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• W2020920210 abstract "The innate inflammatory immune response must be tightly controlled to avoid damage to the host. Here, we showed that the tuberous sclerosis complex-mammalian target of rapamycin (TSC-mTOR) pathway regulated inflammatory responses after bacterial stimulation in monocytes, macrophages, and primary dendritic cells. Inhibition of mTOR by rapamycin promoted production of proinflammatory cytokines via the transcription factor NF-κB but blocked the release of interleukin-10 via the transcription factor STAT3. Conversely, deletion of TSC2, the key negative regulator of mTOR, diminished NF-κB but enhanced STAT3 activity and reversed this proinflammatory cytokine shift. Rapamycin-hyperactivated monocytes displayed a strong T helper 1 (Th1) cell- and Th17 cell-polarizing potency. Inhibition of mTOR in vivo regulated the inflammatory response and protected genetically susceptible mice against lethal Listeria monocytogenes infection. These data identify the TSC2-mTOR pathway as a key regulator of innate immune homeostasis with broad clinical implications for infectious and autoimmune diseases, vaccination, cancer, and transplantation. The innate inflammatory immune response must be tightly controlled to avoid damage to the host. Here, we showed that the tuberous sclerosis complex-mammalian target of rapamycin (TSC-mTOR) pathway regulated inflammatory responses after bacterial stimulation in monocytes, macrophages, and primary dendritic cells. Inhibition of mTOR by rapamycin promoted production of proinflammatory cytokines via the transcription factor NF-κB but blocked the release of interleukin-10 via the transcription factor STAT3. Conversely, deletion of TSC2, the key negative regulator of mTOR, diminished NF-κB but enhanced STAT3 activity and reversed this proinflammatory cytokine shift. Rapamycin-hyperactivated monocytes displayed a strong T helper 1 (Th1) cell- and Th17 cell-polarizing potency. Inhibition of mTOR in vivo regulated the inflammatory response and protected genetically susceptible mice against lethal Listeria monocytogenes infection. These data identify the TSC2-mTOR pathway as a key regulator of innate immune homeostasis with broad clinical implications for infectious and autoimmune diseases, vaccination, cancer, and transplantation. Inflammation, an essential defense mechanism, is a key event in the response to bacterial and viral infections (Kimbrell and Beutler, 2001Kimbrell D.A. Beutler B. The evolution and genetics of innate immunity.Nat. Rev. Genet. 2001; 2: 256-267Crossref PubMed Scopus (465) Google Scholar). The coordinated secretion of pro- and anti-inflammatory cytokines such as IL-12 and IL-10, respectively, by myeloid mononuclear phagocytes including monocytes, macrophages, and dendritic cells (DCs) is indispensable for effective immunity (Trinchieri, 2003Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity.Nat. Rev. Immunol. 2003; 3: 133-146Crossref PubMed Scopus (2781) Google Scholar). Nevertheless, the cellular mechanisms regulating pro- versus anti-inflammatory responses are poorly defined. Phosphatidylinositol 3-kinase (PI3K), which is activated early after bacterial stimulation, was elucidated as an important molecule in innate immune cells that regulates IL-10 and IL-12 production. Genetic or pharmacological ablation of PI3K in monocytes or peripheral DCs inhibits IL-10 and boosts IL-12 production (Fukao et al., 2002Fukao T. Tanabe M. Terauchi Y. Ota T. Matsuda S. Asano T. Kadowaki T. Takeuchi T. Koyasu S. PI3K-mediated negative feedback regulation of IL-12 production in DCs.Nat. Immunol. 