Human Dendritic Cell Subsets Undergo Distinct Metabolic Reprogramming for Immune Response

Toll-like receptor (TLR) agonists induce metabolic reprogramming, which is required for immune activation. We have investigated mechanisms that regulate metabolic adaptation upon TLR-stimulation in human blood DC subsets, CD1c+ myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). We show that TLR-stimulation changes expression of genes regulating oxidative phosphorylation (OXPHOS) and glutamine metabolism in pDC. TLR-stimulation increases mitochondrial content and intracellular glutamine in an autophagy-dependent manner in pDC. TLR-induced glutaminolysis fuels OXPHOS in pDCs. Notably, inhibition of glutaminolysis and OXPHOS prevents pDC activation. Conversely, TLR-stimulation reduces mitochondrial content, OXPHOS activity and induces glycolysis in CD1c+ mDC. Inhibition of mitochondrial fragmentation or promotion of mitochondrial fusion impairs TLR-stimulation induced glycolysis and activation of CD1c+ mDCs. TLR-stimulation triggers BNIP3-dependent mitophagy, which regulates transcriptional activity of AMPKα1. BNIP3-dependent mitophagy is required for induction of glycolysis and activation of CD1c+ mDCs. Our findings reveal that TLR stimulation differentially regulates mitochondrial dynamics in distinct human DC subsets, which contributes to their activation.


INTRODUCTION
Dendritic cells (DCs) regulate the immune homeostasis and development of adaptive immune responses. In human peripheral blood, there are two main subsets of naturally circulating DCs, namely CD1c + myeloid dendritic cells (CD1c + mDCs) and plasmacytoid dendritic cells (pDC) (1,2). These subsets differ in function, localization, and phenotype. CD1c + mDCs are primarily localized in the marginal zone of the lymph nodes and confer immunity against bacteria and fungi (3,4) by inducing Th1 responses via the production of IL-12 (5,6). Conversely, pDCs localize to the T-cell areas in lymph nodes and are proficient in viral antigen recognition (7). Mature pDCs abundantly produce type I IFNs upon activation and induce T cell responses (2,8).
Under non-inflammatory conditions, DCs are poorly immunogenic. However, inflammatory stimuli or pathogenderived products trigger a group of pattern recognition receptors, including Toll-like receptors (TLRs), which results in a process of cellular activation, termed DC maturation, hence making them highly immunogenic (9). DC maturation is a tightly coordinated response, which involves various signaling pathways, molecular trafficking, cytokine production and cytoskeletal remodeling (10)(11)(12). These processes require metabolic adaptations, which are essential for DC survival, migration and eventually the development of immunity. DC activation upon TLR stimulation is associated with metabolic reprogramming and expression of genes encoding cytokines and chemokines, which promote immune response (13,14). Effector functions requires a glycolytic switch in mouse bone-marrow DCs cultured in GM-CSF (14,15), while lipid metabolism and OXPHOS are indispensable for murine pDC immune function (16).
Mitochondrial dynamics and bioenergetics are reciprocally coupled to adjust bioenergetic adaptation to metabolic needs of the cell (17). Mitochondrial dynamics are controlled by a group of dynamin-related GTPases, i.e., mitofusin 1 and 2 (Mfn1/2) and optic atrophy 1 (Opa1) for fusion and dynamin related protein 1 (Drp1) for fission (18). Mfn1 plays a crucial role in mitochondrial fusion, while Mfn2 is central to mitochondrial metabolism, by regulating mitochondrial membrane potential and the OXPHOS system (17). The balanced mitochondrial dynamics is critical for normal mitochondrial function, bioenergetics and quality control via mitophagy (19)(20)(21). Mitophagy is a process by which a cell removes damaged mitochondria to use them as additional fuels during stress (22,23). Upon stress or damage, mitochondria exhibit compromised metabolism, ATP production and reduction in membrane potential, which are characteristics of mitochondrial dysfunction and the initial trigger for mitophagy (24).
