Dendritic cell SIRT1–HIF1α axis programs thedifferentiation of CD4+ T cells through IL-12and TGF-β1Guangwei Liua,b,1, Yujing Bic, Lixiang Xued, Yan Zhanga,b, Hui Yanga,b, Xi Chena,b, Yun Lua,b, Zhengguo Zhanga,b,Huanrong Liua,b, Xiao Wanga,b, Ruoning Wange,1, Yiwei Chua,b, and Ruifu Yangc

aKey Laboratory of Medical Molecular Virology of Ministries of Education and Health, Department of Immunology, School of Basic Medical Sciences, FudanUniversity, Shanghai 200032, China; bBiotherapy Research Center and Institute of Immunobiology, Fudan University, Shanghai 200032, China; cState KeyLaboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China; dDepartment of Biochemistry andMolecular Biology, Health Science Center, Peking University, Beijing 100191, China; and eCenter for Childhood Cancer and Blood Diseases, Hematology/Oncologyand Bone Marrow Transplantation, The Research Institute at Nationwide Children’s Hospital, The Ohio State University, Columbus, OH 43205

Edited by Shizuo Akira, Osaka University, Osaka, Japan, and approved January 28, 2015 (received for review October 24, 2014)

The differentiation of naive CD4+ T cells into distinct lineages playscritical roles in mediating adaptive immunity or maintaining im-mune tolerance. In addition to being a first line of defense, theinnate immune system also actively instructs adaptive immunitythrough antigen presentation and immunoregulatory cytokineproduction. Here we found that sirtuin 1 (SIRT1), a type III histonedeacetylase, plays an essential role in mediating proinflammatorysignaling in dendritic cells (DCs), consequentially modulating thebalance of proinflammatory T helper type 1 (TH1) cells and antiin-flammatory Foxp3+ regulatory T cells (Treg cells). Genetic deletionof SIRT1 in DCs restrained the generation of Treg cells while driv-ing TH1 development, resulting in an enhanced T-cell–mediatedinflammation against microbial responses. Beyond this finding,SIRT1 signaled through a hypoxia-inducible factor-1 alpha (HIF1α)-dependent pathway, orchestrating the reciprocal TH1 and Treg lin-eage commitment through DC-derived IL-12 and TGF-β1. Our stud-ies implicates a DC-based SIRT1–HIF1α metabolic checkpoint incontrolling T-cell lineage specification.

dendritic cells | SIRT1 | innate immunity | T-cell differentiation | HIF1α

CD4+ T cells are essential components of the adaptive im-mune system that regulate immune responses against foreign

antigen. Upon antigen recognition, naive CD4+ T cells undergoactivation and expansion, and, depending on inflammatorycontexts and cytokine milieus, differentiate into functional andphenotypic T helper (TH) subsets characterized by distinct cy-tokine production profile and function (1–3). TH1 cells produceIFN-γ and elicit cellular immunity in responding to intracellularpathogens; TH2 cells produce IL-4 and IL-5 and promote hu-moral immunity in responding to extracellular bacteria andhelminthes; and TH17 cells produce IL-17 and mediate anti-fungal defense and inflammation (4, 5). Additionally, regulatoryT cells, often known as “induced regulatory T cells” (iTreg cells),which act in synergy with naturally occurring Treg cells (nTregcells), produce IL-10 and TGF-β1 and dampen immune responseselicited from TH1, TH2, and TH17 (6–9).Dendritic cells (DCs), an essential component in the innate

immune system, play a critical role in initiating front-line primaryimmune responses and directing subsequent pathogen-specificadaptive immune responses (2). In addition to presenting anti-gens and modulating cell surface costimulatory molecules, DC-derived cytokines and chemokines can result in either a proin-flammatory or antiinflammatory environment, engaging distinctT-cell differentiation programs on naive CD4+ T cells (1, 10–16).For example, DC-producing IL-12 can support TH1 development,whereas DC-producing IL-10 or TGF-β1 can support Treg

development. Recent studies from us and others have shownthat innate signaling in DCs mediated by G protein-coupledreceptor S1P1, the mitogen-activated kinases (MAPKs), and

Wnt–β-catenin plays important roles in stimulating adaptiveimmune responses through directing native CD4+ T-cell differ-entiation (17–20). However, other critical signaling componentsin DCs that may play a role in shaping T-cell lineage engagementremain to be identified.SIRT1 is a mammalian homolog of the yeast NAD+-dependent

protein deacetylase Sirt2, and plays a role in a variety of essentialbiological processes, including cell cycle progression, apoptosis, cellsurvival, gene silencing, heterochromatin formation, tumori-genesis, metabolism, and development (21, 22). SIRT1 has alsobeen implicated in regulating immune responses. In T cells, SIRT1is required to maintain T-cell tolerance (23, 24) and also play arole in inhibiting the function of Treg cells in allograft survival (25).In myeloid cells, SIRT1 limits the inflammatory process by in-hibiting the expression of proinflammatory cytokines (26, 27),while promoting DC maturation and TH2 response in airwayallergy (28). However, whether SIRT1 is involved in bridgingthe innate immune signal to adaptive immune response remainsunexplored.Here, we found that SIRT1 plays a critical role in determining

the T-cell lineage fate by directing DC-derived cytokine pro-duction, which links innate and adaptive immune modulation.Largely through a HIF1α–dependent signaling pathway, SIRT1is required for the reciprocal production of IL-12 and TGF-β1

