NLRP3 (NOD-like receptor pyrin domain-containing protein 3) is an innate immune sensor that contributes 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, and demonstrate that MCC950 closes the 'open' conformation of active NLRP3.
Extracellular proteins with important signalling roles in processes, such as inflammation and angiogenesis, are known to employ unconventional routes of protein secretion. Although mechanisms of ...unconventional protein secretion are beginning to emerge, the precise molecular details have remained elusive for the majority of cargo proteins secreted by unconventional means. Recent findings suggest that for two examples of unconventionally secreted proteins, interleukin 1β (IL-1β) and fibroblast growth factor 2 (FGF2), the common molecular principle of pore formation may be shared. Under specific experimental conditions, secretion of IL-1β and FGF2 is triggered by phosphatidylinositol 4,5-bisphosphate PI(4,5)P
-dependent formation of pores across the plasma membrane. However, the underlying mechanisms are different, with FGF2 known to directly interact with PI(4,5)P
, whereas in the case of IL-1β secretion, it is proposed that the N-terminal fragment of gasdermin D interacts with PI(4,5)P
to form the pore. Thus, although implemented in different ways, these findings suggest that pore formation may be shared by the unconventional secretion mechanisms for FGF2 and IL-1β in at least some cases. In this Opinion article, we discuss the unconventional mechanisms of FGF2 and IL-1β release with a particular emphasis on recent discoveries suggesting the importance of pore formation on the plasma membrane.
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Adenosine triphosphate (ATP) is a molecule that on one hand plays a central role in cellular energetics and which on the other is a ubiquitous signaling molecule when released into ...the extracellular media. Extracellular ATP accumulates in inflammatory environments where it acts as a damage-associated molecular pattern and activates the purinergic P2X receptor 7 (P2X7) in immune cells. P2X7 receptor activation induces the formation of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3) inflammasome and the activation of the inflammatory caspase-1. Caspase-1 causes an inflammatory type of cell death called pyroptosis through the release of pro-inflammatory cytokines and intracellular content. Consequently, intense research efforts have been devoted to the design of novel anti-inflammatory therapies, focusing in particular on the P2X7 receptor and the NLRP3 pathway and the introduction of new blocking molecules in early phase clinical trials.
Increased hepatocyte death contributes to the pathology of acute and chronic liver diseases. However, the role of hepatocyte pyroptosis and extracellular inflammasome release in liver disease is ...unknown.
We used primary mouse and human hepatocytes, hepatocyte-specific leucine 351 to proline Nlrp3KICreA mice, and GsdmdKO mice to investigate pyroptotic cell death in hepatocytes and its impact on liver inflammation and damage. Extracellular NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes were isolated from mutant NLRP3-YFP HEK cells and internalisation was studied in LX2 and primary human hepatic stellate cells. We also examined a cohort of 154 adult patients with biopsy-proven non-alcoholic fatty liver disease (Sir Charles Gairdner Hospital, Nedlands, Western Australia).
We demonstrated that primary mouse and human hepatocytes can undergo pyroptosis upon NLRP3 inflammasome activation with subsequent release of NLRP3 inflammasome proteins that amplify and perpetuate inflammasome-driven fibrogenesis. Pyroptosis was inhibited by blocking caspase-1 and gasdermin D activation. The activated form of caspase-1 was detected in the livers and in serum from patients with non-alcoholic steatohepatitis and correlated with disease severity. Nlrp3KICreA mice showed spontaneous liver fibrosis under normal chow diet, and increased sensitivity to liver damage and inflammation after treatment with low dose lipopolysaccharide. Mechanistically, hepatic stellate cells engulfed extracellular NLRP3 inflammasome particles leading to increased IL-1β secretion and α-smooth muscle actin expression. This effect was abrogated when cells were pre-treated with the endocytosis inhibitor cytochalasin B.
These results identify hepatocyte pyroptosis and release of inflammasome components as a novel mechanism to propagate liver injury and liver fibrosis development.
Our findings identify a novel mechanism of inflammation in the liver. Experiments in cell cultures, mice, and human samples show that a specific form of cell death, called pyroptosis, leads to the release of complex inflammatory particles, the NLRP3 inflammasome, from inside hepatocytes into the extracellular space. From there they are taken up by other cells and thereby mediate inflammatory and pro-fibrogenic stress signals. The discovery of this mechanism may lead to novel treatments for chronic liver diseases in the future.
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•Human and murine hepatocytes undergo pyroptosis and release NLRP3 inflammasome proteins.•Pyroptotic cell death in hepatocytes is dependent on caspase-1 and gasdermin D activation.•Caspase-1 activity is increased in livers and serum from NASH patients.•Nlrp3KICreA mice develop fibrosis and show increased sensitivity to liver damage.•Human hepatic stellate cells internalise extracellular NLRP3-YFP oligomeric particles.•Extracellular NLRP3 oligomeric particles perpetuate inflammation and fibrogenesis.
