Dendritic cells were obtained from human mononuclear cells collected from pooled buffy coats of healthy donors provided by Centro de Hemoterapia de Castilla y León. The differentiation of monocytes was carried out in the presence of GM-CSF and IL-4 for 5 days. Culture was carried out in RPMI 1640 medium containing 11.1 mM d-glucose, 4 mM L-glutamine, and 1 mM pyruvate. 10% FBS was maintained during the differentiation process and reduced to 2% prior to the initiation of the experiments. Glucose starving experiments were carried out in BioWhittaker^® RPMI 1640 medium, containing l-glutamine, without glucose. These conditions were used to replicate the experimental setting used in the Seahorse assays. For the characterization of the cell population as DCs, the expression of CD11c, CD40, and CD86 was assayed. Briefly, cells were centrifuged for 5 min at 350g and resuspended in PBS. Ab was added at the concentration of 0.5 µg for 5 × 10⁵ cells and incubated for 45 min at 4°C. When the Ab was labeled with fluorochrome, cells were washed and fixed in 1% formaldehyde. In the case of non-labeled Ab, indirect immunofluorescence was carried out using a labeled secondary Ab before washing and formaldehyde fixation steps. Isotype-matched irrelevant Ab was used as control. The analysis was performed in a Gallios Flow Cytometer. At least 10,000 cells were analyzed per sample. Kaluza software version 1.1 (Beckman Coulter) was used for quantitative data analysis. Ern1^f/fVav1-Cre mice were obtained by Dr. Cubillos-Ruiz through a collaboration with Prof. Takao Iwawaki. Bone marrow from the femora and tibiae was used to obtain bone marrow-derived dendritic cells (BMDCs) from C57BL/6 [wild type (WT)], Ern1^f/f, and Ern1^f/fVav1-Cre mouse by incubation in media supplemented with 20 ng/mL recombinant murine GM-CSF. Cells were harvested at day 7 of differentiation and used directly for subsequent studies. The inhibitors of the ribonuclease activity of IRE1α, MK8866, and 4μ8C were a gift from Dr. John Patterson from MannKind Corporation, Valencia, CA, USA (28) and purchased to Tocris, respectively. The PERK inhibitors GSK2606414 and AMG PERK 44 (29, 30) were from Sellenckchem and Tocris, respectively. Tunicamycin was from Calbiochem. 2-DG, LPS, thapsigargin, and zymosan were from Sigma.

The study was approved by the Bioethical Committee of the Spanish Council of Research (CSIC) and the written informed consent of all healthy donors was obtained at Centro de Hemoterapia y Hemodonación de Castilla y León Biobank. The participants received written consent according to the regulations of the Biobank and the researchers received the samples in an anonymous way. The process is documented by the Biobank authority according to the specific Spanish regulations. The animal experiments were carried out with permission of the local authority and conform to institutional standards. The ethics committee approved this procedure before starting the study.

Bioenergetic analyses of human DCs were carried out using the Seahorse Bioscience XF24 Extracellular Flux analyzer (Seahorse Bioscience). DCs were stimulated and either adhered immediately to polyornithine-coated Seahorse plates or after 4 h of incubation. To normalize the recording of data, results are referred to μg of protein. Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were analyzed according to the XF Cell Mito Stress Test kit protocol in XF media (non-buffered RPMI medium 1640 containing 11.1 mM glucose, 4 mM l-glutamine, and 1 mM sodium pyruvate), under basal conditions, and in response to 1 µM oligomycin, 1.5 µM FCCP, and 100 nM rotenone plus 1 µM antimycin A.

Lactate was assayed using a colorimetric test in cell supernatants centrifuged at 13,000g for 10 min to remove insoluble material and deproteinized by filtration with 3 kDa MWCO spin filters to remove lactate dehydrogenase. The soluble fraction was directly assayed using a Lactate Assay Kit II from Sigma.

