Targeting the NLRP3 Inflammasome to Reduce Diet-Induced Metabolic Abnormalities in Mice.
ABSTRACT: Although the molecular links underlying the causative relationship between chronic low-grade inflammation and insulin resistance are not completely understood, compelling evidence suggests a pivotal role of the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) inflammasome. Here we tested the hypothesis that either a selective pharmacological inhibition or a genetic downregulation of the NLRP3 inflammasome results in reduction of the diet-induced metabolic alterations. Male C57/BL6 wild-type mice and NLRP3-/- littermates were fed control diet or high-fat, high-fructose diet (HD). A subgroup of HD-fed wild-type mice was treated with the NLRP3 inflammasome inhibitor BAY 11-7082 (3 mg/kg intraperitoneally [IP]). HD feeding increased plasma and hepatic lipids and impaired glucose homeostasis and renal function. Renal and hepatic injury was associated with robust increases in profibrogenic markers, while only minimal fibrosis was recorded. None of these metabolic abnormalities were detected in HD-fed NLRP3-/- mice, and they were dramatically reduced in HD-mice treated with the NLRP3 inflammasome inhibitor. BAY 11-7082 also attenuated the diet-induced increase in NLRP3 inflammasome expression, resulting in inhibition of caspase-1 activation and interleukin (IL)-1? and IL-18 production (in liver and kidney). Interestingly, BAY 11-7082, but not gene silencing, inhibited nuclear factor (NF)-?B nuclear translocation. Overall, these results demonstrate that the selective pharmacological modulation of the NLRP3 inflammasome attenuates the metabolic abnormalities and the related organ injury/dysfunction caused by chronic exposure to HD, with effects similar to those obtained by NLRP3 gene silencing.
Project description:Neuropilin 1 (Nrp1), a coreceptor for class 3 semaphorins and growth factors, is highly expressed in vascular cells and myeloid cells, including macrophages. Unlike well-characterized proangiogenic functions of endothelial cell Nrp1, the contributions of macrophage Nrp1 within the context of metabolic dysfunction remain to be established. The aim of this study was to determine the contributions of macrophage Nrp1 in high-fat diet (HFD)-instigated insulin resistance in vivo. Insulin sensitivity and Nlrp3 inflammasome activation were monitored in wild-type (WT) and myeloid cell-specific Nrp1 knockout (Nrp1<sup>myel-KO</sup>) mice fed an HFD (60% kcal) for 16 weeks. HFD-fed mice exhibited insulin resistance with reduced levels of Nrp1 in macrophages compared with chow-fed mice. Further, HFD-fed Nrp1<sup>myel-KO</sup> mice displayed accentuated insulin resistance, enhanced systemic inflammation, and dramatically increased Nlrp3 inflammasome priming and activation. Importantly, knockout of Nlrp3 ablated HFD-induced insulin resistance and inflammation in Nrp1<sup>myel-KO</sup> mice, indicating that Nrp1 reduction in macrophages instigates insulin resistance by increasing macrophage Nlrp3 inflammasome activation. Mechanistically, Nrp1 deletion activates the nuclear factor-?B pathway, which in turn accentuates the priming of Nlrp3, promotes Nlrp3-ASC inflammasome assembly, and results in the activation of Nlrp3. We conclude that the HFD-instigated Nrp1 reduction in macrophages exacerbates insulin resistance by promoting Nlrp3 inflammasome priming and activation.
