Activation of the NLRP3 inflammasome by diverse stimuli requires a priming signal from TLRs and an activation signal from purinergic receptors or pore-forming toxins. In this study, we demonstrate, ...through detailed analysis of NLRP3 activation in macrophages deficient in key downstream TLR signaling molecules, that MyD88 is required for an immediate early phase, whereas Toll/IL-1R domain-containing adapter inducing IFN-β is required for a subsequent intermediate phase of posttranslational NLRP3 activation. Both IL-1R-associated kinase (IRAK) 1 and IRAK4 are critical for rapid activation of NLRP3 through the MyD88 pathway, but only IRAK1 is partially required in the Toll/IL-1R domain-containing adapter inducing IFN-β pathway. IRAK1 and IRAK4 are also required for rapid activation of NLRP3 by Listeria monocytogenes, as deletion of IRAK1 or IRAK4 led to defective inflammasome activation. These findings define the pathways that lead to rapid NLRP3 activation and identify IRAK1 as a critical mediator of a transcription-independent,inflammasome-dependent early warning response to pathogenic infection.
Background- Inflammation plays a key role in the pathophysiology of myocardial ischemia/reperfusion (I/R) injury; however, the mechanism by which myocardial I/R induces inflammation remains unclear. ...Recent evidence indicates that a sterile inflammatory response triggered by tissue damage is mediated through a multiple-protein complex called the inflammasome. Therefore, we hypothesized that the inflammasome is an initial sensor for danger signal(s) in myocardial I/R injury. Methods and Results- We demonstrate that inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, is crucially involved in the initial inflammatory response after myocardial I/R injury. We found that inflammasomes are formed by I/R and that its subsequent activation of inflammasomes leads to interleukin-1β production, resulting in inflammatory responses such as inflammatory cell infiltration and cytokine expression in the heart. In mice deficient for apoptosis-associated speck-like adaptor protein and caspase-1, these inflammatory responses and subsequent injuries, including infarct development and myocardial fibrosis and dysfunction, were markedly diminished. Bone marrow transplantation experiments with apoptosis-associated speck-like adaptor protein-deficient mice revealed that inflammasome activation in bone marrow cells and myocardial resident cells such as cardiomyocytes or cardiac fibroblasts plays an important role in myocardial I/R injury. In vitro experiments revealed that hypoxia/reoxygenation stimulated inflammasome activation in cardiac fibroblasts, but not in cardiomyocytes, and that hypoxia/reoxygenation-induced activation was mediated through reactive oxygen species production and potassium efflux. Conclusions- Our results demonstrate the molecular basis for the initial inflammatory response after I/R and suggest that the inflammasome is a potential novel therapeutic target for preventing myocardial I/R injury.
TLR2 promotes NLRP3 inflammasome activation via an early MyD88-IRAK1-dependent pathway that provides a priming signal (signal 1) necessary for activation of the inflammasome by a second ...potassium-depleting signal (signal 2). Here we show that TLR3 binding to dsRNA promotes post-translational inflammasome activation through intermediate and late TRIF/RIPK1/FADD-dependent pathways. Both pathways require the scaffolding but not the catalytic function of caspase-8 or RIPK1. Only the late pathway requires kinase competent RIPK3 and MLKL function. Mechanistically, FADD/caspase-8 scaffolding function provides a post-translational signal 1 in the intermediate pathway, whereas in the late pathway it helps the oligomerization of RIPK3, which together with MLKL provides both signal 1 and 2 for inflammasome assembly. Cytoplasmic dsRNA activates NLRP3 independent of TRIF, RIPK1, RIPK3 or mitochondrial DRP1, but requires FADD/caspase-8 in wildtype macrophages to remove RIPK3 inhibition. Our study provides a comprehensive analysis of pathways that lead to NLRP3 inflammasome activation in response to dsRNA.
Abdominal aortic aneurysm (AAA) is considered a chronic inflammatory disease; however, the molecular basis underlying the sterile inflammatory response involved in the process of AAA remains unclear. ...We previously showed that the inflammasome, which regulates the caspase-1-dependent interleukin-1β production, mediates the sterile cardiovascular inflammatory responses. Therefore, we hypothesized that the inflammasome is a key mediator of initial inflammation in AAA formation.
