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• W2534854440 abstract "•IL-4 and CSF-1 promote glucose metabolism during M2, or M(IL-4), macrophage activation.•IL-4 and CSF-1 signal via mTORC2 and IRF4 to induce changes in glucose metabolism•Glucose metabolism supports fatty acid synthesis and oxidation in M2 macrophages•mTORC2- and IRF4-dependent changes in glucose metabolism are critical for M2 activation Macrophage activation status is intrinsically linked to metabolic remodeling. Macrophages stimulated by interleukin 4 (IL-4) to become alternatively (or, M2) activated increase fatty acid oxidation and oxidative phosphorylation; these metabolic changes are critical for M2 activation. Enhanced glucose utilization is also characteristic of the M2 metabolic signature. Here, we found that increased glucose utilization is essential for M2 activation. Increased glucose metabolism in IL-4-stimulated macrophages required the activation of the mTORC2 pathway, and loss of mTORC2 in macrophages suppressed tumor growth and decreased immunity to a parasitic nematode. Macrophage colony stimulating factor (M-CSF) was implicated as a contributing upstream activator of mTORC2 in a pathway that involved PI3K and AKT. mTORC2 operated in parallel with the IL-4Rα-Stat6 pathway to facilitate increased glycolysis during M2 activation via the induction of the transcription factor IRF4. IRF4 expression required both mTORC2 and Stat6 pathways, providing an underlying mechanism to explain how glucose utilization is increased to support M2 activation. Macrophage activation status is intrinsically linked to metabolic remodeling. Macrophages stimulated by interleukin 4 (IL-4) to become alternatively (or, M2) activated increase fatty acid oxidation and oxidative phosphorylation; these metabolic changes are critical for M2 activation. Enhanced glucose utilization is also characteristic of the M2 metabolic signature. Here, we found that increased glucose utilization is essential for M2 activation. Increased glucose metabolism in IL-4-stimulated macrophages required the activation of the mTORC2 pathway, and loss of mTORC2 in macrophages suppressed tumor growth and decreased immunity to a parasitic nematode. Macrophage colony stimulating factor (M-CSF) was implicated as a contributing upstream activator of mTORC2 in a pathway that involved PI3K and AKT. mTORC2 operated in parallel with the IL-4Rα-Stat6 pathway to facilitate increased glycolysis during M2 activation via the induction of the transcription factor IRF4. IRF4 expression required both mTORC2 and Stat6 pathways, providing an underlying mechanism to explain how glucose utilization is increased to support M2 activation. Macrophages are tissue-resident cells that play critical roles in a broad range of immunologic and homeostatic processes (Ginhoux et al., 2016Ginhoux F. Schultze J.L. Murray P.J. Ochando J. Biswas S.K. New insights into the multidimensional concept of macrophage ontogeny, activation and function.Nat. Immunol. 2016; 17: 34-40Crossref PubMed Scopus (469) Google Scholar, Wynn et al., 2013Wynn T.A. Chawla A. Pollard J.W. Macrophage biology in development, homeostasis and disease.Nature. 2013; 496: 445-455Crossref PubMed Scopus (2765) Google Scholar). The ability of these cells to serve multiple functions reflects their ability to express different genes in response to distinct extracellular signals, including pathogen- and damage- associated molecular patterns and cytokines (Glass and Natoli, 2016Glass C.K. Natoli G. Molecular control of activation and priming in macrophages.Nat. Immunol. 2016; 17: 26-33Crossref PubMed Scopus (315) Google Scholar, Wynn et al., 2013Wynn T.A. Chawla A. Pollard J.W. Macrophage biology in development, homeostasis and disease.Nature. 