NLRP3 In Macrophages Research Articles

Lifting the veil on fungal toxins.

Lifting the veil on fungal toxins.

The novel TLR9 antagonist COV08-0064 protects from ischemia/reperfusion injury in non-steatotic and steatotic mice livers

Ischemia/reperfusion (I/R) injury constitutes a major reason for failure of liver surgeries and transplantation. I/R injury is more severe in steatotic livers and limits their use in transplantation. Here, we present a novel and selective Toll-like receptor 9 (TLR9) antagonist COV08-0064 and test its potential to protect from I/R-induced injury in normal and steatotic livers. The in vivo effects of COV08-0064 pretreatment were investigated on normal chow diet (NCD) and high fat diet (HFD)-fed mice subjected to segmental (70%) warm hepatic I/R. Also, the in vitro effects of COV08-0064 were elucidated in murine macrophages and dendritic cells. Mice on a HFD had pronouncedly greater hepatic I/R injury than mice on a NCD. COV08-0064-pretreatment to both NCD and HFD-fed mice reduced hepatic I/R injury. COV08-0064-pretreatment was associated with less production of the liver inflammatory cytokines and mediators TNF-α, IL-1β, IL-6, NLRP3, iNOS and MCP-1. These manifestations were preceded with inhibition of JNK and ERK phosphorylation and TLR9 cleavage in the liver. COV08-0064 enhanced the hepatic expression of the endogenous anti-inflammatory cytokines IL-10 and IL-1Ra at the early phase I/R injury. In vitro, COV08-0064 selectively blocked mRNA upregulation of TNF-α, IL-1β, NLRP3 and MCP-1 in macrophages and IFN-β mRNA in dendritic cells induced by the TLR9 agonist CpG-ODN. These effects were concordant with inhibition of JNK, ERK, IκBα and IKKα/β phosphorylation. In conclusion, TLR9 signaling inhibition by COV08-0064 may be an effective approach in liver surgeries including transplantation to limit I/R-injury and overcome the shortages in the donor pool by incorporating steatotic livers.

Sterile signals generate weaker and delayed macrophage NLRP3 inflammasome responses relative to microbial signals

Inflammation is the host response to microbial infection or sterile injury that aims to eliminate the insult, repair the tissue and restore homeostasis. Macrophages and the NLRP3 inflammasome are key sentinels for both types of insult. Although it is well established that the NLRP3 inflammasome is activated by microbial products and molecules released during sterile injury, it is unclear whether the responses elicited by these different types of signals are distinct. In this study, we used lipopolysaccharide and tumor necrosis factor as prototypical microbial and sterile signal 1 stimuli, respectively, to prime the NLRP3 inflammasome. We then used the bacterial toxin nigericin and a common product released from necrotic cells, ATP, as prototypical microbial and sterile signal 2 stimuli, respectively, to trigger the assembly of the NLRP3 inflammasome complex in mouse and human macrophages. We found that NLRP3 inflammasome responses were weakest when both signal 1 and signal 2 were sterile, but responses were faster and stronger when at least one of the two signals was microbial. Ultimately, the most rapid and potent responses were elicited when both signals were microbial. Together, these data suggest that microbial versus sterile signals are distinct, both kinetically and in magnitude, in their ability to generate inflammasome-dependent responses. This hierarchy of NLRP3 responses to sterile versus microbial stimuli likely reflects the urgent need for the immune system to respond rapidly to the presence of infection to halt pathogen dissemination.

Correcting the NLRP3 inflammasome deficiency in macrophages from autoimmune NZB mice with exon skipping antisense oligonucleotides.

