Extracellular glucose is crucially involved in the fate decision of LPS-stimulated RAW264.7 murine macrophage cells

Pyroptosis, a type of inflammatory cell death, is dependent on the inflammatory caspase-mediated cleavage of gasdermin D (GSDMD), and the subsequent pore formation on plasma membranes through which interleukin (IL)-1β and IL-18 are released from cells. During proinflammatory activation, macrophages shift their metabolism from aerobic oxidative phosphorylation to anaerobic glycolysis. Hypoxia-inducible factor (HIF)1α is involved in the induction of IL-1β gene expression as well as the metabolic shift towards glycolysis. However, the relationships between pyroptosis and glycolysis, as well as between pyroptosis and HIF1α are poorly investigated. Here we show that lipopolysaccharide (LPS) stimulation of RAW264.7 murine macrophage cells results in pyroptosis when cells are cultured in high glucose medium. During pyroptosis, HIF1α activation occurs transiently followed by downregulation to sub-basal levels. HIF1α downregulation and pyroptosis are observed when cells are stimulated with LPS under high glucose conditions. We also found that intracellular levels of methylglyoxal (MGO), a side product of glycolysis, increase when cells are stimulated with LPS under high glucose conditions. The addition of glycolysis inhibitor and rapamycin suppresses HIF1α downregulation and pyroptosis. These results show that glycolysis plays a crucial role not only in pro-inflammatory activation, but also in pyroptosis in LPS-stimulated RAW264.7 macrophages.


Results
LPS induces pro-inflammatory cytokine and M1 marker expression in RAW264.7 cells. RAW264.7 cells cultured in high glucose (4.5 g/l) medium were stimulated with increasing concentrations (1, 10, and 100 ng/ml) of LPS for 6 and 24 h. qPCR analysis was performed to evaluate the gene expressions of pro-and anti-inflammatory cytokines. The expressions of pro-inflammatory IL-1β and IL-18 as well as anti-inflammatory IL-10 were induced by LPS stimulation for 6 h, and tended to return to basal levels after 24 h (Fig. 1A), suggesting that LPS has a mixed or biphasic effect on the inflammatory activation of cells as reported previously 19 . Immunoblot analysis of the intracellular and extracellular levels of IL-1β and IL-18 proteins showed www.nature.com/scientificreports/ that the proteins first increase intracellularly, and are then secreted from the cells in response to LPS stimulation ( Supplementary Fig. 1). In addition, most of the IL-1β and IL-18 released from the cells are in the pro-forms, suggesting that LPS alone causes the increase and release of IL-1β and IL-18, but does not convert them to their active forms sufficiently ( Supplementary Fig. 1). These observations are consistent with previous reports, showing that caspase-1 activation is scarcely observable in RAW264.7 cells due to the lack of an inflammasome component, apoptosis-associated speck-like protein contains a carboxyl-terminal CARD (ASC) 20,21 . The expression of a marker of pro-inflammatory M1 macrophages (classically activated macrophages), the inducible NO synthase (iNOS) gene, was significantly increased after LPS stimulation for 6 and 24 h (Fig. 1B). In contrast, the expression of a marker of anti-inflammatory M2 macrophages (alternatively activated macrophages), arginase-1 (arg-1), was somewhat decreased by stimulation with 10-100 ng/ml LPS for 24 h (Fig. 1B). Taken together, these results show that pro-inflammatory responses take place in LPS-stimulated RAW264.7 cells, although an antiinflammatory reaction appears to occur during the early stage.
