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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...
Citations
... The P2X7 receptor is highly expressed in macrophages and is important in various diseases [97]. Research shows that in macrophages pretreated with LPS, ATP, the second stimulus [98], activates NLRP3 inflammatories through P2X7 receptors, thus mediating inflammation and cell pyroptosis. ...
Background
Neurodegenerative diseases are a common group of neurological disorders characterized by progressive loss of neuronal structure and function leading to cognitive impairment. Recent studies have shown that neuronal pyroptosis mediated by the NLRP3 inflammasome plays a crucial role in the pathogenesis of neurodegenerative diseases.
Objective and method
The NLRP3 inflammasome is a multiprotein complex that, when activated within cells, triggers an inflammatory response, ultimately leading to pyroptotic cell death of neurons. Pyroptosis is a typical pro-inflammatory programmed cell death process occurring downstream of NLRP3 inflammasome activation, characterized by the formation of pores on the cell membrane by the GSDMD protein, leading to cell lysis and the release of inflammatory factors. It has been found that NLRP3 inflammasome-mediated neuronal pyroptosis is closely associated with the development of various neurodegenerative diseases, such as Alzheimer's disease, traumatic brain injury, and Parkinson's disease. Therefore, inhibiting NLRP3 inflammasome activation and attenuating neuronal pyroptosis could potentially serve as novel strategies for the treatment of neurodegenerative diseases.
Results
The aim of this review is to explore the role of NLRP3 activation-mediated neuronal pyroptosis and neuroinflammation in neurodegenerative diseases. Firstly, we extensively discuss the relationship between NLRP3 inflammasome-mediated neuronal pyroptosis and neuroinflammation in various neurodegenerative diseases. Subsequently, we further explore the mechanisms driving NLRP3 activation and assembly, as well as the post-translational modifications regulating NLRP3 inflammasome activation.
Conclusion
Understanding these mechanisms will contribute to a deeper understanding of the link between neuronal pyroptosis and neurodegenerative diseases, and hold significant implications for the treatment and prevention of neurodegenerative diseases.
... The complex promotes the NLRP3 deubiquitination and the NLRP3 inflammasome activation. K + efflux and Ca 2+ influx triggered by P2X7 activation are necessary in mitochondrial ion disruption and mitochondrial reactive oxygen species (mtROS) generation, which activate NLRP3 inflammasome [53]. It has been proposed that low extracellular K + concentrations activate the NLRP3 inflammasome, whereas high extracellular K + concentrations inhibit NLRP3 activation [54]. ...
Neuroinflammation is a key pathological feature in neurological diseases, including Alzheimer's disease (AD). The nucleotide-binding domain leucine-rich repeat-containing proteins (NLRs) belong to the pattern recognition receptors (PRRs) family that sense stress signals, which play an important role in inflammation. As a member of NLRs, the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) is predominantly expressed in microglia, the principal innate immune cells in the central nervous system (CNS). Microglia release proinflammatory cytokines to cause pyroptosis through activating NLRP3 inflammasome. The active NLRP3 inflammasome is involved in a variety of neurodegenerative diseases (NDs). Recent studies also indicate the key role of neuronal NLRP3 in the pathogenesis of neurological disorders. In this article, we reviewed the mechanisms of NLRP3 expression and activation and discussed the role of active NLRP3 inflammasome in the pathogenesis of NDs, particularly focusing on AD. The studies suggest that targeting NLRP3 inflammasome could be a novel approach for the disease modification.
... NLRP1 has also been implicated in AD but its exact role remains unclear. For example, NLRP1 levels have been found to be increased more than 25-fold in the neurons of AD patients, which might relate to a K + /Ca 2+ imbalance due to the neurotoxic effects of Aβ on ion channels [175,176]. Furthermore, Aβ neural stimulation in mice resulted in elevated caspase-1 activity, IL-1β secretion and neural pyroptosis, which was dependent on NLRP1 activity [217]. Congruently, chronic treatment of PC12 cells with Aβ induced NLRP1/caspase-1/GSDMD-dependent pyroptosis followed by the release of IL-1β and IL-18 [218]. ...
Inflammasome complexes and their integral receptor proteins have essential roles in regulating the innate immune response and inflammation at the post-translational level. Yet despite their protective role, aberrant activation of inflammasome proteins and gain of function mutations in inflammasome component genes seem to contribute to the development and progression of human autoimmune and autoinflammatory diseases. In the past decade, our understanding of inflammasome biology and activation mechanisms has greatly progressed. We therefore provide an up-to-date overview of the various inflammasomes and their known mechanisms of action. In addition, we highlight the involvement of various inflammasomes and their pathogenic mechanisms in common autoinflammatory, autoimmune and neurodegenerative diseases, including atherosclerosis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis. We conclude by speculating on the future avenues of research needed to better understand the roles of inflammasomes in health and disease.
