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Tuberous sclerosis 1 (Tsc1)-dependent metaboliccheckpoint controls development of dendritic cellsYanyan Wanga, Gonghua Huanga, Hu Zenga, Kai Yanga, Richard F. Lambb, and Hongbo Chia,1
aDepartment of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105; and bCancer Research UK Centre, University of Liverpool, LiverpoolL3 9TA, United Kingdom
Edited by Peter Cresswell, Yale University School of Medicine, New Haven, CT, and approved November 7, 2013 (received for review May 10, 2013)
Coordination of cell metabolism and immune signals is crucial forlymphocyte priming. Emerging evidence also highlights the im-portance of cell metabolism for the activation of innate immunityupon pathogen challenge, but there is little evidence of how thisprocess contributes to immune cell development. Here we showthat differentiation of dendritic cells (DCs) from bone marrowprecursors is associated with dynamic regulation of mechanistictarget of rapamycin (mTOR) complex 1 (mTORC1) signaling and cellmetabolism. Unexpectedly, enhancing mTORC1 activity via abla-tion of its negative regulator tuberous sclerosis 1 (Tsc1) impairedDC development in vivo and in vitro, associated with defective cellsurvival and proliferation. Moreover, Tsc1 deficiency caused DCspontaneous maturation but a propensity to differentiate intoother lineages, and attenuated DC-mediated effector TH1 responses.Mechanistically, Tsc1-deficient DCs exhibited increased glycolysis,mitochondrial respiration, and lipid synthesis that were partly me-diated by the transcription factor Myc, highlighting a key role ofTsc1 in modulating metabolic programming of DC differentiation.Further, Tsc1 signaled through Rheb to down-regulate mTORC1 forproper DC development, whereas its effect at modulating mTORcomplex 2 (mTORC2) activity was largely dispensable. Our resultsdemonstrate that the interplay between Tsc1-Rheb-mTORC1 sig-naling and Myc-dependent bioenergetic and biosynthetic activi-ties constitutes a key metabolic checkpoint to orchestrate DCdevelopment.
Cell metabolism refers to the intracellular chemical reactionsthat convert nutrients and endogenous molecules into energy
and biomass (proteins, nucleic acids, and lipids). Emerging evi-dence highlights an intimate interaction between metabolismand immunity (1–3). For example, activated T cells are highlyglycolytic and rely on glycolysis to generate ATP (even in thepresence of high levels of oxygen), a phenomenon known asWarburg metabolism, which is unique to cancer cells and acti-vated lymphocytes. Blocking glycolysis impairs activation anddifferentiation of T cells and the outcome of adaptive immuneresponses, thereby indicating a prerequisite role of metabolismin T-cell fate determination (4–6). Other modes of metabolism,such as lipid metabolism and fatty acid oxidation, are also im-portant regulators of T-cell responses (7–10). Although moststudies of metabolic controls of cell fate are focused on T cell-mediated adaptive immunity, we are beginning to appreciate thatactivation of innate immune cells is also metabolically demanding.Engagement of toll-like receptors (TLRs) expressed by dendriticcells (DCs), the specialized antigen-presenting cells for bridginginnate and adaptive immunity, triggers a profound metabolic tran-sition to aerobic glycolysis, similar to Warburg metabolism. Glucoserestriction inhibits the activation and life span of TLR-stimulatedDCs (11, 12). Glucose metabolism is also a limiting step in theactivation of the inflammasome and TLR signaling for the pro-duction of the inflammatory cytokine IL-1β (13, 14). Despiteadvances in our understanding of metabolic regulation of immunecell activation, there is little evidence that cell metabolism is in-volved in the development of immune cells.The evolutionarily conserved mechanistic target of rapamycin
(mTOR) pathway integrates various environmental signals toregulate fundamental physiological functions such as cell growthand proliferation, autophagy, and nutrient sensing and uptake
(15). Whereas the most well-established molecular function ofmTOR is in protein translation, recent studies have identified animportant role of mTOR in activating a metabolic gene-regula-tory network via controlling the respective transcription factorsin glycolysis and lipid synthesis, HIF1α and SREBP (16). mTORexists in two complexes, mTORC1 and mTORC2, both of whichcontribute to T-cell activation and differentiation (17–19). In theinnate immune system, mTOR and the upstream PI3K-AKTpathway have a well-established role in modulating the balancebetween TLR-induced production of pro- and anti-inflammatoryDC cytokines, especially IL-12 and IL-10, thereby affecting DCfunction and immune responses (20–24). Additionally, mTORsignaling promotes the production of type I IFN from plasmacy-toid DCs (pDCs) (25), and regulates other cellular events inducedby TLR stimulation such as survival of activated DCs (12, 26). Theseresults collectively illustrate an important role of mTOR signalingin the activation of both innate and adaptive immune systems.In contrast, the function of mTOR signaling in the de-
velopment of DCs is less understood, with many of the findingsto date obtained via pharmacological approaches. For instance,blocking mTORC1 activity by rapamycin inhibits DC devel-opment and/or maturation, and instead endows DCs with astrong tolerogenic activity to promote T-cell tolerance (19, 27–29). However, rapamycin is not an efficient inhibitor of 4EBP1phosphorylation downstream of mTORC1 activation (30), andmay also inhibit mTORC2 activity with prolonged treatmentand/or at a high dose (31, 32). To conclusively establish the rolesof mTOR in DC development, we have used genetic approachesto eliminate mTOR signaling components, including tuberoussclerosis 1 (Tsc1, a modulator of mTORC1 and mTORC2 ac-tivities), Rheb (a key upstream activator of mTORC1), andRictor (an obligatory component of mTORC2) (15), as well asthe downstream effector Myc, alone or in combination, and
Significance
A fundamental question in immunology is how the coordinationof immune signals and metabolic programs regulates immuneresponses. The identification of metabolic pathways orchestrat-ing the activation of lymphocytes and dendritic cells (DCs) hasadvanced our understanding of immune activation, but whethercell metabolism contributes to development of immune cells isunknown. Here we have genetically defined a crucial metaboliccheckpoint for DC development that is mediated by the interplaybetween Tsc1-mTOR complex 1 signaling and Myc-dependentbioenergetic and biosynthetic programs. Dysregulation ofthis pathway impairs survival, proliferation, and functionaldifferentiation of DCs, thereby highlighting the importance ofmetabolic programming of DC development.
Author contributions: Y.W. and H.C. designed research; Y.W., G.H., H.Z., and K.Y. per-formed research; R.F.L. contributed new reagents/analytic tools; Y.W., G.H., H.Z., K.Y., andH.C. analyzed data; and Y.W. and H.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1308905110/-/DCSupplemental.
E4894–E4903 | PNAS | Published online November 26, 2013 www.pnas.org/cgi/doi/10.1073/pnas.1308905110
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further complemented them with pharmacological approaches.Contrary to our expectations, deletion of Tsc1 and subsequentmTORC1 activation exerted multiple negative effects on DC de-velopment, including survival, proliferation, and lineage differ-entiation. Loss of Tsc1 up-regulated several metabolic programsincluding glycolysis, mitochondrial respiration, and lipid synthesis,partly via a Myc-dependent pathway, and this metabolic pro-gramming contributed to DC survival and differentiation but notproliferation. Further, Tsc1 signaled through Rheb and mTORC1to control DC development, whereas mTORC2 activity was largelydispensable in this process. Importantly, DC differentiation is as-sociated with dynamic regulation of glycolytic and lipogenic pro-grams and the accompanying mTORC1 activity. These data pointto a unique checkpoint active in DC development that is mediatedby the interplay between Tsc1-mTORC1–dependent immunesignaling and Myc-dependent metabolic programming.