2002; 3: 875-881Crossref PubMed Scopus (449) Google Scholar, Martin et al., 2003Martin M. Schifferle R.E. Cuesta N. Vogel S.N. Katz J. Michalek S.M. Role of the phosphatidylinositol 3 kinase-Akt pathway in the regulation of IL-10 and IL-12 by Porphyromonas gingivalis lipopolysaccharide.J. Immunol. 2003; 171: 717-725Crossref PubMed Scopus (213) Google Scholar). However, the molecular mechanisms that control inflammatory responses via PI3K signal transduction in myeloid cells are unknown. The tuberous sclerosis complex (TSC) protein 2 belongs to a group of proteins linked to the PI3K pathway (Wullschleger et al., 2006Wullschleger S. Loewith R. Hall M.N. TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4367) Google Scholar). TSC2 is a tumor suppressor that forms a heterodimeric complex with TSC1. Mutations in TSC1 or TSC2 give rise to the hamartoma-syndrome tuberous sclerosis complex and the proliferative lung disorder lymphangioleiomyomatosis (Paul and Thiele, 2008Paul E. Thiele E. Efficacy of sirolimus in treating tuberous sclerosis and lymphangioleiomyomatosis.N. Engl. J. Med. 2008; 358: 190-192Crossref PubMed Scopus (47) Google Scholar). TSC2 is phosphorylated and inactivated by the protein kinase Akt, which itself is activated by PI3K (Inoki et al., 2002Inoki K. Li Y. Zhu T. Wu J. Guan K.L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling.Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2237) Google Scholar). Importantly, the TSC1-TSC2 complex negatively regulates the mammalian target of rapamycin (mTOR) (Inoki et al., 2002Inoki K. Li Y. Zhu T. Wu J. Guan K.L. TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling.Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2237) Google Scholar). mTOR, an essential serine-threonine kinase, is a central regulator of cell growth and proliferation. mTOR directly phosphorylates the ribosomal p70S6 kinase (p70S6K) and the initiation factor 4E-binding protein 1 (4E-BP1) to initiate translation of distinct mRNAs (Hay and Sonenberg, 2004Hay N. Sonenberg N. Upstream and downstream of mTOR.Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3247) Google Scholar). The prototypic mTOR inhibitor rapamycin, a bacterial macrolide with potent immunosuppressive and antitumor activities, is also currently evaluated to treat tuberous sclerosis and lymphangioleiomyomatosis (Paul and Thiele, 2008Paul E. Thiele E. Efficacy of sirolimus in treating tuberous sclerosis and lymphangioleiomyomatosis.N. Engl. J. Med. 2008; 358: 190-192Crossref PubMed Scopus (47) Google Scholar). Because of the exquisite sensitivity of T cells to the cell-cycle-blocking effects of rapamycin, mTOR inhibition was introduced in clinical transplantation (Huang et al., 2003Huang S. Bjornsti M.A. Houghton P.J. Rapamycins: Mechanism of action and cellular resistance.Cancer Biol. Ther. 2003; 2: 222-232PubMed Google Scholar). Interestingly, the increased usage of rapamycin has been accompanied by distinct inflammatory events including lymphocytic alveolitis, interstitial pneumonitis, and severe forms of glomerulonephritis (Dittrich et al., 2004Dittrich E. Schmaldienst S. Soleiman A. Horl W.H. Pohanka E. Rapamycin-associated post-transplantation glomerulonephritis and its remission after reintroduction of calcineurin-inhibitor therapy.Transpl. Int. 2004; 17: 215-220Crossref PubMed Scopus (120) Google Scholar, Izzedine et al., 2005Izzedine H. Brocheriou I. Frances C. Post-transplantation proteinuria and sirolimus.N. Engl. J. Med. 2005; 353: 2088-2089Crossref PubMed Scopus (84) Google Scholar, Singer et al., 2000Singer S.J. Tiernan R. Sullivan E.J. Interstitial pneumonitis associated with sirolimus therapy in renal-transplant recipients.N. Engl. J. Med. 2000; 343: 1815-1816PubMed Google Scholar, Thaunat et al., 2005Thaunat O. Beaumont C. Chatenoud L. Lechaton S. Mamzer-Bruneel M.F. Varet B. Kreis H. Morelon E. Anemia after Late Introduction of Sirolimus May Correlate with Biochemical Evidence of a Chronic Inflammatory State.Transplantation. 