The TSC-mTOR pathway regulates macrophage polarization.
ABSTRACT: Macrophages are able to polarize to proinflammatory M1 or alternative M2 states with distinct phenotypes and physiological functions. How metabolic status regulates macrophage polarization remains not well understood, and here we examine the role of mTOR (mechanistic target of rapamycin), a central metabolic pathway that couples nutrient sensing to regulation of metabolic processes. Using a mouse model in which myeloid lineage-specific deletion of Tsc1 (Tsc1(?/?)) leads to constitutive mTOR complex 1 (mTORC1) activation, we find that Tsc1(?/?) macrophages are refractory to IL-4-induced M2 polarization, but produce increased inflammatory responses to proinflammatory stimuli. Moreover, mTORC1-mediated downregulation of Akt signalling critically contributes to defective polarization. These findings highlight a key role for the mTOR pathway in regulating macrophage polarization, and suggest how nutrient sensing and metabolic status could be 'hard-wired' to control of macrophage function, with broad implications for regulation of type 2 immunity, inflammation and allergy.
Project description:The liver is a key metabolic organ that controls whole-body physiology in response to nutrient availability. Mammalian target of rapamycin (mTOR) is a nutrient-activated kinase and central controller of growth and metabolism that is negatively regulated by the tumor suppressor tuberous sclerosis complex 1 (TSC1). To investigate the role of hepatic mTOR complex 1 (mTORC1) in whole-body physiology, we generated liver-specific Tsc1 (L-Tsc1 KO) knockout mice. L-Tsc1 KO mice displayed reduced locomotor activity, body temperature, and hepatic triglyceride content in a rapamycin-sensitive manner. Ectopic activation of mTORC1 also caused depletion of hepatic and plasma glutamine, leading to peroxisome proliferator-activated receptor ? coactivator-1? (PGC-1?)-dependent fibroblast growth factor 21 (FGF21) expression in the liver. Injection of glutamine or knockdown of PGC-1? or FGF21 in the liver suppressed the behavioral and metabolic defects due to mTORC1 activation. Thus, mTORC1 in the liver controls whole-body physiology through PGC-1? and FGF21. Finally, mTORC1 signaling correlated with FGF21 expression in human liver tumors, suggesting that treatment of glutamine-addicted cancers with mTOR inhibitors might have beneficial effects at both the tumor and whole-body level.
Project description:Mechanistic target of rapamycin (mTOR, also known as mammalian target of rapamycin) is a ubiquitous serine/threonine kinase that regulates cell growth, proliferation and survival. These effects are cell-type-specific, and are elicited in response to stimulation by growth factors, hormones and cytokines, as well as to internal and external metabolic cues. Rapamycin was initially developed as an inhibitor of T-cell proliferation and allograft rejection in the organ transplant setting. Subsequently, its molecular target (mTOR) was identified as a component of two interacting complexes, mTORC1 and mTORC2, that regulate T-cell lineage specification and macrophage differentiation. mTORC1 drives the proinflammatory expansion of T helper (TH) type 1, TH17, and CD4(-)CD8(-) (double-negative, DN) T cells. Both mTORC1 and mTORC2 inhibit the development of CD4(+)CD25(+)FoxP3(+) T regulatory (TREG) cells and, indirectly, mTORC2 favours the expansion of T follicular helper (TFH) cells which, similarly to DN T cells, promote B-cell activation and autoantibody production. In contrast to this proinflammatory effect of mTORC2, mTORC1 favours, to some extent, an anti-inflammatory macrophage polarization that is protective against infections and tissue inflammation. Outside the immune system, mTORC1 controls fibroblast proliferation and chondrocyte survival, with implications for tissue fibrosis and osteoarthritis, respectively. Rapamycin (which primarily inhibits mTORC1), ATP-competitive, dual mTORC1/mTORC2 inhibitors and upstream regulators of the mTOR pathway are being developed to treat autoimmune, hyperproliferative and degenerative diseases. In this regard, mTOR blockade promises to increase life expectancy through treatment and prevention of rheumatic diseases.
Project description:Macrophage activation/polarization to distinct functional states is critically supported by metabolic shifts. How polarizing signals coordinate metabolic and functional reprogramming, and the potential implications for control of macrophage activation, remains poorly understood. Here we show that IL-4 signaling co-opts the Akt-mTORC1 pathway to regulate Acly, a key enzyme in Ac-CoA synthesis, leading to increased histone acetylation and M2 gene induction. Only a subset of M2 genes is controlled in this way, including those regulating cellular proliferation and chemokine production. Moreover, metabolic signals impinge on the Akt-mTORC1 axis for such control of M2 activation. We propose that Akt-mTORC1 signaling calibrates metabolic state to energetically demanding aspects of M2 activation, which may define a new role for metabolism in supporting macrophage activation.
