Defective endosome-TGN retrograde transport promotes NLRP3 inflammasome activation

Inflammasome complexes are pivotal in the innate immune response to pathogens and other danger signals. The NLRP3 inflammasome is activated in response to a broad variety of cellular stressors. Most of the stimuli act in a potassium efflux-dependent manner but a primary and converging sensing mechanism by the NLRP3 receptor initiating inflammasome assembly remains ill-defined. Here we show that NLRP3 activators disrupt endosome-TGN retrograde transport (ETRT) and lead to localization of NLRP3 to endosomal vesicles. Genetic and pharmacologic perturbation of ETRT leads to accumulation of phosphoinositol-4-phosphate (PI4P) in endosomes to which NLRP3 is recruited. Disruption of ETRT potentiates NLRP3 inflammasome activation in murine and human macrophages in vitro. Mice with defects in ETRT in the myeloid compartment are more susceptible to LPS-induced sepsis showing enhanced mortality and IL-1β serum levels as compared to control animals. Our study thus uncovers that changes in endocytic trafficking mediate NLRP3-dependent inflammatory responses.

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Defective endosome-TGN retrograde transport promotes NLRP3 inflammasome activation

10.21203/rs.3.rs-904605/v1 ◽

2021 ◽

Author(s):

Romeo Ricci ◽

Zhirong Zhang ◽

Li Ran ◽

Rossella Venditti ◽

Zengzhen Liu ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

Inflammatory Responses ◽

Retrograde Transport ◽

Serum Levels ◽

Inflammasome Activation ◽

Dependent Manner ◽

Potassium Efflux ◽

Nlrp3 Inflammasome Activation ◽

Broad Variety

Abstract Inflammasome complexes are pivotal in the innate immune response to pathogens and other danger signals. The NLRP3 inflammasome is activated in response to a broad variety of cellular stressors. Most of the stimuli act in a potassium efflux-dependent manner but a primary and converging sensing mechanism by the NLRP3 receptor initiating inflammasome assembly remains ill-defined. Here we show that NLRP3 activators disrupt endosome-TGN retrograde transport (ETRT) and lead to localization of NLRP3 to endosomal vesicles. Genetic and pharmacologic perturbation of ETRT leads to accumulation of phosphoinositol-4-phosphate (PI4P) in endosomes to which NLRP3 is recruited. Disruption of ETRT potentiates NLRP3 inflammasome activation in murine and human macrophages in vitro. Mice with defects in ETRT in the myeloid compartment are more susceptible to LPS-induced sepsis showing enhanced mortality and IL-1β serum levels as compared to control animals. Our study thus uncovers that changes in endocytic trafficking mediate NLRP3-dependent inflammatory responses.

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Intra-articular Injection of Baicalein Inhibits Cartilage Catabolism and NLRP3 Inflammasome Signaling in a Posttraumatic OA Model

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/6116890 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Hui Bai ◽

Rui Yuan ◽

Zhiheng Zhang ◽

Lin Liu ◽

Xinyu Wang ◽

...

Keyword(s):