2005; 80: 1212-1219Crossref PubMed Scopus (82) Google Scholar). However, the underlying mechanisms of these proinflammatory effects are largely unknown. Some investigators reported an anti-inflammatory property of rapamycin on in vitro-generated DCs, whereas in in vitro-generated macrophages, rapamycin seems to enhance IL-23 production (Monti et al., 2003Monti P. Mercalli A. Leone B.E. Valerio D.C. Allavena P. Piemonti L. Rapamycin impairs antigen uptake of human dendritic cells.Transplantation. 2003; 75: 137-145Crossref PubMed Scopus (144) Google Scholar, Taner et al., 2005Taner T. Hackstein H. Wang Z. Morelli A.E. Thomson A.W. Rapamycin-treated, alloantigen-pulsed host dendritic cells induce ag-specific T cell regulation and prolong graft survival.Am. J. Transplant. 2005; 5: 228-236Crossref PubMed Scopus (199) Google Scholar, Yang et al., 2006Yang C.S. Song C.H. Lee J.S. Jung S.B. Oh J.H. Park J. Kim H.J. Park J.K. Paik T.H. Jo E.K. Intracellular network of phosphatidylinositol 3-kinase, mammalian target of the rapamycin/70 kDa ribosomal S6 kinase 1, and mitogen-activated protein kinases pathways for regulating mycobacteria-induced IL-23 expression in human macrophages.Cell. Microbiol. 2006; 8: 1158-1171Crossref PubMed Scopus (79) Google Scholar). Here, we investigated whether TSC and mTOR are involved in the regulation of inflammatory mediators upon bacterial stimulation of freshly isolated untouched mononuclear phagocytes. We showed that activation of the mTOR pathway limits inflammatory responses by blocking NF-κB and enhancing STAT3 activity. To define a potential role of mTOR in the regulation of pro- versus anti-inflammatory cytokines, we first assessed the effects of prototypic bacterial stimuli, such as lipopolysaccharide (LPS) or Staphylococcus aureus cells (SACs), on human peripheral blood mononuclear cells (PBMCs) in the presence or absence of rapamycin. mTOR inhibition by rapamycin in PBMCs stimulated with LPS or SACs led to a potent upregulation of IL-12p40 production (Figures 1A and 1B). Concurrently, the anti-inflammatory cytokine IL-10 was profoundly suppressed (Figure 1A). Because monocytes and myeloid DCs are the main producers of IL-12 in the blood (Trinchieri, 2003Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity.Nat. Rev. Immunol. 2003; 3: 133-146Crossref PubMed Scopus (2781) Google Scholar), we next investigated the effects of rapamycin on freshly isolated LPS- or SAC-stimulated monocytes and obtained similar results, even at low-nanomolar concentrations of the mTOR inhibitor (Figures 1C and 1D). Activation of monocytes with the intracellular human pathogen Listeria monocytogenes (L.m.), Pam3Cys, or flagellin likewise promoted IL-12p40 and inhibited IL-10 production after rapamycin pretreatment (Figure 1E and data not shown). Importantly, mTOR inhibition increased the production of the biologically active heterodimers IL-12p70 and IL-23 (Figures 1F and 1G), which share the IL-12p40 subunit, and of the proinflammatory cytokines TNF-α and IL-6 (Figures 1H and 1I and Figure S1 available online). When freshly isolated primary myeloid DC were tested (Figure S2), a similar increase in LPS- and SAC-mediated IL-12p40 production and decrease in IL-10 production was observed upon treatment with rapamycin (Figures 1J and 1K). These results indicate that mTOR inhibition alters the cytokine balance of monocytes and myeloid DC toward a proinflammatory phenotype upon microbial stimulation. The ability of rapamycin to affect cytokine production suggested an involvement of the mTOR signaling pathway in the coordinated production of pro- versus anti-inflammatory cytokines. However, the mTOR pathway has not been defined in mononuclear phagocytes, but it is known that bacterial stimuli activate PI3K, which leads to phosphorylation of Akt (Arbibe et al., 2000Arbibe L. Mira J.P. Teusch N. Kline L. Guha M. Mackman N. Godowski P.J. Ulevitch R.J. Knaus U.G. Toll-like receptor 2-mediated NF-kappa B activation requires a Rac1-dependent pathway.Nat. Immunol. 