Science - USA (2020-09-25)

charge transfer. Information about the electronic-
vibronic dynamics on the fastest relevant time
scale is potentially of paramount importance
for the fundamental understanding of chemi-
cal properties and reactions. Because PRI un-
ambiguously monitors the dependence of SXRS
on the properties of the incident radiation, the
method will be particularly fruitful when in-
tense, ultrashort, well-defined, multicolor XFEL
pulses with controlled time delay (see discus-
sion in supplementary text and figs. S7 and
S8) ( 23 ) become available in the near future.

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ACKNOWLEDGMENTS
We thank European XFEL (Schenefeld, Germany) for providing
x-ray free-electron laser beamtime at the SQS instrument, and we
thank its staff for their assistance.Funding:Supported by the
Volkswagen Foundation within a Peter Paul Ewald Fellowship
(M.I. and V.M.) and the Swedish Science Council.Author
contributions:U.E., S.E., H.R., and J.-E.R. proposed the study
and wrote the manuscript with input from all authors. The
experiment was carried out by U.E., H.R., S.M., J.-E.R., J.S., M.A.,
C.S., M.M., T.M.B., R.B., A.D.F., P.G., T.M., Y.O., D.E.R., and
S.E. after adding the pulsed gas source and detection system
by S.M., U.E., H.R., J.S., T.M., and Y.O. and additional help in
preparation and commissioning of the instrument by M.I., J.M., V.M.,
and R.W. The data were analyzed by U.E. and H.R. with input on
simulated XFEL pulses by S.S.Competing interests:None declared.
Data availability:All data needed to evaluate the conclusions in the
paper are in the paper or the supplementary materials, and all data
underlying the figures are accessible online ( 31 ).

SUPPLEMENTARY MATERIALS
science.sciencemag.org/content/369/6511/1630/suppl/DC1
Supplementary Text
Figs. S1 to S8
References ( 32 , 33 )

15 April 2020; accepted 2 July 2020
10.1126/science.abc2622

IMMUNOMETABOLISM

Succination inactivates gasdermin D and

blocks pyroptosis

Fiachra Humphries^1 , Liraz Shmuel-Galia^1 , Natalia Ketelut-Carneiro^1 , Sheng Li^2 , Bingwei Wang^3 ,

Venkatesh V. Nemmara^4 , Ruth Wilson^1 , Zhaozhao Jiang^1 , Farnaz Khalighinejad^5 , Khaja Muneeruddin6,7,

Scott A. Shaffer6,7, Ranjan Dutta^8 , Carolina Ionete^5 , Scott Pesiridis^9 , Shuo Yang^2 ,

Paul R. Thompson^7 , Katherine A. Fitzgerald^1 *

Activated macrophages undergo a metabolic switch to aerobic glycolysis, accumulating Krebs’cycle

intermediates that alter transcription of immune response genes.We extended these observations by defining

fumarate as an inhibitor of pyroptotic cell death. We found that dimethyl fumarate (DMF) delivered to cells

or endogenous fumarate reacts with gasdermin D (GSDMD) at critical cysteine residues to form S-(2-succinyl)-

cysteine. GSDMD succination prevents its interaction with caspases, limiting its processing, oligomerization,

and capacity to induce cell death. In mice, the administration of DMF protects against lipopolysaccharide shock

and alleviates familial Mediterranean fever and experimental autoimmune encephalitis by targeting GSDMD.

Collectively, these findings identify GSDMD as a target of fumarate and reveal a mechanism of action for

fumarate-based therapeutics that include DMF, for the treatment of multiple sclerosis.

C

ell death pathways are important for

host defense. Necroptosis and pyroptosis

also contribute to inflammatory disease

through the release of danger-associated

molecular patterns (DAMPs) ( 1 ). The pore-

forming protein gasdermin D (GSDMD) is the

executioner of pyroptosis. Caspase cleavage

of GSDMD liberates an N-terminal p30 frag-

ment (GSDMD-N), which oligomerizes and

forms pores in the plasma membrane. These

pores serve as a conduit for the release of

interleukin-1b(IL-1b) and IL-18 and, ultimately,

thedemiseofthecell( 1 , 2 ).