This was carried out by RT-PCRs with primers spanning the unspliced regions (Table 1). The PCR conditions were 5 min at 95°C (hot start), 45 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 20 s and elongation at 72°C for 1 min. Final extension was carried out at 72°C for 5 min. Gel electrophoresis was carried out in 3% agarose and spliced XBP1 and unspliced XBP1 bands visualized by GelRed™ staining.

Pyruvate kinase M isoforms are generated by alternative splicing of mutually exclusive exons and differ in that PKM1 mRNA contains exon 9 and lacks exon 10, whereas PKM2 mRNA includes exon 10 and lacks exon 9. RT-PCRs were carried out with primers designed in exon 8 and 11, to yield a 218 bp PKM1 mRNA and a 183 bp PKM2 mRNA. This allows the quantitative assay of each isoform and the identification of the PCR amplicon by digestion with StuI, which recognizes an AGGCT sequence in exon 10 and yields a 149 and a 34 bp product in PKM2.

Proteins were separated by electrophoresis in SDS/PAGE and transferred to nitrocellulose membranes. The membranes were used for the immunodetection of COX-2 (Santa Cruz sc-1745), CHOP (Santa Cruz sc-793), elongation initiation factor 2α (eIF2α, Cell Signaling #5324), P-Y705-STAT3 (Cell Signaling #9131), STAT3 (Cell Signaling #9132), PKM2 (Cell Signaling #3198S), IL-1β (Cell Signaling #12242), histone H3 (abcam ab1791), acetyl-K9-histone H3 (Millipore #4-1003;), P-S10-histone H3 (Millipore #04-817), spliced XBP1 (sXBP1; BioLegend #619501), ATF6 (abcam ab11909), HIF1α (Novus Biologicals NBP1-19779), and P-S52-eIF2α (Invitrogen #44-728G). For immunoblots directed to assay nuclear proteins, the nuclear extracts were obtained by using a nuclear extract kit (Active Motif). Anti-TATA-box-binding protein (Diagenode TBPCSH-100) and anti-histone H3 Ab were used for protein load control.

Dendritic cells were seeded on poly-lysine-coated glass coverslips for 12 h and then stimulated with zymosan particles. Cells were fixed with 10% formaldehyde in PBS and stained with anti-HIF1α Ab or anti-PKM2 Ab and goat anti-rabbit IgG Ab labeled with Alexa-Fluor^®480. The coverslips were observed by laser-scanning confocal fluorescence microscopy using a Leica TCS SP5 apparatus equipped with a white-light laser and a Leica 63PL APO NA 1.40 oil immersion objective. Image analysis and subcellular colocalization fluorograms were generated and analyzed using the Leica confocal software package LAS AF Lite and Adobe Photoshop CS5.1 software.

Total RNA was obtained by TRIzol/chloroform extraction and used for RT reactions. Cycling conditions were adapted to each set of primers. GAPDH was used as a housekeeping gene to assess the relative abundance of the different mRNA using the comparative cycle threshold method. The procedure was used to assay CHOP, IL10, IL23A, IL12A, IL12B, and IL1B mRNA. The sequences of the primers are shown in Table 1.

Lipids were extracted into ethanol from cell supernatants, eluted in methanol using Strata™ C-18E SPE cartridges (Phenomenex^®), and evaporated to dryness under N₂. The chromatographic separation was conducted in an Acquity™ UPLC System equipped with an Acquity UPLC^® BEH C18, 1.7 µm, 2.1 × 100 mm column (Waters). The chromatographic column was directly interfaced into the electrospray ionization source of a mass spectrometer (SYNAPT HDMS G2) from Waters. MS analysis was performed in negative ion mode using a MS^E method that allows simultaneous detection of analytes through a low energy function (full scan) and a high energy function (collision energy) with ion partial fragmentation, as reported (31).

