Toll-Like Receptor 4–Induced Glycolytic Burst in Human Monocyte

Toll-like Receptor 4−Induced Glycolytic Burst in Human Monocyte-Derived Dendritic Cells Results from p38-Dependent Stabilization of HIF-1α and Increased This information is current as Hexokinase II Expression of September 28, 2021. Laure Perrin-Cocon, Anne Aublin-Gex, Olivier Diaz, Christophe Ramière, Francesco Peri, Patrice André and Vincent Lotteau J Immunol published online 23 July 2018 Downloaded from http://www.jimmunol.org/content/early/2018/07/21/jimmun ol.1701522 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2018/07/21/jimmunol.170152 Material 2.DCSupplemental

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 23, 2018, doi:10.4049/jimmunol.1701522 The Journal of Immunology

Toll-like Receptor 4–Induced Glycolytic Burst in Human Monocyte-Derived Dendritic Cells Results from p38-Dependent Stabilization of HIF-1a and Increased Hexokinase II Expression

Laure Perrin-Cocon,* Anne Aublin-Gex,* Olivier Diaz,* Christophe Ramie`re,* Francesco Peri,† Patrice Andre´,* and Vincent Lotteau*

Cell metabolism now appears as an essential regulator of immune cells activation. In particular, TLR stimulation triggers metabolic reprogramming of dendritic cells (DCs) with an increased glycolytic ﬂux, whereas inhibition of glycolysis alters their functional

activation. The molecular mechanisms involved in the control of glycolysis upon TLR stimulation are poorly understood for human Downloaded from DCs. TLR4 activation of human monocyte-derived DCs (MoDCs) stimulated glycolysis with an increased glucose consumption and lactate production. Global hexokinase (HK) activity, controlling the initial rate-limiting step of glycolysis, was also increased. TLR4- induced glycolytic burst correlated with a differential modulation of HK isoenzymes. LPS strongly enhanced the expression of HK2, whereas HK3 was reduced, HK1 remained unchanged, and HK4 was not expressed. Expression of the other rate-limiting glycolytic enzymes was not signiﬁcantly increased. Exploring the signaling pathways involved in LPS-induced glycolysis with various speciﬁc a inhibitors, we observed that only the inhibitors of p38–MAPK (SB203580) and of HIF-1 DNA binding (echinomycin) reduced http://www.jimmunol.org/ both the glycolytic activity and production of cytokines triggered by TLR4 stimulation. In addition, LPS-induced HK2 expression required p38-MAPK–dependent HIF-1a accumulation and transcriptional activity. TLR1/2 and TLR2/6 stimulation increased glucose consumption by MoDCs through alternate mechanisms that are independent of p38–MAPK activation. TBK1 contributed to glycolysis regulation when DCs were stimulated via TLR2/6. Therefore, our results indicate that TLR4-dependent upregulation of glycolysis in human MoDCs involves a p38-MAPK–dependent HIF-1a accumulation, leading to an increased HK activity supported by enhanced HK2 expression. The Journal of Immunology, 2018, 201: 000–000.

oll-like receptors are common sensors expressed by den- stimulate naive T cells (2). TLR4 is a receptor for Gram-negative by guest on September 28, 2021 dritic cells (DCs) recognizing pathogen-associated molec- bacteria LPS (3). This receptor can also be activated by other T ular patterns and damage-associated molecular patterns, microbial ligands such as viral proteins (4–6) and by damage- initiating an appropriate immune response to fight infection (1). associated molecular patterns such as high-mobility group box 1 TLR signaling triggers human DC maturation, promoting the se- (HMGB) (7) or oxidized phospholipids (8). TLR4 is also involved cretion of proinflammatory cytokines and inducing the expression in the sensing of nutrients and metabolic stress. Indeed, saturated of costimulatory molecules so that DCs acquire the ability to free fatty acids can activate TLR4 signaling via their binding to the hepatokine Fetuin A, which is a TLR4 ligand (9). Its increased production in steatotic and inflamed liver contributes to the secre- *Centre International de Recherche en Infectiologie, Biologie Cellulaire des Infections tion of inflammatory cytokines by monocytes and adipose tissue Virales, INSERM, U1111, Universite´ Claude Bernard Lyon 1, CNRS, UMR5308, Ećole (10). By regulating the expression of many genes involved in innate Normale Supe´rieure de Lyon, Hospices Civils de Lyon, Universite´ de Lyon, Lyon, France; and †Department of Biotechnology and Biosciences, University of Milano-Bicocca, 20126 immunity and metabolic reprogramming (11), TLR4 is at the Milano, Italy crossroads of innate immunity and metabolic inflammation (12) and ORCIDs: 0000-0003-0501-6733 (L.P.-C.); 0000-0002-1981-7155 (C.R.); 0000-0002- is involved in the pathogenesis of metabolic diseases such as obesity 5834-7395 (P.A.). and type 2 diabetes (13). Received for publication November 10, 2017. Accepted for publication July 3, 2018. Glucose is a major nutrient for cellular bioenergetics production. This work was supported by European Commission–granted TOLLerant Project Glycolysis converts glucose to pyruvate by a series of enzymatic H2020-MSC-ETN-642157 (to F.P.), INSERM Ebola Task Force I3M, and Fondation reaction steps, generating 2 mol of ATP per mole of glucose. In- pour la Recherche Me´dicale Grant DEQ20160334893 (to V.L.). termediary metabolites of glycolysis are precursors also fueling the Address correspondence and reprint requests to Dr. Laure Perrin-Cocon and Dr. Vincent Lotteau, Inserm U1111 - Centre International de Recherche en Infectio- pentose phosphates, lipids, and amino acid pathways (11). Three rate- logie, 21 Avenue Tony Garnier, 69365 Lyon Cedex 7, France. E-mail addresses: limiting enzymes are controlling the glycolytic flux. The first one, [email protected] (L.P.-C.) and [email protected] (V.L.) the hexokinase (HK), controls the conversion of glucose to glucose The online version of this article contains supplemental material. 6-phosphate. There are four isoenzymes of HK (HK-I, II, III, IV) Abbreviations used in this article: BM-DC, bone marrow–derived DC; DC, dendritic encoded by four different genes (HK1, 2, 3, 4). HK1 and HK3 have a cell; 2-DG, 2-deoxyglucose; Echino, echinomycin; HIF-1, hypoxia-inducible factor 1; HK, hexokinase; MoDC, monocyte-derived DC; mTORC1, mammalian target of rapa- large spectrum of tissue expression, whereas HK2 and HK4 have an mycin complex 1; 2-NBDG, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D- expression profile that is more restricted to metabolically relevant glucose; Pam, Pam3CSK4; PFK, phosphofructokinase; PGN, peptidoglycan; PHD, tissues. HK2 is overexpressed in virtually all cancer cells. Other rate- prolyl-hydroxylase domain enzyme; SB, SB203580. limiting enzymes of glycolysis are the phosphofructokinase (PFK) Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 and pyruvate kinase. PFK is encoded by three different genes