2013; 496: 445-455Crossref PubMed Scopus (2765) Google Scholar). Interleukin 4 (IL-4), which can be made by a variety of innate and adaptive immune cells (Pulendran and Artis, 2012Pulendran B. Artis D. New paradigms in type 2 immunity.Science. 2012; 337: 431-435Crossref PubMed Scopus (323) Google Scholar), induces a signal transducer and activator of transcription 6 (Stat6)-dependent macrophage activation state referred to as M(IL-4), or M2 or “alternative” activation (Murray et al., 2014Murray P.J. Allen J.E. Biswas S.K. Fisher E.A. Gilroy D.W. Goerdt S. Gordon S. Hamilton J.A. Ivashkiv L.B. Lawrence T. et al.Macrophage activation and polarization: nomenclature and experimental guidelines.Immunity. 2014; 41: 14-20Abstract Full Text Full Text PDF PubMed Scopus (3561) Google Scholar). M2 macrophages are important in immunity to parasitic helminths, tissue remodeling and wound repair, adipose tissue homeostasis, and tumor growth and metastasis. Macrophage activation status is intrinsically linked to metabolic remodeling (O’Neill and Pearce, 2016O’Neill L.A. Pearce E.J. Immunometabolism governs dendritic cell and macrophage function.J. Exp. Med. 2016; 213: 15-23Crossref PubMed Scopus (887) Google Scholar). Initial studies established that fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) are enhanced in M2 macrophages and are critical for M2 activation (Huang et al., 2014Huang S.C. Everts B. Ivanova Y. O’Sullivan D. Nascimento M. Smith A.M. Beatty W. Love-Gregory L. Lam W.Y. O’Neill C.M. et al.Cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages.Nat. Immunol. 2014; 15: 846-855Crossref PubMed Scopus (678) Google Scholar, Odegaard and Chawla, 2011Odegaard J.I. Chawla A. Alternative macrophage activation and metabolism.Annu. Rev. Pathol. 2011; 6: 275-297Crossref PubMed Scopus (441) Google Scholar, Vats et al., 2006Vats D. Mukundan L. Odegaard J.I. Zhang L. Smith K.L. Morel C.R. Wagner R.A. Greaves D.R. Murray P.J. Chawla A. Oxidative metabolism and PGC-1beta attenuate macrophage-mediated inflammation.Cell Metab. 2006; 4: 13-24Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar). Integrated metabolomic and transcriptomic studies have revealed that the metabolic reprogramming that occurs during activation is more complex than originally envisaged and have uncovered an enhanced use of glucose for UDP-GlcNAc synthesis as a metabolic signature of M2 macrophages (Jha et al., 2015Jha A.K. Huang S.C. Sergushichev A. Lampropoulou V. Ivanova Y. Loginicheva E. Chmielewski K. Stewart K.M. Ashall J. Everts B. et al.Network integration of parallel metabolic and transcriptional data reveals metabolic modules that regulate macrophage polarization.Immunity. 2015; 42: 419-430Abstract Full Text Full Text PDF PubMed Scopus (1054) Google Scholar). Moreover, a recent report has shown that inhibition of glycolysis prevents the expression of a subset of genes that comprise the M2 activation module (Covarrubias et al., 2016Covarrubias A.J. Aksoylar H.I. Yu J. Snyder N.W. Worth A.J. Iyer S.S. Wang J. Ben-Sahra I. Byles V. Polynne-Stapornkul T. et al.Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation.eLife. 2016; 5Crossref PubMed Scopus (260) Google Scholar). It is now clear that manipulation of metabolic reprogramming in immune cells has therapeutic potential. Depending on context, being able to promote or inhibit M2 activation could have therapeutic benefits, and understanding how glucose metabolism is reprogrammed downstream of stimulation with IL-4, and how this is integrated with changes in FAO, would be an important step toward this goal. Recent work has implicated IL-4-induced signaling through AKT and mechanistic target of rapamycin complex 1 (mTORC1) in the regulation of glucose metabolism for M2 activation (Covarrubias et al., 2016Covarrubias A.J. Aksoylar H.I. Yu J. Snyder N.W. Worth A.J. Iyer S.S. Wang J. Ben-Sahra I. Byles V. Polynne-Stapornkul T. et al.Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation.eLife. 2016; 5Crossref PubMed Scopus (260) Google Scholar), raising the possibility that the mTORC1 pathway might be a target for manipulating alternative activation. However, loss of tuberous sclerosis 1 (Tsc1), a negative regulator of mTORC1, allows enhanced M1 and diminished M2 activation (Byles et al., 2013Byles V. Covarrubias A.J. Ben-Sahra I. Lamming D.W. Sabatini D.M. Manning B.D. Horng T. The TSC-mTOR pathway regulates macrophage polarization.Nat. Commun. 2013; 4: 2834Crossref PubMed Scopus (357) Google Scholar), indicating that the role of mTORC1 in M2 activation is context dependent (Covarrubias et al., 2016Covarrubias A.J. Aksoylar H.I. Yu J. Snyder N.W. Worth A.J. Iyer S.S. Wang J. Ben-Sahra I. Byles V. Polynne-Stapornkul T. et al.Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation.eLife. 2016; 5Crossref PubMed Scopus (260) Google Scholar). Moreover, in brown adipose tissue, mTORC2 is responsible for AKT-induced increases in glycolysis (Albert et al., 2016Albert V. Svensson K. Shimobayashi M. Colombi M. Muñoz S. Jimenez V. Handschin C. Bosch F. Hall M.N. mTORC2 sustains thermogenesis via Akt-induced glucose uptake and glycolysis in brown adipose tissue.EMBO Mol. Med. 2016; 8: 232-246Crossref PubMed Scopus (90) Google Scholar), and mTORC2 is implicated in glycolytic remodeling in tumors (Masui et al., 2015Masui K. Cavenee W.K. Mischel P.S. mTORC2 and Metabolic Reprogramming in GBM: at the Interface of Genetics and Environment.Brain Pathol. 2015; 25: 755-759Crossref PubMed Scopus (19) Google Scholar). Therefore, questions remain about the role of mTOR in M2 activation and the potential for the contribution of mTORC1 and mTORC2 complexes to this process. In light of these accumulated findings, we decided to address the roles of mTORC1 and mTORC2 in the metabolic reprograming that allows M2 activation. Our findings point to an mTORC2-mediated pathway, involving phosphatidyl inositol-3 kinase (PI3K) and AKT, as being essential for accentuated glucose metabolism to promote M2 activation and implicate macrophage colony stimulating factor (M-CSF) as an upstream activator of this pathway. We found that this pathway profoundly influences FAO and that its effects are mediated by interferon regulatory factor 4 (IRF4), a transcription factor that is important for M2 activation (Satoh et al., 2010Satoh T. Takeuchi O. Vandenbon A. Yasuda K. Tanaka Y. Kumagai Y. Miyake T. Matsushita K. Okazaki T. Saitoh T. et al.The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection.Nat. Immunol. 2010; 11: 936-944Crossref PubMed Scopus (853) Google Scholar) and which previously had been shown to play a critical role in metabolic reprogramming toward glycolysis during CD8+ T cell activation (Man et al., 2013Man K. Miasari M. Shi W. Xin A. Henstridge D.C. Preston S. Pellegrini M. Belz G.T. Smyth G.K. Febbraio M.A. et al.The transcription factor IRF4 is essential for TCR affinity-mediated metabolic programming and clonal expansion of T cells.Nat. Immunol. 2013; 14: 1155-1165Crossref PubMed Scopus (266) Google Scholar, Yao et al., 2013Yao S. Buzo B.F. Pham D. Jiang L. Taparowsky E.J. Kaplan M.H. Sun J. Interferon regulatory factor 4 sustains CD8(+) T cell expansion and effector differentiation.Immunity. 