NLRP3 Inflammasome Activation In Macrophages Research Articles

Dual Regulation of Nicotine on NLRP3 Inflammasome in Macrophages with the Involvement of Lysosomal Destabilization, ROS and α7nAChR.

Nicotine, the primary alkaloid in tobacco products, has been shown to have immunoregulatory function in at least 20 diseases. The biological mechanism of action of nicotine immunoregulation is complex, resulting in an improvement of some disease states and exacerbation of others. Given the central role of the NLRP3 inflammasome in macrophages among multiple inflammatory diseases, this study examined how nicotine alters NLRP3 inflammasome activation in macrophages. NLRP3 inflammasome activation was examined mechanistically in the context of different nicotine dosages. We show NLRP3 inflammasome activation, apoptosis-associated speck-like protein (ASC) expression, caspase-1 activity and subsequent IL-1β secretion were positively correlated with nicotine in a dose-dependent relationship, and destabilization of lysosomes and ROS production were also involved. At high concentrations of nicotine surpassing 0.25mM, NLRP3 inflammasome activity declined, along with increased expression of the anti-inflammatory Alpha7 nicotinic acetylcholine receptor (α7nAChR) and the inhibition of TLR4/NF-κB signaling. Consequently, high doses of nicotine also reduced ASC expression, caspase-1 activity and IL-1β secretion in macrophages. Collectively, these results suggest a dual regulatory function of nicotine on NLRP3 inflammasome activation in macrophages, that is involved with the pro-inflammatory effects of lysosomal destabilization and ROS production. We also show nicotine mediates anti-inflammatory effects by activating α7nAChR at high doses.

Melatonin-induced upregulation of telomerase activity interferes with macrophage mitochondrial metabolism and suppresses NLRP3 inflammasome activation in the treatment of Pneumonia

ObjectiveThis study aims to investigate the effects of melatonin-induced upregulation of telomerase activity on mitochondrial metabolism and NLRP3 inflammasome activation in macrophages, with the ultimate goal of elucidating potential therapeutic implications for pneumonia treatment. Materials and methodsMacrophages were treated with melatonin to assess its impact on telomerase activity. Mitochondrial function was evaluated through the measurement of reactive oxygen species (ROS) levels and cellular energy production. NLRP3 inflammasome activation was assessed by examining the production of inflammatory cytokines, such as interleukin-1β (IL-1β). The expression levels of key proteins involved in mitochondrial metabolism and NLRP3 inflammasome signaling were also analyzed. ResultsOur findings demonstrated that melatonin treatment significantly upregulated telomerase activity in macrophages. This was associated with a reduction in ROS levels and enhanced cellular energy production, indicating improved mitochondrial function. Moreover, melatonin treatment suppressed NLRP3 inflammasome activation, resulting in reduced secretion of IL-1β. The expression levels of proteins involved in mitochondrial metabolism and NLRP3 inflammasome signaling were modulated by melatonin. ConclusionThese results suggest that melatonin-induced upregulation of telomerase activity can interfere with mitochondrial metabolism and inhibit NLRP3 inflammasome activation in macrophages. This indicates a potential therapeutic role for melatonin in the treatment of pneumonia. Understanding the molecular mechanisms underlying these effects may lead to the development of novel therapeutic strategies targeting mitochondria and NLRP3 inflammasome activation for the management of pneumonia. Further investigations are warranted to fully uncover the therapeutic potential of melatonin and its implications for pneumonia treatment.

NLRP3 inflammasome activation promotes liver inflammation and fibrosis in experimental biliary atresia.

