NLR Family Pyrin Domain Containing 3 Research Articles

Role of NLRP3 inflammasome in cardiac damage in the setting of an acute cardiorenal syndrome

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ISCIII, Ministerio de Universidades, Education and Research Council of Madrid, Biomedicine Network Comunidad de Madrid Background Acute kidney injury (AKI) is usually associated with an acute cardiac impairment known as cardiorenal syndrome type 3 (CRS3). AKI induces the activation of the immune system being the connection with the disturbance in remote organs as the heart. In response to tissue damage, different inflammatory pathways are activated. In this sense, activation of cardiac nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome has been studied in several cardiac pathologies. Nevertheless, the role of cardiac NLRP3 in ventricular cardiomyocytes as a consequence of renal damage is unknown. Purpose The overall aim of this study was to analyse the involvement of NLRP3 in AKI-induced cardiac damage in CRS3. Firstly, we evaluated the predictive value for mortality of the inflammatory component associated with the NLRP3 in a cohort of AKI patients. Then, we studied whether cardiac NLRP3 activation takes place after AKI induction and whether this may be associated with functional alterations in ventricular cardiomyocytes related to cardiac dysfunction and predisposition to ventricular arrhytmia. Methods We have studied cardiac alterations resulting from AKI induced by folic acid (FA, 250 mg/Kg) after 72 hours in wild-type (WT), Nlrp3-/- and WT AKI mice treated with the NLRP3 inhibitor MCC950 (10 mg/Kg). We examined plasma, renal and cardiac inflammatory components associated with NLRP3, as well as cardiomyocyte intracellular Ca2+ handling by confocal microscopy in all experimental groups. In AKI patients, we analysed systemic NLRP3-related cytokine levels and their possible relationship with markers of renal and cardiac damage and the prediction of mortality. Results FA-induced AKI caused a worsening of contractile function in WT cardiomyocytes due to decreased systolic Ca2+ release after 72 hours. It was accompanied by impaired Ca2+ reuptake via SERCA2a. Nevertheless, in both MCC950-treated and NLRP3-/- mice, these alterations were significantly prevented. During diastole, significant Ca2+ leakage from the sarcoplasmic reticulum was observed after 72 hours of AKI-induction, as well as increased pro-arrhythmogenic events. These alterations were prevented in both NLRP3-/- and MCC950-treated AKI mice. In AKI patients, we found that NLRP3-related cytokines increased from the time of admission to 72 hours, significantly associated with biomarkers of renal and cardiovascular damage, and exhibited a high potential to predict mortality. Conclusions AKI impairs the cytosolic Ca2+ handling in ventricular cardiomyocytes that are translated into contractile dysfunction and increased occurrence of pro-arrhythmogenic events. These cardiac alterations are strongly prevented under NLRP3 blockade, demonstrating the deleterious role of its cardiac activation in CRS3. In addition, results from AKI patients point to NLRP3 as relevant prognostic target in AKI patients at the time of admission to hospital.

Inflammasome activation dynamics within the first 24 hours in patients treated with PCI for myocardial infarction is influenced by the age of patients, preliminary report

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Presented study was financially supported by the Mini-student Grant for medical students of Medical University of Warsaw, No 24/M/MG/N/21 and Grant for Young Researchers of The Medical University of Warsaw, No 2W6/1/M/MBS/N/21. Background Until today, majority of data on the possible role of inflammasome activation in acute coronary syndrome (ACS) patients comes from preclinical studies. Purpose We aimed to determine the dynamics of the expression profile of selected inflammasome genes and inflammasome related factors by quantitative real-time polymerase chain reaction (qRT-PCR) in peripheral blood mononuclear cells (PBMCs) of patients in the acute phase of ACS. Methods In the study group the blood samples for RT-PCR were collected 3 times: at the beginning of the percutaneous coronary intervention (PCI) for ACS, 3h, and 24h after PCI. The study group samples were compared with blood collected from 10 patients with no significant coronary lesions identified during coronary angiography. The relative expression of evaluated genes was analyzed in relation to the β-actin gene in relation to the expression in 0-hour sample using the ΔΔCT method. Based on the literature, we defined a significant change in gene expression at a fold change of 1.5. Results For this preliminary study the population of 19 patients aged 64±13and 84% presented with ST-elevation myocardial infarction (STEMI). Expression of evaluated genes at 3h and 24h relative to 0h sample is presented in picture 1. Gene expression did not differ significantly between the study and control group except for NLR Family Pyrin Domain Containing 6 (NLRP6) (at 3h post-PCI: ΔΔCT=2.5425 vs 0.0005; p=0.002, 24h post-PCI: ΔΔCT=2.1794 vs 0.0005; p=0.009). However, we observed significant upregulation in samples taken at 3h post-PCI relative to 0h level for AIM2 [fold change relative to 0h 1.52 (0.63; 4.67)], IL-1β [2.81 (1.59; 5.93)], IL-18 [2.07 (0.59; 10.14)] and at 24 h for AIM2 [1.90 (0.78; 2.52)], IL-1β [5.16 (2.23; 13.32)] and IL-18 [2.20 (0.59; 5.96)] in the younger subgroup of patients (≤the median age of the study population which was equal 66 years old). We performed a comparison of younger (≤66) vs. older (>66) patients at 3h and 24h post-PCI and observed significantly higher relative gene expression in the younger subgroup compared to the older subgroup of patients at 3h in case of IL-1β (p=0.03), and at 24h in case of NLR Family Pyrin Domain Containing 1(NLRP1)(p=0.016), AIM2 (p=0.04), NLR Family Pyrin Domain Containing 12(NRLP12)(p=0.06), IL-1 (p=0.04), and IL-18 (p=0.03) – the exact fold change values are presented in Table 1. Conclusion NLRP6 is significantly upregulated in ACS patients compared to control subjects in the first 24h post-PCI. We also observed significant dynamics in NLRP3 inflammasome related factors, including IL-1β, IL-18, in the first 24h post-PCI for MI. Also, the activation of the inflammasome genes (NLRP1, NLRP12) and inflammasome effectors may present different dynamics dependent on the age of the patients, and it might be helpful in careful selection of the timepoint for the future administration of inflammasome dedicated inhibitors.Relative genes expressionFold change in gene expression

