Macrophages are activated during microbial infection to coordinate inflammatory responses and host defense. Here we find that in macrophages activated by bacterial lipopolysaccharide (LPS), mitochondrial glycerol 3-phosphate dehydrogenase (GPD2) regulates glucose oxidation to drive inflammatory responses. GPD2, a component of the glycerol phosphate shuttle, boosts glucose oxidation to fuel the production of acetyl coenzyme A, acetylation of histones and induction of genes encoding inflammatory mediators. While acute exposure to LPS drives macrophage activation, prolonged exposure to LPS triggers tolerance to LPS, where macrophages induce immunosuppression to limit the detrimental effects of sustained inflammation. The shift in the inflammatory response is modulated by GPD2, which coordinates a shutdown of oxidative metabolism; this limits the availability of acetyl coenzyme A for histone acetylation at genes encoding inflammatory mediators and thus contributes to the suppression of inflammatory responses. Therefore, GPD2 and the glycerol phosphate shuttle integrate the extent of microbial stimulation with glucose oxidation to balance the beneficial and detrimental effects of the inflammatory response.
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The data supporting the findings of this study are available from the corresponding author upon request.
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J.J. was supported by the British Society for Immunology and T.H. by National Institutes of Health (NIH) grants (nos. R01AI102964 and R21AI119763). N.W.S. was supported by a NIH grant (no. R03 HD092630), X.G. by the Canadian Institutes of Health Research (grant no. 146818) and E.T.C. by the Claudia Adams Barr Program. We thank J.F. Mohan for helpful suggestions for CRISPR–Cas9 genome editing, L.B. Sullivan for technical advice on the GPD2 Seahorse assay, R.L.S. Goncalves for technical advice on the NAD(P)H autofluorescence assay and H. Affronti and K.E. Wellen for sharing reagents.
The authors declare no competing interests.
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Integrated supplementary information
Supplementary Figure 1 ATP-citrate lyase (ACLY) and p300 activity are necessary for supporting pro-inflammatory gene induction during LPS activation in BMDMs.
(a) ChIP-qPCR analysis of histone acetylation in Il6 and Il1b promoter regions in BMDMs stimulated with LPS for 0-1h +/- the ACLY inhibitors BMS 303141 (4 μM) or Medica16 (100 μM) (n=3). (b) qPCR analysis of Il6 and Il1b gene expression in BMDMs stimulated with LPS for 0-3h +/- 4 μM BMS 303141 or 100 μM Medica16. (c) ChIP-qPCR analysis of histone acetylation in Il6 and Il1b promoter regions in BMDMs stimulated with LPS for 0-1h +/- the p300 inhibitor C646 (p300i), 8 μM (n=3). (d) qPCR analysis of Il6 and Il1b gene expression in BMDMs stimulated with LPS for 0-3h +/- 8 μM p300i. Data are from one experiment representative of three independent experiments. Mean values shown.
Supplementary Figure 2 LPS exposure modulates mitochondrial respiration and glucose utilization as a function of time to support inflammatory cytokine production.
(a) Seahorse extracellular flux analysis of maximal mitochondrial oxygen consumption rate (OCR) in BMDMs left unstimulated or stimulated with LPS for the times indicated (n=4). (b) 3H-deoxy-D-glucose uptake (cpm = counts per min) in BMDMs left unstimulated or stimulated with LPS for the times indicated (n=3). (c) ChIP-qPCR analysis of histone acetylation in Il6 and Il1b promoter regions in BMDMs stimulated with LPS for 0-1h in media +/- glucose, following 24h incubation in media +/- glucose (n=3). (d) qPCR analysis of Il6 and Il1b gene expression in BMDMs stimulated with LPS for 0-3h in media +/- glucose, following 24h incubation in media +/- glucose. Data are one experiment representative of ten (a) or three (b-d) independent experiments. Mean values (a-d) +/- s.e.m. (a,b) shown. *p ≤0.05, **p ≤0.01, ***p ≤0.001 (two-tailed Student’s t-test).
