Anti-PD-L1 group. B16-vec + IL-36α group was compared with B16-vec + Anti-PD-L1 group. B16-vec + Anti-PD-L1 + IL-36α group was compared with B16-vec + Anti-PD-L1

Article Snippet: Protein expression levels of IL-36α, IL-36β, and IL-36γ were determined by western blotting using anti-IL-36α (AF2297-SP, R&D Systems), anti-IL-36β (PA5-81415, Invitrogen) and anti-IL-36γ (A10165, ABclonal) antibodies.

Techniques: Tumor Implantation, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: RIPK3 promotes skin inflammation by enhancing IL-36α signaling and necroptosis in keratinocytes

doi: 10.1038/s41419-025-08096-9

Figure Lengend Snippet: A The level of cytokines (TNF-α, IL-6 and IFN-γ) in serum was measured by ELISA. B Heatmap showing mRNA expression levels of cytokines, chemokines and antimicrobial peptides in the skin as measured by RT-PCR. C Percentage of leukocytes (CD45 + ) and neutrophils (CD11b + Gr-1 + ) cells analyzed by flow cytometry in the skin. Immunofluorescence detection of protein levels of S100A8 ( D ) and Gr-1 ( E ) in the skin. Scale bars, 100 μm. RIPK3 f/f ( n = 5), RIPK3 f/f IMQ ( n = 7), RIPK3 ΔKC ( n = 4), RIPK3 ΔKC IMQ ( n = 6). These results are representative of three independent experiments. All dates are shown as means ± SEM, compared as indicated, were measured by one-way ANOVA.

Article Snippet: After treatment with the blocking solution (Beyotime) for 1 h, the sections were treated with RIPK3 (CST, Danvers, MA, USA, 95702), p-MLKL (Abcam, Cambridge, UK, ab196436), S100A8 (Proteintech, Chicago, IL, USA, 15792-1-AP), fluorescein isothiocyanate (FITC)-labeled Gr-1 mAb (BD Biosciences, USA, 553127), and goat anti-IL-36α (R&D, AF2297) at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Immunofluorescence

Journal: Cell Death & Disease

Article Title: RIPK3 promotes skin inflammation by enhancing IL-36α signaling and necroptosis in keratinocytes

doi: 10.1038/s41419-025-08096-9

Figure Lengend Snippet: A Representative western blot assay of necroptotic proteins in primary keratinocytes from RIPK3 f/f and RIPK3 ΔKC mice treated with IL-36α for 6 hours. B RT-PCR detection of mRNA expression levels of S100a8 , S100a9 , Il-1α and Il-36α after TSZ induction in primary keratinocytes. C Representative western blot images assay of necroptotic proteins in IL-36α-induced primary keratinocytes. D RT-PCR detection of mRNA expression levels of cytokines such as Cxcl2 , Lcn2 , S100a9 and Il-36α after IL-36α induction in primary keratinocytes. E Representative images of mice treated with saline and IL-36α at day 5 to demonstrate the scaling of the treated area. Ear thickness ( F ) and ear swelling ( G ) in mice treated with saline and IL-36α in each group. n = 5 mice per group. These results are representative of three independent experiments. All dates are shown as means ± SEM. *** P < 0.001, ns indicates no significance, compared as indicated, were measured by two-way ANOVA. Significant differences were observed between the WT and WT TSZ groups, as well as between the WT and WT IL-36α groups.

Article Snippet: After treatment with the blocking solution (Beyotime) for 1 h, the sections were treated with RIPK3 (CST, Danvers, MA, USA, 95702), p-MLKL (Abcam, Cambridge, UK, ab196436), S100A8 (Proteintech, Chicago, IL, USA, 15792-1-AP), fluorescein isothiocyanate (FITC)-labeled Gr-1 mAb (BD Biosciences, USA, 553127), and goat anti-IL-36α (R&D, AF2297) at 4 °C overnight.

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Saline