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701522 2 TLR4 GLYCOLYTIC BURST INVOLVES A p38–MAPK–HIF-1a AXIS

(PFKL, PFKP, and PFKM) whose expression is tissue dependent. TLR1/2 ligand Pam3CSK4 (Pam), all from InvivoGen (Toulouse, France), Pyruvate kinase is encoded by the PKM gene in immune cells, were dissolved in sterile PBS. FP7 was synthesized from commercially generating two isoforms (PKM1 and PKM2). The pyruvate produced available D-glucose by multistep organic synthesis according to published procedures (32). The purity of the molecule was assessed by nuclear mag- by glycolysis can be converted to lactate that is excreted by specific netic resonance and mass spectrometry analysis. A 5-mM stock solution of monocarboxylate transporters or to oxaloacetate or acetyl-CoA to FP7 was prepared in ethanol/DMSO 1:1 and conserved at 220˚C. fuel the tricarboxylic acid cycle, coupling glycolysis to oxidative The inhibitors SB203580 (SB), SP600125, PD98059, BX795, and phosphorylation, for the generation of high amount of ATP in the LY294002, purchased from InvivoGen, rapamycin, provided by Calbiochem (San Diego, CA), and HIF-1a DNA-binding inhibitor, echinomycin (Echino), presence of oxygen. During hypoxia, oxidative phosphorylation is provided by Cayman Chemical (Ann Arbor, MI), were dissolved in sterile reduced and glycolysis is increased to face energetic needs (14). culture grade DMSO and stored at 220˚C. AZD5363 and SC75741 were However, activation of glycolysis can also occur under aerobic con- purchased from Selleckchem (Houston, TX), prepared in DMSO, and stored ditions, and O. Warburg (15) first discovered that tumor cells have a at 280˚C. Pepinh MYD, pepinh TRIF, and pepinh control, purchased from 2 high rate of glycolysis and that most pyruvate is converted to lactate, InvivoGen, were dissolved in PBS and stored at 20˚C. Glucose uptake was measured by incorporation of 2-deoxy-2-[(7-nitro- even when oxygen is available. Both innate and adaptive immune 2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG; Sigma-Aldrich, cells can shift to aerobic glycolysis upon stimulation. Cell meta- Saint-Quentin Fallavier, France) in culture medium. bolism is now appreciated as a key regulator of T cell differentiation, DC generation and treatment modulating effector and memory functions (16). The transcriptional control of glycolysis in T cells mainly relies on the expression of Monocytes were purified from human peripheral blood of healthy donors hypoxia-inducible factor 1 (HIF-1) and the proto-oncogene MYC (33), obtained from the Etablissement Français du Sang. All experiments were performed in accordance with the guidelines of the World Medical (17). Accumulation of HIF-1a in normoxic conditions has been ob- Downloaded from Association’s Declaration of Helsinki. Experimental procedures were ap- served in several cell types upon LPS stimulation (14, 18, 19). Several proved by the local institutional review committee. Briefly, PBMCs were molecular mechanisms may result in this accumulation, including isolated by standard density gradient centrifugation on Ficoll–Hypaque activation of the mammalian target of rapamycin complex 1 (Eurobio, Courtaboeuf, France). Mononuclear cells were separated from (mTORC1) (20). In primary human plasmacytoid DCs, increased PBLs by centrifugation on a 50% Percoll solution (GE Healthcare, Velizy, France). Monocytes were purified by immunomagnetic depletion using glycolysis was found to play a key role in the regulation of antiviral Pan Mouse IgG Dynabeads (Thermo Fisher Scientific, Villebon sur Yvette, functions, including IFN-a production (21). Tolerogenic DCs also France) with a mixture of mAbs anti-CD19 (4G7 hybridoma), anti-CD3 http://www.jimmunol.org/ show a metabolic signature characterized by high mitochondrial (OKT3 hybridoma; American Type Culture Collection, Manassas, VA), respiration and glycolytic capacity (22). TLR4 stimulation of DCs or and anti-CD56 (NKH1; Beckman Coulter, Villepinte, France). Monocyte purity was .90% as assessed by CD14 labeling without CD3+, CD19+, macrophages results in increased glycolytic activity, an essential + and CD56 contaminating cells (data not shown). process for their proper activation (23–29). Indeed, a metabolic DCs were differentiated from monocytes cultured at 37˚C under 5% CO2 reprogramming to aerobic glycolysis was described to be associated atmosphere for 6 d at 106 cells/ml in RPMI 1640 medium with GlutaMAX to a proinflammatory M1 phenotype of murine macrophages, (Thermo Fisher Scientific) supplemented with 10% FCS (PAN-Biotech, m whereas glycolytic activity was unchanged in alternate activated M2 Aidenbach, Germany), 40 g/ml gentamicin, human recombinant GM-CSF, and IL-4 (PeproTech, Neuilly-Sur-Seine, France). Six days macrophages (11, 30). The molecular mechanisms involved in gly- later, DCs were harvested, washed twice, and resuspended for treatment at colysis upregulation upon TLR4 stimulation remain to be clarified 106 cells/ml fresh RPMI 1640 medium supplemented with 40 mg/ml by guest on September 28, 2021 and may differ according to cell types and functions. gentamicin and 10% FCS (complete medium). DCs were .95% pure as In murine bone marrow–derived DCs (BM-DCs), two different assessed by CD14 and CD1a labeling. molecular pathways have been depicted for glycolytic reprogramming DCs were treated with inhibitors or control solvent for 30 min before ε addition of TLR ligands or the same volume of PBS as control. Pretreatment upon TLR4 stimulation. Activation of TBK1-IKK and AKT kinases of DCs with inhibitory peptides, pepinh MYD, TRIF, or control was carried was found to control the early increase of glycolysis, favoring mito- out 6 h before LPS addition. For quantitative PCR analysis, cells were chondrial translocation of HK-II, fueling the tricarboxylic acid cycle collected after 6 or 24 h incubation. Supernatants and cells were collected and fatty acids synthesis (24). In these DCs, the late increase in gly- after 24 h for glucose assay, cytokines assay, phenotyping, or cell lysate preparation for Western blot and HK assay. colytic metabolism was proposed to be a survival response to maintain ATP production despite the inhibition of oxidative phosphorylation by Phenotype analysis NO produced by the inducible NO synthase (25). This pathway is Monocyte and DC phenotypes were determined after labeling with FITC- unlikely to be involved in human DCs because this enzyme is not labeled anti-CD14, -CD19, –HLA-DR, -CD80, -CD54 and PE-labeled anti- expressed (31), and NO production could not be detected in these cells CD3, -CD56, -CD1a, -CD86, -CD83, and -CD40 (Beckman Coulter). Flow (27). Studies exploring the molecular mechanisms involved in the cytometry analysis was carried out using a FACSCanto II (BD Biosciences, regulation of glycolysis in primary human DCs are lacking for a better Le Pont de Claix, France). For each double labeling, at least 5000 events in the live cell gate were acquired. Viability of cells was monitored using understanding of the reciprocal interactions between cellular metabolic propidium iodide staining (BD Biosciences) of dead cells. activity and the functional DC activation state. Therefore, we explored the pathways involved in the control of Cytokine assays glycolytic activity upon TLR4 stimulation of human monocyte- Clarified culture supernatants were collected 24 h after treatment and stored derived DCs (MoDCs). In contrast to murine DCs, increased at 220˚C. IL-8, MIP-1b, IL-6, IL-10, IL-12p40, and TNF-a were assayed glycolysis did not rely on the TBK1- or AKT-dependent pathways using quantitative cytokine-specific Cytometric Bead Array Flex Sets (BD but on the engagement of a TLR4–p38–MAPK axis stabilizing Biosciences). a HIF-1 and upregulating HK2 expression. Interestingly, this Glucose consumption and lactate production pathway does not appear to be involved in the activation of glycolysis triggered by TLR1/2 or TLR2/6 stimulation. Metabolites were quantified in freshly clarified cell supernatants and control medium using glucose (HK) and lactate assay kits (Sigma-Aldrich) according to the manufacturers’ instructions. Quantifications were normalized to DC cell number at day 6. Materials and Methods Reagents Quantitative real-time PCR Ultrapure TLR4 ligand LPS from Escherichia coli O111:B4, TLR2/6 For total RNA isolation, 6- or 24-h treated DCs were collected and washed ligand peptidoglycan (PGN) from Staphylococcus aureus, and synthetic twice with PBS. RNA extraction was performed using the NucleoSpin The Journal of Immunology 3

