Activated macrophages undergo a metabolic switch to aerobic glycolysis accumulating Krebs cycle intermediates that alter transcription of immune response genes. Here we extend 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 LPS shock and alleviates familial Mediterranean fever and experimental autoimmune encephalitis (EAE) by targeting GSDMD. Collectively, these findings identify GSDMD as a target of fumarate and reveal a mechanism of action for fumarate-based therapeutics including DMF used to treat multiple sclerosis.

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In this study, Li et al. demonstrate that gasdermin D in peripheral myeloid cells promotes the activation and differentiation of T cell in the secondary lymphoid organs, thus driving T cell–mediated neuroinflammation and demyelination in the CNS of EAE mice.

The NLRP3 inflammasome has an important function in inflammation by promoting the processing of pro-IL-1β and pro-IL-18 to their mature bioactive forms, and by inducing cell death via pyroptosis. Here we show a critical function of the E3 ubiquitin ligase Pellino2 in facilitating activation of the NLRP3 inflammasome. Pellino2-deficient mice and myeloid cells have impaired activation of NLRP3 in response to toll-like receptor priming, NLRP3 stimuli and bacterial challenge. These functions of Pellino2 in the NLRP3 pathway are dependent on Pellino2 FHA and RING-like domains, with Pellino2 promoting the ubiquitination of NLRP3 during the priming phase of activation. We also identify a negative function of IRAK1 in the NLRP3 inflammasome, and describe a counter-regulatory relationship between IRAK1 and Pellino2. Our findings reveal a Pellino2-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome.

Thiamine (vitamin B1) deficiency (TD) causes region selective neuronal loss in the brain; it has been used to model neurodegeneration that accompanies mild impairment of oxidative metabolism. The mechanisms for TD-induced neurodegeneration remain incompletely elucidated. Inhibition of protein glycosylation, perturbation of calcium homeostasis and reduction of disulfide bonds provoke the accumulation of unfolded proteins in the endoplasmic reticulum (ER), and cause ER stress. Recently, ER stress has been implicated in a number of neurodegenerative models. We demonstrated here that TD up-regulated several markers of ER stress, such as GRP78, GADD153/Chop, phosphorylation of eIF2α and cleavage of caspase-12 in the cerebellum and the thalamus of mice. Furthermore, ultrastructural analysis by electron microscopic study revealed an abnormality in ER structure. To establish an in vitro model of TD in neurons, we treated cultured cerebellar granule neurons (CGNs) with amprolium, a potent inhibitor of thiamine transport. Exposure to amprolium caused apoptosis and the generation of reactive oxygen species in CGNs. Similar to the observation in vivo, TD up-regulated markers for ER stress. Treatment of a selective inhibitor of caspase-12 significantly alleviated amprolium-induced death of CGNs. Thus, ER stress may play a role in TDinduced brain damage.

The innate immune system is equipped with pattern-recognition receptors that recognize pathogen-associated molecular patterns 1 . Pattern-recognition receptors include transmembrane Toll-like receptors (TLRs) and cytosolic Nod-like receptors 2 . Nod1 and Nod2 recognize structures in bacterial peptidoglycan 3 . Loss-of-function mutants of Nod2 are associated with Crohn's disease 4-6 , whereas gain-offunction mutants result in predisposition to the development of earlyonset sarcoidosis and Blau syndrome 7,8 . Nod2 responds to muramyl dipeptide (MDP), a derivative of peptidoglycan 9,10 . Nod2 consists of two amino-terminal caspase-recruitment domains (CARDs), a central self-oligomerization NACHT region and multiple carboxyterminal leucine-rich repeats 11 . Engagement of the leucine-rich repeats by MDP promotes a conformational change that exposes the NACHT domain, which allows self-oligomerization of Nod2 and the binding of its CARDs to the CARD-containing kinase RIP2 (refs. 12,13). RIP2 interacts with the kinase TAK1, which leads to activation of the transcription factor NF-κB and mitogen-activated protein kinases (MAPKs) and induction of the expression of proinflammatory cytokines [14][15][16][17][18] . Ubiquitination of RIP2 is critical for Nod2 signaling pathways 15,16 .The attachment of polyubiquitin chains to RIP2 serves to recruit TAK1 via the adaptors TAB2 and TAB3 (ref. 19), and that facilitates TAK1-induced phosphorylation and activation of IκB kinases (IKKs) that induce phosphorylation of inhibitory IκB proteins 20 . Phosphorylated IκB proteins undergo proteasome-mediated degradation 21 that allows NF-κB to translocate to the nucleus and induce proinflammatory gene expression 22 . Studies have investigated the enzymes responsible for catalyzing the ubiquitination of RIP2. The Ubc13-Uev1a dimer acts as the E2 conjugating enzyme in the Lys63 (K63)-linked polyubiquitination of RIP2 (refs. 15,16), but the identity of the E3 ubiquitin ligase(s) that directly ubiquitinate(s) RIP2 to mediate Nod2-induced activation of NF-κB remains unclear. TRAF6 has been proposed as the main E3 ligase for RIP2 (ref. 15), but the ubiquitination of RIP2 is intact in TRAF6-deficient cells 16 and knockdown of TRAF6 does not affect RIP2-mediated activation of NF-κB 9,14 . Three members of the 'IAP' family of E3 ubiquitin ligases (XIAP, cIAP1 and cIAP2) have been proposed to regulate RIP2 ubiquitination 23,24 . Although the conclusions of the last two studies differ about the functional importance of cIAP1 and cIAP2 in mediating Nod2-induced ubiquitination of RIP2, one demonstrated that XIAP promotes the ubiquitination of RIP2 and recruitment of the linear ubiquitin chain-assembly complex (LUBAC) to Nod2 (ref. 23). However, the XIAP-mediated polyubiquitination of RIP2 is not K63 linked, a type of linkage associated with RIP2-induced activation of NF-κB. The E3 ligase Itch can also directly ubiquitinate RIP2 to negatively regulate Nod2-induced activation of NF-κB 25 . Thus, it remains unclear which E3 ubiquitin ligase directly cataly...