Significance

Naive CD4+ T cells differentiate into diverse effector and regu-latory subsets to orchestrate immunity and tolerance. Whereasthe mechanism of T-cell intrinsic signals has been extensivelystudied, how T-cell lineage differentiation is controlled by in-nate immune signals remains unknown. Here we used loss-of-function mouse systems, combined with other complementaryapproaches and models, to define the role of dendritic cell (DC)sirtuin 1 (SIRT1) as a key regulator in orchestrating the orien-tation of T-cell differentiation via HIF1α signaling in a mamma-lian target of rapamycin–independent manner. DC-expressedSIRT1, a type III histone deacetylase, programmed reciprocal Thelper 1 (TH1) and regulatory T-cell (Treg) differentiation bymodulating IL-12–STAT4 and TGF-β1–SMAD3 axes and cytokinereceptor expressions at the DC–T-cell interface.

Author contributions: G.L. and Y.B. designed research; G.L., Y.B., Y.Z., H.Y., X.C., and X.W.performed research; L.X. contributed new reagents/analytic tools; G.L., Y.B., L.X., Y.L.,Z.Z., H.L., R.W., Y.C., and R.Y. analyzed data; and G.L. and R.W. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence may be addressed. Email: liugw@fudan.edu.cn or ruoning.wang@nationwidechildren.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1420419112/-/DCSupplemental.

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production in DCs as well as the expression of IL-12Rβ2and TGF-βR2 in responding T cells, resulting in a differentiallineage engagement of TH1 and iTreg in the microbial-inducedinflammation.

ResultsSIRT1 Is Highly Expressed in DCs and the Deficiency of SIRT1 in CD11c+

Cells Does Not Change DC Homeostasis. To systematically profileSIRT1 expression in the immune system, we purified the mouseimmune cells based on surrogate cell surface markers: macro-phages (CD11b+F4/80+ cells), DCs (CD11c+MHCII+F4/80−Ly6G−NK1.1−CD19−TCR− cells), neutrophils (CD11b+ Ly6G+

cells), natural killer (NK) cells (NK1.1+ CD49b+ cells), B cells(CD19+ cells), and T cells (TCR+ cells) (SI Appendix, Fig. S1A).Subsequent qPCR and immunoblot analysis showed that SIRT1is highly expressed in DC cells (SI Appendix, Fig. S1 B–D). To gaininsights into the role of SIRT1 in DCs, we generated a CD11c+

cell-specific SIRT1-deficient mouse model by crossing SIRT1fl/fl

with CD11c-Cre (referred to as SIRT1CD11c−/− hereafter). In ad-dition to DCs, the expression of CD11c in monocytes/macrophages,neutrophils, and some B cells has also been reported (29, 30). Toexamine the specificity and efficacy of SIRT1 deletion, we iso-lated various cell populations (indicated in SI Appendix, Fig. S1A)from wild-type (WT) and SIRT1CD11c−/− mice and examined theexpression of SIRT1. We revealed a reduction of SIRT1 proteinin DCs but not in other immune cells (SI Appendix, Fig. S1C). Also,we observed a reduction of SIRT1 mRNA expression in CD11c+

DC cells but not in other immune cells isolated from SIRT1CD11c−/−

mice compared with their WT counterparts (SI Appendix, Fig.S1D). Notably, the deletion of SIRT1 in DCs is incomplete (about5-fold reduction in protein level and about 2.5-fold reduction inmRNA level). We reason that both the potential limited efficacyof CD11c-Cre and impurity of DCs (95% in purity, SI Appendix, Fig.S1A) may contribute to the residue level of SIRT1 in our experi-

ment. Nevertheless, these data suggested that the CD11c-Cre ac-tivity is largely restrained in DCs with a relatively limited efficacy.Next, we examined the composition of DC subsets in vivo and

revealed a comparable percentage and cell number of CD11c+

cells as well as CD8α+, CD11b+, and mPDCA1+ subsets in thespleen isolated from WT and SIRT1CD11c−/− mice (SI Appendix,Fig. S2A). Also, the expression of costimulatory molecules:CD80, CD86, CD40, and CD54 in CD11c+ cells isolated fromWT and Sirt1CD11c−/−mice were comparable (SI Appendix, Fig.S2B). Moreover, acute deletion of SIRT1 in bone marrow cells(Sirt1CreER model described in detail below) resulted in a simi-lar number of bone marrow-derived DCs (BMDCs) in vitro (SIAppendix, Fig. S3). These data suggested that SIRT1 deficiencydoes not affect DC development and homeostasis.

SIRT1 Signaling in DCs Is Involved in Regulating T-Cell Differentiation.As professional antigen-presenting cells (APCs), DCs presentforeign antigens with major histocompatibility complexes (MHCs)to T cells and modulate T-cell immune function. We then exam-ined whether SIRT1 regulates antigen presentation in DCs. Forthe analysis of antigen presentation in DC, we transferred naiveCD4+ T cells isolated from OT-II TCR-transgenic donors intoWT and Sirt1CD11c−/− recipients, which were subsequently im-munized with ovalbumin (OVA) and complete Freund’s adju-vant (CFA). At day 7 after immunization, draining lymph node(DLN) cells were isolated and stimulated with OVA ex vivo for3 d. CD4+ T donor cells isolated from WT and Sirt1CD11c−/−

recipients display a comparable proliferation rate, indicating asimilar capability of antigen presentation in both groups (Fig. 1A).However, our parallel assays on T-cell differentiation markersrevealed increased IFN-γ+ but not IL-17A+ or IL-4+ donor CD4+

