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.
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The datasets generated during the current study are available from the corresponding author on reasonable request.
References
Cassetta L, Pollard JW. A timeline of tumour-associated macrophage biology. Nat Rev Cancer. 2023;23:238–57.
Onkar S, Cui J, Zou J, Cardello C, Cillo AR, Uddin MR, et al. Immune landscape in invasive ductal and lobular breast cancer reveals a divergent macrophage-driven microenvironment. Nat Cancer. 2023;4:516–34.
Cao X, Li B, Chen J, Dang J, Chen S, Gunes EG, et al. Effect of cabazitaxel on macrophages improves CD47-targeted immunotherapy for triple-negative breast cancer. J Immunother Cancer. 2021;9:e002022.
Cassetta L, Fragkogianni S, Sims AH, Swierczak A, Forrester LM, Zhang H, et al. Human tumor-associated macrophage and monocyte transcriptional landscapes reveal cancer-specific reprogramming, biomarkers, and therapeutic targets. Cancer Cell. 2019;35:588–602.e10.
Wei YT, Wang XR, Yan C, Huang F, Zhang Y, Liu X, et al. Thymosin alpha-1 reverses M2 polarization of tumor-associated macrophages during efferocytosis. Cancer Res. 2022;82:1991–2002.
Vadevoo SMP, Gunassekaran GR, Lee C, Lee N, Lee J, Chae S, et al. The macrophage odorant receptor Olfr78 mediates the lactate-induced M2 phenotype of tumor-associated macrophages. Proc Natl Acad Sci USA. 2021;118:e2102434118.
Mantovani A, Locati M. Macrophage metabolism shapes angiogenesis in tumors. Cell Metab. 2016;24:887–8.
Tang K, Ma J, Huang B. Macrophages' M1 or M2 by tumor microparticles: lysosome makes decision. Cell Mol Immunol. 2022;19:1196–7.
Barreira da Silva R, Leitao RM, Pechuan-Jorge X, Werneke S, Oeh J, Javinal V, et al. Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity. Nat Immunol. 2022;23:568–80.
Muller AK, Kohler UA, Trzebanski S, Vinik Y, Raj HM, Girault JA, et al. Mouse modeling dissecting macrophage-breast cancer communication uncovered roles of PYK2 in macrophage recruitment and breast tumorigenesis. Adv Sci. 2022;9:e2105696.
Nishiga Y, Drainas AP, Baron M, Bhattacharya D, Barkal AA, Ahrari Y, et al. Radiotherapy in combination with CD47 blockade elicits a macrophage-mediated abscopal effect. Nat Cancer. 2022;3:1351–66.
Li J, Ye Y, Liu Z, Zhang G, Dai H, Li J, et al. Macrophage mitochondrial fission improves cancer cell phagocytosis induced by therapeutic antibodies and is impaired by glutamine competition. Nat Cancer. 2022;3:453–70.
Arkee T, Hostager BS, Houtman JCD, Bishop GA. TRAF3 in T cells restrains negative regulators of LAT to promote TCR/CD28 signaling. J Immunol. 2021;207:322–32.
Shi JH, Sun SC. Tumor necrosis factor receptor-associated factor regulation of nuclear factor kappaB and mitogen-activated protein kinase pathways. Front Immunol. 2018;9:1849.
Guan K, Wei C, Zheng Z, Song T, Wu F, Zhang Y, et al. MAVS promotes inflammasome activation by targeting ASC for K63-linked ubiquitination via the E3 ligase TRAF3. J Immunol. 2015;194:4880–90.
Parvatiyar K, Pindado J, Dev A, Aliyari SR, Zaver SA, Gerami H, et al. A TRAF3-NIK module differentially regulates DNA vs RNA pathways in innate immune signaling. Nat Commun. 2018;9:2770.
Shen Y, Liu WW, Zhang X, Shi JG, Jiang S, Zheng L, et al. TRAF3 promotes ROS production and pyroptosis by targeting ULK1 ubiquitination in macrophages. FASEB J. 2020;34:7144–59.
