TLR4-NLRP3 Inflammasome Research Articles

BMP-7 attenuates TLR4-NLRP3 inflammasome mediated pyroptosis in vascular smooth muscle cells in atherosclerotic plaques

BMP-7 attenuates TLR4-NLRP3 inflammasome mediated pyroptosis in vascular smooth muscle cells in atherosclerotic plaques

Anti-hyperuricemia effect of hesperetin is mediated by inhibiting the activity of xanthine oxidase and promoting excretion of uric acid

Hesperetin is a natural flavonoid with many biological activities. In view of hyperuricemia treatment, the effects of hesperetin in vivo and in vitro, and the underlying mechanisms, were explored. Hyperuricemia models induced by yeast extract (YE) or potassium oxonate (PO) in mice were created, as were models based on hypoxanthine and xanthine oxidase (XOD) in L-O2 cells and sodium urate in HEK293T cells. Serum level of uric acid (UA), creatinine (CRE), and urea nitrogen (BUN) were reduced significantly after hesperetin treatment in vivo. Hesperetin provided hepatoprotective effects and inhibited xanthine oxidase activity markedly, altered the level of malondialdehyde (MDA), glutathione peroxidase (GSH-PX) and catalase (CAT), downregulated the XOD protein expression, toll-like receptor (TLR)4, nucleotide binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, interleukin-18 (IL-18), upregulated forkhead box O3a (FOXO3a), manganese superoxide dismutase (MnSOD) in a uric acid-synthesis model in mice. Protein expression of organic anion transporter 1 (OAT1), OAT3, organic cationic transporter 1 (OCT1), and OCT2 was upregulated by hesperetin intervention in a uric acid excretion model in mice. Our results proposal that hesperetin exerts a uric acid-lowering effect through inhibiting xanthine oxidase activity and protein expression, intervening in the TLR4-NLRP3 inflammasome signaling pathway, and up-regulating expression of FOXO3a, MnSOD, OAT1, OAT3, OCT1, and OCT2 proteins. Thus, hesperetin could be a promising therapeutic agent against hyperuricemia.

Isoflurane Attenuates Cerebral Ischaemia-Reperfusion Injury via the TLR4-NLRP3 Signalling Pathway in Diabetic Mice.

Ischaemic stroke is a severe disease worldwide. Restoration of blood flow after ischaemic stroke leads to cerebral ischaemia–reperfusion injury (CIRI). Various operations, such as cardiac surgery with deep hypothermic circulatory arrest, predictably cause cerebral ischaemia. Diabetes is related to the occurrence of perioperative stroke and exacerbates neurological impairment after stroke. Therefore, the choice of anaesthetic drugs has certain clinical significance for patients with diabetes. Isoflurane (ISO) exerts neuroprotective and anti-neuroinflammatory effects in patients without diabetes. However, the role of ISO in cerebral ischaemia in the context of diabetes is still unknown. Toll-like receptor 4 (TLR4) and NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome activation play important roles in microglia-mediated neuroinflammatory injury. In this study, we treated a diabetic middle cerebral artery occlusion mouse model with ISO. We found that diabetes exacerbated cerebral ischaemia damage and that ISO exerted neuroprotective effects in diabetic mice. Then, we found that ISO decreased TLR4-NLRP3 inflammasome activation in microglia and the excessive autophagy induced by CIRI in diabetic mice. The TLR4-specific agonist CRX-527 reversed the neuroprotective effects of ISO. In summary, our study indicated that ISO exerts neuroprotective effects against the neuroinflammation and autophagy observed during diabetic stroke via the TLR4-NLRP3 signalling pathway.

BMP-7 Attenuates Inflammation-Induced Pyroptosis and Improves Cardiac Repair in Diabetic Cardiomyopathy.