T cells isolated from Sirt1CD11c−/− recipients (Fig. 1B). Also, theamount of secreted IFN-γ but not IL-17 was higher in donorT cells isolated from the immunized Sirt1CD11c−/− recipients

Fig. 1. DC SIRT1 is required for regulating T-cell differentiation. (A–D) Naive OT-II (Thy1.1+) T cells were transferred into WT and SIRT1CD11c−/− mice andimmunized with OVA + CFA. DLN cells were analyzed at 7 d after immunization. (A) Proliferation of DLN cells after ex vivo stimulation with OVA for 72 h.(B) Expression of IFN-γ and IL-17A in donor-derived T cells after PMA + ionomycin stimulation. (Right) The proportion of IFN-γ+ population among donor cells.(C) Secreted IFN-γ and IL-17A after stimulation with 5 μg/mL OVA for 72 h. (D, Left) The intracellular expression of Foxp3. (Middle) The proportion of Foxp3+

population among donor T cells. (Right) The mRNA expression of Foxp3 in donor-derived CD4+T cells. (E) Naive OT-II (Thy1.1+) T cells were transferred into WTand SIRT1CD11c−/− mice and then fed with OVA in the drinking water for 5 d. (Left) Foxp3+ population in donor-derived CD4+ T cells from MLN. (Middle) theproportion of Foxp3+ population among donor T cells. Right, the mRNA expression of Foxp3 in donor-derived CD4+ T cells. Data are shown as mean ± SD, n = 4,from one of three (A–D) or four (E) independent experiments. **P < 0.01 and ***P < 0.001 compared with the indicated groups.

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than from WT recipients, indicative of enhanced TH1 differ-entiation (Fig. 1C). By contrast, we observed fewer Foxp3+

cells and lower level of Foxp3 mRNA expression in T cells fromSirt1CD11c−/− recipients compared with those in WT recipients,indicative of decreased Treg differentiation (Fig. 1D). Also, weobserved similar alterations on Treg differentiation in mesentericlymph nodes (MLNs) from a model of oral antigen-induced iTreggeneration, whereby Sirt1CD11c−/− recipients were fed the antigenin the drinking water following adoptive transfer of OT-II T cells(Fig. 1E). Together, these results indicate that Sirt1 signaling inDCs may restrain inflammatory responses through modulatingT-cell differentiation.

DC SIRT1 Deletion Enhances Microbial Infection-Induced Inflammation.Next, we examined the expression of SIRT1 in DCs in respondingto various proinflammatory or antiinflammatory stimuli such asLPS, IFN-γ, TNFα, TGF-β1, and IL-10 (7). The proinflammatoryand antiinflammatory stimuli readily suppress and promoteSIRT1 expression, respectively (Fig. 2A and SI Appendix, Fig. S4).To further explore the role of SIRT1 in relevant in vivo con-

texts, we examined the pathological progression and T-cell dif-ferentiation of WT and SIRT1CD11c−/− mice in an inflammatorymodel. We challenged mice with Gram-positive bacteria Listeriamonocytogenes, a prototypic model eliciting a strong TH1 cell-polarized response (31). In this model, we found L. monocytogenesinfection resulted in more TH1 cells, comparable TH17 cells, butfewer Treg cells in splenic CD4+ T cells isolated from Sirt1CD11c−/−

mice compared with T cells isolated from WT mice, as demon-strated by FACS, qPCR, and ELISA analysis of IFN-γ, IL-17A,and Foxp3 (Fig. 2 B and C and SI Appendix, Fig. S5 A and B).Furthermore, we tested the role of DC–SIRT1 in a colitis

model, the pathogenic progression of which is largely determinedby the balance of TH1 and iTreg in vivo (32–34). For this test, we

transferred naive WT T cells (CD4+TCR+CD45RbhiCD25−)into Rag1−/− or Sirt1CD11c−/−Rag1−/− mice. Sirt1CD11c−/−Rag1−/−

recipients exhibited markedly accelerated weight loss (Fig. 2D)and pathological inflammatory injuries (Fig. 2E) compared withRag1−/− recipients. Moreover, we observed more IFN-γ+ cells,comparable IL-17+ cells, and fewer Treg cells in Sirt1CD11c−/−Rag1−/−

recipients than their WT counterparts (Fig. 2F). Altogether,our data from two different in vivo models indicate that theablation of SIRT1 in DCs results in an altered balance of TH1and Treg cells.

The Role of SIRT1 Signaling in DCs in Directing Antigen-Specific TH1and Treg Differentiation. To determine how DC–SIRT1 directsantigen-specific T-cell differentiation, we first primed DCs fromWT and Sirt1CD11c−/− with OVA and LPS, and subsequentlyinjected these cells into two groups of recipient mice, whichpreviously received naive OT-II T cells. Then, we analyzed theintracellular expression of T-cell differentiation markers in do-nor T cells 7 d after DC transfer. Evidently, Sirt1CD11c−/−DC transferinduced more IFN-γ+, comparable IL-17A+, and fewer iTreg donorT cells than those in the WT DC transferred group (Fig. 3A). Next,we extended experiments in the CD11c-Cre model to the acutedeletion model. We generated Sirt1fl/flRosa26-Cre-ERT2 mice (re-ferred to as Sirt1CreER mice hereafter) by crossing Sirt1fl/fl mice withRosa26-Cre-ERT2 mice (CreERT2 fusion gene in the ubiquitouslyexpressed Rosa26 locus). This model allows us to acutely deletetargeted gene in a wide range of cell lineages by treating mice in vivoor treating isolated cells in vitro with tamoxifen. Also, the acutedeletion of SIRT1 may avoid any potential adaptive and compen-satory effects caused by CD11c-Cre–mediated deletion, a chronicprocess started at early development stage. In addition, the as-sessment of phenotypes following the deletion of the SIRT1 inCreER system will allow us to determine whether the phenotypes