ResultsTsc1 Plays an Important Role in DC Development in Vitro. DCsoriginate from hematopoietic stem cells (HSCs) in the bonemarrow (BM) through multiple steps of differentiation, and thiscan be recapitulated in the in vitro differentiation system mediatedby FLT3L (33–35). To determine the importance of mTORC1regulation in DC development, we focused on Tsc1, an upstreamregulator of mTOR signaling (15), by crossing Tsc1flox/flox
mice with Rosa26-Cre-ERT2 mice (a Cre-ER fusion gene wasrecombined into the ubiquitously expressed Rosa26 locus) togenerate Tsc1flox/floxRosa26-Cre-ERT2 mice (Tsc1CreER mice). Tocircumvent the detrimental effects of chronic loss of Tsc1 on thefunction of HSCs (36, 37), we determined the requirement ofTsc1 during DC development through in vitro acute deletion.Specifically, we cultured BM cells with FLT3L in the presence of4-hydroxytamoxifen (4-OHT), which resulted in efficient deletionof Tsc1 after 2 d of treatment (Fig. S1A). Under these conditions,Tsc1CreER BM cells showed a considerable defect to generateCD11c+ cells (Fig. 1A). Analysis of DC-specific transcripts in-cluding Zbtb46, Ccr7, Flt3, and c-Kit (35) in sorted CD11c+ cellsconfirmed them as DCs (Fig. S1B). The impairment in DC gen-erationwas apparent in both conventionalDC (cDC;CD11c+B220–)and pDC (CD11c+B220+) subsets (Fig. 1B). Thus, Tsc1 is importantfor DC development in vitro.We next determined the temporal requirement of Tsc1 in DC
development. First, we assessed the generation of DCs from
Lin–Sca-1+c-Kit+ cells (LSKs; a heterogeneous mixture of HSCsand multipotent progenitors) and macrophage and DC precursors(MDPs; one of the earliest precursor populations identified forDCs, defined as Lin–CD11c−Sca-1–CSF1R+) (38–40). Both pro-genitor cultures lacking Tsc1 were impaired from developing intoCD11c+ DCs after FLT3L stimulation (Fig. S1 C and D), in-dicating the involvement of Tsc1 in DC development from mul-tipotent and DC-committed precursors. Second, we cultured BMcells from Tsc1CreER mice with 4-OHT added at various timesafter the initiation of DC differentiation. Compared with wild-type(WT) control, Tsc1CreER cells were impaired from developing intoCD11c+ DCs even when 4-OHT was added at 5 d after the ini-tiation of DC differentiation (Fig. S1E). These results illustratea direct role of Tsc1 in programming DC development.
A Cell-Autonomous Role of Tsc1 in the Generation of DCs andPrecursors in Vivo. To determine the role of Tsc1 in DC de-velopment in vivo, we treated WT and Tsc1CreER mice withtamoxifen for 5 d and then rested the mice for 5–10 d. Suchtreatment resulted in efficient deletion of Tsc1 in splenic DCs fromTsc1CreER mice (Fig. S1F). Compared with WT control, Tsc1CreERmice contained a reduced number of cDCs (CD11c+MHC-II+),including both CD8+ and CD11b+ subsets (Fig. 1C). The pDC(CD11clomPDCA-1+) population was also diminished (Fig. 1D).Similar defects in the cDC population were observed in mesen-teric lymph nodes and nonlymphoid organs including liver andkidney (Fig. 1E). Therefore, Tsc1 plays an important role inmaintaining DC populations in vivo.To address whether the reduction of DCs in Tsc1CreER mice
was an intrinsic defect, we generated mixed-BM chimeras. Spe-cifically, the recipients (CD45.1.1+) were lethally irradiated andreconstituted with tamoxifen-treated WT or Tsc1CreER BM cells(CD45.2.2+; donor) along with WT BM cells (CD45.1.2+; spike)at a 1:1 ratio. After reconstitution, we analyzed the contributionsof donor and spike cells to various immune compartments. Be-cause CD4 T cells are relatively resistant to Tsc1 deletion (41), weused the chimerism of CD4 T cells for normalization (Fig. S1G).DCs, including various subsets, derived from Tsc1-deficient donorcells were greatly underrepresented relative to the spike cells(Fig. 1F). Thus, Tsc1 has a key cell-autonomous role in regulatingDC development in vivo. Notably, other myeloid cells lackingTsc1, such as macrophages and neutrophils, were also decreasedin the chimeras (Fig. S1 H and I).
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Fig. 1. Loss of Tsc1 disrupts DC development in vitro andin vivo. (A and B) BM cells from WT or Tsc1CreER mice werecultured with FLT3L (input, 3 × 106) in the presence of4-hydroxytamoxifen, followed by analysis of the percent-age (Left) and cell number of total CD11c+ DCs (Right)(A) and the number of cDCs (CD11c+B220–) and pDCs(CD11c+B220+) (B). (C and D) WT or Tsc1CreER mice weretreated with tamoxifen for 5 d, followed by resting for5–10 d, and the percentage and number of splenic cDCsand cDC subsets (C) or pDCs (D) were analyzed. (E) DCpercentage (Left) and number (Right) in mesenteric lymphnodes (MLNs), liver, and kidney of WT or Tsc1CreER micewere analyzed as in C. (F) BM cells from tamoxifen-treatedWT or Tsc1CreER CD45.2.2+ mice were mixed with cells fromCD45.1.2+ mice at a 1:1 ratio and transferred into irradi-ated CD45.1.1+ recipients. The contributions of spike BM-derived (CD45.1.2+) and WT or Tsc1CreER donor BM-derived(CD45.2.2+) cells among total CD11c+ cDCs, CD8+ cDCs,CD11b+ cDCs, and pDCs in the reconstituted mixed chi-meras were analyzed (Upper). (Lower) Normalized ratiosagainst the percentages of CD4+ T cells. Error bars indicateSEM. Data are representative of two to four independentexperiments.
Wang et al. PNAS | Published online November 26, 2013 | E4895
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We further determined whether Tsc1 regulates DC developmentpartly by impacting the generation of early precursor MDPs. Fol-lowing tamoxifen treatment in vivo, the percentage and cell num-ber of MDPs in Tsc1CreER mice were significantly decreasedcompared with those in WT mice (Fig. S2A). Furthermore, morecommitted precursors, including common dendritic precursors(CDPs; defined as Lin−CD11c−Sca-1−CSF1R+Flt3+CD117lo) andcommitted precursors of cDCs (precDCs; defined as Lin−IA/E−CD11c+Flt3+CD172αint) (33), were also reduced upon Tsc1deletion (Fig. S2 B and C). Associated with the numerical re-duction of these precursor populations was a decreased expressionof GM-CSFR (CD116), although Flt3 expression was largely nor-mal (Fig. S2D). These data indicate a requirement of Tsc1 in thegeneration of DC precursors, which may act in synergy with itseffect at driving the differentiation from these precursors to matureDCs (Fig. S1D) to promote DC development.
Tsc1 Promotes DC Survival and Proliferation. The overall size of DCpopulations is dependent upon the rates of apoptosis and pro-liferation. We first evaluated the effects of Tsc1 on the survival ofDCs. Caspase activity, a hallmark of apoptotic cell death, was in-creased in freshly isolated splenic DCs of tamoxifen-treatedTsc1CreER mice and of the chimeras reconstituted with Tsc1CreER
BM cells (Fig. 2 A and B). In vitro FLT3L-derived DCs from4-OHT–treated Tsc1CreER BM cells also showed significantlyincreased apoptosis as indicated by the loss of the live AnnexinV− 7-AAD− population, and this increase was observed when4-OHT was added at 0, 24, and 48 h after the initiation of DCdifferentiation (Fig. 2C). These data indicate an important roleof Tsc1 in promoting the survival of DCs.We then dissected molecular mechanisms by which Tsc1 reg-
ulates DC survival. Bcl-2 family proteins are important regu-lators of apoptotic cell death, and the balance of anti- andproapoptotic Bcl-2 family members is crucial to dictating thedecision between survival and apoptosis (42). The expression ofthe proapoptotic Bim and Puma was increased in 4-OHT–treated Tsc1CreER DCs, whereas the antiapoptotic protein Bcl-2was diminished. In contrast, the activity of p38 MAPK, anotherimportant cell death regulator, was comparable between WT andTsc1-deficient DCs (Fig. 2D). Aside from Bcl-2 family proteins,production of reactive oxygen species (ROS) also contributes toapoptotic cell death (42). Tsc1-deficient DCs produced greatlyincreased levels of ROS (Fig. 2E). Therefore, the increased
apoptosis of Tsc1-deficient DCs is associated with dysregulatedBcl-2 family proteins and elevated oxidative stress.We next tested the possibility that the decreased DC pop-
ulation in the absence of Tsc1 was also partly ascribed todefective DC proliferation. We labeled freshly isolated BM cellswith carboxyfluorescein diacetate succinimidyl ester (CFSE) andcultured the cells with FLT3L in the presence of 4-OHT. Theproliferation of Tsc1-deficient CD11c+ DCs was similar to thatof WT cells when examined at day 3.5. However, the mutant cellsshowed diminished CFSE dilution when analyzed at day 7.5 (Fig.2F). Furthermore, a bromodeoxyuridine (BrdU) incorporationassay showed that Tsc1-deficient CD11c+ DCs incorporated lessBrdU than did WT cells at day 7.5 (Fig. 2G). Thus, Tsc1 isdispensable for initial DC proliferation but its absence com-promises continuous DC proliferation.