Shi JH, Ling C, Wang TT, Zhang LN, Liu WW, Qin Y, et al. TRK-fused gene (TFG) regulates ULK1 stability via TRAF3-mediated ubiquitination and protects macrophages from LPS-induced pyroptosis. Cell Death Dis. 2022;13:93.
Zhou C, Lu C, Pu H, Li D, Zhang L. TRAF6 promotes IL-4-induced M2 macrophage activation by stabilizing STAT6. Mol Immunol. 2020;127:223–9.
Yu T, Gan S, Zhu Q, Dai D, Li N, Wang H, et al. Modulation of M2 macrophage polarization by the crosstalk between Stat6 and Trim24. Nat Commun. 2019;10:4353.
Kamerkar S, Leng C, Burenkova O, Jang SC, McCoy C, Zhang K, et al. Exosome-mediated genetic reprogramming of tumor-associated macrophages by exoASO-STAT6 leads to potent monotherapy antitumor activity. Sci Adv. 2022;8:eabj7002.
Xiong X, Yang C, He WQ, Yu J, Xin Y, Zhang X, et al. Sirtuin 6 maintains epithelial STAT6 activity to support intestinal tuft cell development and type 2 immunity. Nat Commun. 2022;13:5192.
Zhao M, Ren K, Xiong X, Xin Y, Zou Y, Maynard JC, et al. Epithelial STAT6 O-GlcNAcylation drives a concerted anti-helminth alarmin response dependent on tuft cell hyperplasia and Gasdermin C. Immunity. 2022;55:623–38.e5.
Liang CL, Jiang H, Feng W, Liu H, Han L, Chen Y, et al. Total glucosides of paeony ameliorate pristane-induced lupus nephritis by inducing PD-1 ligands(+) macrophages via activating IL-4/STAT6/PD-L2 signaling. Front Immunol. 2021;12:683249.
Rahal OM, Wolfe AR, Mandal PK, Larson R, Tin S, Jimenez C, et al. Blocking interleukin (IL)4- and IL13-mediated phosphorylation of STAT6 (Tyr641) decreases M2 polarization of macrophages and protects against macrophage-mediated radioresistance of inflammatory breast cancer. Int J Radiat Oncol Biol Phys. 2018;100:1034–43.
Wang Y, Malabarba MG, Nagy ZS, Kirken RA. Interleukin 4 regulates phosphorylation of serine 756 in the transactivation domain of Stat6. Roles for multiple phosphorylation sites and Stat6 function. J Biol Chem. 2004;279:25196–203.
Gu F, Wang C, Wei F, Wang Y, Zhu Q, Ding L, et al. STAT6 degradation and ubiquitylated TRIML2 are essential for activation of human oncogenic herpesvirus. PLoS Pathog. 2018;14:e1007416.
Waqas SFH, Hoang AC, Lin YT, Ampem G, Azegrouz H, Balogh L, et al. Neuropeptide FF increases M2 activation and self-renewal of adipose tissue macrophages. J Clin Invest. 2017;127:2842–54.
Gao P, Ma X, Yuan M, Yi Y, Liu G, Wen M, et al. E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3. Nat Commun. 2021;12:1194.
Chiang N, de la Rosa X, Libreros S, Pan H, Dreyfuss JM, Serhan CN. Cysteinyl-specialized proresolving mediators link resolution of infectious inflammation and tissue regeneration via TRAF3 activation. Proc Natl Acad Sci USA. 2021;118:e2013374118.
Sui H, Dongye S, Liu X, Xu X, Wang L, Jin CQ, et al. Immunotherapy of targeting MDSCs in tumor microenvironment. Front Immunol. 2022;13:990463.
Hegde S, Leader AM, Merad M. MDSC: markers, development, states, and unaddressed complexity. Immunity. 2021;54:875–84.