Project description:<b>Background:</b> Both PCSK9 and NLRP3 inflammasome play important roles in atherogenesis. This study was designed to test the hypothesis that NLRP3 inflammasome via IL-1β induces PCSK9 secretion. The inter-twined relationship between NLRP3 inflammasome, IL-1β and PCSK9 may be relevant in atherogenesis. <b>Methods:</b> We studied NLRP3 inflammasome-mediated PCSK9 secretion in mouse peritoneal macrophages and in a variety of tissues, such as liver, kidney and small intestine. Macrophages were derived from wild-type (WT) and a variety of gene deletion mice to define the mechanistic basis of NLRP3 inflammasome -mediated PCSK9 secretion. Additional studies were performed in high-fat diet fed mice. <b>Results:</b> We observed that NLRP3 and its downstream signals ASC, Caspase-1, IL-18, and IL-1β all participate in PCSK9 secretion. IL-1β seems to be more important than IL-18 in the induction of PCSK9 secretion. Further, there appears to be significant involvement of MAPKs in this process. Lastly, we observed that mice fed high fat diet have high expression of NLRP3 and a greater secretion of PCSK9 than mice fed a standard diet, and this increased secretion of PCSK9 in high fat diet-fed mice was attenuated in <i>IL-1β<sup>-/-</sup></i> mice. Conclusions: This study based on extensive in vitro and in vivo data provides evidence that NLRP3 inflammasome via IL-1β plays an important role in determining PCSK9 secretion, particularly in the presence of high-fat diet.
Project description:Treatment for traumatic brain injury (TBI) remains elusive despite compelling evidence from animal models for a variety of therapeutic targets. The activation of the NLRP3 (Nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3) inflammasome has been proposed as key point in the brain damage associated with TBI. NLRP3 was tested as potential target for reducing neuronal loss and promoting functional recovery in a mouse model of TBI. Male NLRP3-/- (n = 20) and wild type (n = 27) mice were used. A closed TBI model was performed and inflammatory and apoptotic markers were evaluated. A group of WT mice also received BAY 11-7082, a NLRP3 inhibitor, to further evaluate the role of this pathway. At 24 h following TBI NLRP3-/- animals demonstrated a preserved cognitive function as compared to WT mice, additionally brain damage was less severe and the inflammatory mediators were reduced in brain lysates. The administration of BAY 11-7082 in WT animals subjected to TBI produced overlapping results. At day 7 histology revealed a more conserved brain structure with reduced damage in TBI NLRP3-/- animals compared to WT. Our data indicate that the NLRP3 pathway might be exploited as molecular target for the short-term sequelae of TBI.
Project description:Activation of the inflammasome generates the pro-inflammatory cytokines interleukin-1 beta and -18, which are important mediators of inflammation. Abnormal activation of the inflammasome leads to many inflammatory diseases, including gout, silicosis, neurodegeneration, and genetically inherited periodic fever syndromes. Therefore, identification of small molecule inhibitors that target the inflammasome is an important step toward developing effective therapeutics for the treatment of inflammation. Here, we show that the herbal NF-kappaB inhibitory compound parthenolide inhibits the activity of multiple inflammasomes in macrophages by directly inhibiting the protease activity of caspase-1. Additional investigations of other NF-kappaB inhibitors revealed that the synthetic I kappaB kinase-beta inhibitor Bay 11-7082 and structurally related vinyl sulfone compounds selectively inhibit NLRP3 inflammasome activity in macrophages independent of their inhibitory effect on NF-kappaB activity. In vitro assays of the effect of parthenolide and Bay 11-7082 on the ATPase activity of NLRP3 demonstrated that both compounds inhibit the ATPase activity of NLRP3, suggesting that the inhibitory effect of these compounds on inflammasome activity could be mediated in part through their effect on the ATPase activity of NLRP3. Our results thus elucidate the molecular mechanism for the therapeutic anti-inflammatory activity of parthenolide and identify vinyl sulfones as a new class of potential therapeutics that target the NLRP3 inflammasome.