YOD1 protects against MRSA sepsis-induced DIC through Lys33-linked deubiquitination of NLRP3

Disseminated intravascular coagulation (DIC) is considered to be the most common and lethal complication of sepsis. NLR-family pyrin domain-containing-3 (NLRP3) inflammasome plays an important role in host defense against microbial pathogens, and its deregulation may cause coagulation cascade and should be strictly managed. Here, we identified the deubiquitinase YOD1, which played a vital role in regulating coagulation in a NLRP3 inflammasome-dependent manner in sepsis induced by methicillin-resistant Staphylococcus aureus (MRSA). YOD1 interacted with NLRP3 to remove K33-linked ubiquitination of NLRP3 based on its deubiquitinating enzyme activity and specifically inhibited expression of NLRP3 as well as activation of NLRP3 inflammasome. Deficiency of YOD1 expression enhanced NLRP3 inflammasome activation and coagulation both in vitro and in vivo. In addition, pharmacological inhibition of the NLRP3 effectively improved coagulation and alleviated organ injury in Yod1−/− mice infected with MRSA. Thus, our study reported that YOD1 is a key regulator of coagulation during MRSA infection, and provided YOD1 as a potential therapeutic target for the treatment of NLRP3 inflammasome-related diseases, especially MRSA sepsis-induced DIC.

Inflammation in HIV and Its Impact on Atherosclerotic Cardiovascular Disease.

People living with HIV have a 1.5- to 2-fold increased risk of developing cardiovascular disease. Despite treatment with highly effective antiretroviral therapy, people living with HIV have chronic inflammation that makes them susceptible to multiple comorbidities. Several factors, including the HIV reservoir, coinfections, clonal hematopoiesis of indeterminate potential (CHIP), microbial translocation, and antiretroviral therapy, may contribute to the chronic state of inflammation. Within the innate immune system, macrophages harbor latent HIV and are among the prominent immune cells present in atheroma during the progression of atherosclerosis. They secrete inflammatory cytokines such as IL (interleukin)-6 and tumor necrosis-α that stimulate the expression of adhesion molecules on the endothelium. This leads to the recruitment of other immune cells, including cluster of differentiation (CD)8+ and CD4+ T cells, also present in early and late atheroma. As such, cells of the innate and adaptive immune systems contribute to both systemic inflammation and vascular inflammation. On a molecular level, HIV-1 primes the NLRP3 (NLR family pyrin domain containing 3) inflammasome, leading to an increased expression of IL-1β, which is important for cardiovascular outcomes. Moreover, activation of TLRs (toll-like receptors) by HIV, gut microbes, and substance abuse further activates the NLRP3 inflammasome pathway. Finally, HIV proteins such as Nef (negative regulatory factor) can inhibit cholesterol efflux in monocytes and macrophages through direct action on the cholesterol transporter ABCA1 (ATP-binding cassette transporter A1), which promotes the formation of foam cells and the progression of atherosclerotic plaque. Here, we summarize the stages of atherosclerosis in the context of HIV, highlighting the effects of HIV, coinfections, and antiretroviral therapy on cells of the innate and adaptive immune system and describe current and future interventions to reduce residual inflammation and improve cardiovascular outcomes among people living with HIV.