Understanding of metabolic changes underpinning human DC-subsets immune function are less known and insights into these changes can help develop new strategies for controlling immunogenicity. Given the distinct ontogeny and functional specializations of CD1c + mDC and pDC, we aimed at identifying metabolic adaptations engaged by human DC-subsets for effector function. We here demonstrate that TLR-stimulation in CD1c + mDC and pDC results in differential mitochondrial rewiring and metabolic adaptations. TLR stimulation results in increased glutaminolysis and OXPHOS in pDC, while it promotes mitophagy and glycolysis in CD1c + mDC. Notably, these metabolic adaptations are indispensable for activation of CD1c + mDC and pDC. Our data provides novel insights into subset-specific regulation of mitochondrial metabolism, which impacts DC function.

DC Isolation and Culture
For functional assays, DCs were isolated from buffy coats of healthy volunteers (Sanquin, Nijmegen, The Netherlands). Written informed consent per the Declaration of Helsinki and according to institutional guidelines, were obtained from healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were isolated by using Ficoll density centrifugation (Lymphoprep; Axis-Shield PoC AS, Oslo, Norway). CD1c isolation kit (Miltenyi Biotec, Bergisch-Gladbach, Germany) was used to isolate CD1c + mDCs, as per manufacturer's instructions. Next, monocytes were depleted by either plastic adhesion, or by the use of CD14 microbeads (Miltenyi Biotec). Consequently, pDCs were purified by positive selection using anti-BDCA-4-conjugated magnetic microbeads (Miltenyi Biotec). DCs were cultured in X-VIVO-15 medium (Lonza, Basel, Switzerland) supplemented with 2% human serum (Sanquin). DCs were stimulated with: pRNA (15 µg/ml) freshly prepared 5-10 min before adding to the cell culture. pDCs were cultured with IL-3 (10 ng/mL) (Cellgenix, Freiburg, Germany) as a survival factor in addition to the stimuli.
Protamine-RNA Complexes pRNA complexes were made freshly before adding to the cells. Protamine (protaminehydrochloride MPH 5000 IE/ml; Meda Pharma BV Amstelveen, The Netherlands) was diluted to 0.5 mg/ml in RNase free water and mixed with 2-kbp-long singlestranded mRNA (coding for gp100). It was extensively mixed and incubated for 5-10 min at room temperature, before added to the cells.

RNA Sequencing
Total RNA was isolated from CD1c + mDCs and pDCs using Trizol (Invitrogen, MA, USA). RNA sequencing and read alignment were performed by BGI TECH SOLUTIONS (Hong Kong). Reads were aligned to human genome version 19. RNA sequencing data is deposited at the Gene Expression Omnibus (GEO; accession number: GSE89442). Data was analyzed using the R platform package "edgeR, " version 3.12, to analyze whole transcriptome principal coordinates analysis (using the "plotMDS" command), differential expression analysis, and GO term analysis. Differential expression was determined by fitting a generalized linear model using the "glmFit" command, and significance was determined using the likelihood ratio test provided by the "glmLRT" command (27).

RESULTS
Mitochondrial Dynamics Is Differentially Regulated in CD1c + mDC and pDC Upon TLR7/8 Stimulation To investigate changes in metabolism, human CD1c + mDC and pDC were stimulated with a complex of protamine and mRNA (pRNA) that acts as a TLR7/8 ligand. pRNA has been shown to activate CD1c + mDCs and pDCs and induces them to release IL-12 and IFN-α, respectively (28). Previously, we analyzed the whole-transcriptome of human CD1c + mDC and pDC upon TLR7/8 stimulation (27). Our data demonstrated that pRNA upregulated cytokines and migration-related genes in CD1c + mDCs as well as type I and III interferons (IFN-α and IFN-λ) related genes in pDC. Moreover, we demonstrated that pRNA stimulation increased expression of maturation markers (i.e., CD80, PD-L1 & CD40) in both CD1c + mDC and pDC, in addition to increase in immunostimulatory cytokines i.e., TNFα and INFα for CD1c + mDC and pDC, respectively (27). To investigate whether changes in metabolism are required for human DC-subsets immune response, we analyzed expression of OXPHOS related genes in human CD1c + mDC and pDC. OXPHOS related genes were significantly downregulated in CD1c + mDCs upon pRNA-stimulation ( Figure 1A). Conversely pRNA-stimulation increased expression of NDUFAF1, NDUFA9, COX7A2, ATP5H, and ATP6V1F in pDC ( Figure 1B) suggesting up-regulation of OXPHOS in pDC.
