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Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory syndrome caused by mutations of NLRP3 gene encoding cryopyrin. Familial cold autoinflammatory syndrome, the mildest form of CAPS, is characterized by cold-induced inflammation induced by the overproduction of IL-1β. However, the molecular mechanism of how mutated NLRP3 causes inflammasome activation in CAPS remains unclear. Here, we found that CAPS-associated NLRP3 mutants form cryo-sensitive aggregates that function as a scaffold for inflammasome activation. Cold exposure promoted inflammasome assembly and subsequent IL-1β release triggered by mutated NLRP3. While K+ efflux was dispensable, Ca2+ was necessary for mutated NLRP3- mediated inflammasome assembly. Notably, Ca2+ influx was induced during mutated NLRP3- mediated inflammasome assembly. Furthermore, caspase-1 inhibition prevented Ca2+ influx and inflammasome assembly induced by the mutated NLRP3, suggesting a feed- forward Ca2+ influx loop triggered by mutated NLRP3. Thus, the mutated NLRP3 forms cryo- sensitive aggregates to promote inflammasome assembly distinct from canonical NLRP3 inflammasome activation. [ABSTRACT FROM AUTHOR]

Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory syndrome caused by mutations of NLRP3 gene encoding cryopyrin. Familial cold autoinflammatory syndrome, the mildest form of CAPS, is characterized by cold-induced inflammation induced by the overproduction of IL-1β. However, the molecular mechanism of how mutated NLRP3 causes inflammasome activation in CAPS remains unclear. Here, we found that CAPS-associated NLRP3 mutants form cryo-sensitive aggregates that function as a scaffold for inflammasome activation. Cold exposure promoted inflammasome assembly and subsequent IL-1β release triggered by mutated NLRP3. While K+ efflux was dispensable, Ca2+ was necessary for mutated NLRP3-mediated inflammasome assembly. Notably, Ca2+ influx was induced during mutated NLRP3-mediated inflammasome assembly. Furthermore, caspase-1 inhibition prevented Ca2+ influx and inflammasome assembly induced by the mutated NLRP3, suggesting a feed-forward Ca2+ influx loop triggered by mutated NLRP3. Thus, the mutated NLRP3 forms cryo-sensitive aggregates to promote inflammasome assembly distinct from canonical NLRP3 inflammasome activation. [ABSTRACT FROM AUTHOR]

Cigarette smoking is a major risk factor for aortic aneurysm and dissection; however, no causative link between smoking and these aortic disorders has been proven. In the present study, we investigated the mechanism by which cigarette smoke affects vascular wall cells and found that cigarette smoke extract (CSE) induced a novel form of regulated cell death termed ferroptosis in vascular smooth muscle cells (VSMCs). CSE markedly induced cell death in A7r5 cells and primary rat VSMCs, but not in endothelial cells, which was completely inhibited by specific ferroptosis inhibitors [ferrostatin-1 (Fer-1) and Liproxstatin-1] and an iron chelator (deferoxamine). CSE-induced VSMC death was partially inhibited by a GSH precursor (N-acetyl cysteine) and an NADPH oxidase inhibitor [diphenyleneiodonium chloride (DPI)], but not by inhibitors of pan-caspases (ZVAD), caspase-1 (Z-YVAD), or necroptosis (necrostatin-1). CSE also upregulated IL-1, IL-6, TNF-, matrix metalloproteinase (MMP)-2, MMP-9, and TIMP-1 (tissue inhibitor of metalloproteinase)in A7r5 cells, which was inhibited by Fer-1. Furthermore, CSE induced the upregulation of Ptgs2 mRNA, lipid peroxidation, and intracellular GSH depletion, which are key features of ferroptosis. VSMC ferroptosis was induced by acrolein and methyl vinyl ketone, major constituents of CSE. Furthermore, CSE caused medial VSMC loss in ex vivo aortas. Electron microscopy analysis showed mitochondrial damage and fragmentation in medial VSMCs of CSE-treated aortas. All of these manifestations were partially restored by Fer-1. These findings demonstrate that ferroptosis is responsible for CSE-induced VSMC death and suggest that ferroptosis is a potential therapeutic target for preventing aortic aneurysm and dissection. [ABSTRACT FROM AUTHOR]