Apoptosis-associated speck-like protein containing a caspase recruitment domain is highly expressed in adventitial macrophages in human and murine AAA tissues. Using an established mouse model of AAA induced by continuous infusion of angiotensin II in Apoe(-/-) mice, NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency in Apoe(-/-) mice were shown to decrease the incidence, maximal diameter, and severity of AAA along with adventitial fibrosis and inflammatory responses significantly, such as inflammatory cell infiltration and cytokine expression in the vessel wall. NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency in Apoe(-/-) mice also reduced elastic lamina degradation and metalloproteinase activation in the early phase of AAA formation. Furthermore, angiotensin II stimulated generation of mitochondria-derived reactive oxygen species in the adventitial macrophages, and this mitochondria-derived reactive oxygen species generation was inhibited by NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1 deficiency. In vitro experiments revealed that angiotensin II stimulated the NLRP3 inflammasome activation and subsequent interleukin-1β release in macrophages, and this activation was mediated through an angiotensin type I receptor/mitochondria-derived reactive oxygen species-dependent pathway.
Our results demonstrate the importance of the NLRP3 inflammasome in the initial inflammatory responses in AAA formation, indicating its potential as a novel therapeutic target for preventing AAA progression.
► Caspase-1 deficiency reduced the atherosclerosis in Apoe−/− mice fed the Western diet. ► Caspase-1 deficiency reduced vascular inflammation in Apoe−/− mice fed the Western diet. ► Calcium crystals ...activate caspase-1 and release IL-1β and IL-1α in macrophages. ► IL-1β release by calcium crystals was caspase-1-dependent. ► IL-1 α release by calcium crystals was caspase-1-independent.
Recent investigations have suggested that the inflammasome plays a role in the development of vascular inflammation and atherosclerosis; however, its precise role remains controversial. We produced double-deficient mice for apolipoprotien E (Apoe) and caspase-1 (Casp1), a key component molecule of the inflammasome, and investigated the effect of caspase-1 deficiency on vascular inflammation and atherosclerosis.
Atherosclerotic plaque areas in whole aortas and aortic root of Western diet (WD)-fed Apoe−/−Casp1−/− mice were significantly reduced compared to those in Apoe−/− mice. The amount of macrophages and vascular smooth muscle cells in the plaques was also reduced in Apoe−/−Casp1−/− mice. No significant differences in plasma lipid profiles and body weight change were observed between these mice. Expression of interleukin (IL)-1β in the plaques as well as plasma levels of IL-1β, IL-1α, IL-6, CCL2, and TNF-α, in Apoe−/−Casp1−/− mice were lower than those in Apoe−/− mice. In vitro experiments showed that calcium phosphate crystals induced caspase-1 activation and secretion of IL-1β and IL-1α in macrophages.
Our findings suggest that caspase-1 plays a critical role in vascular inflammation and atherosclerosis, and that modulation of caspase-1 could be a potential target for prevention and treatment of atherosclerosis.
Inflammation plays a key role in the pathophysiology of hepatic ischemia-reperfusion (I/R) injury. However, the mechanism by which hepatic I/R induces inflammatory responses remains unclear. Recent ...evidence indicates that a sterile inflammatory response triggered by I/R is mediated through a multiple-protein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in hepatic I/R injury and found that hepatic I/R stimuli upregulated the inflammasome-component molecule, nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), but not apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). NLRP3(-/-) mice, but not ASC(-/-) and caspase-1(-/-) mice, had significantly less liver injury after hepatic I/R. NLRP3(-/-) mice showed reduced inflammatory responses, reactive oxygen species production, and apoptosis in I/R liver. Notably, infiltration of neutrophils, but not macrophages, was markedly inhibited in the I/R liver of NLRP3(-/-) mice. Bone marrow transplantation experiments showed that NLRP3 not only in bone marrow-derived cells, but also in non-bone marrow-derived cells contributed to liver injury after I/R. In vitro experiments revealed that keratinocyte-derived chemokine-induced activation of heterotrimeric G proteins was markedly diminished. Furthermore, NLRP3(-/-) neutrophils decreased keratinocyte-derived chemokine-induced concentrations of intracellular calcium elevation, Rac activation, and actin assembly formation, thereby resulting in impaired migration activity. Taken together, NLRP3 regulates chemokine-mediated functions and recruitment of neutrophils, and thereby contributes to hepatic I/R injury independently of inflammasomes. These findings identify a novel role of NLRP3 in the pathophysiology of hepatic I/R injury.
Inflammation plays a crucial role in the pathophysiological characteristics of chronic kidney disease; however, the inflammatory mechanisms underlying the chronic kidney disease process remain ...unclear. Recent evidence indicates that sterile inflammation triggered by tissue injury is mediated through a multiprotein complex called the inflammasome. Therefore, we investigated the role of the inflammasome in the development of chronic kidney disease using a murine unilateral ureteral obstruction (UUO) model. Inflammasome-related molecules were up-regulated in the kidney after UUO. Apoptosis-associated speck-like protein containing a caspase recruitment domain deficiency significantly reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, and improved subsequent renal injury and fibrosis. Furthermore, apoptosis-associated speck-like protein containing a caspase recruitment domain was specifically up-regulated in collecting duct (CD) epithelial cells of the UUO-treated kidney. In vitro experiments showed that extracellular adenosine triphosphate (ATP) induced inflammasome activation in CD epithelial cells through P2X7 -potassium efflux and reactive oxygen species–dependent pathways. These results demonstrate the molecular basis for the inflammatory response in the process of chronic kidney disease and suggest the CD inflammasome as a potential therapeutic target for preventing chronic kidney disease progression.