(a) Depiction of the spatial and biochemical position of GPD2 at the nexus between glycolysis and electron transport. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) generates NADH from oxidation of glucose in the cytosol, supplying electrons for reduction of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate (G3P) by cytosolic glycerol 3-phosphate dehydrogenase (GPD1). Electrons from G3P are passed directly to the electron transport chain (ETC) through the FAD cofactor of mitochondrial glycerol 3-phosphate dehydrogenase (GPD2), reducing ubiquinone (Q) to ubiquinol (QH2) thus contributing to the proton motive force for oxidative phosphorylation. As the glycerol phosphate shuttle (GAPDH-GPD1-GPD2) directly links glycolysis to the ETC and resupplies NAD+ to the former, increased GPD2 activity serves as a metabolic node by which the ETC may regulate the rate of glucose utilization. LPS activation increases GPS flux, permitting the acquisition of a high metabolic state characterized by increased glucose oxidation and GPD2-driven mitochondrial respiration. (b) Immunoblot analysis of GPD2 protein in WT and Gpd2−/− BMDMs unstimulated or stimulated with LPS for the times indicated. (c) Seahorse extracellular flux analysis of OCR in WT BMDMs stimulated with LPS for the indicated times, permeabilized, and treated with 10 mM glycerol 3-phosphate, 2 μM rotenone, and 1 mM ADP. Mean values shown. (d) FACS analysis of F4/80 and CD11b expression on BMDMs from WT and Gpd2−/− mice. Numbers indicate percent of cells in gate. Data are from one experiment representative of three independent experiments.
13C6-glucose tracing into intermediates of glycolysis and TCA cycle in WT and Gpd2−/− BMDMs stimulated with LPS for indicated times, presented as LPS-induced fold change in percent m+6 (1,2), m+3 (3-7), or m+2 (8-11) enrichment (1,6,8-11 n=3; 2-5,7 n=4). Data are from one experiment representative of two independent experiments. Means +/- s.e.m. shown. *p ≤0.05, **p ≤0.01, ***p ≤0.001 (two-tailed Student’s t-test).
Supplementary Figure 5 Integrated models of the metabolic adaptations underpinning the biphasic functional response to LPS exposure.
(a) GPD2-dependent transport of electrons from the glycerol phosphate shuttle (GPS) through the electron transport chain (ETC) is upregulated by LPS activation to support an increase in glucose-derived carbon flux through glycolysis and the TCA cycle, providing increased availability of citrate and acetyl-CoA. Coupled with an LPS-induced increase in ACLY activity, enhanced production of acetyl-CoA drives histone acetylation and induction of the inflammatory genes Il6 and Il1b. As oxidation of carbon substrates in the TCA cycle depends on the availability of electron acceptors such as NAD+, the cyclic reduction and oxidation of these molecules serves as an important link between oxidative metabolism in the TCA cycle and ETC activity. (b) Duration of LPS exposure regulates the transition from inflammatory gene induction to suppression (blue histogram) in macrophages. Acute LPS exposure, or activation, induces an increase in GPD2-dependent oxidative metabolism, which enhances the production of acetyl-CoA from glucose to fuel histone acetylation and thus promote expression of the inflammatory genes Il6 and Il1b. Prolonged LPS exposure produces a state of tolerance in which induction of Il6 and Il1b expression is attenuated due in part to decreased oxidative metabolism, which limits flux of glucose through the TCA cycle into citrate and acetyl-CoA for histone acetylation.