Kumar S, Torres MP, Kaur S, Rachagani S, Joshi S, Johansson SL, et al. Smoking accelerates pancreatic cancer progression by promoting differentiation of MDSCs and inducing HB-EGF expression in macrophages. Oncogene. 2015;34:2052–60.
Zhang N, Gao X, Zhang W, Xiong J, Cao X, Fu ZF, et al. JEV infection induces M-MDSC differentiation into CD3(+) macrophages in the brain. Front Immunol. 2022;13:838990.
Bishop GA, Stunz LL, Hostager BS. TRAF3 as a multifaceted regulator of B lymphocyte survival and activation. Front Immunol. 2018;9:2161.
Czimmerer Z, Halasz L, Daniel B, Varga Z, Bene K, Domokos A, et al. The epigenetic state of IL-4-polarized macrophages enables inflammatory cistromic expansion and extended synergistic response to TLR ligands. Immunity. 2022;55:2006–26.e6.
Gordon SR, Maute RL, Dulken BW, Hutter G, George BM, McCracken MN, et al. PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity. Nature. 2017;545:495–9.
Jin J, Xiao Y, Hu H, Zou Q, Li Y, Gao Y, et al. Proinflammatory TLR signalling is regulated by a TRAF2-dependent proteolysis mechanism in macrophages. Nat Commun. 2015;6:5930.
Vallabhapurapu S, Matsuzawa A, Zhang W, Tseng PH, Keats JJ, Wang H, et al. Nonredundant and complementary functions of TRAF2 and TRAF3 in a ubiquitination cascade that activates NIK-dependent alternative NF-kappaB signaling. Nat Immunol. 2008;9:1364–70.
Newman AC, Kemp AJ, Drabsch Y, Behrends C, Wilkinson S. Autophagy acts through TRAF3 and RELB to regulate gene expression via antagonism of SMAD proteins. Nat Commun. 2017;8:1537.
Hornick EL, Wallis AM, Bishop GA. TRAF3 enhances type I interferon receptor signaling in T cells by modulating the phosphatase PTPN22. Sci Signal. 2022;15:eabn5507.
Perkins DJ, Polumuri SK, Pennini ME, Lai W, Xie P, Vogel SN. Reprogramming of murine macrophages through TLR2 confers viral resistance via TRAF3-mediated, enhanced interferon production. PLoS Pathog. 2013;9:e1003479.
Zarnegar BJ, Wang Y, Mahoney DJ, Dempsey PW, Cheung HH, He J, et al. Noncanonical NF-kappaB activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK. Nat Immunol. 2008;9:1371–8.
Pohl C, Dikic I. Cellular quality control by the ubiquitin-proteasome system and autophagy. Science. 2019;366:818–22.
Hu H, Sun SC. Ubiquitin signaling in immune responses. Cell Res. 2016;26:457–83.
Liu Q, Aminu B, Roscow O, Zhang W. Targeting the ubiquitin signaling cascade in tumor microenvironment for cancer therapy. Int J Mol Sci. 2021;22:791.
Senft D, Qi J, Ronai ZA. Ubiquitin ligases in oncogenic transformation and cancer therapy. Nat Rev Cancer. 2018;18:69–88.
Wu G, Li D, Liang W, Sun W, Xie X, Tong Y, et al. PP6 negatively modulates LUBAC-mediated M1-ubiquitination of RIPK1 and c-FLIP(L) to promote TNFalpha-mediated cell death. Cell Death Dis. 2022;13:773.
Aalto A, Martinez-Chacon G, Kietz C, Tsyganova N, Kreutzer J, Kallio P, et al. M1-linked ubiquitination facilitates NF-kappaB activation and survival during sterile inflammation. FEBS J. 2022;289:5180–97.
Sanjo H, Zajonc DM, Braden R, Norris PS, Ware CF. Allosteric regulation of the ubiquitin:NIK and ubiquitin:TRAF3 E3 ligases by the lymphotoxin-beta receptor. J Biol Chem. 2010;285:17148–55.