2013; 39: 833-845Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Our data show that IRF4 expression requires both mTORC2 and Stat6 pathways and provide an underlying mechanism to explain how glucose utilization is increased to support M2 activation. Macrophages grown from bone marrow were unstimulated (M0) or activated with IL-4 (M2), and glucose consumption and changes in extracellular acidification rates ([ECARs] a measure of the production of lactic acid, an end product of cytoplasmic glucose metabolism) were assessed. Glucose consumption, expression of genes encoding enzymes in the glycolysis pathway, and basal ECARs were higher in M2 macrophages than in M0 macrophages (Figures 1A–1C), and M2 macrophages exhibited increased glycolytic reserve ([GR] defined as the ability to upregulate aerobic glycolysis, measureable as increased ECAR, after inhibition of mitochondrial ATP synthesis by oligomycin and inner membrane depolarization by FCCP, Figure 1C). High GR is a measure of the increased ability of M2 cells to route pyruvate to lactate in order to meet their ATP needs through aerobic glycolysis. We asked whether increased glucose utilization is important for alternative activation by stimulating cells with IL-4 in the presence of 2-deoxyglucose (2-DG). Expression of the M2 activation markers resistin-like alpha (RELMα) and programmed cell death 1 ligand 2 (PD-L2) was markedly inhibited by 2-DG (Figure 1D). Similar results were obtained when medium that lacked glucose was used (Figure S1). As expected, 2-DG caused a reduction in basal ECARs and GR (Figure 1E). These data confirm and expand recent findings on the importance of glucose for M2 activation (Covarrubias et al., 2016Covarrubias A.J. Aksoylar H.I. Yu J. Snyder N.W. Worth A.J. Iyer S.S. Wang J. Ben-Sahra I. Byles V. Polynne-Stapornkul T. et al.Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation.eLife. 2016; 5Crossref PubMed Scopus (260) Google Scholar). For comparison, macrophages classically activated with interferon (IFN)-γ plus lipopolysaccharide (LPS), known as M1 macrophages, were also examined in these experiments. As expected (O’Neill and Pearce, 2016O’Neill L.A. Pearce E.J. Immunometabolism governs dendritic cell and macrophage function.J. Exp. Med. 2016; 213: 15-23Crossref PubMed Scopus (887) Google Scholar), M1 macrophages consumed significantly more glucose than M0 cells (Figure 1A), exhibited an elevated basal ECAR, and had very little GR given that they effectively run maximal glycolysis at baseline (Figure 1C). The fact that M2 macrophages have a large GR (Figure 1C) indicated that glucose-derived pyruvate is entering mitochondria and being used to fuel the tricarboxylic acid cycle and support mitochondrial ATP synthesis in these cells. Consistent with this, we found that 2-DG and UK5099 (which inhibits the mitochondrial pyruvate carrier [MPC-1]; Halestrap, 1975Halestrap A.P. The mitochondrial pyruvate carrier. Kinetics and specificity for substrates and inhibitors.Biochem. J. 1975; 148: 85-96Crossref PubMed Scopus (365) Google Scholar) caused declines in ATP in M2 macrophages (Figure 1F). In addition, like 2-DG, UK5099 caused a reduction in the IL-4-induced expression of PD-L2 and RELMα (Figure 1G). However, it had no effect on nitric oxide synthase, inducible nitric oxide synthase (iNOS) expression in M1 macrophages, in which pyruvate is largely converted to lactate due to the inhibition of the electron transport chain by nitric oxide (Figure 1H) (Everts et al., 2012Everts B. Amiel E. van der Windt G.J. Freitas T.C. Chott R. Yarasheski K.E. Pearce E.L. Pearce E.J. Commitment to glycolysis sustains survival of NO-producing inflammatory dendritic cells.Blood. 