Biliary atresia (BA) is characterized by a progressive fibroinflammatory cholangiopathy in early infants with unknown etiology. Although innate immune disorder is involved in its mechanism, role of NLRP3 inflammasome in BA remains largely undefined. To explore the role of NLRP3 inflammasome in BA. The expressions of NLRP3 inflammasome-related genes were determined in BA patients. Role of NLRP3 inflammasome was evaluated using MCC950 in experimental BA. Furthermore, gadolinium chloride, a macrophage scavenger, was applied to validate the inflammasome's cellular localization. Finally, the effects of NLRP3 inflammasome activation on liver fibrosis were explored in vivo and vitro in experimental BA. The components of NLRP3 inflammasome were up-regulated in BA patients. Inflammasome-related genes showed positively correlated with liver inflammation and fibrosis in BA patients. In experimental BA, inflammasome-related genes were up-regulated, and their expressions were inhibited by MCC950, which promoted mice growth, protected liver function, alleviated obstructive jaundice, inhibited liver inflammation, and reduced serum IL-1β level. NLRP3 inflammasome was expressed in macrophages, and macrophage elimination exerted the same protective roles as MCC950 did in BA. Additionally, NLRP3 inflammasome activation promoted liver fibrosis in experimental BA. NLRP3 inflammasome activation in macrophages promoted liver inflammation and fibrosis in experimental BA.

Integrating network pharmacology and experimental models to identify notoginsenoside R1 ameliorates atherosclerosis by inhibiting macrophage NLRP3 inflammasome activation.

Atherosclerosis is a cardiovascular disease, accounting for the most common mortality cause worldwide. Notoginsenoside R1 (NGR1) is a characteristic saponin of Radix notoginseng that exhibits anti-inflammatory and antioxidant effects while modulating lipid metabolism. Evidence suggests that NGR1 exerts cardioprotective, neuroprotective, and anti-atherosclerosis effects. However, underlying NGR1 mechanisms alleviating atherosclerosis (AS) have not been examined. This study used a network pharmacology approach to construct the drug-target-disease correlation and protein-protein interaction (PPI) network of NGR1 and AS. Moreover, functional annotation and pathway enrichment analyses deciphered the critical biological processes and signaling pathways potentially regulated by NGR1. The protective effect of NGR1 against AS and the underlying mechanism(s) was assessed in an atherogenic apolipoprotein E-deficient (ApoE-/-) mice in vivo and an oxidized low-density lipoprotein (ox-LDL)-induced macrophage model in vitro. The network pharmacology and molecular docking analyses revealed that NGR1 protects against AS by targeting the NLRP3/caspase-1/IL-1β pathway. NGR1 reduced foam cell formation in ox-LDL-induced macrophages and decreased atherosclerotic lesion formation, serum lipid metabolism, and inflammatory cytokines in AS mice in vivo. Therefore, NGR1 downregulates the NLRP3 inflammasome complex gene expression of NLRP3, caspase-1, ASC, IL-1β, and IL-18, in vivo and in vitro.

The Angiotensin II Receptor Neprilysin Inhibitor LCZ696 Inhibits the NLRP3 Inflammasome By Reducing Mitochondrial Dysfunction in Macrophages and Alleviates Dextran Sulfate Sodium-induced Colitis in a Mouse Model.

The intracellular sensor protein complex known as the NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome plays a crucial role in regulating inflammatory diseases by overseeing the production of interleukin (IL)-1β and IL-18. Targeting its abnormal activation with drugs holds significant promise for inflammation treatment. This study highlights LCZ696, an angiotensin receptor-neprilysin inhibitor, as an effective suppressor of NLRP3 inflammasome activation in macrophages stimulated by ATP, nigericin, and monosodium urate. LCZ696 also reduces caspase-11 and GSDMD activation, lactate dehydrogenase release, propidium iodide uptake, and the extracellular release of NLRP3 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in ATP-activated macrophages, suggesting a potential mitigation of pyroptosis. Mechanistically, LCZ696 lowers mitochondrial reactive oxygen species and preserves mitochondrial integrity. Importantly, it does not significantly impact NLRP3, proIL-1β, inducible nitric oxide synthase, cyclooxygenase-2 expression, or NF-κB activation in lipopolysaccharide-activated macrophages. LCZ696 partially inhibits the NLRP3 inflammasome through the induction of autophagy. In an in vivo context, LCZ696 alleviates NLRP3-associated colitis in a mouse model by reducing colonic expression of IL-1β and tumor necrosis factor-α. Collectively, these findings suggest that LCZ696 holds significant promise as a therapeutic agent for ameliorating NLRP3 inflammasome activation in various inflammatory diseases, extending beyond its established use in hypertension and heart failure treatment.