Fig. 2. Dendritic cell SIRT1 alters the differentiation of CD4+T-cell lineage against microbial-induced inflammation. (A) The protein level of SIRT1 in the DCcells following stimulation with LPS (10 ng/mL), TNFα (100 ng/mL), IL-12 (10 ng/mL), IFN-γ (50 ng/mL), IL-10 (20 ng/mL) and TGF-β1 (5 ng/mL). (B and C)SIRT1CD11c−/− mice were infected with L. monocytogenes. The intracellular staining of IFN-γ, IL-17A and Foxp3 in the spleen was determined following LLO189–201

stimulation at day 7. (D–F) T cells (CD4+TCR+CD45RbhiCD25−) were transferred into Rag1−/− and SIRT1CD11c−/−Rag1−/− mice, and body weight was measuredweekly (D) and representative colon histology (E). The intracellular staining of IFN-γ, IL-17A and Foxp3 in donor cells (CD45.1+ cells) isolated from the MLNs (F).Data (B–F) are shown as mean ± SD, from one of four (B–F) independent experiments (at least four mice per group). **P < 0.01 and ***P < 0.001 comparedwith the indicated groups.

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in CD11c-CRE are due to a CRE-specific effect. Both WT andSirt1CreER mice were pretreated with tamoxifen for 3 d, whichresulted in an efficient deletion of SIRT1 in DCs (SI Appendix,Fig. S6). Then DCs from Sirt1CreER and WT mice were isolated

and treated, and these cells were injected into recipient mice asdescribed above. Acute deletion of SIRT1 in DCs promotes TH1but suppresses Treg differentiation as demonstrated by IFN-γ, IL-17A, and Foxp3 intracellular staining of donor T cells (Fig. 3B).Collectively, these results suggested that SIRT1 deficiency inDCs alters antigen-specific TH1 and iTreg cell differentiation ina reciprocal manner in vivo.

SIRT1 Is Involved in a DC-Dependent Regulation of TH1 and Treg CellDifferentiation in Vitro. Next, we applied an in vitro coculturesystem (composed of purified naive OT-II T cells, WT, orSirt1CD11c−/− splenic DCs and stimuli LPS and OVA) to de-termine the functional interaction between antigen-specificOTII T cells and DCs. We observed more IFN-γ+ T cells, higherexpression of IFN-γ mRNA, fewer Foxp3+ T cells, and lowerexpression of Foxp3 mRNA in T cells cocultured with Sirt1CD11c−/−

DCs than in T cells cocultured with WT DCs (Fig. 4 A and B).Also, polyclonal T cells activated by α-CD3 and SIRT1-deficientDCs displayed similar alterations in TH1 and iTreg differentia-tion (Fig. 4 C and D). Notably, the expression of TH2 and TH17cell differentiation markers are comparable in both groups (Fig.4 B and D). Thus, SIRT1 signaling in DCs directly inhibits TH1,whereas it instructs iTreg cell differentiation in vitro.

SIRT1 Modulates DC-Derived T-Cell Polarizing Cytokines. We nextsought to measure DC-derived cytokines that are known to reg-ulate TH1 and iTreg cell differentiation, including IL-12 and TGF-β1. LPS stimulation of SIRT1CD11c−/− DCs in vitro resulted in anincreased intracellular Il-12p40 protein but decreased TGF-β1protein compared with WT DCs (Fig. 5A). Also, a similar alter-ation of IL-12 (IL-12p70) and TGF-β1 in mRNA and proteinlevel was observed in DCs after LPS stimulation in vitro (Fig. 5B)and in vivo (Fig. 5C and SI Appendix, Fig. S7). On the other hand,the expression of IL-12p35 and IL-23p19, two known partners ofIL-12p40, was not significantly altered in SIRT1CD11c−/− DCs

Fig. 3. SIRT1 is required for adoptively transferred DCs to direct TH1 andiTreg immunity. Indicated splenic DCs were pulsed with LPS and OVA andinjected s.c. into SIRT1CD11c−/− (A) or Sirt1CreER (B) recipient mice, whichpreviously received naive OT-II (Thy1.1+) T cells 24 h before, as described inMaterials and Methods. The intracellular staining of IFN-γ and IL-17A wasdetermined in donor-derived CD4+T cells. (Right) The proportion of IFN-γ+,IL-17+, and Foxp3+ population among donor cells. Data are shown as mean ±SD, from one of three independent experiments (at least five mice pergroup). **P < 0.01 and ***P < 0.001 compared with the indicated groups.