Therapeutic Targeting of PKM2 Ameliorates NASH Fibrosis Progression in a Macrophage-Specific and Liver-Specific Manner

Nonalcoholic steatohepatitis (NASH) may soon become the leading cause of end-stage liver disease worldwide with limited treatment options. Liver fibrosis, which is driven by chronic inflammation and hepatic stellate cell (HSC) activation, critically determines morbidity and mortality in patients with NASH. Pyruvate kinase M2 (PKM2) is involved in immune activation and inflammatory liver diseases; however, its role and therapeutic potential in NASH-related fibrosis remain largely unexplored. Bioinformatics screening and analysis of human and murine NASH livers indicated that PKM2 was upregulated in nonparenchymal cells (NPCs), especially macrophages, in the livers of patients with fibrotic NASH. Macrophage-specific PKM2 knockout (PKM2FL/FLLysM-Cre) significantly ameliorated hepatic inflammation and fibrosis severity in three distinct NASH models induced by a methionine- and choline-deficient (MCD) diet, a high-fat high-cholesterol (HFHC) diet, and a western diet plus weekly carbon tetrachloride injection (WD/CCl4). Single-cell transcriptomic analysis indicated that deletion of PKM2 in macrophages reduced profibrotic Ly6Chigh macrophage infiltration. Mechanistically, PKM2-dependent glycolysis promoted NLR family pyrin domain containing 3 (NLRP3) activation in proinflammatory macrophages, which induced HSC activation and fibrogenesis. A pharmacological PKM2 agonist efficiently attenuated the profibrotic crosstalk between macrophages and HSCs in vitro and in vivo. Translationally, ablation of PKM2 in NPCs by cholesterol-conjugated heteroduplex oligonucleotides, a novel oligonucleotide drug that preferentially accumulates in the liver, dose-dependently reversed NASH-related fibrosis without causing observable hepatotoxicity. The present study highlights the pivotal role of macrophage PKM2 in advancing NASH fibrogenesis. Thus, therapeutic modulation of PKM2 in a macrophage-specific or liver-specific manner may serve as a novel strategy to combat NASH-related fibrosis.

Xanthoangelol alleviates ischemic stroke injury by inhibiting the inflammatory activation of microglia

BackgroundLocal inflammation induced by microglial activation plays a significant role in the pathological process of cerebral ischemia. Angelica keiskei (Miq.) Koidz., a traditional botanical drug, can be used as a diuretic, laxative or galactagogue. Xanthoangelol (XA), an active chalcone compound from the aerial part of Angelica keiskei, has anti-inflammatory effects in the peripheral tissues. However, its effect against neuroinflammation is yet unclear. ObjectiveThe present study aims to investigate whether XA could mitigate ischemic stroke damage through attenuating neuroinflammation due to microglia activation. MethodsMiddle cerebral artery occlusion/reperfusion (MCAO/R) induced cerebral ischemia and oxygen-glucose deprivation/reperfusion (OGD/R) or lipopolysaccharide (LPS)-stimulated BV2 microglia cells were utilized to evaluate XA's protection against ischemic injury and neuroinflammation. The severity of brain injury was assessed using 2, 3, 5-triphenyltetrazolium chloride (TTC) staining and neurological assessment. The expressions of inflammatory cytokines were quantified by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). Reactive oxygen species (ROS) were assessed using dichlorodihydrofluorescein diacetate (DCF-DA) staining. NF-κB p65 nuclear translocation was confirmed by immunofluorescence (IF) staining. The expressions of proteins were quantified by Western blotting. ResultsXA was efficacious in reducing infarct size and improving neurological function in MCAO/R mice. In ischemic brain tissue, XA reduced microglial activation and proinflammatory cytokine expression. In lipopolysaccharide (LPS) and OGD/R-induced cell models, XA suppressed the production of ROS and decreased the secretion of inflammatory cytokines. Additionally, XA suppressed the nuclear translocation and phosphorylation of NF-κB p65 and blocked the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome. The protection of XA against MCAO/R-induced damages was not attenuated in TLR4−/− and MD2−/− mice. Mito-TEMPO treatment reversed XA's anti-inflammatory properties in OGD/R-induced BV2 cells. ConclusionXA attenuates ischemic stroke injury by suppressing microglial inflammatory responses. This efficacy is tied to its antioxidant activity and is independent of Toll-like receptor 4 (TLR4) or myeloid differentiation protein 2 (MD2).

Tributyltin chloride induces chondrocyte damage through the activation of NLRP3‑mediated inflammation and pyroptosis.

