Succinate is an inflammatory signal that induces IL-1β through HIF-1α

Abstract

Macrophages activated by the Gram-negative bacterial product lipopolysaccharide switch their core metabolism from oxidative phosphorylation to glycolysis. Here we show that inhibition of glycolysis with 2-deoxyglucose suppresses lipopolysaccharide-induced interleukin-1β but not tumour-necrosis factor-α in mouse macrophages. A comprehensive metabolic map of lipopolysaccharide-activated macrophages shows upregulation of glycolytic and downregulation of mitochondrial genes, which correlates directly with the expression profiles of altered metabolites. Lipopolysaccharide strongly increases the levels of the tricarboxylic-acid cycle intermediate succinate. Glutamine-dependent anerplerosis is the principal source of succinate, although the 'GABA (γ-aminobutyric acid) shunt' pathway also has a role. Lipopolysaccharide-induced succinate stabilizes hypoxia-inducible factor-1α, an effect that is inhibited by 2-deoxyglucose, with interleukin-1β as an important target. Lipopolysaccharide also increases succinylation of several proteins. We therefore identify succinate as a metabolite in innate immune signalling, which enhances interleukin-1β production during inflammation.

Fig. 1. Glycolysis is necessary for LPS-induced IL-1β expression

(A, B) IL-1β and TNFα mRNA in 100ng/ml LPS- or 1×10⁵ B pertussis- stimulated BMDMs ± 2DG (1mM) pretreatment for 3h (n=3). (C) Mice i.p. injected ± 2DG (2g/kg) or PBS for 3h, then 15 mg/kg LPS or PBS for 1.5h. Serum levels of IL-1β and TNFα (LPS n=15; LPS+2DG n=14; 2DG n=8; vehicle n=5). Error bars, s.e.m, * p < 0.05; ** p < 0.01.

Fig. 2. HIF-1α is responsible for LPS-induced IL-1β expression

(A) LPS-induced HIF-1α and IL-1β protein expression ± 2DG (1, 5, 10mM). (B) IL-1β in BMDMs incubated in normoxia or hypoxia for 24h then LPS for 24h. (C) IL-1β mRNA in LPS-stimulated BMDMs pretreated ± αKG derivative (1mM). (D) LPS-induced HIF-1β and IL-1β in BMDMs pretreated ± αKG (0.01, 1, 100, 1000μM). (E) LPS-induced IL-1β protein in WT and HIF-1α-deficient BMDMs. (F) ChIP-PCR using HIF-1α antibody and primers specific for −300 position of il1b in LPS-treated BMDMs ±2DG (1mM). (G) Reporter activity in RAW-264 cells transfected with il1b- or −357 il1b. Representative of 3 experiments. Error bars, s.d. (I) ChIP-PCR as above in BMDMs treated with LPS ±αKG (1mM). Error bars, s.e.m, ** p < 0.01.

Fig. 3. Succinate is induced by LPS to drive HIF-1α-induced IL-1β expression

(A) Glucose utilization over time, as a ratio of ECAR:OCR in LPS-treated BMDMs, analysed on the Seahorse XF-24. (B) Succinate abundance in LPS-stimulated BMDMs pretreated ± 2DG (1, 5, 10mM). (C, D) LPS-stimulated BMDMs pretreated ± 5mM diethylsuccinate or butylmalonate (1mM) for 3 h. n=3. (E) Succinyl-lysine expression and corresponding IL-1β in LPS-stimulated BMDMs ± 2DG (1, 5, 10mM) pretreatment for 3h. (J) Average emPAI values (exponentially modified protein abundance index) as a measure of relative abundance of proteins listed in Supplementary Table 1. Error bars, s.e.m, * p < 0.05; ** p < 0.01.

Fig. 4. Glutamine is the source of LPS-induced succinate

(A) SLC3A2 mRNA in LPS-treated BMDMs for 8 and 24h. (B) IL-1β in human PBMCs transfected with 100nM SLC3A2 siRNA compared non-silencing control. Representative of 3 experiments. (C) GABA abundance in serum-deprived BMDMs stimulated with 10ng/ml LPS for 24h. (D) Schematic representing tracing of ¹³C₅,¹⁵N₂ labelled glutamine in BMDMs treated with LPS for 24h. (E) LPS-stimulated BMDMs pretreated ± vigabatrin (500μM) for 20h then 1mM ¹³C₅,¹⁵N₂ glutamine for 4h. Table represents ratio of ¹³C₄-succinate to the sum of all measured succinate isotopomers as measured by LC-MS. The values shown are mean ± s.e.m, n=4. (F, G) LPS-induced HIF-1α and IL-1β protein and IL-1β mRNA (n=7) in BMDMs pretreated ± vigabatrin for 30 min. (H) IL-1β in serum from mice i.p. injected ± vigabatrin (400mg/kg) or PBS for 1.5h, then 15 mg/kg LPS or PBS solution for 1.5h (LPS n=16; LPS+vigabatrin (LPS + V) n=14; vigabatrin (V) n=3; vehicle n=3). (I) IL-1β in serum from mice i.p. injected ± vigabatrin (400mg/kg) or PBS for 1.5h then infected with 1×10⁶ Salmonella Typhimurium UK1 i.p. for 2h (J) Survival of mice i.p. injected ± vigabatrin (400mg/kg) or PBS for 1.5h, then 60 mg/kg LPS or PBS (PBS n=10 (not shown), vigabatrin n=10, LPS n=10, LPS + vigabatrin n=10). Error bars, s.e.m, * p < 0.05; ** p < 0.01. (K) Proposed model: LPS induces high levels of succinate to drive HIF-1α-dependent IL-1β expression and protein succinylation.