Dimethyl fumarate targets GAPDH and aerobic glycolysis to modulate immunity

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Science  27 Apr 2018:
Vol. 360, Issue 6387, pp. 449-453
DOI: 10.1126/science.aan4665

Immunometabolism as therapeutic target

Dimethyl fumarate (DMF) is an immunomodulatory compound used to treat multiple sclerosis and psoriasis whose mechanisms of action remain only partially understood. Kornberg et al. found that DMF and its metabolite, monomethyl fumarate, succinate the glycolytic enzyme GAPDH (see the Perspective by Matsushita and Pearce). After DMF treatment, GAPDH was inactivated, and aerobic glycolysis was down-regulated in both myeloid and lymphoid cells. This resulted in down-modulated immune responses because inflammatory immune-cell subsets require aerobic glycolysis. Thus, metabolism can serve as a viable therapeutic target in autoimmune disease.

Science, this issue p. 449; see also p. 377


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.

Pro-inflammatory stimuli induce a metabolic switch in both myeloid and lymphoid cells, leading to a Warburg-like up-regulation of aerobic glycolysis that regulates the balance between inflammatory and regulatory immune phenotypes (1, 2). Classically activated macrophages and effector lymphocytes such as T helper 1 (TH1) and TH17 cells require glycolysis for their survival, differentiation, and effector functions (39), whereas oxidative metabolism favors the differentiation of alternatively activated (M2) macrophages and regulatory T (Treg) cells (10, 11).

Dimethyl fumarate (DMF) is a serendipitously discovered immunomodulatory drug used to treat psoriasis and multiple sclerosis (MS) (12). Although its mechanisms of action remain incompletely understood, it is known to covalently modify cysteine residues in a process termed succination (not to be confused with lysine succinylation) (fig. S1) (13, 14). DMF succinates kelch-like ECH-associated protein 1 (KEAP1), which activates nuclear factor (erythroid-derived 2)–related factor 2 (Nrf2) to produce antioxidant and anti-inflammatory effects that nonetheless fail to fully account for the drug’s actions (15). Endogenous fumarate also succinates proteins, with a primary target being the active-site cysteine of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (16). Here, we show that DMF and its clinically relevant metabolite monomethyl fumarate (MMF) target GAPDH and inactivate its enzyme activity, both in vitro and after oral treatment in mice and humans. In turn, GAPDH inhibition down-regulates aerobic glycolysis in myeloid and lymphoid cells, preventing immune activation and shifting the balance between inflammatory and regulatory cell types.

To determine whether GAPDH was succinated by DMF and MMF, we performed liquid chromatography–tandem mass spectrometry (LC–MS/MS). Treatment of recombinant human GAPDH with MMF led to monomethyl succination (2-monomethyl succinyl-cysteine) at its active-site cysteine (Cys152 in human) as well as at Cys156 and Cys247, whereas DMF produced a combination of dimethyl succination (2-dimethyl succinyl-cysteine) and monomethyl succination at the same cysteines (table S1 and fig. S2). Neither of these modifications was observed at any cysteine in vehicle-treated GAPDH. In mice, oral treatment with DMF led to both monomethyl and dimethyl succination exclusively of the active-site cysteine (Cys150 in mouse) of GAPDH purified from the spleen and brain, with no such modifications observed on other cysteines or in vehicle-treated mice (Fig. 1A and table S1). Although only the monomethyl form of the drug is detected in serum (17), our finding that dimethyl succination occurred after oral administration was consistent with prior work (18, 19). Monomethyl succination of GAPDH Cys152 was also identified in peripheral blood mononuclear cells (PBMCs) from MS patients treated with DMF (Fig. 1B and table S1) but not on other cysteines or in PBMCs from MS patients not treated with DMF. GAPDH succination by endogenous fumarate occurred physiologically, as we identified modification by fumarate (2-succinyl-cysteine) at the active-site cysteine in vehicle-treated mice and healthy human controls (fig. S3).

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 (Cys150 in mouse) by either MMF (2-monomethyl succinyl-cysteine, +130 Da) or DMF (2-dimethyl succinyl-cysteine, +144 Da) in GAPDH immunoprecipitated from splenic lysates of mice treated orally with DMF (100 mg/kg) daily for 5 days. Because samples were reduced and treated with iodoacetate, nonsuccinated cysteines were modified with carbamidomethyl (+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 (Cys152 in human) of GAPDH immunoprecipitated from PBMC lysates of MS patients treated with DMF for 3 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. See fig. S4C for associated Kitz-Wilson plots and kinetic parameters. (D) Peritoneal macrophages were treated overnight with DMF. Cell 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 5 days by oral gavage. On day 5, mice were killed 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. *P < 0.05, **P < 0.01 (two-tailed Student t test).