Project description:TSC1 is a tumor suppressor that associates with TSC2 to inactivate Rheb, thereby inhibiting signaling by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). mTORC1 stimulates cell growth by promoting anabolic cellular processes, such as translation, in response to growth factors and nutrient signals. To test roles for TSC1 and mTORC1 in ?-cell function, we utilized Rip2/Cre to generate mice lacking Tsc1 in pancreatic ?-cells (Rip-Tsc1cKO mice). Although obesity developed due to hypothalamic Tsc1 excision in older Rip-Tsc1cKO animals, young animals displayed a prominent gain-of-function ?-cell phenotype prior to the onset of obesity. The young Rip-Tsc1cKO animals displayed improved glycemic control due to mTOR-mediated enhancement of ?-cell size, mass, and insulin production but not determinants of ?-cell number (proliferation and apoptosis), consistent with an important anabolic role for mTOR in ?-cell function. Furthermore, mTOR mediated these effects in the face of impaired Akt signaling in ?-cells. Thus, mTOR promulgates a dominant signal to promote ?-cell/islet size and insulin production, and this pathway is crucial for ?-cell function and glycemic control.
Project description:Macrophages can be polarized into the inflammatory M1 lineage or the immunomodulatory M2 lineage, depending on the differential tissue microenvironment signaling, specific pathogens or cytokine stimulation. Tumor necrosis factor ??induced protein 8?like protein 2 (TIPE2) has been demonstrated to negatively regulate inflammation by inhibiting the Toll?like receptor (TLR) pathway. The present study utilized murine bone marrow derived macrophages (BMDMs) as the model of undifferentiated (M0) macrophages to study the roles of TIPE2 in the differential polarization status of BMDMs. It was observed that the expression levels of TIPE2 were diminished in M1 macrophages treated with lipopolysaccharide/interferon ?, and elevated in M2 macrophages treated with interleukin (IL)?4. BMDMs with TIPE2 overexpression exhibited defective M1 polarization and enhanced responses to IL?4 stimulation. TIPE2 impeded M1 polarization by interfering with mitogen?activated protein kinase kinase kinase 7?inhibitor of nuclear factor??B kinase subunit ? and B cell receptor?associated protein?serine/threonine?protein kinase mTOR complex 1 (mTORC1) activation. TIPE2 overexpression accelerated IL?4 induced M2 polarization by dampening mTORC1 activation via the accelerated process of arginine to urea. Overall, these results define a key role for TIPE2 in macrophage polarization by impeding mTORC1 response.
Project description:The tuberous sclerosis complex (TSC) 1/2 is a negative regulator of the nutrient-sensing kinase mechanistic target of rapamycin complex (mTORC1), and its function is generally associated with tumor suppression. Nevertheless, biallelic loss of function of TSC1 or TSC2 is rarely found in malignant tumors. Here, we show that TSC1/2 is highly expressed in Burkitt's lymphoma cell lines and patient samples of human Burkitt's lymphoma, a prototypical MYC-driven cancer. Mechanistically, we show that MYC induces TSC1 expression by transcriptional activation of the TSC1 promoter and repression of miR-15a. TSC1 knockdown results in elevated mTORC1-dependent mitochondrial respiration enhanced ROS production and apoptosis. Moreover, TSC1 deficiency attenuates tumor growth in a xenograft mouse model. Our study reveals a novel role for TSC1 in securing homeostasis between MYC and mTORC1 that is required for cell survival and tumor maintenance in Burkitt's lymphoma. The study identifies TSC1/2 inhibition and/or mTORC1 hyperactivation as a novel therapeutic strategy for MYC-driven cancers.
Project description:Coordination of cell metabolism and immune signals is crucial for lymphocyte priming. Emerging evidence also highlights the importance of cell metabolism for the activation of innate immunity upon pathogen challenge, but there is little evidence of how this process contributes to immune cell development. Here we show that differentiation of dendritic cells (DCs) from bone marrow precursors is associated with dynamic regulation of mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signaling and cell metabolism. Unexpectedly, enhancing mTORC1 activity via ablation of its negative regulator tuberous sclerosis 1 (Tsc1) impaired DC development in vivo and in vitro, associated with defective cell survival and proliferation. Moreover, Tsc1 deficiency caused DC spontaneous maturation but a propensity to differentiate into other lineages, and attenuated DC-mediated effector TH1 responses. Mechanistically, Tsc1-deficient DCs exhibited increased glycolysis, mitochondrial respiration, and lipid synthesis that were partly mediated by the transcription factor Myc, highlighting a key role of Tsc1 in modulating metabolic programming of DC differentiation. Further, Tsc1 signaled through Rheb to down-regulate mTORC1 for proper DC development, whereas its effect at modulating mTOR complex 2 (mTORC2) activity was largely dispensable. Our results demonstrate that the interplay between Tsc1-Rheb-mTORC1 signaling and Myc-dependent bioenergetic and biosynthetic activities constitutes a key metabolic checkpoint to orchestrate DC development.
