TRAF3/STAT6 axis regulates macrophage polarization and tumor progression

Abstract

Converting tumor-associated macrophages (TAMs) from the M2 to the M1 phenotype is considered an effective strategy for cancer therapy. TRAF3 is known to regulate NF-κB signaling. However, the role of TRAF3 in TAM polarization has not yet been completely elucidated. Here, we found that ablation of TRAF3 increased M1 markers, iNOS, FGR and SLC4A7, while down-regulated M2 markers, CD206, CD36 and ABCC3, expression levels in macrophages. Moreover, TRAF3 deficiency enhanced LPS-induced M1 and abolished IL-4-induced macrophage polarization. Next, quantitative ubiquitomics assays demonstrated that among the quantitative 7618 ubiquitination modification sites on 2598 proteins, ubiquitination modification of IL-4 responding proteins was the most prominently reduced according to enrichment analysis. STAT6, a key factor of IL-4 responding protein, K450 and K129 residue ubiquitination levels were dramatically decreased in TRAF3-deficient macrophages. Ubiquitination assay and luciferase assay demonstrated that TRAF3 promotes STAT6 ubiquitination and transcriptional activity. Site mutation analysis revealed STAT6 K450 site ubiquitination played a vital role in TRAF3-mediated STAT6 activation. Finally, B16 melanoma mouse model demonstrated that myeloid TRAF3 deficiency suppressed tumor growth and lung metastasis in vivo. Taken together, TRAF3 plays a vital role in M2 polarization via regulating STAT6 K450 ubiquitination in macrophages.

Fig. 1. Combined analysis of transcriptome and proteome revealed TRAF3 regulates M1/M2 macrophage polarization.

A BMDMs were generated by cultivating bone marrow cells isolated from the femurs of 6–8 weeks old WT and TRAF3^MKO mice. Total RNA was extracted and RNA-sequencing analysis was performed using Illumina Hiseq2500/X platform. Cell proteome expression profile was analyzed using LC-MS/MS analysis. B Volcano map of RNA-sequencing different expression genes. M1/M2 macrophage marker genes are provided. Fold change >1.5, FDR < 0.05, n = 3 for each group. C Volcano map of proteome expression profile. M1/M2 macrophage markers are provided. Fold change >1.5, p < 0.05, n = 3 for each group. D Heat map of M1/M2 macrophage marker gene mRNA and protein levels in WT and TRAF3^MKO BMDMs. E BMDMs were harvested and lysed with RIPA buffer. Immunoblotting assays were performed to analyze M1 and M2 marker expression.

Fig. 2. TRAF3 deficiency decreased IL-4-induced M2 macrophage polarization and promotes LPS-induced M1 macrophage polarization.

A BMDMs from WT and TRAF3^MKO mice were treated with IL-4 (10 ng/ml) or LPS (1 μg/ml) for 48 h. Total RNA was extracted and qRT-PCR analysis of IL-6, CD206 and iNOS were performed. Bar graphs with error bars are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001; n = 3 for each group. B, C BMDMs from WT and TRAF3^MKO mice were treated with IL-4 (10 ng/ml) or LPS (1 μg/ml) for 48 h. Gr-1, CD206 and CD86 levels were analyzed by flow cytometric analysis. B Representative FACS plots are shown; C Summary graphs of MFI median based on multiple mice (n = 6/group) are shown. Scattered graphs with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. Quantitative ubiquitomics assays demonstrated that TRAF3 regulates IL-4 pathway.

BMDMs from WT and TRAF3^MKO mice were treated with MG132 for 2 h before harvest and quantitative ubiquitomics assays were performed. A Principal component analysis (PCA) was done to evaluate the quantitative repeatability of ubiquitination modification. B Bar graph shows the significant different proteins or sites number of ubiquitin modification. Regulation means TRAF3^MKO/WT up-regulated or down-regulated. Fold change >1.5, p < 0.05. C Functional enrichment of Gene Ontology (GO) analysis of biological process was done based on down-regulated ubiquitination modifying protein. For each category, a two-tailed Fisher’s exact test was employed to test the enrichment of the differentially modified protein against all identified proteins. The GO with a corrected p value < 0.05 is considered significant. D Volcano map of ubiquitin-modified sites profile. Blue, TRAF3^MKO/WT down-regulated sites; red, TRAF3^MKO/WT up-regulated sites. Fold change >1.5, p < 0.05, n = 3 for each group. E STAT6 K450 and K129 residue ubiquitination scores in WT and TRAF3^MKO BMDMs. Student’s t test, *p < 0.05, **p < 0.01, n = 3 for each group.

