Succinate Dehydrogenase Supports Metabolic Repurposing of Mitochondria to Drive Inflammatory Macrophages

Abstract

Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state.

Keywords: immunometabolism; innate immunity; macrophage; reverse electron transport; succinate; succinate dehydrogenase; toll-like receptors.

Figure 1. Succinate drives IL-1β production and limits the production of IL-1RA and IL-10.

BMDMs were pretreated for 3 h with succinate (Suc; 5 mM; A-I, or 0.2 – 5 mM, J) before being stimulated with LPS (100 ng/ml) for 48 h (A - C, G, J), or the indicated times (D – F, H, I). mRNA was extracted from total cell lysates and analyzed by qPCR for IL-1β (A), TNF-α (D) IL-1RA (G) and IL-10 (H) expression. Whole cell lysates were analyzed by western blotting for pro-IL-1β, HIF-1α, phospho-p65, total p65, IκBα and β-actin (B, C, F). Supernatants were analyzed by ELISA for TNF-α (E) and IL-10 production (I, J). The data in (A, D, E, G - J) represent mean ± S.E.M., n=3, *p<0.05, **p < 0.01. The blots in (B, C F) are representative of 3 independent experiments.

Figure 1. Succinate drives IL-1β production and limits the production of IL-1RA and IL-10.

BMDMs were pretreated for 3 h with succinate (Suc; 5 mM; A-I, or 0.2 – 5 mM, J) before being stimulated with LPS (100 ng/ml) for 48 h (A - C, G, J), or the indicated times (D – F, H, I). mRNA was extracted from total cell lysates and analyzed by qPCR for IL-1β (A), TNF-α (D) IL-1RA (G) and IL-10 (H) expression. Whole cell lysates were analyzed by western blotting for pro-IL-1β, HIF-1α, phospho-p65, total p65, IκBα and β-actin (B, C, F). Supernatants were analyzed by ELISA for TNF-α (E) and IL-10 production (I, J). The data in (A, D, E, G - J) represent mean ± S.E.M., n=3, *p<0.05, **p < 0.01. The blots in (B, C F) are representative of 3 independent experiments.

Figure 2. Inhibition of succinate dehydrogenase impairs LPS-induced IL-1β production and boosts IL-1RA and IL-10.

BMDMs were pretreated for 3 h with dimethyl malonate (DMM; 10 mM) prior to stimulation with LPS (100ng/ml) for 24 h (A), 48 h (B, C, E, G, H), or 4 h (D, F). SDHB-proficient (SDHB^fl/fl EtOH) and SDHB-deficient (SDHB^fl/fl Tamox) BMDMs were untreated (Ctl) or treated with LPS (100 ng/μl) for 24 h (I, J). Lysed cells were analyzed by liquid chromatography-mass spectrometry (LC-MS) to determine succinate levels (A). mRNA from total cell lysates was analyzed by qPCR for IL-1β (B), TNF-α (D), IL-1RA (F) and IL-10 (G) expression. Whole cell lysates were analyzed by Western blotting for pro-IL-1β, HIF-1α and β-actin (C, I). Supernatants were analyzed by ELISA for TNF-α (E, J) and IL-10 production (H). The data in (A, B, D - H) represent mean ± S.E.M., n=3. The data in (J) represent mean ± S.E.M., n=6, *p < 0.05, **p < 0.01, ***p < 0.001. The blots are representative of 3 (C) or 5 (J) independent experiments.

Figure 2. Inhibition of succinate dehydrogenase impairs LPS-induced IL-1β production and boosts IL-1RA and IL-10.

BMDMs were pretreated for 3 h with dimethyl malonate (DMM; 10 mM) prior to stimulation with LPS (100ng/ml) for 24 h (A), 48 h (B, C, E, G, H), or 4 h (D, F). SDHB-proficient (SDHB^fl/fl EtOH) and SDHB-deficient (SDHB^fl/fl Tamox) BMDMs were untreated (Ctl) or treated with LPS (100 ng/μl) for 24 h (I, J). Lysed cells were analyzed by liquid chromatography-mass spectrometry (LC-MS) to determine succinate levels (A). mRNA from total cell lysates was analyzed by qPCR for IL-1β (B), TNF-α (D), IL-1RA (F) and IL-10 (G) expression. Whole cell lysates were analyzed by Western blotting for pro-IL-1β, HIF-1α and β-actin (C, I). Supernatants were analyzed by ELISA for TNF-α (E, J) and IL-10 production (H). The data in (A, B, D - H) represent mean ± S.E.M., n=3. The data in (J) represent mean ± S.E.M., n=6, *p < 0.05, **p < 0.01, ***p < 0.001. The blots are representative of 3 (C) or 5 (J) independent experiments.