Based on these findings, we hypothesized that TLR7/8stimulation alters mitochondrial content in CD1c + mDC. To test this, CD1c + mDC were stained with MitoTracker TM Green FM, a fluorescent dye that localizes to mitochondria in a mitochondrial membrane potential independent manner. Indeed, TLR7/8stimulation significantly decreased mitochondrial content in CD1c + mDC ( Figure 1D). By comparison, staining of pDC with MitoTracker TM Green FM showed a significant increase in mitochondrial content upon TLR7/8-stimulation ( Figure 1D) consistent with increased Mfn2 and PGC1α levels. To confirm the involvement of mitochondrial dynamics in regulating mitochondrial mass, we stimulated CD1c + mDC with pRNA in the presence or absence of a fusion promoter (15oxospiramilactone, S3) (38) or a fission inhibitor (Mdivi-1) (39). Interestingly, S3 increased Mfn2 expression and Mdivi-1 reduced both endogenous and pRNA-induced Drp1 levels in CD1c + mDC (Figures 1E,F). Of note, S3 and Mdivi-1 significantly prevented loss of mitochondrial content in CD1c + mDC upon TLR7/8-stimulation ( Figure 1G). Collectively, these data indicate that TLR7/8-stimulation results in mitochondrial fragmentation and reduced mitochondrial content in CD1c + mDCs and increased mitochondrial biogenesis, fusion and content in pDCs.

pDC Stimulated via TLR7/8 have Increased Glutaminolysis and OXPHOS Which Are Crucial For Activation
We next asked whether increased mitochondrial fusion and content along with upregulation of NDUFA10 and OXPHOS related genes in TLR7/8-stimulated pDCs was associated with metabolic changes. OXPHOS is driven by NADH and FADH 2, produced by the tricarboxylic acid (TCA) cycle (40,41) and the amino acid glutamine is among the key metabolites that support the TCA cycle. Glutaminolysis is a metabolic pathway, which requires deamination of glutamine by glutaminase (GLS), generating glutamate, which in turn is converted to α-KG, a TCA cycle intermediate (42,43). To determine whether glutaminolysis contributes to increased OXPHOS upon TLR7/8stimulation in pDC, we examined expression of genes related to amino acid metabolism. pRNA-stimulation significantly increased expression of GLS and SLC1A3 in pDC (Figure 2A). GLS catalyzes the conversion of glutamine to glutamate (44) while SLC1A3 is a glutamate transporter (45,46). Upregulation of these genes suggests increased glutaminolysis in pDCs upon TLR-stimulation. To test this, we measured intracellular glutamine levels in pDC. pRNA-stimulation significantly increased intracellular glutamine in pDC, which could be inhibited by 6-Diazo-5-oxo-L-norleucine (DON) (Figure 2B), a glutamine antagonist, which inhibits glutamine utilizing enzymes by irreversible alkylation of L-cysteinyl residues (47).