The functional relevance and mechanistic basis of the effects of the pyroptosis executioner Gasdermin D (GSDMD) on colitis remain unclear. In this study, we observed that GSDMD protein was activated during intestinal inflammation in a model of chemically induced colitis. GSDMD deficiency exacerbated experimental colitis independent of changes in the microbiota and without affecting the production of antimicrobial peptides. GSDMD deficiency in macrophages, but not epithelial cells, was sufficient to drive this exacerbated experimental colitis. We further demonstrate that GSDMD functions in macrophages as a negative regulator to control cyclic GMP–AMP synthase (cGAS)–dependent inflammation, thereby protecting against colitis. Moreover, the administration of cGAS inhibitor can rescue the colitogenic phenotype in GSDMD-deficient mice. Collectively, these findings provide the first demonstration of GSDMD’s role in controlling colitis and a detailed delineation of the underlying mechanism.

0 5 5Recognition of pathogen-associated molecules in microbes by TLRs leads to activation of transcription factors such as NF-κB that promote increased transcription of proinflammatory cytokines and interferons 1 . All mammalian TLRs, with the exception of TLR3, use the adaptor MyD88 as the receptor-proximal signaling molecule to trigger downstream activation of NF-κB 2 . The association of MyD88 with TLRs facilitates recruitment of members of the IRAK family of kinases that in turn activate the E3 ubiquitin ligase TRAF6 (refs. 3-5). The formation of polyubiquitin chains by TRAF6 serves to bring TAK1 into close proximity with its substrates, including IκB kinases (IKKs). The TAK1-induced phosphorylation and activation of IKKα and IKKβ promotes IKK-induced phosphorylation of IκB proteins 6 that normally sequester NF-κB in an inactive form in the cytoplasm. Phosphorylated forms of IκB are subject to polyubiquitination and subsequently proteasome-dependent degradation, thus liberating NF-κB to translocate to the nucleus and transcriptionally upregulate the expression of a plethora of genes 7 . Most TLRs use this MyD88-dependent pathway to activate NF-κB, but TLR4 can additionally deploy another adaptor protein, TRIF, to trigger a MyD88-independent pathway that also activates NF-κB 8 . Among TLRs, TLR3 uses TRIF as its exclusive receptor-proximal adaptor protein. TRIF interacts with RIP1 kinase to trigger downstream IKK-mediated activation of NF-κB 9,10 . TRAF6 has been reported to associate with TRIF and mediate activation of NF-κB 11-13 , but other studies had concluded that TRAF6 is dispensable for TLR3 signaling 14,15 . Such discrepancies in relation to the role of TRAF6 in TRIF signaling may be due to cell-specific roles for TRAF6 and/or functional redundancy of TRAF6 with other members of the TRAF family 11 . In addition to activation of NF-κB, TRIF can also trigger activation of interferon-regulatory factor (IRF) transcription factors. Thus, TRIF forms a complex with the kinases TBK1 and IKKi (also known as IKKε) and both kinases can catalyze phosphorylation and activation of IRF3 and IRF7, leading to their nuclear translocation and induction of type I interferons 1,16 . The latter are key antiviral molecules that block viral replication 17,18 .It is clear from the above that ubiquitination is important in TLR signal transduction. Additionally, there is an emerging appreciation of the roles of the E3 ubiquitin ligase family of Pellino proteins in TLR signaling. The mammalian Pellino family consists of four members: Pellino1, Pellino2 and splice variants of Pellino3 termed Pellino3 long (Pellino3L; also known as Pellino3a) and Pellino3 short (Pellino3S; also known as Pellino3b) 19,20 . Each Pellino family member contains an N-terminal forkhead-associated (FHA) domain that recognizes phosphothreonine residues and mediates association with IRAKs 21 , and a C-terminal RING-like domain that confers E3 ubiquitin ligase activity and an ability to catalyze lysine 63 (Lys63)-linked polyubiquitination of IRAKs [22][23]...