High Mitochondrial Respiration and Glycolytic Capacity Represent a Metabolic Phenotype of Human Tolerogenic Dendritic Cells This information is current as of October 1, 2021. Frano Malinarich, Kaibo Duan, Raudhah Abdull Hamid, Au Bijin, Wu Xue Lin, Michael Poidinger, Anna-Marie Fairhurst and John E. Connolly J Immunol 2015; 194:5174-5186; Prepublished online 27 April 2015; Downloaded from doi: 10.4049/jimmunol.1303316 http://www.jimmunol.org/content/194/11/5174 Supplementary http://www.jimmunol.org/content/suppl/2015/04/25/jimmunol.130331 http://www.jimmunol.org/ Material 6.DCSupplemental References This article cites 35 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/194/11/5174.full#ref-list-1 Why The JI? Submit online. by guest on October 1, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology High Mitochondrial Respiration and Glycolytic Capacity Represent a Metabolic Phenotype of Human Tolerogenic Dendritic Cells Frano Malinarich,*,† Kaibo Duan,† Raudhah Abdull Hamid,*,† Au Bijin,*,† Wu Xue Lin,*,† Michael Poidinger,† Anna-Marie Fairhurst,† and John E. Connolly*,†,‡ Human dendritic cells (DCs) regulate the balance between immunity and tolerance through selective activation by environmental and pathogen-derived triggers. To characterize the rapid changes that occur during this process, we analyzed the underlying met- abolic activity across a spectrum of functional DC activation states, from immunogenic to tolerogenic. We found that in contrast to the pronounced proinflammatory program of mature DCs, tolerogenic DCs displayed a markedly augmented catabolic pathway, related to oxidative phosphorylation, fatty acid metabolism, and glycolysis. Functionally, tolerogenic DCs demonstrated the highest mitochondrial oxidative activity, production of reactive oxygen species, superoxide, and increased spare respiratory capacity. Fur- Downloaded from thermore, assembled, electron transport chain complexes were significantly more abundant in tolerogenic DCs. At the level of gly- colysis, tolerogenic and mature DCs showed similar glycolytic rates, but glycolytic capacity and reserve were more pronounced in tolerogenic DCs. The enhanced glycolytic reserve and respiratory capacity observed in these DCs were reflected in a higher met- abolic plasticity to maintain intracellular ATP content. Interestingly, tolerogenic and mature DCs manifested substantially different expression of proteins involved in the fatty acid oxidation (FAO) pathway, and FAO activity was significantly higher in tolerogenic DCs. Inhibition of FAO prevented the function of tolerogenic DCs and partially restored T cell stimulatory capacity, demonstrating http://www.jimmunol.org/ their dependence on this pathway. Overall, tolerogenic DCs show metabolic signatures of increased oxidative phosphorylation pro- graming, a shift in redox state, and high plasticity for metabolic adaptation. These observations point to a mechanism for rapid genome-wide reprograming by modulation of underlying cellular metabolism during DC differentiation. The Journal of Immu- nology, 2015, 194: 5174–5186. endritic cell (DC) maturation status plays a key role in processes require energetic metabolic adaptations important for regulating the balance between immunity and tolerance DC survival, migration, and ultimately the development of pathogen- D (1, 2). DC maturation is a tightly coordinated response specific immunity. Understanding the metabolic changes underpin- by guest on October 1, 2021 involving multiple signaling pathways, molecular trafficking, cy- ning DC immune function could help develop new strategies for tokine production, and cytoskeletal remodeling (3–5). These controlling immunogenicity. Studies in mice have demonstrated that T cell activation and differentiation from naive into effector and memory T cells are processes highly dependent on energetic *Institute of Molecular and Cell Biology, Agency for Science, Technology and metabolic adaptations. This knowledge is currently being con- Research, Singapore 138673; †Singapore Immunology Network, Singapore 138648; and ‡Institute of Biomedical Studies, Baylor University, Waco, TX 76798 sidered for potential novel immune therapies (6–8). However, knowledge surrounding the immune-metabolic states of DCs is Received for publication December 11, 2013. Accepted for