Notably, extracellular flux analysis (EFA) revealed increased basal oxygen consumption rate (OCR), maximal OCR ( Figure 2C; Supplementary Figures 1B,C), ATP-linked OCR, mitochondrial OCR and spare respiratory capacity (SRC) in pRNA-stimulated pDC compared to unstimulated pDC (Figures 2D-F). To explore whether increased OXPHOS activity in pRNA-stimulated pDC is due to increased glutaminolysis, we pharmacologically attenuated Glutaminase, an enzyme responsible for conversion of glutamine into glutamate. pDC were stimulated with pRNA in the presence or absence of BPTES, a chemical inhibitor of GLS. BPTES inhibited in pDC the pRNA-induced increase in basal OCR (Figure 2C;  Supplementary Figures 1A-C), ATP-linked OCR (Figure 2D), maximal OCR (Supplementary Figure 1C) mitochondrial OCR ( Figure 2E) and SRC ( Figure 2F). These results indicate that pRNA stimulation of pDC results in increased OXPHOS due to increased glutaminolysis. Intriguingly, we did not observe an increase in ECAR (Supplementary Figure 1D) and 2-NBDG uptake ( Figure 2G) upon pRNA-stimulation.
We next asked whether these metabolic changes are required for pDC activation. Activation of these cells was assessed by measuring secretion of immunostimulatory cytokine IFNα and membrane expression of co-stimulatory molecule CD80 and coinhibitory molecule PD-L1. A reduced secretion of IFNα by pRNA-stimulated in pDC was observed when Rotenone (ROT), Antimycin A (AA), BPTES and DON were added to the culture medium ( Figure 2H). Addition of these factors also significantly reduced the pRNA-mediated upregulation of CD80 and PD-L1 on pDC ( Figure 2I). By comparison, we observed no effect of ROT, AA, BPTES and DON on pRNA-stimulated TNFα (Supplementary Figure 3B) and CD80 and PD-L1 in CD1c + mDC (Supplementary Figure 3C).
Of note, TLR stimulation triggers autophagy in pDC, which is required to produce type I IFN (48)(49)(50)(51)(52). Consistently, we observed significant increase in autophagosomes upon pRNA-stimulation in pDC ( Figure 3A). Intriguingly, autophagy has been reported to supply metabolic substrates to preserve mitochondrial function (53)(54)(55)(56)(57). We hypothesized that increased glutamine and glutaminolysis in TLR7/8stimulated pDCs is provided by autophagy. To investigate this, autophagy inhibitor 3-MA was added during the pRNA stimulation of pDC. 3-MA significantly reduced the pRNAinduced increase in glutamine levels in pDC ( Figure 3B). Consistently, 3-MA significantly reduced pRNA-induced increase in basal OCR ( Figure 3C; Supplementary Figure 1E Figure 1H) and mitochondrial OCR (Figure 3D) indicating the requirement of autophagy for optimal induction of OXPHOS upon TLRstimulation of pDC. Notably, 3-MA significantly reduced both IFNα secretion ( Figure 3E) as well as expression of CD80 and PD-L1 upon pRNA-stimulation of pDCs ( Figure 3F). Since, TLR7/8 stimulated pDC activation was prevented by pharmacological attenuation of OXPHOS, glutaminolysis and autophagy, we next asked whether the observed reduction was due to effect on cell viability. Analysis of cell viability revealed that BPTES, DON, 3-MA, ROT and AA did not affect viability of pDC alone or in combination with pRNA (Supplementary Figures 7,  8). Together, these data show that TLR7/8-stimulated pDC activation requires autophagy-supplemented glutaminolysis to fuel OXPHOS.