Project description:mTOR senses nutrient and energy status to regulate cell survival and metabolism in response to environmental changes. Surprisingly, targeted mutation of Tsc1, a negative regulator of mTORC1, caused a broad reduction in miRNAs due to Drosha degradation.In contrast, when the activated mTOR level is repressed by its inhibitor rapamycin, the repression of miRNA is released. mTOR activation increased expression of Mdm2, which is hereby identified as the necessary and sufficient ubiquitin E3 ligase for Drosha. Drosha was induced by nutrient and energy deprivation and conferred resistance to glucose deprivation. Using a high throughput screen of a miRNA library, we identified 4 miRNAs that were necessary and sufficient to protect cells against glucose deprivation-induced apoptosis. These miRNA was regulated by glucose through the mTORC1-MDM2- Drosha axis. Taken together, our data reveal an mTOR-Mdm2-Drosha pathway in mammalian cells that broadly regulates miRNA biogenesis as a response to alteration in cellular environment. mTOR activation by Tsc1 knockout cause repression of both miRNA and pre-miRNA in mouse embryonic fibroblasts (MEFs) Overall design: Tsc1-/- MEF cells were treated with vehicle or rapamycin (R, 5nM) for 4 days before harvesting, while Tsc1+/+ cells were only treated with vehicle for 4 days. Total RNA isolation was performed with TRIzol (Invitrogen) according to manufacturer’s instructions. RNA labeling and hybridization for miRNA expression profiling on miRNA microarray chips were performed as described (Liu, C.G., Calin, G.A., Volinia, S., and Croce, C.M. (2008). MicroRNA expression profiling using microarrays. Nat Protoc 3, 563-578). genotype: Tsc1+/+: Tsc1+/+_1, Tsc1+/+_2, Tsc1+/+_3, Tsc1+/+_4, Tsc1+/+_5 genotype: Tsc1-/-:Tsc1-/-_1, Tsc1-/-_2, Tsc1-/-_3, Tsc1-/-_4, Tsc1-/-_5, Tsc1-/-_R_1, Tsc1-/-_R_2, Tsc1-/-_R_3, Tsc1-/-_R-4, Tsc1-/-_R_5 biological replicate: Tsc1+/+_1, Tsc1+/+_2, Tsc1+/+_3, Tsc1+/+_4, Tsc1+/+_5 biological replicate: Tsc1-/-_1, Tsc1-/-_2, Tsc1-/-_3, Tsc1-/-_4, Tsc1-/-_5 biological replicate: Tsc1-/-_R_1, Tsc1-/-_R_2, Tsc1-/-_R_3, Tsc1-/-_R_4, Tsc1-/-_R_5
Project description:The metabolism of glucose and glutamine, primary carbon sources utilized by mitochondria to generate energy and macromolecules for cell growth, is directly regulated by mTORC1. We show that glucose and glutamine, by supplying carbons to the TCA cycle to produce ATP, positively feed back to mTORC1 through an AMPK-, TSC1/2-, and Rag-independent mechanism by regulating mTORC1 assembly and its lysosomal localization. We discovered that the ATP-dependent TTT-RUVBL1/2 complex was disassembled and repressed by energy depletion, resulting in its decreased interaction with mTOR. The TTT-RUVBL complex was necessary for the interaction between mTORC1 and Rag and formation of mTORC1 obligate dimers. In cancer tissues, TTT-RUVBL complex mRNAs were elevated and positively correlated with transcripts encoding proteins of anabolic metabolism and mitochondrial function-all mTORC1-regulated processes. Thus, the TTT-RUVBL1/2 complex responds to the cell's metabolic state, directly regulating the functional assembly of mTORC1 and indirectly controlling the nutrient signal from Rags to mTORC1.
Project description:Tuberous sclerosis complex 1 (TSC1) forms a heterodimmer with tuberous sclerosis complex 2, to inhibit signalling by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). The mTORC1 stimulates cell growth by promoting anabolic cellular processes, such as gene transcription and protein translation, in response to growth factors and nutrient signals. Originally designed to test the role of TSC1 in adipocyte function, mice in which the gene for TSC1 was specifically deleted by the fatty acid binding protein 4 (FABP4)-Cre (Fabp4-Tsc1cKO mice) died prematurely within 48 h after birth. The Fabp4-Tsc1cKO mouse revealed a much smaller phenotype relative to the wild-type littermates. Maternal administration of rapamycin, a classical mTOR inhibitor, significantly increased the survival time of Fabp4-Tsc1cKO mice for up to 23 days. Both macroscopic and microscopic haemorrhages were observed in the lungs of Fabp4-Tsc1cKO mice, while other tissues showed no significant changes. Levels of surfactant proteins A and B demonstrated a significant decrease in the Fabp4-Tsc1cKO mice, which was rescued by maternal injection of rapamycin. Co-localization of FABP4 or TSC1 with surfactant protein B was also detected in neonatal pulmonary tissues. Our study suggests that TSC1-mTORC1 may be critical for the synthesis of surfactant proteins A and B.