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P2X7-induced potassium efflux regulates NLRP3 inflammasome assembly and pyroptotic cell death. (a) Immunoblot analysis of procaspase-1 p45 and activated p10 fragments, and proIL-1β (34 kD) and mature (17 kD) fragments in the lysates and concentrated supernatants of J774A.1 cells primed for 4 h with 1 μg/ml LPS and stimulated with 3 mM ATP for 30 min with or without addition of 130 mM extracellular KCl or 25 μM of the P2X7 antagonist A438079. (b) Propidium iodide in J774A.1 cells primed with LPS for 4 h and stimulated with ATP in the presence or absence of 130 mM extracellular KCl. (c) Caspase-1 FLICA staining (green) in J774A.1 cells untreated or primed for 4 h with 1 μg/ml LPS and subsequently stimulated with 3 mM ATP for 30 min with or without 130 mM extracellular KCl or 25 μM A438079. Arrows: caspase-1 specks indicative of inflammasome assembly. Scale bar represents 50 μM. Nuclei are stained with NucBlue Fixed DAPI solution (blue). (d) Image cytometry analysis of inflammasomes detected by Caspase-1 FLICA. J774A.1 cells untreated or primed for 4 h with 1 μg/ml LPS and subsequently stimulated with 3 mM ATP for 30 min with or without 130 mM extracellular KCl or 25 μM A438079. Bar graph represents mean counts and standard error from at least 4000 cells for each condition from at a minimum of three fields in two independent experiments. Statistics were calculated by one-way ANOVA with Tukey’s post hoc analysis
Source publication
P2X7 purinergic receptor engagement with extracellular ATP induces transmembrane potassium and calcium flux resulting in assembly of the NLRP3 inflammasome in LPS-primed macrophages. The role of potassium and calcium in inflammasome regulation is not well understood, largely due to limitations in existing methods for interrogating potassium in real...
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Background
Neuroinflammation is an essential player in many neurological diseases including traumatic brain injury (TBI). Recent studies have identified that inflammasome complexes are responsible for inflammatory responses in many pathological conditions. Inflammasomes are intracellular multiprotein complexes which regulate the innate immune respo...
The protein NLRP3 and its complexes are associated with an array of inflammatory pathologies, among which neurodegenerative, autoimmune, and metabolic diseases. Targeting the NLRP3 inflammasome represents a promising strategy for easing the symptoms of pathologic neuroinflammation. When the inflammasome is activated, NLRP3 undergoes a conformationa...
Besides being recognized by membrane receptor TLR4, lipopolysaccharide (LPS) can also be internalized into the cytosol and activate Caspase-4/11 pyroptotic pathways to further amplify inflammation in sepsis. The objective of this study was to investigate whether Galectin-3 (Gal3) could promote the uptake of LPS by governing RAGE or administering en...
The NLRP3 inflammasome is a multiprotein complex that regulates the activation of caspase-1 leading to the maturation of the pro-inflammatory cytokines IL-1β and IL-18, and promoting pyroptosis. Classically, the NLRP3 inflammasome in murine macrophages is activated by the recognition of pathogen-associated molecular patterns and by many structurall...
Citations
... It was observed that Nano-spheres, ellipses, and rods restrained the intracellular calcium in iBMDM cells similar to the LPS and Nigericin (positive control) treated iBMDMs. Potassium efflux is associated with calcium influx through the cell membrane channel 75 . Nigericin is a known NLRP3 signal 2 molecule that increases potassium efflux through the cellular membrane, resulting in calcium influx upstream of NLRP3 inflammasomes. ...
Localized delivery of inflammasome inhibitors in phagocytic macrophages could be promising for psoriasis treatment. The present work demonstrates the development of non-spherical lipid nanoparticles, mimicking pathogen-like shapes, consisting of an anti-inflammatory inflammasome inhibiting lipid (pyridoxine dipalmitate) as a trojan horse. The nanorods inhibit inflammasome by 3.8- and 4.5-fold compared with nanoellipses and nanospheres, respectively. Nanorods reduce apoptosis-associated speck-like protein and lysosomal rupture, restrain calcium influx, and mitochondrial reactive oxygen species. Dual inflammasome inhibitor (NLRP3/AIM-2-IN-3) loaded nanorods cause synergistic inhibition by 21.5- and 59-folds compared with nanorods and free drug, respectively alongside caspase-1 inhibition. The NLRP3/AIM-2-IN-3 nanorod when transformed into a polymeric scaffold, simultaneously and effectively inhibits RNA levels of NLRP3, AIM2, caspase-1, chemokine ligand-2, gasdermin-D, interleukin-1β, toll-like receptor 7/ 8, and IL-17A by 6.4-, 1.6-, 2.0-, 13.0-, 4.2-, 24.4-, 4.3-, and 1.82-fold, respectively in psoriatic skin in comparison to Imiquimod positive control group in an in-vivo psoriasis-like mice model.