2012; 120: 1422-1431Crossref PubMed Scopus (372) Google Scholar). Targeting MPC-1 with a short hairpin RNA (shRNA) also resulted in decreased commitment to the M2 phenotype, as measured by RELMα expression (Figure 1I). Previous work has shown a requirement for OXPHOS for M2 activation (Vats et al., 2006Vats D. Mukundan L. Odegaard J.I. Zhang L. Smith K.L. Morel C.R. Wagner R.A. Greaves D.R. Murray P.J. Chawla A. Oxidative metabolism and PGC-1beta attenuate macrophage-mediated inflammation.Cell Metab. 2006; 4: 13-24Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar). We found that 2-DG, UK5099, and Mpc-1-shRNA all inhibited IL-4-induced elevations in OXPHOS, as measured by oxygen consumption rates (OCRs) and/or spare respiratory capacity (SRC) (Figures 1J–1L). We postulated previously that de novo fatty acid synthesis (FAS) could be contributing to the fueling of FAO in M2 macrophages (Huang et al., 2014Huang S.C. Everts B. Ivanova Y. O’Sullivan D. Nascimento M. Smith A.M. Beatty W. Love-Gregory L. Lam W.Y. O’Neill C.M. et al.Cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages.Nat. Immunol. 2014; 15: 846-855Crossref PubMed Scopus (678) Google Scholar). In this scenario, we assumed that, because lipolysis is necessary for FAO and M2 activation, de novo synthesized fatty acids (FAs) would first need to be incorporated into triacylglycerols (TAGs) prior to use for FAO. Indeed, expression of both Fasn and Acaca, which encode FAS enzymes, were increased in M2 macrophages (Figure S1B). We tested whether glycolysis and mitochondrial pyruvate import could be contributing to this pathway by measuring the effects of 2-DG and UK5099 on TAGs in M2 macrophages and found that TAGs were diminished when glucose use was inhibited (Figure 1M). Moreover, the FAS inhibitor C75 prevented IL-4-induced increases in RELMα expression but had no effect on iNOS expression in M1 cells (Figures S2C and S2D), and Acaca-shRNA inhibited IL-4-induced increases in PD-L2 and RELMα expression and OCR (Figure S2E). Together, these data indicate that glycolysis and mitochondrial pyruvate import are essential for M2 activation, possibly because they are being used to fuel FAS for increased FAO and OXPHOS. Infection of mice with the gastrointestinal helminth parasite Heligmosomoides polygyrus bakeri (H. polygyrus) evokes a Th2 response in the mesenteric LN and M2 activation of peritoneal macrophages (pMacs) (Huang et al., 2014Huang S.C. Everts B. Ivanova Y. O’Sullivan D. Nascimento M. Smith A.M. Beatty W. Love-Gregory L. Lam W.Y. O’Neill C.M. et al.Cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages.Nat. Immunol. 2014; 15: 846-855Crossref PubMed Scopus (678) Google Scholar, Reynolds et al., 2012Reynolds L.A. Filbey K.J. Maizels R.M. Immunity to the model intestinal helminth parasite Heligmosomoides polygyrus.Semin. Immunopathol. 2012; 34: 829-846Crossref PubMed Scopus (144) Google Scholar). To determine whether glucose metabolism is critical for M2 development in vivo, we injected mice infected with H. polygyrus with 2-DG intraperitoneally (i.p.) and examined pMac activation status 3 hr later (Figure 1N). Although we found that the total number of peritoneal cells, which increased as a result of infection, was not affected by 2-DG, (Figure 1O), the percentage of pMacs (defined as CD11b+F4/80+ cells) that expressed RELMα was significantly suppressed (Figure 1P). Furthermore, 2-DG suppressed pMac proliferation in infected mice (Figure 1Q). Taken together, our data suggest that enhanced glucose metabolism is essential for M2 macrophage activation. mTOR is a component of two functionally distinct protein complexes (mTORC1 and mTORC 2) that are key regulatory molecules in the control of immune cell function and energy homeostasis (Weichhart et al., 2015Weichhart T. Hengstschläger M. Linke M. Regulation of innate immune cell function by mTOR.Nat. Rev. Immunol. 