RNA Mini kit from Macherey-Nagel according to the manufacturers’ instruc- Fisher Scientific). Chemiluminescent signals were acquired and quantified tions. cDNA were synthetized using RNA-to-cDNA kit (Applied Biosystems, with a LAS4000 Imager (GE Healthcare). Villebon sur Yvette, France) before quantitative PCR experiment using the following primers: for HK1 DNA forward 59-GCCTCTTATTTGAAGGGCGG- Statistical analysis 39,reverse59-GACACAGTCATCATCGGACG-39; for HK2 DNA forward 59- TCCCCTGCCACCAGACTA-39,reverse59-TGGACTTGAATCCCTTGGTC- Data are expressed as mean 6 SEM. The Student t test was used for 39; for HK3 DNA forward 59-CATCGTGGACTTCCAGCAGAAG-39,reverse comparisons of two sample means. Multiple group comparisons were 59-CTTGGTCCAGTTCAGGAGGATG-39; for HK4 DNA forward 59- performed by one-way ANOVA followed by Bonferroni multiple com- CAGAAGGCTCAGAAGTCGGG-39,reverse59-TGGTGTTTGGTCTTCAC- parisons test and was applied to the analysis of all dose-dependent ex- GCT-39; for PKM1 DNA forward 59-CGAGCCTCAAGTCACTCCAC-39, periments. A p value ,0.05 was considered statistically significant. reverse 59-GTGAGCAGACCTGCCAGACT-39; for PKM2 DNA forward 59- ATTATTTGAGGAACTCCGCCGCCT-39,reverse59-ATTCCGGGTCACA- GCAATGATGG-39 (34); for PFKL DNA forward 59-AAGAAGTAGG- Results CTGGCACGACGT-39,reverse59-GCGGATGTTCTCCACAATGGAC-39; TLR4 stimulation induces glycolytic burst and upregulation of 9 9 for PFKP DNA forward 5 -AGGCAGTCATCGCCTTGCTAGA-3 , re- HK2 expression and activity verse 59-ATCGCCTTCTGCACATCCTGAG-39; for PFKM DNA forward 59-GCTTCTAGCTCATGTCAGACCC-39, reverse 59-CCAATCCTCA- MoDCs stimulated by LPS for 24 h showed an increased glucose 9 9 CAGTGGAGCGAA-3 ; for TBP DNA forward 5 -CCACGAACCAC- consumption and lactate production compared with nonstimulated GGCACTGATTT-39, reverse 59-CAGTCTGGACTGTTCTTCACTCTT- 39; and SYBR Green master mix on a CFX96 Touch Real-Time PCR MoDCs (Fig. 1A, 1B), indicative of an increased glycolytic activity. Detection System (Bio-Rad, Marnes-la-Coquette, France). Levels of The amplitude of this glycolytic burst was dependent on the dose of mRNA for specific genes are reported as relative gene expression nor- LPS and was statistically significant from 10 to 1000 ng/ml. We malized to the housekeeping gene TBP. previously characterized a selective TLR4 antagonist, FP7, inhib- Downloaded from HK activity assay iting the secretion of proinflammatory cytokines by LPS-stimulated MoDCs with an IC50 below 1 mM (27). FP7 also prevented LPS- The method used for extracting HK from DCs was adapted from Kuang induced secretion of IFN-b (Supplemental Fig. 1), suggesting that et al. (35), and the assay was a modification of that described by Ramie`re 6 FP7 blocked both endosomal TRIF–dependent and surface et al. (36). 280˚C frozen pelleted cells were homogenized (100 ml/2 3 10 cells) in precooled homogenization buffer (50 mM Tris–HCl, 250 mM MyD88–dependent TLR4 signaling (37). Because FP7 competes sucrose, 5 mM EDTA [pH 7.4], 5 mM 2-ME, 10 mM glucose, 0.2% Triton with LPS for binding to the TLR4 MD-2 subunit (32, 38) and http://www.jimmunol.org/ X-100). After a 20 min incubation on ice, homogenates were pulse- prevents ligand-mediated TLR4 internalization (F.A. Facchini, sonicated 5 s at half power of the device (EpiShear Probe Sonicator; D. Di Fusco, S. Barresi, A. Minotti, F. Granucci, G. Monteleone, Active Motif, La Hulpe, Belgium). Homogenates were then centrifuged at 500 3 g for 20 min at 4˚C. Supernatants were immediately used for F. Peri, and I. Monteleone, manuscript in preparation), these results determination of HK activity. HK activity was measured spectrophoto- suggest that FP7 may not directly inhibit endosomal TLR4 metrically through NADP+ reduction in the glucose 6-phosphate dehy- signaling but rather inhibits upstream activation of TLR4 receptor drogenase–coupled reaction. HK activity was assayed in medium by LPS. Treatment of cells with 10 mM FP7 prior to LPS containing 50 mM triethanolamine (pH = 7.6), 550 mM D-Glucose, + stimulation prevented the increase in glucose consumption and 100 mM MgCl2, 14 mM NADP , and 125 U/ml glucose 6-phosphate de- hydrogenase (Saccharomyces cerevisiae), equilibrated to 37˚C. The reac- lactate production triggered by LPS from 0.1 to 100 ng/ml by guest on September 28, 2021 tion was started by addition of ATP (final concentration 19 mM), and (Fig. 1A, 1B), showing that this glycolytic regulation was TLR4 absorbance was continuously recorded for 30 min at 340 nm (TECAN dependent. At the highest LPS concentration (1000 ng/ml), FP7 did Infinite M200). Dilutions of purified recombinant HK (Sigma-Aldrich) not totally block the increase of the glycolytic flux, consistent with were used as standards, and results were expressed as international unit per milligram of proteins. previous data (27). Using a fluorescent deoxyglucose as a non- hydrolyzable substrate of HK (2-NBDG), we measured the capacity Phosphoprotein detection of glucose uptake by the cells after 6 and 24 h LPS stimulation. No Day 6 DCs were pretreated in water bath at 37˚C with inhibitors for 30 min difference in the uptake could be detected between control and LPS- before TLR ligand addition for 15 min. TLR stimulation was stopped on stimulated MoDCs (Fig. 1C), indicating that increased glycolytic ice before two cold PBS washes. Then, cells were fixed with Cytofix so- activity in TLR4-stimulated MoDCs was not due