Fig. 4. DC–SIRT1 signaling directs TH1 and Treg differentiation in vitro. (A and B) Naive OT-II T cells were cocultured with WT or SIRT1CD11c−/− DCs in thepresence of antigen for 5 d. (A) Intracellular staining of IFN-γ and Foxp3 expression in cultured T cells. (Right) The proportions of IFN-γ+ and Foxp3+ proportionin CD4+ T cells. (B) The mRNA expression of indicated genes in T cells restimulated with α-CD3 for 5 h. (C and D) Naive T cells from C57BL/6 mice werecocultured with WT or SIRT1CD11c−/− DCs in the presence of α-CD3 for 5 d. (C) The proportions of IFN-γ+ and Foxp3+ proportion in CD4+ T cells. (D) The mRNAexpression of indicated genes in OT-II T cells. Data are representative of two (A and B) and three (C and D) independent experiments (three to five mice pergroup), **P < 0.01 and ***P < 0.001 compared with the indicated groups.

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(SI Appendix, Fig. S8A). Also, the expression of TGF-β2 andTGF-β3 is comparable in SIRT1CD11c−/− DCs and WT DCs afterLPS stimulation in vitro (SI Appendix, Fig. S8B). Moreover, wewere unable to find any significant changes of IL-12 and TGF-β1in other immune cells isolated from Sirt1CD11c−/− or WT micefollowing in vitro LPS stimulation (SI Appendix, Fig. S9).Next, we applied a DC–T-cell coculture system (as described

above) to determine whether SIRT1 signaling in DCs modulatesT-cell differentiation through intercellular cytokine signaling. InT cells cocultured with SIRT1CD11c−/− DCs, we observed an in-creased STAT4 phosphorylation and decreased SMAD3 phos-phorylation compared with T cells cocultured with WT DCs (SIAppendix, Fig. S10). The alteration of these phosphorylationevents is consistent with the fact that STAT4 and SMAD3 aredownstream signaling targets of IL-12 and TGF-β1, respectively.Moreover, we were able to reverse the impact of SIRT1CD11c−/−

on TH1 cell differentiation and Treg differentiation in our co-culture system by adding anti–IL-12 neutralizing antibodies andadding TGF-β1, respectively (Fig. 5D). Together, our data sug-gested that enhanced IL-12 and diminished TGF-β1 productionin SIRT1CD11c−/− DCs contributes to the alteration of TH1 andTreg differentiation.

HIF1α Mediates SIRT1-Dependent Regulation of IL-12 and TGF-β1in DCs. To gain more mechanistic insights on SIRT1-dependentregulation of DC-derived cytokines, we assessed the pre-dominant signaling molecules in DCs that are involved in LPS-mediated proinflammatory signaling pathways. DCs isolatedfrom SIRT1CD11c−/− and WT display a comparable level ofphosphorylation on ErK, JNK, p38, and AKT, indicative of un-altered signaling. By contrast, SIRT1 deficiency in DCs resultsin an enhanced S6 phosphorylation (pS6), indicative of themammalian target of rapamycin (mTOR) signaling, and an ac-cumulated transcriptional factor, HIF1α (Fig. 6A). Conversely,pharmacological stimulation of SIRT1 activity by SRT1720 (anactivator of SIRT1) significantly decreases HIF1α expression(Fig. 6A).

To determine whether mTOR signaling is involved in SIRT1-dependent regulation on DC-derived cytokines, we applieda pharmacological approach (rapamycin) to block mTOR ac-tivity in DCs. Whereas rapamycin treatment is sufficient to re-duce the level of pS6 in SIRT1CD11c−/− DCs to a comparablelevel as in WT DCs following LPS stimulation, it failed to reversethe cytokine production and HIF1α level (Fig. 6B). Thus, ourresults suggested that mTOR is unlikely involved in mediatingSIRT1-dependent regulation on cytokines and HIF1α in DCs.Next, we sought to assess the role of HIF1α in DCs. The

treatment of LPS-stimulated DCs with CoCl2, which resulted inan accumulation of HIF1α, readily increases IL-12 and decreasesTGF-β1 (SI Appendix, Fig. S11A). Conversely, 2-ME2, an in-hibitor of HIF1α, significantly reversed the up-regulated IL-12expression and down-regulated TGF-β1 expression, which resultedfrom SIRT1 deficiency in DCs (SI Appendix, Fig. S11B). Fur-thermore, the genetic deletion of HIF1α in SIRT1CD11c−/− DCs(DKO) reversed the alterations on IL-12 and TGF-β1 causedby SIRT1 deficiency following LPS stimulation (Fig. 6 C–E).Together, our results suggested that HIF1α signaling mediatesSIRT1 effects on IL-12 and TGF-β1 in DCs.

DC SIRT1 Signaling Modulates IL-12Rβ2 and TGF-βR2 Expression inT Cells. T-cell polarizing cytokines often induce the expression oftheir corresponding cytokine receptors on T cells, resulting in arobust programming of cell fate determination (1, 16, 20, 35). Thisled us to investigate the expression of cytokine receptors of IL-12and TGF-β in T cells activated by WT and SIRT1CD11c−/− DCs.Expression of IL-12Rβ1, TGF-βR1, and TGF-βR3 protein andmRNA is comparable in T cells induced by WT or SIRT1CD11c−/−

DCs (SI Appendix, Fig. S12). In contrast, SIRT1CD11c−/− DCs in-duced more IL-12Rβ2 but less TGF-βR2 in T cells compared withWT DCs. Also, either the genetic deletion of HIF1α (SI Appendix,Fig. S13) and the IL-12 neutralizing antibodies or TGF-β1 treat-ment could reverse the changes of IL-12Rβ2 or TGF-βR2 in T cellscocultured with SIRT1CD11c−/− DCs, respectively (SI Appendix,Fig. S14).

