recombinant mouse il 36α  (R&D Systems)

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: Then, the macrophages were stimulated for 6 h with recombined murine IL-36α (100 ng/mL) (7059-ML-010/CF, R&D Systems), and the expression of cytokines was detected using real-time quantitative reverse transcription PCR (RT-qPCR) assays.

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: Then, the macrophages were stimulated for 6 h with recombined murine IL-36α (100 ng/mL) (7059-ML-010/CF, R&D Systems), and the expression of cytokines was detected using real-time quantitative reverse transcription PCR (RT-qPCR) assays.

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: Then, the macrophages were stimulated for 6 h with recombined murine IL-36α (100 ng/mL) (7059-ML-010/CF, R&D Systems), and the expression of cytokines was detected using real-time quantitative reverse transcription PCR (RT-qPCR) assays.

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: Then, the macrophages were stimulated for 6 h with recombined murine IL-36α (100 ng/mL) (7059-ML-010/CF, R&D Systems), and the expression of cytokines was detected using real-time quantitative reverse transcription PCR (RT-qPCR) assays.

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: Then, the macrophages were stimulated for 6 h with recombined murine IL-36α (100 ng/mL) (7059-ML-010/CF, R&D Systems), and the expression of cytokines was detected using real-time quantitative reverse transcription PCR (RT-qPCR) assays.

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: Then, the macrophages were stimulated for 6 h with recombined murine IL-36α (100 ng/mL) (7059-ML-010/CF, R&D Systems), and the expression of cytokines was detected using real-time quantitative reverse transcription PCR (RT-qPCR) assays.

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: Then, the macrophages were stimulated for 6 h with recombined murine IL-36α (100 ng/mL) (7059-ML-010/CF, R&D Systems), and the expression of cytokines was detected using real-time quantitative reverse transcription PCR (RT-qPCR) assays.

Techniques: Tumor Implantation, MANN-WHITNEY