Chromatin immunoprecipitation assays were conducted with Ab against P-T71-ATF2 (Cell Signaling #9221), ATF4 (Santa Cruz sc-7583), C/EBPβ (Santa Cruz sc-150), and sXBP1 as previously reported (21). Briefly, cells were stimulated and then washed twice with PBS and fixed with 1% formaldehyde. Cross-linking was terminated by 0.125 M glycine. Crude nuclear extracts were collected by microcentrifugation and resuspended in a lysis buffer containing a high salt concentration. Chromatin sonication was carried out using a Bioruptor device from Diagenode. The chromatin solution was precleared by adding Protein A/G PLUS-Agarose for 30 min at 4°C under continuous rotation. After elimination of the beads, Ab was added for overnight incubation at 4°C, and then Protein A/G PLUS-Agarose was added and incubated for an additional period of 2 h at 4°C. Beads were harvested by centrifugation at 4,000g and sequentially washed with lysis buffer high salt, wash buffer, and elution buffer. Cross-links were reversed by heating at 67°C in a water bath, and the DNA bound to the beads isolated by extraction with phenol/chloroform/isoamylalcohol. Irrelevant Ab and sequences of the IL12A promoter were used as control of binding specificity. The sequences of the primers are shown in Table 2. Results are expressed as percentage of input.

Data are represented as the mean ± SEM and were analyzed with the Prism 4.0 statistical program (GraphPad Software). Comparison between experimental groups was carried out using the two-tailed Student’s t-test and ANOVA. Differences were considered significant for p < 0.05.

Stimulation of DCs with LPS produced a concentration-dependent increase of the ECAR that could be detected immediately after the addition of the stimuli in the XF Mito Stress Test (Figure 1A, right panel). Assessment of the OCR showed stable values in the case of LPS (Figure 1B) and a tendency to increase in the case of zymosan that did not reach statistical significance (Figure 1C). The ECAR values were somewhat higher in response to zymosan than in the case of LPS and showed a more reproducible pattern of response (Figure 1C). These results are consistent with a fast and lasting increase of glycolysis upon a wide range of PAMPs concentrations, as judged from the net decrease of the OCR/ECAR ratio (Figures 1B,C, bottom panels).

One of the mechanisms promoting glycolysis in the presence of normoxia, usually termed Warburg effect or aerobic glycolysis, is the activation of HIF1 by the stabilization of its HIF1α subunit. This explains the induction of glycolytic enzyme expression, which in macrophages depends on the formation of a complex with pyruvate kinase M2 (PKM2) (32) that translocates to the nucleus and regulates the expression of proglycolytic enzymes (33). PKM2 may act as a protein kinase that phosphorylates STAT3 at Y705 (34) and histone H3 (35). This explains a positive feedback loop between HIF1 and STAT3 since Y705-STAT3 activates HIF1α transcription (36, 37). In a previous study, we observed that both zymosan and LPS are robust activators of Y705-STAT3 phosphorylation through the induction of secondary mediators (38). In keeping with that report, the assay of P-Y705-STAT3 in nuclear extracts showed a time-course compatible with its involvement in HIF1α expression (Figure 2A). As a correlate of enhanced transcriptional activity, zymosan induced global phosphorylation of S10-histone H3 and acetylation of K9-histone H3 (Figure 2B). The nuclear translocation of HIF1α was confirmed in immunofluorescence confocal microscopy studies (Figure 2C). The preferential expression of PKM2 was confirmed by Western blot (Figure 2D) and by immunofluorescence confocal microscopy, which showed the enzyme in the nuclei and in cytoplasm areas surrounding phagocytosed zymosan particles (Figure 2E), as well by analysis of its mRNA by RT-PCR. Assays with sets of primers that generate products distinguishable by the number of bp, showed a 149 and a 34 bp product in PKM2 upon treatment with StuI, whereas the PKM1 amplicon was not digested (Figure 2F). Taken collectively, these data show the presence of P-Y705-STAT3 and HIF1α in the nuclei of zymosan- and LPS-stimulated DCs, as well as a high expression of PKM2 protein. However, the temporal pattern of HIF1α protein induction does not parallel the early changes of both OCR and ECAR, thus suggesting that other mechanisms may underlie the early rewiring of the bioenergetic profile of DCs, i.e., the association of hexokinase II with voltage-dependent anion channels in the outer mitochondrial membrane (39).