Covalent modification of its catalytic cysteine should irreversibly inactivate GAPDH. We found that both DMF and MMF decreased the catalytic activity of recombinant GAPDH in a dose- and time-dependent manner (Fig. 1C). This inhibition was irreversible, as desalting failed to restore activity (fig. S4A). Additionally, this inhibition was mediated by active-site binding of DMF or MMF, as the drug effect was blocked by pre-incubation with 10-fold excess of GAPDH substrates (fig. S4B). GAPDH inhibition was biphasic, with an initial fast phase and a secondary slow phase. We used the Kitz-Wilson method (20) to calculate the kinetics of inhibition for both phases (fig. S4C). GAPDH activity was similarly inhibited in cultured mouse peritoneal macrophages (mPMs) treated overnight with 25 μM DMF (Fig. 1D). In mice, oral treatment with DMF decreased GAPDH activity measured from both the spleen and small intestine (Fig. 1E). This effect was particularly profound in the small intestine, which may be relevant given the role of the gut immune system in autoimmune disorders such as MS (21).

We next asked whether GAPDH inhibition by DMF affected aerobic glycolysis in activated immune cells. Using lactate production as a proxy measure, co-treatment with DMF significantly impaired glycolysis in mPMs stimulated in vitro for 24 hours with lipopolysaccharide (LPS; 1 μg/ml) (Fig. 2A). MMF had a similar effect but with lower potency. The influence on glycolysis was not due to cytotoxicity (fig. S5). Measurements of the extracellular acidification rate revealed a similar inhibition of aerobic glycolysis by DMF and MMF in LPS-stimulated mPMs (Fig. 2B). In activated mouse and human CD4+ lymphocytes, DMF and MMF decreased basal glycolysis, with an even greater effect on maximal glycolytic capacity (Fig. 2C and fig. S6). In LPS-stimulated mPMs, treatment with DMF produced a blockade in glycolytic flux at GAPDH (Fig. 2D and fig. S7), providing evidence that GAPDH inactivation mediates the down-regulation of glycolysis by DMF.

Fig. 2 GAPDH inactivation by DMF and MMF inhibits glycolysis in activated, but not resting, macrophages and lymphocytes.

(A) mPMs were treated with LPS with or without 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 naïve CD4+ lymphocytes activated overnight with antibodies to CD3 and CD28 with or without DMF or MMF. Data represent mean ± SEM of four experiments performed in triplicate. (D) mPMs were stimulated with LPS for 24 hours with or without 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 or 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 = 2 experiments performed in duplicate) or HIF-1α levels (N = 3 experiments) in LPS-stimulated mPMs. Data are quantified in fig. S8. *P < 0.05, **P < 0.01 [one-way analysis of variance (ANOVA) with Dunnett multiple-comparisons test].

DMF had no effect on glycolysis in unstimulated mPMs (Fig. 2E), raising the possibility that DMF acts not only on GAPDH but also on the signaling pathway required for glycolytic up-regulation. However, DMF had no effect on the activity of mechanistic target of rapamycin (mTOR, as measured via p70-S6 kinase phosphorylation) or levels of hypoxia-inducible factor 1α (HIF-1α) (Fig. 2F and fig. S8). This selective inhibition of glycolysis in LPS-stimulated mPMs is consistent with recent evidence that GAPDH only becomes a rate-limiting enzyme when glycolysis is up-regulated in the setting of Warburg physiology (2224), as it is in cancer and activated immune cells. This likely explains why DMF is not generally toxic.

We also examined whether DMF affected oxidative phosphorylation (OXPHOS). DMF increased OXPHOS in mPMs under both resting and LPS-stimulated conditions (fig. S9A). The inhibition of glycolysis did not depend on the up-regulation of OXPHOS (fig. S9, B and C).

