Assembly of the NLRP3 inflammasome activates caspase-1 and mediates the processing and release of the leaderless cytokine IL-1β and thereby serves a central role in the inflammatory response and in diverse human diseases. Here we found that upon activation of caspase-1, oligomeric NLRP3 inflammasome particles were released from macrophages. Recombinant oligomeric protein particles composed of the adaptor ASC or the p.D303N mutant form of NLRP3 associated with cryopyrin-associated periodic syndromes (CAPS) stimulated further activation of caspase-1 extracellularly, as well as intracellularly after phagocytosis by surrounding macrophages. We found oligomeric ASC particles in the serum of patients with active CAPS but not in that of patients with other inherited autoinflammatory diseases. Our findings support a model whereby the NLRP3 inflammasome, acting as an extracellular oligomeric complex, amplifies the inflammatory response.

Cell volume regulation is a primitive response to alterations in environmental osmolarity. The NLRP3 inflammasome is a multiprotein complex that senses pathogen- and danger-associated signals. Here, we report that, from fish to mammals, the basic mechanisms of cell swelling and regulatory volume decrease (RVD) are sensed via the NLRP3 inflammasome. We found that a decrease in extracellular osmolarity induced a K(+)-dependent conformational change of the preassembled NLRP3-inactive inflammasome during cell swelling, followed by activation of the NLRP3 inflammasome and caspase-1, which was controlled by transient receptor potential channels during RVD. Both mechanisms were necessary for interleukin-1β processing. Increased extracellular osmolarity prevented caspase-1 activation by different known NLRP3 activators. Collectively, our data identify cell volume regulation as a basic conserved homeostatic mechanism associated with the formation of the NLRP3 inflammasome and reveal a mechanism for NLRP3 inflammasome activation.

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Background: The inflammasome is a multimolecular complex that regulates the processing of the pro-inflammatory cytokine interleukin-1β.Results: Inhibitors of deubiquitinase (DUB) enzymes inhibited the release of interleukin-1β.Conclusion: DUBs regulate assembly of the inflammasome.Significance: DUBs may represent new anti-inflammatory drug targets for the treatment of inflammatory disease.

Pannexin 1 (Panx1) is expressed in various mammalian tissues including the brain and immune cells. Here, we present evidence that Panx1 when expressed in mammalian cells, forms anion-selective channels, with a rank order of permeabilities: NO (3) (-)> I(-) > Br (-)> Cl (-) > F (-)>> aspartate (-)≈ glutamate (-)≈ gluconate(-). Single-channel Panx1-mediated currents have a unitary conductance around 68 pS. Our results show that Panx1 assembles into a membrane anion channel with a relatively low single-channel conductance.

Purpose: Glioblastoma (GBM) is the most common and malignant form of primary human brain tumor in adults, with an average survival at diagnosis of 18 months. Metabolism is a new attractive therapeutic target in cancer; however, little is known about metabolic heterogeneity and plasticity within GBM tumors. We therefore aimed to investigate metabolic phenotyping of primary cultures in the context of molecular tumor heterogeneity to provide a proof of concept for personalized metabolic targeting of GBM.Experimental Design: We have analyzed extensively several primary GBM cultures using transcriptomics, metabolic phenotyping assays, and mitochondrial respirometry.Results: We found that metabolic phenotyping clearly identifies 2 clusters, GLN High and GLN Low , mainly based on metabolic plasticity and glutamine (GLN) utilization. Inhibition of glutamine metabolism slows the in vitro and in vivo growth of GLN High GBM cultures despite metabolic adaptation to nutrient availability, in particular by increasing pyruvate shuttling into mitochondria. Furthermore, phenotypic and molecular analyses show that highly proliferative GLN High cultures are CD133 neg and display a mesenchymal signature in contrast to CD133 pos GLN Low GBM cells. Conclusions: Our results show that metabolic phenotyping identified an essential metabolic pathway in a GBM cell subtype, and provide a proof of concept for theranostic metabolic targeting.

The transport of pyruvate into mitochondria requires a specific carrier, the mitochondrial pyruvate carrier (MPC). The MPC represents a central node of carbon metabolism, and its activity is likely to play a key role in bioenergetics. Until now, investigation of the MPC activity has been limited. However, the recent molecular identification of the components of the carrier has allowed us to engineer a genetically encoded biosensor and to monitor the activity of the MPC in real time in a cell population or in a single cell. We report that the MPC activity is low in cancer cells, which mainly rely on glycolysis to generate ATP, a characteristic known as the Warburg effect. We show that this low activity can be reversed by increasing the concentration of cytosolic pyruvate, thus increasing oxidative phosphorylation. This biosensor represents a unique tool to investigate carbon metabolism and bioenergetics in various cell types.

Apoptosis-associated speck-like protein containing a CARD (ASC) is a key adaptor molecule required for inflammatory processes. ASC acts by bridging NLRP proteins, such as NLRP3, with pro-caspase-1 within the inflammasome complex that subsequently results in the activation of caspase-1 and the secretion of interleukin (IL)-1β and IL-18. In response to bacterial infection, ASC also forms specks by self-oligomerization to activate caspase-1 and induce pyroptosis. Hitherto the role of these specks in NLRP3 inflammasome activation in response to danger signals is largely unexplored. Here we report that under hypotonic conditions, ASC formed specks independently of NLRP3 that did not activate caspase-1. These specks were not associated with pyroptosis and were controlled by Transient Receptor Potential Vanilloid 2 channel mediated signaling. However, interaction with NLRP3 enhanced ASC speck formation leading to fully functional inflammasomes and caspase-1 activation. This study reveals that the ASC speck could present different oligomerization assemblies and represents an essential step in the activation of functional NLRP3 inflammasomes.