... For example, ATP mobilized Ca 2+ influx weakly via the P2X7 receptor that was coordinated with K + efflux (Di et al. 2018). This in turn promoted the release of ER-linked Ca 2+ stores that was followed by the opening of membrane Ca 2+ channels (Murakami et al. 2012;Yaron et al. 2015). ...
Chronic pain is not only one of the most common health problems, it is often challenging to treat adequately. Chronic pain has a high prevalence globally, affecting approximately 20% of the adult population. Chronic inflammatory pain and neuropathic (nerve) pain conditions are areas of large unmet medical need because analgesic/adjuvant agents recommended for alleviation of these types of chronic pain often lack efficacy and/or they produce dose-limiting side effects. Recent work has implicated the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome in the pathobiology of chronic pain, especially neuropathic and inflammatory pain conditions. NLRP3 is activated by damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). This in turn leads to recruitment and activation of caspase-1 an enzyme that cleaves the inactive IL-1β and IL-18 precursors to their respective mature pro-inflammatory cytokines (IL-1β and IL-18) for release into the cellular milieu. Caspase-1 also cleaves the pyroptosis-inducing factor, gasdermin D, that leads to oligomerization of its N-terminal fragment to form pores in the host cell membrane. This then results in cellular swelling, lysis and release of cytoplasmic contents in an inflammatory form of cell death, termed pyroptosis. The ultimate outcome may lead to the development of neuropathic pain and/or chronic inflammatory pain. In this review, we address a role for NLRP3 inflammasome activation in the pathogenesis of various chronic pain conditions.
... Thus, stimulation of PLB-985 cells differentiated with DMSO (1.3%, 3 days) released O 2 ·− upon treatment with PMA for 10 min, an effect that was enhanced by priming the cells with interferon-γ (IFN-γ), which upregulated the expression of the NADPH oxidase 2 proteins gp91phox, p47phox and p22phox [56]. In contrast, nigericin is a K + ionophore, which promotes the influx of Ca 2+ by inducing the efflux of mitochondrial K + [57]. This induces NET release independently of PKC and NADPH oxidase activation [11]. ...
... This induces NET release independently of PKC and NADPH oxidase activation [11]. However, it is possible that nigericin may also induce some NET release via an NADPH oxidase-dependent pathway, owing to the potential activation of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome as a result of the influx of K + [57]. Inflammasome activation would induce the release of interleukin 1β (IL-1β), which could then trigger the release of NETs from other neutrophils in a similar manner to PMA [9]. ...
Extracellular traps are released by neutrophils and other immune cells as part of the innate immune response to combat pathogens. Neutrophil extracellular traps (NETs) consist of a mesh of DNA and histone proteins decorated with various anti-microbial granule proteins, such as elastase and myeloperoxidase (MPO). In addition to their role in innate immunity, NETs are also strongly linked with numerous pathological conditions, including atherosclerosis, sepsis and COVID-19. This has led to significant interest in developing strategies to inhibit NET release. In this study, we have examined the efficacy of different antioxidant approaches to selectively modulate the inflammatory release of NETs. PLB-985 neutrophil-like cells were shown to release NETs on exposure to phorbol myristate acetate (PMA), hypochlorous acid or nigericin, a bacterial peptide derived from Streptomyces hygroscopicus. Studies with the probe R19-S indicated that treatment of the PLB-985 cells with PMA, but not nigericin, resulted in the production of HOCl. Therefore, studies were extended to examine the efficacy of a range of antioxidant compounds that modulate HOCl production by MPO to prevent NETosis. It was shown that thiocyanate, selenocyanate and various nitroxides could prevent NETosis in PLB-985 neutrophils exposed to PMA and HOCl, but not nigericin. These results were confirmed in analogous experiments with freshly isolated primary human neutrophils. Taken together, these data provide new information regarding the utility of supplementation with MPO inhibitors and/or HOCl scavengers to prevent NET release, which could be important to more specifically target pathological NETosis in vivo.