publication March 18, 2015. rather limited. This work was supported by Agency for Science, Technology and Research Core Maturation of murine bone marrow–derived DCs (BMDCs) is Funding (to J.E.C.). highly dependent on metabolic changes. In the immature state, The microarray data presented in this article have been submitted to the National oxidative phosphorylation (OXPHOS) and fatty acid oxidation Center for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi. (FAO) are the main energy sources. DC maturation coincides with nlm.nih.gov/geo/query/acc.cgi?token=arwhicsyrnchxmb&acc=GSE52894) under ac- cession number GSE52894. an increase in mitochondrial OXPHOS followed by a pronounced Address correspondence and reprint requests to Dr. John E. Connolly, Institute of switch to aerobic glycolysis (9–11). Importantly, pharmacological Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Room 7-17B, Singapore inhibition of glycolysis prevents BMDC maturation, suggesting 138673. E-mail address: [email protected] the importance of this metabolic switch for DC immunogenicity The online version of this article contains supplemental material. (10, 11). Abbreviations used in this article: ACADM, acyl-CoA dehydrogenase, C-4 to C-12 Consistent with the studies in mice, downregulation of OXPHOS straight chain; BMDC, bone marrow–derived DC; CM-H2DCFDA, 5-(and-6)-chlor- omethyl-29,79-dichlorodihydrofluorescein diacetate, acetyl ester; COXII, cyclooxy- gene expression has been reported during human monocyte-derived genase II; DC, dendritic cell; DEG, differentially expressed gene; 2-DG, 2-deoxy-D- DC (moDC) maturation (12). In contrast to mature moDCs, pro- glucose; ECAR, extracellular acidification rate; FAO, fatty acid oxidation; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; HADHA, hydroxyacyl-CoA teomic analysis of tolerogenic moDCs, which are resistant to dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, a subunit; HADHB, maturation (13), has shown a predominance of OXPHOS pathway hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase, b subunit; iNOS, inducible NO synthase; IPA, ingenuity pathway analysis; LDHB, lactate proteins (14). Therefore, a comparative analysis across the spec- dehydrogenase B; moDC, monocyte-derived DC; OCR, oxygen consumption rate; trum of moDCs is required to explain the metabolic changes be- OXPHOS, oxidative phosphorylation; ROS, reactive oxygen species; SO, superoxide; tween immunogenic and tolerogenic moDC states. SRC, spare respiratory capacity. In this study, we compared the changes in energetic metabolism Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 among human moDCs. Protein expression and functional analyses www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303316 The Journal of Immunology 5175 demonstrated that tolerogenic moDCs have a prominent and stable panel kits (Millipore), respectively, using a FLEXMAP 3D system OXPHOS program with high reactive oxygen species (ROS) and (Luminex). superoxide (SO) production, as well as a robust mitochondrial spare Mitochondrial DNA content determination respiratory capacity (SRC). Although tolerogenic and immature moDCs displayed similar levels of FAO activity, glycolytic ca- Total DNA from moDCs was isolated by phenol-chloroform extraction as pacity, and reserve, these catabolic pathways were maintained at previously described (15). Real-time PCR was performed in the Stratagene Mx3005P quantitative PCR system using the SYBR Green/ROX FAST a low level in mature moDCs. Finally, tolerogenic moDCs, in contrast Mastermix kit (Qiagen) with GAPDH (forward, 59-CCTAGGGCTGCT- to mature moDCs, demonstrate a high bioenergetic plasticity for CACATATTC-39, reverse: 59-CGCCCAATACGACCAAATCTA-39)and metabolic adaptation in terms of ATP production. cyclooxygenase II (COXII; forward: 59-GTTGTAGCCCACTTCCAC- TATG, reverse: TAGGGTGTAGCCTGAGAATAGG-39)primers.The mitochondrial/nuclear DNA content was calculated by using the formula: Materials and Methods 22CT mitochondrial COXII/22CT nuclear GAPDH. Data were analyzed by MxPro moDC generation quantitative PCR software (Agilent Technologies). Monocytes were purified from PBMCs of healthy donors (Institutional Real-time metabolic characterization Review Board approval NUS-IRB 10-250) by density centrifugation (Ficoll–Paque, GE Healthcare), followed by magnetic