to an increased lution (BD Biosciences) for 10 min at 37˚C before permeabilization with availability of intracellular glucose. We thus analyzed the expres- methanol 90% for 30 min on ice. Phosphoprotein staining was performed sion of the three rate-limiting glycolytic enzymes, HK, PFK, and by incubation 1 h at room temperature with the following anti-human Abs: purified anti–phospho-p38 MAPK (Thr180/Tyr182; Cell Signaling Tech- PKM, at the mRNA level. Compared with control cells, HK2 was nology, Leiden, the Netherlands), PE-conjugated anti-phospho–NF-kB strongly induced in LPS-stimulated MoDCs, whereas HK3 (Ser536; Cell Signaling Technology), purified anti–phospho-AKT (Ser473; was reduced (Fig. 1D). HK1 remained unchanged, whereas HK4 was Cell Signaling Technology), PE-conjugated anti–phospho-SAPK/JNK not expressed in MoDCs. In contrast to PFKM and PKM1, PFKL, (Thr183/Tyr185; Cell Signaling Technology). When unlabeled primary Ab was used, cells were further incubated 1 h at room temperature with PFKP, and PKM2 were highly expressed in MoDCs but not signifi- secondary Alexa Fluor 488–conjugated anti-rabbit Ab (Thermo Fisher cantly augmented upon LPS stimulation (Fig. 1E, 1F). The data in- Scientific). Cells were analyzed using a FACSCanto II (BD Biosciences). dicate that the expression profile of HK isoenzymes is modified upon TLR4 stimulation. This new expression profile correlates with a SDS-PAGE and Western blotting major increase in HK activity in total lysates of LPS-stimulated DCs were lysed in buffer containing 20 mM Tris-HCl, 180 mM NaCl, 1 mM MoDCs compared with unstimulated cells (Fig. 1G). EDTA, 0.5% Nonidet P-40, and 13 mixture of protease inhibitors (Sigma- Aldrich). Lysate proteins were separated by NuPAGE 4–12% Bis-Tris Gel TLR4-induced glycolytic burst involves p38–MAPK activation (Thermo Fisher Scientific) in MOPS running buffer. Gels were transferred and HIF-1a transcriptional activity to nitrocellulose membranes with the Trans-Blot Turbo Transfer system (Bio-Rad). Membranes were blocked for 1 h with PBS buffer containing To investigate the molecular mechanisms involved in the regulation 0.1% Tween 20 and 5% nonfat dry skim milk and incubated 1 h with the of glycolysis upon TLR4 stimulation, we screened a panel of in- primary Ab against HIF-1a (Novus Biologicals, Lille, France) or actin hibitors described to target main actors of the TLR4 signaling (clone AC-40; Sigma-Aldrich). Membranes were washed and incubated pathway. A scheme positioning the inhibitors and their target is 1 h with HRP-coupled goat polyclonal anti-rabbit IgG or HRP-coupled goat polyclonal anti-mouse IgG (Jackson ImmunoResearch Laboratories, presented on a simplified view of the TLR4 signaling pathway Suffolk, U.K.). Western blotting experiments were developed using (Fig. 2). TLR4 signaling in MoDCs results in the phenotypic and SuperSignal West Femto or Pico Chemiluminescent Substrate (Thermo functional maturation of these cells, inducing cytokine secretion 4 TLR4 GLYCOLYTIC BURST INVOLVES A p38–MAPK–HIF-1a AXIS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. TLR4 stimulation of glucose consumption and lactate production correlates with enhanced expression of HK2 and increased HK activity. DCs were differentiated from human peripheral blood monocytes for 6 d. DCs were seeded at 1 3 106 cells/ml in fresh culture medium before treatment. (A and B) DCs were treated at 37˚C with 10 mM FP7 (+FP7) or control solvent (2FP7) 30 min prior to stimulation with increasing amounts of LPS for 24 h. Glucose consumption and lactate production were monitored using enzymatic detection kits. Means 6 SEM from 11 (A)or5(B) independent experiments are shown. (C) DCs were stimulated with 10 ng/ml LPS for 6 or 24 h. Uptake of 2-NBDG was allowed for 15 min at 37˚C. Internalization of the fluorescent probe was analyzed by flow cytometry. (D–F) DCs were stimulated or not with 10 ng/ml LPS for 24 h. Gene expression was analyzed by quantitative RT- PCR and normalized to housekeeping gene TBP expression. (G) DCs, stimulated or not with 10 ng/ml LPS for 24 h, were washed and lysed in homogenization buffer for HK activity assay. Results are expressed as unit per milligram of proteins. and enhancing the expression of surface maturation markers. CD40, and the CD54 adhesion molecule was analyzed by FACS MoDCs were treated with these drugs prior to LPS stimulation for (Fig. 3B, 3C). SB significantly inhibited the expression of 24 h, and secretion of proinflammatory cytokines, expression of CD86 and CD54 and reduced (although not significantly) CD40 phenotypic maturation marker, and glucose consumption were expression compared with LPS-stimulated MoDCs. SC75741 and measured. AZD5363 mainly reduced CD54 expression. The other inhibitors As expected, p38–MAPK inhibitor SB inhibited the secretion of did not induce significant changes in MoDC phenotype. IL-6, IL-8, and MIP-1b (Fig. 3A) (39, 40). Echino strongly re- Among the tested inhibitors, p38–MAPK inhibitor SB was the duced IL-8 secretion, and the inhibitor of TBK1, BX795, inhibited most potent inhibitor of the glycolytic burst triggered by LPS MIP-1b secretion induced by LPS. Surface expression of the (Fig. 4A). Echino, which prevents DNA binding of HIF-1a to MHC molecule HLA-DR, costimulation molecules CD86 and hypoxia response elements, also significantly reduced the action The Journal of Immunology 5