... As observed during PLC inhibition, EGTA chelation of extracellular Ca 2+ significantly reduced LTB 4 production compared to untreated BMNs ( Fig 4A; p�0.0001). Influx of extracellular Ca 2+ also requires the cell to maintain a membrane potential by efflux of intracellular potassium (K + ) [67,68]. Therefore, if extracellular Ca 2+ is required, disrupting the K + gradient should also inhibit LTB 4 synthesis. ...
Leukotriene B4 (LTB4) is an inflammatory lipid produced in response to pathogens that is critical for initiating the inflammatory cascade needed to control infection. However, during plague, Yersinia pestis inhibits the timely synthesis of LTB4 and subsequent inflammation. Using bacterial mutants, we previously determined that Y. pestis inhibits LTB4 synthesis via the action of the Yop effector proteins that are directly secreted into host cells through a type 3 secretion system (T3SS). Here, we show that the T3SS is the primary pathogen associated molecular pattern (PAMP) required for production of LTB4 in response to both Yersinia and Salmonella. However, we also unexpectantly discovered that T3SS-mediated LTB4 synthesis by neutrophils and macrophages require the activation of two distinctly different host signaling pathways. We identified that phagocytosis and the NLRP3/CASP1 inflammasome significantly impact LTB4 synthesis by macrophages but not neutrophils. Instead, the SKAP2/PLC signaling pathway is required for T3SS-mediated LTB4 production by neutrophils. Finally, while recognition of the T3SS is required for LTB4 production, we also discovered that a second unrelated PAMP-mediated signal activates the MAP kinase pathway needed for synthesis. Together, these data demonstrate significant differences in the host factors and signaling pathways required by macrophages and neutrophils to quickly produce LTB4 in response to bacteria. Moreover, while macrophages and neutrophils might rely on different signaling pathways for T3SS-dependent LTB4 synthesis, Y. pestis has evolved virulence mechanisms to counteract this response by either leukocyte to inhibit LTB4 synthesis and colonize the host.
... The intricacies of NLRP3 activation are influenced by multiple upstream events, which may act in tandem or independently. These events include ion fluxes, such as potassium efflux (Yaron et al., 2015), calcium efflux (Murakami et al., 2012;Uryash et al., 2024), and chloride efflux (Swanson et al., 2019;Green et al., 2018), and organelle damage, including lysosomal rupture (Hornung et al., 2008), mitochondrial dysfunction, and Golgi disintegration . Importantly, NIMA-associated kinase 7 (NEK7), a kinase involved in mitosis, has been identified as a critical requirement for the activation of the NLRP3 inflammasome (Sharif et al., 2019). ...
Postoperative cognitive dysfunction (POCD) poses a significant threat to patients undergoing anesthesia and surgery, particularly elderly patients. It is characterized by diminished cognitive functions post surgery, such as impaired memory and decreased concentration. The potential risk factors for POCD include age, surgical trauma, anesthetic type, and overall health condition; however, the precise mechanisms underlying POCD remain elusive. Recent studies suggest that neuroinflammation might be a primary pathogenic factor. NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasomes are implicated in exacerbating POCD by promoting the release of inflammatory factors and proteins that initiate pyroptosis, further influencing the disease process. The regulation of NLRP3 inflammasome activity, including its activation and degradation, is tightly controlled through multiple pathways and mechanisms. In addition, autophagy, a protective mechanism, regulates the NLRP3 inflammasome to control the progression of POCD. This review reviews recent findings on the role of the NLRP3 inflammasome in POCD pathogenesis and discusses therapeutic strategies aimed at reducing NLRP3 sources, inhibiting cellular pyroptosis, and enhancing autophagy.