Oxidative Stress ◽

Rat Model ◽

Nlrp3 Inflammasome ◽

Type Ii Collagen ◽

Cartilage Degeneration ◽

Inflammasome Activation ◽

Dependent Manner ◽

Nlrp3 Inflammasome Activation ◽

Immunochemical Staining

Baicalein has been shown to have chondroprotective potential in vitro. However, its effect on disease modification in osteoarthritis (OA) is largely unknown. The present study is aimed at determining whether baicalein could slow the progression of OA and inhibit OA-related inflammation in a rat model of destabilization of the medial meniscus (DMM) and the underlying mechanisms. The rats subjected to DMM surgery were treated with baicalein (0.8, 1.6, and 3.2 μg/L, 50 μL, once a week) by intra-articular injection for 6 weeks. Dexamethasone (0.4 mg/mL, 50 μL, once a week) was used as a positive control. Histologic grading of cartilage degeneration was performed using the Osteoarthritis Research Society International (OARSI) recommended grading system (on a scale of 0-6). The expression levels of molecules associated with cartilage homeostasis and inflammatory cytokines were analyzed; moreover, the NLRP3 inflammasome activation and cartilage oxidative stress-associated molecules were determined. Baicalein treatment reduced the OARSI score and slowed OA disease progression in a dose-dependent manner within a certain range. Compared with DMM rats, intra-articular injection of baicalein led to (1) reduced levels of inflammatory mediates such as IL-1β and TNF-α, (2) reduced immunochemical staining of MMP-13 and ADAMTS-5, (3) suppressed immunochemical staining loss of type II collagen, (4) reduced expression of cartilage degradation markers including CTX-II and COMP in urine, and (5) inhibited NLRP3 inflammasome activation rather than regulated expression of SOD, GSH, and MDA. In contrast to the administration of baicalein, dexamethasone injection showed similar effects to slow OA progression, while dexamethasone inhibited NLRP3 inflammasome partly through decreasing levels of SOD, GSH, and MDA. This study indicated that baicalein may have the potential for OA prevention and exerts anti-inflammatory effects partly via suppressing NLRP3 inflammasome activation without affecting oxidative stress-associated molecules, and inhibition of cartilage catabolism enzymes in an OA rat model.

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Calycosin Alleviates Doxorubicin-Induced Cardiotoxicity and Pyroptosis by Inhibiting NLRP3 Inflammasome Activation

Oxidative Medicine and Cellular Longevity ◽

10.1155/2022/1733834 ◽

2022 ◽

Vol 2022 ◽

pp. 1-15

Author(s):

Lei Zhang ◽

Cundong Fan ◽

Hua-Chen Jiao ◽

Qian Zhang ◽

Yue-Hua Jiang ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

Cardiac Injury ◽

H9c2 Cell ◽

Inflammasome Activation ◽

Dependent Manner ◽

H9c2 Cells ◽

Stress Injury ◽

Nlrp3 Inflammasome Activation

Calycosin (CAL) is the main active component present in Astragalus and reportedly possesses diverse pharmacological properties. However, the cardioprotective effect and underlying mechanism of CAL against doxorubicin- (DOX-) induced cardiotoxicity need to be comprehensively examined. Herein, we aimed to investigate whether the cardioprotective effects of CAL are related to its antipyroptotic effect. A cardiatoxicity model was established by stimulating H9c2 cells and C57BL/6J mice using DOX. In vitro, CAL increased H9c2 cell viability and decreased DOX-induced pyroptosis via NLRP3, caspase-1, and gasdermin D signaling pathways in a dose-dependent manner. In vivo, CAL-DOX cotreatment effectively suppressed DOX-induced cytotoxicity as well as inflammatory and cardiomyocyte pyroptosis via the same molecular mechanism. Next, we used nigericin (Nig) and NLRP3 forced overexpression to determine whether CAL imparts antipyroptotic effects by inhibiting the NLRP3 inflammasome in vitro. Furthermore, CAL suppressed DOX-induced mitochondrial oxidative stress injury in H9c2 cells by decreasing the generation of reactive oxygen species and increasing mitochondrial membrane potential and adenosine triphosphate. Likewise, CAL attenuated the DOX-induced increase in malondialdehyde content and decreased superoxide dismutase and glutathione peroxidase activities in H9c2 cells. In vivo, CAL afforded a protective effect against DOX-induced cardiac injury by improving myocardial function, inhibiting brain natriuretic peptide, and improving the changes of the histological morphology of DOX-treated mice. Collectively, our findings confirmed that CAL alleviates DOX-induced cardiotoxicity and pyroptosis by inhibiting NLRP3 inflammasome activation in vivo and in vitro.