regulation of HK2 expression (41). It is constitutively expressed, and the regulation of its degradation controls its accumulation and activity (42, 43). In total lysates from MoDCs, we observed that LPS stimulation for 24 h resulted in an important accumulation of HIF-1a that was prevented by SB treatment (Fig 5C, 5D). Therefore, HIF-1a accumulation triggered by LPS appeared to be dependent on p38–MAPK activation. p38–MAPK–HIF-1a axis is not engaged in the glycolytic burst induced by TLR1/2 and TLR2/6 stimulation As other bacterial ligands can induce MoDCs functional activation, we tested the effect of PGN and Pam, the ligands for TLR2/6 and TLR1/2, respectively, on glucose consumption. Both TLR ligands signiﬁcantly increased glucose consumption of MoDCs, although to a different level (Fig. 6A). PGN, like LPS, induced a strong increase of glucose consumption, whereas Pam was less efﬁcient (50% of the LPS increase). Inhibition of glucose consumption by SB reached 70 and 65% when it was induced by LPS–TLR4 and

Pam–TLR1/2, respectively, whereas it was only 35% after PGN– Downloaded from TLR2/6 stimulation. p38–MAPK phosphorylation was detected upon stimulation of all these TLRs and was inhibited by SB (Fig. 6B). The inhibition of this kinase by SB strongly impacted the secretion of cytokines triggered by TLR4. Cytokine secretion elicited by TLR2/6 or TLR1/2 tended to be reduced by SB but to a

FIGURE 2. Schematic representation of TLR4 signaling pathways. lesser extent (Fig. 6C, 6D). Therefore, the amplitude of glycolytic http://www.jimmunol.org/ Drug targets are indicated by T marks on a simplified view of TLR4 burst and of its inhibition by SB indicates that glycolysis regula- signaling pathways. tion by TLRs is unlikely to result from a common mechanism. Under the same conditions, we analyzed cells content in HIF-1a of LPS on glucose consumption (Fig. 4A). The other drugs did not and observed that TLR1/2 and TLR2/6 stimulation of MoDCs, by significantly reduce glucose consumption by LPS-stimulated cells. Pam or PGN, respectively, resulted in a weak accumulation of a The inhibitors showed no significant toxicity (Fig. 4B). These HIF-1 compared with TLR4 stimulation by LPS (Fig. 6E). SB a results indicate that p38–MAPK and HIF-1a are major actors of treatment did not modify the amount of HIF-1 upon TLR2/6 and the increased glucose consumption triggered by LPS and suggest TLR1/2, in contrast to TLR4 stimulation. In line with this ob-