Zhang L, Li Q, Yang J, Xu P, Xuan Z, Xu J, et al. Cytosolic TGM2 promotes malignant progression in gastric cancer by suppressing the TRIM21-mediated ubiquitination/degradation of STAT1 in a GTP binding-dependent modality. Cancer Commun. 2022;43:123–49.
Ruan HH, Zhang Z, Wang SY, Nickels LM, Tian L, Qiao JJ, et al. Tumor necrosis factor receptor-associated factor 6 (TRAF6) mediates ubiquitination-dependent STAT3 activation upon salmonella enterica serovar typhimurium infection. Infect Immun. 2017;85:e00081–17.
Precious B, Young DF, Andrejeva L, Goodbourn S, Randall RE. In vitro and in vivo specificity of ubiquitination and degradation of STAT1 and STAT2 by the V proteins of the paramyxoviruses simian virus 5 and human parainfluenza virus type 2. J Gen Virol. 2005;86:151–8.
Zuo Y, Feng Q, Jin L, Huang F, Miao Y, Liu J, et al. Regulation of the linear ubiquitination of STAT1 controls antiviral interferon signaling. Nat Commun. 2020;11:1146.
Ren Y, Zhao P, Liu J, Yuan Y, Cheng Q, Zuo Y, et al. Deubiquitinase USP2a sustains interferons antiviral activity by restricting ubiquitination of activated STAT1 in the nucleus. PLoS Pathog. 2016;12:e1005764.
Engblom C, Pfirschke C, Pittet MJ. The role of myeloid cells in cancer therapies. Nat Rev Cancer. 2016;16:447–62.
Ruffell B, Chang-Strachan D, Chan V, Rosenbusch A, Ho CM, Pryer N, et al. Macrophage IL-10 blocks CD8+ T cell-dependent responses to chemotherapy by suppressing IL-12 expression in intratumoral dendritic cells. Cancer Cell. 2014;26:623–37.
Ma S, Sun B, Duan S, Han J, Barr T, Zhang J, et al. YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8(+) T cells. Nat Immunol. 2023;24:255–66.
Klug F, Prakash H, Huber PE, Seibel T, Bender N, Halama N, et al. Low-dose irradiation programs macrophage differentiation to an iNOS(+)/M1 phenotype that orchestrates effective T cell immunotherapy. Cancer Cell. 2013;24:589–602.
Gardam S, Sierro F, Basten A, Mackay F, Brink R. TRAF2 and TRAF3 signal adapters act cooperatively to control the maturation and survival signals delivered to B cells by the BAFF receptor. Immunity. 2008;28:391–401.
Funding
This study was supported by National Natural Science Foundation of China (Nos. 82273463, 82103181, 31971304), Natural Science Foundation of Hebei Province (Nos. H2022201065, H2022201067, C2020201052, H2021201028), Hebei Province Foundation for Returned Overseas Scholars (No. C20200305), Government Foundation of Clinical Medicine Talents Training Program of Hebei Province (No. 361007), Foreign Intelligence Introduction Project of Hebei Province (No. 360601), CAMS Innovation Fund for Medical Sciences (No. 2019-I2M-5-055), Science and Technology Research Project of Colleges in Hebei Province (No. QN2023008), Foundation of President of Hebei University under grant (No.202204) and Tumor Microecological Metabolism Regulation Research Innovation Team of Hebei University.
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All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by J-HS, L-NL, D-DS, W-WL, CL, F-XW and T-TW. BL, N-PC, YQ and J-HS analyzed and interpreted the data. The first draft of the manuscript was written by J-HS and Z-YN and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
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Shi, JH., Liu, LN., Song, DD. et al. TRAF3/STAT6 axis regulates macrophage polarization and tumor progression. Cell Death Differ 30, 2005–2016 (2023). https://doi.org/10.1038/s41418-023-01194-1
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DOI: https://doi.org/10.1038/s41418-023-01194-1
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