LPS-induced pyroptosis is accompanied by extracellular acidosis in RAW264.7 cells. The activation of macrophages in the pro-inflammatory direction is associated with a metabolic polarization into glycolysis, and the increased glycolytic rate was observed as an increase in extracellular acidification. In addition, macrophage activation often leads to subsequent cell death through pyroptosis, during which GSDMD cleavage releasing the p30 fragment is observed. We thus examined these two events after LPS stimulation for 72 h. A concentration-dependent decrease in the extracellular pH, which reached around 5.7 after 10-100 ng/ml LPS stimulation for 72 h, was observed when the cells were cultured in high glucose medium ( Fig. 2A). The decrease in extracellular pH saturated at around 10 ng/ml LPS ( Fig. 2A). LDH release assays showed that cell death was already observed at concentrations as low as 1 ng/ml LPS (Fig. 2B). Immunoblot analysis showed intracellular LDH levels to be almost constant at 24 h and rather decreased at 72 h of LPS stimulation, justifying the use of the LDH release assay as a cell death assay ( Supplementary Fig. 2). Immunoblot analysis showed GSDMD cleavage to p30 in cells stimulated with 10-100 ng/ml LPS. In contrast, cleaved caspase3 was observed only in cells stimulated with 1 ng/ml LPS (Fig. 2C). Caspase3 activation in 1 ng/ml LPS-stimulated cells was further confirmed by the presence of the p43 and p30 fragments of GSDMD and DFNA5 (GSDME), respectively, which have been shown to be caspase-3-dependent fragments [22][23][24] (Fig. 2C). Thus, cells stimulated with 1 ng/ml LPS seemed to undergo apoptosis, which should be followed secondary necrosis. Morphological analysis by microscopy indicated plasma membrane ballooning, a characteristic feature of pyroptosis, in cells stimulated with 10 ng/ml LPS (Fig. 2D). These results indicate an LPS concentration-dependent change in cell death from apoptosis to pyroptosis, and show that 10 ng/ml LPS is sufficient to induce the phenotypic polarization into oxygen-independent glycolysis and subsequent pyroptosis. Interestingly, we observed significant downregulation of OXPHOS proteins (complex I-IV) in cells treated with 10 or 100 ng/ml LPS for 72 h, further supporting the metabolic shift towards glycolysis in LPS-stimulated cells (Fig. 2E, F).
HIF1α activation is followed by its downregulation to below basal level in RAW264.7 cells stimulated with LPS. We next examined autophagy and HIF1α, both of which play crucial roles in the activation of macrophages 16,25 , in RAW264.7 cells stimulated with LPS for 6, 24, and 72 h. At every time point tested, LC3-II tended to decrease and p62 to increase in cells stimulated with 10-100 ng/ml LPS (Fig. 3). Thus, autophagy appears to be suppressed below the basal level in LPS-stimulated cells. A concentration-dependent increase in HIF1α levels was observed 6 h after LPS stimulation, but the HIF1α levels were downregulated significantly 24 h after 10-100 ng/ml LPS stimulation (Fig. 3). This downregulation of HIF1α levels was not observed 12 h after LPS stimulation, suggesting that the downregulation takes place between 12 and 24 h ( Supplementary  Fig. 3). Corresponding to the downregulation of HIF1α, PKM2, a coactivator HIF1α 18 , was also downregulated in cells stimulated with 10-100 ng/ml LPS for 72 h. Taken together, autophagy is downregulated during LPS administration. During LPS treatment, HIF1α is upregulated at 6 h, but downregulated at 24 h of stimulation.

Relative levels of intracellular metabolites in RAW264.7 cells stimulated with LPS.
To obtain further evidence of the metabolic shift toward glycolysis in LPS-stimulated cells, we examined the relative levels of several metabolic intermediates involved in glycolysis and the TCA cycle in LPS-stimulated RAW264.7 cells. Cells were stimulated with 1, 10, or 100 ng/ml LPS for 6 and 24 h, and cell extracts were analyzed by GC-MS. A greater than threefold increase in intracellular lactate levels was observed in LPS-stimulated cells as compared to non-stimulated cells (Fig. 4). Correspondingly, intracellular glucose levels dropped to 20-30% (1 ng/ ml) and approx. 10% (10 and 100 ng/ml) in LPS (24 h)-stimulated cells as compared to those in non-stimulated cells (Fig. 4). Expression of a glucose transporter (GLUT1), which is involved in the metabolic shifting of macrophages 26 , was induced by LPS within 6 h and sustained for at least 24 h ( Supplementary Fig. 4). Thus, the drop in intracellular glucose levels in response to LPS stimulation should result from an increased conversion of glucose to glucose-6-phosphate due to the increase in glycolysis. In cells stimulated with 10-100 ng/ml LPS, we observed significant decreases in aspartate and glutamate after 6 h of stimulation, as well as significant increases in α-ketoglutarate and malate after 24 h of stimulation (Fig. 4). Since these four metabolites are components of the malate-aspartate shuttle that transports glycolysis-derived NADH to the mitochondrial respiratory complex across the mitochondrial membrane, the balance between glycolysis and oxidative phosphorylation appears to be altered in LPS-stimulated cells as compared to non-stimulated cells. These results confirm that LPS stimulation leads to an M1 phenotype, which is associated with enhanced glycolysis and impaired oxidative phosphorylation. It should be noted that we also observed a slight but significant increase in succinate (Fig. 4), which has been shown to be upregulated in macrophages activated in the pro-inflammatory direction 27 . www.nature.com/scientificreports/ www.nature.com/scientificreports/

Methylglyoxal accumulates in LPS-stimulated RAW264.7 cells, and is involved in the downregulation of HIF1α.