Proteins that belong to the protein phosphatase 1 and actin regulator (phactr) family are involved in cell motility and morphogenesis. However, the mechanisms that regulate the actin cytoskeleton are ...poorly understood. We have previously shown that phactr3, also known as scapinin, localizes to the plasma membrane, including lamellipodia and membrane ruffles. In the present study, experiments using deletion and point mutants showed that the basic and hydrophobic residues in the N-terminus play crucial roles in the localization to the plasma membrane. A BH analysis (http://helixweb.nih.gov/bhsearch) is a program developed to identify membrane-binding domains that comprise basic and hydrophobic residues in membrane proteins. We applied this program to phactr3. The results of the BH plot analysis agreed with the experimentally determined region that is responsible for the localization of phactr3 to the plasma membrane. In vitro experiments showed that the N-terminal itself binds to liposomes and acidic phospholipids. In addition, we showed that the interaction with the plasma membrane via the N-terminal membrane-binding sequence is required for phactr3-induced morphological changes in Cos7 cells. The membrane-binding sequence in the N-terminus is highly conserved in all members of the phactr family. Our findings may provide a molecular basis for understanding the mechanisms that allow phactr proteins to regulate cell morphogenesis.
Many members of the nucleotide-binding and oligomerization domain (NOD)- and leucine-rich-repeat-containing protein (NLR) family play important roles in pathogen recognition and inflammation. ...However, we previously reported that human PYNOD/NLRP10, an NLR-like protein consisting of a pyrin domain and a NOD, inhibits inflammatory signal mediated by caspase-1 and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) in reconstitution experiments using HEK293 cells. In this study, we investigated the molecular mechanism of PYNOD's anti-inflammatory activity in vitro and its expression and function in mice. Human PYNOD inhibited the autoprocessing of caspase-1 and caspase-1-mediated IL-1beta processing and suppressed the aggregation of ASC, a hallmark of ASC activation. Interestingly, the NOD of human PYNOD was sufficient to inhibit caspase-1-mediated IL-1beta secretion, whereas its pyrin domain was sufficient to inhibit ASC-mediated NF-kappaB activation and apoptosis and to reduce ASC's ability to promote caspase-1-mediated IL-1beta production. Mouse PYNOD protein was detected in the skin, tongue, heart, colon, peritoneal macrophages, and several cell lines of hematopoietic and myocytic lineages. Mouse PYNOD colocalized with ASC aggregates in LPS + R837-stimulated macrophages; however, unlike human PYNOD, mouse PYNOD failed to inhibit ASC aggregation. Macrophages and neutrophils from PYNOD-transgenic mice exhibited reduced IL-1beta processing and secretion upon microbial infection, although mouse PYNOD failed to inhibit caspase-1 processing, which was inhibited by caspase-4 inhibitor z-LEED-fluoromethylketone. These results suggest that mouse PYNOD colocalizes with ASC and inhibits caspase-1-mediated IL-1beta processing without inhibiting caspase-4 (mouse caspase-11)-mediated caspase-1 processing. Furthermore, PYNOD-transgenic mice were resistant to lethal endotoxic shock. Thus, PYNOD is the first example of an NLR that possesses an anti-inflammatory function in vivo.
Despite the increasing commercial use of nanoparticles, little is known about their effects on placental inflammation and pregnancy complications. In this study, nanosilica (NS) particles upregulated ...the inflammasome component nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) and induced placental inflammation and reactive oxygen species (ROS) generation, resulting in pregnancy complications. Furthermore, NS-induced pregnancy complications were markedly improved in Nlrp3
−/−
mice but not in component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-deficient (Asc
−/−
) mice, indicating the independence of NLRP3 inflammasomes. Pregnancy complications in Nlrp3
−/−
and Asc
−/−
mice phenotypes were dependent on the balance between interleukin (IL)-1α and IL-10. NS-induced pregnancy complications were completely prevented by either inhibition of ROS generation or forced expression of IL-10. Our findings provide important information about NS-induced placental inflammation and pregnancy complications and the novel pathophysiological roles of NLRP3 and ASC in pregnancy.
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