In the present study, we investigated a novel signaling target in diabetic cardiomyopathy where inflammation induces caspase-1-dependent cell death, pyroptosis, involving Nek7-GBP5 activators to activate the NLRP3 inflammasome, destabilizes cardiac structure and neovascularization. Furthermore, we explored the therapeutic ability of bone morphogenetic protein-7 (BMP-7) to attenuate these adverse effects. C57BL/6J mice (n = 16 mice/group) were divided into: control (200 mg/kg, 0.9% saline intraperitoneal injection, i.p.); Streptozotocin (STZ) and STZ-BMP-7 groups (STZ, 200 mg/kg, i.p. injection). After 6 weeks, heart function was examined with echocardiography, and mice were sacrificed. Immunostaining, Western blotting, H&E, and Masson’s trichrome staining was performed on heart tissues. STZ-induced diabetic cardiomyopathy significantly increased inflammasome formation (TLR4, NLRP3, Nek7, and GBP5), pyroptosis markers (caspase-1, IL-1β, and IL-18), inflammatory cytokines (IL-6 and TNF-α), MMP9, and infiltration of monocytes (CD14), macrophage (iNOS), and dendritic cells (CD11b and CD11c) (p < 0.05). Moreover, a significant endothelial progenitor cells (EPCs) dysfunction (c-Kit/FLk-1, CD31), adverse cardiac remodeling, and reduction in left ventricular (LV) heart function were observed in STZ versus control (p < 0.05). Treatment with BMP-7 significantly reduced inflammasome formation, pyroptosis, and inflammatory cytokines and infiltrated inflammatory cells. In addition, BMP-7 treatment enhanced EPC markers and neovascularization and subsequently improved cardiac remodeling in a diabetic heart. Moreover, a significant improvement in LV heart function was achieved after BMP-7 administration relative to diabetic mice (p < 0.05). In conclusion, BMP-7 attenuated inflammation-induced pyroptosis, adverse cardiac remodeling, and improved heart function via the TLR4-NLRP3 inflammasome complex activated by novel signaling Nek7/GBP5. Our BMP-7 pre-clinical studies of mice could have significant potential as a future therapy for diabetic patients.

Isoorientin exerts a urate-lowering effect through inhibition of xanthine oxidase and regulation of the TLR4-NLRP3 inflammasome signaling pathway.

Isoorientin (ISO), a natural flavonoid compound, has been identified in several plants and its biological activity is determined and the study on lowering uric acid has not been reported. In view of the current status of treatment of hyperuricemia, we evaluated the hypouricemic effects of ISO in vivo and in vitro, and explored the underlying mechanisms. Yeast extract-induced hyperuricemia animal model as well as hypoxanthine and xanthine oxidase (XOD) co-induced high uric acid L-O2 cell model and enzymatic experiments in vitro were selected. The XOD activity and uric acid (UA) level were inhibited after the treatment of ISO in vitro and in vivo. Furthermore, serum creatinine (CRE) and blood urea nitrogen (BUN) levels were also significantly reduced and liver damage was recovered in pathological histology after the ISO administration in hyperuricemia animal model. The results of mechanism illustrated that protein expressions such as XOD, toll-like receptor 4 (TLR4), cathepsin B (CTSB), NLRP3, and its downstream caspase-1 as well as interleukin-18 (IL-18) were markedly downregulated by ISO intervention in vitro and in vivo. Our results suggest that ISO exerts a urate-lowering effect through inhibiting XOD activity and regulating TLR4-NLRP3 inflammasome signal pathway, thus representing a promising candidate therapeutic agent for hyperuricemia. Both animal models and in vitro experiments suggested that ISO may effectively lower uric acid produce. The mechanism might be the inhibition of XOD activity and NLRP3 inflammasome of upregulation.

Procyanidins exhibits neuroprotective activities against cerebral ischemia reperfusion injury by inhibiting TLR4-NLRP3 inflammasome signal pathway.

Ischemic stroke is a serious cardiovascular disease with high morbidity and mortality rates that affects millions of people worldwide.Currently, the only therapy with proven efficacy for acute ischemic stroke is alteplase, however, it still has many shortcomings and limitations. Therefore,we screen new compounds from traditional Chinese medicine to explore their efficacy against ischemic reperfusion injury. Procyanidins, a natural productextracted from grapes seed, which have been shown can ameliorate cerebral ischemic injury. However, the underlying mechanism is still not very clear. Theaim of this study was to investigate the effect of procyanidins on middle cerebral artery occlusion/reperfusion (MCAO/R)-mediated cerebral ischemic injuryand its underlying possible mechanisms. SD rats were used to evaluate the effect of procyanidins on MCAO/R induced cerebral ischemic injury in vivo. Histological analysis was used toassess neuronal apoptosis. Cell signaling was assayed by Western blot. In this study, we found that procyanidins can significantly ameliorate the middle cerebral artery occlusion/reperfusion (MCAO/R)-mediatedneurological deficits, and relieved brain edema, cerebral infarction volume, histopathological damage and apoptosis in rats. In addition, procyanidins canalso markedly inhibit MCAO/R and oxygen-glucose deprivation/reoxygenation (OGD/R)-mediated activation of TLR4-p38-NF-κB-NLRP3 signalingpathway in vitro and in vivo. Moreover, procyanidins can inhibit MCAO/R and OGD/R-induced the production of inflammatory cytokines such asinterleukin-1β (IL-1β) in vitro and in vivo. Besides, treatment with TLR4 inhibitor (Cli-095) in BV2 cell also shows the same effect. Altogether, these data suggested that procyanidins exerted a potential neuroprotective effect may by inhibit the TLR4-p38-NF-κB-NLRP3signaling pathway in the brain in MCAO/R rats.