2015; 15: 599-614Crossref PubMed Scopus (478) Google Scholar). mTORC1 is implicated in the regulation of glucose metabolism, but although mTORC1 has been implicated in the expression of a subset of M2 genes, constitutive activation of mTORC1 has been shown to negatively regulate alternative activation (Byles et al., 2013Byles V. Covarrubias A.J. Ben-Sahra I. Lamming D.W. Sabatini D.M. Manning B.D. Horng T. The TSC-mTOR pathway regulates macrophage polarization.Nat. Commun. 2013; 4: 2834Crossref PubMed Scopus (357) Google Scholar, Covarrubias et al., 2016Covarrubias A.J. Aksoylar H.I. Yu J. Snyder N.W. Worth A.J. Iyer S.S. Wang J. Ben-Sahra I. Byles V. Polynne-Stapornkul T. et al.Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation.eLife. 2016; 5Crossref PubMed Scopus (260) Google Scholar), raising the possibility that mTORC2 could also be playing a role in metabolic reprogramming for M2 activation. We found that N-myc downstream regulated gene 1 (NDRG1) and AKTs473, downstream targets in the mTORC2-dependent signaling pathway (García-Martínez and Alessi, 2008García-Martínez J.M. Alessi D.R. mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1).Biochem. J. 2008; 416: 375-385Crossref PubMed Scopus (707) Google Scholar), and ribosomal S6 kinase (S6K)T389, a downstream target in the mTORC1 pathway, were phosphorylated in M2 macrophages, indicating that both mTORC1 and mTORC2 pathways are active in these cells (Figure 2A). The mTOR inhibitor, Torin 1 (Liu et al., 2010Liu Q. Chang J.W. Wang J. Kang S.A. Thoreen C.C. Markhard A. Hur W. Zhang J. Sim T. Sabatini D.M. Gray N.S. Discovery of 1-(4-(4-propionylpiperazin-1-yl)-3-(trifluoromethyl)phenyl)-9-(quinolin-3-yl)benzo[h][1,6]naphthyridin-2(1H)-one as a highly potent, selective mammalian target of rapamycin (mTOR) inhibitor for the treatment of cancer.J. Med. Chem. 2010; 53: 7146-7155Crossref PubMed Scopus (183) Google Scholar), which inhibits both mTORC1 and mTORC2, as indicated by decreased phosphorylation of their respective targets, S6K and AKTs473, in macrophages responding to IL-4 (Figure S2A), effectively suppressed M2 activation (Figure S2B). In contrast, rapamycin (at 20 nM, at which it selectively inhibits mTORC1 [Figure S2A]) did not inhibit M2 activation (Figure S2B). Moreover, Torin, but not rapamycin, inhibited increased uptake of glucose by M2 macrophages, in comparison to that by M0 macrophages, as measured via flow cytometry to detect uptake of the fluorescent glucose analog 2-NBDG (Figure S2D). We next examined the response to IL-4 of macrophages in which key components of the mTORC2 and mTORC1 complexes, namely rictor and raptor, were deleted. Phosphorylation of NDRG1 (García-Martínez and Alessi, 2008García-Martínez J.M. Alessi D.R. mTOR complex 2 (mTORC2) controls hydrophobic motif phosphorylation and activation of serum- and glucocorticoid-induced protein kinase 1 (SGK1).Biochem. J. 2008; 416: 375-385Crossref PubMed Scopus (707) Google Scholar) was lost in macrophages from Rictorfl/flLysMcre (RictorΔMΦ) mice, but increased in IL-4-stimulated macrophages from Rptorfl/flLysMcre (RaptorΔMΦ) mice (Figure 2A), suggesting that mTORC1 might restrain mTORC2 activation in M2 macrophages. We also found that phosphorylation of AKTs473 was greatly diminished in RictorΔMΦ macrophages but not in RaptorΔMΦ macrophages (Figure 2A). As anticipated, S6K phosphorylation was diminished in RaptorΔMΦ macrophages but not in RictorΔMΦ macrophages (Figure 2A). Deletion of neither Raptor nor Rictor impaired IL-4-induced Stat6 phosphorylation (Figure 2A), indicating that Stat6 activation in M2 macrophages is mTOR independent. We found that RictorΔMΦ M2 macrophages had lower GR and basal ECAR and RaptorΔMΦ M2 macrophages had higher GR and basal ECARs than control M2 macrophages (Figures 2B and 2C). These changes