Inflammasomes are molecular complexes activated by infection and cellular stress, leading to caspase-1 activation and subsequent interleukin-1β (IL-1β) processing and cell death. The autoimmune NZB mouse strain does not express NLRP3, a key inflammasome initiator mediating responses to a wide variety of stimuli including endogenous danger signals, environmental irritants and a range of bacterial, fungal and viral pathogens. We have previously identified an intronic point mutation in the Nlrp3 gene from NZB mice that generates a splice acceptor site. This leads to inclusion of a pseudoexon that introduces an early termination codon and is proposed to be the cause of NLRP3 inflammasome deficiency in NZB cells. Here we have used exon skipping antisense oligonucleotides (AONs) to prevent aberrant splicing of Nlrp3 in NZB macrophages, and this restored both NLRP3 protein expression and NLRP3 inflammasome activity. Thus, the single point mutation leading to aberrant splicing is the sole cause of NLRP3 inflammasome deficiency in NZB macrophages. The NZB mouse provides a model for addressing a splicing defect in macrophages and could be used to further investigate AON design and delivery of AONs to macrophages in vivo.

Bisphosphonate Induces Osteonecrosis of the Jaw in Diabetic Mice via NLRP3/Caspase-1-Dependent IL-1β Mechanism.

Diabetes mellitus is an established risk factor associated with bisphosphonate-related osteonecrosis of the jaw (BRONJ). Sustained activation of Nod-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome contributes to the persistent inflammation and impaired cutaneous wound healing in diabetic mice and human. We have recently demonstrated a compelling linkage between M1 macrophages and BRONJ conditions in both murine and human diseases. The aim of this study was to determine whether NLRP3 inflammasome activation is involved in BRONJ development in diabetic mice. We showed an increased incidence of delayed oral wound healing and bone necrosis of extraction sockets in db/db mice compared with those in nondiabetic db/+ controls, which correlated with an elevated expression of NLRP3, caspase-1, and IL-1β in macrophages residing at local wounds. Constitutively, bone marrow-derived macrophages from db/db mice (db/db BMDMs) secrete a relatively higher level of IL-1β than those from db/+ mice (db/+ BMDMs). Upon stimulation by NLRP3 activators, the secretion of IL-1β by db/db BMDMs was 1.77-fold higher than that by db/+ BMDMs (p < 0.001). Systemic treatment of mice with zoledronate (Zol), a nitrogen-containing bisphosphonate, resulted in a 1.86- and 1.63-fold increase in NLRP3/caspase-1-dependent IL-1β secretion by db/+ and db/db BMDMs, respectively, compared with BMDMs derived from nontreated mice (p < 0.001). Importantly, systemic administration of pharmacological inhibitors of NLRP3 activation improved oral wound healing and suppressed BRONJ formation in db/db mice. Mechanistically, we showed that supplementation with intermediate metabolites of the mevalonate pathway, inhibitors of caspase-1 and NLRP3 activation, an antagonist for P2X7 R, or a scavenger of reactive oxygen species (ROS), robustly abolished Zol-enhanced IL-1β release from macrophages in response to NLRP3 activation (p < 0.001). Our findings suggest that diabetes-associated chronic inflammatory response may have contributed to impaired socket wound healing and rendered oral wound susceptible to the development of BRONJ via NLRP3 activation in macrophages.

Strain- and host species-specific inflammasome activation, IL-1β release, and cell death in macrophages infected with uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1β (IL-1β) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1β secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1β secretion pathway in human macrophages. This has important implications for understanding UTI in humans.

Abstract 176: Dietary PUFAs Enhance Macrophage Autophagy, Attenuate NLRP3 Inflammasome Activation, and Reduce Atherosclerosis