(a) Schematic of workflow for in vitro LPS tolerance experiments. Naive BMDMs were left unstimulated (U) or challenged with LPS for 24h to induce tolerance (T). LPS responsiveness was assessed by LPS-stimulating naive (U+LPS) or tolerant (T+LPS) BMDMs. In the T+L+2DG condition, 2-deoxyglucose (2DG) was added during tolerance induction and washed out prior to LPS restimulation in the absence of inhibitor. (b) qPCR analysis of Il6 and Il1b gene expression in BMDMs treated as described in a (n=3). Data are from one experiment representative of four experiments. Means +/- s.e.m. shown (b). ***p ≤0.001 (two-tailed Student’s t-test).
Supplementary Figure 7 Pharmacological inhibition of GPD2 modulates LPS activation and tolerance in BMDMs.
(a) Seahorse extracellular flux analysis of maximal mitochondrial OCR in BMDMs stimulated with LPS for 1h +/- the GPD2 inhibitor iGP-1 (GPD2i), 300 μM (n=4). (b) ChIP-qPCR analysis of histone acetylation in Il6 and Il1b promoter regions in BMDMs treated as in a (n=3). (c) qPCR analysis of Il6 and Il1b gene expression in BMDMs stimulated with LPS for 0-3h +/- 300 μM GPD2i. (d) Seahorse analysis of maximal mitochondrial OCR in BMDMs stimulated with LPS for 12h +/- 300 μM GPD2i (n=3). (e) ChIP-qPCR analysis of histone acetylation in Il6 and Il1b promoter regions in unstimulated (U) or LPS-stimulated (U+LPS) naive BMDMs and unstimulated (T) or LPS-stimulated (T+LPS) tolerant BMDMs (n=3). Tolerance was induced by 24h LPS challenge +/- 300 μM GPD2i (T+LPS+GPD2i), which were both washed off before stimulation. (f) qPCR analysis of Il6 and Il1b gene expression in BMDMs treated as in e (n=3). Data are from one experiment representative of three independent experiments. Mean (a-f) +/- s.e.m. (a,d,f) shown. *p ≤0.05, **p ≤0.01, ***p ≤0.001 (two-tailed Student’s t-test).
Supplementary Figure 8 GPD2 deficiency protects against induction of reverse electron transport (RET) through a mechanism independent of known negative regulators of oxidative metabolism in LPS-stimulated BMDMs.
(a) qPCR analysis of Irg1, Nos2, and Idh1 gene expression in BMDMs unstimulated or stimulated with LPS for 12h. (b) Steady-state metabolomic analysis of itaconate levels, shown as relative to unstimulated, in BMDMs stimulated with LPS for the indicated times (n=3). Data are from one experiment representative of three independent experiments (a) or from one experiment (b). Mean (a-b) +/- s.e.m. (b) shown. (c) Schematic depicting FET and RET during LPS activation and tolerance. During acute LPS exposure (LPS activation), electrons from oxidation of metabolic substrates (that is glucose) flow forward through the ETC (green dotted line), creating a proton motive force for ATP production and also returning electron acceptor molecules (that is NAD+) for continued oxidation of metabolic substrates. LPS-induced GPD2 activity initially boosts forward electron transport (FET; left) to support an increase in glucose oxidation for acetyl-CoA synthesis and induction of inflammatory genes by enhanced histone acetylation. However, sustained GPD2 activity may overwhelm the ubiquinone pool (Q), the common sink for electrons from Complex I, Complex II, and GPD2, leading to a thermodynamic environment that permits electron backflow (red dotted line). Such reverse electron transport (RET) decreases return of NAD+ molecules to the TCA cycle, impairing glucose oxidation for acetyl-CoA synthesis and histone acetylation at inflammatory genes. Therefore, we propose that GPD2-GPS activity acts as a rheostat for inflammatory gene induction in BMDMs, linking the duration of LPS exposure to the directionality of electron transport to control glucose oxidation and balance induction and suppression of inflammatory responses.
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Langston, P.K., Nambu, A., Jung, J. et al. Glycerol phosphate shuttle enzyme GPD2 regulates macrophage inflammatory responses. Nat Immunol 20, 1186–1195 (2019). https://doi.org/10.1038/s41590-019-0453-7
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