Fig. 5. SIRT1-dependent IL-12 and TGF-β1 production in DCs regulates TH1 and Treg cell differentiation. (A) The intracellular staining of indicated genes in WTand SIRT1CD11c−/− splenic DCs 5 h following LPS stimulation in vitro. (Right) The proportion of IL-12p40+ and intracellular TGF-β1 expression (mean fluores-cence intensity, MFI) among CD11c+ cells. (B and C) The mRNA expression of IL-12p70 and TGF-β1 in WT and SIRT1CD11c−/− splenic DCs following LPS stim-ulation in vitro (B) and in vivo (C) were detected. (D) The intracellular staining of IFN-γ and Foxp3 in T cells cocultured with WT or SIRT1CD11c−/− splenic DCswith indicated treatment. (Right) The proportion of IFN-γ+ and Foxp3+ proportion in CD4+ T cells. Data are representative of four independent experiments(three to five mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the indicated groups.

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Next, we sought to determine whether the alteration of cytokinereceptors mediates the change of TH1 and Treg differentiation.For this inquiry, we applied shRNA approaches to knockdownIL-12Rβ2 and TGF-βR2 expression in OTII T cells (SI Appendix,Fig. S15), which were subsequently stimulated by either WT orSIRT1CD11c−/− DCs. Then, we determined mRNA expression ofIFN-γ and Foxp3, indicative of TH1 and iTreg differentiation.Knockdown of IL-12Rβ2 or TGF-βR2 in OT II T cells selectivelyreversed the alteration of TH1 or iTreg differentiation caused bySIRT1 deficiency in DCs, respectively (SI Appendix, Fig. S16).Collectively, IL-12Rβ2 and TGF-βR2 expression in T cells isrequired for the alteration of TH1 and iTreg cell differentiationinduced by SIRT1CD11c−/− DCs.

Pharmacologically Targeting SIRT1 in Mouse and Human DCsModulates T-Cell Differentiation. Next, we sought to apply a phar-macological approach to target SIRT1 in both mouse and humanDCs and determine whether we can recapitulate our finding ingenetic targeting SIRT1. We first applied Ex527, a selective in-hibitor for SIRT1 but not for other histone deacetylases (36, 37)to the mouse DC–T-cell coculture system. In consistent with theresults from genetic targeting SIRT1, we observed that SIRT1inhibitor significantly enhanced TH1 but suppressed iTreg dif-ferentiation (Fig. 7 A–C). Then, we extended our inhibitor ex-periment to the human DC–T-cell coculture system, wherebyDCs were derived from human peripheral blood monocytes andT cells were isolated from human cord blood. The pharmaco-logical inhibition of SIRT1 in human DCs largely recapitulatedwhat we observed in genetic and pharmacological targeting ofmouse DCs in terms of the alterations of human T-cell IFN-γ,Foxp3, IL-12, TGF-β1, IL-12Rβ2, and TGF-βR2 expression (Fig.7 D–F). Thus, our data indicated that SIRT1 mediated an evo-lutionarily conserved signaling pathway in both mouse andhuman DCs.

DiscussionDCs play a central role in initiating front-line innate immunityand inducing subsequent adaptive immunity in the process ofhost defense against infection (38, 39). Particularly, DCs shapeantigen-specific adaptive immune response through presentingantigens, modulating cell surface costimulatory molecules, andproducing cytokines and chemokines (40, 41). Fine tuning amyriad of DC intrinsic signaling pathways is required for elicitingan effective adaptive immune response without triggeringinflammation-induced host damage (41, 42). Our current studyrevealed that an integrated SIRT1–HIF1α signaling axis in DCsdirects the generation of two particular subsets of T cells, TH1and iTreg cells, under infectious inflammation. Whereas SIRT1 isnot involved in regulating antigen presentation in DCs, SIRT1–HIF1α axis in DCs instructs TH1 and iTreg differentiationthrough modulating the production of DC-derived T-cell polar-izing cytokines, including IL-12 and TGF-β1. The altered IL-12Rβ2/TGF-βR2 expression and downstream STAT4/SMAD3signaling in responding T cells further confer a robust DC–T-cellcross-talk, dictating the programming of TH1 and iTreg differ-entiation (SI Appendix, Fig. S17).Recent studies suggested that SIRT1 is involved in regulating

immune response in various inflammatory models (23–28, 43,44). Mechanistically, transcriptional factor has been implicatedas a critical proinflammatory-signaling module in myeloid leu-kocytes (45–50). In consistent with recent findings that SIRT1 isresponsible for the deacetylation and destabilization of HIF1α(51, 52), we found that HIF1α is not only regulated by SIRT1 butalso is involved in mediating the effects of SIRT1 deficiencyon DCs.Metabolic regulation and cell signaling are tightly and ubiquitously

linked with immune responses (53–55). The effective immuneresponse requires DCs to function in various conditions, in-cluding the alteration of extracellular or intracellular metabolicstates due to the migration to a nutrient and/or oxygen-deficient

Fig. 6. SIRT1 regulates IL-12 and TGF-β1 production through HIF1α but independent of mTOR. (A) Intracellular staining and immunoblot of indicatedproteins in DCs following LPS stimulation in vitro. (B) Intracellular staining of IL-12p40 and TGF-β1 in LPS-stimulated WT and SIRT1CD11c−/− splenic DCspretreated with rapamycin (100 μM) for 1 h. (Left) The proportions of IL-12p40+ and intracellular TGF-β1 expression (MFI) in DCs. (Right) Intracellularstaining of phosphorylation of S6 and HIF1α. (C–E) The intracellular staining of IL-12p40 and TGF-β1 in DCs isolated from indicated mice 4 h following i.p.injection of 10 mg/kg LPS (C). The proportion of IL-12p40 and MFI of TGF-β1 in DCs (D). The serum IL-12 and TGF-β1 levels indicated in mice (E ). Data arerepresentative of four (A and E) and two (B–D) independent experiments (three to five mice per group). *P < 0.05, **P < 0.01, and ***P < 0.001 comparedwith the indicated groups.