... Additionally, we found that blocking the PLC pathway inhibited inflammasome activation induced by MSU and CPP crystals. While the exact mechanisms by which increased intracellular calcium leads to inflammasome activation remain to be fully elucidated, it has been proposed that calcium could directly promote NLRP3-ASC association 50 , causes mitochondrial Ca 2+ overload and mitochondrial dysfunction leading to mitochondrial reactive oxygen species (mtROS) production 26,51,52 . In any case, P2Y2 and P2Y6 are necessary, but other key cellular events, such as K + and Cl − efflux, are also required to trigger inflammasome activation in coordination with purinergic signaling and calcium mobilization. ...
Deposition of monosodium urate and calcium pyrophosphate (MSU and CPP) micro-crystals is responsible for painful and recurrent inflammation flares in gout and chondrocalcinosis. In these pathologies, the inflammatory reactions are due to the activation of macrophages responsible for releasing various cytokines including IL-1β. The maturation of IL-1β is mediated by the multiprotein NLRP3 inflammasome. Here, we find that activation of the NLRP3 inflammasome by crystals and concomitant production of IL-1β depend on cell volume regulation via activation of the osmo-sensitive LRRC8 anion channels. Both pharmacological inhibition and genetic silencing of LRRC8 abolish NLRP3 inflammasome activation by crystals in vitro and in mouse models of crystal-induced inflammation. Activation of LRRC8 upon MSU/CPP crystal exposure induces ATP release, P2Y receptor activation and intracellular calcium increase necessary for NLRP3 inflammasome activation and IL-1β maturation. We identify a function of the LRRC8 osmo-sensitive anion channels with pathophysiological relevance in the context of joint crystal-induced inflammation.
... K + efflux is crucial in most inflammasome activations, facilitating NLRP3-NEK7 oligomerization (He et al., 2016;Sharif et al., 2019). The relationship between K + efflux and Ca 2+ flux in inflammasome has hardly been studied, despite their potential link (Yaron et al., 2015). ...
We studied lysosomal Ca ²⁺ in inflammasome. Lipopolysaccharide (LPS) + palmitic acid (PA) decreased lysosomal Ca ²⁺ ([Ca ²⁺ ] Lys ) and increased [Ca ²⁺ ] i through mitochondrial ROS, which was suppressed in Trpm2 -KO macrophages. Inflammasome activation and metabolic inflammation in adipose tissue of high-fat diet (HFD)-fed mice were ameliorated by Trpm2 KO. ER→lysosome Ca ²⁺ refilling occurred after lysosomal Ca ²⁺ release whose blockade attenuated LPS + PA-induced inflammasome. Subsequently, store-operated Ca ²⁺ entry (SOCE) was activated whose inhibition suppressed inflammasome. SOCE was coupled with K ⁺ efflux whose inhibition reduced ER Ca ²⁺ content ([Ca ²⁺ ] ER ) and impaired [Ca ²⁺ ] Lys recovery. LPS + PA activated KCa3.1 channel, a Ca ²⁺ -activated K ⁺ channel. Inhibitors of KCa3.1 channel or Kcnn4 KO reduced [Ca ²⁺ ] ER , attenuated increase of [Ca ²⁺ ] i or inflammasome activation by LPS + PA, and ameliorated HFD-induced inflammasome or metabolic inflammation. Lysosomal Ca ²⁺ release induced delayed JNK and ASC phosphorylation through CAMKII-ASK1. These results suggest a novel role of lysosomal Ca ²⁺ release sustained by ER → lysosome Ca ²⁺ refilling and K ⁺ efflux through KCa3.1 channel in inflammasome activation and metabolic inflammation.
... We have previously characterized the in vitro activity of A438079, a potent small molecule inhibitor of P2X7 (25), and reported that its use for P2X7 inhibition results in suppression of both potassium and calcium fluxes and abrogation of mROS generation thereby potently suppressing NLRP3 inflammasome activation in macrophages in culture (26). Here, we develop a biomaterial-based platform to act as a depot for sustained delivery of A438079 in vivo and evaluate its efficacy in a model of impaired diabetic wound healing. ...