2000; 1: 533-540Crossref PubMed Scopus (545) Google Scholar). In other cell types, it has been further shown that activation of Akt promotes the phosphorylation and inactivation of TSC2 (Manning et al., 2002Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway.Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1187) Google Scholar). We found that LPS induced Akt-dependent phosphorylation of TSC2 on Ser939 in human monocytes (Figure 2A). Moreover, LPS also activated the downstream mediators of mTOR, 4E-BP1, p70S6K, and S6, whereas rapamycin inhibited their phosphorylation (Figures 2A and 2B). In addition, blockade of Akt inhibited phosphorylation of 4E-BP1 and S6, confirming that the mTOR pathway is downstream of Akt in monocytes (Figure 2C and Figure S3). Murine bone-marrow-derived macrophages (BMDMs) similarly activated the mTOR pathway after in vitro infection with live L.m. and rapamycin inhibited this activation (Figure 2D). Ser2448 is a well-known phosphorylation site of mTOR and sensitive to PI3K inhibition (Sekulic et al., 2000Sekulic A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. A direct linkage between the phosphoinositide 3-kinase-AKT signaling pathway and the mammalian target of rapamycin in mitogen-stimulated and transformed cells.Cancer Res. 2000; 60: 3504-3513PubMed Google Scholar). Ser2448 phosphorylation of mTOR was evident in LPS-stimulated monocytes (Figure 2E) and could be prevented by pharmacological inhibition of PI3K with wortmannin (Figure 2E), demonstrating that PI3K signaling is upstream of mTOR in human monocytes. Accordingly, PI3K inhibition augmented IL-12 but suppressed IL-10 production (Figure 2F). Taken together, these results provide evidence that the mTOR pathway is downstream of PI3K and that this pathway can be activated by bacterial stimuli in human and murine myeloid cells. Rapamycin associates with FK506-binding protein 12 (FKBP12) to selectively bind and inhibit a complex consisting of mTOR, mLST8, and Raptor (mTORC1) but not a complex of mTOR, mLST8, and Rictor (mTORC2) (Wullschleger et al., 2006Wullschleger S. Loewith R. Hall M.N. TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4367) Google Scholar). However, prolonged treatment with rapamycin has been shown to inhibit mTORC2 signaling in distinct cell lines (Rosner and Hengstschlager, 2008Rosner M. Hengstschlager M. Cytoplasmic and nuclear distribution of the protein complexes mTORC1 and mTORC2: Rapamycin triggers dephosphorylation and delocalisation of the mTORC2 components rictor and sin1.Hum. Mol. Genet. 2008; (Published online July 8, 2008)https://doi.org/10.1093/hmg/ddn192Crossref PubMed Scopus (190) Google Scholar, Sarbassov et al., 2006Sarbassov D.D. Ali S.M. Sengupta S. Sheen J.H. Hsu P.P. Bagley A.F. Markhard A.L. Sabatini D.M. Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB.Mol. Cell. 2006; 22: 159-168Abstract Full Text Full Text PDF PubMed Scopus (2059) Google Scholar). Pre-exposure of monocytes to rapamycin and subsequent washout of the inhibitor similarly deviated cytokine production after LPS stimulation indicating mTORC1-specfic effects (Figure 3A). Similar to rapamycin, the immunosuppressive macrolide FK506 engages FKBP12 and blocks calcineurin phosphatase instead of mTOR. We observed that FK506 did not significantly modulate IL-12 or IL-10 production (Figure 3B) but was able to outcompete the rapamycin effects (Figure 3C). Because rapamycin regulates the balance of IL-12 and IL-10, hyperactivation of mTOR after bacterial stimulation ought to reverse the pattern of cytokine production. Accordingly, amino acids or the nucleotide ATP, which have been shown to activate mTOR signaling (Yang and Guan, 2007Yang Q. Guan K.L. Expanding mTOR signaling.Cell Res. 2007; 17: 666-681Crossref PubMed Scopus (426) Google Scholar), but not UTP inhibited IL-12 and simultaneously enhanced IL-10 production (Figures 3D and 3E). To directly address whether the mTOR pathway controls inflammatory cytokine production, we made use of murine embryonic fibroblasts (MEFs) lacking TSC2, a key negative regulator of mTOR. TSC2 deficiency led to constitutive activation of the mTOR pathway (Figure 4A). We next examined whether loss of TSC2 might affect cytokine production in a reciprocal manner to rapamycin. Because MEF do not produce IL-12p40, we transfected MEF with an IL-12p40 promoter plasmid to assess IL-12p40 induction. Indeed, IL-12p40 was inhibited markedly in Tsc2−/− cells and could not be induced by LPS stimulation (Figure 4B), whereas IL-10 was increased in Tsc2−/− cells (Figure 4C). Moreover, IL-6 production was partly inhibited in Tsc2−/− cells (Figure 4D). To confirm the involvement of TSC2 also in myeloid immune cells, we employed RNA interference in THP-1 cells to knock down TSC2 (Figure 4E). IL-12p40 and TNF-α production was reduced in TSC2-silenced cells upon LPS stimulation, whereas IL-10 was increased (Figures 4F, 4G, and 4H). In addition to the data obtained with the mTOR inhibitor rapamycin, these results demonstrate that the TSC2-mTOR pathway critically controls inflammatory cytokine production. We went on to elucidate the downstream signals of mTOR, which regulate the IL-12 and IL-10 balance. IL-10 is a well-known endogenous inhibitor of IL-12 production. However, the exogenous addition of IL-10 or neutralizing anti-IL-10 did not change the effect of rapamycin on IL-12p40 production, indicating an IL-10-independent upregulation of IL-12p40 by mTOR inhibition (Figure S4). Moreover, the protein-synthesis inhibitor cycloheximide did not reverse the rapamycin-induced cytokine alteration, indicating that new protein synthesis is not required for the proinflammatory phenotype of mTOR-inhibited cells (Figure S5). Next, we examined the activation of the kinases p38, ERK, and JNK, which are well-known regulators of IL-12 expression (Dong et al., 2002Dong C. Davis R.J. Flavell R.A. MAP kinases in the immune response.Annu. Rev. Immunol. 2002; 20: 55-72Crossref PubMed Scopus (1306) Google Scholar). Phosphorylation of p38 and ERK was not influenced by rapamycin (Figure 2D and Figure 5A). Although rapamycin enhanced JNK activity (Figure 5A), the main target of JNK, the transcription factor complex AP-1, was not active in monocytes, as demonstrated by EMSA and luciferase reporter studies (Figure 5C and Figure S6). These findings suggest a distinct mTOR-sensitive pathway. The transcription factor NF-κB is a master regulator of proinflammatory responses and a major regulator of IL-12p40. We observed enhanced NF-κB activation in primary human monocytes, when they were treated with rapamycin and LPS compared to LPS alone, indicating that mTOR negatively regulates NF-κB (Figure 5B). Luciferase reporter assays in THP-1 and U937 cells confirmed this hyperactivation of NF-κB, but not of CRE or AP-1, after mTOR inhibition (Figure 5C and Figure S7). In line with these results, rapamycin not only enhanced but also