Tributyltin chloride (TBTC) is known to have effects and mechanisms in various diseases; however, whether TBTC is detrimental to joints and causes osteoarthritis (OA), as well as its underlying mechanism, has not yet been fully elucidated. The present study explored the effects of TBTC on rat chondrocytes, as well as on mouse OA. The toxicity of TBTC toward rat chondrocytes was detected using a lactate dehydrogenase (LDH) leakage assay and cell viability was evaluated using the Cell Counting Kit‑8 assay. The results showed that TBTC decreased the viability of rat chondrocytes and increased the LDH leakage rate in a concentration‑dependent manner. Moreover, compared with in the control group, TBTC increased the expression levels of interleukin (IL)‑1β, IL‑18, matrix metalloproteinase (MMP)‑1, MMP‑13, NLR family pyrin domain containing 3 (NLRP3), caspase‑1, PYD and CARD domain containing, and gasdermin D in chondrocytes. Furthermore, knockdown of NLRP3 reversed the TBTC‑induced increases in LDH leakage and NLRP3 inflammasome‑associated protein levels. In vivo, TBTC exacerbated cartilage tissue damage in mice from the OA group, as evidenced by the attenuation of safranin O staining. In conclusion, TBTC may aggravate OA in mice by promoting chondrocyte damage and inducing pyroptosis through the activation of NLRP3 and caspase‑1 signaling. The present study demonstrated that TBTC can cause significant damage to the articular cartilage; therefore, TBTC contamination should be strictly monitored.

N-3 Polyunsaturated Fatty Acids Protect against Alcoholic Liver Steatosis by Activating FFA4 in Kupffer Cells.

Supplementation with fish oil rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs) effectively reduces acute and chronic alcohol-induced hepatic steatosis. We aimed to find molecular mechanisms underlying the effects of n-3 PUFAs in alcohol-induced hepatic steatosis. Because free fatty acid receptor 4 (FFA4, also known as GPR120) has been found as a receptor for n-3 PUFAs in an ethanol-induced liver steatosis model, we investigated whether n-3 PUFAs protect against liver steatosis via FFA4 using AH7614, an FFA4 antagonist, and Ffa4 knockout (KO) mice. N-3 PUFAs and compound A (CpdA), a selective FFA4 agonist, reduced the ethanol-induced increase in lipid accumulation in hepatocytes, triglyceride content, and serum ALT levels, which were not observed in Ffa4 KO mice. N-3 PUFAs and CpdA also reduced the ethanol-induced increase in lipogenic sterol regulatory element-binding protein-1c expression in an FFA4-dependent manner. In Kupffer cells, treatment with n-3 PUFA and CpdA reversed the ethanol-induced increase in tumor necrosis factor-α, cyclooxygenase-2, and NLR family pyrin domain-containing 3 expression levels in an FFA4-dependent manner. In summary, n-3 PUFAs protect against ethanol-induced hepatic steatosis via the anti-inflammatory actions of FFA4 on Kupffer cells. Our findings suggest FFA4 as a therapeutic target for alcoholic hepatic steatosis.

Nlrp6 protects from corticosterone-induced NSPC ferroptosis by modulating RIG-1/MAVS-mediated mitophagy

Hippocampal neural stem/progenitor cells (NSPCs) are highly vulnerable to different stress stimuli, resulting in adult neurogenesis decline and eventual cognitive defects. Our previous study demonstrated that NOD-like receptor family pyrin domain-containing 6 (Nlrp6) highly expressed in NSPCs played a critical role in sustaining hippocampal neurogenesis to resist stress-induced depression, but the underlying mechnistms are still unclear. Here, we found that Nlrp6 depletion led to cognitive defects and hippocampal NSPC loss in mice. RNA-sequencing analysis of the primary NSPCs revealed that Nlrp6 deficiency altered gene expression profiles of mitochondrial energy generation and ferroptotic process. Upon siNlrp6 transfection, as well as corticosterone (CORT) exposure, downregulation of Nlrp6 suppressed retinoic acid-inducible gene I (RIG-1)/mitochondrial antiviral signaling proteins (MAVS)-mediated autophagy, but drove NSPC ferroptotic death. More interesting, short chain fatty acids (SCFAs) upregulated Nlrp6 expression and promoted RIG-1/MAVS-mediated mitophagy, preventing CORT-induced NSPC ferroptosis. Our study further demonstrates that Nlrp6 should be a sensor for RIG-1/MAVS-mediated mitophagy and play a critical role in maintain mitochondrial homeostasis of hippocampal NSPCs. These results suggests that Nlrp6 should be a potential drug target to combat neurodegenerative diseases relative with chronic stress.

Hydrogen gas inhalation ameliorates LPS-induced BPD by inhibiting inflammation via regulating the TLR4–NFκB–IL6/NLRP3 signaling pathway in the placenta