a by guest on September 28, 2021 an unexpected link between them. By blocking dimerization of servation, the inhibition of HIF-1 transcriptional activity by MyD88 or TRIF, we observed that both TLR adaptors were Echino did not significantly impact glucose consumption triggered recruited to trigger the glycolytic burst upon TLR4 stimulation by PGN and Pam (Fig. 7), suggesting that this pathway does not (Supplemental Fig. 2), pointing to a common downstream play a major role in glycolysis regulation by TLR2/6 and TLR1/2. pathway that may involve TRAF6, a key component upstream of As expected, glycolysis inhibition by 2-DG reduced glucose p38–MAPK. consumption triggered by PGN and Pam. The screening of other BX795 altered the secretion of cytokines and/or the expression inhibitors showed a weak contribution of TBK1 in the upregula- of phenotypic marker without affecting LPS-triggered regulation of tion of glucose consumption by PGN, whereas PI3K, AKT, and glycolysis (Figs. 3A–C, 4A). In contrast, treatment by the HIF-1a mTOR inhibition did not significantly alter the glycolytic burst antagonist Echino resulted in both reduced LPS-induced glyco- (Fig. 7). The screening did not allow the identification of a lytic activity and IL-8 secretion (Figs 3A, 4A), indicating that pathway involved in the glycolytic regulation upon TLR1/2 reduced glycolytic burst may contribute to the reduction of cyto- stimulation by Pam. a kines secretion or maturation markers expression. Accordingly, Therefore, the regulation of HIF-1 stabilization upon PGN or we previously reported that inhibition of the glycolytic burst Pam stimulation did not depend on p38–MAPK activation. TLR2/6 by 2-deoxyglucose (2-DG) treatment affected both cytokine and TLR1/2 stimulation resulted in a slight accumulation of a secretion and phenotypic marker expression induced by TLR4 p38-independent HIF-1 and increased glucose consumption. In stimulation (27). contrast, TLR4 regulation of glycolysis in human MoDCs impli- cates p38-MAPK–dependent stabilization of HIF-1a (Fig. 8). LPS-induced HK2 expression requires p38-MAPK–dependent HIF-1a stabilization Discussion MoDC stimulation by LPS resulted in a strong increase of HK2 The present study aimed at characterizing the molecular mecha- expression that can be detected as soon as 6 h after LPS addition nisms involved in the metabolic reprogramming of human MoDCs (Fig. 5A). At this early time, HK3 expression was unchanged triggered by TLR4 stimulation. We showed that TLR4-dependent compared with control cells, whereas it was decreased 24 h glycolysis activation in these cells was correlated to an increased poststimulation (Fig. 1D). HK1 remained constant (Fig. 5A). global HK activity and an increased expression of HK2. HIF-1a,a Using the inhibitors that reduced glucose consumption by LPS- master transcriptional regulator of glycolytic enzymes, accumu- treated MoDCs, we found that SB and Echino strongly reduced lated in TLR4-stimulated MoDCs. An inhibitor of the transcrip- HK2 transcription induced by LPS (Fig. 5A). SB treatment of tional activity of HIF-1a inhibited both TLR4-induced HK2 MoDCs inhibited the phosphorylation of p38–MAPK without expression and glucose consumption. Data are highlighting the affecting the phosphorylation of NF-kB and JNK, as expected role of p38–MAPK activation in HIF-1a stabilization and (Fig. 5B). HIF-1a is an important transcription factor for the glycolysis regulation upon TLR4 stimulation. Our study has 6 TLR4 GLYCOLYTIC BURST INVOLVES A p38–MAPK–HIF-1a AXIS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. Drug effects on DC phenotypic and functional activation by LPS. DCs were seeded at 1 3 106 cells/ml in fresh culture medium and treated at 37˚C for 30 min with 20 mM SB, 10 mM SP600125, 40 mM PD98059, 5 mM SC75741, 100 nM rapamycin, 5 mM BX795, 10 mM LY294002, 10 mM AZD5363, 10 nM Echino, or control solvent (solv) prior to stimulation with 10 ng/ml LPS for 24 h. Control cells (Ctl) received PBS instead of LPS. Cells and supernatants were collected. (A) Cytokine secretion was assayed in DC supernatants by Cytometric Bead Array. Data represents mean cytokine secretion from at least three independent experiments. (B and C) Surface expression of DC maturation markers was monitored by flow cytometry, and means 6 SEM of fluorescence intensity from at least three independent experiments are shown. analyzed in detail the molecular pathways of glycolysis regulation Previous results with mouse DCs showed that activation of TBK1- in this model of immunostimulatory DCs (44, 45). Malinarich IKKε and AKT kinases controlled the early increase in glycolysis et al. (22) compared the metabolic profile of tolerogenic DCs and triggered by TLR4 (24). In our experimental conditions, TBK1 immunostimulatory MoDCs and found that tolerogenic DCs had and AKT inhibitors did not impact the glycolytic activity of the highest glycolytic capacity upon ATP synthase inhibition, LPS-stimulated MoDCs (Fig. 4A). Moreover, no phosphorylation whereas they had a similar glycolytic rate to that of LPS- of AKT could be detected after LPS stimulation of MoDCs stimulated MoDCs. Similarly to us, they found that LPS stimu- (cf. Supplemental Fig. 3). Pam induced AKT phosphorylation lation enhanced lactate secretion and glycolytic rate of MoDCs (cf. Supplemental Fig. 3); however, inhibiting PI3K or AKT kinases without affecting glucose uptake. did not significantly alter the glycolytic reprogramming of MoDCs The Journal of Immunology 7

Previous works indicated that LPS stimulation of mouse BM-DCs and human MoDCs resulted in HIF-1a accumulation (14, 18, 19). In normoxia, HIF-1a degradation is regulated by hydroxylation of proline and asparagine residues by prolyl- hydroxylase domain enzymes (PHDs). The interaction of the von Hippel–Lindau factor with these hydroxylated residues recruits an E3 ubiquitin–ligase that targets HIF-1a to the proteasome for degradation (48). Hypoxia induces HIF-1a accumulation via PHD inhibition due to the lack of its oxygen cosubstrate (18). However, data describing the molecular mechanisms controlling HIF-1a accumulation in primary cells upon inﬂammation in normoxic conditions are sparse. In murine macrophages, LPS- induced HIF-1a accumulation seems to require NF-kB– and ERK-dependent transcriptional events (49, 50). In other studies, reactive oxygen species production and succinate accumulation upon LPS stimulation inhibit PHD activity, thus increasing HIF- 1a stability (51, 52). In murine BM-DCs, sequestration of iron, a PHD cofactor, stabilizes HIF-1a upon LPS stimulation (18). The

AKT–mTOR–HIF-1a pathway was found to upregulate glycolysis Downloaded from in murine monocytes (20). In human MoDCs, our results point to a different molecular mechanism involving a p38-MAPK–dependent HIF-1a stabilization. This is crucial to the glycolytic regulation upon TLR4 activation, but p38 activity is not involved in the stabilization of HIF-1a upon TLR2/6 stimulation. Although p38–MAPK