TLR7/8 Stimulated Alterations in Mitochondrial Dynamics Triggers Glycolysis Which Is Required For CD1c + mDC Activation
Our data show that TLR7/8-stimulation reduces expression of OXPHOS related genes and mitochondrial content in CD1c + mDCs, which is associated with metabolic changes with a shift toward glycolysis (58) to compensate for the reduced activity of the respiratory chain to generate ATP (17). In this sense, we wondered whether mitochondrial alterations induced by TLR7/8-stimulation led to a metabolic shift in CD1c + mDC. To this end, analysis of glycolysis related genes showed significant upregulation of ENO2 ( Figure 4A). ENO2 encodes a dimeric enzyme, Enolase, which catalyzes the second last step in glycolysis i.e., interconverting 2phosphoglycerate (2-PGA) and phosphoenolpyruvate (PEP) (59). Next, we monitored EFA in pRNA-stimulated CD1c + mDC. We found that TLR7/8-stimulation significantly reduced OCR (Figure 4B; Supplementary Figure 2C). To test our hypothesis that mitochondrial fragmentation leads to induction of glycolysis in CD1c + mDC upon TLR7/8-stimulation, we monitored EFA in the presence of S3 and Mdivi-1. Interestingly, S3 and Mdivi-1 significantly prevented the pRNA-induced decrease in OCR ( Figure 4B; Supplementary Figures 2A-C), SRC (Figure 4C), mitochondrial OCR ( Figure 4D) ATP-linked OCR ( Figure 4E) and maximal OCR (Supplementary Figure 2D) in CD1c + mDCs.
To investigate the induction of glycolysis, we monitored pRNA-induced ECAR in CD1c + mDC. Importantly, pRNA stimulation significantly increased ECAR in CD1c + mDC ( Figure 4F). Of note, S3 and Mdivi-1 significantly reduced the pRNA-induced increase in ECAR (Figure 4F), indicating that indeed mitochondrial fragmentation induced by TLR7/8stimulation leads to a shift toward glycolysis in CD1c + mDC.
To specify the involvement of BNIP3, the effect of olomoucine, a transcriptional inhibitor of BNIP3 (69) on TLR7/8-induced mitophagy in CD1c + mDC was examined. Olomoucine significantly reduced steady state BNIP3 ( Figure 5D) and the pRNA-induced increase of BNIP3 in CD1c + mDC ( Figure 5D). Niclosamide is a transcriptional inhibitor of S100A4 (70), which is transcriptional repressor of of BNIP3 (71). Niclosamide increased BNIP3 expression in CD1c + mDC ( Figure 5D). To quantitatively asses mitophagy in CD1c + mDC cells, we employed flow cytometry based method (72). This approach is suitable to robustly assess Frontiers in Immunology | www.frontiersin.org mitophagy without need to perform traditional fluorescence microscopy of mitochondrial-autophagosome colocalization in BNIP3 transfected cells, in order to avoid transfection and prolonged culture-induced cell death in rare human CD1c + mDC cells. The reversal in alteration in MitoTracker upon mitophagy inhibitors (i.e., olomoucine and 3-MA) indicates induction of mitophagy and can be used to calculate mitophagic flux (72). Of note, loss of pRNA-induced mitochondrial content in CD1c + mDC cells was significantly potentiated by niclosamide, which augments BNIP3 expression (Figure 5E). On other hand, loss of pRNA-induced mitochondrial content in CD1c + mDC cells was significantly reversed by olomoucine and 3-MA ( Figure 5E) indiacating induction of mitophagy. Furthermore, analysis of mitophagic flux, revealed that pRNA stimulation significantly increased mitophagic flux in CD1c + mDC ( Figure 5F). This data indicates that TLR7/8stimulation triggers BNIP3-dependent mitophagy in CD1c + mDC cells.

DISCUSSION
Changes in metabolism following TLR stimulation are indispensable for DC activation. However, the metabolic signature generated in naturally occurring human DCs in response to TLR-stimulation is not known in detail. Herein, we investigated TLR-induced metabolic changes in two human blood DC-subsets, CD1c + mDC and pDC. Our data show that TLR stimulation results in a differential mitochondrial rewiring in pDC and CD1c + mDC. We have focused on mitochondria as metabolic hubs critical for signals downstream of innate receptors in myeloid cells (75). Promotion of mitochondrial fusion results in increased OXPHOS activity via formation of supercomplexes (76). Supercomplex reorganization in macrophages is also driven by innate sensing of microbes, regulating macrophage cytokine production (77). Conversely, mitochondrial fission results in decreased OXPHOS activity and induction of glycolysis (21). Interestingly, mitochondrial dynamics play an important role in differentiation and migration of immature DC (78). Mitochondrial fusion proteins are upregulated during differentiation of bone marrow progenitors to immature DC. Mitochondrial fusion-related proteins i.e., Mfn2 and Opa1 have been shown to be required for migration of immature DC (78).
