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SIRT1 Limits the Function and Fate of Myeloid-Derived Suppressor Cells In Author Manuscript Published OnlineFirst on December 18, 2013; DOI: 10.1158/0008-5472.CAN-13-2584 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. SIRT1 limits the function and fate of myeloid-derived suppressor cells in tumors by orchestrating HIF1α-dependent glycolysis Guangwei Liu1,4,†, Yujing Bi2, Bo Shen3, Hui Yang1,4, Yan Zhang1,4, Xiao Wang1,4, Huanrong Liu1,4, Yun Lu1,4, Jiongbo Liao1,4, Xi Chen1,4, Yiwei Chu1,4 1Key Laboratory of Medical Molecular Virology of MOE/MOH, Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai, China; 2State Key laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing, China; 3Institute of Radiomedicine, 4Biotherapy Research Center, Fudan University, Shanghai, China Running title: SIRT1 negatively control tumor MDSC differentiation † Corresponding Authors: Dr. Guangwei Liu, Fudan University, Yixueyuan Road 138, Shanghai, China 200032; Phone: (86) 21-54237563; Fax: (86) 21-54237563 Email: [email protected] Key words: Myeloid-derived suppressive cells; SIRT1; Innate immunity; Glycolytic metabolism; mTOR; HIF-1α 1 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 18, 2013; DOI: 10.1158/0008-5472.CAN-13-2584 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Myeloid-derived suppressor cells (MDSCs) display an immature phenotype that may assume a classically activated (M1) or alternatively activated phenotype (M2) in tumors. In this study, we investigated metabolic mechanisms underlying the differentiation of MDSC into M1 or M2 myeloid lineage and their effect on cancer pathophysiology. We found that SIRT1 deficiency in MDSCs directs a specific switch to M1 lineage when cell enter the periphery from bone marrow, decreasing suppressive function in favor of a pro-inflammatory M1 phenotype associated with tumor cell attack. Glycolytic activation through the mTOR-HIF1α pathway was required for differentiation to the M1 phenotype, which conferred protection against tumors. Our results define the essential nature of a SIRT1-mTOR/HIF1α glycolytic pathway in determining MDSC differentiation, with implications for metabolic reprogramming as a cancer therapeutic approach. 2 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 18, 2013; DOI: 10.1158/0008-5472.CAN-13-2584 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Myeloid-derived suppressor cells (MDSCs) are one of the major components of the immune-suppressive network responsible for immune cell tolerance in cancer(1-6). Polarized MDSC lineages can be distinguished as M1 and M2 cells. M2 can be induced by IL-4 or IL-13, produce arginase 1 (Arg1) and anti-inflammatory cytokines, eventually converging to facilitate tumorgenesis (5-9). In marked contrast, M1 could be induced by LPS or/and IFN-γ, produce inducible nitric oxide synthase (iNOS), NO and pro-inflammatory cytokines, leading to their anti-tumor effects(1, 2, 7). Whereas the roles of distinct lineage of MDSC have long been extensively known, it remains elusive how the mechanism of M1 and M2 MDSC lineage differentiation. SIRT1, the leading family member, the human orthology of yeast Sir2, has marked anti-inflammtory effects in several systems(10, 11). SIRT1 may limit the inflammatory process by inhibiting the expression of pro-inflammatory cytokines such as TNF-α (11, 12). And, SIRT1 could functions as an anergic factor to maintain peripheral CD4+ T tolearance(13), prolong allograft survival (14) and inhibit TH2 response in airway allergy(15). However, basic regulatory roles of SIRT1 on the myeloid cell differentiation remain unclear. A defining feature of immune cell activation is highly anabolic and demonstrate a striking increase in glycolysis, as well as an increase in glucose and amino 3 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 18, 2013; DOI: 10.1158/0008-5472.CAN-13-2584 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. acid uptake(16, 17). In support of this notion, proper regulation of glucose metabolism is required for the development of immune responses(18, 19). Although a role for the metabolic pathways in immune cell activation and responses is beginning to be appreciated (20-22), little information exists about whether the basic metabolic machinery is actively regulated and contributes to MDSC differentiation. In the present study, we reported that SIRT1 is a key factor in the regulation of MDSC differentiation into M1 or M2 phenotypes through HIF1α induced glycolytic metabolic reprogramming and has an impact on MDSC functions in both immune suppression and promotion of tumor progression. 