Project description:Low-grade chronic inflammation plays a pivotal role in the pathogenesis of insulin resistance (IR), and skeletal muscle has a central role in this condition. NLRP3 inflammasome activation pathways promote low-grade chronic inflammation in several tissues. However, a direct link between IR and NLRP3 inflammasome activation in skeletal muscle has not been reported. Here, we evaluated the NLRP3 inflammasome components and their role in GLUT4 translocation impairment in skeletal muscle during IR. Male C57BL/6J mice were fed with a normal control diet (NCD) or high-fat diet (HFD) for 8 weeks. The protein levels of NLRP3, ASC, caspase-1, gasdermin-D (GSDMD), and interleukin (IL)-1β were measured in both homogenized and isolated fibers from the flexor digitorum brevis (FDB) or soleus muscle. GLUT4 translocation was determined through GLUT4<i>myc</i>-eGFP electroporation of the FBD muscle. Our results, obtained using immunofluorescence, showed that adult skeletal muscle expresses the inflammasome components. In the FDB and soleus muscles, homogenates from HFD-fed mice, we found increased protein levels of NLRP3 and ASC, higher activation of caspase-1, and elevated IL-1β in its mature form, compared to NCD-fed mice. Moreover, GSDMD, a protein that mediates IL-1β secretion, was found to be increased in HFD-fed-mice muscles. Interestingly, MCC950, a specific pharmacological NLRP3 inflammasome inhibitor, promoted GLUT4 translocation in fibers isolated from the FDB muscle of NCD- and HFD-fed mice. In conclusion, we found increased NLRP3 inflammasome components in adult skeletal muscle of obese insulin-resistant animals, which might contribute to the low-grade chronic metabolic inflammation of skeletal muscle and IR development.
Project description:BACKGROUND AND PURPOSE:There are no medications currently available to treat metabolic inflammation. Bruton's tyrosine kinase (BTK) is highly expressed in monocytes and macrophages and regulates NF-?B and NLRP3 inflammasome activity; both propagate metabolic inflammation in diet-induced obesity. EXPERIMENTAL APPROACH:Using an in vivo model of chronic inflammation, high-fat diet (HFD) feeding, in male C57BL/6J mice and in vitro assays in primary murine and human macrophages, we investigated if ibrutinib, an FDA approved BTK inhibitor, may represent a novel anti-inflammatory medication to treat metabolic inflammation. KEY RESULTS:HFD-feeding was associated with increased BTK expression and activation, which was significantly correlated with monocyte/macrophage accumulation in the liver, adipose tissue, and kidney. Ibrutinib treatment to HFD-fed mice inhibited the activation of BTK and reduced monocyte/macrophage recruitment to the liver, adipose tissue, and kidney. Ibrutinib treatment to HFD-fed mice decreased the activation of NF-?B and the NLRP3 inflammasome. As a result, ibrutinib treated mice fed HFD had improved glycaemic control through restored signalling by the IRS-1/Akt/GSK-3? pathway, protecting mice against the development of hepatosteatosis and proteinuria. We show that BTK regulates NF-?B and the NLRP3 inflammasome specifically in primary murine and human macrophages, the in vivo cellular target of ibrutinib. CONCLUSION AND IMPLICATIONS:We provide "proof of concept" evidence that BTK is a novel therapeutic target for the treatment of diet-induced metabolic inflammation and ibrutinib may be a candidate for drug repurposing as an anti-inflammatory agent for the treatment of metabolic inflammation in T2D and microvascular disease.
Project description:<b>Background:</b> The NLRP3 inflammasome is one of the key contributors to impaired wound healing in diabetes. In this study, we assessed the role of rapamycin on high glucose-induced inflammation in THP-1-derived macrophages and investigated the underlying signaling mechanisms. <b>Methods:</b> THP-1-derived macrophages were treated with high glucose to induce NLRP3 inflammasome activation. The cells were pretreated with rapamycin, BAY 11-7082, or PDTC before exposure to HG. mTOR, NF-?B, and NLRP3 inflammasome expression were measured by western blotting. <b>Results:</b> We found that rapamycin reduced NLRP3 inflammasome activation in macrophages. Rapamycin reduced NLRP3 inflammasome activation by inhibiting mTOR phosphorylation and NF-?B activation. Moreover, mTOR siRNA inhibited NF-?B activation, leading to the suppression of NLRP3 inflammasome activation. <b>Conclusion:</b> Rapamycin can ameliorate high glucose-induced NLRP3 inflammasome activation by attenuating the mTOR/NF-?B signaling pathway in macrophages. Rapamycin may act as a possible therapeutic option for high glucose-induced inflammatory response in impaired wound healing in the future.