IntroductionHydrogen (H2) is regarded as a novel therapeutic agent against several diseases owing to its inherent biosafety. Bronchopulmonary dysplasia (BPD) has been widely considered among adverse pregnancy outcomes, without effective treatment. Placenta plays a role in defense, synthesis, and immunity, which provides a new perspective for the treatment of BPD. This study aimed to investigate if H2 reduced the placental inflammation to protect the neonatal rat against BPD damage and potential mechanisms.MethodsWe induced neonatal BPD model by injecting lipopolysaccharide (LPS, 1 µg) into the amniotic fluid at embryonic day 16.5 as LPS group. LPS + H2 group inhaled 42% H2 gas (4 h/day) until the samples were collected. We primarily analyzed the neonatal outcomes and then compared inflammatory levels from the control group (CON), LPS group and LPS + H2 group. HE staining was performed to evaluate inflammatory levels. RNA sequencing revealed dominant differentially expressed genes. Bioinformatics analysis (GO and KEGG) of RNA-seq was applied to mine the signaling pathways involved in protective effect of H2 on the development of LPS-induced BPD. We further used qRT-PCR, Western blot and ELISA methods to verify differential expression of mRNA and proteins. Moreover, we verified the correlation between the upstream signaling pathways and the downstream targets in LPS-induced BPD model.ResultsUpon administration of H2, the inflammatory infiltration degree of the LPS-induced placenta was reduced, and infiltration significantly narrowed. Hydrogen normalized LPS-induced perturbed lung development and reduced the death ratio of the fetus and neonate. RNA-seq results revealed the importance of inflammatory response biological processes and Toll-like receptor signaling pathway in protective effect of hydrogen on BPD. The over-activated upstream signals [Toll-like receptor 4 (TLR4), nuclear factor kappa-B p65 (NF-κB p65), Caspase1 (Casp1) and NLR family pyrin domain containing 3 (NLRP3) inflammasome] in LPS placenta were attenuated by H2 inhalation. The downstream targets, inflammatory cytokines/chemokines [interleukin (IL)-6, IL-18, IL-1β, C–C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 1 (CXCL1)], were decreased both in mRNA and protein levels by H2 inhalation in LPS-induced placentas to rescue them from BPD. Correlation analysis displayed a positive association of TLR4-mediated signaling pathway both proinflammatory cytokines and chemokines in placenta.ConclusionH2 inhalation ameliorates LPS-induced BPD by inhibiting excessive inflammatory cytokines and chemokines via the TLR4–NFκB–IL6/NLRP3 signaling pathway in placenta and may be a potential therapeutic strategy for BPD.

Exosomal HMGB3 released by glioma cells confers the activation of NLRP3 inflammasome and pyroptosis in tumor-associated macrophages

BackgroundPrevious evidences has highlighted the pivotal role of NOD-like receptor family pyrin domain-containing 3 (NLRP3)-mediated inflammasomes and pyroptosis activation in driving tumor malignancy and shaping the tumor microenvironment. Herein, we aimed to elucidate the impact of high-mobility group box 3 (HMGB3) released in glioma-derived exosomes on macrophage infiltration in gliomas, NLRP3 inflammasome activation and polarization. MethodsTranscripts and protein levels of HMGB3, and cytokines associated with macrophage phenotypes and pyroptosis were assessed in glioma tissues and cell lines (U251, LN229, T98G, A172) using qRT-PCR and/or Western blot analysis. Exosomes secreted from LN229 and NHA cells were isolated via differential ultracentrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and analysis of exosome-related markers. PKH67 staining was employed to examine exosomes uptake by THP-1 differentiated macrophages. Flow cytometry was utilized to assess macrophage pyroptotic rates and polarization-related markers. ResultsHMGB3 expression was elevated in glioma tissues, serum samples and tumor cell lines. Kaplan-Meier curves revealed a positive correlation between higher HMGB3 expression and poor overall survival and recurrence-free survival. Moreover, glioma tissues with increased HMGB3 expression exhibited significant upregulation of M2 macrophages markers (CD68, CD206, Arg1) and NLRP3 inflammasome components (NLRP3, IL-1β, ASC), suggesting that HMGB3 was closely associated with macrophage infiltration and NLRP3 inflammasome activation. Notably, HMGB3 was found to be enriched in glioma cell- secreted exosomes and could be internalized by macrophages. Knockdown of HMGB3 in glioma cell exosomes could restrain M2 macrophage polarization, NLRP3 inflammasome activation and pyroptosis. ConclusionThese findings suggested that glioma cells secreted exosomal HMGB3 could facilitate macrophage M2 polarization, pyroptosis and inflammatory infiltration, indicating HMGB3 might be a poor prognosis factor for glioma.