Here, we investigated the role of mitochondrial dynamics in regulating immune function of human DC subsets. We find that stimulation of pDCs with TLR7/8 agonist increases expression of PGC1α and Mfn2, which suggests increase in mitochondrial mass. Indeed, we observed that TLR7/8-stimulation resulted in increased mitochondrial mass in pDC, as demonstrated by MitoTracker Green and Porin levels. Moreover, PGC-1α positively regulates mitochondrial fusion by stimulating Mfn2 expression via targeting the Mfn2 promoter in an ERRα-binding element-dependent manner (79). Importantly, increased Mfn2 expression results in increased glucose oxidation and expression of OXPHOS complex I, IV and V (80). Consistently, we observed increased expression of OXPHOS related genes and protein levels of NDUFA10 upon TLR7/8-stimulation in pDC, indicating upregulation of OXPHOS. Taken together, these data indicate that TLR7/8 stimulation increases mitochondrial fusion, mass and increased OXPHOS activity in pDC. Conversely, pRNA stimulation of CD1c + mDCs results in increased expression of Drp1, which contributes to mitochondrial fission (81,82), which lead to decrease in mitochondrial mass as shown by decreased levels of MitoTracker Green and Porin. Mitochondrial fission promotes a shift to aerobic glycolysis (58,83,84). Our data shows that TLR-stimulation leads to increased glycolysis in CD1c + mDC. Increased expression of Drp1 together with decreased expression of NDUFA10 and mitochondrial mass, in CD1c + mDC indicates induction of mitochondrial fission, which is linked to glycolysis (21,85,86). Intriguingly, Drp1 has been demonstrated to be required for the activation of bone marrow-derived DCs upon LPS-stimulation (87). It has been reported that TLR-stimulated metabolic reprogramming is required to meet the energy demand for the activation process in DC (14,16,88). Of note, our data show that mitochondrial dynamics modulate expression of inflammatory mediators (i.e., TNFα, CD80, and PD-L1) in human DC-subsets. Our data highlights the importance of mitochondrial remodeling in innate sensing.
Both fission and fusion proteins also play a key role in mitophagy regulation. Upon stress, Drp1 specifically splits a mitochondrion into a healthy fraction and a damaged fraction, to promote degradation of damaged fraction via mitophagy (23). To this end, our data show that TLR-stimulation induces BNIP3-dependent mitophagy in CD1c + mDC. Additionally, we demonstrate that TLR-stimulated mitophagy and glycolysis are essential for CD1c + mDC activation. We further demonstrate induction of Enolase-dependent glycolysis in CD1c + mDC upon TLR-stimulation. Consistently, ENO2 inhibition impairs CD1c + mDC maturation and activation. These results implicate increased glycolysis for proficient antigen processing and presentation by CD1c + mDC to induce a robust immune response. Previously, Chlamydia infection was shown to increase mitochondrial permeability in parallel with mitochondrial remodeling in Enolase1 (ENO1)-dependent manner in mouse bone marrow-derived DCs (89). Intriguingly, BNIP3-dependent mitophagy contributes to mitochondrial elimination during polarization toward pro-inflammatory and glycolytic macrophages (90).