Figure 3. Limiting succinate oxidation induces an anti-inflammatory response.

BMDMs were pretreated with dimethyl malonate (DMM; 10 mM) or succinate (5 mM) for 3 h before being stimulated with LPS (100 ng/μl) for 4 or 48 h. RNA was isolated and RNA sequencing was performed. The gene expression in the different stimulations and time-points was modeled with a generalized linear model, and fold-changes and FDR-adjusted p-values were calculated. Fig 3(A) shows the distribution of the fold changes (log₂FC) and the FDR – adjusted p-values (log FDR) for the comparison between LPS-treated samples and control – without and with pretreatment with succinate and DMM. A large number of genes were found to be oppositely regulated when pretreated with succinate as compared to when they were pretreated with DMM. Significant changes are coloured red, while the insignificant changes are coloured grey. Fig 3(B) shows the difference in fold changes when the BMDMs were pretreated with either DMM or succinate, as compared to when they were not. The genes are annotated for the immune pathways they belong to. Fig 3(C) shows the results of functional enrichment of our gene expression analysis in the Kegg and Reactome pathway databases. The heatmap represents the statistical significance (FDR-adjusted p-value) of the different pathways found to be enriched in our analysis, with the darker colours denoting pathways enriched with higher confidence.

Figure 4. Inhibition of succinate dehydrogenase in vivo is anti-inflammatory.

Mice were injected intraperitoneally (i.p.) with DMM (160 mg/kg) or PBS for 3 h, followed by PBS or LPS (15 mg/kg) for 2 h. Serum was isolated from whole blood and IL-1β (A), IL-10 (B) and TNF-α (C) production were measured by ELISA. Spleens were isolated and IL-1β (D) and PHD3 (F) expression were analyzed by qPCR and pro-IL-1β and β-actin were measured by Western blotting (E). The data in (A-D, F) represent mean ± S.E.M., n=5 per group, *p < 0.05, **p < 0.01, ***p < 0.001. Blots are representative of 1 sample from each treatment group.

Figure 5. Glycolytic ATP production facilitates an increase in mitochondrial membrane potential that is required for the pro-inflammatory effects of LPS.

(A – E) BMDMs were stimulated with LPS (100 ng/ml) for 48 h (A, B) or 24 h (C, D). Oxygen consumption rate (OCR) and proton production rate (PPR) were analyzed as read-outs for for oxidative phosphorylation and glycolysis, respectively, using the Seahorse XF-24. The NAD⁺/NADH ratio in cell lysates was determined using an NAD⁺/NADH assay kit (C). The ATP/ADP ratio in cell lysates was determined using an ATP/ADP assay kit (D). BMDMs were untreated (Ctl) or stimulated with LPS (100 ng/ml) for 24 h before OCR analysis using the Seahorse XF-24 (E). During the Seahorse run, BMDMs were first injected with oligomycin (Oligo; 10 μM) or vehicle (EtOH), and OCR was measured for the following 6 h. At this point, rotenone (Rot; 100 nM) and antimycin A (4 μM) were injected to all wells to abolish OCR. (F – I) BMDMs were untreated (Ctl), treated with LPS (100 ng/ml) for the indicated times (F, G) or pretreated with 2-deoxyglucose (2DG; 1 mM) for 3 h prior to LPS for 24 h (I). Cells were costained with TMRM (20 nM) and MitoTracker Green (50 nM) for 30 min and then analyzed by FACS to quantify the membrane potential. The intensity of TMRM staining reflects the membrane potential. To analyse the membrane potential by confocal microscopy BMDMs were untreated (Ctl), treated with LPS (100 ng/ml) for 24 h, oligomycin (Oligo: 5 μM) for 1 h or with carbonylcyanide m-chlorophenylhydrazone (CCCP; 10 μM) for 2 min (H). Cells were stained with TMRM (20 nM) for 30 min prior to imaging. The intensity of TMRM staining reflects the membrane potential. (J - M) BMDMs were also pretreated with CCCP (0.5-10 μM; J - M), or 2DG (1 mM; N) for 3 h before being stimulated with LPS (100 ng/ml) for 4 h (J - M) or 48 h (N). mRNA was extracted from total cell lysates and analyzed by qPCR for IL-1β expression (J) and whole cell lysates were analyzed by Western blotting for pro-IL-1β and β-actin (K). Supernatants were analyzed by ELISA for TNF-α (L) and IL-10 (M, N) production. The data in (A-D, J - M) represent mean ± S.E.M., n=3, *p=0.05, **p < 0.01, ***p < 0.001. The data in (F) shows quantification of TMRM high cells and represents mean ± S.E.M., *p<0.05. The cytometric dot plots in (G, I) are representative from 3 (G) or 4 (I) separate experiments. Images in (H) are representative from 5 separate experiments. The blots in (K) are representative of three independent experiments. The Seahorse OCR data in (E) is representative of 4 independent experiments.