Protective effects of paeonol against cognitive impairment in lung diseases

Pulmonary inflammation may lead to neuroinflammation resulting in neurological dysfunction, and it is associated with a variety of acute and chronic lung diseases. Paeonol is a herbal phenolic compound with anti-inflammatory and anti-oxidative properties. The aim of this study is to understand the beneficial effects of paeonol on cognitive impairment, pulmonary inflammation and its underlying mechanisms. Pulmonary inflammation-associated cognitive deficit was observed in TNFα-stimulated mice, and paeonol mitigated the cognitive impairment by reducing the expressions of interleukin (IL)-1β, IL-6, and NOD-like receptor family pyrin domain-containing 3 (NLRP3) in hippocampus. Moreover, elevated plasma miR-34c-5p in lung-inflamed mice was also reduced by paeonol. Pulmonary inflammation induced by intratracheal instillation of TNFα in mice resulted in immune cells infiltration in bronchoalveolar lavage fluid, pulmonary edema, and acute fibrosis, and these inflammatory responses were alleviated by paeonol orally. In MH-S alveolar macrophages, tumor necrosis factor (TNF) α- and phorbol myristate acetate (PMA)-induced inflammasome activation was ameliorated by paeonol. In addition, the expressions of antioxidants were elevated by paeonol, and reactive oxygen species production was reduced. In this study, paeonol demonstrates protective effects against cognitive deficits and pulmonary inflammation by exerting anti-inflammatory and anti-oxidative properties, suggesting a powerful benefit as a potential therapeutic agent.

Honokiol Exhibits Anti-NLRP3 Inflammasome and Antimicrobial Properties in Neisseria gonorrhoeae-Infected Macrophages.

The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, crucial in infectious and inflammatory diseases by regulating IL-1β, presents a target for disease management. Neisseria gonorrhoeae causes gonorrhea in over 87 million people annually, with previous research revealing NLRP3 inflammasome activation in infected macrophages. No natural products have been reported to counteract this activation. Exploring honokiol, a phenolic compound from Chinese herbal medicine, we investigated its impact on NLRP3 inflammasome activation in N. gonorrhoeae-infected macrophages. Honokiol's impact on the protein expression of pro-inflammatory mediators was analyzed using ELISA and Western blotting. The generation of intracellular H2O2 and mitochondrial reactive oxygen species (ROS) was detected through specific fluorescent probes (CM-H2DCFDA and MitoSOX, respectively) and analyzed by flow cytometry. Mitochondrial membrane integrity was assessed using specific fluorescent probes (MitoTracker and DiOC2(3)) and analyzed by flow cytometry. Additionally, the effect of honokiol on the viability of N. gonorrhoeae was examined through an in vitro colony-forming units assay. Honokiol effectively inhibits caspase-1, caspase-11 and GSDMD activation and reduces the extracellular release of IL-1β, NLRP3, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in N. gonorrhoeae-infected macrophages. Detailed investigations have demonstrated that honokiol lowers the production of H2O2 and the phosphorylation of ERK1/2 in N. gonorrhoeae-infected macrophages. Importantly, the phosphorylation of JNK1/2 and p38 and the activation of NF-κB remain unaffected. Moreover, honokiol reduces the N. gonorrhoeae-mediated generation of reactive oxygen species within the mitochondria, preserving their integrity. Additionally, honokiol suppresses the expression of the pro-inflammatory mediator IL-6 and inducible nitric oxide synthase induced by N. gonorrhoeae independently of NLRP3. Impressively, honokiol exhibits in vitro anti-gonococcal activity against N. gonorrhoeae. Honokiol inhibits the NLRP3 inflammasome in N. gonorrhoeae-infected macrophages and holds great promise for further development as an active ingredient in the prevention and treatment of symptoms associated with gonorrhea.

Puerarin mitigated LPS-ATP or HG-primed endothelial cells damage and diabetes-associated cardiovascular disease via ROS-NLRP3 signalling.

The occurrence and development of diabetic vascular diseases are closely linked to inflammation-induced endothelial dysfunction. Puerarin (Pue), the primary component of Pueraria lobata, possesses potent anti-inflammatory properties. However, its vasoprotective role remains elusive. Therefore, we investigated whether Pue can effectively protect against vascular damage induced by diabetes. In the study, Pue ameliorated lipopolysaccharide-adenosine triphosphate (LPS-ATP) or HG-primed cytotoxicity and apoptosis, while inhibited reactive oxygen species (ROS)-mediated NLR family pyrin domain containing 3 (NLRP3) inflammasome in HUVECs, as evidenced by significantly decreased ROS level, NOX4, Caspase-1 activity and expression of NLRP3, GSDMD, cleaved caspase-1, IL-1β and IL-18. Meanwhile, ROS inducer CoCI2 efficiently weakened the effects of Pue against LPS-ATP-primed pyroptosis. In addition, NLRP3 knockdown notably enhanced Pue's ability to suppress pyroptosis in LPS-ATP-primed HUVECs, whereas overexpression of NLRP3 reversed the inhibitory effects of Pue. Furthermore, Pue inhibited the expression of ROS and NLRP3 inflammasome-associated proteins on the aorta in type 2 diabetes mellitus rats. Our findings indicated that Pue might ameliorate LPS-ATP or HG-primed damage in HUVECs by inactivating the ROS-NLRP3 signalling pathway.

FGF21 overexpression alleviates VSMC senescence in diabetic mice by modulating the SYK-NLRP3 inflammasome-PPARγ-catalase pathway.