Progression of atherosclerosis leads to progressive macrophage autophagy deficiency, which enhances inflammasome activation and atherosclerosis. We previously showed that dietary enrichment with n-3 and n-6 polyunsaturated fatty acids (PUFAs) derived from botanical oils (Echium oil (EO) containing 18:4 n-3 and borage oil (BO) containing 18:3 n-6), reduced atherosclerosis similar to that observed with fish oil (FO) consumption, compared to the saturated/monounsaturated-enriched botanical palm oil (PO). We hypothesized that dietary PUFAs enhance autophagy, which in turn, dampens macrophage NLRP3 (Nucleotide-binding oligomerization domain leucine-rich repeat containing receptor protein 3) inflammasome activation, reducing macrophage inflammation and atherosclerosis. To test the hypothesis, we fed female low density lipoprotein receptor (LDLr) knockout mice diets containing 10% (calories) PO and 0.2% cholesterol, supplemented with an additional 10% of calories as PO, EO, BO or FO for 10-16 weeks before measurement of atherosclerosis, autophagy and inflammasome activation. Compared to their PO-fed counterparts, mice fed PUFAs enriched diets (EO, BO or FO) had increased LC3-II expression in peritoneal macrophage, aorta and liver, suggesting autophagic activation. Consistent with enhanced autophagy, plasma reactive oxygen species (ROS) were significantly lower in PUFA diet-fed mice, relative to PO-fed animals. Since ROS activates, but autophagy attenuates, NLRP3 inflammasome activation, we examined whether dietary PUFAs attenuate NLRP3 inflammasome activation in mice. We found that dietary PUFAs markedly inhibited inflammasome activation, shown by: 1) less IL-1β secretion from peritoneal macrophages after ATP, oxidized LDL, or palmitate-induced NLRP3 inflammasome activation, 2) less IL-1β secretion from liver explants in response to lipopolysaccharide (LPS), and 3) deceased caspase-1 cleavage in peritoneal macrophages and liver and attenuated caspase-1 activity in blood monocytes. In conclusion, our data suggest that dietary n-3 and n-6 PUFAs are equally effective in reducing atherosclerosis, in part, by activation of macrophage autophagy and attenuation of NLRP3 inflammasome activation.

Brief report: Granulocyte-macrophage colony-stimulating factor drives monosodium urate monohydrate crystal-induced inflammatory macrophage differentiation and NLRP3 inflammasome up-regulation in an in vivo mouse model.

To determine the role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the differentiation of inflammatory macrophages in an in vivo model of monosodium urate monohydrate (MSU) crystal-induced inflammation. C57BL/6J mice were treated with either clodronate liposomes to deplete peritoneal macrophages or GM-CSF antibody and were then challenged by intraperitoneal injection of MSU crystals. Peritoneal lavage fluid was collected, and cellular infiltration was determined by flow cytometry. Purified resident and MSU crystal-recruited monocyte/macrophages were stimulated ex vivo with MSU crystals. The interleukin-1β (IL-1β) levels in lavage fluids and ex vivo assay supernatants were measured. GM-CSF-derived and macrophage colony-stimulating factor (M-CSF)-derived macrophages were generated in vitro from bone marrow cells. Protein expression of IL-1β, caspase 1, NLRP3, and ASC by in vitro- and in vivo-generated monocyte/macrophages was analyzed by Western blotting. Depletion of resident macrophages lowered MSU crystal-induced IL-1β and GM-CSF levels in vivo as well as IL-1β production by MSU crystal-recruited monocytes stimulated ex vivo. GM-CSF neutralization in vivo decreased MSU crystal-induced IL-1β levels and neutrophil infiltration. MSU crystal-recruited monocyte/macrophages from GM-CSF-neutralized mice expressed lower levels of the macrophage marker CD115 and produced less IL-1β following ex vivo stimulation. These monocytes exhibited decreased expression of NLRP3, pro/active IL-1β, and pro/active caspase 1. In vitro-derived GM-CSF-differentiated macrophages expressed higher levels of NLRP3, pro/active IL-1β, and pro/active caspase 1 compared to M-CSF-differentiated macrophages. GM-CSF plays a key role in the differentiation of MSU crystal-recruited monocytes into proinflammatory macrophages. GM-CSF production may therefore contribute to the exacerbation of inflammation in gout.

DPP-4 inhibitors repress NLRP3 inflammasome and interleukin-1beta via GLP-1 receptor in macrophages through protein kinase C pathway.

