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

Abstract

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.

Figure 1

Expressions of cytokines and phenotypic markers in RAW264.7 cells stimulated by LPS. (A) The gene expressions of pro- and anti-inflammatory cytokines in response to LPS. The cells were treated with the indicated concentrations of LPS for 6 and 24 h, and qPCR analysis was performed to evaluate the levels of IL-1β, IL-18, and IL-10 relative to GAPDH. (B) M1 and M2 phenotypic marker expressions in response to LPS. The cells were treated with the indicated concentrations of LPS for 6 and 24 h, and qPCR analysis was performed to evaluate the levels of iNOS and arginase-1 (Arg-1) relative to GAPDH. The 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).

Figure 2

Pyroptosis in LPS-stimulated RAW264.7 cells is accompanied by extracellular acidosis as well as downregulation of mitochondrial respiratory proteins. (A) Extracellular pH (medium pH) of cells treated with the indicated concentrations of LPS for 72 h. Upper panel shows the color of the medium. (B) LDH release assay from cells treated with the indicated concentrations of LPS for 48 h. (C) Immunoblot analysis of cleaved-caspase3, GSDMD, and DFNA5 in cells treated with the indicated concentrations of LPS for 72 h. FL, full length. (D) Representative phase contrast images of cells stimulated with or without LPS (10 ng/ml, 48 h). The lower panel is an enlarged image of the framed portion of the upper right image. Arrows indicate ballooning of the plasma membrane. (E, F) Immunoblot analysis of mitochondrial respiratory proteins (OXPHOS proteins) in 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).

Figure 3

Levels of autophagy markers and HIF1α protein in RAW264.7 cells after treatment with LPS. The cells were treated with the indicated concentrations of LPS for 6, 24, and 72 h. Total cell lysates were extracted, and the levels of the indicated proteins (LC3, p62, HIF1α and PKM2) were determined by immunoblotting. #, 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).

Figure 4

Comparative analysis of several metabolites involved in glycolysis, the TCA cycle, and the malate-aspartate shuttle in RAW264.7 cells after treatment with LPS. The cells were treated with the indicated concentrations of LPS for 6 or 24 h. Total cell lysates were extracted, and the levels of the indicated metabolites were examined by GC-MS. 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).

Figure 5

Pyroptosis in LPS-stimulated RAW264.7 cells is not observed under low glucose conditions. (A) Comparison of the rates of cell death between high and low glucose supplementation. The cells were treated with the indicated concentrations of LPS for 48 h in media containing high (4.5 g/l, HG) or low (1 g/l, LG) concentrations of glucose. Cell viabilities were determined by CCK8 assay. The mean viability of the control group (0 ng/ml LPS) was set as 100%. The data represent means and S.E., n = 4, **P < 0.01 (one-way ANOVA followed by Dunnett’s post-hoc multiple comparison test). (B, C) Immunoblot analysis of HIF1α and GSDMD in cells treated with the indicated concentrations of 10 ng/ml LPS for 24 (B) or 48 (C) hours. Actin was also examined as an internal standard. (D) Intracellular levels of methylglyoxal (MGO) in cells treated with 10 ng/ml LPS for 24 h. Total cellular lysates were extracted, and the MGO levels were examined by LC-MS. The mean level of the control group (0 ng/ml LPS, LG) was set as 100%. The data represent means and S.E., n = 4, *P < 0.05, **P < 0.01 (one-way ANOVA followed by Tukey–Kramer post-hoc multiple comparison test). (E) Extracellularly added MGO downregulates HIF1α. The cells were treated with 1 mM MGO for 1 h and the relative HIF1α levels were determined by immunoblotting.

Figure 6

Effects of inhibitors and activators on LPS-stimulated RAW264.7 cells. The cells were pre-treated with the indicated concentrations of cobalt chloride (CoCl₂), 2-deoxy-d-glucose (2-DG), diethyl succinate (suc), or dimethyl malonate (malo) for 30 min, and then treated with 10 ng/ml LPS for 48 h. (A, C) Total cellular lysates were extracted, and the levels of the indicated proteins were examined by immunoblotting. (B, D) Cell viabilities were also determined by CCK8 assay. Mean viability of the control group (0 ng/ml LPS) was set as 100%. The data represent means and S.E., n = 4, *P < 0.05, **P < 0.01 (one-way ANOVA followed by Tukey–Kramer post-hoc multiple comparison test).

Figure 7

Effects of rapamycin on LPS-stimulated RAW264.7 cells. (A, B) Rapamycin inhibits pyroptosis. The cells were pre-treated with 10 µM rapamycin (rap) for 30 min, and then treated with 10 ng/ml LPS for 48 h. Cell viabilities were determined by CCK8 assay (A). Mean viability of the control group (0 ng/ml LPS) was set as 100%. Total cellular lysates were also extracted, and the levels of GSDMD-p30 were examined by immunoblotting. (B). (C) Rapamycin suppresses the induction of cytokine expression by LPS. qPCR analysis was performed to evaluate the levels of IL-1β and IL-10 relative to GAPDH. (D) Rapamycin suppresses downregulation of HIF1α. Immunoblot analysis of HIF1α and p62 is shown. (E) Effect of rapamycin on OXPHOS proteins. Immunoblot analysis of OXPHOS (E, F). The data represent means and S.E., n = 3 or 4, *P < 0.05, **P < 0.01 (one-way ANOVA followed by Tukey–Kramer post-hoc multiple comparison test).