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

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Cancer Immunology, Immunotherapy Aims and scope Submit manuscript

Abstract

Interleukin-36α (IL-36α) is essential for various inflammatory conditions, such as psoriasis and rheumatoid arthritis, whereas its role in tumor immunity is unclear. In this study, it was demonstrated that IL-36α could activate the NF-κB and MAPK signaling pathways in macrophages, leading to the expression of IL-1β, IL-6, TNF-α, CXCL1, CXCL2, CXCL3, CXCL5 and iNOS. Importantly, IL-36α has significant antitumor effects, altering the tumor microenvironment and promoting the infiltration of MHC IIhigh macrophages and CD8+ T cells while decreasing the levels of monocyte myeloid-derived suppressor cells, CD4+ T cells and regulatory T cells. This ultimately results in the inhibition of tumor growth and migration. Furthermore, IL-36α synergized with the PD-L1 antibody increased the immune cells infiltration and enhanced the anti-tumor effect of the PD-L1 antibody on melanoma. Collectively, this study reveals a new role for IL-36α in promoting anti-tumor immune responses in macrophages and suggests its potential for cancer immunotherapy.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Abbreviations

B16-vec:

B16-vector

BMDMs:

Bone marrow-derived macrophages

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

i.p.:

Intraperitoneal

i.v.:

Intravenous

IL-36α:

Interleukin-36α

MAPK:

Mitogen-activated protein kinases

MDSCs:

Myeloid-derived suppressor cells

NF-κB:

Nuclear factor kappa B

PBS:

Phosphate-buffered solution

PCR:

Polymerase chain reaction

RT-qPCR:

Real-time quantitative reverse transcription PCR

s.c.:

Subcutaneous

TAM:

Tumor-associated macrophages

TCM:

Tumor-conditioned medium

TGF-β:

Transforming growth factor beta

TME:

Tumor microenvironment

Tregs:

Regulatory T cells

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (82073858, 81973329, 82273934, 82173821, 82104186), Translational Medicine Research Program of Anhui Province (202204295107020038), and Anhui Scientific Project (2022AH040214).

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Authors and Affiliations

  1. Shanghai Frontiers Science Center for Drug Target Identification and Delivery, School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, People’s Republic of China

    Xinyi Lou, Shixin Duan, Mengkai Li, Yao Yuan, Shiyi Chen, Zhiming Wang, Lei Sun & Feng Qian

  2. Anhui Province Key Laboratory of Translational Cancer Research, Department of Medical Oncology, First Affiliated Hospital of Bengbu Medical College, Bengbu Medical College, Bengbu, Anhui, 233004, People’s Republic of China

    Zishu Wang & Feng Qian

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  1. Xinyi Lou
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  2. Shixin Duan
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  3. Mengkai Li
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  4. Yao Yuan
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  8. Lei Sun
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Contributions

XL and SD did conception and design and acquisition of data. XL, SD, ML, YY, ZW, ZW done development of methodology. XL, SD and ML were involved in analysis and interpretation of data. XL and SC wrote the manuscript. LS and FQ done conceiving the study and writing the paper.

Corresponding authors

Correspondence to Lei Sun or Feng Qian.

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262_2023_3477_MOESM1_ESM.pdf

Supplementary data 1 Immunoblotting detects IL-36 family expression. a Immunoblotting assay was used to detect IL-36α expression in melanoma cells overexpressing IL-36α and control melanoma cells. b Immunoblotting assay was used to detect IL-36β expression in melanoma cells overexpressing IL-36β and control melanoma cells. c Immunoblotting assay was used to detect IL-36γ expression in melanoma cells overexpressing IL-36γ and control melanoma cells. Supplementary data 2 IL-36α has no effect on B16-vec melanoma cells. a 1 × 106 B16-vec cells were stimulated with PBS, 100 ng/mL IL-36α or 20 ng/mL TGF-β, respectively. And the migration of cells was counted after 24 h. b Quantitative assay of wound-healing experiment in these three groups. The experiment was repeated three times, and the scratch area was counted using ImageJ software. c Clone formation assay was used to detect the proliferation of B16-vec, B16-IL-36α and B16-vec stimulated with 100 ng/mL IL-36α. d Quantitative assay of clone formation experiment in these three groups. This experiment was repeated three times and the area of clone formation was counted using ImageJ software. e The proliferation of cells in 5 × 105 B16-vec cells, B16-IL-36α cells or 100 ng/mL IL-36α stimulated B16-vec cells after 24 h were labeled with EdU, respectively, indicating the proliferation of cells in these experiments. f Quantitative assay of cell proliferation experiment in these three groups. The experiment was repeated three times and the expression of EdU was assayed by Flow cytometry. g The migrated B16-vec cells, B16-IL-36α cells and B16-vec cells with 100 ng/mL IL-36α stimulation were measured through Transwell assay. h Quantitative assay of Transwell experiment in these three groups. The experiment was repeated three times, and the migrated cells were counted using ImageJ software. Data (mean ± SEM) are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, determined by one-way ANOVA with Dunn’s post hoc analysis. Supplementary data 3 IL-36α elevates macrophages proliferation. a Proliferating cells in 5 × 105 BMDM cells after 24 h stimulation with PBS or 100 ng/mL IL-36α, respectively, were labeled with EdU to indicate the level of cell proliferation in these experiments. b Quantitative assay of cell proliferation experiment in these three groups. The experiment was repeated three times and the expression of EdU was assayed by Flow cytometry. Data (mean±SEM) are representative of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, determined by two-tailed unpaired student’s t test. Supplementary data 4 IL-36α does not change the PD-L1 expression both in cells and in tumor tissues. a 1 × 106 B16-vec cells and B16-IL-36α cells were plated into 6-well plates, and some of B16-vec cells were stimulated by 100 ng/mL IL-36α for 6 h. Then these cells were collected and stained with PE-anti mouse PD-L1 antibody. PE-Rat IgG2a, λ antibody was used as isotype control. And the PD-L1 expression in these cells were measured by flow cytometry. b The mean fluorescence intensity (MFI) of PD-L1 expression in these cells was counted. c, d Immunoblotting analysis of PD-L1 expression in tumor tissues with different treatment. 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 test. Supplementary data 5 IL-36α inhibits LLC tumor growth through altering immune cell infiltration. a Schematic protocol of the mice Lewis lung model. Mice were challenged with 5×105 LLC-vec cells or LLC-IL-36α cells s.c. On day 16, Mice LLC tumors are removed and analyzed. b, c Mice LLC-vec and LLC-IL-36α tumor tissues were removed on day 16, 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 two-tailed unpaired Student’s t test. LLC-vec group was compared with LLC-IL-36α group. d Mice weights were measured every 2 days from the fourth day after implantation of tumors. e Tumor volumes of mice were measured every 2 days from day 6 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. LLC-vec group was compared with LLC-IL-36α group. 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; ****p < 0.0001, determined by two-tailed unpaired student’s t test. LLC-vec group was compared with LLC-IL-36α group. m The survival of mice was monitored. Thirteen mice were in each group. The p value was based on a log rank test. LLC-vec group was compared with LLC-IL-36α group (PDF 520 KB)

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Lou, X., Duan, S., Li, M. et al. IL-36α inhibits melanoma by inducing pro-inflammatory polarization of macrophages. Cancer Immunol Immunother 72, 3045–3061 (2023). https://doi.org/10.1007/s00262-023-03477-5

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