Dysregulated DC Maturation and Functional Differentiation in theAbsence of Tsc1. DCs are the most potent antigen-presentingcells for the activation and differentiation of naïve T cells. To inves-tigate the effect of Tsc1 signaling on DC functions at mediatingT-cell responses, we first cocultured WT and Tsc1-deficient DCswith naïve CD4+ T cells from OT-II mice [a T cell receptor(TCR)-transgenic model with T cells recognizing ovalbumin(OVA) amino acids 323–339] in the presence of antigen but noexogenous cytokines to mimic the physiological interaction be-tween DCs and T cells. After 5–6 d, T cells activated with Tsc1-deficient DCs secreted decreased IFN-γ, but similar levels of IL-4,compared with those activated with WT DCs (Fig. 3A). We nextused freshly isolated DC subsets from in vivo tamoxifen-treatedWT and Tsc1CreER mice and cocultured them with OT-II naïveT cells. All DC subsets lacking Tsc1 showed a defective ability todrive TH1 but not TH2 differentiation (Fig. 3B). Finally, we de-termined the role of Tsc1 in mediating DC-dependent T-cellresponses in vivo. To this end, we generated WT and Tsc1CreER
BM chimeras and then transferred OVA-specific CD4+ T cellsinto the chimeras, followed by immunization with the cognate an-tigen. T cells isolated from Tsc1CreER chimera hosts produced lessIFN-γ (Fig. 3C). These results demonstrate that Tsc1 signaling inDCs is required for the differentiation of antigen-specific naïveT cells into TH1 cells.To explore the mechanistic basis, we analyzed the effects of
Tsc1 deficiency on DC maturation, cytokine production, andlineage differentiation. First, we examined the expression of
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Fig. 2. Tsc1 deficiency impairs DC survival and pro-liferation. (A and B) Caspase activity in freshly isolatedsplenic DCs from tamoxifen-treated WT or Tsc1CreER mice(A), or WT or Tsc1CreER BM-derived cells in mixed chimeras(generated as described in Fig. 1F) (B), assessed with FITC-VAD-FMK staining. “Control” indicates no staining withFITC-VAD-FMK. (Right) Percentage of caspase-positivecells. (C) BM cells fromWT or Tsc1CreER mice were culturedwith FLT3L, with 4-OHT added at 0, 24, and 48 h after theinitiation of DC differentiation. The apoptosis of CD11c+
cells was analyzed by flow cytometry at day 7.5. (Right)Percentage of live cells (Annexin V– 7-AAD–). (D) WT orTsc1CreER BM cells were cultured with FLT3L and 4-OHT,followed by purification of CD11c+ cells and immunoblotanalysis. (E) ROS production in 4-OHT–treated WT orTsc1CreER BM-derived CD11c+ cells. (F) BM cells fromWT orTsc1CreER mice were labeled with CFSE and cultured withFLT3L and 4-OHT for 3.5 or 7.5 d. (G) BM cells from WT orTsc1CreER mice were cultured with FLT3L and 4-OHT for3.5 and 7.5 d and pulse-labeled with BrdU for 3 h beforestaining. Error bars indicate SEM. Data are representativeof two or three independent experiments.
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various surface markers associated with DC maturation, in-cluding CD86, CD80, and CD40. Surprisingly, Tsc1-deficientDCs showed more elevated expression of CD86, CD80, andCD40 than WT cells did (Fig. 3D). Moreover, CD86 was up-regulated on DCs derived from Tsc1-deficient donor cells in themixed chimeras, although CD80 and CD40 expression in vivowas largely normal (Fig. 3E). Thus, Tsc1 deficiency resulted inspontaneous maturation of DCs, with a more pronounced effecton CD86 up-regulation. However, this was unlikely to cause di-minished effector T-cell responses, as these DC molecules generallypromote effector responses (43). Second, we examined the ex-pression of proinflammatory cytokines, and found that Il12a (IL-12p35) was decreased in Tsc1-deficient DCs, whereas the anti-inflammatory cytokine Il10 was comparable betweenWT and Tsc1-deficient DCs (Fig. 3F). Given the importance of IL-12 formediating TH1 differentiation (44), the impaired cytokine pro-duction from Tsc1-deficient DCs, together with the survival de-fect and numerical reduction, likely contributed to decreasedTH1 responses in responding T cells.Third, we determined the effects of Tsc1 deficiency on the
integrity of DC differentiation. Although CD11c+ DCs de-veloped in the absence of Tsc1 expressed normal levels of DC-specific transcripts as described above (Fig. S1B), they aberrantlyacquired expression of macrophage and neutrophil markers F4/80and Ly6G, respectively. This defect was exacerbated with moreextended duration of culture (Fig. 3G). To determine whether thisaltered differentiation was functionally relevant to DC cytokineproduction, we purified F4/80+CD11c+ cells and measured theircytokine expression levels. Loss of Tsc1 resulted in a profounddown-regulation of Il12a in these populations. In contrast, WT andTsc1-deficient F4/80–CD11c+ cells expressed comparable levels ofIl12a (Fig. 3H). Therefore, Tsc1 maintains the integrity of DCdifferentiation, the loss of which results in aberrant up-regulationof other lineage markers and impaired DC cytokine expression.
Tsc1 Coordinates Cell Growth and Metabolism in DCs. We next ex-plored the molecular mechanism underlying Tsc1-mediated con-trol of DC development. We noticed that DCs developed in theabsence of Tsc1 exhibited an increased cell size. This alterationwas evident in BM chimeras (Fig. 4A), indicative of an intrinsicdefect. Additionally, cell size was increased in Tsc1-deficient DCsdeveloped from total BM cells, LSKs, or MDPs in vitro (Fig. 4B),
and this was also observed when Tsc1 deletion was initiated at 3–5 d after the start of culture (Fig. S3A). Further, the mutant DCscontained more protein and RNA content on a per-cell basis (Fig.S3B). These results indicated that Tsc1-deficient DCs underwentmore cell growth. Cell growth is dependent on the regulated ex-pression of amino acid transporters that include CD98 as a keycomponent, and of CD71, the transferrin receptor that mediatesiron uptake. Flow cytometry analysis of surface expression ofCD98 and CD71 revealed elevated levels of CD98 and CD71 uponloss of Tsc1 (Fig. 4C). This effect was also observed in DCs derivedfrom Tsc1-deficient MDPs (Fig. S4A), indicating an important roleof Tsc1 in cell-growth regulation during DC development.Cell growth is coupled with cell metabolism, which consists of
bioenergetic and biosynthetic activities (1, 17). Two major modesof bioenergetics are glycolysis and mitochondrial respiration. Todetermine the role of Tsc1 in DC metabolism, we first measuredthe glycolytic activity of WT and Tsc1-deficient cells by thegeneration of 3H-labeled H2O from [3-3H]glucose. Comparedwith FLT3L-derived WT DCs, those lacking Tsc1 displayed up-regulated glycolysis (Fig. 4D). This was verified by the elevatedextracellular acidification rate (ECAR) as measured by theSeahorse extracellular flux analyzer (Fig. 4E). Further, mRNAexpression of glycolytic enzymes, including Hk2, Ldha, and Tpi1,as well as Hif1a, the transcription factor that regulates glucosemetabolism, were all elevated in DCs developed from Tsc1-deficient BM cells (Fig. 4F) or MDPs (Fig. S4B). Moreover, oxygenconsumption rate (OCR), indicative of mitochondrial respirationactivity, was increased in Tsc1-deficient cells (Fig. 4G), and thiswas associated with increased mitochondrial mass (Fig. 4H).Therefore, Tsc1-deficient cells exhibit increased glycolysis andmitochondrial respiration.Having established an important role of Tsc1 in DC bio-
energetics, we next determined whether it also contributes to cellbiosynthetic activity. In particular, de novo synthesis of lipids(cholesterol and fatty acids) has been shown to depend uponmTOR activity (45). However, the role of Tsc1 in this processremains unclear because it can either promote or inhibit lipidsynthesis in a context-dependent manner (16, 46, 47). Wetherefore measured the incorporation of [1-14C]acetate intochloroform/methanol-soluble lipids in DCs with normal or ab-sent Tsc1 expression; [1-14C]acetate was used here to bypass therequirement of Tsc1 in glycolysis or mitochondrial activity, thereby
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Fig. 3. Tsc1 deficiency causes spontaneous mat-uration but aberrant differentiation of DCs thatprevents induction of effector T-cell responses. (A)WT or Tsc1CreER BM cells were cultured with FLT3Land 4-OHT for 7–8 d and sorted for CD11c+ cells,which were then cultured with naïve OT-II T cellsin the presence of OVA323–339 and LPS, followedby IFN-γ and IL-4 detection. (B) Freshly isolatedsplenic DC subsets from tamoxifen-treated WT orTsc1CreER mice were cocultured with OT-II T cellsand analyzed as described in A. (C ) Antigen-specific T cells from OT-II TCR-transgenic mice(Thy1.1+) were transferred into complete WTand Tsc1CreER chimera mice, followed by antigenimmunization in the presence of CFA. At day 7after immunization, draining lymph node cellswere isolated for IFN-γ expression analysis. (D) BMcells from WT or Tsc1CreER mice were cultured withFLT3L and 4-OHT, followed by analysis of CD86,CD80, and CD40 expression on CD11c+ cells. (E)Expression of CD86, CD80, and CD40 on splenic DCsfrom WT or Tsc1CreER BM-derived cells in mixedchimeras (generated as described in Fig. 1F). (F andG) BM cells from WT or Tsc1CreER mice were cul-tured with FLT3L and 4-OHT, followed by analysisof CD11c+ cells for cytokine mRNA (F) and F4/80and Ly6G expression (G). (H) WT or Tsc1CreER BM cells were cultured with FLT3L and 4-OHT, and CD11c+F4/80– cells and CD11c+F4/80+ cells were sorted for RNAanalysis. Error bars indicate SEM. Data are representative of two to four independent experiments.