... This allosteric site is ideal to facilitate druggability of the P2X7 receptor, as allosteric sites are commonly more selective, allow lower target-based toxicity, fewer side effects, and accessible physicochemical properties (43,44). We previously investigated the inhibitory function of A438079 and reported a potent ability to ameliorate extracellular ATP-evoked potassium and calcium flux and subsequent inflammasome activation in mouse macrophages (26). We performed UV-VIS spectrophotometric analysis of A438079 in PBS ( Figure 2B) and identified two diagnostic absorption peaks at 226 nm and 258 nm which exhibit linear response from 40 µM to 2500 µM ( Figure 2C). ...
... Without inhibitor treatment, approximately 6% of the population exhibited perinuclear ASC specks. We note these levels of ASC speck positivity are in agreement with prior work by us (26) and others (45,46), but are likely an underestimation due to repeated wash steps during immunofluorescence processing. When treated with conditioned medium exposed to A438079-loaded silk films, <0.5% of the population exhibited ASC specks. ...
The inflammasome is a multiprotein complex critical for the innate immune response to injury. Inflammasome activation initiates healthy wound healing, but comorbidities with poor healing, including diabetes, exhibit pathologic, sustained activation with delayed resolution that prevents healing progression. In prior work, we reported the allosteric P2X7 antagonist A438079 inhibits extracellular ATP-evoked NLRP3 signaling by preventing ion flux, mitochondrial reactive oxygen species generation, NLRP3 assembly, mature IL-1β release, and pyroptosis. However, the short half-life in vivo limits clinical translation of this promising molecule. Here, we develop a controlled release scaffold to deliver A438079 as an inflammasome-modulating wound dressing for applications in poorly healing wounds. We fabricated and characterized tunable thickness, long-lasting silk fibroin dressings and evaluated A438079 loading and release kinetics. We characterized A438079-loaded silk dressings in vitro by measuring IL-1β release and inflammasome assembly by perinuclear ASC speck formation. We further evaluated the performance of A438079-loaded silk dressings in a full-thickness model of wound healing in genetically diabetic mice and observed acceleration of wound closure by 10 days post-wounding with reduced levels of IL-1β at the wound edge. This work provides a proof-of-principle for translating pharmacologic inhibition of ATP-induced inflammation in diabetic wounds and represents a novel approach to therapeutically targeting a dysregulated mechanism in diabetic wound impairment.
... The former, induced by lipopolysaccharide (LPS), facilitates mRNA expression of Nlrp3 and Il1β through NF-κB pathway. The latter, stimulated with extracellular ATP, pore-forming toxins, etc., thereby drives mitochondrial (mt) dysfunction [24,25] and concurrently impairs ion homeostasis [26,27], resulting in the assembly of NLRP3 components, namely NLRP3 inflammasome activation. According to earlier reports, Dioscin suppresses NF-κB activation [20] and mtROS generation [28]. ...
... K + efflux and excessive generation of ROS could trigger the assembly of NLRP3 inflammasome [43][44][45]. Earlier literature documented that K + efflux facilitated mtROS generation in BMDMs [26,46]. Therefore, the influence of K + efflux on NLRP3 activation and the regulatory role of Dioscin in K + efflux was further investigated. ...
... A prior investigation concluded that mtROS generation could be enhanced by impaired ion homeostasis [55]. Interestingly, recent studies reported that K + efflux is implicated in mtROS production [26,46,56]. Specifically, K + efflux could activate NLRP3 inflammasome and augment IL-1β secretion [43,46,57]. ...
Gingival inflammation and alveolar bone loss are characteristic manifestations of periodontitis. Interleukin (IL)-1β, the maturation of which is mainly regulated by NOD-like receptor protein (NLRP) 3 inflammasome, not only amplifies the inflammatory response but also triggers osteoclastogenesis, thereby accelerating the progression of periodontitis. Dioscin, a natural steroid saponin, has been shown to inhibit NLRP3 inflammasome. Nevertheless, research on the effectiveness of Dioscin for the management of periodontitis remains scarce. In this study, Dioscin was found to dramatically reduce the integral components of NLRP3 inflammasome, ultimately limiting IL-1β secretion. Notably, the inhibitory impact of Dioscin on NLRP3 inflammasome might be exerted by curbing the generation of mitochondrial (mt) reactive oxygen species (ROS) and oxidized (ox) mtDNA, which were mediated by inhibition of K⁺ efflux. Furthermore, Dioscin effectively alleviated periodontitis in mice. Overall, the results established that Dioscin could alleviate periodontitis by inhibiting NLRP3 inflammasome via modulation of the K⁺ efflux-mtROS-ox-mtDNA pathway, holding the potential to treat periodontitis and other NLRP3-driven inflammatory diseases.