Diabetes accelerates vascular senescence, which is the basis for atherosclerosis and stiffness. The activation of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and oxidative stress are closely associated with progressive senescence in vascular smooth muscle cells (VSMCs). The vascular protective effect of FGF21 has gradually gained increasing attention, but its role in diabetes-induced vascular senescence needs further investigation. In this study, diabetic mice and primary VSMCs are transfected with an FGF21 activation plasmid and treated with a peroxisome proliferator-activated receptor γ (PPARγ) agonist (rosiglitazone), an NLRP3 inhibitor (MCC950), and a spleen tyrosine kinase (SYK)-specific inhibitor, R406, to detect senescence-associated markers. We find that FGF21 overexpression significantly restores the level of catalase (CAT), vascular relaxation, inhibits the intensity of ROSgreen fluorescence and p21 immunofluorescence, and reduces the area of SA-β-gal staining and collagen deposition in the aortas of diabetic mice. FGF21 overexpression restores CAT, inhibits the expression of p21, and limits the area of SA-β-gal staining in VSMCs under high glucose conditions. Mechanistically, FGF21 inhibits SYK phosphorylation, the production of the NLRP3 dimer, the expression of NLRP3, and the colocalization of NLRP3 with PYCARD (ASC), as well as NLRP3 with caspase-1, to reverse the cleavage of PPARγ, preserve CAT levels, suppress ROSgreen density, and reduce the expression of p21 in VSMCs under high glucose conditions. Our results suggest that FGF21 alleviates vascular senescence by regulating the SYK-NLRP3 inflammasome-PPARγ-catalase pathway in diabetic mice.

Sex- Specific Effects of Chronic Unpredictable Stress on Renal Mitochondrial Function in C57BL/6 Mice

Background: The kidneys play a central role in long term control of blood pressure. Chronic psychological stress has been linked to hypertension, inflammation, and is associated with both an increased prevalence and progression of renal disease. However, the molecular and cellular mechanisms by which chronic stress alters renal function are not fully understood, and data on sex specific differences in renal and blood pressure responses to stress are limited. The impact of stress to cause local inflammation and mitochondrial dysfunction in the kidneys is not clear and represents a potential mechanism leading to renal dysfunction and hypertension. The chronic unpredictable stress (CUS) model induces psychological stress in rodents thus allowing the study of integrated physiological changes to the kidney in response to stress. Hypothesis: Exposing mice to CUS will lead to increased blood pressure that is associated with decreased renal mitochondrial function and renal inflammation. Methods: To investigate the link between stress, renal mitochondrial function, inflammation, and blood pressure, we exposed adult male and female C57BL/6 mice to CUS for 28 consecutive days. Blood pressure was measured via tail cuff on the 28th day of stress. 24 hours later, mice were euthanized and estrous cycle phase in females was determined by vaginal cytology. Kidney mitochondrial oxygen consumption was assessed using the Oroboros respirometer on the pole ends of the left kidneys. NLR family pyrin domain containing 3 (NLRP3) expression was measured in the right kidney using Real-Time TaqMan PCR. Results: Mean arterial pressure was elevated in both stressed males (in mmHg, 106±4 vs 87±4, n<11, p<0.01) and females (131± 6 vs 94±11 n<4, p<0.01) compared to non-stressed mice. While maximal electron transport capacity (ETS), a measure of mitochondrial function, was unchanged in CUS male kidneys, stressed females in the receptive (estrus and proestrus) phase of the estrous cycle exhibited a significant decrease in renal ETS compared to non-stressed females (in pmol O2/mg protein, 14.4±0.9 vs 11.4±0.7, n=11, p<0.05). Renal mRNA expression of NLRP3 was increased in CUS males (35%± 7.7, n=8, p<0.01) but unchanged in CUS females. Conclusion: These data suggest potential mechanistic differences in stress-induced hypertension in renal mitochondria (decreased ETS in stressed females) and inflammasome level (increased NLRP3 in stressed males) between male and female mice. This work was supported by P20GM109091 (F.H.), R01HL130972-01A1, R01HL5949, BX000168-10A1, BX005320 (F.G.S.), R01DK132948 (C.W. & F.P.) R01 MH129798 (SKW); VA VISN7 RDA (F.H.),, 1U54HL169191-01 & BX002604 (M.J.R.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the abstract. This is the full abstract presented at the American Physiology Summit 2024 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.