Tumor Cell-Derived IL1β Promotes Desmoplasia and Immune Suppression in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignancy typified by a highly stromal and weakly immunogenic tumor microenvironment that promotes tumor evolution and contributes to therapeutic resistance. Here, we demonstrate that PDA tumor cell-derived proinflammatory cytokine IL1β is essential for the establishment of the protumorigenic PDA microenvironment. Tumor cell-derived IL1β promoted the activation and secretory phenotype of quiescent pancreatic stellate cells and established an immunosuppressive milieu mediated by M2 macrophages, myeloid-derived suppressor cells, CD1dhiCD5+ regulatory B cells, and Th17 cells. Loss of tumor cell-derived IL1 signaling in tumor stroma enabled intratumoral infiltration and activation of CD8+ cytotoxic T cells, attenuated growth of pancreatic neoplasia, and conferred survival advantage to PDA-bearing mice. Accordingly, antibody-mediated neutralization of IL1β significantly enhanced the antitumor activity of α-PD-1 and was accompanied by increased tumor infiltration of CD8+ T cells. Tumor cell expression of IL1β in vivo was driven by microbial-dependent activation of toll-like receptor 4 (TLR4) signaling and subsequent engagement of the NLRP3 inflammasome. Collectively, these findings identify a hitherto unappreciated role for tumor cell-derived IL1β in orchestrating an immune-modulatory program that supports pancreatic tumorigenesis. SIGNIFICANCE: These findings identify a new modality for immune evasion in PDA that depends on IL1β production by tumor cells through TLR4-NLRP3 inflammasome activation. Targeting this axis might provide an effective PDA therapeutic strategy.

Human and Pathogen Derived Ndpks Act As Novel Damps and PAMPs to Drive Leukemia Cell Survival and Progression through Signaling Via the TLR4-Mediated Alternative NLRP3 Inflammasome Pathway

Elevated plasma levels of the nucleoside diphosphate kinase (NDPK) NM23-H1 are associated with poorer prognosis in acute myeloid leukemia (AML). We previously demonstrated that leukemic blasts release NM23-H1, which binds to more differentiated myeloid cells inducing their secretion of inflammatory cytokines, including IL-1β, that promote survival and proliferation of leukemic blasts1. Both AML and myelodysplastic syndrome (MDS) patients are prone to infections due to impaired hematopoiesis that is worsened by treatment. NDPKs are highly evolutionarily conserved raising the possibility that bacterial/fungal NDPKs could mediate the same survival effect on malignant AML/MDS blasts and exacerbate disease progression. To test this, we generated recombinant NDPKs (rNDPKs) from bacteria and fungi associated with common infections in these patients (E. coli, S. aureus, S. pneumoniae, K. pneumoniae, C. albicans). Cytokine production and survival responses of primary AMLs to these proteins were indistinguishable from their response to rNM23-H1. This activity was independent of NDPK enzyme activity since mutant rNM23-H1 and bacterial and fungal rNDPKs with impaired oligomerization, kinase or exonuclease activity elicited the same cytokine and survival response. Toll like receptors (TLRs) are the major family of human DAMP/PAMP receptors and IL-1β secretion is closely associated with TLR-4 mediated activation of the NLRP3 inflammasome in monocytes. We therefore postulated that NM23-H1 and pathogen derived NDPKs act as novel damage- and pathogen- associated molecular pattern (DAMP, PAMP) molecules. We confirmed that fluorescently labelled rNM23-H1 and S. pneumoniae rNDPK bound selectively to monocytes in peripheral blood. Using in vitro generated monocytes (vitamin D3 differentiated THP-1 cells) we demonstrated that both wild type and mutant rNM23-H1 and bacterial/fungal rNDPKs induced activation of caspase-1 and cleavage of pro-IL-1β into its active form. Secretion of IL-1β was inhibited by antagonists/inhibitors of TLR4, NLRP3 and caspase-1 indicating the involvement of the TLR4-NLRP3 inflammasome axis is mediating the NDPK response. Unlike the canonical NLRP3-inflammasome pathway that leads to monocyte cell death by pyroptosis, rNM23-H1 and rNDPKs did not lead to cell death indicating that rNDPKs are responsible for the activation of the alternative inflammasome. In our earlier studies, and those of others, we demonstrated that not all AML primary samples responsed to NM23-H1 in vitro. We have observed that non responders to NM23-H1 also do not respond to pathogen derived rNDPKs. In contrast, we have observed uniform responses in terms of cytokine release in all normal peripheral blood. We hence hypothesized that the non-rNDPK-responding AML samples may reflect the absence of monocytes in culture. To test this, we generated conditioned media using normal donor leukocytes, in presence or absence of a TLR-4 antagonist to inhibit the IL-1β production. The conditioned media was then used to culture primary AML samples, in parallel with rNDPK in unconditioned media. All the samples analyzed showed increased survival in rNDPK conditioned media even whilst some did not respond to rNDPK in unconditioned media. In summary, our data demonstrate for the first time that NM23-H1 and bacterial/fungal NDPKs are novel DAMPs/PAMPS that signal via TLR4 in monocytes. We further demonstrate that this interaction results in activation of the alternative NLRP3 inflammasome and subsequent cleavage and secretion of IL-1β without death by pyroptosis. Our data showing that bacterial/fungal NDPKs can promote survival of AML blasts indicates that rather than just being a consequence of AML associated immunosuppression, infections may drive the progression and AML. These findings have important implications in the clinical management of AML and its precursor myelodysplastic syndromes (MDS). Lilly AJ, et al.Cancer Res. 2011;71(3):1177-86.Gaidt MM, et al. Immunity. 2016;44(4):833-46 Disclosures Drayson: Abingdon Health: Consultancy, Equity Ownership.