prolonged IL-12p40 and TNF-α production in monocytes for up to 20 hr after LPS stimulation (Figure S8). Because the mTOR pathway is constitutively active in Tsc2−/− cells (Figure 4A), these cells should have a decreased ability to activate NF-κB. Accordingly, Tsc2−/− cells displayed a significantly reduced LPS-inducible and basal NF-κB activity (Figure 5D). Noticeably, rapamycin rescued NF-κB activity in LPS-triggered Tsc2−/− cells to a significant extent (Figure 5D). In agreement, nuclear translocation of NF-κB was reduced in Tsc2−/− cells (Figure 5E). Supershift assays revealed that the composition of NF-κB family members on the NF-κB promoter was not altered by rapamycin, and p65 was mainly occupying the NF-κB binding site (Figure S9), suggesting that mTOR might negatively modulate p65 activity to dampen NF-κB activation. The transactivation domain (TAD) of p65 recruits cofactors to enhance transcription (Perkins, 2007Perkins N.D. Integrating cell-signalling pathways with NF-kappaB and IKK function.Nat. Rev. Mol. Cell Biol. 2007; 8: 49-62Crossref PubMed Scopus (1809) Google Scholar). Moreover, PTEN and PI3K might negatively control NF-κB activity through p65(TAD) (Mayo et al., 2002Mayo M.W. Madrid L.V. Westerheide S.D. Jones D.R. Yuan X.J. Baldwin Jr., A.S. Whang Y.E. PTEN blocks tumor necrosis factor-induced NF-kappa B-dependent transcription by inhibiting the transactivation potential of the p65 subunit.J. Biol. Chem. 2002; 277: 11116-11125Crossref PubMed Scopus (108) Google Scholar). To test whether mTOR controls the activity of p65(TAD), we utilized a p65(TAD)-Gal4 fusion protein in luciferase reporter assays (Rocha et al., 2003Rocha S. Campbell K.J. Perkins N.D. p53- and Mdm2-independent repression of NF-kappa B transactivation by the ARF tumor suppressor.Mol. Cell. 2003; 12: 15-25Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). p65(TAD) was hyperactivated when mTOR was inhibited in human THP-1 and murine MEF cells (Figures 5F and 5G). Moreover, p65(TAD) activity was not induced by LPS in Tsc2−/− cells, whereas rapamycin restored LPS-dependent p65(TAD) activity in these cells (Figure 5G). To directly show that the enhanced NF-κB activity is responsible for the observed effects of mTOR inhibition, we blocked NF-κB signaling with SN50, a cell-permeable peptide, which inhibits NF-κB translocation to the nucleus (Lin et al., 1995Lin Y.Z. Yao S.Y. Veach R.A. Torgerson T.R. Hawiger J. Inhibition of nuclear translocation of transcription factor NF-kappa B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence.J. Biol. Chem. 1995; 270: 14255-14258Crossref PubMed Scopus (836) Google Scholar). SN50 abolished the rapamycin-induced augmentation of IL-12p40 and TNF-α production in human monocytes (Figures 5H and 5I). Collectively, these results demonstrate that the mTOR pathway limits NF-κB activity in myeloid cells by modulation of the p65 transactivation domain to regulate proinflammatory cytokine production. Because NF-κB hyperactivation cannot explain IL-10 blockade induced by rapamycin, we investigated the effects of rapamycin on STAT3, given that STAT3 is a major transcription factor for IL-10 in human monocytes (Benkhart et al., 2000Benkhart E.M. Siedlar M. Wedel A. Werner T. Ziegler-Heitbrock H.W. Role of Stat3 in lipopolysaccharide-induced IL-10 gene expression.J. Immunol. 2000; 165: 1612-1617PubMed Google Scholar, Cheng et al., 2003Cheng F. Wang H.W. Cuenca A. Huang M. Ghansah T. Brayer J. Kerr W.G. Takeda K. Akira S. Schoenberger S.P. et al.A critical role for Stat3 signaling in immune tolerance.Immunity. 