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Juglone Suppresses LPS-induced Inflammatory Responses and NLRP3 Activation in Macrophages

Molecules ◽

10.3390/molecules25133104 ◽

2020 ◽

Vol 25 (13) ◽

pp. 3104 ◽

Cited By ~ 3

Author(s):

Nam-Hun Kim ◽

Hong-Ki Kim ◽

Ji-Hak Lee ◽

Seung-Il Jo ◽

Hye-Min Won ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

Inflammatory Responses ◽

Inflammasome Activation ◽

No Production ◽

Dependent Manner ◽

Inhibitory Effects ◽

Concentration Dependent Manner ◽

Inflammatory Cytokine Production ◽

Caspase 1 ◽

Nlrp3 Inflammasome Activation

The NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome has been implicated in a variety of diseases, including atherosclerosis, neurodegenerative diseases, and infectious diseases. Thus, inhibitors of NLRP3 inflammasome have emerged as promising approaches to treat inflammation-related diseases. The aim of this study was to explore the effects of juglone (5-hydroxyl-1,4-naphthoquinone) on NLRP3 inflammasome activation. The inhibitory effects of juglone on nitric oxide (NO) production were assessed in lipopolysaccharide (LPS)-stimulated J774.1 cells by Griess assay, while its effects on reactive oxygen species (ROS) and NLRP3 ATPase activity were assessed. The expression levels of NLRP3, caspase-1, and pro-inflammatory cytokines (IL-1β, IL-18) and cytotoxicity of juglone in J774.1 cells were also determined. Juglone was non-toxic in J774.1 cells when used at 10 μM (p < 0.01). Juglone treatment inhibited the production of ROS and NO. The levels of NLRP3 and cleaved caspase-1, as well as the secretion of IL-1β and IL-18, were decreased by treatment with juglone in a concentration-dependent manner. Juglone also inhibited the ATPase activities of NLRP3 in LPS/ATP-stimulated J774.1 macrophages. Our results suggested that juglone could inhibit inflammatory cytokine production and NLRP3 inflammasome activation in macrophages, and should be considered as a therapeutic strategy for inflammation-related diseases.

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Inhibition of IP3R/Ca2+ Dysregulation Protects Mice From Ventilator-Induced Lung Injury via Endoplasmic Reticulum and Mitochondrial Pathways

Frontiers in Immunology ◽

10.3389/fimmu.2021.729094 ◽

2021 ◽

Vol 12 ◽

Author(s):

Liu Ye ◽

Qi Zeng ◽

Maoyao Ling ◽

Riliang Ma ◽

Haishao Chen ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Lung Injury ◽

Er Stress ◽

Mitochondrial Dysfunction ◽

Nlrp3 Inflammasome ◽

Inflammatory Responses ◽

Inflammasome Activation ◽

Dependent Manner ◽

Ventilator Induced Lung Injury ◽

Nlrp3 Inflammasome Activation

RationaleDisruption of intracellular calcium (Ca2+) homeostasis is implicated in inflammatory responses. Here we investigated endoplasmic reticulum (ER) Ca2+ efflux through the Inositol 1,4,5-trisphosphate receptor (IP3R) as a potential mechanism of inflammatory pathophysiology in a ventilator-induced lung injury (VILI) mouse model.MethodsC57BL/6 mice were exposed to mechanical ventilation using high tidal volume (HTV). Mice were pretreated with the IP3R agonist carbachol, IP3R inhibitor 2-aminoethoxydiphenyl borate (2-APB) or the Ca2+ chelator BAPTA-AM. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected to measure Ca2+ concentrations, inflammatory responses and mRNA/protein expression associated with ER stress, NLRP3 inflammasome activation and inflammation. Analyses were conducted in concert with cultured murine lung cell lines.ResultsLungs from mice subjected to HTV displayed upregulated IP3R expression in ER and mitochondrial-associated-membranes (MAMs), with enhanced formation of MAMs. Moreover, HTV disrupted Ca2+ homeostasis, with increased flux from the ER to the cytoplasm and mitochondria. Administration of carbachol aggravated HTV-induced lung injury and inflammation while pretreatment with 2-APB or BAPTA-AM largely prevented these effects. HTV activated the IRE1α and PERK arms of the ER stress signaling response and induced mitochondrial dysfunction-NLRP3 inflammasome activation in an IP3R-dependent manner. Similarly, disruption of IP3R/Ca2+ in MLE12 and RAW264.7 cells using carbachol lead to inflammatory responses, and stimulated ER stress and mitochondrial dysfunction.ConclusionIncrease in IP3R-mediated Ca2+ release is involved in the inflammatory pathophysiology of VILI via ER stress and mitochondrial dysfunction. Antagonizing IP3R/Ca2+ and/or maintaining Ca2+ homeostasis in lung tissue represents a prospective treatment approach for VILI.