Figure 5. Glycolytic ATP production facilitates an increase in mitochondrial membrane potential that is required for the pro-inflammatory effects of LPS.

(A – E) BMDMs were stimulated with LPS (100 ng/ml) for 48 h (A, B) or 24 h (C, D). Oxygen consumption rate (OCR) and proton production rate (PPR) were analyzed as read-outs for for oxidative phosphorylation and glycolysis, respectively, using the Seahorse XF-24. The NAD⁺/NADH ratio in cell lysates was determined using an NAD⁺/NADH assay kit (C). The ATP/ADP ratio in cell lysates was determined using an ATP/ADP assay kit (D). BMDMs were untreated (Ctl) or stimulated with LPS (100 ng/ml) for 24 h before OCR analysis using the Seahorse XF-24 (E). During the Seahorse run, BMDMs were first injected with oligomycin (Oligo; 10 μM) or vehicle (EtOH), and OCR was measured for the following 6 h. At this point, rotenone (Rot; 100 nM) and antimycin A (4 μM) were injected to all wells to abolish OCR. (F – I) BMDMs were untreated (Ctl), treated with LPS (100 ng/ml) for the indicated times (F, G) or pretreated with 2-deoxyglucose (2DG; 1 mM) for 3 h prior to LPS for 24 h (I). Cells were costained with TMRM (20 nM) and MitoTracker Green (50 nM) for 30 min and then analyzed by FACS to quantify the membrane potential. The intensity of TMRM staining reflects the membrane potential. To analyse the membrane potential by confocal microscopy BMDMs were untreated (Ctl), treated with LPS (100 ng/ml) for 24 h, oligomycin (Oligo: 5 μM) for 1 h or with carbonylcyanide m-chlorophenylhydrazone (CCCP; 10 μM) for 2 min (H). Cells were stained with TMRM (20 nM) for 30 min prior to imaging. The intensity of TMRM staining reflects the membrane potential. (J - M) BMDMs were also pretreated with CCCP (0.5-10 μM; J - M), or 2DG (1 mM; N) for 3 h before being stimulated with LPS (100 ng/ml) for 4 h (J - M) or 48 h (N). mRNA was extracted from total cell lysates and analyzed by qPCR for IL-1β expression (J) and whole cell lysates were analyzed by Western blotting for pro-IL-1β and β-actin (K). Supernatants were analyzed by ELISA for TNF-α (L) and IL-10 (M, N) production. The data in (A-D, J - M) represent mean ± S.E.M., n=3, *p=0.05, **p < 0.01, ***p < 0.001. The data in (F) shows quantification of TMRM high cells and represents mean ± S.E.M., *p<0.05. The cytometric dot plots in (G, I) are representative from 3 (G) or 4 (I) separate experiments. Images in (H) are representative from 5 separate experiments. The blots in (K) are representative of three independent experiments. The Seahorse OCR data in (E) is representative of 4 independent experiments.