Two-pore potassium channel TREK-1 (K2P2.1) regulates NLRP3 inflammasome activity in macrophages.

Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages (AMs) and bone marrow-derived macrophages (BMDMs) from wild-type (wt) and TREK-1-/- mice, we measured responses to inflammasome priming [using lipopolysaccharide (LPS)] and activation (LPS + ATP). We measured IL-1β, caspase-1, and NLRP3 via ELISA and Western blot. A membrane-permeable potassium indicator was used to measure potassium efflux during ATP exposure, and a fluorescence-based assay was used to assess changes in membrane potential. Inflammasome activation induced by LPS + ATP increased IL-1β secretion in wt AMs, whereas activation was significantly reduced in TREK-1-/- AMs. Priming of BMDMs using LPS was not affected by either genetic deficiency or pharmacological inhibition of TREK-1 with Spadin. Cleavage of caspase-1 following LPS + ATP treatment was significantly reduced in TREK-1-/- BMDMs. The intracellular potassium concentration in LPS-primed wt BMDMs was significantly lower compared with TREK-1-/- BMDMs or wt BMDMs treated with Spadin. Conversely, activation of TREK-1 with BL1249 caused a decrease in intracellular potassium in wt BMDMs. Treatment of LPS-primed BMDMs with ATP caused a rapid reduction in intracellular potassium levels, with the largest change observed in TREK-1-/- BMDMs. Intracellular K+ changes were associated with changes in the plasma membrane potential (Em), as evidenced by a more depolarized Em in TREK-1-/- BMDMs compared with wt, and Em hyperpolarization upon TREK-1 channel opening with BL1249. These results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.NEW & NOTEWORTHY Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages and bone marrow-derived macrophages from wild-type and TREK-1-/- mice, we measured responses to inflammasome priming (using LPS) and activation (LPS + ATP). Our results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.

Flow-Induced Shear Stress Primes NLRP3 Inflammasome Activation in Macrophages via Piezo1.

The NLRP3 inflammasome is a crucial component of the innate immune system, playing a pivotal role in initiating and regulating the body's inflammatory response to various pathogens and cellular damage. Environmental stimuli, such as temperature, pH level, and nutrient availability, can influence the behavior and functions of innate immune cells, including immune cell activity, proliferation, and cytokine production. However, there is limited understanding regarding how mechanical forces, like shear stress, govern the intrinsic inflammatory reaction, particularly the activation of the NLRP3 inflammasome, and how shear stress impacts NLRP3 inflammasome activation through its capacity to induce alterations in gene expression and cytokine secretion. Here, we investigated how shear stress can act as a priming signal in NLRP3 inflammasome activation by exposing immortalized bone marrow-derived macrophages (iBMDMs) to numerous physiologically relevant magnitudes of shear stress before chemically inducing inflammasome activation. We demonstrated that shear stress of large magnitudes was able to prime iBMDMs more effectively for inflammasome activation compared to lower shear stress magnitudes, as quantified by the percentage of cells where ASC-CFP specks formed and IL-1β secretion, the hallmarks of inflammasome activation. Testing this in NLRP3 and caspase-1 knockout iBMDMs showed that the NLRP3 inflammasome was primarily primed for activation due to shear stress exposure. Quantitative polymerase chain reaction (qPCR) and a small-molecule inhibitor study mechanistically determined that shear stress regulates the NLRP3 inflammasome by upregulating Piezo1, IKKβ, and NLRP3. These findings offer insights into the mechanistic relationship among physiological shear stresses, inflammasome activation, and their impact on the progression of inflammatory diseases and their interconnected pathogenesis.