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Intracellular NAD+ Depletion Confers a Priming Signal for NLRP3 Inflammasome Activation

Frontiers in Immunology ◽

10.3389/fimmu.2021.765477 ◽

2021 ◽

Vol 12 ◽

Author(s):

Do-Wan Shim ◽

Hyo-Joung Cho ◽

Inhwa Hwang ◽

Taek-Yeol Jung ◽

Hyun-Seok Kim ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

Specific Inhibitor ◽

Retrograde Transport ◽

Poly I:C ◽

Inflammasome Activation ◽

Mouse Bone Marrow ◽

Caspase 1 ◽

Nicotinamide Mononucleotide ◽

Nlrp3 Inflammasome Activation

Nicotinamide adenine dinucleotide (NAD+) is an important cofactor in many redox and non-redox NAD+-consuming enzyme reactions. Intracellular NAD+ level steadily declines with age, but its role in the innate immune potential of myeloid cells remains elusive. In this study, we explored whether NAD+ depletion by FK866, a highly specific inhibitor of the NAD salvage pathway, can affect pattern recognition receptor-mediated responses in macrophages. NAD+-depleted mouse bone marrow-derived macrophages (BMDMs) exhibited similar levels of proinflammatory cytokine production in response to LPS or poly (I:C) stimulation compared with untreated cells. Instead, FK866 facilitated robust caspase-1 activation in BMDMs in the presence of NLRP3-activating signals such as ATP and nigericin, a potassium ionophore. However, this FK866-mediated caspase-1 activation was completely abolished in Nlrp3-deficient macrophages. FK866 plus nigericin stimulation caused an NLRP3-dependent assembly of inflammasome complex. In contrast, restoration of NAD+ level by supplementation with nicotinamide mononucleotide abrogated the FK866-mediated caspase-1 cleavage. FK866 did not induce or increase the expression levels of NLRP3 and interleukin (IL)-1β but drove mitochondrial retrograde transport into the perinuclear region. FK866-nigericin-induced mitochondrial transport is critical for caspase-1 cleavage in macrophages. Consistent with the in vitro experiments, intradermal coinjection of FK866 and ATP resulted in robust IL-1β expression and caspase-1 activation in the skin of wild-type, but not Nlrp3-deficient mice. Collectively, our data suggest that NAD+ depletion provides a non-transcriptional priming signal for NLRP3 activation via mitochondrial perinuclear clustering, and aging-associated NAD+ decline can trigger NLRP3 inflammasome activation in ATP-rich environments.

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MCL Plays an Anti-Inflammatory Role inMycobacterium tuberculosis-Induced Immune Response by Inhibiting NF-κB and NLRP3 Inflammasome Activation

Mediators of Inflammation ◽

10.1155/2017/2432904 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 4

Author(s):

Qingwen Zhang ◽

Xinru Jiang ◽

Weigang He ◽

Kailin Wei ◽

Jinxia Sun ◽

...