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allowing us to directly evaluate the role of Tsc1 in de novo lipidbiosynthesis rate (46). Compared with WT DCs, those lackingTsc1 showed a markedly increased lipid synthesis rate (Fig. 4I).Furthermore, expression of many genes in cholesterol and fattyacid metabolism, including Hmgcs1, Hmgcr, and Scd2, was greatlyelevated in DCs derived from Tsc1-deficient BM cells (Fig. 4J) orMDPs (Fig. S4C). Altogether, these results indicate that Tsc1 hasa key role in negatively controlling cell growth, bioenergetics, andlipid biosynthesis, thereby coupling cell growth and metabolism.
The Tsc1–Myc Axis Orchestrates Metabolic Programming of DCs. Thetranscription factor Myc is an established regulator of cell metab-olism, especially glycolytic activity, and plays a key role in metabolicreprogramming of activated T cells (4). We hypothesized that theincreased metabolic activity of Tsc1-deficient DCs is partly de-pendent upon Myc. Indeed, Myc expression was up-regulated inTsc1-deficient BM-derived CD11c+ DCs or freshly isolated DCsfollowing in vivo tamoxifen treatment (Fig. 5A). To determine thefunctional relevance of Myc in Tsc1-mediated control of DCmetabolism, we generated Tsc1flox/floxMycflox/floxRosa26-Cre-ERT2
(Tsc1/MycCreER) mice. Remarkably, loss of Myc nearly completelyblocked elevated glycolysis and de novo lipid synthesis in Tsc1-deficient DCs, even though Myc deficiency alone did not causemajor alteration of DC metabolism (Fig. 5 B and C). This rescueeffect was associated with the restoration of glycolytic andlipogenic gene expression in DCs deficient in both Tsc1 and Myc(Fig. 5D). Moreover, the increased cell size and CD71 and CD98expression in Tsc1-deficient DCs were partly rescued by the si-multaneous loss of Myc (Fig. 5E). Therefore, the aberrant in-duction of Myc in Tsc1-deficient DCs accounted, at least partially,for the dysregulated growth and metabolism.We next evaluated the extent to which Myc-dependent me-
tabolism contributes to DC development. Importantly, the ex-acerbated apoptosis in Tsc1-deficient DCs was partly blocked byMyc deficiency (Fig. 5F). This blocking was associated with thepartial rescue of Bcl-2 (Fig. 5G) and Bim dysregulation (Fig.S5A), although ROS production was not corrected (Fig. S5B). Incontrast, Myc deletion alone diminished DC proliferation, andDCs developed in the absence of both molecules were still de-fective in proliferation and total DC production (Fig. 5 H and I).Thus, aberrant induction of Myc contributes to defective survivalbut not proliferation in Tsc1-deficient DCs. Further, Myc de-letion partially blocked the spontaneous maturation of DCslacking Tsc1, as the increased expression of the maturation markers
CD86, CD80, and CD40 in these cells was down-regulated by thedeletion of Myc (Fig. 5J). Analysis of BM chimeras furthershowed that Myc deficiency partially blocked the aberrant up-regulation of CD86 on Tsc1-deficient DCs in vivo (Fig. S5C).Finally, the increased F4/80 and Ly6G expression in the absenceof Tsc1 was partly rescued by simultaneous loss of Tsc1 and Myc(Fig. 5K). Taken together, Myc plays a key role in mediating thedysregulated metabolism and contributes to the impaired sur-vival and maturation of Tsc1-deficient DCs. However, othereffects, such as the defective proliferation and in vivo differen-tiation in Tsc1-deficient cells, occur independent of Myc.
Rapamycin-Sensitive mTORC1 Signaling Contributes to Tsc1-DeficientDC Defects. We investigated the signaling mechanisms by whichTsc1 controls DC development. Although Tsc1 is known tonegatively regulate mTORC1 (15), it also regulates mTORC2activity (48). Indeed, the defective survival of Tsc1-deficientT cells is insensitive to rapamycin treatment in vitro (49), and hasbeen suggested to be ascribed to diminished mTORC2 activity(50). To determine the biochemical functions of Tsc1 in DCs, wefirst used immunoblotting to examine canonical targets ofmTORC1, namely the phosphorylation of S6, S6K1, and 4EBP1(15). DCs developed in the absence of Tsc1 showed increasedactivities of these mTORC1 targets (Fig. 6A). In contrast, thephosphorylation of Akt (Ser473), which is a target of mTORC2,was diminished in the mutant cells (Fig. 6A). Thus, Tsc1 serves asa negative regulator of mTORC1 in DCs but promotes mTORC2activity. To further examine the role of Tsc1 in restrainingmTORC1 activity at the single-cell level, we used flow cytometryto measure phosphorylation of S6 at different time points of DCdifferentiation. Increased phosphorylation of S6 was observed at4 d after the initiation of 4-OHT treatment (Fig. 6B), which waslargely in agreement with the timing of Tsc1 RNA deletion (Fig.S1A). Moreover, in vivo tamoxifen-treated Tsc1CreER splenic DCshad higher levels of S6 activation than WT controls (Fig. 6C).Similarly, DCs derived from Tsc1CreER BM donor cells in mixedchimeras also had increased p-S6 than those derived from WTBM or spike BM cells (Fig. 6D). These results indicate that Tsc1plays an important role in inhibiting mTORC1 activation in DCsin vitro and in vivo.To determine whether elevated mTORC1 activation contrib-
utes to the defects in Tsc1-deficient DCs, we treated BM cells invitro with the mTORC1 inhibitor rapamycin. As expected, rapa-mycin blocked the elevated mTORC1 activity in Tsc1-deficient
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Fig. 4. Loss of Tsc1 up-regulates cell growth and mul-tiple metabolic programs including glycolysis, mito-chondrial respiration, and lipid biosynthesis. (A) Cell sizeof splenic DCs from spike BM and WT or Tsc1CreER donorBM-derived cells in the mixed chimeras (generated asdescribed in Fig. 1F). FSC, forward scatter. (B) Size ofCD11c+ cells derived from 4-OHT–treated WT or Tsc1CreER
total BM cells (Left), LSKs (Center), or MDPs (Right). (C–J)BM cells from WT or Tsc1CreER mice were cultured withFLT3L and 4-OHT, followed by analysis of CD71 and CD98expression (C), glycolytic activity through measurementof the generation of 3H2O from [3-3H]glucose (DPM,disintegrations per minute) (D), extracellular acidifica-tion rate (mpH, milli-pH units) (E), mRNA expression ofglycolysis-related genes (F), oxygen consumption rate(G), mitochondria mass as determined by MitoTrackerGreen (H), de novo lipogenesis assay (I), and mRNA ex-pression of lipogenic genes (J). Error bars indicate SEM.Data are representative of two to five independentexperiments.
