Activation Of NLRP3 Inflammasome Pathway Research Articles

Foxo3a-dependent Bim transcription protects mice from a high fat diet via inhibition of activation of the NLRP3 inflammasome by facilitating autophagy flux in Kupffer cells.

BackgroundThe role of Foxo3a in the regulation of autophagy flux and activation of the NLRP3 inflammasome in KCs suffering from HFD conditions is unknown.ResultsUp-regulation of Foxo3a restored autophagy flux and dampened the activation of the NLRP3 inflammasome in KCs stimulated with PA and LPS. In contrast, down-regulation of Foxo3a increased blockage of autophagy flux and promoted NLRP3 inflammasome activation. Additionally, mRNA levels of Bim were significantly changed with the alteration of Foxo3a in KCs under PA and LPS stimulation among foxo3a targeted genes. Overexpression of Bim restored autophagy influx and attenuated NLRP3 inflammasome pathway activation. In addition, autophagy formation was restored, and activation of NLRP3 inflammasome was inhibited in KCs isolated from mice treated with Iturin A and fed with a HFD.Materials and methodsAutophagy flux in KCs and activation levels of NLRP3 inflammasome were evaluated after altering the expression of Foxo3a in KCs before stimulation with PA and LPS. Additionally, various target genes of Foxo3a were measured in KCs pretreated with an agonist (Iturin A) or inhibitor (SC97) of Foxo3a after KCs stimulation with PA and LPS in order to hunt for targets of Foxo3a. Activation levels of NLRP3 inflammasome in isolated KCs, as well as autophagy flux, were measured after mice were treated with Iturin A and fed with a HFD for 16 weeks.ConclusionsFoxo3a restores autophagy flux and attenuates the activation of the NLRP3 inflammasome by promoting the transcription of Bim, suggesting a potential therapeutic target in NAFLD and other obesity-related diseases.

Differences in Inflammatory Response Induced by Two Representatives of Clades of the Pandemic ST258 Klebsiella pneumoniae Clonal Lineage Producing KPC-Type Carbapenemases.

ST258-K. pneumoniae (ST258-KP) strains, the most widespread multidrug-resistant hospital-acquired pathogens, belong to at least two clades differing in a 215 Kb genomic region that includes the cluster of capsule genes. To investigate the effects of the different capsular phenotype on host-pathogen interactions, we studied representatives of ST258-KP clades, KKBO-1 and KK207-1, for their ability to activate monocytes and myeloid dendritic cells from human immune competent hosts. The two ST258-KP strains strongly induced the production of inflammatory cytokines. Significant differences between the strains were found in their ability to induce the production of IL-1β: KK207-1/clade I was much less effective than KKBO-1/clade II in inducing IL-1β production by monocytes and dendritic cells. The activation of NLRP3 inflammasome pathway by live cells and/or purified capsular polysaccharides was studied in monocytes and dendritic cells. We found that glibenclamide, a NLRP3 inhibitor, inhibits more than 90% of the production of mature IL-1β induced by KKBO1 and KK207-1. KK207-1 was always less efficient compared to KKBO-1 in: a) inducing NLRP3 and pro-IL-1β gene and protein expression; b) in inducing caspase-1 activation and pro-IL-1β cleavage. Capsular composition may play a role in the differential inflammatory response induced by the ST258-KP strains since capsular polysaccharides purified from bacterial cells affect NLRP3 and pro-IL-1β gene expression through p38MAPK- and NF-κB-mediated pathways. In each of these functions, capsular polysaccharides from KK207-1 were significantly less efficient compared to those purified from KKBO-1. On the whole, our data suggest that the change in capsular phenotype may help bacterial cells of clade I to partially escape innate immune recognition and IL-1β-mediated inflammation.

Involvement of matrix metalloproteinases (MMPs) and inflammasome pathway in molecular mechanisms of fibrosis.

Fibrosis is a basic connective tissue lesion defined by the increase in the fibrillar extracellular matrix (ECM) components in tissue or organ. Matrix metalloproteinases (MMPs) are a major group of proteases known to regulate the turn-over of ECM and so they are suggested to be important in tissue remodelling observed during fibrogenic process associated with chronic inflammation. Tissue remodelling is the result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of ECM components markedly controlled by the MMPs/TIMP imbalance. We previously showed an association of the differences in collagen deposition in the lungs of bleomycin-treated mice with a reduced molar pro-MMP-9/TIMP-1 ratio. Using the carbon tetrachloride (CCl4) preclinical model of liver fibrosis in mice, we observed a significant increase in collagen deposition with increased expression and release of tissue inhibitors of metalloproteinase (TIMP)-1 both at 24 h and 3 weeks later. This suggests an early altered regulation of matrix turnover involved in the development of fibrosis. We also demonstrated an activation of NLRP3-inflammasome pathway associated with the IL-1R/MyD88 signalling in the development of experimental fibrosis both in lung and liver. This was also associated with an increased expression of purinergic receptors mainly P2X7. Finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of tissue remodelling and fibrosis.

