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Dimethyl Fumarate Targets GAPDH and Aerobic Glycolysis to Modulate Immunity

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Dimethyl Fumarate Targets GAPDH and Aerobic Glycolysis to Modulate Immunity

Michael D Kornberg et al. Science.

Abstract

Activated immune cells undergo a metabolic switch to aerobic glycolysis akin to the Warburg effect, thereby presenting a potential therapeutic target in autoimmune disease. Dimethyl fumarate (DMF), a derivative of the Krebs cycle intermediate fumarate, is an immunomodulatory drug used to treat multiple sclerosis and psoriasis. Although its therapeutic mechanism remains uncertain, DMF covalently modifies cysteine residues in a process termed succination. We found that DMF succinates and inactivates the catalytic cysteine of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in mice and humans, both in vitro and in vivo. It thereby down-regulates aerobic glycolysis in activated myeloid and lymphoid cells, which mediates its anti-inflammatory effects. Our results provide mechanistic insight into immune modulation by DMF and represent a proof of concept that aerobic glycolysis is a therapeutic target in autoimmunity.

Conflict of interest statement

Competing interests:

P.A.C. has received research funding in the past from Biogen, the company that sells DMF (trade name Tecfidera) as a therapy for MS. He received a consulting honorarium from Biogen in 2015 for work related to the compound opicinumab. The other authors declare no competing interests.