Effect of tubastatin A on NLRP3 inflammasome activation in macrophages under hypoxia/reoxygenation conditions

Effect of tubastatin A on NLRP3 inflammasome activation in macrophages under hypoxia/reoxygenation conditions

A novel TWIK2 channel inhibitor binds at the bottom of the selectivity filter and protects against LPS-induced experimental endotoxemia in vivo

TWIK2 channel plays a critical role in NLRP3 inflammasome activation and mice deficient in TWIK2 channel are protected from sepsis and inflammatory lung injury. However, inhibitors of TWIK2 channel are currently in an early stage of development, and the molecular determinants underlying the chemical modulation of TWIK2 channel remain unexplored. In this study, we identified NPBA and the synthesized derivative NPBA-4 potently and selectively inhibited TWIK2 channel by using whole-cell patch clamp techniques. Furthermore, the mutation of the last residues of the selectivity filter in both P1 and P2 (i.e., T106A, T214A) of TWIK2 channel substantially abolished the effect of NPBA on TWIK2 channel. Our data suggest that NPBA blocked TWIK2 channel through binding at the bottom of the selectivity filter, which was also supported by molecular docking prediction. Moreover, we found that NPBA significantly suppressed NLRP3 inflammasome activation in macrophages and alleviated LPS-induced endotoxemia and organ injury in vivo. Notably, the protective effects of NPBA against LPS-induced endotoxemia were abolished in Kcnk6-/- mice. In summary, our study has uncovered a series of novel inhibitors of TWIK2 channel and revealed their distinct molecular determinants interacting TWIK2 channel. These findings provide new insights into the mechanisms of pharmacological action on TWIK2 channel and opportunities for the development of selective TWIK2 channel modulators to treat related inflammatory diseases.

Mechanisms of NLRP3 inflammasome activation in macrophages by air pollution fine particulate matter (PM2.5)

Mechanisms of NLRP3 inflammasome activation in macrophages by air pollution fine particulate matter (PM2.5)

Endoplasmic Reticulum Stress Mediated NLRP3 Inflammasome Activation and Pyroptosis in THP-1 Macrophages Infected with Bacillus Calmette-Guérin.

Tuberculosis (TB) is a zoonotic infectious disease caused by Mycobacterium tuberculosis (Mtb). Mtb is a typical intracellular parasite, and macrophages are its main host cells. NLRP3 inflammasome-mediated pyroptosis is a form of programmed cell death implicated in the clearance of pathogenic infections. The bidirectional regulatory effect of endoplasmic reticulum stress (ERS) plays a crucial role in determining cell survival and death. Whether ERS is involved in macrophage pyroptosis with Mtb infection remains unclear. This article aims to explore the regulation of the NLRP3 inflammasome and pyroptosis by ERS in THP-1 macrophages infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). The results showed that BCG infection induced THP-1 macrophage ERS, NLRP3 inflammasome activation and pyroptosis, which was inhibited by ERS inhibitor TUDCA. NLRP3 inhibitor MCC950 inhibited THP-1 macrophage NLRP3 inflammasome activation and pyroptosis caused by BCG infection. Compared with specific Caspase-1 inhibitor VX-765, pan-Caspase inhibitor Z-VAD-FMK showed a more significant inhibitory effect on BCG infection-induced pyroptosis of THP-1 macrophages. Taken together, this study demonstrates that ERS mediated NLRP3 inflammasome activation and pyroptosis after BCG infection of THP-1 macrophages, and that BCG infection of THP-1 macrophages induces pyroptosis through canonical and noncanonical pathways.