Keyword(s):

Immune Response ◽

Inflammatory Response ◽

Nlrp3 Inflammasome ◽

Inflammatory Responses ◽

Inhibitory Effect ◽

Drug Candidate ◽

Inflammasome Activation ◽

Dependent Manner ◽

Nlrp3 Inflammasome Activation

Mycobacterium tuberculosis(Mtb) remains a significant menace to global health as it induces granulomatous lung lesions and systemic inflammatory responses during active tuberculosis (TB). Micheliolide (MCL), a sesquiterpene lactone, was recently reported to have a function of relieving LPS-induced inflammatory response, but the regulative role of MCL on the immunopathology of TB still remains unknown. In this experiment, we examined the inhibitory effect of MCL on Mtb-induced inflammatory response in mouse macrophage-like cell line Raw264.7 by downregulating the activation of nuclear factor kappa B (NF-κB) and NLRP3 inflammasome. Evidences showed that MCL decreased the secretion of Mtb-induced inflammatory cytokines (IL-1βand TNF-α) in a dose-dependent manner. Meanwhile, MCL dramatically suppressed Mtb-induced activation of iNOS and COX2 as well as subsequent production of NO. Furthermore, MCL inhibited Mtb-induced phosphorylation of Akt (Ser 473) in Raw264.7. According to our results, MCL plays an important role in modulating Mtb-induced inflammatory response through PI3K/Akt/NF-κB pathway and subsequently downregulating the activation of NLRP3 inflammasome. Therefore, MCL may represent as a potential drug candidate in the adjuvant treatment of TB by regulating host immune response.

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Anthocyanin Extract from Purple Sweet Potato Exacerbate Mitophagy to Ameliorate Pyroptosis in Klebsiella pneumoniae Infection

International Journal of Molecular Sciences ◽

10.3390/ijms222111422 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11422

Author(s):

Guokai Dong ◽

Nana Xu ◽

Meng Wang ◽

Yunyun Zhao ◽

Fei Jiang ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Sweet Potato ◽

Nlrp3 Inflammasome ◽

Inflammatory Responses ◽

Klebsiella Pneumonia ◽

Inflammasome Activation ◽

Nlrp3 Inflammasome Activation ◽

Nrf2 Signaling Pathway ◽

Purple Sweet Potato

Given the rise of morbidity and mortality caused by Klebsiella pneumoniae (KP), the increasing number of strains resistant to antibiotics, and the emergence of hypervirulent Klebsiella pneumonia, treatment of KP infection becomes difficult; thus, novel drugs are necessary for treatment. Anthocyanins, or natural flavonoids, have an extensive effect against bacterial infection. However, few studies on anti-KP are identified. Here, we evaluated the therapeutic effect of purple sweet potato anthocyanins (PSPAs) on KP, containing 98.7% delphinidin 3-sambubioside. Results showed that KP-infected mice after PSPAs treatment manifested decreased mortality, weakened lung injury, dampened inflammatory responses, and reduced bacterial systemic dissemination in vivo. In Vitro, PSPAs significantly suppressed pyroptosis and restricted NLRP3 inflammasome activation in alveolar macrophages infected with KP. As for the mechanism, PSPAs promote mitophagy by recruiting Parkin to the mitochondria. PSPAs-conferred mitophagy increased mitochondrial membrane potential and decreased mitochondrial reactive oxygen species and mitochondrial DNA, resulting in impaired NLRP3 inflammasome activation. In addition, the promotion of mitophagy by PSPAs required the Nrf2 signaling pathway. Collectively, these findings suggest that PSPAs are a potential option for the treatment of KP infection.