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DCs (Fig. 6E). Whereas rapamycin treatment had a modest effectin reducing DC production from WT cells, it largely restored thepercentage and total number of Tsc1-deficient DCs (Fig. 6F).Rapamycin also blocked excessive expression of CD86, CD80, andCD40 (Fig. 6G), and rescued the aberrant induction of F4/80 andLy6G in the mutant cells (Fig. 6H). At the cellular and molecularlevels, rapamycin blocked the up-regulation of apoptosis, cell size,ROS production, and mitochondrial mass (Fig. 6I), as well as Mycexpression (Fig. 6J), as observed in Tsc1-deficient DCs. Takentogether, the increased mTORC1 activity is required to drive thedevelopmental and metabolic defects of Tsc1-deficient DCs.One caveat remains, however, because the extended treatment of
rapamycin can also inhibit mTORC2 activity (31, 32). To determinethe effects of mTORC2 on DC development, we used a similarstrategy as Tsc1 deletion and generated Rictorflox/floxRosa26-Cre-ERT2 mice (RictorCreER), which allowed us to acutely delete Rictor,the mTORC2-defining component. BM cells with deletion of Rictorin vitro developed normally into DCs, although total DC numbertrended lower (Fig. S6A). This finding is in agreement with a recentstudy showing a dispensable role of Rictor in GM-CSF–derived DCdevelopment (29). The mutant DCs showed comparable cell death,cell size, and expression of DC maturation markers as WT cells(Fig. S6 B–D). Therefore, deficiency of Rictor did not alter DCdevelopment, suggesting that loss of mTORC2 activity in Tsc1-deficient DCs is insufficient to drive the disrupted immune homeo-stasis. These results collectively indicate that aberrant activation ofmTORC1 largely accounts for impaired DC development.
Tsc1 Signals Through Rheb to Control mTORC1 and DC Development.One of the important inputs to mTORC1 is mediated by thesmall G protein Rheb, although Rheb-independent pathways
have also been identified (17). For example, the kinase AMPKcan directly phosphorylate Raptor to mediate a metaboliccheckpoint (51). Having established a role for Tsc1 in DC de-velopment via mTORC1 inhibition, we next determined whetherthis effect is mediated by Rheb. To this end, we used the Rosa26-Cre-ERT2 system and generated mice lacking both Tsc1 andRheb (Tsc1/RhebCreER). We cultured BM cells from the mutantmice and proper controls with FLT3L and examined DC gen-eration following acute deletion in vitro. The diminished gen-eration of DCs in the absence of Tsc1 was restored to WT levelsby the simultaneous loss of Rheb (Fig. 7A). We then determinedthe effects of Rheb deficiency on the altered apoptosis andproliferation of Tsc1-deficient DCs. The increased apoptosis ofTsc1-deficient DCs was nearly completely restored in Tsc1/Rheb-deficient DCs (Fig. 7B), associated with the reversal ofROS overproduction and dysregulated expression of Bcl-2 familyproteins (Fig. 7 C and D). Additionally, the defective pro-liferation of Tsc1-deficient DCs was similarly rescued by the lossof Rheb (Fig. 7E). Furthermore, the altered maturation markersas observed in Tsc1-deficient DCs were restored to WT levels inTsc1/Rheb-deficient cells (Fig. 7F). These results indicate thatTsc1 signals to mTORC1 inhibition in a strictly Rheb-dependentmanner. Surprisingly, deficiency of Rheb alone did not havea strong effect on DC development, survival, or proliferation(Fig. 7 A–F). Thus, whereas an excessive Rheb-mTORC1 activityis detrimental to DC generation, loss of function of this pathwayappears to be compatible with DC development.Given the importance of cell growth and metabolism in DC
development, we further measured the role of the Tsc1–Rhebaxis in this process. The increased cell growth and nutrient receptorexpression in Tsc1-deficient DCs were blocked by the simultaneous
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Fig. 5. Tsc1 controls metabolic programming of DCsthrough inhibition of Myc expression. (A) Immunoblotanalysis of Myc expression in BM-derived CD11c+ cellsfrom WT or Tsc1CreER mice cultured with FLT3L and 4-OHT(Left) or freshly isolated splenic CD11c+ cells from tamox-ifen-treated WT or Tsc1CreER mice (Right). (B–D) BM cellsfrom WT, Tsc1CreER, MycCreER, or Tsc1/MycCreER mice werecultured with FLT3L and 4-OHT, followed by analyses ofmetabolic parameters including glycolysis (B), de novo li-pogenesis (C), and mRNA expression of glycolytic andlipogenic genes (D). (E–K) BM cells from the indicatedmice were cultured with FLT3L and 4-OHT, followed byanalysis of CD11c+ cells for cell size and CD71 and CD98expression (E), Annexin V and 7-AAD staining (F), ex-pression of Bcl-2 (G), cell proliferation as determined byBrdU incorporation (H), DC production (input BM cells, 3 ×106) (I), expression of maturation markers (J), and ex-pression of F4/80 and Ly6G (K). NS, not significant. Errorbars indicate SEM. Data are representative of two or threeindependent experiments.
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loss of Rheb (Fig. 7G). Further, the elevated mRNA expressionof glycolytic (Hk2, Ldha, and Tpi1) and lipogenic genes (Hmgcs1,Hmgcr, and Scd2), as observed in Tsc1-deficient DCs, wascompletely restored in Tsc1/Rheb-deficient cells (Fig. 7H). Fi-nally, Rheb deficiency restored the aberrant induction of p-S6and p-4EBP1 observed in Tsc1-deficient cells (Fig. 7 I and J).We next explored how mTORC1 activity is regulated during DC
development. We cultured BM cells with FLT3L and examinedphosphorylation of S6 and 4EBP1 at different time points. Phos-phorylation of these molecules reached a high level at day 5 andthen abated (Fig. 7K). Importantly, the cell size and glycolytic andlipogenic activities followed a similar biphasic pattern of regulation,with the highest levels observed at days 3–5 (Fig. 7 L–N). Therefore,mTORC1 activity and mTORC1-dependent metabolism are dy-namically regulated during DC development, whereas uncontrolledmTORC1 activation impairs DC development (Fig. 7O).
DiscussionA fundamental question in immunology is how the developmentof DCs is regulated by cellular and molecular processes. Therecent identification of distinct DC precursors and transcriptionfactors orchestrating differentiation of DCs and various subsetshas revolutionized our understanding of DC biology (33–35). Incontrast, we have limited information on how intracellular sig-naling networks program DC development. Moreover, whereasemerging evidence highlights a role of cell metabolism in the
activation of innate and adaptive immunity (1–3), its involvementin the development of immune cells, and in particular DCs,remains undefined. Here we describe that the interplay betweenTsc1-mTORC1 signaling and Myc-dependent metabolism orches-trates a previously unappreciated metabolic checkpoint to controlDC development. Surprisingly, loss of Tsc1 and the ensuingmTORC1 up-regulation disrupt development of DCs by impairingtheir survival, proliferation, and differentiation. These defects impairTH1 effector responses, despite the elevated expression of mat-urationmarkers.Mechanistically, Tsc1 controls DC developmentin part by actively repressing Myc-mediated metabolic programs,especially glycolysis and lipid synthesis. This process requiresTsc1 to signal through Rheb to inhibit mTORC1 activity. Ourresults therefore identify Tsc1-Rheb-mTORC1 as a central path-way to program DC development by mediating the interplay be-tween immune signaling and Myc-mediated metabolic programs.Emerging evidence from mouse genetic models highlights
a role of mTOR in DC development and activation. Specifically,deletion of Pten facilitates FLT3L but not GM-CSF–driven DCdevelopment in vitro and the expansion of CD8+ DCs in vivo(27). Further, deficiency of Tsc1 has been recently shown toimpair TLR-induced activation and maturation of DCs derivedfrom GM-CSF, although Tsc1 loss does not affect DC de-velopment in response to GM-CSF (52) or DC terminal differ-entiation or maintenance (27). In contrast, our results haveidentified a detrimental effect of Tsc1 deficiency on the gener-ation of DCs in vivo and in response to FLT3L stimulation in
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Fig. 6. Rapamycin-sensitive mTORC1 signaling contributes to Tsc1-deficient DC defects. (A) BM cells from WT or Tsc1CreER mice were cultured with FLT3L and4-OHT, followed by immunoblot analysis of p-S6, p-S6K, p-4EBP1, and p-AKT (Ser473) in purified CD11c+ cells. (B) BM cells from WT or Tsc1CreER mice werecultured with FLT3L and 4-OHT, followed by flow cytometry analysis of p-S6 at the indicated time points of DC differentiation. (C and D) Phosphorylation of S6was determined in splenic DCs from tamoxifen-treated WT or Tsc1CreER mice (C) or from WT or Tsc1CreER donor BM-derived cells (Left) and spike BM cells(Right) in the mixed chimeras (generated as described in Fig. 1F) (D). (E–J) BM cells from WT or Tsc1CreER mice were cultured with FLT3L and 4-OHT in thepresence of rapamycin or control, followed by analyses of phosphorylation of S6 (E), CD11c+ DC percentage (Left) and number (Right) (input BM cells, 2 × 106)(F), maturation marker expression (G), F4/80 and Ly6G expression (H), Annexin V staining, cell size, ROS production, and mitochondrial mass (I), and Mycexpression (the numbers below the lanes indicate the band intensity relative to that of the loading control β-actin) (J). Error bars indicate SEM. Data arerepresentative of two to four independent experiments.