Given the increases of the ECAR induced by PAMPs, lactate levels were assayed. Lactate production was strongly blocked in glucose-starved cells and to a lesser extent in the presence of 10 mM 2-DG (Figure 3A). Consistent with seminal studies on non-opsonic phagocytosis, in which 2-DG did not reduce zymosan uptake even when ATP production was blocked (40, 41), DCs showed robust phagocytosis in the presence of 2-DG and also exhibited a robust ability to release arachidonic acid (Figure 4), thus suggesting that the effect of 2-DG cannot be explained by an effect on the earliest steps of DCs activation. Unexpectedly, the mRNA levels of various cytokines showed notable differences under glucose-limiting conditions and in the presence of 2-DG. The most striking effect was observed on the induction of IL23A, which increased ~ 20-fold 24 h after stimulation (Figure 3B). The assay of IL-23 protein showed a net increase in the case of LPS, but not in the case of zymosan (Figure 3C). A likely explanation is that the high production of IL-23 protein induced by zymosan under these conditions could not increase because, unlike LPS, zymosan triggers low transcription of the gene encoding the p40 chain (IL12B), which is a limiting step for the production of this heterodimeric cytokine. The assay of other cytokines showed prominent changes in response to LPS, including an increase of IL12A mRNA and a reduction of IL10 and IL1B mRNA. The expression of pro-IL-1β was also blunted in the presence of 2-DG (Figure 2D). Given that these findings could not be explained by a simple blockade of glucose metabolism, alternative mechanisms were postulated, focusing on the effect of 2-DG on mannose metabolism, N-glycosylation reactions, and the possible activation of the UPR as a result of potential protein misfolding. Consistent with this hypothesis, stimulation of DCs with zymosan and LPS in the presence of 2-DG induced robust splicing of the XBP1 mRNA (Figure 3D), whereas the sole addition of 2-DG or PAMP did not. In sharp contrast, XBP1 splicing was not observed in DCs starved of glucose for up to 18 h and stimulated with PAMPs (not shown). Given the prominent enhancement of aerobic glycolysis induced by PAMPs and a recent report underscoring that this process reduces the supply of fructose-6-phosphate to glutamine-fructose-6-phosphate transaminase 1 (GFPT1), the enzyme that transfers the amino group of l-glutamine to yield glucosamine-6-phosphate for N-glycan biosynthesis (42), our data point out that the coincidental inhibition of mannose metabolism by 2-DG and of the hexosamine biosynthetic pathway by aerobic glycolysis show optimal conditions for N-glycosylation blockade. XBP1 splicing was observed after 2 h of incubation (Figure 3E) and was blunted by 1 mM mannose and the inhibitor of the endonuclease activity of IRE1α MKC8866 (Figure 3F). Notably, these treatments also inhibited the effect of 2-DG on the induction of IL23A elicited by zymosan (Figure 3G), whereas this effect was less definite in the case of LPS. The effect of 2-DG on IL10 mRNA induction was less sensitive to mannose and IRE1α inhibitors (Figure 3G, right panel). These data suggest that the effect of 2-DG on IL23A expression can be explained by interference with mannose metabolism and associated with the activation of the UPR, whereas the effect on IL10 mRNA expression seems dependent on other mechanisms.

Since the distinct effects observed on IL23A and IL10 mRNA expression elicited by 2-DG could not be explained by a sole mechanism and given that 2-DG reduced lactate production (Figure 3A) and blocked pro-IL-1β expression (Figure 2D), the effect of 2-DG on the lactate/HIF1 route was addressed. 2-DG inhibited the expression of HIF1α induced by zymosan (Figure 5A). An effect countered by lactate (Figure 5B). Stimulation of DCs in the presence of sodium lactate did not influence the expression of IL1B mRNA. In contrast, IL10 and IL23A mRNA expression showed a trend to increase (Figure 5C). Treatment with LPS and zymosan in the presence of either sodium lactate or lactic acid did not induce XBP1 splicing (Figure 5D). These results confirm the involvement of the lactate/HIF1 system in pro-IL-1β expression and show that the effect of lactate on the mRNA expression of IL10 and IL23A is independent of XBP1 splicing.