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environment (tumor microenvironment and inflammatory sites)or an ongoing metabolic reprogramming (resulted from in-flammatory stimulation), respectively. The adaptation of DCsto changing metabolic states resulted from a mechanism of“metabolic checkpoint,” an active signaling process involved insensing metabolic alteration and subsequently signaling trans-action and execution (56). Recent studies indicated that a Toll-like receptor signaling-mediated metabolic reprogramming isrequired for DC maturation and antigen presentation (57–59).Moreover, the dysregulated mTORC1 or the adenosine mono-phosphate (AMP)-activated protein kinase (AMPK) activityresults in an impaired DC development and maturation, in-dicating a metabolic checkpoint sensing amino acids and in-tracellular ATP during DC development and maturation (58,60). Our current study further implicated a metabolic checkpointin DCs requiring the interplay of SIRT1 and HIF1α, two meta-bolic sensors of redox and oxygen states, respectively (61–63).Finally, the above scenarios indicate that the DC-directedadaptive immunity requires the coupling of two evolutionarilyconserved stress responses, inflammatory response, and meta-bolic stress response.

Materials and MethodsMice and Bacterial Infection Model. All animal experiments were performedwith the approval of the Animal Ethics Committee of FudanUniversity. C57BL/6Sirt1fl/fl, CD11c-Cre, Rosa26-Cre-ERT2, and HIF1αfl/fl mice were obtainedfrom The Jackson Laboratory. Thy1.1 and OT-II TCR-transgenic mice wereobtained from the Center of Model Animal Research at Nanjing University.Rag1−/− and CD45.1 mice were obtained from the Beijing University Exper-imental Animal Center. C57BL/6 mice were obtained from the Fudan Uni-versity Experimental Animal Center. All mice had been backcrossed to theC57BL/6 background for at least eight generations and were used at an ageof 6–12 wk. WT control mice were of the same genetic background and,where relevant, included Cre+ mice to account for the effects of Cre (noadverse effects due to Cre expression itself were observed in vitro or in vivo).For bacterial infection, mice were injected i.v. with 4 × 104 L. monocytogenes-expressing OVA. After 7 d, splenocytes were harvested and stimulated withlisteriolysin O189–120 (LLO189–120) for 5 h for intracelluar staining.

Cell Adoptive Transfer. Naive T cells 2 × 106 (CD4+CD62LhiCD44loCD25−) fromOT-II TCR-transgenic mice were sorted and transferred into Sirt1fl/fl-CD11c-Cre− (WT) and Sirt1fl/fl-CD11c-Cre+ (Srit1CD11c−/−) mice. After 24 h, they wereinjected s.c. with OVA323–339 in the presence of CFA (Difco) and LPS (Sigma).At day 7–8 after immunization, DLN cells were harvested and stimulatedwith the cognate peptide for 2–3 d for cytokine mRNA and secretion anal-yses, or pulsed with phorbol 12-myristate 13-acetate (PMA) and ionomycinfor 5 h for intracellular staining from donor-derived T cells. For oral antigenstimulation, after adoptive transfer of 2 × 106 naive OT-II T cells, mice werefed with water supplemented with 20 mg/mL OVA protein (grade VI; Sigma-Aldrich) for 5 d. For T-cell transfer-induced colitis, 3 × 105 T cells (CD4+TCR+

CD45RbhiCD25−) from WT mice were transferred into Rag1−/− or SIRT1CD11c−/−

Rag1−/− mice and body weight was measured weekly. For DC transfer,splenic DCs (2 × 105) were pulsed with 50 μg/mL OVA and 500 ng/mL LPSfor 8 h, washed, and injected s.c. into C57BL/6 mice that had received 2 × 106

naive OT-II T cells 24 h before. Mice were killed 7–8 d later for analysis.

Cell Cultures and Flow Cytometry. Spleens were digested with collagenase D,and DCs (CD11c+TCR−CD19−NK1.1−F4/80−Ly6G−) were sorted on a FACSAriaII (Becton Dickinson). Lymphocytes were sorted to enrich for naive T cells. ForDC–T-cell cocultures, DCs and T cells (1:10) were mixed in the presence of1 μg/mL OVA323–339 peptide and 100 ng/mL LPS. After 5 d of culture, live T cellswere stimulated with PMA and ionomycin for intracellular cytokine stainingor with plate-bound α-CD3 to measure cytokine secretion and mRNAexpression. T-cell proliferation was determined by pulsing of cells with3H-thymidine for the final 12–16 h of culture. For drug treatments, cells wereincubated with vehicle, CoCl2 (200 μM; Calbiochem), 2-ME2 (2 μM; Calbio-chem), rapamycin (100 μM; Calbiochem), or EX-527 (10 μM; Sigma) for 0.5–1 hbefore stimulation. For antibody or cytokine treatment, cultures were sup-plemented with 5 ng/mL TGF-β1 (R&D Systems), anti–IL-12 mAb (R&DSystems). Flow cytometry was performed with antibodies from eBioscienceor BD Biosciences. Anti-mouse TGF-βR3 (ab78421) is from Abcam. Anti-mouse HIF1αmAb (241812), anti-mouse TGF-β1 mAb (9016), anti-mouse TGF-βR1 mAb (141231), anti-mouse TGF-βR2 (21813), anti-mouse IL-12Rβ1(16161),and anti-mouse IL-12Rβ2 (16162) are from R&D Systems. Flow cytometrydata were acquired on a FACSCalibur (Becton Dickinson) or an Epics XLbench-top flow cytometer (Beckman Coulter) and data were analyzed withFlowJo (Tree Star). For inducible deletion of loxP-flanked alleles in vivo, micewere treated with 2 mg tamoxifen (Sigma-Aldrich) daily for 3 d.

