P2X 7 receptors are ATP-gated cation channels; their activation in macrophage also leads to rapid opening of a membrane pore permeable to dyes such as ethidium, and to release of the pro-inflammatory cytokine, interleukin-1b (IL-1b). It has not been known what this dye-uptake path is, or whether it is involved in downstream signalling to IL-1b release. Here, we identify pannexin-1, a recently described mammalian protein that functions as a hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin-1 is required for processing of caspase-1 and release of mature IL-1b induced by P2X 7 receptor activation.

The gasdermins are a new family of pore-forming cell death effectors that cause membrane permeabilization and pyroptosis, a lytic pro-inflammatory type of cell death. Gasdermins consist of a cytotoxic N-terminal domain and a C-terminal repressor domain connected by a flexible linker. Proteolytic cleavage between these two domains releases the intramolecular inhibition on the cytotoxic domain, allowing it to insert into cell membranes and to form large oligomeric membrane pores, which disrupt ion homeostasis and induce cell death. In this review, we discuss the recent developments in gasdermin research with a focus on the mechanisms that control gasdermin activation, pore formation and the consequences of gasdermin-induced membrane permeabilization.

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.

The proinflammatory IL-1 cytokines IL-1α, IL-1β, and IL-18 are key mediators of the acute immune response to injury and infection. Mechanisms underlying their cellular release remain unclear. Activation of purinergic P2X7 receptors (P2X7R) by extracellular ATP is a key physiological inducer of rapid IL-1β release from LPS-primed macrophage. We investigated patterns of ATP-mediated release of IL-1 cytokines from three macrophage types in attempts to provide direct evidence for or against distinct release mechanisms. We used peritoneal macrophage from P2X7R−/− mice and found that release of IL-1α, IL-18, as well as IL-1β, by ATP resulted exclusively from activation of P2X7R, release of all these IL-1 cytokines involved pannexin-1 (panx1), and that there was both a panx1-dependent and -independent component to IL-1β release. We compared IL-1-release patterns from LPS-primed peritoneal macrophage, RAW264.7 macrophage, and J774A.1 macrophage. We found RAW264.7 macrophage readily release pro-IL-1β independently of panx1 but do not release mature IL-1β because they do not express apoptotic speck-like protein with a caspase-activating recruiting domain and so have no caspase-1 inflammasome activity. We delineated two distinct release pathways: the well-known caspase-1 cascade mediating release of processed IL-1β that was selectively blocked by inhibition of caspase-1 or panx1, and a calcium-independent, caspase-1/panx1-independent release of pro-IL-1β that was selectively blocked by glycine. None of these release responses were associated with cell damage or cytolytic effects. This provides the first direct demonstration of a distinct signaling mechanism responsible for ATP-induced release of pro-IL-1β.

Pannexin (Panx) 1 is a widely expressed protein that shares structural, but not amino acid, homology with gap junction proteins, the connexins. Panx1 does not form gap junctions in mammalian cells, but it may function as a plasma membrane hemichannel. Little is known of the pharmacological properties of panx1 expression in mammalian cells. Here, we identify three variants in the human PANX1 gene. We expressed these variants and mouse Panx1 in mammalian cells and compared Panx1-induced currents. All human Panx1 variants and the mouse Panx1 showed identical protein expression levels, localization patterns, and functional properties, although the frequency of functional expression was species-dependent. Panx1 currents were independent of changes in extracellular or intracellular calcium or phospholipase C transduction. We found compounds that inhibited Panx1 currents with a rank order of potency: carbenoxolone Ͼ disodium 4,4Ј-diisothio-Triphosphate nucleotides (ATP, GTP, and UTP) rapidly and reversibly inhibited Panx1 currents via mechanism(s) independent of purine receptors. When Panx1 was coexpressed with purinergic P2X 7 receptor (P2X 7 R), DIDS was found to act as a P2X 7 R antagonist to inhibit ATP-evoked currents, but none of the other compounds inhibited P2X 7 R currents. This is the first detailed pharmacological characterization of Panx1-mediated currents in mammalian cells and sheds new, although contradictory, light on the hypothesis that Panx1 acts as a hemichannel to allow passage of large molecules in response to P2X 7 R activation.Pannexins are a three-membered family of membrane proteins (Panx1-3) that bear topological similarity, but minimal amino acid homology, to the large family of gap junction channels, the connexins (Panchin, 2005;Barbe et al., 2006). Unlike connexins, pannexins do not form gap junctions when expressed in mammalian cells (Huang et al., 2007a) but do lead to the appearance of ionic currents whose properties resemble "undocked" gap junction hemichannels (Scemes et al., 2007). Panx1 has distinct but generally ubiquitous expression in excitable and nonexcitable cells and has generated increasing interest as a likely hemichannel conduit for nonvesicular release of ATP from erythrocytes and taste receptor cells (Locovei et al., 2006a;Huang et al., 2007b). Panx1 has also been shown to form protein-protein association with the purinergic P2X 7 receptor (P2X 7 R), whose activation by extracellular ATP opens a typical cationic channel within milliseconds followed seconds later by an opening, or activation, of a large pore permeable to molecules up to 900 Da (Pelegrin and Surprenant, 2006). The initial phase of this P2X 7 R-activated dye-permeable pore is inhibited by blockade of Panx1 using siRNA knockdown techniques, by a Panx1-mimetic inhibitory peptide, and by the relatively nonselective gap junction channel blocker, carbenoxolone (CBX) Surprenant, 2006, 2007). These results have provided the evidence for the hypothesis that Panx1 hemichannels open in response to conformati...

Pannexin-1 is a recently identified membrane protein that can act as a nonselective pore permeable to dyes such as ethidium when ectopically expressed. Blockade of pannexin-1 in macrophage endogenously expressing the ATP-gated P2X 7 receptor (P2X 7 R) blocks the initial dye uptake, but not the ionic current, and also blocks processing and release of interleukin-1␤ (IL-1␤) in response to P2X 7 R activation. These results suggest that pannexin-1 may be a hemichannel activated by the P2X 7 R to provide the conduit for dye uptake and downstream signaling to processing and release of IL-1␤. We have pursued this hypothesis by measuring dye uptake and IL-1␤ processing and release in mouse J774 macrophage in response to P2X 7 R activation and to maitotoxin and nigericin, two agents considered to evoke IL-1␤ release via the same mechanism. The experiments were carried out over time periods during which no lactate dehydrogenase was released from cells to examine only noncytolytic pathways. P2X 7 R activation evoked dye uptake that could be separated into two components by pannexin-1 inhibition: an initial rapid phase and a slower pannexin-1-independent phase. Maitotoxin-evoked dye uptake was unaltered by pannexin-1 inhibition. Nigericin did not induce dye uptake. Inhibition of pannexin-1 blocked caspase-1 and IL-1␤ processing and release in response to all three stimuli. Thus, although pannexin-1 is required for IL-1␤ release in response to maitotoxin, nigericin, and ATP, a mechanism distinct from pannexin-1 hemichannel activation must underlie the former two processes.

NLRP3 is an innate immune sensor contributing to the development of different diseases including monogenic autoinflammatory syndromes, gout, atherosclerosis, and Alzheimer’s disease. The molecule sulfonylurea MCC950 is a NLRP3 inflammasome inhibitor with potential clinical utility. However, the mechanism of action of MCC950 remains unknown. Here, we characterize the mechanism of action of MCC950 in both wild-type and autoinflammatory-related NLRP3 mutants, demonstrating that MCC950 closes the ‘open’ conformation of active NLRP3.