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.

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...

Obesity underpins the development of numerous chronic diseases, such as type II diabetes mellitus. It is well established that obesity negatively alters immune cell frequencies and functions. Mucosal-associated invariant T (MAIT) cells are a population of innate T cells, which we have previously reported are dysregulated in obesity, with altered circulating and adipose tissue frequencies and a reduction in their IFN-g production, which is a critical effector function of MAIT cells in host defense. Hence, there is increased urgency to characterize the key molecular mechanisms that drive MAIT cell effector functions and to identify those which are impaired in the obesity setting. In this study, we found that MAIT cells significantly upregulate their rates of glycolysis upon activation in an mTORC1-dependent manner, and this is essential for MAIT cell IFN-g production. Furthermore, we show that mTORC1 activation is dependent on amino acid transport via SLC7A5. In obese patients, using RNA sequencing, Seahorse analysis, and a series of in vitro experiments, we demonstrate that MAIT cells isolated from obese adults display defective glycolytic metabolism, mTORC1 signaling, and SLC7A5 aa transport. Collectively, our data detail the intrinsic metabolic pathways controlling MAIT cell cytokine production and highlight mTORC1 as an important metabolic regulator that is impaired in obesity, leading to altered MAIT cell responses.

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The immune suppressive and anti-inflammatory capabilities of bone marrow-derived mesenchymal stromal cells (MSCs) represent an innovative new tool in regenerative medicine and immune regulation. The potent immune suppressive ability of MSC over T cells, dendritic cells, and natural killer cells has been extensively characterized, however, the effect of MSC on B cell function has not yet been clarified. In this study, the direct effect of MSC on peripheral blood B cell function is defined and the mechanism utilized by MSC in enhancing B cell survival in vitro identified. Human MSC supported the activation, proliferation, and survival of purified CD19(+) B cells through a cell contact-dependent mechanism. These effects were not mediated through B cell activating factor or notch signaling. However, cell contact between MSC and B cells resulted in increased production of vascular endothelial growth factor (VEGF) by MSC facilitating AKT phosphorylation within the B cell and inhibiting caspase 3-mediated apoptosis. Blocking studies demonstrated that this cell contact-dependent effect was not dependent on signaling through CXCR4-CXCL12 or through the epidermal growth factor receptor (EGFR). These results suggest that direct cell contact between MSC and B cells supports B cell viability and function, suggesting that MSC may not represent a suitable therapy for B cell-mediated disease.

Aims/hypothesis Mucosal-associated invariant T cells (MAIT cells) are an abundant population of innate T cells. When activated, MAIT cells rapidly produce a range of cytokines, including IFNγ, TNF-α and IL-17. Several studies have implicated MAIT cells in the development of metabolic dysfunction, but the mechanisms through which this occurs are not fully understood. We hypothesised that MAIT cells are associated with insulin resistance in children with obesity, and affect insulin signalling through their production of IL-17. Methods In a cross-sectional observational study, we investigated MAIT cell cytokine profiles in a cohort of 30 children with obesity and 30 healthy control participants, of similar age, using flow cytometry. We then used a cell-based model to determine the direct effect of MAIT cells and IL-17 on insulin signalling and glucose uptake. Results Children with obesity display increased MAIT cell frequencies (2.2% vs 2.8%, p=0.047), and, once activated, these produced elevated levels of both TNF-α (39% vs 28%, p=0.03) and IL-17 (1.25% vs 0.5%, p=0.008). The IL-17-producing MAIT cells were associated with an elevated HOMA-IR (r=0.65, p=0.001). The MAIT cell secretome from adults with obesity resulted in reduced glucose uptake when compared with the secretome from healthy adult control (1.31 vs 0.96, p=0.0002), a defect that could be blocked by neutralising IL-17. Finally, we demonstrated that recombinant IL-17 blocked insulin-mediated glucose uptake via inhibition of phosphorylated Akt and extracellular signal-regulated kinase. Conclusions/interpretations Collectively, these studies provide further support for the role of MAIT cells in the development of metabolic dysfunction, and suggest that an IL-17-mediated effect on intracellular insulin signalling is responsible. Graphical abstract

Mucosal Associated Invariant T (MAIT) cells are an abundant population of innate T cells which recognise bacterial ligands presented by the MHC class-I like molecule MR1. MAIT cells play a key role in host protection against bacterial and viral pathogens. Upon activation MAIT cells undergo proliferative expansion and increased production of effector molecules such as cytokines. The molecular and metabolic mechanisms controlling MAIT cell effector functions are still emerging. In this study, we found that expression of the key metabolism regulator and transcription factor MYC is upregulated in MAIT cells upon immune stimulation. Using quantitative mass spectrometry, we identified the activation of two MYC controlled metabolic pathways; amino acid transport and glycolysis, both of which are critical for MAIT cell proliferation. Finally, we show that MYC expression in response to immune activation is diminished in MAIT cells isolated from people with obesity, resulting in defective MAIT cell proliferation and functional responses. Collectively our data details for the first time the importance of MYC regulated metabolism for MAIT cell proliferation, and provides additional insight into the molecular defects underpinning functional failings of MAIT cells in obesity.

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells which recognize a limited repertoire of ligands presented by the MHC class-I like molecule MR1. In addition to their key role in host protection against bacterial and viral pathogens, MAIT cells are emerging as potent anti-cancer effectors. With their abundance in human, unrestricted properties, and rapid effector functions MAIT cells are emerging as attractive candidates for immunotherapy. In the current study, we demonstrate that MAIT cells are potent cytotoxic cells, rapidly degranulating and inducing target cell death. Previous work from our group and others has highlighted glucose metabolism as a critical process for MAIT cell cytokine responses at 18 h. However, the metabolic processes supporting rapid MAIT cell cytotoxic responses are currently unknown. Here, we show that glucose metabolism is dispensable for both MAIT cell cytotoxicity and early (<3 h) cytokine production, as is oxidative phosphorylation. We show that MAIT cells have the machinery required to make (GYS-1) and metabolize (PYGB) glycogen and further demonstrate that that MAIT cell cytotoxicity and rapid cytokine responses are dependent on glycogen metabolism. In summary, we show that glycogen-fueled metabolism supports rapid MAIT cell effector functions (cytotoxicity and cytokine production) which may have implications for their use as an immunotherapeutic agent.