Given the evidence that enhanced glycolysis leads to a drop in the intracellular glucose level within 24 h (Fig. 4), we examined the effect of altering the glucose concentration of the culture medium from 4.5 g/l (high glucose) to 1 g/l (low glucose). As shown in Fig. 5A, there was a smaller decrease in viability observed in LPS-stimulated cells cultured in low glucose medium as compared to high glucose medium. We next compared HIF1α as well as GSDMD between cells cultured in high and low glucose medium. As shown in Fig. 5B, C, no significant downregulation of HIF1α or GSDMD cleavage into p30 was observed in LPS-stimulated cells cultured in low glucose medium. These results suggest that the pyroptosis observed in LPS-stimulated cells cultured in high glucose medium did not result from a shortage of glucose. We also compared the mRNA and protein levels of LPS-induced IL-1β, and observed higher IL-1β expression in high glucose medium as compared to low glucose medium, confirming the pro-inflammatory properties of cells cultured in high glucose medium ( Supplementary Fig. 5). Using LC-MS, we next examined the intracellular levels of methylglyoxal (MGO). MGO is a dicarbonyl compound generated as a by-product of glycolysis and the pentose phosphate pathway, and is highly reactive to proteins including HIF1 28 . MGO is also suggested as an exaggerating factor in hyperglycemia-aggravated illnesses 29 including diabetes 28 , cardiac ischemia 30 , degenerative brain disorders 29 , and sepsis 31 . LPS significantly increased MGO levels only in cells cultured in high glucose medium (Fig. 5D). Immunoblot analysis using anti-MG-H1 antibody, which reacts with MGO-modified arginine residues in proteins, also showed increased MGO-modified protein levels in the cells cultured in high glucose medium (Supplementary Fig. 5). Furthermore, the addition of MGO to cells, which resulted in a significant increase in intracellular MGO levels ( Supplementary Fig. 5), rapidly downregulated HIF1α (Fig. 5E), as reported previously 32 . The addition of iodoacetamide, a frequently-used inhibitor of glycolysis 33 , resulted in a significant increase in MGO, confirming that MGO is mainly produced from glycolytic intermediates ( Supplementary Fig. 5). Collectively, the intracellular accumulation of MGO should be involved, at least in part, in the downregulation of HIF1α in LPS-stimulated cells cultured in high glucose medium.

Suppression of pyroptosis by 2-deoxyglucose and succinate.
We further examined the possible role of enhanced glycolysis as well as decreased oxidative phosphorylation in the downregulation of HIF1α and pyroptosis in RAW264.7 cells. CoCl 2 , an HIF1α activator, could not suppress pyroptosis in LPS-stimulated cells (Fig. 6A), and 400 µM CoCl 2 showed significant cytotoxicity (Fig. 6B) that involved GSDMD cleavage into p30 (Fig. 6A). In contrast, the glycolysis inhibitor 2-deoxy-d-glucose (2-DG) suppressed all of the PKM2 and HIF1α downregulation, decrease in viability, and GSDMD cleavage (Fig. 6A, B). In addition, we found that succinate, which is involved in the activation of PKM2 34 and HIF1α 35 , suppressed the downregulation of PKM2 and HIF1α as well as the decrease in viability and cleavage of GSDMD when applied at 10 mM to cells as a cell permeable analogue, diethyl succinate (Fig. 6C, D). In contrast, the complex II inhibitor diethyl malonate had no effect on LPS-stimulated cells (Fig. 6C, D). Taken together, the re-activation of HIF1α is not sufficient for the suppression of pyroptosis. In contrast, the inhibition of glycolysis and the addition of succinate are sufficient to prevent HIF1α downregulation as well as pyroptosis.