We next asked whether inhibition of GAPDH and aerobic glycolysis mediated the immunologic actions of DMF. We first addressed this question in macrophages. We reproduced previous findings that both DMF and glycolytic blockade prevent classical macrophage activation (fig. S10, A to D) (3, 25) and then determined that the inhibition of cytokine production by DMF was unrelated to its effects on OXPHOS (fig. S10E). We next measured the production of interleukin (IL)–1β under low (0.5 mM) or high (10 mM) glucose concentrations and found that DMF was much less effective in the presence of high glucose (Fig. 3A); this result suggests that its anti-inflammatory effect can be overcome by driving glycolysis higher with saturating concentrations of glucose. DMF augmented the IL-4–induced expression of arginase-1 (Arg-1), a marker of M2 alternative activation (fig. S10F), but the relative importance of DMF’s effects on aerobic glycolysis versus OXPHOS in promoting alternative activation was not examined.

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 enzyme-linked immunosorbent assay. Data represent mean ± SEM of three experiments performed once or in 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 once or in 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. DMSO, dimethyl sulfoxide. (E) Overexpression of wild-type GAPDH (GAPDH-WT), but not a Cys150 → Ser mutant (GAPDH-C150S), mitigated the effect of DMF on IL-1β secretion in mPMs. Data represent the mean of two experiments performed in duplicate. *P < 0.05, **P < 0.01 [two-tailed Student t test in (A), one-way ANOVA with Dunnett multiple-comparisons test in (B) and (E)]; ns, nonsignificant.

Heptelidic acid (also known as koningic acid) is a GAPDH inhibitor that binds the active site and covalently modifies the catalytic cysteine (23, 26). The treatment of mPMs with heptelidic acid replicated the effects of DMF on IL-1β secretion (Fig. 3B), inducible nitric oxide synthase (iNOS) expression (Fig. 3C and fig. S11A), and nuclear translocation of nuclear factor κB (NF-κB) (Fig. 3D and fig. S11B). Conversely, the overexpression of wild-type GAPDH, but not catalytically inactive GAPDH mutated at Cys150, mitigated the effect of DMF on IL-1β production (Fig. 3E and fig. S12). Thus, the immunologic actions of DMF were replicated by GAPDH inhibition and reversed by increasing GAPDH expression.

We next examined the effects of DMF and MMF on lymphocyte differentiation and function. We activated mouse naïve CD4+ T cells under TH1, TH17, or Treg cell–polarizing conditions for 4 days with or without DMF or MMF, with treatment at the start of polarization. Consistent with the known effects of glycolytic blockade (7, 8), DMF had a disproportionate impact on the survival of TH1 and TH17 versus Treg cells (Fig. 4A and fig. S13A). Similarly, DMF and MMF inhibited both differentiation and cytokine production under TH1- and TH17-polarizing conditions, an effect replicated by heptelidic acid (Fig. 4, B to D, and fig. S13B). As reported with HIF-1α deficiency and 2-deoxyglucose treatment (11), DMF promoted Treg cell differentiation under Treg-polarizing conditions (Fig. 4E and fig. S13B), and both DMF and MMF reciprocally inhibited TH17 and promoted Treg cell development under TH17-polarizing conditions (Fig. 4F). When added after 3 days of polarization, DMF, MMF, and low-dose heptelidic acid had no effect on the viability (fig. S14A) or differentiation (fig. S14B) of TH1 or TH17 cells. However, all three drugs inhibited the expression of interferon-γ (IFN-γ) and IL-17 (Fig. 4G), suggesting an effect on cytokine production independent of survival and differentiation.

Fig. 4 Impacts of DMF and MMF on survival, differentiation, and effector function of metabolically distinct lymphocyte subsets.

(A to F) Mouse naïve CD4+ lymphocytes were activated for 4 days with antibodies to CD3 and CD28 under TH1 cell–, TH17 cell–, or Treg cell–polarizing conditions. Cells were treated with the 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– versus Treg cell–polarizing conditions, as assessed by viability staining. Data represent mean ± SEM of three experiments performed in duplicate or triplicate. DMF and MMF decreased the proportion of Tbet+ and IFN-γ+ 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 or MMF on IFN-γ and IL-17 expression under TH1 cell– 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 or MMF produced a reciprocal increase in FoxP3+ cells under TH17 cell–polarizing conditions. Bar graph (left) and representative flow cytometric plot (right) show results from a triplicate experiment. Data represent mean ± SEM. (G) Mouse naïve CD4+ lymphocytes were activated under TH1 cell– or TH17 cell–polarizing conditions for 3 days and then treated overnight with the indicated drug. Expression of IFN-γ and IL-17 was then assessed by flow cytometry. Data represent mean ± SEM of a triplicate experiment. (H) Daily intraperitoneal 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, **P < 0.01 [one-way ANOVA with Tukey multiple-comparisons test in (A); one-way ANOVA with Dunnett multiple-comparisons in (B), (C), (E), (F), and (G); Mann-Whitney U test in (H)].