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vitro, thereby highlighting an intrinsic role of Tsc1 in DC de-velopment. The simplest interpretation is that the mTOR regu-lators exert pathway- and context-specific regulation of DCbiology. For instance, Pten is an inhibitor of both mTORC1 andmTORC2 activities by repressing upstream PI3K activity (17).Pten also has functions independent of PI3K/mTOR, such asacting in the nucleus to maintain chromosomal stability (53). Incontrast, loss of Tsc1 up-regulates mTORC1 but diminishesmTORC2 activity, although the reduction of mTORC2 activityalone (as achieved by Rictor deletion) is not sufficient to alterDC development. Consistent with a dominant role of mTORC1in this process, the defect in Tsc1-deficient DCs can be rescuedby rapamycin and, more importantly, by Rheb deficiency. Fur-thermore, deletion of Pten but not Tsc1 using CD11c-Cre miceaffects homeostasis of DCs (27). These results collectively sup-port an important but complex role of mTORC1 signaling inDC development.Aside from a reduction in overall DC populations, DCs that
were able to develop in the absence of Tsc1 (as indicated by thenormal expression of DC-specific transcripts) aberrantly up-regulate markers for macrophages and neutrophils. These resultsindicate that Tsc1 deficiency results in a loss of DC de-velopmental integrity by failing to repress diversion into alter-native lineages. Further, these mutant DCs undergo spontaneousmaturation, as shown by the excessive induction of costimulatorymolecules, especially CD86, that was obvious in Tsc1-deficientDCs in vivo and in vitro. This finding is in agreement with theobservation that rapamycin treatment during DC developmentreduces DC maturation (19, 28, 29), thereby providing geneticevidence for a role of mTORC1 in promoting DC maturation.Despite the increased CD86 expression, DCs developed in the
absence of Tsc1 were unable to effectively mediate the differ-entiation of TH1 cells, and such a functional loss is associatedwith aberrant lineage development. Therefore, the aberrantdifferentiation of Tsc1-deficient DCs, likely acting in synergywith the survival defect and numerical reduction, contributes tothe loss of DC functional fitness, further highlighting the im-portance of active control of mTORC1 activity for DC de-velopment and function.We further link Tsc1-mTORC1 functions to metabolic pro-
gramming of DC development. Remarkably, loss of Tsc1 resultsin extensive changes of multiple metabolic programs. First, DCsdeveloped in the absence of Tsc1 up-regulate glycolysis and ex-pression of key glycolytic enzymes. Second, the mutant cells showincreased mitochondrial metabolism as shown by increased rateof mitochondrial respiration, excessive ROS production, andelevated mitochondrial mass. Third, lipid synthesis is markedlyincreased as well, associated with up-regulated genes in bothfatty acid and cholesterol metabolic pathways. Further, the dys-regulated metabolism in Tsc1-deficient cells is contingent uponRheb function, thereby highlighting an important role of Tsc1-Rheb-mTORC1 to coordinately regulate metabolic programsduring DC development. However, DC development proceedsnormally in the absence of Rheb, suggesting the presence ofa Rheb-independent pathway [e.g., the compensation from thehomolog Rhebl1/Rheb2 (54) or a distinct GTPase that is yet tobe identified] to mediate mTORC1 activation during early DCdevelopment. Importantly, expression of the key metabolictranscription factor Myc is enhanced in Tsc1-deficient cells, anddeletion of Myc has a marked effect to block the dysregulatedmetabolism and also partly rectifies abnormal DC survival andmaturation. These results point to a unique metabolic check-
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Fig. 7. Tsc1 signals through Rheb to controlmTORC1 activation in DC development. (A–D) BM cells from WT, Tsc1CreER, RhebCreER, orTsc1/RhebCreER mice were cultured withFLT3L and 4-OHT, followed by analysis ofCD11c+ DC number (input BM cells, 2 × 106)(A), Annexin V and 7-AAD staining (B), ROSproduction (C), and immunoblot analysis ofBim, Puma, and Bcl-2 in purified CD11c+ DCs(D). (E) BM cells from WT or Tsc1CreER micewere labeled with CFSE and cultured withFLT3L and 4-OHT, followed by analysis ofCFSE dilution at day 7.5. (F–J) BM cells werecultured with FLT3L and 4-OHT, followedby analysis of CD11c+ DCs for maturationmarker expression (F), cell size and CD71and CD98 expression (G), mRNA analysis ofglycolytic and lipogenic genes (H), immu-noblot of p-S6, p-S6K, and p-4EBP1 (I), andintracellular staining of p-S6 (J). (K–N) WTBM cells were cultured with FLT3L for theindicated times, followed by analysis of p-S6by immunoblot (the numbers below thelanes indicate the band intensity relative tothat of the loading control β-actin) (K), cellsize (L), glycolysis (M), and de novo lipo-genesis (N). Error bars indicate SEM. Data arerepresentative of two or three independentexperiments. (O) The interplay between Tsc1-Rheb-mTORC1 signaling and Myc-dependentmetabolic programming in DC development.
Wang et al. PNAS | Published online November 26, 2013 | E4901
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point mediated by the interplay between Tsc1-Rheb-mTORC1signaling and Myc-dependent bioenergetic and biosynthetic ac-tivities that actively orchestrates DC development. Our studyhas established a key metabolic checkpoint in immune cell de-velopment, and this is reminiscent of the recent identification ofan Lkb1-dependent but mTORC1-independent metabolic con-trol mechanism required for HSC development (55–57).Despite the general role of mTORC1 and metabolism in
promoting cell proliferation, we had an unexpected finding thatDCs developed in the absence of Tsc1 were impaired in con-tinuous proliferation whereas the initial proliferation was nor-mal. Additionally, this effect was independent of Myc. In fact,deficiency of Myc alone reduced DC proliferation. Although theprecise mechanism remains to be established, we noticed thatdeletion of another mTOR inhibitor, Pten, depletes HSCs byinducing the expression of p53 and other tumor suppressors (58).We speculate that the aberrant cell growth and metabolism dueto mTORC1 hyperactivation in the absence of Tsc1 inducesmetabolic stress that, once accumulated, leads to cell-cycle arrestand apoptosis induction, possibly via a tumor suppressor re-sponse. Future work is warranted to identify the molecularcomponents involved.In summary, we have identified a unique metabolic checkpoint
crucial for DC development (Fig. 7O). Although emerging evi-dence highlights a role of glucose metabolism in the activation ofinnate immunity, especially in response to TLR stimulation (11–14), whether cell metabolism contributes to DC developmentremains unclear. Similarly, whereas the role of mTOR signaling,including the Tsc1/Tsc2 pathway, in TLR responses in macro-phages, DCs, and other cells is beginning to be recognized (20,52, 59), its involvement in the development of DCs has not beenappreciated. Given the evolutionarily conserved function ofmTOR signaling, we speculate that mTORC1-dependent meta-bolic programming of DC development represents a previouslyunappreciated paradigm of metabolic control of immune celldevelopment that can be applied to the generation of othermyeloid cells and additional immune lineages.
Materials and MethodsMice and BM Chimeras. C57BL/6, CD45.1, Thy1.1, and OT-II mice were pur-chased from The Jackson Laboratory. Tsc1flox/flox,Mycflox/flox, Rhebflox/flox, andRictorflox/flox mice were bred with Rosa26-Cre-ERT2 mice, and had beenbackcrossed to the C57BL/6 background for at least eight generations. WTcontrols were Cre+ mice in the same genetic background to account for Creeffects. For in vivo tamoxifen treatment, WT or Tsc1CreER mice were injectedi.p. with 2 mg tamoxifen (Sigma) per mouse for 5 consecutive days andrested for 5–10 d before analyses. For mixed-BM experiments, BM cells fromin vivo tamoxifen-treated WT or Tsc1CreER (CD45.2.2+) mice were mixed withcells from CD45.1.2+ mice at a 1:1 ratio and transferred into lethally irradi-ated (11 Gy) CD45.1.1+ mice, as described previously (60). For complete BMexperiments, BM cells from tamoxifen-treated WT or Tsc1CreER CD45.2.2+
mice were transferred into lethally irradiated CD45.1.1+ mice. Animal pro-tocols were approved by the Institutional Animal Care and Use Committeeof St. Jude Children’s Research Hospital.