Rapamycin is an antibiotic that acts in various ways including as an immunosuppressant, autophagy activator, and glycolysis inhibitor 36 . Thus, rapamycin negatively regulates macrophage activation by inhibiting glycolytic metabolism and stimulating autophagy. The addition of rapamycin restored the viability that was lost in LPSstimulated cells (Fig. 7A) and suppressed GSDMD-p30 formation (Fig. 7B). Moreover, rapamycin suppressed the inductions of both IL-1β and IL-10 ( Fig. 7C), as well as the downregulation of HIF1α (Fig. 7D). Rapamycin also tended to prevent the downregulation of OXPHOS proteins in LPS-stimulated cells (Fig. 7E, F). The effectiveness of rapamycin on the suppression of IL induction, recovery from the downregulation of HIF1α, and suppression of pyroptosis further confirms that glycolysis is a key event not only in macrophage activation, but also in pyroptosis.

Discussion
At first, we suspected metabolic acidosis as the primary cause of pyroptosis during LPS stimulation under high glucose supplementation. Indeed, LPS stimulation of RAW264.7 cells cultured in high glucose medium resulted in a significant increase in intracellular lactate within 24 h (Fig. 4). Further stimulation for 72 h led to a severe decline in the extracellular pH (~ 5.7). Since lactate is excluded from cells through monocarboxylate transporters (MCTs) 37,38 , the extracellular acidosis should be caused mainly by the excretion of intracellular lactate. A previous report demonstrated that acidosis itself could activate inflammasomes 39 . However, we observed neither a significant decrease in viability nor an increase in LDH release even in cells treated with up to 30 mM lactate ( Supplementary Fig. 6). Thus, lactic acidosis seems not be the main cause of pyroptosis in LPS-stimulated RAW264.7 cells.
cells treated with the indicated concentrations of LPS for 72 h (E). C-I ~ V indicate proteins of mitochondrial respiratory complex I (NDUFB8), II (SDHB), III (UQCRC2), IV (MTCO1), and V (ATP5A). Levels of C-I ~ V to relative to GAPDH are shown in (F). All data represent means and S.E., n = 3 or 4, *P < 0.05, **P < 0.01 versus 0 ng/ml LPS (one way ANOVA followed by Dunnett's post-hoc multiple comparison test). www.nature.com/scientificreports/  #, a band corresponding to the molecular weight of PKM2 (~ 60 kDa) was used to quantify PKM2. ##, a smaller band (~ 40 kDa), which showed essentially the same behavior as the 60 kDa band, was also observed in RAW264.7 cells. Actin was also measured and used as an internal standard. The data represent means and S.E., n = 3 or 4, *P < 0.05, **P < 0.01 (one way ANOVA followed by Dunnett's post-hoc multiple comparison test). www.nature.com/scientificreports/ HIF1α activation is required for cells to maintain homeostasis under low-oxygen conditions. Indeed, in a low oxygen environment, HIF1α deficiency results in cell death 40 . The accumulation of MGO under hyperglycemia and the subsequent degradation of HIF1α are proposed as mechanisms underlying the detrimental effects of high glucose on various injuries, 41 such as hypoxic cardiomyocyte injuries 42 . MGO modifies HIF1α at its proline residues, leading to ubiquitination by the C-terminus of the HSC70-interacting protein (CHIP) and subsequent proteasome-dependent degradation 32 . Therefore, our current findings that intracellular MGO levels not only correlate with the induction of pyroptosis, but also inversely correlate with the levels of HIF1α (Fig. 5B, C) imply that the detrimental effect of high glucose on LPS-induced RAW264.7 cells might be derived, at least in part, from the MGO-dependent degradation of HIF1α. HIF1α downregulation has also been reported in cells during prolonged hypoxia 43 . Interestingly, the downregulation of HIF1α by prolonged hypoxia is also mediated by CHIP 44 . Thus, the downregulation of HIF1α below the basal level that is observed in LPS-stimulated cells (Fig. 3) might result from an enhanced downregulation by CHIP.