Of note, metabolic reprogramming toward glycolysis is regulated by mitophagy, as mitophagy inhibition reduced expression of glycolysis regulators e.g., PFKFB3, HK2, GAPDH, and PKM2 (90). Therefore, it is conceivable that BNIP3dependent mitophagy similarly controls glycolysis regulators in CD1c + mDC. We found that BNIP3 regulates transcriptional activity of AMPKα1. AMPK is a negative regulator of aerobic glycolysis (91). Intriguingly, AMPK activation has been reported to antagonize glycolytic switch in DCs (14). Our data shows that TLR7/8-stimulation decreases AMPKα1 which can be restored upon BNIP3 inhibition. In contrast, loss of BNIP3 has been reported to reduce AMPK activity in liver (92). However, recent studies have demonstrated that AMPK activation can also be regulated via reactive oxygen species (ROS) (93). Of note, mitophagy regulates ROS (19), which in turn can act as transcription factor to control gene expression (94). Therefore, it is possible that BNIP3 inhibition reduces mitophagy, which in turn suppresses ROS levels to modulate AMPKα1 in CD1c + mDC. Glycolysis is also required for canonical activation of the inflammasome in macrophages (95,96). Interestingly, TLR-stimulation has been shown to induce inflammasome activation in CD1c + mDC (97). Intriguingly, autophagy negatively regulates NLRP3 inflammasome activation in macrophages and bone marrow derived DC (98,99). Moreover, mitophagy prevents hyperinflammation triggered by NLRP3 inflammasome activation in macrophages (100). Our data show that mitophagy is indispensable for CD1c + mDC activation. Collectively, our data suggest a scenario in which TLR-stimulation results in mitochondrial fission leading to induction of mitophagy, which in turn regulates glycolysis via AMPKα1 to activate CD1c + mDC.
It has been demonstrated that autophagy is required for production of type I IFNs in pDC following TLR7 signaling in vitro and in vivo (48)(49)(50)(51)(52). To this end, TLR7-stimulated autophagy deficient pDCs are unable to produce IFNα, in comparison to their autophagy proficient counterparts (48,49). We here demonstrate that autophagy serves to provide glutamine to fuel OXPHOS in pDC upon TLRstimulation, similar to mechanisms previously shown in tumor cells (54)(55)(56). Our data show that TLR-stimulation in pDCs increases cellular glutamine levels in an autophagy dependent-manner. Additionally, autophagy inhibition abrogates glutamine fueled OXPHOS in pDCs upon TLR stimulation. Autophagy is involved in regulating several DC functions e.g., DC maturation, antigen presentation, cytokine production, DC migration and T-cell activation (101). Herein, we provide novel insight into pDC innate sensing mechanism by providing link between autophagy and type I IFN production by demonstrating that autophagy serves to provide glutamine, which is required for IFNα production. Conversely, selective autophagy i.e., mitophagy is required for induction of glycolysis via AMPKα1 regulation. Thus, our data provides novel mechanistic insight in differential role of autophagy in human DC subsets that can lead to immunostimulatory phenotype.
TLR stimulation triggers a shift in metabolism toward aerobic glycolysis, in human mDCs and mouse bone-marrow derived DCs (BMDCs), which is indispensable for the immune effector function and survival of DCs (14,15,102,103). This shift toward glycolysis is required to support the metabolic requirements coupled with increased protein synthesis, which contributes to DC immunogenicity. This TLR-induced surge in glycolysis initiates de novo fatty acid synthesis through glucose-dependent citrate metabolism, which sustains the synthesis and secretion of inflammatory cytokines (103,104). Furthermore, disrupting the glucose-to-citrate pathway reduces DC maturation, cytokine secretion and in turn T cell stimulatory capacity. Influenza virus (flu), Rhinovirus (RV) and a TLR7 agonist induce early glycolysis in human pDC, which is required for type I IFN production and upregulation of HLA-DR, CD80, CD86 (105). However, the generated type I IFN can in turn signal through IFNAR in a paracrine way to trigger FAO and OXPHOS in pDC (16). We find increased glutamine levels after TLR-stimulation in pDC. Of note, glutaminase inhibition in pDCs attenuated OXPHOS, suggesting that glutaminolysis drives OXPHOS induction in response to TLR stimulation in pDC. The requirement of glutamine for various immune effector functions has been demonstrated, e.g., LPS-driven inflammatory response in succinate-dependent anaplerosis (106,107). However, these reports show that activity of glutamine depends on glycolysis. In contrast, it has also been reported that glutamine drives glucose-independent TCA cycle (108). Additionally, glutamine has been demonstrated to be required for trained immunity in monocytes (109), for activated T cells to fuel metabolism (110) and cytokine production by lymphocytes and macrophages (111). Tumor associated M2-like macrophages utilize glutamine for TCA cycle activity, which is required for M2 polarization (112). Moreover, tumor associated macrophages in glioblastoma show increased glutamate transport and metabolism (113). Intriguingly, glutaminolysis has been reported to be dispensable for mouse bone marrow-derived DCs cultured in the presence of GM-CSF for activation upon TLR-stimulation (114). Moreover, it is possible that type I IFN paracrine signaling in TLR-stimulated pDC contributes to the induction of fatty acid oxidation, as shown for CpG stimulated murine pDC (16).