Seasonal coronavirus infections trigger NLRP3 inflammasome activation in macrophages but is therapeutically targetable

Seasonal coronaviruses widely circulate in the global population, and severe complications can occur in specific vulnerable populations. Little is known on their pathogenic mechanisms and no approved treatment is available. Here, we present anecdotal evidence that the level of IL-1β, a hallmark of inflammasome activation, appears elevated in a subset of seasonal coronavirus infected patients. We found that cultured human macrophages support the full life cycle of three cultivatable seasonal coronaviruses. Their infections effectively activate NLRP3 inflammasome activation through TLR4 ligation and NF-κB activation. This activation can be attenuated by specific pharmacological inhibitors and clinically used medications including dexamethasone and flufenamic acid. Interestingly, combination of antiviral and anti-inflammatory drugs simultaneously inhibit seasonal coronavirus-triggered inflammatory response and viral replication. Collectively, these findings show that the TLR4/NF-κB/NLRP3 axis drives seasonal coronavirus triggered-inflammatory response, which in turn represents a viable therapeutic target.

1,8-cineole ameliorates colon injury by downregulating macrophage M1 polarization via inhibiting the HSP90-NLRP3-SGT1 complex

Ulcerative colitis (UC) is characterized by chronic relapsing intestinal inflammation. Currently, there is no effective treatment for the disease. According to our preliminary data, 1,8-cineole, which is the main active compound of Amomum compactum Sol. ex Maton volatile oil and an effective drug for the treatment of pneumonia, showed remarkable anti-inflammatory effects on colitis pathogenesis. However, its mechanism of action and direct targets remain unclear. This study investigated the direct targets and mechanism through which 1,8-cineole exerts its anti-inflammatory effects using a dextran sulfate sodium salt-induced colitis mouse model. The effects of 1,8-cineole on macrophage polarization were investigated using activated bone marrow-derived macrophages and RAW264.7 cells. In addition, 1,8-cineole targets were revealed by drug affinity responsive target stability, thermal shift assay, cellular thermal shift assay, and heat shock protein 90 (HSP90) adenosine triphosphatases (ATPase) activity assays. The results showed that 1,8-cineole exhibited powerful anti-inflammatory properties in vitro and in vivo by inhibiting the macrophage M1 polarization and protecting intestinal barrier function. Mechanistically, 1,8-cineole directly interacted with HSP90 and decreased its ATPase activity, also inhibited nucleotide-binding and oligomerization domain-, leucine rich repeat-, and pyrin domain-containing 3 (NLRP3) binding to HSP90 and suppressor of G-two allele of SKP1 (SGT1) and suppressed NLRP3 inflammasome activation in macrophages. These results demonstrated that 1,8-cineole is a potential drug candidate for UC treatment.

Endosomal trafficking of two-pore K+ efflux channel TWIK2 to plasmalemma mediates NLRP3 inflammasome activation and inflammatory injury.

Potassium efflux via the two-pore K+ channel TWIK2 is a requisite step for the activation of NLRP3 inflammasome, however, it remains unclear how K+ efflux is activated in response to select cues. Here, we report that during homeostasis, TWIK2 resides in endosomal compartments. TWIK2 is transported by endosomal fusion to the plasmalemma in response to increased extracellular ATP resulting in the extrusion of K+. We showed that ATP-induced endosomal TWIK2 plasmalemma translocation is regulated by Rab11a. Deleting Rab11a or ATP-ligated purinergic receptor P2X7 each prevented endosomal fusion with the plasmalemma and K+ efflux as well as NLRP3 inflammasome activation in macrophages. Adoptive transfer of Rab11a-depleted macrophages into mouse lungs prevented NLRP3 inflammasome activation and inflammatory lung injury. We conclude that Rab11a-mediated endosomal trafficking in macrophages thus regulates TWIK2 localization and activity at the cell surface and the downstream activation of the NLRP3 inflammasome. Results show that endosomal trafficking of TWIK2 to the plasmalemma is a potential therapeutic target in acute or chronic inflammatory states.