Although CoCl 2 caused the upregulation of HIF1α, it did not suppress pyroptosis in LPS-stimulated cells (Fig. 6A, B). Rather, CoCl 2 alone seemed to induce GSDMD cleavage into p30 when applied at a concentration of 400 µM (Fig. 6A). It should be noted that CoCl 2 did not upregulate PKM2 levels even though it upregulated HIF1α (Fig. 6A). In contrast to CoCl 2 , 2-DG not only suppressed the downregulations of HIF1α and PKM2, www.nature.com/scientificreports/ but also ameliorated GSDMD cleavage and the loss of viability in LPS-stimulated cells (Fig. 6A, B). Thus, the re-activation of HIF1α is not sufficient to inhibit pyroptosis. Other molecules such as PKM2 might be required for the transcriptional activation of HIF1, as well as survival during prolonged LPS stimulation in macrophages. The mitochondrial complex II inhibitor diethyl malonate had no effect on any of the LPS-induced changes tested, while dimethyl succinate suppressed all of the downregulations of HIF1α as well as PKM2, GSDMD cleavage, and loss of viability, when applied at a concentration of 10 mM (Fig. 6C, D). Succinate has been shown to accumulate during the proinflammatory activation of macrophages, and is believed to act as a mediator of HIF1α activation 27,45 . In contrast, malonate has been shown to suppress LPS-induced HIF1α activation 46 . Therefore, our finding that succinate reactivates HIF1α while malonate does not is in agreement with the previous reports. However, our results also indicate that succinate acts to suppress pyroptosis (Fig. 6C, D). The role of succinate in macrophage activation might differ between the early and late stages of pyroptosis.
In conclusion, we show that LPS stimulation of RAW264.7 macrophages in high glucose medium leads to pyroptosis, during which HIF1α is downregulated to below the basal level. This downregulation of HIF1α is caused, at least in part, by the cellular accumulation of MGO in LPS-stimulated cells cultured in high glucose medium. These results reveal that pyroptosis, like the inflammasome, is crucially regulated by glycolysis. www.nature.com/scientificreports/ tively) at 37 °C under a 5% CO 2 atmosphere. During stimulation with LPS, cells were cultured in 1 ml of high glucose DMEM containing FBS on 3.5 diameter dishes. In some experiments, low glucose (1 g/l) DMEM was used instead of high glucose DMEM. The pH of the medium was measured by a portable pH meter (LAQUAtwin, Horiba, Tokyo, Japan). Immunoblotting. Total cellular lysates were extracted from the cells, separated by SDS-PAGE, and transferred to a PVDF membrane. After blocking with 3% milk, blots were probed with primary antibodies (Supplementary Table 2) and HRP-conjugated secondary antibodies (Promega), developed with an ECL system, and www.nature.com/scientificreports/ images were captured by image capture (LuminoGraph III, ATTO, Tokyo, Japan). ImageJ (1.47v) was used to quantify band intensities.

Determination of cell death and cell viability.
GC-MS analysis of metabolites. Cells grown on 3.5 cm diameter dishes were collected in PBS, and centrifuged at 1,000 rpm for 1 min at 4 °C. The cell pellets were disrupted by ultrasonication in 500 µl methanol, followed by further centrifugation at 15,000 rpm for 15 min at 4 °C. After removing the supernatants, water (100 µl) was added to the pellets to extract the highly hydrophilic metabolites and added to the supernatants (methanol solution). 2-Isopropylmalic acid was added as an internal standard. Metabolites were dried by vacuum evaporation, dissolved in 100 µl pyridine containing 20 mg/ml methoxyamine, and incubated at 37 °C for 90 min. After trimethylsilylation with MSTFA (N-methyl-N-trimethylsilyltrifluoroacetamide), the metabolites were subjected to GC-MS analysis (GCMS-TQ8030, Shimadzu, Kyoto, Japan).