Caspase-8 promotes NLRP3 inflammasome activation mediates eye development defects in zebrafish larvae exposed to perfulorooctane sulfonate (PFOS)

Epidemiological evidence showed that serum high perfluorooctane sulfonate (PFOS) levels are associated with multiple eye related diseases, but the potential underlying molecular mechanisms remain poorly understood. Zebrafish and photoreceptor cell (661w) models were used to investigate the molecular mechanism of PFOS induced eye development defects. Our results showed a novel molecular mechanism of PFOS-induced inflammation response-mediated photoreceptor cell death associated with eye development defects. Inhibition of Caspase-8 activation significantly decreased photoreceptor cell death in PFOS exposure. Mechanistically, Toll-like receptor 4 (TLR4) mediates activation of Caspase-8 promote activation of NLR family pyrin domain-containing 3 (NLRP3) inflammasome to elicit maturation of interleukin-1 beta (IL-1β) via Caspase-1 activation, facilitating photoreceptor cell inflammation damage in PFOS exposure. In addition, we also made a novel finding that Caspase-3 activation was increased via Caspase-8 activation and directly intensified cell death. Our results show the important role of Caspase-8 activation in PFOS induced eye development defects and highlight Caspase-8 mediated activation of the NLRP3 inflammation triggers activation of Caspase-1 and promote the maturation of IL-1β in retinal inflammatory injury.

Cinnamaldehyde, A Bioactive Compound from the Leaves of Cinnamomum osmophloeum Kaneh, Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice by Inhibiting the NLRP3 Inflammasome.

Inflammatory bowel disease (IBD) comprises a group of idiopathic intestinal disorders, including ulcerative colitis and Crohn's disease, significantly impacting the quality of life for affected individuals. The effective management of these conditions remains a persistent challenge. The NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, a complex molecular structure, regulates the production of pro-inflammatory cytokines such as interleukin-1β. Abnormal activation of the NLRP3 inflammasome plays a pivotal role in the development of IBD, making it a compelling target for therapeutic intervention. Our research revealed that cinnamaldehyde (CA), a major bioactive compound found in the leaves of Cinnamomum osmophloeum kaneh, demonstrated a remarkable ability to alleviate colitis induced by dextran sulfate sodium (DSS) in a mouse model. This effect was attributed to CA's ability to downregulate the activation of the NLRP3 inflammasome and reduce the expression of pro-inflammatory mediators in the colon. In the mechanism study, we observed that CA inhibited the NLRP3 inflammasome in macrophages, at least partially, by enhancing the autophagic response, without reducing mitochondrial damage. These findings collectively suggest that CA holds significant potential as a therapeutic agent for enhancing the management of IBD, offering a promising avenue for further research and development.

Melatonin suppresses TLR4-mediated RSV infection in the central nervous cells by inhibiting NLRP3 inflammasome formation and autophagy.

Respiratory syncytial virus (RSV) infects neuronal cells in the central nervous system (CNS), resulting in neurological symptoms. In the present study, we intended to explore the mechanism of RSV infection-induced neuroinflammatory injury from the perspective of the immune response and sought to identify effective protective measures against the injury. The findings showed that toll-like receptor 4 (TLR4) was activated after RSV infection in human neuronal SY5Y cells. Furthermore, TLR4 activation induced autophagy and apoptosis in neuronal cells, promoted the formation of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, and increased the secretion of downstream inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-18 (IL-18) and tumour necrosis factor-α (TNF-α). Interestingly, blockade of TLR4 or treatment with exogenous melatonin significantly suppressed TLR4 activation as well as TLR4-mediated apoptosis, autophagy and immune responses. Therefore, we infer that melatonin may act on the TLR4 to ameliorate RSV-induced neuronal injury, which provides a new therapeutic target for RSV infection.

Ganoderma lucidum spore extract improves sleep disturbances in a rat model of sporadic Alzheimer's disease.

Introduction: Ganoderma lucidum (G. lucidum, Lingzhi) has long been listed as a premium tonic that can be used to improve restlessness, insomnia, and forgetfulness. We previously reported that a rat model of sporadic Alzheimer's disease (sAD) that was induced by an intracerebroventricular injection of streptozotocin (ICV-STZ) showed significant learning and cognitive deficits and sleep disturbances. Treatment with a G. lucidum spore extract with the sporoderm removed (RGLS) prevented learning and memory impairments in sAD model rats. Method: The present study was conducted to further elucidate the preventive action of RGLS on sleep disturbances in sAD rats by EEG analysis, immunofluorescence staining, HPLC-MS/MS and Western blot. Results: Treatment with 720mg/kg RGLS for 14days significantly improved the reduction of total sleep time, rapid eye movement (REM) sleep time, and non-REM sleep time in sAD rats. The novelty recognition experiment further confirmed that RGLS prevented cognitive impairments in sAD rats. We also found that RGLS inhibited the nuclear factor-κB (NF-κB)/Nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammatory pathway in the medial prefrontal cortex (mPFC) in sAD rats and ameliorated the lower activity of γ-aminobutyric acid (GABA)-ergic neurons in the parabrachial nucleus (PBN). Discussion: These results suggest that inhibiting the neuroinflammatory response in the mPFC may be a mechanism by which RGLS improves cognitive impairment. Additionally, improvements in PBN-GABAergic activity and the suppression of neuroinflammation in the mPFC in sAD rats might be a critical pathway to explain the preventive effects of RGLS on sleep disturbances in sAD.