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 > disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) approximately disodium 4-acetamido-4'-isothiocyanato-stilben-2,2'-disulfonate approximately 5-nitro-2-(3-phenylpropylamino)benzoic acid > indanyloxyacetic acid 94 >> probenecid >> flufenamic acid = niflumic acid. 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.
The gasdermins are a family of recently identified pore-forming effector proteins that cause membrane permeabilization and pyroptosis, a lytic pro-inflammatory type of cell death. Gasdermins contain ...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 form large oligomeric pores, which disrupts ion homeostasis and induces cell death. Gasdermin-induced pyroptosis plays a prominent role in many hereditary diseases and (auto)inflammatory disorders as well as in cancer. In this Review, we discuss recent developments in gasdermin research with a focus on mechanisms that control gasdermin activation, pore formation and functional consequences of gasdermin-induced membrane permeabilization.
Inflammasomes are multiprotein complexes that signal by oligomerizing the apoptosis speck‐like protein with caspase recruitment and activator domain (ASC) and are involved in multiple inflammatory, ...metabolic and degenerative diseases. Pharmacological targeting of specific inflammasomes with small molecules is leading to the development of novel drugs for most common diseases. The targeting of ASC oligomers will result in a pan‐inflammasome treatment. In their study, Bertheloot et al (2022) developed specific anti‐ASC nanobodies and showed their efficacy to disaggregate already formed ASC oligomers and to treat inflammatory diseases in animal models. This approach represents a novel biologic‐based treatment for inflammasomes‐initiated inflammatory diseases.
P. Pelegrin & A. Sahil discuss the study by Bertheloot et al (in this issue of EMBO Mol Med) that reports specific anti‐ASC nanobodies able to disaggregate ASC oligomers and treat inflammatory diseases in animal models.
The NLRP3 inflammasome is an important regulator of inflammation and immunity. It is a multimolecular platform formed within cells that facilitates the activation of proinflammatory caspases to drive ...secretion of cytokines such as interleukin-1β (IL-1β). Knowledge of the mechanisms regulating formation of the NLRP3 inflammasome is incomplete. Here we report Cl⁻ channel-dependent formation of dynamic ASC oligomers and inflammasome specks that remain inactive in the absence of K⁺ efflux. Formed after Cl⁻ efflux exclusively, ASC specks are NLRP3 dependent, reversible, and inactive, although they further prime inflammatory responses, accelerating and enhancing release of IL-1β in response to a K⁺ efflux-inducing stimulus. NEK7 is a specific K⁺ sensor and does not associate with NLRP3 under conditions stimulating exclusively Cl⁻ efflux, but does after K⁺ efflux, activating the complex driving inflammation. Our investigation delivers mechanistic understanding into inflammasome activation and the regulation of inflammatory responses.
Pharmacological inhibition of NLRP3 inflammasome activation may offer a new option in the treatment of inflammatory bowel disease. In this work, we report the design, synthesis, and biological ...screening of a series of acrylate derivatives as NLRP3 inhibitors. The in vitro determination of reactivity, cytotoxicity, NLRP3 ATPase inhibition, and antipyroptotic properties allowed the selection of 11 (INF39), a nontoxic, irreversible NLRP3 inhibitor able to decrease interleukin-1β release from macrophages. Bioluminescence resonance energy transfer experiments proved that this compound was able to directly interfere with NLRP3 activation in cells. In vivo studies confirmed the ability of the selected lead to alleviate the effects of colitis induced by 2,4-dinitrobenzenesulfonic acid in rats after oral administration.
NLRP3 is a cytosolic sensor triggered by different pathogen- and self-derived signals that plays a central role in a variety of pathological conditions, including sterile inflammation. The ...leucine-rich repeat domain is present in several innate immune receptors, where it is frequently responsible for sensing danger signals and regulation of activation. Here we show by reconstitution of truncated and chimeric variants into Nlrp3
macrophages that the leucine-rich repeat domain is dispensable for activation and self-regulation of NLRP3 by several different triggers. The pyrin domain on the other hand is required to maintain NLRP3 in the inactive conformation. A fully responsive minimal NLRP3 truncation variant reconstitutes peritonitis in Nlrp3
mice. We demonstrate that in contrast to pathogen-activated NLRC4, the constitutively active NLRP3 molecule cannot engage wild-type NLRP3 molecules in a self-catalytic oligomerization. This lack of signal amplification is likely a protective mechanism to decrease sensitivity to endogenous triggers to impede autoinflammation.
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