Cell Purification and Cultures. Mouse spleens were digested with CollagenaseD (Roche), and DCs (CD11c+TCR–CD19–DX5–), CD8+ cDCs (CD11c+CD8+CD11b–TCR–
CD19–DX5–), CD11b+ cDCs (CD11c+CD11b+CD8–TCR–CD19–DX5–), or pDCs(CD11c+mPDCA-1+TCR–CD19–DX5–) were sorted on a Reflection (iCyt). DCs fromnonlymphoidorganswere isolatedasdescribed (61). Lymphocyteswere sorted fornaïve T cells (CD4+CD62LhiCD44loCD25–). For DC culture, BM cells (2–4 × 106) werecultured in RPMI-1640 medium containing 10% (vol/vol) FBS and mouseFLT3L (200 ng/mL), in the presence of 4-hydroxytamoxifen (0.5 μM) whenindicated. Unless otherwise noted, FLT3L-stimulated cells were analyzed atdays 7–8 for DC generation or at days 5–6 for metabolic or molecularmechanisms. For labeling with CFSE (Life Technologies), BM cells were in-cubated in RPMI-1640 mediumwith 5% (vol/vol) FBS and 4 μM CFSE at 37 °Cfor 25 min, followed by extensive washes. The BM precursor populationswere isolated as follows: LSK cells were defined as Lin– (CD3, CD45R, Ter119,CD11b, and Gr1) Sca-1hic-Kithi, and MDPs as Lin–Sca-1–CSF1R+. Rapamycin(50 nM) was added from day 0 of culture. For DC and T-cell cocultures, BMcells were culturedwith FLT3L and 4-OHT for 7 d and sorted for CD11c+ DCs,
which were then mixed with naïve OT-II T cells at a 1:10 ratio in the pres-ence of OVA323–339 peptide and 100 ng/mL LPS; alternatively, freshly iso-lated DCs from spleen were used for cocultures with T cells. After 5–6 d ofculture, live T cells were collected and stimulated with PMA (phorbol 12-myristate 13-acetate) and ionomycin for intracellular cytokine staining.
Antigen Challenge. Antigen-specific T cells from OT-II TCR-transgenic mice(Thy1.1+) were sorted and transferred into complete WT or Tsc1CreER chimeramice. Twenty-four hours later, the mice were injected s.c. with OVA323–339
(100 μg) in the presence of complete Freund’s adjuvant (CFA; Difco). At day 7after immunization, draining lymph node cells were isolated and stimulatedwith PMA and ionomycin for intracellular cytokine staining.
Flow Cytometry. Flow cytometry was performed as described previously (61,62). For intracellular cytokine detection, cells were stimulated for 5 h withPMA and ionomycin in the presence of monensin before staining, accordingto the manufacturer’s instructions (BD Biosciences). For caspase activity de-tection, cells were stained with FITC-VAD-FMK according to the manu-facturer’s instructions (Promega). ROS was measured by incubation withCM-H2DCFDA (10 μM; Invitrogen) at 37 °C for 30 min after staining of sur-face markers. To stain mitochondria, lymphocytes were incubated withMitoTracker Green (20 nM; Invitrogen) at 37 °C for 20 min after staining ofsurface markers. BrdU labeling was performed according to the manu-facturer’s instructions (BD Biosciences) (41). For detection of phosphory-lated signaling proteins, cells were fixed with Phosflow Lyse/Fix Buffer,followed by permeabilization with Phosflow Perm Buffer III (BD Biosciences)and staining with antibodies to S6 phosphorylated at Ser235 and Ser236(D57.2.2E; Cell Signaling Technology). Bim staining was done accordingto the manufacturer’s instructions (Cell Signaling Technology). Flowcytometry data were acquired on an LSR II or LSR Fortessa (BD Biosciences)and analyzed using FlowJo software (Tree Star). Precursor populations weregated as follows: MDPs, Lin– (CD3–, CD45R–, Ter119–, CD11b–, and Gr1–)CD11c−Sca-1–CSF1R+; CDPs, Lin−CD11c−Sca-1−CSF1R+Flt3+CD117lo; and precDCs,Lin−MHC-II−CD11c+Flt3+CD172αint.
De Novo Lipogenesis, Glycolysis, and Bioenergetics Assays. Bone marrow cul-tures were incubated with 4 μCi/mL [1-14C]acetic acid (PerkinElmer) for 4 h.Following incubation, cells were collected, washed twice with PBS, and lysedin 0.5% (vol/vol) Triton X-100. Lipids were extracted by the addition ofa chloroform and methanol mixture (2:1, vol/vol) with vortexing, followedby the addition of water with vortexing. After centrifugation (350 × g,15 min), the lipid-containing phase (at the bottom) was separated and 14Cincorporation was measured using a Beckman LS6500 scintillation counter.Glycolytic flux was determined by measuring the detritiation of [3-3H]glucose,as we have previously described (5). The bioenergetic activities of the ECARand OCR pathways were measured using the Seahorse XF24-3 ExtracellularFlux Analyzer per the manufacturer’s instructions (Seahorse Bioscience).
RNA and Protein Analyses. Real-time PCR analysis was performed withprimers and probe sets from Applied Biosystems, or using Power SYBRGreen Master Mix from Life Technologies. The cycling threshold value ofthe endogenous control gene (HPRT or actin) was subtracted from thecycling threshold value of each target gene to generate the change incycling threshold (ΔCT). The relative expression of each target gene isexpressed as the fold change relative to that of WT samples (2−ΔΔCT), asdescribed (61, 62), and the values of biological replicates are presented.Immunoblotting was performed as described (61, 62) using the followingantibodies: p-S6, p70 S6K (p-S6K), p-AKT (Ser473), p-4EBP1, Myc (all fromCell Signaling Technology), Bcl-2 (Santa Cruz), Bim (Abcam), Puma, andβ-actin (both from Sigma). Protein concentration was measured by the BCAProtein Assay Kit (Thermo Scientific Pierce), and RNA concentration wasmeasured with a NanoDrop spectrophotometer.
Statistical Analysis. P values were calculated using Student t test or ANOVA(GraphPad Prism). P values of less than 0.05 were considered significant. Allerror bars in the graphs represent SEM.
ACKNOWLEDGMENTS. We acknowledge D. Green for Mycflox mice, C. Cloerand N. Brydon for animal colony management, and the St. Jude ImmunologyFACS core facility for cell sorting. This work was supported by the NationalInstitutes of Health (Grants R21 AI094089, R01 NS064599, and R01 AI101407;to H.C.), a research grant from the National Multiple Sclerosis Society (RG4691-B-2; to H.C.), and the American Lebanese Syrian Associated Charities (H.C.).
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1. Wang R, Green DR (2012) Metabolic checkpoints in activated T cells. Nat Immunol13(10):907–915.
2. MacIver NJ, Michalek RD, Rathmell JC (2013) Metabolic regulation of T lymphocytes.Annu Rev Immunol 31:259–283.
3. Pearce EL, Pearce EJ (2013) Metabolic pathways in immune cell activation and qui-escence. Immunity 38(4):633–643.
4. Wang R, et al. (2011) The transcription factor Myc controls metabolic reprogrammingupon T lymphocyte activation. Immunity 35(6):871–882.
5. Shi LZ, et al. (2011) HIF1alpha-dependent glycolytic pathway orchestrates a metaboliccheckpoint for the differentiation of TH17 and Treg cells. J Exp Med 208(7):1367–1376.
6. Michalek RD, et al. (2011) Cutting edge: Distinct glycolytic and lipid oxidative meta-bolic programs are essential for effector and regulatory CD4+ T cell subsets. J Immunol186(6):3299–3303.
7. Bensinger SJ, et al. (2008) LXR signaling couples sterol metabolism to proliferation inthe acquired immune response. Cell 134(1):97–111.
8. Kidani Y, et al. (2013) Sterol regulatory element-binding proteins are essential for themetabolic programming of effector T cells and adaptive immunity. Nat Immunol14(5):489–499.
9. Pearce EL, et al. (2009) Enhancing CD8 T-cell memory by modulating fatty acid me-tabolism. Nature 460(7251):103–107.
10. van der Windt GJ, et al. (2012) Mitochondrial respiratory capacity is a critical regulatorof CD8+ T cell memory development. Immunity 36(1):68–78.
11. Everts B, et al. (2012) Commitment to glycolysis sustains survival of NO-producinginflammatory dendritic cells. Blood 120(7):1422–1431.
12. Krawczyk CM, et al. (2010) Toll-like receptor-induced changes in glycolytic metabo-lism regulate dendritic cell activation. Blood 115(23):4742–4749.
13. Masters SL, et al. (2010) Activation of the NLRP3 inflammasome by islet amyloidpolypeptide provides a mechanism for enhanced IL-1β in type 2 diabetes. Nat Im-munol 11(10):897–904.
14. Tannahill GM, et al. (2013) Succinate is an inflammatory signal that induces IL-1βthrough HIF-1α. Nature 496(7444):238–242.