phosphorylation is induced by TLR4, TLR1/2, and TLR2/6 http://www.jimmunol.org/ (Fig. 6B), the consequence on HIF-1a accumulation varies. This differential regulation points to an indirect control of HIF-1a stability by p38–MAPK. The molecular mechanism explaining the differential regulation of HIF-1a accumulation in MoDCs according to TLR stimulation remains to be discovered. In tumor cells, previous studies showed that p38–MAPK could contribute to FIGURE 4. LPS-triggered increase in glucose consumption involves HIF-1a stabilization (53, 54). One possible mechanism proposed p38–MAPK activation and HIF-1a transcriptional activity. DCs were by Khurana et al. (55) in mouse embryonic fibroblasts is that the seeded at 1 3 106 cells/ml in fresh culture medium. Cells were treated at RING finger ubiquitin ligase Siah2 can be phosphorylated by p38– by guest on September 28, 2021 37˚C for 30 min with 20 mM SB, 10 mM SP600125, 40 mM PD98059, MAPK, increasing the degradation of PHD3, therefore reducing 5 mM SC75741, 100 nM rapamycin, 5 mM BX795, 10 mM LY294002, HIF-1a degradation. 10 mM AZD5363, 10 nM Echino, or control solvent prior to stimulation with In both mouse and human models, HIF-1a stabilization alone A 10 ng/ml LPS for 24 h. ( ) Glucose consumption was monitored using was not sufficient to induce DC maturation, but it positively enzymatic detection kits. To measure the effect of inhibitors, results were 6 contributes to their functionality upon TLR stimulation (14, 19). normalized to control LPS–treated cells from the same experiment. Means a SEM from 3 to 11 independent experiments are shown. (B) Cells were In mouse BM-DCs, HIF-1 induction by the combined effects of collected and analyzed by flow cytometry for propidium iodide labeling. LPS and hypoxia plays a key role in the regulation of glucose consumption, DC maturation, and their ability to stimulate allo- geneic T cells (14). In human MoDCs, HIF-1a stabilization could upon TLR1/2 or TLR2/6 stimulation (Fig. 7). TLR2/6 stimulation by synergize with TLR stimulation to favor maturation (19). PGN induced an important increase of glucose consumption by The HIF-1a DNA-binding inhibitor, Echino, reduced the se- MoDCs, which was significantly reduced by TBK1 inhibition (Fig. 7). cretion of IL-8 by LPS-stimulated DCs (Fig. 3A). IL-8 secretion This is consistent with recent data showing that TBK1 is involved in highly depends on enhanced gene transcription, and IL-8 promoter TLR2 signaling (46). Our results indicate that TBK1 contributed to mainly recruits NF-kB, AP-1, and NRF (56) but does not contain this metabolic regulation by mechanisms different from those de- HIF response elements. The reduction of IL-8 secretion by Echino scribed for murine BM-DCs (24) because AKT was not involved. treatment may thus result from the limitation of the glycolytic Thus, glycolysis activation by TLR4, TLR2/6, and TLR1/2 appears to burst of MoDCs. Indeed, previous results indicate that HK inhi- engage distinct signaling pathways in human MoDCs. bition by 2-DG suppressed increased glycolysis and reduced the Major differences in the regulation of glycolytic activity have secretion of many cytokines, and especially IL-8, upon LPS- been reported between mouse and human DCs. mTOR was shown stimulation (27, 29). By reducing the glycolytic burst triggered to be an important regulator of LPS-induced murine DC activation by LPS, Echino may impede optimal maturation and especially and glucose consumption, but not in human myeloid DCs (47). In impact maturation processes that require active gene transcription, agreement with these results, we observed that the mTORC1 in- such as cytokine neosynthesis. Reduced expression of phenotypic hibitor rapamycin did not impact the upregulation of glycolysis maturation markers of MoDCs was observed with 2-DG, which upon LPS stimulation in MoDCs (Fig. 4). Moreover, human strongly inhibits glycolysis (27), but not with Echino, which only MoDCs and macrophages do not produce any NO (27, 31), an partially reduces LPS-induced glycolytic burst (Fig. 3B, 3C). inhibitor of the oxidative phosphorylation that was shown to boost Glycolysis inhibition by 2-DG strongly reduced the secretion the glycolytic pathway in mouse DCs (25). Therefore, results of inflammatory cytokines and altered the expression of some obtained from murine BM-DCs or macrophages cannot be readily phenotypic maturation markers triggered by PGN and Pam extrapolated to human DCs. (Supplemental Fig. 4), reinforcing previous results highlighting 8 TLR4 GLYCOLYTIC BURST INVOLVES A p38–MAPK–HIF-1a AXIS Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. LPS-induced HK2 expression requires p38-dependent HIF-1a stabilization. DCs were seeded at 1 3 106 cells/ml in fresh culture medium. Cells were treated at 37˚C for 30 min with 20 mM SB or 10 nM Echino prior to stimulation with 10 ng/ml LPS for 6 h (A), 15 min (B), or 24 h (C and D). (A) Gene expression was analyzed by quantitative RT-PCR and normalized to housekeeping gene TBP expression. Results are presented as fold induction to control (Ctl) cells that received solvent. (B) After fixation and permeabilization, cells were labeled for phospho-p38–MAPK (pp38), phospho–NF-kB(pNF-kB), or phospho-JNK (pJNK) and analyzed by flow cytometry. (C and D) Cell lysates were analyzed by Western blot to detect HIF-1a and actin. Chemiluminescent signals from three independent experiments were quantified, and means 6 SEM are shown. the importance of the glycolytic burst for TLR-induced DC mat- metabolic state (11). The signaling of many receptors can result uration (27, 29). Reduction by Echino of both the glycolytic burst in the regulation of metabolic pathways by controlling the ac- (Fig. 7) and the secretion of proinflammatory cytokines tivation of transcription factors and the expression of metabolic (Supplemental Fig. 4) triggered by PGN and Pam was not statis- enzymes. Some transcription factors such as HIF-1a regulate the tically significant. Although we cannot totally exclude a contri- expression of both metabolic enzymes and proinflammatory bution of HIF-1a, this pathway did not appear to play a major role cytokines such as IL-1b in mouse M1 macrophages (52) and in glycolysis regulation by TLR2/6 and TLR1/2. TNF-a in human monocytes (20). The concentration of metab- Increasing evidence indicates that metabolic pathways play olites varying according to the metabolic state of the cells can important roles in the fine tuning of immune cell functions. also impact their immune function. These metabolic signals can Detailed studies have been conducted on T cell differentiation and be detected by various sensors and are important regulators of mouse macrophage functional polarization according to their inflammation (57). The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. p38-MAPK–dependent HIF-1a stabilization is not engaged by TLR2/6 or TLR1/2 stimulation. DCs were seeded at 1 3 106 cells/ml in fresh culture medium. Cells were treated at 37˚C with 20 mM SB or control solvent (Ctl) 30 min prior to stimulation with 10 ng/ml LPS, 10 mg/ml PGN, or Pam or PBS for 15 min (B)or24h(A and C–E). (A) Glucose consumption was monitored using enzymatic detection kits. Means 6 SEM from seven independent experiments are shown. (B) After fixation and permeabilization, cells were labeled for phospho-p38–MAPK (pp38) and analyzed by flow cytometry. (C and D) Cytokine secretion was assayed in DC supernatants by Cytometric Bead Array. Data represent mean cytokine secretion from at least five independent experiments. (E) Cell lysates were analyzed by Western blot to detect HIF-1a and actin. Spliced image is shown from a single experiment and a single exposition. Chemiluminescent signals from three independent experiments were quantified, and means 6 SEM are shown. 10 TLR4 GLYCOLYTIC BURST INVOLVES A p38–MAPK–HIF-1a AXIS

FIGURE 7. Screening of inhibitor effects on glucose consumption upon TLR2/6 (PGN) or TLR1/2 (Pam) ligands. DCs were seeded at 1 3 106 cells/ml in fresh culture medium and treated at 37˚C for 30 min with 40mM PD68059, 10 mM SP600125, 5 mM SC75741, 100 nM rapamycin, 5 mM BX795, 10 mM LY294002, 10 mM AZD5363, 10 nM Echino, 5 mM 2DG, or control solvent (solv) prior to stimulation with 10 mg/ml PGN or Pam for 24 h. Glucose consumption was monitored using enzymatic detection kits. Results were normalized to control ligand-stimulated cells from the same experiment. Means 6 SD from two to ﬁve independent experiments are shown. *p , 0.05 compared to the solvent.