Tetramethylpyrazine ameliorates depression by inhibiting TLR4-NLRP3 inflammasome signal pathway in mice.

Depression is a common but serious mental illness; meanwhile, it is also an inflammatory disorder. Toll-like receptor 4 (TLR4), as the pattern recognition receptor, has been shown to play a vital role in neuroinflammation. The nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome acts as an important signaling molecule downstream of TLR4 and can promote the maturation of inflammatory cytokines, such as interleukin-1β (IL-1β). Tetramethylpyrazine (TMP) is a natural compound with neuroprotective effects but with unknown mechanisms on its antidepressant-like effect. In this study, we hypothesized that TMP ameliorates depression may be through the inhibition of the TLR4-NF-κB-NLRP3 signal pathway. Our results have shown that chronic unpredictable mild stress (CUMS) that induced the decreased sucrose preference and increased immobile time was prominently reversed by TMP and fluoxetine. Additionally, we also found that CUMS induced the upregulation of proinflammatory cytokines; TLR4 and NLRP3-associated proteins were significantly suppressed by TMP in the prefrontal cortex and hippocampus. TMP also exhibited potent antioxidant effects and increased the monoamine levels in the serum and brain, such as increasing the activity of SOD and GSH-Px, and reducing the activity of MDA in the serum, and elevating the 5-HT and NE concentration in the serum and brain. Moreover, treatment with Cli-095 (TLR4 inhibitor) also markedly inhibited CUMS-induced depression-like behaviors. Taken together, our findings suggested that TMP exerted a potential antidepressant-like effect in CUMS mice, and the molecular mechanisms may relate to inhibit the TLR4-NF-κB-NLRP3 signaling pathway in the brain.

Soluble uric acid increases PDZK1 and ABCG2 expression in human intestinal cell lines via the TLR4-NLRP3 inflammasome and PI3K/Akt signaling pathway

BackgroundIn addition to the kidney, the intestine is one of the most important organs involved in uric acid excretion. However, the mechanism of urate excretion in the intestine remains unclear. Therefore, the relationship between soluble uric acid and the gut excretion in human intestinal cells was explored. The relevant signaling molecules were then also examined.MethodsHT-29 and Caco-2 cell lines were stimulated with soluble uric acid. Western blotting and qRT-PCR were used to measure protein and mRNA levels. Subcellular fractionation methods and immunofluorescence were used to quantify the proteins in different subcellular compartments. Flow cytometry experiments examined the function of ATP-binding cassette transporter, subfamily G, member 2 (ABCG2). Small interfering RNA transfection was used to assess the interaction between ABCG2 and PDZ domain-containing 1 (PDZK1).ResultsSoluble uric acid increased the expression of PDZK1 and ABCG2. The stimulation of soluble uric acid also facilitated the translocation of ABCG2 from the intracellular compartment to the plasma membrane and increased its transport activity. Moreover, the upregulation of PDZK1 and ABCG2 by soluble uric acid was partially decreased by either TLR4-NLRP3 inflammasome inhibitors or PI3K/Akt signaling inhibitors. Furthermore, PDZK1 knockdown significantly inhibited the expression and transport activity of ABCG2 regardless of the activation by soluble uric acid, demonstrating a pivotal role for PDZK1 in the regulation of ABCG2.ConclusionsThese findings suggest that urate upregulates the expression of PDZK1 and ABCG2 for excretion in intestinal cells via activating the TLR4-NLRP3 inflammasome and PI3K/Akt signaling pathway.