Role of GPER in the expression and secretion of pro-inflammatory cytokines and calcium signaling in bone marrow-derived macrophages

Macrophages play an important role in mediating inflammation in numerous disease states by secreting pro-inflammatory cytokines including IL-1β and IL-18. Macrophages are driven into the M1-proinflammatory phenotype upon detection of pathogen-associated molecular patterns (PAMPs), or damage-associated molecular patterns (DAMPs). As macrophages are polarized into the M1 phenotype, they upregulate the expression of pro-inflammatory cytokines and components of the NLRP3 inflammasome. An additional activating stimulus is required to induce the assembly of the NLRP3 inflammasome, which facilitates the activation of pro-caspase 1 and the subsequent conversion of the pro-forms of both IL-18 and IL-1β into their mature forms for their secretion through gasdermin D membrane pores. A canonical activator of the NLRP3 inflammasome is extracellular ATP. ATP activation of the P2X7 receptor induces potassium efflux and calcium influx, both of which are necessary for NLRP3 inflammasome activation. Previous studies suggest that the G protein-coupled receptor (GPER) agonist G-1 reduced inflammation in animal disease models. However, the GPER-dependent effect of G-1 on pro-inflammatory cytokine expression and NLRP3 inflammasome activation in macrophages is unknown. In the current study, we utilized bone marrow-derived macrophages (BMDMs) from either WT or GPER-/- mice to test the hypothesis that G-1 reduces inflammatory cytokine production and NLRP3 inflammasome expression in a GPER-dependent manner. We found that the treatment of WT BMDMs with 100 nM G-1 for 24 hrs inhibited LPS-induced NLRP3, pro-IL-1β, IL-6, CCL5 and expression, and IL-1β secretion. Importantly, G-1 had no effect on GPER-/- BMDMs. To begin to determine if GPER modulates NLRP3 inflammasome activation, we characterized ATP-induced Ca2+ signals in both WT and GPER BMDMs with and without LPS pretreatment. No differences were observed between unprimed WT and GPER-/- BMDMs with respect to basal Ca2+ level, ATP-induced intracellular Ca2+ release, and subsequent bulk Ca2+ signal upon reestablishment of extracellular Ca2+. However, priming with LPS significantly increased ATP-induced intracellular Ca2+ release in both WT and GPER-/- BMDMs. Compared to WT cells, LPS-primed GPER-/- BMDMs exhibit significantly higher basal Ca2+ yet significantly lower ATP-induced Ca2+ release. Consistently, 15-minute treatment with 100 nM G-1 agonist strongly reduces ATP-induced Ca2+ release in WT BMDMs but had no effect in GPER-/- BMDMs. These results suggest a role for GPER in mediating Ca2+ uptake into IP3-sensitive intracellular compartments in BMDMs during LPS priming. In keeping with the known role for mobilization of intracellular Ca2+ in ATP-induced secretion of pro-inflammatory cytokines, preliminary data suggests that short-term G-1 pretreatment reduced IL-1β secretion induced by ATP in LPS-primed BMDMs. Overall, these data indicate a role for GPER in the priming and activation of the NLRP3 inflammasome. This research was supported by Des Moines University IOER Research & Grant Award #03-22-10. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Monosodium urate crystals alter the circadian clock in macrophages leading to loss of NLRP3 inflammasome repression: Implications for timing of the gout flare.

Gout is caused by monosodium urate (MSU) crystal deposition within joints. This leads to acute episodes of inflammation ("gout flares") driven by NLRP3 inflammasome activation in macrophages. Gout flares are frequently present during late night/early morning. The reason for this timing is unclear. Recent evidence suggests the NLRP3 inflammasome is under circadian control. The purpose of this study was to determine whether MSU crystals cause changes in the circadian clock in macrophages leading to time-of-day differences in NLRP3 inflammasome activation. Levels of circadian clock components were measured in undifferentiated "monocytic" and PMA-differentiated "macrophagic" THP-1 cells cultured with/without MSU crystals. Caspase-1 activity was measured to assess NLRP3 inflammasome activity. MSU crystal exposure resulted in minimal effects on clock genes in THP-1 monocytes but BMAL1, CRY1, PER2, and REV-ERBα showed altered expression with reduced protein levels of BMAL1 and REV-ERBα in THP-1 macrophages. REV-ERBα activation or BMAL1 over-expression resulted in reduced MSU crystal-induced caspase-1 activity. BMAL1 knockdown resulted in a further increase in MSU crystal-induced caspase-1 activity, but only at times of day when BMAL1 levels were naturally high. MSU crystal-induced NLRP3 inflammasome activation was greatest at the time of day when BMAL1 levels were naturally low. MSU crystals alter the expression of circadian clock components in THP-1 macrophages leading to loss of BMAL1 and REV-ERBα-mediated repression of NLRP3 inflammasome activity and time-of-day differences in susceptibility to inflammasome activation. Our findings suggest that the nocturnal risk of gout flare is at least partially a consequence of altered circadian control of immune cell function.