The effect of l-glutamine starvation was addressed given its involvement in both the hexosamine biosynthesis pathway and the tricarboxylic acid cycle. In addition, glutaminolysis contributes to restore metabolic homeostasis by enhancing the XBP1- and α-ketoglutarate-dependent expression of GFPT1 (43). l-Glutamine removal induced a significant increase of lactate production in resting DCs after 24 h of incubation. In contrast, lactate levels were not significantly affected in the absence of l-glutamine after stimulation with zymosan, oligomycin or LPS (Figure 6A). IL10 mRNA expression showed a trend to reduction in the absence of l-glutamine that reached statistical difference at 24 h (Figure 6B). Stimulation of DCs in the absence of l-glutamine did not induce XBP1 splicing, although the effect of 2-DG was somewhat more prominent in the absence of l-glutamine (Figure 6C).

Given the association of XBP1 splicing with the enhanced expression of IL23A mRNA, our experiments then focused on whether the UPR induced by other mechanisms showed similar results and the possible involvement of the PERK/eIF2α/ATF4/CHOP branch of the UPR. P-S52-eIF2α was detected to a low extent in resting DCs and did not increase upon 2-DG treatment and PAMPs stimulation (Figure 7A), in agreement with a recent report showing that LPS did not affect eIF2α phosphorylation (44). The mRNA expression of DDIT3/CHOP was enhanced by 2-DG and blunted by mannose in the presence of PAMPs (Figure 7B), thus suggesting that mannose may counter some of the mechanisms triggered by the stimuli by acting as a substrate for the formation of dolichol-linked oligosaccharide. Tunicamycin and thapsigargin induced similar effects to those elicited by 2-DG on IL23A and IL10 mRNA expression (Figure 7C) as well as on XBP1 splicing (Figure 7D). Consistent with the inhibitory activity of 2-DG on COX-2 N-glycosylation (Figure 2D), tunicamycin, a robust inhibitor of this posttranslational modification, caused the same effect. In contrast, thapsigargin, which elicits the UPR via SERCA inhibition and does not interfere with N-glycosylation reactions, failed to do so (Figure 7E). These data indicate that ER stress responses, rather than N-glycosylation inhibition alone, underpin the cytokine signature induced by ER stressors. In keeping with previous results (21), the expression of CHOP protein was detectable in the nuclear fractions of resting DCs and showed a net trend to decrease after stimulation by zymosan (Figure 7F). A recent report has disclosed that PERK inhibition decreases UPR-dependent inflammation via attenuation of eIF2α phosphorylation and reduction of the transcription of inflammatory genes as a result of translational repression (45). Consistent with this mechanism, PERK inhibition with GSK2606414 reduced the induction of DDIT3 and IL23A mRNA elicited by LPS in the presence of 2-DG. A similar result was obtained with the pyrazole-based PERK inhibitor AMG PERK 44, which has been found recently to lack the RIPK1-inhibitory effect recently disclosed for GSK2606414 (30). The effect of both compounds was less evident in zymosan-treated DCs and suggests a stimulus-dependent recruitment of the different arms of the UPR (Figure 7G). Consistent with the selectivity of GSK2006414 on the PERK route, XBP1 splicing was not inhibited (Figure 7H). Stimulation with zymosan induced ATF6 cleavage to an extent higher than that produced by the sole addition of 2-DG, as well as a noticeable change in the mobility of full-length ATF6, most likely due to the underglycosylation of the protein before cleavage by site-1 proteases (46). Combination of zymosan with 2-DG did not enhance ATF6 cleavage and the effect of LPS was similar to the one elicited by the sole addition of 2-DG (Figure 7I).