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Panax notoginseng saponins attenuate neuroinflammation through TXNIP-mediated NLRP3 inflammasome activation in aging rats

Current Pharmaceutical Biotechnology ◽

10.2174/1389201021999201110204735 ◽

2020 ◽

Vol 21 ◽

Author(s):

Zhiyong Zhou ◽

Menghan He ◽

Qingqing Zhao ◽

Dongfan Wang ◽

Changcheng Zhang ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

Panax Notoginseng ◽

Control Group ◽

Inflammasome Activation ◽

Panax Notoginseng Saponins ◽

Aging Rats ◽

Nlrp3 Inflammasome Activation ◽

Bv2 Microglia ◽

Old Rats

Introduction:: Microglia-mediated inflammatory responses play a crucial role in aging-related neurodegenerative diseases. The TXNIP/NLRP3 pathway is a key pathway leading to microglial activation. Panax notoginseng saponins (PNS) have been widely used for the treatment of stroke in China. Objective:: This study evaluates the anti-neuroinflammatory effect of PNS and investigates the mechanism via TXNIPmediated NLRP3 inflammasome activation in aging rats. Materials and Methods:: Eighteen-month-old Sprague-Dawley rats were randomly divided into the aging control group and PNS treated groups (n=15 each group). For PNS-treated groups, rats were administrated food with PNS at the doses of 10 mg/kg and 30 mg/kg for consecutive 6 months until they were 24-month old. Rats from the aging control group were given the same food without PNS. Two-month-old rats were purchased and given the same food until 6-month old as the adult control group (n = 15). Then, the cortex and hippocampus were rapidly harvested and deposited. H&E staining was used to assess histo-morphological changes. Western blotting was carried out to detect the protein expression. Immunofluorescence was employed to measure the co-localization of NLRP3, TXNIP and Iba-1. In vitro model was established by LPS+ATP coincubation in the BV2 microglia cell line. Results:: Aging rats exhibited increased activation of microglia, accompanied by a high level of IL-1β expression. Meanwhile, aging rats showed enhanced protein expression of TXNIP and NLRP3 related molecules, which co-localized with microglia. PNS treatment effectively reduced the number of degenerated neurons and reversed the activation of the TXNIP/NLRP3 inflammatory pathway. In vitro results showed that PNS up to 100 μg / ml had no significant toxicity on BV2 microglia. Discussion:: PNS (25, 50 μg/ml) effectively reduced the inflammatory response induced by LPS and ATP co-stimulation, thus inhibiting the expression of TXNIP/NLRP3 pathway-related proteins. Conclusion:: PNS treatment improved aging-related neuronal damage through inhibiting TXNIP mediated NLRP3 inflammasome activation, which provided a potential target for the treatment of inflammatory-related neurodegenerative diseases.

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SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation

Nature Communications ◽

10.1038/s41467-021-25015-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Pan Pan ◽

Miaomiao Shen ◽

Zhenyang Yu ◽

Weiwei Ge ◽

Keli Chen ◽

...

Keyword(s):

Lung Injury ◽

Nlrp3 Inflammasome ◽

Cultured Cells ◽

Inflammatory Responses ◽

Specific Inhibitor ◽

Inflammasome Activation ◽

N Protein ◽

Distinct Mechanism ◽

Nlrp3 Inflammasome Activation ◽

Proinflammatory Responses

AbstractExcessive inflammatory responses induced upon SARS-CoV-2 infection are associated with severe symptoms of COVID-19. Inflammasomes activated in response to SARS-CoV-2 infection are also associated with COVID-19 severity. Here, we show a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation. N protein facilitates maturation of proinflammatory cytokines and induces proinflammatory responses in cultured cells and mice. Mechanistically, N protein interacts directly with NLRP3 protein, promotes the binding of NLRP3 with ASC, and facilitates NLRP3 inflammasome assembly. More importantly, N protein aggravates lung injury, accelerates death in sepsis and acute inflammation mouse models, and promotes IL-1β and IL-6 activation in mice. Notably, N-induced lung injury and cytokine production are blocked by MCC950 (a specific inhibitor of NLRP3) and Ac-YVAD-cmk (an inhibitor of caspase-1). Therefore, this study reveals a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.

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