Formaldehyde aggravates allergic contact dermatitis by facilitating NLRP3 inflammasome activation in macrophages.

Formaldehyde (FA) is known to be an environmental pollutant and contact sensitizer at 1% or 2% concentrations, which can induce inflammatory diseases such as allergic contact dermatitis (ACD). However, the aggravative effects of FA on ACD at legitimate low concentrations in cosmetics have not been studied. The activation of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome in ACD was recently identified, and the inflammatory responses were attenuated by NLRP3 inhibition. Since non-cytotoxic concentrations of FA at 50 and 100μM were found to reinforce inflammatory responses in macrophages, the 0.05% low concentration of FA was applied to ACD mice induced by 2,4-dinitro-1-fluorobenzene. FA significantly exacerbated inflammatory responses and NLRP3 inflammasome activation, which was confirmed in RAW264.7 macrophages treated with FA at 50 and 100μM in vitro. Induction of mitochondrial reactive oxygen species, the common activation signal for NLRP3 inflammasome, was also observed in FA-treated macrophages. Inhibition of NLRP3 by MCC950 significantly attenuated the NLRP3 inflammasome activation induced by 100μM FA in vitro and alleviated FA-enhanced inflammatory responses in ACD mice. These results not only demonstrated that FA was able to aggravate the inflammatory responses of ACD by facilitating NLRP3 inflammasome activation in macrophages, which was likely to play important roles in FA-related sensitization, but also indicated that NLRP3 could be targeted to relieve FA-induced inflammation.

Atranorin inhibits NLRP3 inflammasome activation by targeting ASC and protects NLRP3 inflammasome-driven diseases.

Aberrant NLRP3 activation has been implicated in the pathogenesis of numerous inflammation-associated diseases. However, no small molecular inhibitor that directly targets NLRP3 inflammasome has been approved so far. In this study, we show that Atranorin (C19H18O8), the secondary metabolites of lichen family, effectively prevents NLRP3 inflammasome activation in macrophages and dendritic cells. Mechanistically, Atranorin inhibits NLRP3 activation induced cytokine secretion and cell pyroptosis through binding to ASC protein directly and therefore restraining ASC oligomerization. The pharmacological effect of Atranorin is evaluated in NLRP3 inflammasome-driven disease models. Atranorin lowers serum IL-1β and IL-18 levels in LPS induced mice acute inflammation model. Also, Atranorin protects againstMSU crystal induced mice gouty arthritis model and lowers ankle IL-1β level. Moreover, Atranorin ameliorates intestinal inflammation and epithelial barrier dysfunction in DSS induced mice ulcerative colitis and inhibits NLRP3 inflammasome activation in colon. Altogether, our study identifies Atranorin as a novel NLRP3 inhibitor that targets ASC protein and highlights the potential therapeutic effects of Atranorin in NLRP3 inflammasome-driven diseases including acute inflammation, gouty arthritis and ulcerative colitis.

P120-Catenin suppresses NLRP3 inflammasome activation in macrophages.