Cigarette smoking is a major risk factor for aortic aneurysm and dissection; however, no causative link between smoking and these aortic disorders has been proven. In the present study, we investigated the mechanism by which cigarette smoke affects vascular wall cells and found that cigarette smoke extract (CSE) induced a novel form of regulated cell death termed ferroptosis in vascular smooth muscle cells (VSMCs). CSE markedly induced cell death in A7r5 cells and primary rat VSMCs, but not in endothelial cells, which was completely inhibited by specific ferroptosis inhibitors [ferrostatin-1 (Fer-1) and Liproxstatin-1] and an iron chelator (deferoxamine). CSE-induced VSMC death was partially inhibited by a GSH precursor (N-acetyl cysteine) and an NADPH oxidase inhibitor [diphenyleneiodonium chloride (DPI)], but not by inhibitors of pan-caspases (ZVAD), caspase-1 (Z-YVAD), or necroptosis (necrostatin-1). CSE also upregulated IL-1, IL-6, TNF-, matrix metalloproteinase (MMP)-2, MMP-9, and TIMP-1 (tissue inhibitor of metalloproteinase)in A7r5 cells, which was inhibited by Fer-1. Furthermore, CSE induced the upregulation of Ptgs2 mRNA, lipid peroxidation, and intracellular GSH depletion, which are key features of ferroptosis. VSMC ferroptosis was induced by acrolein and methyl vinyl ketone, major constituents of CSE. Furthermore, CSE caused medial VSMC loss in ex vivo aortas. Electron microscopy analysis showed mitochondrial damage and fragmentation in medial VSMCs of CSE-treated aortas. All of these manifestations were partially restored by Fer-1. These findings demonstrate that ferroptosis is responsible for CSE-induced VSMC death and suggest that ferroptosis is a potential therapeutic target for preventing aortic aneurysm and dissection. [ABSTRACT FROM AUTHOR]

Inflammation plays a pivotal role in the pathophysiology of gastric aspiration-induced acute lung injury (ALI). However, its mechanism remains unclear. In this study, we investigated the role of NLRP3 inflammasome-driven IL-1β production in a mouse model of acid aspiration-induced inflammation and ALI. Acid aspiration-induced inflammatory responses and ALI in wild-type mice were significantly attenuated in IL-1β-/- mice, but not NLRP3-/- mice. In vitro experiments revealed that severe acidic stress (pH 1.75) induced the processing of pro-IL-1β into its 18-kDa mature form (p18-IL-1β), which was different from the caspase-1-processed 17-kDa form (p17-IL-1β), in human THP-1 macrophages and primary murine macrophages. Deficiency of NLRP3 and caspase-1 had no effect on acidic stress-produced IL-1β. The production of IL-1β by severe acidic stress was prevented by inhibitors of serine proteases [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], but not of cysteine proteases (E-64), cathepsin G, or inflammasome. The cathepsin D inhibitor pepstatin A inhibited IL-1β production induced by mild acidic stress (pH 6.2) or lactic acid, but not severe acidic stress. Using mass spectrometry and processing-site mutants of pro-IL-1β, we identified D109 as a novel cleavage site of pro-IL-1β in response to severe acidic stress and calculated the theoretical molecular mass of the mature form to be 18.2 kDa. The bioactivity of acidic stress-produced IL-1β was confirmed by its ability to promote p38 phosphorylation and chemokine upregulation in alveolar epithelial cells. These findings demonstrate a novel mechanism of acid-induced IL-1β production and inflammation independent of NLRP3 inflammasome and provide new insights into the therapeutic strategies for aspiration pneumonitis and ALI. [ABSTRACT FROM AUTHOR]