... The above changes will further promote the activation step. In the activation step, some activators, including adenosine triphosphate (ATP), protoxin, viral RNA, and particulate matter [36], cause changes in intracellular ion flow [37][38][39][40][41][42][43], mitochondrial dysfunction [44][45][46], and lysosomal rupture [47][48][49][50]. Then, the oligomerization and activation of NLRP3 inflammasome start. ...
... The priming stage aims to upregulate production and post-translational modifications (PTMs) of NLRP3 inflammasome-related components (Fig. 2). Primarily, a few transcription factors, like nuclear factor kappa B (NF-κB), are triggered after PRRs recognizing stimulation by PAMPs or DAMPs [37,51,52]. Subsequently, proteins NLRP3, procaspase-1, and pro-IL-1β/18 are up-regulated in the nucleus [34,53,54]. ...
... K + efflux is a crucial mechanism behind ATP-induced NLRP3 inflammasome activation. After extracellular ATP interacts with P2X7 in LPS-primed macrophages, K + and Cl − efflux leads to the formation of NLRP3 inflammasome [37]. In macrophages, NLRP3 inflammasome is enhanced by K + efflux via the two-pore domain weak inwardly rectifying the K + channel 2 (TWIK2) channel to promote inflammation. ...
Background
The NLRP3 inflammasome is a vital player in the emergence of inflammation. The priming and activation of the NLRP3 inflammasome is a major trigger for inflammation which is a defense response against adverse stimuli. However, the excessive activation of the NLRP3 inflammasome can lead to the development of various inflammatory diseases. Cannabidiol, as the second-most abundant component in cannabis, has a variety of pharmacological properties, particularly anti-inflammation. Unlike tetrahydrocannabinol, cannabidiol has a lower affinity for cannabinoid receptors, which may be the reason why it is not psychoactive. Notably, the mechanism by which cannabidiol exerts its anti-inflammatory effect is still unclear.
Methods
We have performed a literature review based on published original and review articles encompassing the NLRP3 inflammasome and cannabidiol in inflammation from central databases, including PubMed and Web of Science.
Results and conclusions
In this review, we first summarize the composition and activation process of the NLRP3 inflammasome. Then, we list possible molecular mechanisms of action of cannabidiol. Next, we explain the role of the NLRP3 inflammasome and the anti-inflammatory effect of cannabidiol in inflammatory disorders. Finally, we emphasize the capacity of cannabidiol to suppress inflammation by blocking the NLRP3 signaling pathway, which indicates that cannabidiol is a quite promising anti-inflammatory compound.
... Again, the evidence points towards cellular energy: i) endolysosomal acidification requires ATP, ii) intracellular ATP has been shown to decrease in response to NLRP3 stimuli through K + -and Ca 2+ -mediated mitochondrial dysfunction, and iii) artificial decrease of intracellular ATP through inhibition of glycolysis has been shown to trigger NLRP3 (148). Moreover, triggering of NLRP3 through P2X 7 involves mobilisation of mitochondrial potassium (24). Recently, ATP-generation in mitochondria was found to be driven to a large extent by the secondary K + gradient (42) (generated by the mitochondrial H + /K + antiporter (58)), providing a possible mechanistic explanation to how potassium outflux could lead to a strong and immediate reduction of ATP production. ...