in metabolism in RictorΔMΦ cells were linked to reduced IL-4-induced expression of genes encoding glycolysis pathway enzymes (Figure S2E). We showed that glycolysis and mitochondrial pyruvate import are essential for increased OXPHOS in M2 cells (Figure 1). We reasoned that if mTORC2 is controlling glucose usage, RictorΔMΦ macrophages should exhibit diminished changes in OXPHOS after stimulation with IL-4. Consistent with this, control and RaptorΔMΦ M2 macrophages behaved similarly to each other in a mitochondrial fitness test, but RictorΔMΦ M2 macrophages had a significantly diminished baseline OCR and SRC (Figures 2D–2F). Moreover, the sensitivity of SRC to etomoxir (ETO), which inhibits mitochondrial carnitine palmitoyl transferase-1 (Cpt1) and was apparent in control and RaptorΔMΦ M2 macrophages, was largely lost in RictorΔMΦ M2 macrophages (Figure 2D), suggesting that FAO was diminished in the absence of Rictor. As expected, ETO inhibited M2 activation, a result we recapitulated when Cpt1a expression was suppressed with a Cpt1a-hpRNA (Figure S2G). Finally, we asked whether deletion of Rictor affects the expression of M2 genes. We found that IL-4-induced expression of CD301, RELMα, Arg1, Chil3 (Ym1), Il10, Lipa, Cd36, Fabp4, Pparg, and Ppargc1b was diminished when Rictor was deleted (Figures 2G and S2H). In contrast, expression of CD301 and RELMα was increased over that seen in control M2 cells when RaptorΔMΦ macrophages were stimulated with IL-4 (Figure 2G). Raptor deletion had no effect on Ym1, Il10, Lipa, Cd36, Pparg, or Ppargc1b expression, but did diminish expression of Arg1 and Fabp4 (Figure S2H). Our data indicate that mTORC2 controls M2 activation by regulating glucose metabolism, and this in turn has effects on FAO. To further assess this, we asked whether enforcing expression of the facilitated glucose transporter Glut1 (Slc2a1) would rescue the ability of RictorΔMΦ macrophages to become alternatively activated. We found that enforced expression of Glut1 (Figure 2H) reversed the phenotype of RictorΔMΦ macrophages, allowing them to express amounts of PD-L2 and RELMα in response to IL-4 that were equivalent to those expressed by IL-4-stimulated wild-type (WT) macrophages (Figure 2I). Overexpression of Glut1 resulted in increased GR and SRC (Figures 2J and 2K). In recent work, we found that AKT is essential for regulating glycolytic metabolism in dendritic cells (Everts et al., 2014Everts B. Amiel E. Huang S.C. Smith A.M. Chang C.H. Lam W.Y. Redmann V. Freitas T.C. Blagih J. van der Windt G.J. et al.TLR-driven early glycolytic reprogramming via the kinases TBK1-IKKε supports the anabolic demands of dendritic cell activation.Nat. Immunol. 2014; 15: 323-332Crossref PubMed Scopus (670) Google Scholar), and previous work has shown that AKT is important for M2 activation (Byles et al., 2013Byles V. Covarrubias A.J. Ben-Sahra I. Lamming D.W. Sabatini D.M. Manning B.D. Horng T. The TSC-mTOR pathway regulates macrophage polarization.Nat. Commun. 2013; 4: 2834Crossref PubMed Scopus (357) Google Scholar, Rückerl et al., 2012Rückerl D. Jenkins S.J. Laqtom N.N. Gallagher I.J. Sutherland T.E. Duncan S. Buck A.H. Allen J.E. Induction of IL-4Rα-dependent microRNAs identifies PI3K/Akt signaling as essential for IL-4-driven murine macrophage proliferation in vivo.Blood. 2012; 120: 2307-2316Crossref PubMed Scopus (144) Google Scholar) and increased glycolysis in these cells (Covarrubias et al., 2016Covarrubias A.J. Aksoylar H.I. Yu J. Snyder N.W. Worth A.J. Iyer S.S. Wang J. Ben-Sahra I. Byles V. Polynne-Stapornkul T. et al.Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation.eLife. 