Inflammasome activation is of central importance for the process of generation of overwhelming inflammatory response and the pathogenesis of sepsis. The intrinsic molecular mechanism for controlling inflammasome activation is still poorly understood. Here we investigated the role of p120-catenin expression in macrophages in regulating nucleotide-binding oligomerization domain (NOD) and leucine-rich repeat (LRR)- and pyrin domain-containing proteins 3 (NLRP3) inflammasome activation. Depletion of p120-catenin in murine bone marrow-derived macrophages enhanced caspase-1 activation and secretion of active interleukin (IL)-1β in response to ATP stimulation following LPS priming. Coimmunoprecipitation analysis showed that p120-catenin deletion promoted NLRP3 inflammasome activation by accelerating the assembly of the inflammasome complex comprised of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1. Depletion of p120-catenin also increased the production of mitochondrial reactive oxygen species. Pharmacological inhibition of mitochondrial reactive oxygen species nearly completely abolished NLRP3 inflammasome activation, caspase-1 activation, and the production of IL-1β in p120-catenin-depleted macrophages. Furthermore, p120-catenin ablation significantly disrupted mitochondrial function, evidenced by decreased mitochondrial membrane potential and lower production of intracellular ATP. In alveolar macrophage-depleted mice challenged with cecal ligation and puncture, pulmonary transplantation of p120-catenin-deficient macrophages dramatically enhanced the accumulation of IL-1β and IL-18 in bronchoalveolar lavage fluid. These results demonstrate that p120-catenin prevents NLRP3 inflammasome activation in macrophages by maintaining mitochondrial homeostasis and reducing the production of mitochondrial reactive oxygen species in response to endotoxin insult. Thus, inhibition of NLRP3 inflammasome activation by stabilization of p120-catenin expression in macrophages may be a novel strategy to prevent an uncontrolled inflammatory response in sepsis.

TNFα-induced NLRP3 inflammasome mediates adipocyte dysfunction and activates macrophages through adipocyte-derived lipocalin 2

Background and aimsObesity is a state of chronic low-grade systemic inflammation. Recent studies showed that NLRP3 inflammasome initiates metabolic dysregulation in adipose tissues, primarily through activation of adipose tissue infiltrated macrophages. However, the mechanism of NLRP3 activation and its role in adipocytes remains elusive. Therefore, we aimed to examine the activation of TNFα-induced NLRP3 inflammasome in adipocytes and its role on adipocyte metabolism and crosstalk with macrophages. MethodsThe effect of TNFα on adipocyte NLRP3 inflammasome activation was measured. Caspase-1 inhibitor (Ac-YVAD-cmk) and primary adipocytes from NLRP3 and caspase-1 knockout mice were utilized to block NLRP3 inflammasome activation. Biomarkers were measured by using real-time PCR, western blotting, immunofluorescence staining, and enzyme assay kits. Conditioned media from TNFα-stimulated adipocytes was used to establish the adipocyte-macrophage crosstalk. Chromatin immunoprecipitation assay was used to identify the role of NLRP3 as a transcription factor. Mouse and human adipose tissues were collected for correlation analysis. ResultsTNFα treatment induced NLRP3 expression and caspase-1 activity in adipocytes, partly through autophagy dysregulation. The activated adipocyte NLRP3 inflammasome participated in mitochondrial dysfunction and insulin resistance, as evidenced by the amelioration of these effects in Ac-YVAD-cmk treated 3T3-L1 cells or primary adipocytes isolated from NLRP3 and caspase-1 knockout mice. Particularly, the adipocyte NLRP3 inflammasome was involved in glucose uptake regulation. Also, TNFα induced expression and secretion of lipocalin 2 (Lcn2) in a NLRP3-dependent manner. NLRP3 could bind to the promoter and transcriptionally regulate Lcn2 in adipocytes. Treatment with adipocyte conditioned media revealed that adipocyte-derived Lcn2 was responsible for macrophage NLRP3 inflammasome activation, working as a second signal. Adipocytes isolated from high-fat diet mice and adipose tissue from obese individuals showed a positive correlation between NLRP3 and Lcn2 gene expression. ConclusionsThis study highlights the importance of adipocyte NLRP3 inflammasome activation and novel role of TNFα-NLRP3-Lcn2 axis in adipose tissue. It adds rational for the current development of NLRP3 inhibitors for treating obesity-induced metabolic diseases.