Project description:NLRP3 inflammasome is a vital player in macrophages pyroptosis, which is a type of proinflammatory cell-death and takes part in the pathogenesis of atherosclerosis. In this study, we used apoE<sup>-/-</sup> mice and ox-LDL induced THP-1 derived macrophages to explore the mechanisms of MCC950, a selective NLRP3 inhibitor in treating atherosclerosis. For the in vivo study, MCC950 was intraperitoneal injected to 8-week-old apoE<sup>-/-</sup> mice fed with high-fat diet for 12 weeks. For the in vitro study, THP-1 derived macrophages were treated with ox-LDL and MCC950 for 48 h. MCC950 administration reduced plaque areas and macrophages contents, but did not improve the serum lipid profiles in aortic root of apoE<sup>-/-</sup> mice. MCC950 inhibited the activation of NLRP3/ASC/Caspase-1/GSDMD-N axis, and alleviated macrophages pyroptosis and the production of IL-1β and IL-18 both in aorta and in cell lysates. However, MCC950 did not affect the expression of TLR4 or the mRNA levels of NLRP3 inflammasome and its downstream proteins, suggesting that MCC950 had no effects on the priming of NLRP3 inflammasome activation in macrophages. The anti-atherosclerotic mechanisms of MCC950 on attenuating macrophages inflammation and pyroptosis involved in inhibiting the assembly and activation of NLRP3 inflammasome, rather than interrupting its priming.
Project description:Background: Obesity is an increasingly prevalent metabolic disorder in the modern world and is associated with structural and functional changes in the heart. The NLRP3 inflammasome is an innate immune sensor that can be activated in response to endogenous danger signals and triggers activation of interleukin (IL)-1? and IL-18. Increasing evidence points to the involvement of the NLRP3 inflammasome in obesity-induced inflammation and insulin resistance, and we hypothesized that it also could play a role in the development of obesity induced cardiac alterations. Methods and Results: WT, Nlrp3 -/-, and ASC -/- (Pycard -/-) male mice were exposed to high fat diet (HFD; 60 cal% fat) or control diet for 52 weeks. Cardiac structure and function were evaluated by echocardiography and magnetic resonance imaging, respectively. Whereas, NLRP3 and ASC deficiency did not affect the cardiac hypertrophic response to obesity, it was preventive against left ventricle concentric remodeling and impairment of diastolic function. Furthermore, whereas NLRP3 and ASC deficiency attenuated systemic inflammation in HFD fed mice; long-term HFD did not induce significant cardiac fibrosis or inflammation, suggesting that the beneficial effects of NLRP3 inflammasome deficiency on myocardial remodeling at least partly reflect systemic mechanisms. Nlrp3 and ASC (Pycard) deficient mice were also protected against obesity-induced systemic metabolic dysregulation, as well as lipid accumulation and impaired insulin signaling in hepatic and cardiac tissues. Conclusions: Our data indicate that the NLRP3 inflammasome modulates cardiac concentric remodeling in obesity through effects on systemic inflammation and metabolic disturbances, with effect on insulin signaling as a potential mediator within the myocardium.
Project description:Aging and lipotoxicity are two major risk factors for gout that are linked by the activation of the NLRP3 inflammasome. Neutrophil-mediated production of interleukin-1? (IL-1?) drives gouty flares that cause joint destruction, intense pain, and fever. However, metabolites that impact neutrophil inflammasome remain unknown. Here, we identified that ketogenic diet (KD) increases ?-hydroxybutyrate (BHB) and alleviates urate crystal-induced gout without impairing immune defense against bacterial infection. BHB inhibited NLRP3 inflammasome in S100A9 fibril-primed and urate crystal-activated macrophages, which serve to recruit inflammatory neutrophils in joints. Consistent with reduced gouty flares in rats fed a ketogenic diet, BHB blocked IL-1? in neutrophils in a NLRP3-dependent manner in mice and humans irrespective of age. Mechanistically, BHB inhibited the NLRP3 inflammasome in neutrophils by reducing priming and assembly steps. Collectively, our studies show that BHB, a known alternate metabolic fuel, is also an anti-inflammatory molecule that may serve as a treatment for gout.