Given that the trans-activation of IL23A induced by zymosan depends on at least NF-κB proteins and ATF2 (21), the interaction of P-T71-ATF2 with the IL23A promoter was assayed. The binding was partially inhibited in the presence of 2-DG, thus suggesting that other transcription factors might be engaged under these conditions (Figure 8A). The involvement of sXBP1 was then considered the leading hypothesis because of (i) the robust splicing of XBP1 and the effect of IRE1α endonuclease inhibitors; (ii) the presence of three X-boxes in the IL23A promoter identified using the TRANSFAC database (21); and (iii) the role of XBP1 in the regulation of diverse targets exhibiting a wide array of sequence motifs (12, 47). sXBP1 protein was detected in the nuclear extract of DCs, but its expression did not show significant changes upon various treatments nor in the presence of MKC8866 (Figure 8B). 2-DG increased the binding of sXBP1 to the three X-boxes in the IL23A promoter (Figure 8C), which suggests the involvement of this transcription factor in the enhancement of IL23A expression and that in addition to the expression of the protein, either posttranslational modifications (48, 49) or formation of an enhanceosome with other factors may be necessary for full development of transcriptional activity (50). Since sXBP1 also plays a central role in the control of the hexosamine biosynthetic pathway by regulating the expression of GFPT1 (51), the expression of this enzyme and the binding of sXBP1 to an X-box placed 147 bp upstream of the transcription initiation nucleotide were addressed. Resting DCs showed a high expression of GFPT1 mRNA, which was significantly enhanced when DCs were stimulated with either zymosan or LPS in the presence of 2-DG (Figure 8E). Notably, binding of sXBP1 to the X-box of GFPT1 showed a parallel enhancement under these conditions (Figure 8F). These results confirm the presence of detectable amounts of sXBP1 protein in resting DCs in the absence of definite XBP1 mRNA splicing and suggest that in addition to the expression of protein, posttranslational modifications or interaction with other factors may be necessary for its transcriptional activity.

IL23A trans-activation by LPS has been associated with a set of factors including NF-κB, cAMP response element-binding protein (CREB), and C/EBPβ. Consistent with this notion and the finding that inhibition of the IRE1α and PERK routes showed stimulus-dependent effects, the binding of C/EBPβ to three sites in the IL23A promoter, two of which are close to two X-boxes and to a κB-site (21), was addressed. Unlike zymosan, 2-DG increased robustly the binding of C/EBPβ to the three sites (Figure 8D), thus suggesting the association of this factor with the enhancing effect of 2-DG on the expression of IL23A induced by LPS.

Since the biochemical and pharmacological experiments suggested that the effect of the UPR on IL23A expression could be explained by a stimulus-dependent recruitment of the IRE1α and the PERK routes, experiments were carried out in mice with hematopoietic system-specific deletion of Ern1, i.e., the gene encoding Ire1α (Ern1^f/fVav1-Cre mice). As shown in Figure 9A, the sole addition of 2-DG induced Xbp1 splicing in BMDCs from Ern1^f/f mice, which is a salient difference as compared to human DCs, in which both 2-DG and PAMPs are required for XBP1 splicing. Ern1^f/fVav1-Cre mice did not show Xbp1 splicing, whereas Ddit3 mRNA expression was not significantly affected (Figure 9B). Notably, a substantial reduction in Il23a expression was observed in the Ern1^f/fVav1-Cre mice upon treatment with both LPS and zymosan in the presence of 2-DG, as compared to the Ern1^f/f mice (Figure 9C). Consistent with the results observed in DCs, pharmacological blockade of the PERK route with GSK2606414 inhibited Il23a mRNA expression in WT mice stimulated with LPS (Figure 9D). These results suggest the involvement of the Ire1α route in response to both zymosan and LPS and of the PERK route in the case of LPS.