2016; 5Crossref PubMed Scopus (260) Google Scholar). In keeping with these results, the AKT inhibitor triciribine suppressed M2 activation, as assessed by PD-L2 and RELMα expression (Figure 3A), and simultaneously blocked increases in glucose uptake and ECAR (Figures 3B and 3C). Consistent with the functional link between glucose usage and OXPHOS in M2 activation, triciribine inhibited IL-4-induced increases in basal OCR and SRC (Figure 3D). Our data collectively point to a pathway in which mTORC2-mediated phosphorylation of AKT is critical for M2 activation. PI3K is implicated in M2 activation, and PI3K has been shown to directly activate mTORC2 (Weichhart et al., 2015Weichhart T. Hengstschläger M. Linke M. Regulation of innate immune cell function by mTOR.Nat. Rev. Immunol. 2015; 15: 599-614Crossref PubMed Scopus (478) Google Scholar, Zinzalla et al., 2011Zinzalla V. Stracka D. Oppliger W. Hall M.N. Activation of mTORC2 by association with the ribosome.Cell. 2011; 144: 757-768Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar). We therefore asked whether PI3K plays a role in activating mTORC2 in M2 activation. We found that the PI3K inhibitor LY294002 strongly suppressed IL-4-induced expression of PD-L2 and RELMα (Figure 3E) and associated elevations in basal ECAR and basal OCR and SRC (Figures 3F and 3G). mTORs2481 phosphorylation, a marker of mTORC2 activation (Copp et al., 2009Copp J. Manning G. Hunter T. TORC-specific phosphorylation of mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker for intact mTOR signaling complex 2.Cancer Res. 2009; 69: 1821-1827Crossref PubMed Scopus (345) Google Scholar), was higher in M2 macrophages than in M0 macrophages, and this effect was diminished by inhibition of PI3K (Figure 3H). Moreover, induced phosphorylation of AKTs473 in M2 macrophages was prevented by inhibition of PI3K (Figure 3H). We also found that phosphorylation of the mTORC2 target NDRG1 was inhibited by LY294002 (Figure 3H). AKT inhibition had no effect on NRDG1 phosphorylation (Figure 3I), confirming that AKT activation is occurring downstream of mTORC2 in M2 macrophages. Consistent with previous reports, inhibition of neither PI3K nor AKT had any measurable effect on Stat6 phosphorylation (Figure 3I) (Covarrubias et al., 2016Covarrubias A.J. Aksoylar H.I. Yu J. Snyder N.W. Worth A.J. Iyer S.S. Wang J. Ben-Sahra I. Byles V. Polynne-Stapornkul T. et al.Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation.eLife. 2016; 5Crossref PubMed Scopus (260) Google Scholar, Munugalavadla et al., 2005Munugalavadla V. Borneo J. Ingram D.A. Kapur R. p85alpha subunit of class IA PI-3 kinase is crucial for macrophage growth and migration.Blood. 2005; 106: 103-109Crossref PubMed Scopus (55) Google Scholar), indicating that the PI3K-mTORC2-AKT pathway is occurring in parallel to the Stat6 pathway after IL-4 stimulation. Collectively, our findings suggest a pathway in which, after stimulation with IL-4, mTORC2 is activated by PI3K and then itself activates AKT and that this pathway is important for the changes in metabolism that are essential to M2 activation. Given the recognized role of PI3K in growth-factor-initiated signaling (Zinzalla et al., 2011Zinzalla V. Stracka D. Oppliger W. Hall M.N. Activation of mTORC2 by association with the ribosome.Cell. 2011; 144: 757-768Abstract Ful" @default.
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• W2534854440 date "2016-10-01" @default.
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• W2534854440 title "Metabolic Reprogramming Mediated by the mTORC2-IRF4 Signaling Axis Is Essential for Macrophage Alternative Activation" @default.
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• W2534854440 doi "https://doi.org/10.1016/j.immuni.2016.09.016" @default.
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