2003; 19: 425-436Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar) and mTOR has been implicated in the regulation of STAT3 (Karras et al., 1997Karras J.G. Wang Z. Huo L. Howard R.G. Frank D.A. Rothstein T.L. Signal transducer and activator of transcription-3 (STAT3) is constitutively activated in normal, self-renewing B-1 cells but only inducibly expressed in conventional B lymphocytes.J. Exp. Med. 1997; 185: 1035-1042Crossref PubMed Scopus (130) Google Scholar). Indeed, rapamycin inhibited tyrosine phosphorylation of STAT3 and reduced STAT3 activity (Figure 5J and Figure S10). Moreover, in Tsc2−/− cells, STAT3 was hyperactivated (Figure 5K). Inhibition of STAT3 via the selective inhibitor Stattic (Schust et al., 2006Schust J. Sperl B. Hollis A. Mayer T.U. Berg T. Stattic: A small-molecule inhibitor of STAT3 activation and dimerization.Chem. Biol. 2006; 13: 1235-1242Abstract Full Text Full Text PDF PubMed Scopus (695) Google Scholar) reduced IL-10 production of LPS-treated monocytes but did not enhance production of TNF-α and IL-12p40 (Figure 5L). In agreement, Stattic failed to upregulate NF-κB activity in Tsc2+/+ as well as Tsc2−/− cells (Figure 5M). Importantly, STAT3-deficient BMDMs (Stat3Lys−/−) showed diminished IL-10 production compared to control cells (Stat3fl/fl) after LPS stimulation, whereas rapamycin was still able to enhance IL-12p40 in these cells (Figure 5N). These results demonstrate that mTOR activates STAT3 to induce IL-10 production and independently inhibits NF-κB to restrain proinflammatory responses. mTOR is considered to control primarily posttranscriptional events by modulating translation and mRNA stability (Hay and Sonenberg, 2004Hay N. Sonenberg N. Upstream and downstream of mTOR.Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3247) Google Scholar). However, the NF-κB hyperactivation described above (Figure 5) strongly indicated that mTOR regulates transcriptional processes in human monocytes. Accordingly, increased IL-12p40 and decreased IL-10 mRNA expression were observed in mTOR-inhibited human monocytes (Figures 6A and 6B). Rapamycin did not influence the stability of IL-12p40 or IL-10 mRNA (Figures 6C and 6D), substantiating the transcriptional control of IL-12p40 and IL-10 by mTOR. To determine whether mTOR affects the transcription of other immunomodulatory molecules in myeloid cells, we performed microarray analysis of LPS-stimulated monocytes with or without rapamycin. After 4 hr of LPS stimulation, 290 genes were markedly modulated with rapamycin treatment (Table S1). Of these, 35 or 38 genes were more than 1.5-fold upregulated or downregulated, respectively. Analysis of transcription-factor binding sites revealed that genes containing NF-κB promoters were substantially overrepresented among the rapamycin-upregulated genes, corroborating with the finding that mTOR controls NF-κB activation on a genome-wide level (Figure S11). To validate upregulation and downregulation of some of the differentially regulated genes, we assessed protein expression of genes that are important for the regulation of immune responses. Rapamycin prevented the upregulation of the negative T cell regulator PD-L1 (B7-H1) and the HIV coreceptor CCR5 (Figures 6E and 6F) but superinduced the costimulatory molecule CD86 on monocytes and peripheral myeloid DCs (Figure 6G and Figure S2B). CCL22, a T cell-attracting chemokine, was significantly upregulated by rapamycin, whereas the chemokine MCP-1, which is important for neoangiogenesis and induction of Th2 cell responses (Salcedo et al., 2000Salcedo R. Ponce M.L. Young H.A. Wasserman K. Ward J.M. Kleinman H.K. Oppenheim J.J. Murphy W.J. Human endothelial cells express CCR2 and respond to MCP-1: Direct role of MCP-1 in angiogenesis and tumor progression.Blood. 