15. Laplante M, Sabatini DM (2012) mTOR signaling in growth control and disease. Cell149(2):274–293.
16. Düvel K, et al. (2010) Activation of a metabolic gene regulatory network downstreamof mTOR complex 1. Mol Cell 39(2):171–183.
17. Chi H (2012) Regulation and function of mTOR signalling in T cell fate decisions. NatRev Immunol 12(5):325–338.
18. Powell JD, Pollizzi KN, Heikamp EB, Horton MR (2012) Regulation of immune re-sponses by mTOR. Annu Rev Immunol 30:39–68.
19. Thomson AW, Turnquist HR, Raimondi G (2009) Immunoregulatory functions ofmTOR inhibition. Nat Rev Immunol 9(5):324–337.
20. Weichhart T, et al. (2008) The TSC-mTOR signaling pathway regulates the innate in-flammatory response. Immunity 29(4):565–577.
21. Ohtani M, et al. (2008) Mammalian target of rapamycin and glycogen synthase kinase3 differentially regulate lipopolysaccharide-induced interleukin-12 production indendritic cells. Blood 112(3):635–643.
22. Schmitz F, et al. (2008) Mammalian target of rapamycin (mTOR) orchestrates thedefense program of innate immune cells. Eur J Immunol 38(11):2981–2992.
23. Haidinger M, et al. (2010) A versatile role of mammalian target of rapamycin in hu-man dendritic cell function and differentiation. J Immunol 185(7):3919–3931.
24. Ohtani M, et al. (2012) Cutting edge: mTORC1 in intestinal CD11c+ CD11b+ dendriticcells regulates intestinal homeostasis by promoting IL-10 production. J Immunol188(10):4736–4740.
25. Cao W, et al. (2008) Toll-like receptor-mediated induction of type I interferon inplasmacytoid dendritic cells requires the rapamycin-sensitive PI(3)K-mTOR-p70S6Kpathway. Nat Immunol 9(10):1157–1164.
26. Amiel E, et al. (2012) Inhibition of mechanistic target of rapamycin promotes den-dritic cell activation and enhances therapeutic autologous vaccination in mice.J Immunol 189(5):2151–2158.
27. Sathaliyawala T, et al. (2010) Mammalian target of rapamycin controls dendritic celldevelopment downstream of Flt3 ligand signaling. Immunity 33(4):597–606.
28. Turnquist HR, et al. (2010) mTOR and GSK-3 shape the CD4+ T-cell stimulatory anddifferentiation capacity of myeloid DCs after exposure to LPS. Blood 115(23):4758–4769.
29. Rosborough BR, et al. (2013) Murine dendritic cell rapamycin-resistant and rictor-independent mTOR controls IL-10, B7-H1, and regulatory T-cell induction. Blood121(18):3619–3630.
30. Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J (2008) Rapamycin differentially inhibitsS6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation. ProcNatl Acad Sci USA 105(45):17414–17419.
31. Sarbassov DD, et al. (2006) Prolonged rapamycin treatment inhibits mTORC2 assemblyand Akt/PKB. Mol Cell 22(2):159–168.
32. Delgoffe GM, et al. (2011) The kinase mTOR regulates the differentiation of helper Tcells through the selective activation of signaling by mTORC1 and mTORC2. Nat Im-munol 12(4):295–303.
33. Liu K, et al. (2009) In vivo analysis of dendritic cell development and homeostasis.Science 324(5925):392–397.
34. Liu K, Nussenzweig MC (2010) Origin and development of dendritic cells. ImmunolRev 234(1):45–54.
35. Merad M, Sathe P, Helft J, Miller J, Mortha A (2013) The dendritic cell lineage: On-togeny and function of dendritic cells and their subsets in the steady state and theinflamed setting. Annu Rev Immunol 31:563–604.
36. Chen C, et al. (2008) TSC-mTOR maintains quiescence and function of hematopoieticstem cells by repressing mitochondrial biogenesis and reactive oxygen species. J ExpMed 205(10):2397–2408.
37. Gan B, et al. (2008) mTORC1-dependent and -independent regulation of stemcell renewal, differentiation, and mobilization. Proc Natl Acad Sci USA 105(49):19384–19389.
38. Fogg DK, et al. (2006) A clonogenic bone marrow progenitor specific for macro-phages and dendritic cells. Science 311(5757):83–87.
39. Varol C, et al. (2007) Monocytes give rise to mucosal, but not splenic, conventionaldendritic cells. J Exp Med 204(1):171–180.
40. Waskow C, et al. (2008) The receptor tyrosine kinase Flt3 is required for dendritic celldevelopment in peripheral lymphoid tissues. Nat Immunol 9(6):676–683.
41. Yang K, Neale G, Green DR, He W, Chi H (2011) The tumor suppressor Tsc1 enforcesquiescence of naive T cells to promote immune homeostasis and function. Nat Im-munol 12(9):888–897.
42. Chen M, Wang J (2010) Programmed cell death of dendritic cells in immune regula-tion. Immunol Rev 236(1):11–27.
43. Joffre O, Nolte MA, Spörri R, Reis e Sousa C (2009) Inflammatory signals in dendriticcell activation and the induction of adaptive immunity. Immunol Rev 227(1):234–247.
44. Zhu J, Yamane H, Paul WE (2010) Differentiation of effector CD4 T cell populations.Annu Rev Immunol 28:445–489.
45. Porstmann T, et al. (2008) SREBP activity is regulated by mTORC1 and contributes toAkt-dependent cell growth. Cell Metab 8(3):224–236.
46. Yecies JL, et al. (2011) Akt stimulates hepatic SREBP1c and lipogenesis through par-allel mTORC1-dependent and independent pathways. Cell Metab 14(1):21–32.
47. Kenerson HL, Yeh MM, Yeung RS (2011) Tuberous sclerosis complex-1 deficiency at-tenuates diet-induced hepatic lipid accumulation. PLoS One 6(3):e18075.
48. Huang J, Dibble CC, Matsuzaki M, Manning BD (2008) The TSC1-TSC2 complex is re-quired for proper activation of mTOR complex 2. Mol Cell Biol 28(12):4104–4115.
49. O’Brien TF, et al. (2011) Regulation of T-cell survival and mitochondrial homeostasisby TSC1. Eur J Immunol 41(11):3361–3370.
50. Zhang L, et al. (2012) TSC1/2 signaling complex is essential for peripheral naïve CD8+ Tcell survival and homeostasis in mice. PLoS One 7(2):e30592.
51. Gwinn DM, et al. (2008) AMPK phosphorylation of raptor mediates a metaboliccheckpoint. Mol Cell 30(2):214–226.
52. Pan H, et al. (2013) Critical role of the tumor suppressor tuberous sclerosis complex 1in dendritic cell activation of CD4 T cells by promoting MHC class II expression via IRF4and CIITA. J Immunol 191(2):699–707.
53. Shen WH, et al. (2007) Essential role for nuclear PTEN in maintaining chromosomalintegrity. Cell 128(1):157–170.
54. Zou J, et al. (2011) Rheb1 is required for mTORC1 and myelination in postnatal braindevelopment. Dev Cell 20(1):97–108.
55. Gan B, et al. (2010) Lkb1 regulates quiescence and metabolic homeostasis of hae-matopoietic stem cells. Nature 468(7324):701–704.
56. Gurumurthy S, et al. (2010) The Lkb1 metabolic sensor maintains haematopoieticstem cell survival. Nature 468(7324):659–663.
57. Nakada D, Saunders TL, Morrison SJ (2010) Lkb1 regulates cell cycle and energy me-tabolism in haematopoietic stem cells. Nature 468(7324):653–658.
58. Lee JY, et al. (2010) mTOR activation induces tumor suppressors that inhibit leuke-mogenesis and deplete hematopoietic stem cells after Pten deletion. Cell Stem Cell7(5):593–605.
59. Pan H, O’Brien TF, Zhang P, Zhong XP (2012) The role of tuberous sclerosis complex 1in regulating innate immunity. J Immunol 188(8):3658–3666.
60. Huang G, Wang Y, Shi LZ, Kanneganti TD, Chi H (2011) Signaling by the phosphataseMKP-1 in dendritic cells imprints distinct effector and regulatory T cell fates. Immunity35(1):45–58.
61. Wang Y, et al. (2012) Transforming growth factor beta-activated kinase 1 (TAK1)-dependent checkpoint in the survival of dendritic cells promotes immune homeostasisand function. Proc Natl Acad Sci USA 109(6):E343–E352.
62. Huang G, et al. (2012) Signaling via the kinase p38α programs dendritic cells to driveTH17 differentiation and autoimmune inflammation. Nat Immunol 13(2):152–161.
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