Ligustrazine disrupts lipopolysaccharide-activated NLRP3 inflammasome pathway associated with inhibition of Toll-like receptor 4 in hepatocytes

Intestine microbial products may translocate into the liver via portal vein and trigger or exacerbate hepatocyte inflammatory responses during liver injury. The NLRP3 inflammasome pathway plays a key role in regulation of inflammatory cytokines in response to bacterial products. The present study was aimed to investigate the effects of ligustrazine, a natural alkaloid compound, on the NLRP3 inflammasome pathway activation and interleukin-1β (IL-1β) generation in hepatocytes. We cultured human LO2 hepatocytes and treated them with lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria, for mimicking hepatic exposure to microbial products in vitro. The results demonstrated that LPS upregulated NLRP3 and cleaved-caspase-1, and promoted the expression and secretion of IL-1β in LO2 cells. Ligustrazine was found to reduce NLRP3 and cleaved-caspase-1, prevented IL-1β cleavage, and decreased IL-1β secretion into extracellular environment. Further examinations showed that LPS upregulated the expression of Toll-like receptor 4 (TLR4), but ligustrazine repressed TLR4 expression in LPS-treated hepatocytes. Moreover, pharmacological inhibition of TLR4 by its specific inhibitor TAK-242 downregulated NLRP3 and cleaved-caspase-1, and combination treatment with TAK-242 and ligustrazine led to more significant inhibitory effects on the NLRP3 pathway. TAK-242 also reduced cleaved-IL-1β, and this reducing effect was enhanced by ligustrazine. Collectively, the current results revealed that ligustrazine interrupted LPS-activated NLRP3 inflammasome signaling and reduced generation of IL-1β in hepatocytes, which was associated with inhibition of TLR4. This study uncovered a novel mechanism for ligustrazine as a potential hepatoprotective agent.

NLRP3 Inflammasome Activation in the Brain after Global Cerebral Ischemia and Regulation by 17β-Estradiol.

17β-Estradiol (E2) is a well-known neuroprotective factor in the brain. Recently, our lab demonstrated that the neuroprotective and cognitive effects of E2 require mediation by the estrogen receptor (ER) coregulator protein and proline-, glutamic acid-, and leucine-rich protein 1 (PELP1). In the current study, we examined whether E2, acting via PELP1, can exert anti-inflammatory effects in the ovariectomized rat and mouse hippocampus to regulate NLRP3 inflammasome activation after global cerebral ischemia (GCI). Activation of the NLRP3 inflammasome pathway and expression of its downstream products, cleaved caspase-1 and IL-1β, were robustly increased in the hippocampus after GCI, with peak levels observed at 6-7 days. Expression of P2X7 receptor, an upstream regulator of NLRP3, was also increased after GCI. E2 markedly inhibited NLRP3 inflammasome pathway activation, caspase-1, and proinflammatory cytokine production, as well as P2X7 receptor expression after GCI (at both the mRNA and protein level). Intriguingly, the ability of E2 to exert these anti-inflammatory effects was lost in PELP1 forebrain-specific knockout mice, indicating a key role for PELP1 in E2 anti-inflammatory signaling. Collectively, our study demonstrates that NLRP3 inflammasome activation and proinflammatory cytokine production are markedly increased in the hippocampus after GCI, and that E2 signaling via PELP1 can profoundly inhibit these proinflammatory effects.

IL-1β production is dependent on the activation of purinergic receptors and NLRP3 pathway in human macrophages.

The Nod-like receptor family protein 3 (NLRP3)-inflammasome pathway is known to be activated by danger signals such as monosodium urate (MSU). We investigated the role of P2 purinergic receptors in the activation of NLRP3-inflammasome pathway after MSU treatment of primary human monocyte-derived macrophages (MDMs). After initial stimulation with a low concentration of LPS (0.1 µg/ml), a 6 h treatment with MSU crystals (250, 500, and 1000 µg/ml) induced the MDMs to release IL-1β, IL-1α, and IL-6 in a dose-dependent manner. Moreover, the caspase 1 inhibitor Z-YVAD-FMK and the cathepsin B inhibitor CA-074Me reduced production of IL-1β in a dose-dependent manner after LPS + MSU treatment. We used real-time reverse transcription-quantitative PCR to show that treatment with LPS and MSU (500 µg/ml) induced significantly greater expression of NLRP3 and IL-1β than after treatment with LPS. We also found that MSU treatment induced P2X purinergic receptor 7 (P2X7R) mRNA and protein expression. Furthermore, addition of the P2X7 purinergic receptor antagonist A-740003 significantly impeded IL-1β production and pro-IL-1β cleavage after treatment with LPS + MSU. Remarkably, RNA silencing of P2X7R (but not P2X4R) inhibited the release of IL-1β and other M1 macrophage cytokines (such as IL-1α, IL-6, and TNF-α) from MDMs stimulated with LPS + MSU. Taken as a whole, our results show that P2 purinergic receptors and the NLRP3 inflammasome pathway are involved in the secretion of IL-1β from MSU-stimulated human macrophages. This pathway may constitute a novel therapeutic target for controlling the inflammatory process in several associated pathologies.

Porphyromonas gingivalis-impaired innate immune response via NLRP3 proteolysis in endothelial cells

Porphyromonas gingivalis (Pg) is involved in the link between periodontal diseases and atherosclerosis worsening. In periodontal cells, Pg modifies IL-1β expression via the NLRP3 inflammasome pathway activation. Our aim was to investigate NLRP3 inflammasome activation in endothelial cells (ECs) after Pg infection and Pg-LPS stimulation. In both situations, RT-PCR experiments demonstrated an increase of the NLRP3 mRNA level that can be potentiated by pre-treatment of ECs with 5 mM ATP. However, Western blotting analysis revealed that Pg infection induced a proteolysis of NLRP3 protein and a major decrease of the native protein. After ATP pre-treatment and/or Pg-LPS stimulation, this proteolysis was not observed, while NLRP3 protein levels were increased. Proteolysis of the NLRP3 protein was not observed with heat-killed Pg and inhibition of ECs protein synthesis with cycloheximide did not abolish the NLRP3 protein degradation induced by Pg infection in ATP pre-treated cells. Additionally, significant increases of secreted IL-1β were measured after ATP pre-treatment and/or Pg-LPS stimulation, but not after Pg infection. These data showed that Pg and Pg-LPS differentially controlled the NLRP3 inflammasome pathway in ECs, and suggested a novel potential mechanism developed by Pg to reduce IL-1β secretion and to escape host immune response.