Glycerol phosphate shuttle enzyme GPD2 regulates macrophage inflammatory responses

Abstract

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.

Figure 1

ACLY activity supports inflammatory gene induction in LPS-activated macrophages. (a) Immunoblot analysis of ACLY phosphorylation at Ser455 in BMDMs stimulated with LPS for the indicated times. (b) ChIP-qPCR analysis of histone acetylation in Il6 and Il1b promoter regions in BMDMs stimulated with LPS for 0–1h +/− the ACLY inhibitor SB-204990 (ACLYi), 80 μM (n=3). (c) qPCR analysis of Il6 and Il1b gene expression in BMDMs stimulated with LPS for 0–3h +/− 80- μM ACLYi (n=2). Data are from one experiment representative of three independent experiments (a-c). Mean (b-c) shown.

Figure 2

Glucose oxidation supports inflammatory gene induction in LPS-activated macrophages by providing carbon substrate for histone acetylation. (a) Seahorse extracellular flux analysis of oxygen consumption rate (OCR) in BMDMs unstimulated or stimulated with LPS for 1h +/− 2-deoxyglucose (2DG), 10 mM (n=8). Injections were 1 μM oligomycin (O), 1.5 μM FCCP (F), and 2 μM rotenone and 2 μM antimycin A (R/AA). Basal and maximal mitochondrial OCR are shown at right. (b,c) ¹³C₆-glucose tracing into citrate-isocitrate and acetyl-CoA, presented as abundance of m+2 isotopologue relative to all other isotopologues, in BMDMs stimulated with LPS for the indicated times (n=4). (d) Tracing of ¹⁴C₆-glucose-derived carbons into histones in BMDMs stimulated with LPS for 0–2h (n=2). (e) ChIP-qPCR analysis of histone acetylation in Il6 and Il1b promoter regions in BMDMs stimulated with LPS for 0–1h +/− 10 mM 2DG (n=3). (f) qPCR analysis of Il6 and Il1b gene expression in BMDMs stimulated with LPS for 0–3h +/− 10 mM 2DG. (g) ELISA for IL-6 production in culture supernatants of BMDMs stimulated and treated as in f (n=4). Data are from one experiment representative of ten (a), three (b-d), or four (e-g) independent experiments. Mean (a-g) +/− s.e.m. (a-c,g) shown. *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001 (two-tailed Student’s t-test).

Figure 3

The GPS enzyme GPD2 regulates glucose oxidation in LPS-activated macrophages. (a) Global steady-state metabolite profiling of BMDMs unstimulated or LPS-stimulated for 3h (n=4). Enriched pathways are shown, ranked by p value. (b) Schematic depicting the spatial and biochemical position of the GPS and GPD2 at the nexus between glycolysis and electron transport. (c) qPCR analysis of Gpd2 gene expression in BMDMs stimulated with LPS for 1.5h. (d) Immunoblot analysis of GPD2 in unstimulated wild-type (WT) and Gpd2^−/− BMDMs. (e) Seahorse extracellular flux analysis of OCR in permeabilized WT and Gpd2^−/− BMDMs (n=6). Injections were 10 mM glycerol 3-phosphate, 2 μM rotenone, 1 mM ADP (G/R/A); 1 μM oligomcyin (O); and 2 μM antimycin A (AA). Bar graph shows fold change in OCR after G/R/A injection. (f) ¹³C₆-glucose tracing into glycerol 3-phosphate (presented as peak areas for each isotopologue) in WT and Gpd2^−/− BMDMs (n=3). (g) Seahorse analysis of basal and maximal mitochondrial OCR in WT and Gpd2^−/− BMDMs unstimulated or stimulated with LPS for 1h (n=6). (h) Uptake of ³H-deoxy-D-glucose in WT and Gpd2^−/− BMDMs stimulated with LPS for the indicated times (n=3). (i) Seahorse analysis of extracellular acidification rate (ECAR) in WT and Gpd2^−/− BMDMs stimulated with LPS for 0–2h (n=6). Data are from one experiment representative of two (a), three (c,d,f-i), or four (e) independent experiments. Mean (c,e-i) +/− s.e.m. (e-i) shown. *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001 (two-tailed Student’s t-test).

Figure 4

GPD2 activity influences inflammatory gene induction in LPS-activated macrophages by regulating acetyl-CoA production and histone acetylation. (a,b) ¹³C₆-glucose tracing into citrate-isocitrate (n=3) and acetyl-CoA (n=4) in WT and Gpd2^−/− BMDMs, stimulated with LPS for 0–1h, presented as LPS-induced fold change in percent m+2 enrichment. (c) ChIP-qPCR analysis of histone acetylation in Il6 and Il1b promoter regions in WT and Gpd2^−/− BMDMs stimulated with LPS for 0–1h (n=3). (d) qPCR analysis of Il6 and Il1b gene expression in WT and Gpd2^−/− BMDMs stimulated with LPS for 0–3h (n=2). (e) ELISA for IL-6 production in culture supernatants of WT and Gpd2^−/− BMDMs stimulated as in d (n=4). Data are from one experiment representative of three (a,b) or four (c-e) independent experiments. Mean (a-e) +/− s.e.m. (a,b,e) shown. *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001 (two-tailed Student’s t-test).