Anti-atherosclerotic effects of dipeptidyl peptidase-4 (DPP-4) inhibitors have been shown in many studies. Since inflammation and immune response play a key role in atherogenesis, we examined the effect of DPP-4 inhibitors on the expression of nod-like receptor family, pyrin domain containing 3 (NLRP3) Inflammasome and Interleukin-1beta (IL-1β) in human macrophages. THP-1 macrophages were incubated with oxidized low density lipoprotein (ox-LDL) with or without DPP-4 inhibitors (sitagliptin and NVPDPP728). The effects of DPP-4 inhibitors on the expression of NLRP3, toll-like receptor 4 (TLR4) and pro-inflammatory cytokine IL-1β were studied. Both DPP-4 inhibitors induced a significant reduction in NLRP3, TLR4 and IL-1β expression; concurrently, there was an increase in glucagon like peptide 1 receptor (GLP-1R) expression. Simultaneously, DPP-4 inhibitors reduced phosphorylated-PKC, but not PKA, levels. To determine the role of PKC activation in the effects of DPP-4 inhibitors, cells were treated with PMA- which blocked the effect of DPP-4 inhibitors on NLRP3 and IL-1β as well as TLR4 and GLP-1R. Over-expression of GLP-1R in macrophages with its agonist liraglutide also blocked the effects of PMA. DPP-4 inhibitors suppress NLRP3, TLR4 and IL-1β in human macrophages through inhibition of PKC activity. This study provides novel insights into the mechanism of inhibition of inflammatory state and immune response in atherosclerosis by DPP-4 inhibitors.

3,4-Methylenedioxy-β-nitrostyrene Inhibits NLRP3 Inflammasome Activation by Blocking Assembly of the Inflammasome

The NLRP3 inflammasome is a critical component of the innate immune system. NLRP3 activation is induced by diverse stimuli associated with bacterial infection or tissue damage, but its inappropriate activation is involved in the pathogenesis of inherited and acquired inflammatory diseases. However, the mechanism by which NLRP3 is activated remains poorly understood. In this study, we explored the role of kinases in NLRP3 inflammasome activation by screening a kinase inhibitor library and identified 3,4-methylenedioxy-β-nitrostyrene (MNS) as an inhibitor for NLRP3 inflammasome activation. Notably, MNS did not affect the activation of the NLRC4 or AIM2 (absent in melanoma 2) inflammasome. Mechanistically, MNS specifically prevented NLRP3-mediated ASC speck formation and oligomerization without blocking potassium efflux induced by NLRP3 agonists. Surprisingly, Syk kinase, the reported target of MNS, did not mediate the inhibitory activity of MNS on NLRP3 inflammasome activation. We also found that the nitrovinyl group of MNS is essential for the inhibitory activity of MNS. Immunoprecipitation, mass spectrometry, and mutation studies suggest that both the nucleotide binding oligomerization domain and the leucine-rich repeat domain of NLRP3 were the intracellular targets of MNS. Administration of MNS also inhibited NLRP3 ATPase activity in vitro, suggesting that MNS blocks the NLRP3 inflammasome by directly targeting NLRP3 or NLRP3-associated complexes. These studies identified a novel chemical probe for studying the molecular mechanism of NLRP3 inflammasome activation which may advance the development of novel strategies to treat diseases associated with abnormal activation of NLRP3 inflammasome.

Abstract 464: Tumor derived vacuolar ATPase subunit promotes M2 type polarization of macrophages.

Abstract Tumors and tumor associated macrophages (TAMs) express a unique surface vacuolar ATPase (V-ATPase) on the plasma membrane. In tumor cells, a 20 kd peptide is cleaved from a2 isoform of V-ATPase and is referred to as a2NTD; it is associated with in vitro induction of pro-tumorigenic properties in monocytes and IL-β secretion. In this study, we evaluated the immuno-modulatory properties of a2NTD in inducing murine peritoneal macrophage polarization and activation of inflammasome components both ex vivo and in vivo. Peritoneal macrophages from BALB/c mice were stimulated with recombinant a2NTD and the mRNA and protein expression of M1 (CD11c, TNF-α), M2 (Mannose receptor-1, Arginase-1, IL-10) markers, IL-1β and NLRP3 (inflammasome marker) were analyzed using quantitative real time PCR and flow cytometry. Further, mice were injected with a2NTD i.p. and peritoneal cells were analyzed for M1/M2 specific markers. The results of both in vivo and ex vivo analysis showed an elevated population of M2 subtype in a2NTD-treated mice compared to control (P&amp;lt;0.05). a2NTD upregulated the expression of IL-1β and NLRP3 in macrophages. These data demonstrated that a2NTD modulates macrophage polarization towards the M2 subtype functionally similar to tumor associated macrophages (TAMs). Since, tumor progression requires sterile inflammation and maturation of resident macrophages into TAMs; our data provide evidence that the surface expression of V-ATPase in tumor cells has a role in inducing the unique inflammatory environment of tumors by modulating macrophage phenotype and inducing IL-1β production through the action of a2NTD. The study also provides indication for the involvement of a2NTD in NLRP3 mediated inflammasome activation. Citation Format: Gajendra K. Katara, Mukesh K. Jaiswal, Alice Gilmam-Sacks, Kenneth D. Beaman. Tumor derived vacuolar ATPase subunit promotes M2 type polarization of macrophages. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 464. doi:10.1158/1538-7445.AM2013-464