The NLRP3 inflammasome is a key regulator of inflammation that responds to a broad range of stimuli. The exact mechanism of activation has not been determined, but there is a consensus on cellular potassium efflux as a major common denominator. Once NLRP3 is activated, it forms high-order complexes together with NEK7 that trigger aggregation of ASC into specks. Typically, there is only one speck per cell, consistent with the proposal that specks form – or end up at – the centrosome. ASC polymerisation in turn triggers caspase-1 activation, leading to maturation and release of IL-1β and pyroptosis, i.e., highly inflammatory cell death. Several gain-of-function mutations in the NLRP3 inflammasome have been suggested to induce spontaneous activation of NLRP3 and hence contribute to development and disease severity in numerous autoinflammatory and autoimmune diseases. Consequently, the NLRP3 inflammasome is of significant clinical interest, and recent attention has drastically improved our insight in the range of involved triggers and mechanisms of signal transduction. However, despite recent progress in knowledge, a clear and comprehensive overview of how these mechanisms interplay to shape the system level function is missing from the literature. Here, we provide such an overview as a resource to researchers working in or entering the field, as well as a computational model that allows for evaluating and explaining the function of the NLRP3 inflammasome system from the current molecular knowledge. We present a detailed reconstruction of the molecular network surrounding the NLRP3 inflammasome, which account for each specific reaction and the known regulatory constraints on each event as well as the mechanisms of drug action and impact of genetics when known. Furthermore, an executable model from this network reconstruction is generated with the aim to be used to explain NLRP3 activation from priming and activation to the maturation and release of IL-1β and IL-18. Finally, we test this detailed mechanistic model against data on the effect of different modes of inhibition of NLRP3 assembly. While the exact mechanisms of NLRP3 activation remains elusive, the literature indicates that the different stimuli converge on a single activation mechanism that is additionally controlled by distinct (positive or negative) priming and licensing events through covalent modifications of the NLRP3 molecule. Taken together, we present a compilation of the literature knowledge on the molecular mechanisms on NLRP3 activation, a detailed mechanistic model of NLRP3 activation, and explore the convergence of diverse NLRP3 activation stimuli into a single input mechanism.
... This is referred to as "macropore formation," and this enables permeation to membrane-impermeable large molecules such as ethidium and TO-PRO3 (11)(12)(13). This macropore formation and resulting K þ efflux are critical steps for the formation and activation of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain-containing (NLRP)-3 inflammasome complex (14,15). Based on these backgrounds, many recent studies have focused on the mechanism of pore formation in P2X7 receptors and its downstream signaling pathways (12). ...
... In macrophages, P2X7 receptors are known to play critical roles in the formation of NLRP3 inflammasome by promoting both Ca 2 þ influx and K þ efflux (15). In the setting, macrophages secrete cleaved and matured IL-1b and promote inflammatory responses (9). ...
The ionotropic purinergic P2X7 receptor responds to extracellular ATP and can trigger pro-inflammatory immune signaling in macrophages. Caveolin-1 (Cav-1) is known to modulate functions of macrophages and innate immunity. However, it is unknown how Cav-1 modulates P2X7 receptor activity in macrophages. We herein examined P2X7 receptor activity and macrophage functions using bone marrow-derived macrophages (BMDMs) from wild-type (WT) and Cav-1 knockout (KO) mice. ATP (1 mM) application caused biphasic increase in cytosolic [Ca ²⁺ ] and sustained decrease in cytosolic [K ⁺ ]. A specific P2X7 receptor blocker, A-740003, inhibited the maintained cytosolic [Ca ²⁺ ] increase and cytosolic [K ⁺ ] decrease. Total internal reflection fluorescent imaging and proximity ligation assays revealed a novel molecular complex formation between P2X7 receptors and Cav-1 in WT BMDMs that were stimulated with lipopolysaccharides. This molecular coupling was increased by ATP application. Specifically, the ATP-induced Ca ²⁺ influx and K ⁺ efflux through P2X7 receptors were increased in Cav-1 KO BMDMs, even though the total and surface protein levels of P2X7 receptors in WT and Cav-1 KO BMDMs were unchanged. Cell-impermeable dye (TO-PRO3) uptake analysis revealed that macro-pore formation of P2X7 receptors was enhanced in Cav-1 KO BMDMs. Cav-1 KO BMDMs increased ATP-induced IL-1b secretion, reactive oxygen species production, Gasdermin D (GSDMD) cleavage, and lactate dehydrogenase release indicating pyroptosis. A-740003 completely prevented ATP-induced pyroptosis. In combination, these data sets show that Cav-1 has a negative effect on P2X7 receptor activity in BMDMs and that Cav-1 in macrophages may contribute to finely tuned immune responses by preventing excessive IL-1b secretion and pyroptosis.