Our results also show that the increased HK activity after 24 h adipose tissue, and skeletal muscle, and it is also upregulated in Downloaded from stimulation by TLR4 of human MoDCs is correlated to increased many types of tumors associated with enhanced aerobic gly- HK2 gene expression, whereas HK3 was downregulated, HK1 did colysis (Warburg effect), suggesting that it is a major regulator of not change, and HK4 was not expressed. These results differ from the glycolytic rate in these cells (59). HK-II activity is upregu- those obtained in murine bone marrow–derived macrophages, in lated by its binding to the outer mitochondrial membrane protein which HK1 but not HK2 was upregulated via mTORC1 activation voltage-dependent anion channel 1 (VDAC1). Our results indi- upon LPS and ATP stimulation (58). This difference, however, is cate that HK2 upregulation also plays a key role in metabolic http://www.jimmunol.org/ in agreement with the lack of effect of the mTORC1 inhibitor regulation of human primary DCs. Although the molecular rapamycin on glycolysis upregulation in MoDCs. HKs catalyze mechanisms triggered by TLR4 to stimulate glycolysis are dif- the first step of glucose metabolism, producing glucose 6-phos- ferent in humans MoDCs and mouse BM-DCs, they can both phate, which is a precursor for glycolysis but also for the pentose involve HK-II protein modulation (24). In the future, HK-II in- phosphate and hexosamine biosynthetic pathways. Thus, HK plays tracellular localization should be investigated in MoDCs in re- a key role in glycolysis and biosynthetic pathways. HK-II is the sponse to TLR4 stimulation. Moreover, the contribution of other predominant isoenzyme in insulin-sensitive tissues such as heart, nonenzymatic functions of HK-II should also be analyzed. In- deed, overexpression and mitochondrial association of HK-II confer protection to apoptotic or necrotic stimuli in different by guest on September 28, 2021 cell types by several mechanisms (59). Increased expression of HK2 upon LPS stimulation may contribute to prosurvival effects of LPS stimulation of MoDCs. HK-II, but not other HKs, bind and inhibit mTORC1 in the absence of glucose, facilitating autophagy in response to glucose starvation. Thus, HK-II can protect cells from cellular damage and provide energy by recy- cling intracellular constituents (60). Metabolomics of immune cells recently established that TLR4 induces glycolytic reprogramming and a drastic rewiring of tricarboxylic acid cycle to generate inflammatory intermediates that contribute to macrophages and DCs activation. Dysregulated TLR4-triggered inflammatory response can be involved in pathologic conditions such as sepsis (61), neuropathic pain (62), amyotrophic lateral sclerosis (63), some autoimmune diseases such as rheumatoid arthritis (64), or obesity-associated type 2 diabetes (65). Our results unravel a novel molecular mechanism linking TLR4 signaling and glycolysis activation and, together with previous reports, reinforce the idea that a better understanding of mechanism underlying TLR4-induced glycolytic reprogramming should offer innovative therapeutic windows.

Acknowledgments We acknowledge the contribution of Me´lanie Tonnerre for technical assis- FIGURE 8. TLR4 major pathway to induce a glycolytic burst in human tance, of the Etablissement Français du Sang Auvergne-Rhoˆne-Alpes, and Mo-DCs. The molecular actors involved in the glycolytic burst triggered SFR Biosciences (UMS3444/CNRS, US8/Inserm, ENS de Lyon, Univer- by TLR4 in human MoDCs are indicated in gray. LPS stimulation of the site´ Claude Bernard Lyon 1) facility AniRA-Cytome´trie. TLR4 signaling pathway resulted in the phosphorylation of p38–MAPK that plays a major role in the accumulation of HIF-1a. HIF-1a strongly enhanced the expression of HK2. HK activity and glycolytic activity were Disclosures increased in LPS-stimulated MoDCs. The authors have no financial conflicts of interest. The Journal of Immunology 11

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(pg/ml) 20

15 IFN 10 5 0 LPS 0 10 100 0 10 100 (ng/ml) solv FP7

Supplementary Fig S1: FP7 antagonizes LPS-induced IFNβ secretion. DCs were seeded at 1 x 106 cells/ml in fresh culture medium, treated at 37°C with 10 µM FP7 or control solvent (solv) 30 min prior to stimulation with increasing amounts of LPS for 24h. IFNβ secretion was assayed in DC supernatants by high sensitivity ELISA kit (PBL Assay Science, Piscataway, NJ, USA). Data represents mean cytokine secretion from 2 independent experiments.

Supplementary Fig S2: Inhibition of MyD88- and TRIF-dependent signaling prevents LPS- induced glucose consumption increase. DCs were seeded at 1 x 106 cells/ml in fresh culture medium, pretreated at 37°C with the indicated doses of inhibitory peptides pepinh MYD, pepinh TRIF or control pepinh (Ctl) for 6h prior stimulation with 10 ng/ml LPS for 24h. Cells and supernatants were collected. (A) Glucose consumption was monitored using enzymatic detection kits. (B) Cytokine secretion was assayed by Cytometric Bead Array. (C-D) Surface expression of DC maturation markers was monitored by flow cytometry. Means ± SEM with non-toxic doses of pepinh are shown. *p<0.05;***p<0.001 compared to pepinh Ctl.

Supplementary Fig S3: Pam but not PGN nor LPS induces AKT phosphorylation. DCs were seeded at 1 x 106 cells/ml in fresh culture medium. Cells were treated at 37°C with 10 ng/ml LPS, 10 µg/ml PGN or 10 µg/ml Pam or PBS (Ctrl) for 15 min. Cells were fixed, permeabilized and labeled with anti-phospho-AKT (pAKT). Cells were analyzed by flow cytometry.

Supplementary Fig S4: Effect of Echinomycin and 2-DG on DC cytokine secretion and phenotype upon TLR1/2 and 2/6 stimulation. DCs were seeded at 1 x 106 cells/ml in fresh culture medium. Cells were treated at 37°C for 30 min with 10 nM Echinomycin (Echino) or 2,5 mM 2-deoxy-glucose (2-DG) or control solvent prior to stimulation or not (Ctrl) with 10 µg/ml PGN or Pam for 24h. (A) Cytokine secretion was assayed in DC supernatants by Cytometric Bead Array. Data represents mean cytokine secretion ± SEM from at least 3 independent experiments. (B-C) Surface expression of DC maturation markers was monitored by flow cytometry and means ± SEM of fluorescence intensity from at least 3 independent experiments are shown. * p<0,05 compared to PGN or Pam-stimulated cells, paired t-test.