Figure 6. Inhibition of ROS production by impairing complex I or II activity or by dissipating the membrane potential limits IL-1β production in LPS-activated macrophages.

(A, B, E, F) BMDMs were pretreated for 3 h with dimethyl malonate (DMM; 10 mM; A), carbonylcyanide m-chlorophenylhydrazone (CCCP; 7.5 μM; E), or rotenone (Rot; 0.5 μM; F) before being stimulated with LPS (1 μg/ml; A, E, F) for 24 h (A, F) or 4 h (E), or were treated with succinate (Suc; 1 - 5 mM) for 24 h (B). Live cells were analyzed by FACS and mean fluorescence intensity (MFI) was quantified as a measure of cellular reactive oxygen species production. (C, D, G, H, I) BMDMs were pretreated with MitoQ (500 nM; C) or MitoTEMPO (Mt.T; 0.5 - 1 mM; D) for 1 h prior to the addition of succinate (Suc; 5 mM) for 3 h before stimulation with LPS (100 ng/ml) for 48 h. BMDMS were also pretreated for 3 h with rotenone (Rot; 0.1 – 1 μM; G - I) prior to stimulation with LPS (100 ng/ml) for 24 h. (J – N) Wild-type and AOX-expressing BMDMs were untreated (Ctl) or pretreated for 3 h with succinate (Suc; 5 mM; J, M) before being stimulated with LPS (100 ng/μl; J, M-O or 1 μg/ml; K, L) for 48 h. Whole cell lysates were analyzed by Western blotting for pro-IL-1β, HIF-1α, β-actin and AOX (C, D, H, J, O). mRNA was extracted from total cell lysates and analyzed by qPCR for IL-1β expression (G). Supernatants were analyzed by ELISA for TNF-α production (I). Live cells were analyzed by FACS and mean fluorescence intensity (MFI) was quantified as a measure of cellular reactive oxygen species production (K, L) or cells were costained with TMRM (20 nM) and MitoTracker Green (50 nM) for 30 min and then analyzed by FACS to quantify the membrane potential (M, N). The intensity of TMRM staining reflects the membrane potential. The cytometric dot plots in (N) are representative from 5 independent experiments. (P) Wild-type (WT) and alternative oxidase (AOX)-expressing mice were injected i.p. with LPS (10 mg/kg); survival rate was monitored. AOX group n=11, WT group n=12. The data in (A, E – G, I) represents mean ± S.E.M., n=3, or n=5 for (L, M) *p<0.05, **p < 0.01, ***p < 0.001. The blots in (C, D, H, J, K, O) are representative of 3 independent experiments. (Q) shows a schematic diagram illustrating the proposed mechanism by which metabolic alterations govern the inflammatory phenotype of macrophages.

Figure 6. Inhibition of ROS production by impairing complex I or II activity or by dissipating the membrane potential limits IL-1β production in LPS-activated macrophages.