Figures

Fig. 1
Fig. 1
DMF and MMF succinate and inactivate GAPDH in vitro and after oral treatment. (A) Representative LC-MS/MS spectra demonstrating covalent modification of the catalytic cysteine (Cys-150 in mouse) by either monomethyl (2-monomethyl succinyl-cysteine, +130 Da) or dimethyl (2-dimethyl succinyl-cysteine, +144 Da) fumarate in GAPDH immunoprecipitated from splenic lysates of mice treated orally with 100 mg/kg DMF daily for five days. Because samples were reduced and treated with iodoacetate, non-succinated cysteines were modified with carbamidomethyl (Carb, +57 Da). Pooled samples were analyzed from two vehicle-treated and two DMF-treated animals. (B) Representative LC-MS/MS spectrum demonstrating monomethyl succination of the catalytic cysteine (Cys-152 in human) of GAPDH immunoprecipitated from PBMC lysates of MS patients treated with DMF for three months. Pooled samples were analyzed from three DMF-treated and two non-DMF-treated patients. (C) Dose- and time-dependent inactivation of GAPDH enzyme activity in vitro. Recombinant GAPDH was treated with the indicated drug concentrations or vehicle alone. Aliquots were removed at the specified time points, followed by enzyme activity assay. Data were pooled from four experiments performed in duplicate and represent mean ± SEM for each time point. Associated Kitz–Wilson plots and kinetic parameters are shown in fig. S4C. (D) Peritoneal macrophages were treated overnight with DMF. Cells lysates were used for GAPDH enzyme activity assay. Data represent mean ± SEM of three experiments performed in duplicate. (E) Mice were treated with DMF 100 mg/kg daily for five days by oral gavage. On Day 5, mice were sacrificed, and lysates from spleen and small intestine were used for GAPDH enzyme activity assay. Data represent mean ± SEM of five mice per group, with assays run in triplicate. Da = daltons. *P < 0.05 and **P < 0.01 by two-tailed Student’s t-test.
Fig. 2
Fig. 2
GAPDH inactivation by DMF and MMF inhibits glycolysis in activated, but not resting, macrophages and lymphocytes. (A) Mouse peritoneal macrophages (mPMs) were treated with LPS ± DMF or MMF for 24 hours followed by measurement of lactate (a proxy measure of glycolysis) in culture media by colorimetric assay. Data represent mean ± SEM of three experiments performed in duplicate. (B) mPMs were treated as in (A), and glycolysis was measured as extracellular acidification rate (ECAR) using a Seahorse extracellular flux analyzer. Data represent mean ± SEM of five experiments performed in quadruplicate. (C) Glycolysis was measured via Seahorse extracellular flux analyzer in mouse naive CD4+ lymphocytes activated overnight with anti-CD3/CD28 antibodies ± DMF/MMF. Data represent mean ± SEM of four experiments performed in triplicate. (D) mPMs were stimulated with LPS for 24 hours ± 25 μM DMF, in triplicate. Cells were then labeled with U13C-glucose, and 13C-labeling of glycolytic intermediates was measured from lysates via LC-MS. Heat map shows blockade of glycolytic flux at the level of GAPDH. (E) DMF/MMF had no effect on glycolysis in unstimulated mPMs, measured as ECAR. Data represent mean ± SEM of four experiments performed in quadruplicate. (F) Representative immunoblots showing no effect of DMF on phospho-S6K (a marker of mTOR activity) (N = two experiments performed in duplicate) or HIF-1α levels (N = three experiments) in LPS-stimulated mPMs. Data are quantified in fig. S8. *P < 0.05 and **P < 0.01 by one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison.
Fig. 3
Fig. 3
Inhibition of GAPDH and aerobic glycolysis mediates anti-inflammatory effects of DMF in macrophages. (A) mPMs were treated with LPS ± DMF for 24 hours in either limiting (0.5 mM) or saturating (10 mM) concentrations of glucose, followed by measurement of IL-1β secretion by ELISA. Data represent mean ± SEM of three experiments performed in singlet or duplicate. (B) Treating LPS-stimulated mPMs with 30 μM heptelidic acid, a selective GAPDH inhibitor, replicated the effect of DMF on IL-1β secretion. Data represent mean ± SEM of three experiments performed in singlet or duplicate. (C and D) Representative immunoblots from three experiments showing that heptelidic acid replicated the effects of DMF on iNOS expression (C) and nuclear translocation of NF-κB (D) in LPS-stimulated mPMs. Data are quantified in fig. S11. (E) Overexpression of wild-type GAPDH (GAPDH-WT), but not Cys-150 mutant (GAPDH-C150S), mitigated the effect of DMF on IL-1β secretion in mPMs. Data represent the mean of two experiments performed in duplicate. ns = non-significant. *P < 0.05 and **P < 0.01 by two-tailed Student’s t-test (A) and one-way ANOVA with Dunnett’s multiple comparison (B and E).
Fig. 4
Fig. 4
DMF and MMF differentially impact survival, differentiation, and effector function of metabolically distinct lymphocyte subsets. (A to F) Mouse naïve CD4+ lymphocytes were activated for four days with anti-CD3/CD28 antibodies under Th1-, Th17-, or Treg-cell-polarizing conditions. Cells were treated with indicated doses of DMF, MMF, or heptelidic acid on day 0 and assayed by flow cytometry on day 4. (A) DMF disproportionately decreased survival under Th1- and Th17-cell vs. Treg-cell polarizing conditions, as assessed by LIVE/DEAD aqua stain. Data represent mean ± SEM of three experiments performed in duplicate or triplicate. (B and C) DMF/MMF decreased the proportion of Tbet+ and IFNγ+ cells under Th1-cell-polarizing conditions (B) and of IL-17+ cells under Th17-cell-polarizing conditions (C, left). DMF (25 μM) produced variable results under Th17-cell-polarizing conditions, likely due to high toxicity at that dose, but nonetheless caused a significant decrease in total IL-17+ cell count (C, right). Data represent mean ± SEM of three experiments performed in duplicate or triplicate. (D) Representative flow cytometric plots demonstrating that low-dose (0.5 μM) heptelidic acid replicated the effect of DMF/MMF on IFNγ and IL-17 expression under Th1- and Th17-cell-polarizing conditions, respectively. Toxicity limited the testing of higher doses. Values represent mean ± SEM of a triplicate experiment. (E) In contrast to effects on Th1 and Th17 cells, DMF increased the proportion of FoxP3+ cells under Treg-cell-polarizing conditions. Data represent mean ± SEM of three experiments performed in duplicate or triplicate. (F) DMF/MMF produced a reciprocal increase in FoxP3+ cells under Th17-cell-polarizing conditions. Bar graph (left) and representative flow cytometric plot (right) from a triplicate experiment. Data represent mean ± SEM. (G) Mouse naïve CD4+ lymphocytes were activated under Th1- or Th17-cell-polarizing conditions for three days and then treated overnight with the indicated drug. Expression of IFNγ and IL-17 was then assessed by flow cytometry. Data represent mean ± SEM of a triplicate experiment. (H) Daily I.P. treatment with heptelidic acid attenuated the course of EAE. Data were pooled from five mice per group and represent mean ± SEM for each time point. (I) Proposed model of immune modulation by DMF, which may exploit a physiologic negative feedback function of endogenous fumarate. *P < 0.05 and **P < 0.01 by one-way ANOVA with Tukey’s multiple comparison (A); one-way ANOVA with Dunnett’s multiple comparison (B, C, E–G); and Mann–Whitney U test (H).

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