... Thus, blockade with high extracellular K + or P2X7 inhibition suppresses ATP-induced IL-1B and caspase-1 release in mouse macrophage J774A.1 cells. These results suggest a mechanism whereby P2X7 activation triggered by ATP regulates K + efflux and subsequent activation of NLRP3 inflammasome (Yaron et al. 2015). A decrease in intracellular K + concentration facilitates a structural change in NLRP3 during its activation in BMDMs. ...
The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is an oligomeric complex that assembles in response to exogenous signals of pathogen infection and endogenous danger signals of non-microbial origin. When NLRP3 inflammasome assembly activates caspase-1, it promotes the maturation and release of the inflammatory cytokines interleukin-1B and IL-18. Aberrant activation of the NLRP3 inflammasome has been implicated in various diseases, including chronic inflammatory, metabolic, and cardiovascular diseases. The NLRP3 inflammasome can be activated through several principal mechanisms, including K+ efflux, lysosomal damage, and the production of mitochondrial reactive oxygen species. Interestingly, metabolic danger signals activate the NLRP3 inflammasome to induce metabolic diseases. NLRP3 contains three crucial domains: an N-terminal pyrin domain, a central nucleotide-binding domain, and a C-terminal leucine-rich-repeat domain. Protein-protein interactions act as a "pedal or brake" to control the activation of the NLRP3 inflammasome. In this review, we present the mechanisms underlying NLRP3 inflammasome activation after induction by metabolic danger signals or via the protein-protein interactions with NLRP3 that likely occur in metabolic diseases. Understanding these mechanisms will enable the development of specific inhibitors to treat NLRP3-related metabolic diseases.
... Anders et al. discovered that calcium oxalate crystal accumulation triggers the NLRP3-inflammasome via the TGFβ signaling pathway rather than the IL-1β signaling cascade, as previously assumed [148]. In addition, a study found that K + efflux mediates Ca 2+ influx, resulting in mitochondrial Ca 2+ excess and mitochondrial malfunction [149]. Another study, on the other hand, claimed that K + efflux activates NLRP3 in a Ca 2+ influx-independent manner [150]. ...
Chronic kidney disease (CKD) is associated with a variety of distinct disease processes that permanently change the function and structure of the kidney across months or years. CKD is characterized as a glomerular filtration defect or proteinuria that lasts longer than three months. In most instances, CKD leads to end-stage kidney disease (ESKD), necessitating kidney transplantation. Mitochondrial dysfunction is a typical response to damage in CKD patients. Despite the abundance of mitochondria in the kidneys, variations in mitochondrial morphological and functional characteristics have been associated with kidney inflammatory responses and injury during CKD. Despite these variations, CKD is frequently used to define some classic signs of mitochondrial dysfunction, including altered mitochondrial shape and remodeling, increased mitochondrial oxidative stress, and a marked decline in mitochondrial biogenesis and ATP generation. With a focus on the most significant developments and novel understandings of the involvement of mitochondrial remodeling in the course of CKD, this article offers a summary of the most recent advances in the sources of procured mitochondrial dysfunction in the advancement of CKD. Understanding mitochondrial biology and function is crucial for developing viable treatment options for CKD.
... In the case of NLRP3 activation by ATP, ATP was shown to induce weak influx of Ca 21 through its receptor P2X 7 , which increased K 1 efflux via TWIK2. K 1 efflux in turn stimulated Ca 21 mobilization by opening plasma membrane Ca 21 channels and by releasing endoplasmic reticulum Ca 21 (Murakami et al., 2012;Yaron et al., 2015). ...
The nucleotide-binding, oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a multiprotein complex that combines sensing, regulation, and effector functions to regulate inflammation in health and disease. NLRP3 is activated by a diverse range of inflammation-instigating signals known as pathogen associated molecular patterns and danger associated molecular patterns. Upon activation, NLRP3 oligomerizes and recruits partner proteins to form a supramolecular platform to process the maturation and release of interleukin (IL)-1β, IL-18, and gasdermin D, major mediators of inflammation and inflammatory cell death termed pyroptosis. The NLRP3 inflammasome has been implicated in the pathogenesis of a wide range of disease conditions, including chronic inflammatory disease that are associated with lifestyle and dietary changes, aging, and environmental exposures, and have become the leading cause of death worldwide. Pharmacological targeting of NLRP3 and signaling demonstrated promising efficacy in ameliorating a list of disease conditions in animal models. These findings underscore the potential and importance of NLRP3 as a druggable target for treating a range of diseases. In this review, recent progress in understanding the structure and mechanism of action of the NLRP3 inflammasome is discussed with focus on pharmacological inhibition of NLRP3 by small molecule inhibitors. New structural and mechanistic insights into NLRP3 activation and inhibitor-NLRP3 interactions would aid in the rational design and pharmacological evaluation of NLRP3 inhibitors for treatment of human disease. SIGNIFICANCE STATEMENT: The NLRP3 inflammasome plays central role in innate immune sensing and control of inflammation. Pharmacological inhibition of NLRP3 demonstrated promising efficacy in a range of diseases in animal models. Recent elucidation of the structure and inhibitor binding of NLRP3 generated new insights into its mode of action and inhibitor-NLRP3 interaction at molecular levels, providing new framework for developing small chemical inhibitors of NLRP3 with improved efficacy and specificity against chronic disease that has become major health concerns worldwide.