(A, B, E, F) BMDMs were pretreated for 3 h with dimethyl malonate (DMM; 10 mM; A), carbonylcyanide m-chlorophenylhydrazone (CCCP; 7.5 μM; E), or rotenone (Rot; 0.5 μM; F) before being stimulated with LPS (1 μg/ml; A, E, F) for 24 h (A, F) or 4 h (E), or were treated with succinate (Suc; 1 - 5 mM) for 24 h (B). Live cells were analyzed by FACS and mean fluorescence intensity (MFI) was quantified as a measure of cellular reactive oxygen species production. (C, D, G, H, I) BMDMs were pretreated with MitoQ (500 nM; C) or MitoTEMPO (Mt.T; 0.5 - 1 mM; D) for 1 h prior to the addition of succinate (Suc; 5 mM) for 3 h before stimulation with LPS (100 ng/ml) for 48 h. BMDMS were also pretreated for 3 h with rotenone (Rot; 0.1 – 1 μM; G - I) prior to stimulation with LPS (100 ng/ml) for 24 h. (J – N) Wild-type and AOX-expressing BMDMs were untreated (Ctl) or pretreated for 3 h with succinate (Suc; 5 mM; J, M) before being stimulated with LPS (100 ng/μl; J, M-O or 1 μg/ml; K, L) for 48 h. Whole cell lysates were analyzed by Western blotting for pro-IL-1β, HIF-1α, β-actin and AOX (C, D, H, J, O). mRNA was extracted from total cell lysates and analyzed by qPCR for IL-1β expression (G). Supernatants were analyzed by ELISA for TNF-α production (I). Live cells were analyzed by FACS and mean fluorescence intensity (MFI) was quantified as a measure of cellular reactive oxygen species production (K, L) or cells were costained with TMRM (20 nM) and MitoTracker Green (50 nM) for 30 min and then analyzed by FACS to quantify the membrane potential (M, N). The intensity of TMRM staining reflects the membrane potential. The cytometric dot plots in (N) are representative from 5 independent experiments. (P) Wild-type (WT) and alternative oxidase (AOX)-expressing mice were injected i.p. with LPS (10 mg/kg); survival rate was monitored. AOX group n=11, WT group n=12. The data in (A, E – G, I) represents mean ± S.E.M., n=3, or n=5 for (L, M) *p<0.05, **p < 0.01, ***p < 0.001. The blots in (C, D, H, J, K, O) are representative of 3 independent experiments. (Q) shows a schematic diagram illustrating the proposed mechanism by which metabolic alterations govern the inflammatory phenotype of macrophages.

Figure 6. Inhibition of ROS production by impairing complex I or II activity or by dissipating the membrane potential limits IL-1β production in LPS-activated macrophages.

(A, B, E, F) BMDMs were pretreated for 3 h with dimethyl malonate (DMM; 10 mM; A), carbonylcyanide m-chlorophenylhydrazone (CCCP; 7.5 μM; E), or rotenone (Rot; 0.5 μM; F) before being stimulated with LPS (1 μg/ml; A, E, F) for 24 h (A, F) or 4 h (E), or were treated with succinate (Suc; 1 - 5 mM) for 24 h (B). Live cells were analyzed by FACS and mean fluorescence intensity (MFI) was quantified as a measure of cellular reactive oxygen species production. (C, D, G, H, I) BMDMs were pretreated with MitoQ (500 nM; C) or MitoTEMPO (Mt.T; 0.5 - 1 mM; D) for 1 h prior to the addition of succinate (Suc; 5 mM) for 3 h before stimulation with LPS (100 ng/ml) for 48 h. BMDMS were also pretreated for 3 h with rotenone (Rot; 0.1 – 1 μM; G - I) prior to stimulation with LPS (100 ng/ml) for 24 h. (J – N) Wild-type and AOX-expressing BMDMs were untreated (Ctl) or pretreated for 3 h with succinate (Suc; 5 mM; J, M) before being stimulated with LPS (100 ng/μl; J, M-O or 1 μg/ml; K, L) for 48 h. Whole cell lysates were analyzed by Western blotting for pro-IL-1β, HIF-1α, β-actin and AOX (C, D, H, J, O). mRNA was extracted from total cell lysates and analyzed by qPCR for IL-1β expression (G). Supernatants were analyzed by ELISA for TNF-α production (I). Live cells were analyzed by FACS and mean fluorescence intensity (MFI) was quantified as a measure of cellular reactive oxygen species production (K, L) or cells were costained with TMRM (20 nM) and MitoTracker Green (50 nM) for 30 min and then analyzed by FACS to quantify the membrane potential (M, N). The intensity of TMRM staining reflects the membrane potential. The cytometric dot plots in (N) are representative from 5 independent experiments. (P) Wild-type (WT) and alternative oxidase (AOX)-expressing mice were injected i.p. with LPS (10 mg/kg); survival rate was monitored. AOX group n=11, WT group n=12. The data in (A, E – G, I) represents mean ± S.E.M., n=3, or n=5 for (L, M) *p<0.05, **p < 0.01, ***p < 0.001. The blots in (C, D, H, J, K, O) are representative of 3 independent experiments. (Q) shows a schematic diagram illustrating the proposed mechanism by which metabolic alterations govern the inflammatory phenotype of macrophages.