Activation of NLRP3 Inflammasome in Alveolar Macrophages Contributes to Mechanical Stretch-Induced Lung Inflammation and Injury

Mechanical ventilation of lungs is capable of activating the innate immune system and inducing sterile inflammatory response. The proinflammatory cytokine IL-1β is among the definitive markers for accurately identifying ventilator-induced lung inflammation. However, mechanisms of IL-1β release during mechanical ventilation are unknown. In this study, we show that cyclic stretch activates the nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasomes and induces the release of IL-1β in mouse alveolar macrophages via caspase-1- and TLR4-dependent mechanisms. We also observed that NADPH oxidase subunit gp91(phox) was dispensable for stretch-induced cytokine production, whereas mitochondrial generation of reactive oxygen species was required for stretch-induced NLRP3 inflammasome activation and IL-1β release. Further, mechanical ventilation activated the NLRP3 inflammasomes in mouse alveolar macrophages and increased the production of IL-1β in vivo. IL-1β neutralization significantly reduced mechanical ventilation-induced inflammatory lung injury. These findings suggest that the alveolar macrophage NLRP3 inflammasome may sense lung alveolar stretch to induce the release of IL-1β and hence may contribute to the mechanism of lung inflammatory injury during mechanical ventilation.

Ventilator-induced Lung Injury Is Mediated by the NLRP3 Inflammasome

The innate immune response is important in ventilator-induced lung injury (VILI) but the exact pathways involved are not elucidated. The authors studied the role of the intracellular danger sensor NLRP3 inflammasome. NLRP3 inflammasome gene expression was analyzed in respiratory epithelial cells and alveolar macrophages obtained from ventilated patients (n = 40). In addition, wild-type and NLRP3 inflammasome deficient mice were randomized to low tidal volume (approximately 7.5 ml/kg) and high tidal volume (approximately 15 ml/kg) ventilation. The presence of uric acid in lung lavage, activation of caspase-1, and NLRP3 inflammasome gene expression in lung tissue were investigated. Moreover, mice were pretreated with interleukin-1 receptor antagonist, glibenclamide, or vehicle before start of mechanical ventilation. VILI endpoints were relative lung weights, total protein in lavage fluid, neutrophil influx, and pulmonary and systemic cytokine and chemokine concentrations. Data represent mean ± SD. Mechanical ventilation up-regulated messenger RNA expression levels of NLRP3 in alveolar macrophages (1.0 ± 0 vs. 1.70 ± 1.65, P less than 0.05). In mice, mechanical ventilation increased both NLRP3 and apoptosis-associated speck-like protein messenger RNA levels, respectively (1.08 ± 0.55 vs. 3.98 ± 2.89; P less than 0.001 and 0.95 ± 0.53 vs. 6.0 ± 3.55; P less than 0.001), activated caspase-1, and increased uric acid levels (6.36 ± 1.85 vs. 41.9 ± 32.0, P less than 0.001). NLRP3 inflammasome deficient mice displayed less VILI due to high tidal volume mechanical ventilation compared with wild-type mice. Furthermore, treatment with interleukin-1 receptor antagonist or glibenclamide reduced VILI. Mechanical ventilation induced a NLRP3 inflammasome dependent pulmonary inflammatory response. NLRP3 inflammasome deficiency partially protected mice from VILI.