Numerous studies have shown that cell me-

tabolism affects inflammatory responses. Lipo-

polysaccharide (LPS)–activated macrophages

switch from oxidative phosphorylation to aero-

bic glycolysis. Krebs’cycle intermediates, such

as succinate and itaconate, accumulate and

moonlight as positive and negative regulators

of inflammatory gene expression ( 3 – 8 ). To de-

termine whether Krebs’cycle intermediates

modulate pyroptosis, we tested their effect

on inflammasome responses. Bone marrow–

derived macrophages (BMDMs) were primed

with LPS for 2 hours before being exposed to

metabolites and then exposed to nigericin

(Nig). Inflammasome activation and pyrop-

tosis were then measured. The metabolites

were added after LPS to avoid any impact on

transcription. Dimethyl fumarate (DMF) po-

tently blocked LPS-Nig–induced release of

lactate dehydrogenase (LDH) (Fig. 1A) and

IL-1b(Fig. 1B) but not tumor necrosis factor–a

(TNF-a) (Fig. 1C). DMF also blocked the for-

mation of GSDMD-N (Fig. 1D). Monomethyl

fumarate (MMF), a cell-impermeable derivative

of fumarate, had no inhibitory effect. DMF

impaired kinetic cell death over 6 hours (Fig.

1E and fig. S1A). DMF even impaired cell death

when added after Nig treatment (fig. S1, B and

C). The inhibitory effect of DMF was also ob-

served in BMDMs after transfected LPS (Fig.

1F), Salmonella infection (fig. S1, D and E), or

poly(deoxyadenylic-thymidylic) [poly(dA:dT)]

(fig. S1, F and G). DMF also blocked LPS-Nig–

induced cell death and LDH release in human

THP1 (fig. S2, A to C) and CD14+monocytes

(fig. S2, D and E). BMDMs pretreated with

DMF had decreased GSDMD oligomerization

in response to LPS-Nig, indicating that DMF

blocks pore formation (Fig. 1G).

Fumarate accumulated in LPS-activated

macrophages (fig. S2F), similar to prior studies

( 5 , 7 ). To assess the effect of endogenous fu-

marate, we induced its accumulation in cells

by blocking fumarate hydratase with FHIN1

(Fig. S2F). FHIN1 impaired cell death (Fig. 1H

and fig. S2G) and reduced the formation of

GSDMD-N (Fig. 1I). DMF has previously been

reported to exhibit antiinflammatory activity

through nuclear respiratory factor 2 (NRF2)

or glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) ( 9 , 10 ). Cells treated with the GAPDH

inhibitor heptilidic acid (HA) ( 11 ) or the NRF2

inhibitor ML385 ( 12 ) had no impact on cell

death (fig. S3, A and B), LDH release (fig. S3C),

IL-1brelease (fig. S3D), or GSDMD-N forma-

tion (fig. S3, E and F). HA and ML385 inhibited

SCIENCEsciencemag.org 25 SEPTEMBER 2020•VOL 369 ISSUE 6511 1633

(^1) Program in Innate Immunity, Department of Medicine,
University of Massachusetts Medical School, Worcester, MA
01605, USA.^2 Department of Immunology, Key Laboratory of
Immunological Environment and Disease, State Key Laboratory
of Reproductive Medicine, Nanjing Medical University, Nanjing,
China.^3 Department of Pharmacology, Nanjing University of
Chinese Medicine, Nanjing, China.^4 Department of Chemistry
and Biochemistry, Rowan University, Glassboro, NJ 08028, USA.
(^5) Department of Neurology, University of Massachusetts Medical
School,Worcester,MA01605,USA.^6 Mass Spectrometry Facility,
University of Massachusetts Medical School, Shrewsbury, MA
01545, USA.^7 Department of Biochemistry and Molecular
Pharmacology, University of Massachusetts Medical School,
Worcester, MA 01605, USA.^8 Department of Neurosciences,
Lerner Research Institute, Cleveland Clinic, Case Western Reserve
University,Cleveland,OH44106,USA.^9 Innate Immunity Research
Unit, GlaxoSmithKline, Collegeville, PA 19426, USA.
*Corresponding author. Email: [email protected]
RESEARCH | REPORTS