4 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 18, 2013; DOI: 10.1158/0008-5472.CAN-13-2584 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Materials and Methods Mice, chimeras and tumor model C57BL/6 SIRT1fl/fl and Lysm-Cre and HIF-1αfl/fl mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All mice were used at an age of 6-12 weeks. Reconstitution of lethally irradiated CD45.1+ congenic mice with CD45.2+ bone marrow cells (BMs) from SIRT1-deficient (SIRT1flox/flox; Cre+/-) or WT (SIRT1flox/flox; Cre-/-) mice(23-25). To establish s.c. tumors, 5 x 105 EL-4 or B16.F10 tumor cells were injected into C57BL/6 mice. This number of cells formed a tumor with a 1-2 cm diameter within 2-3 weeks of injection. Experimental protocols were approved by the Animal Ethics Committee of Fudan University, Shanghai, China. Monoclonal antibody (mAbs) and flow cytometry Flow cytometry (FCM) was used to perform the analysis of cell surface markers, phosphorylated or intracellular signaling proteins, as described (25). FCM data were acquired on a FACSCalibur (Becton Dickinson, San Diego, CA), and data were analyzed with FlowJo (TreeStar, San Carlos, CA). MDSC cell isolation Single cell suspensions were prepared from spleens and bone marrow. MDSCs were isolated by cell sorting on a FACSAria (BD) after cell staining with anti-Gr1-APC and anti-CD11b-PE. Tumors were dissected and digested with 0.7 mg/mL collagenase XI (Sigma-Aldrich) and 30 mg/mL of type IV bovine pancreatic Dnase (Sigma-Aldrich) for 45 min at 37 0C. Gr1+ 5 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 18, 2013; DOI: 10.1158/0008-5472.CAN-13-2584 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. cells were isolated by using biotinylated anti-Gr1 antibody and streptavidin microbeads with MiniMACS columns (Miltenyi Biotech). MDSCs were isolated from tumor tissues as described (26). Arginase activity and NO production. Arginase activity was measured in cell lysates, arginine hydrolysis was conducted by incubating the lysate with L-arginine. For NO production, culture supernatant or serum was incubated with Greiss reagent, as described (24, 27). Real-time PCR and Immunoblot. RNA was extracted with an RNeasy kit (Qiagen), and cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen). An ABI 7900 real-time PCR system was used for quantitative PCR.The expression of each target gene is presented as the ‘fold change’ relative to that of WT, as described(23, 28). Immunoblot analysis was performed as described (25) using anti-HIF1α (Cayman Chemicals), anti-Acetyl-NF-κB (Cell signaling) and anti-NF-κB (Cell signaling) antibody. MDSC suppression assay. For the in vitro suppression assay, sorted CD11b+Gr1+ cells were added in the mixed lymphocyte reaction (MLR) system for 96 h in 96-well plate, as described(8, 25). 18 h before harvesting, cells were pulsed with 3H-thymidine (GE Healthcare). 3H-thymidine uptake was counted using a liquid scintillation counter and expressed as cpm. 6 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 18, 2013; DOI: 10.1158/0008-5472.CAN-13-2584 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ELISAs. Cytokines concentrations in culture supernatants were measured with mouse IL-10, TGF-β1, TNFα and IL-12 ELISA kits (eBioscience) as per the manufacturer’s instructions. Tumor killing activity. Sorted MDSCs were isolated from tumor or spleen and coincubated with tumor cells (target cells) at 20:1, 10:1 and 5:1 for 4 h. Tumor-killing activities were measured as percentage of tumor cell death. Glycolysis flux assay. Glycolysis of MDSCs was determined by measuring the detritiation of [3-3H]-glucose. In brief, the assay was initiated by adding 1 μCi [3-3H]-glucose (Perkin Elmer) and, 2 h later, medium was transferred to microcentrifuge tubes containing 50 μl 5N HCL. The microcentrifuge tubes were then placed in 20 ml scintillation vials containing 0.5 ml water and the 3 vials capped and sealed. H2O was separated from un-metabolized [3-3H]-glucose by evaporation diffusion for 24 h at room temperature. Statistical analysis. All data are presented as the mean ± SD. Student’s unpaired t test was applied for comparison of means was used to compare differences between groups. Comparison of the survival curves was performed using the Log-Rank (Mantel-Cox) test. A P value (alpha-value) of less than 0.05 was considered to be statistically significant. 7 Downloaded from cancerres.aacrjournals.org on October 2, 2021.
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