Our study provides several novel insights into TLRstimulated metabolic adaptations in human DC subsets. Our data demonstrate that different DC-subsets engage distinct metabolic adaptations in a mitochondrial dynamics-dependent manner following TLR stimulation. Furthermore, our study provides novel mechanistic insights in human DC-subset metabolism by demonstrating the involvement of mitophagy dependentglycolysis in CD1c + mDC and autophagy supplemented glutaminolysis for OXPHOS in pDC ( Figure 6G). As metabolic manipulation results in modulation of DC activation, our results may have important implications in development of DC-based therapies.  Figure 2C but are shown separately for clarity. Data represents mean ± SEM of three independent experiments. * * * p < 0.001 (Student's t-test).

AUTHOR CONTRIBUTIONS
(E-H) Data were collected within the same experiments as Figure 3C but are shown separately for clarity. Data represents mean ± SEM of three independent experiments. * * p < 0.01; * * * p < 0.001 (Student's t-test).

Supplementary Figure 2 | (A-D)
Data were collected within the same experiments as Figure 4B but are shown separately for clarity. Data represent mean ± SEM of three independent experiments. * * * p < 0.001 (Student's t-test). (E) Relative gene expression of mitophagy related genes in CD1c + mDC. Data represents mean ± SEM of three independent experiments. * p < 0.05; * * p < 0.01 (Student's t-test).

Supplementary Figure 3 | (A)
Depicted is the mean fluorescence intensity of cells stained with 2-NBDG as percentage of the mean fluorescence intensity of control cells ± SEM of four independent experiments * p < 0.05 (Student's t-test). (B) TNF-α levels on protein level were measured in the supernatant of the CD1c + mDC stimulated for 6 h in the presence or absence of 10 nM rotenone or 10 nM antimycin A. Data represents mean ± SEM of three independent experiments * * p < 0.01 (Student's t-test). (C) Percentage mean fluorescence intensity of maturation markers (CD80 and PD-L1) in CD1c + mDCs stimulated for 6 h in the presence or absence of 10 nM rotenone or 10 nM antimycin A. Data represents mean ± SEM of three independent experiments. * * p < 0.01 (Student's t-test). (D) IFN-α levels on protein level were measured in the supernatant of the pDC stimulated for 6 h in the presence or absence of either 5 µM S3 or 1 µM Mdivi-1 or 10 µM olomycine. Data represents mean ± SEM of three independent experiments * * * p < 0.001 (Student's t-test). (E) Percentage mean fluorescence intensity of maturation markers (CD80 and PD-L1) in pDC stimulated for 6 h in the presence or absence of either 5 µM S3 or 1 µM Mdivi-1 or 10 µM olomycine. Data represents mean ± SEM of three independent experiments. * * p < 0.01; * p < 0.05 (Student's t-test).