Fig. 4. TRAF3 promotes STAT6 ubiquitination and enhances IL-4 induced STAT6 transcriptional activity.

A HEK293 cells were co-transfected with His-STAT6, Flag-TRAF3 and HA-Ub expression plasmids. Ubiquitination assay of STAT6 was performed using anti-His antibody for IP and anti-Ub antibody for immunoblotting of ubiquitinated STAT6. B Schematic representation of the mouse STAT6 protein, showing the potential TRAF3-related ubiquinated lysine (K) residues (K129 and K450) highlighted in red. C Amino acid sequence alignment of STAT6 among the indicated species, showing K129 and K450 highlighted in red. D HEK293 cells were transfected with indicated plasmids and luciferase assay was performed to determine transcriptional activity of STAT6 or indicated STAT6 mutants (K129R and K450R) with or without TRAF3 expression. Cells were stimulated with IL-4 (10 ng/ml) for 24 h before harvest. Data with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. One-way ANOVA, *p < 0.05, **p < 0.01, n = 3 for each group. E His-STAT6 WT or His-STAT6 K450R were transfected into HEK293 cells together with Flag-TRAF3 and HA-Ub K63. Cells were treated with (+) or without (−) IL-4 (10 ng/ml) for 2 h. Nuclear and cytosol fractions were extracted and p-STAT6 levels were detected by immunoblotting. LaminB shows nuclear fraction and Tubulin shows cytosol fraction.

Fig. 5. TRAF3 deficiency suppressed tumor induced M2 TAM polarization.

A–D BMDMs were co-cultured with B16 conditional media (B16-CM) for 48 h. Total RNA was extracted and qRT-PCR analysis of ARG-1 (A), CD206 (B), IL-6 (C) and iNOS (D) was performed. E, F BMDMs were co-cultured with E0771, B16 or MC38 conditional media (E0771-CM, B16-CM or MC38-CM) for 48 h. The concentration of IL-6 (E) and iNOS (F) in the supernatant of cell culture medium was determined using ELISA assay. Bar graphs with error bars are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 3 for each group.

Fig. 6. Myeloid TRAF3 deficiency suppressed B16 melanoma tumorigenesis and lung metastasis in mouse model.

A–C WT and TRAF3^MKO mice were injected s.c. with B16 melanoma cells at 5 × 10⁵ cells per site. Tumor volume was calculated by the formula: V = 1/2 × a (length) × b² (width). Tumors were harvested from the mice 21 days after tumor injection. Tumor growth curve (A), tumor weight (B) and presentative photograph of tumors (C) are shown. n = 8 for each group. D–H WT and TRAF3^MKO mice were dosed by tail-veil injection with 0.5 × 10⁵ B16 cells. D Presentative photograph of lungs are shown; E Summary graphs of the number of pulmonary metastases at day 14 are shown (n = 16 for WT group and n = 13 for TRAF3^MKO group); F H&E staining of lung tissue (Upper, magnification ×20, bar = 2 mm; Lower, magnification ×100, bar = 300 μm); G Lung area occupied by tumor, n = 6 for each group; H Tumor size, n = 6 for each group. Data are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. Two-way ANOVA (A), Student’s t test (B, E, G, H). **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 7. TRAF3 regulates immune cell infiltration in B16 tumor model.

Flow cytometric analysis of immune cells (CD45+) infiltrated to day 21 tumor tissues. A Frequency of tumor infiltrated F4/80+CD11b+ TAMs and Gr-1+CD11b+ MDSCs. B Summary of flow cytometry data of (A) based on multiple mice, showing the frequency of the indicated cell populations within total tumor cells. C Frequency of tumor infiltrated CD86+CD11b+ M1 TAMs and CD206+CD11b+ M2 TAMs. D Summary of flow cytometry data of (C) based on multiple mice. E Frequency of tumor infiltrated CD4+ T cells and CD8a+ T cells. F Summary of flow cytometry data of (E) based on multiple mice. G Multiplexed immunofluorescence staining of infiltrating immune cells in B16 tumor tissue. Presentative images are shown. Bar = 200 μm. CD8, pink; CD86, green; CD206, red. Data are represented as mean ± SD. Each panel is a representative experiment of at least three independent biological replicates. Student’s t test, *p < 0.05, n = 6 for each group.