... MAVS is a key sensor of mtROS, contributing to the production of type I interferon (IFN) and activation of caspase-1, and is responsible for host immunity and inflammation (Buskiewicz et al., 2016). Furthermore, ATP and K + efflux are regarded as two key NLRP3 activators caused by impaired mitochondria and induce Ca 2+ influx and direct damage to mitochondrial Ca 2+ homeostasis (Yaron et al., 2015;Katsnelson et al., 2016), further affecting Krebs cycle enzyme activity, leading to a loss of MMP and mitochondrial network fragmentation (Hawkins et al., 2007;Liu et al., 2018). Mitochondrial Ca 2+ homeostasis plays a crucial role in plaque calcification and unstable lesions in AS (Forrester et al., 2018). ...
Atherosclerosis (AS) is the main cause of cardiovascular disease (CVD) and is characterized by endothelial damage, lipid deposition, and chronic inflammation. Gut microbiota plays an important role in the occurrence and development of AS by regulating host metabolism and immunity. As human mitochondria evolved from primordial bacteria have homologous characteristics, they are attacked by microbial pathogens as target organelles, thus contributing to energy metabolism disorders, oxidative stress, and apoptosis. Therefore, mitochondria may be a key mediator of intestinal microbiota disorders and AS aggravation. Microbial metabolites, such as short-chain fatty acids, trimethylamine, hydrogen sulfide, and bile acids, also affect mitochondrial function, including mtDNA mutation, oxidative stress, and mitophagy, promoting low-grade inflammation. This further damages cellular homeostasis and the balance of innate immunity, aggravating AS. Herbal medicines and their monomers can effectively ameliorate the intestinal flora and their metabolites, improve mitochondrial function, and inhibit atherosclerotic plaques. This review focuses on the interaction between gut microbiota and mitochondria in AS and explores a therapeutic strategy for restoring mitochondrial function and intestinal microbiota disorders using herbal medicines, aiming to provide new insights for the prevention and treatment of AS.
... It is further understood that K + efflux constitutes an important stage of priming the microglial inflammasome, which is necessary for maturating proinflammatory cytokines such as IL-1β (Xu et al., 2020). Along these lines, it is increasingly recognized that P2X7 and P2X4 conduct K + countercurrent when activated (Yaron et al., 2015;Nguyen et al., 2020); moreover, changes in K + -channel expression upon microglial activation may contribute to these responses (Nguyen et al., 2017). ...
Adenosine triphosphate (ATP) and its metabolites drive microglia migration and cytokine production by activating P2X- and P2Y- class purinergic receptors. Purinergic receptor activation gives rise to diverse intracellular calcium (Ca2+ signals, or waveforms, that differ in amplitude, duration, and frequency. Whether and how these characteristics of diverse waveforms influence microglia function is not well-established. We developed a computational model trained with data from published primary murine microglia studies. We simulate how purinoreceptors influence Ca2+ signaling and migration, as well as, how purinoreceptor expression modifies these processes. Our simulation confirmed that P2 receptors encode the amplitude and duration of the ATP-induced Ca2+ waveforms. Our simulations also implicate CD39, an ectonucleotidase that rapidly degrades ATP, as a regulator of purinergic receptor-induced Ca2+ responses. Namely, it was necessary to account for CD39 metabolism of ATP to align the model’s predicted purinoreceptor responses with published experimental data. In addition, our modeling results indicate that small Ca2+ transients accompany migration, while large and sustained transients are needed for cytokine responses. Lastly, as a proof-of-principal, we predict Ca2+ transients and cell membrane displacements in a BV2 microglia cell line using published P2 receptor mRNA data to illustrate how our computer model may be extrapolated to other microglia subtypes. These findings provide important insights into how differences in purinergic receptor expression influence microglial responses to ATP.