Fig. 7. Pharmacologically targeting SIRT1 in mouse and human DCs. As described in Materials and Methods, mouse DCs (A–C) or human DCs (D–F) pulsedwith LPS (100 ng/mL) were cocultured with mouse or human T cells, respectively, in the absence or presence of EX-527 (10 μM). The intracellular staining ofIFN-γ and Foxp3 in T cells (A or D); the mRNA expression of indicated genes in T cells (B or E); the level of IL-12 and TGF-β1 in indicated supernatant (C or F)were detected. Data are shown as mean ± SD, n = 3–5, from one of two independent experiments. ***P < 0.001 compared with the indicated groups.

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RNA and Protein Analysis. RNA was extracted with an RNeasy kit (Qiagen),and cDNA was synthesized using SuperScrip III reverse transcriptase (Invi-trogen). An ABI 7900 real-time PCR system was used for quantitative PCR,with primer and probe sets obtained from Applied Biosystems. Results wereanalyzed using SDS 2.1 software (Applied Biosystems). The cycling thresholdvalue of the endogenous control gene (Hprt1, encoding hypoxanthineguanine phosphoribosyl transferase) was subtracted from the cyclingthreshold (ΔCT). The expression of each target gene is presented as the foldchange relative to that of control samples (2-ΔΔCT), as described previously(64). For detection of phosphorylated signaling proteins, purified cells wereactivated with LPS (Sigma), immediately fixed with Phosflow Perm buffer(BD Biosciences), and stained with phycoerythrin or allophycocyanin directlyconjugated to antibody to Erk phosphorylated at Thr202 and Tyr204 (20A;BD Biosciences), p38MAPK phosphorylated at Thr180 and Thr182 (D3F9; CellSignaling Technology), JNK phosphorylated at Thr183 and Tyr185 (G9; CellSignaling Technology), STAT4 phosphorylated at Tyr701 and Ser727 (58D6;Cell Signaling Technology), SMAD3 phosphorylated at Tyr705 and Ser727(D3A7; Cell Signaling Technology), AKT phosphorylated at Ser473 andThr308 (587F11; Cell Signaling Technology), or S6 phosphorylated at Ser235and Ser236 (D57.2.2E; Cell Signaling Technology), as described previously(9). Immunoblot analysis was performed as described (64) using anti-HIF1α(H1α67; Sigma-Aldrich), anti-Sirt1 (D60e1; Cell Signaling Technology), andanti–β-actin (AC-15; Sigma-Aldrich) antibodies.

IL-12Rβ2 and TGF-βR2 Knockdown with RNA Interference. A gene-knockdownlentiviral construct was generated by subcloning gene-specific shorthairpin RNA (shRNA) sequences into lentiviral shRNA expression plasmids(pMagic4.1) as described before (65). Lentiviruses were harvested fromculture supernatant of 293T cells transfected with shRNA vector. Sorted

OT-II CD4+ T cells were infected with recombinant lentivirus, and greenfluorescent protein expressing cells were isolated using fluorescencesorting 48 h later. The IL-12Rβ2 and TGF-βR2 expression was confirmedusing real-time PCR. The sorted T cells with either control or shRNAvectors were used for functional assay.

Human DC and T-Cell Cultures. For assays of human DC-mediated T-cell acti-vation and differentiation, normal human DCs (CC-2701; Lonza) were cul-tured and their populations were expanded for 5 d with human granulocyte-macrophage colony-stimulating factor and IL-4 (R&D), followed by treatmentwith EX-527 and stimulation for 24 h with LPS. DCs were washed extensivelyand cultured with human cord blood CD4+ T cells (2C-200; Lonza) at a ratioof 1:10. After 7 d of culture, live T cells were purified and then stimulatedeither with PMA and ionomycin for intracellular cytokine staining for 5 h orwith plate-bound anti-CD3 for analysis of mRNA expression.

Statistical Analysis. All data are presented as the mean ± SD. Student’s un-paired t test was applied for comparison of means and to compare differ-ences between groups. Comparison of the survival curves was performedusing the log-rank (Mantel–Cox) test. A P value (alpha-value) of less than0.05 was considered to be statistically significant.

ACKNOWLEDGMENTS. The authors’ research is supported by the NationalNatural Science Foundation for General Programs of China Grants 31171407and 81273201 (to G.L.) and Grant 81271907 (to Y.B.), Key Basic ResearchProject of the Science and Technology Commission of Shanghai MunicipalityGrant 12JC1400900 (to G.L.), Innovation Program of Shanghai MunicipalEducation Commission Grant 14Z Z009 (to G.L.), and Excellent Youth Foun-dation of Chinese Academy of Sciences Grant KSCX2-EW-Q-7-1 (to G.L.).

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