2000; 96: 34-40Crossref PubMed Google Scholar), was downregulated (Figures 6H and 6I). Conversely, in Tsc2−/− cells MCP-1 was upregulated (Figure S12). Thus, mTOR signaling controls the expression of a broad range of immunologically relevant genes in human monocytes. The ability of mTOR to differentially regulate distinct cytokines, chemokines, and costimulatory molecules in myeloid cells suggest an impact on subsequent T cell activation. By employing an allogeneic T cell-activation model, we observed that rapamycin pretreatment of LPS-stimulated monocytes enhanced their T cell-stimulatory capacity (Figure 6J). Strikingly, analysis of T cell cytokine production revealed that IFN-γ and IL-17 production was significantly higher in allogeneic T cells challenged with mTOR-inhibited monocytes (Figures 6K and 6L). Nevertheless, rapamycin suppressed T cell proliferation and IFN-γ production when added directly to the mixed lymphocyte culture, reflecting its cell-cycle-blocking effect on T lymphocytes (data not shown). These results indicate that in myeloid immune cells mTOR negatively regulates the priming of Th1 and Th17 T cells. We found that BMDMs from BALB/c and C57BL/6 mice, similarly to human cells, differentially regulated IL-12 and IL-10 production upon L.m. or LPS challenge under mTOR blockade in vitro (Figure 7A and Figure S13). To examine the physiological relevance of altered cytokine production after mTOR inhibition in vivo, we tested the effect of rapamycin on the immune response to L.m. in infected BALB/c mice. The increased susceptibility of BALB/c mice to L.m. results from an inherent inability to express sufficient amounts of IL-12, IFN-γ, and IL-6 while producing excessive amounts of IL-10 (Dai et al., 1997Dai W.J. Kohler G. Brombacher F. Both innate and acquired immunity to Listeria monocytogenes infection are increased in IL-10-deficient mice.J. Immunol. 1997; 158: 2259-2267PubMed Google Scholar, Seki et al., 2002Seki E. Tsutsui H. Tsuji N.M. Hayashi N. Adachi K. Nakano H. Futatsugi-Yumikura S. Takeuchi O. Hoshino K. Akira S. et al.Critical roles of myeloid differentiation factor 88-dependent proinflammatory cytokine release in early phase clearance of Listeria monocytogenes in mice.J. Immunol. 2002; 169: 3863-3868PubMed Google Scholar). Consequently, it has been shown that modifying the early cytokine imbalance in BALB/c mice is critical in controlling L.m. infection, even in mice lacking T and B cells (Tripp et al., 1994Tripp C.S. Gately M.K. Hakimi J. Ling P. Unanue E.R. Neutralization of IL-12 decreases resistance to Listeria in SCID and C.B-17 mice. Reversal by IFN-gamma.J. Immunol. 1994; 152: 1883-1887PubMed Google Scholar). Remarkably, administration of rapamycin for 3 days before receiving an LD100 or LD50 dose of L.m. significantly improved the survival of these mice compared with that of control mice given L.m. only (Figures 7B and 7C), even though the L.m.-specific T cell response was alleviated as expected (Figure S14). The T cell immunosuppressant FK506 did not modulate IL-12 or IL-10 production in vitro (Figure 3B) and also did not improve survival after L.m. infection in vivo (Figure 7B). Furthermore, depletion of macrophages and DCs with clodronate in vivo abolished the enhanced rapamycin-mediated resistance after L.m. infection (data not shown), indicating that myeloid immune cells are critical for the observed in vivo effects. Improved survival of rapamycin-treated mice infected with L.m. was confirmed by" @default.
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• W2020920210 date "2008-10-01" @default.
• W2020920210 modified "2023-10-17" @default.
• W2020920210 title "The TSC-mTOR Signaling Pathway Regulates the Innate Inflammatory Response" @default.
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