Because GAPDH binding to mRNA underlies posttranscriptional regulation of cytokine production (9), we tested the effect of GAPDH succination on RNA binding (fig. S15). Pretreatment with DMF or heptelidic acid decreased GAPDH-RNA binding, as reported with other modifications of the active-site cysteine (27). This effect was small, however, as a similar decrease was produced by nicotinamide adenine dinucleotide (NAD+) at one-tenth the concentration. Thus, the alteration of such binding does not appear to underlie the immunologic actions of DMF.

Finally, to ascertain whether GAPDH inhibition produced anti-inflammatory actions in vivo, we examined the effect of heptelidic acid in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, and found that it attenuated the disease in these mice (Fig. 4H).

By demonstrating that a known immunomodulatory drug acts by inhibiting aerobic glycolysis, our findings provide a proof of concept that metabolism is a viable therapeutic target in autoimmunity. They may also explain important observations of DMF therapy in patients. DMF differentially affects distinct lymphocyte subsets, producing lymphopenia that selectively depletes highly glycolytic effector T cells while sparing oxidative naïve T cells and Treg cells (28, 29). Our findings suggest that the inhibition of aerobic glycolysis underlies these selective effects. It is also notable that DMF is simply a derivative of fumarate, which is a metabolic intermediate of the Krebs cycle, lying downstream of glycolysis in cellular energy production. We hypothesize that fumarate-induced inactivation of GAPDH represents an endogenous negative feedback loop. DMF—a more cell-permeable and electrophilic derivative of fumarate—may simply exploit this physiologic pathway to produce its immunologic actions (Fig. 4I). However, additional targets of succination (in addition to KEAP1) are likely relevant to both the therapeutic and toxic effects of the drug.

Supplementary Materials

Materials and Methods

Table S1

Figs. S1 to S15

References (3038)

References and Notes

Acknowledgments: We thank J. Liu and J. Stivers for insights regarding enzyme kinetics experiments; Z. Zhou, W. Chen, and A. Hoke for providing access to the Seahorse extracellular flux analyzer and support with its use; L. DeVine and R. Cole from the Johns Hopkins Mass Spectrometry Core Facility for their assistance and helpful discussion; B. Wipke and R. Scannevin for insightful discussion and for providing a protocol for preparation of DMF suspension; B. Paul for help with mPM isolation and GAPDH-RNA binding; C. Darius for drawing blood samples; and J. Bo for preparation of PBMCs. Funding: Supported by National Institute of Neurological Disorders and Stroke (NINDS) R25 grant RFA-NS-12-003, National Multiple Sclerosis Society (NMSS)–American Academy of Neurology (AAN) Clinician Scientist Development Award FAN 17107-A-1, and a Conrad N. Hilton Foundation Marilyn Hilton Bridging Award (M.D.K.); an AAN John F. Kurtzke Clinician Scientist Development Award, NMSS Career Transition Award TA-1503-03465, and a Race to Erase MS Young Investigator Award (P.B.); CPRIT Core Facility Support Award RP170005, NCI Cancer Center Support Grant P30CA125123, and intramural funds from the Dan L. Duncan Cancer Center (V.P. and N.P.); American Cancer Society grant 127430-RSG-15-105-01-CNE and NIH grants R01CA220297 and R01CA216426 (N.P.); NINDS grant R37NS041435 (P.A.C.); and USPHS grant MH18501 (S.H.S.). Author contributions: M.D.K., P.B., P.A.C., and S.H.S. contributed to overall project design. M.D.K., P.B., P.M.K., V.P., N.P., and A.M.S. performed the research. M.D.K., P.B., P.M.K., V.P., and N.P. analyzed the data. M.D.K. and P.B. prepared the figures. M.D.K. and S.H.S. wrote the manuscript. P.B., P.A.C., and N.P. edited the manuscript. Competing interests: P.A.C. has received research funding 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. Data and materials availability: Data in this paper are presented and/or tabulated in the main text and supplementary materials.
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