Figure 5

Glucose utilization impairs oxidative metabolism and limits inflammatory gene induction in tolerant macrophages. (a,b) ¹³C₆-glucose tracing into citrate-isocitrate and acetyl-CoA, presented as abundance of m+2 isotopologue relative to all other isotopologues, in unstimulated (U) or LPS-stimulated (U+LPS) naïve BMDMs and unstimulated (T) or LPS-stimulated (T+LPS) BMDMs tolerized by 24h LPS stimulation (n=4). (c) Seahorse extracellular flux analysis of OCR in BMDMs stimulated with LPS for 12h +/− 5 mM 2DG (n=8). Injections were 1 μM oligomycin (O), 1.5 μM FCCP (F), and 2 μM rotenone and 2 μM antimycin A (R/AA). Basal and maximal mitochondrial OCR are shown at right. (d) ChIP-qPCR analysis of histone acetylation in Il6 and Il1b promoter regions in unstimulated (U) or LPS-stimulated (U+LPS) naïve BMDMs and unstimulated (T) or LPS-stimulated BMDMs tolerized by 24h LPS stimulation +/− 5mM 2DG treatment (T+LPS or T+LPS+2DG) (n=3). (e) qPCR analysis of Il6 and Il1b gene expression in BMDMs treated as in d (n=2). (f) ELISA for IL-6 production in culture supernatants of BMDMs treated as in d (n=2). (g,h) Immunoblot analysis of IκBα degradation and IRF3 phosphorylation at the indicated times following LPS challenge of unstimulated (U) BMDMs or BMDMs tolerized by 24h LPS stimulation +/− 5mM 2DG treatment (T or T+2DG). Data are from one experiment representative of three (a-d,g,h) or four (e,f) independent experiments. Mean (a-f) +/− s.e.m. (a-c) shown. *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001 (two-tailed Student’s t-test).

Figure 6

GPD2 activity influences suppression of inflammatory responses in tolerant macrophages. (a) Seahorse extracellular flux analysis of OCR in WT and Gpd2^−/− BMDMs stimulated with LPS for 12h (n=8). Injections were 1 μM oligomycin (O), 1.5 μM FCCP (F), and 2 μM rotenone and 2 μM antimycin A (R/AA). Basal and maximal mitochondrial OCR are shown at right. (b) ChIP-qPCR analysis of histone acetylation in Il6 and Il1b promoter regions in unstimulated (U) or LPS-stimulated (U+LPS) naïve wild-type (WT) and Gpd2^−/− BMDMs and unstimulated (T) or LPS-stimulated (T+LPS) tolerant WT and Gpd2^−/− BMDMs (n=3). Tolerance was induced by 24h challenge with LPS, which was washed off before stimulation. (c) qPCR analysis of Il6 and Il1b gene expression in BMDMs treated as in b (n=2). (d) ELISA for IL-6 production in culture supernatants of BMDMs treated as in b (n=2). (e,f) Immunoblot analysis of IκBα degradation and IRF3 phosphorylation at the indicated times following LPS challenge of WT and Gpd2^−/− naive (Unstim) BMDMs and BMDMs tolerized by 24h treatment with LPS (Tolerant). Data are from one experiment representative of three independent experiments (a-f). Mean (a-d) +/− s.e.m. (a) shown. *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001 (two-tailed Student’s t-test).

Figure 7

GPD2-dependent glucose oxidation contributes to reverse electron transport in LPS tolerant BMDMs. (a,b) MitoSox labeling of mitochondrial superoxide levels in WT and Gpd2^−/− BMDMs unstimulated or stimulated with LPS for 12h +/− 5 mM 2DG and +/− 1.5 μM rotenone (Rot), shown as histograms in a (numbers are mean fluorescence intensities, MFIs) and as percent change in MitoSox MFI after Rot in b (n=6). (c) Real-time fluorescence microscopy analysis of NAD(P)H autofluorescence intensity in BMDMs unstimulated or stimulated with LPS for 12h, followed by injection of 1.5 μM Rot (n=100 individual cells). (d) NAD(P)H autofluorescence sensitivity to Rot treatment, as measured in c, in WT and Gpd2^−/− BMDMs unstimulated or stimulated with LPS for 12h +/− 5 mM 2DG. Data are from three (b) or two (d) independent experiments or from one experiment representative of three independent experiments (a,c). Mean (b-d) +/− s.e.m. (b,c) shown. *p ≤0.05, **p ≤0.01, ***p ≤0.001, ****p ≤0.0001 (two-tailed Student’s t-test).

Figure 8

GPD2 activity supports LPS tolerance in vivo. (a) ELISA analysis of IL-6 amounts in sera of WT and Gpd2^−/− mice 6h after a lethal dose of LPS (30 mg/kg), preceded by injection with vehicle (saline) or sublethal LPS (3 mg/kg) 24h before (mean +/− s.d. shown). p=0.0155 (n=9) and p=0.0237 (n=10) respectively for mice without or with sublethal LPS pretreatment; 2way ANOVA with Sidak’s multiple comparisons test (*p ≤0.05). (b) Mouse internal body temperature 6h after lethal LPS challenge (mean +/− s.d. shown). (c) Mouse survival after lethal LPS challenge 24h after sublethal LPS injection. p =0.0012; WT n=20, Gpd2^−/− n=19; Mantel-Cox test. Data are from two independent experiments (a-c).