The nucleotide-binding oligomerization domain--like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a molecular platform that induces caspase-1 activation and subsequent IL-1b maturation, and is implicated in inflammatory diseases; however, little is known about the negative regulation of NLRP3 inflammasome activation. In this article, we identified an E3 ligase, Ariadne homolog 2 (ARIH2), as a posttranslational negative regulator of NLRP3 inflammasome activity in macrophages. ARIH2 interacted with NLRP3 via its NACHT domain (aa 220-575) in the NLRP3 inflammasome complex. In particular, we found that while using mutants of ARIH2 and ubiquitin, the really interesting new gene 2 domain of ARIH2 was required for NLRP3 ubiquitination linked through K48 and K63. Deletion of endogenous ARIH2 using CRISPR/Cas9 genome editing inhibited NLRP3 ubiquitination and promoted NLRP3 inflammasome activation, resulting in apoptosis-associated speck-like protein containing a caspase recruitment domain oligomerization, pro--IL-1β processing, and IL-1β production. Conversely, ARIH2 overexpression promoted NLRP3 ubiquitination and inhibited NLRP3 inflammasome activation. Our findings reveal a novel mechanism of ubiquitination-dependent negative regulation of the NLRP3 inflammasome by ARIH2 and highlight ARIH2 as a potential therapeutic target for inflammatory diseases. [ABSTRACT FROM AUTHOR]

Caspase-1 is a cysteine protease responsible for the processing of the proinflammatory cytokine interleukin-1β and activated by the formation of inflammasome complexes. Although several investigations have found a link between diet-induced obesity and caspase-1, the relationship remains controversial. Here, we found that mice deficient in caspase-1 were susceptible to high-fat diet-induced obesity with increased adiposity as well as normal lipid and glucose metabolism. Caspase-1 deficiency clearly promoted the infiltration of inflammatory macrophages and increased the production of C-C motif chemokine ligand 2 (CCL2) in the adipose tissue. The dominant cellular source of CCL2 was stromal vascular fraction rather than adipocytes in the adipose tissue. These findings demonstrate a critical role of caspase-1 in macrophage-driven inflammation in the adipose tissue and the development of obesity. These data provide novel insights into the mechanisms underlying inflammation in the pathophysiology of obesity. [ABSTRACT FROM AUTHOR]

Intestinal ischemia/reperfusion (I/R) injury is a life-threatening complication that leads to inflammation and remote organ damage. The NLRP3 inflammasome regulates the caspase-1-dependent release of IL-1b, an early mediator of inflammation after I/R injury. In this study, we investigated the role of the NLRP3 inflammasome in mice with intestinal I/R injury. Deficiency of NLRP3, ASC, caspase-1/11, or IL-1β prolonged survival after intestinal I/R injury, but neither NLRP3 nor caspase-1/11 deficiency affected intestinal inflammation. Intestinal I/R injury caused acute lung injury (ALI) characterized by inflammation, reactive oxygen species generation, and vascular permeability, which was markedly improved by NLRP3 deficiency. Bone marrow chimeric experiments showed that NLRP3 in non-bone marrow-derived cells was the main contributor to development of intestinal I/R-induced ALI. The NLRP3 inflammasome in lung vascular endothelial cells is thought to be important to lung vascular permeability. Using mass spectrometry, we identified intestinal I/R-derived lipid mediators that enhanced NLRP3 inflammasome activation in lung vascular endothelial cells. Finally, we confirmed that serum levels of these lipid mediators were elevated in patients with intestinal ischemia. To our knowledge, these findings provide new insights into the mechanism underlying intestinal I/R-induced ALI and suggest that endothelial NLRP3 inflammasome-driven IL-1b is a novel potential target for treating and preventing this disorder. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "Anti-inflammatory and Antiatherogenic Effects of the Inflammasome NLRP3 Inhibitor Arglabin in ApoE2.Ki Mice Fed a High-Fat Diet," by A. Abderrazak and colleagues in the 2015 issue.

Accumulating evidence suggests that IL-1β plays a pivotal role in the pathophysiology of hepatic ischemia--reperfusion (I/R) injury; however, the mechanism by which I/R triggers IL-1β production in the liver remains unclear. Recent data have shown that neutrophils contribute to hepatic I/R injury independently of the inflammasomes regulating IL-1β maturation. Thus, we investigated the role of neutrophils in IL-1β maturation and tissue injury in a murine model of hepatic I/R. IL-1b was released from the I/R liver and its deficiency reduced reactive oxygen species generation, apoptosis, and inflammatory responses, such as inflammatory cell infiltration and cytokine expression, thereby resulting in reduced tissue injury. Depletion of either macrophages or neutrophils also attenuated IL-1β release and hepatic I/R injury. In vitro experiments revealed that neutrophil-derived proteinases process pro--IL-1β derived from macrophages into its mature form independently of caspase-1. Furthermore, pharmacological inhibition of serine proteases attenuated IL-1β release and hepatic I/R injury in vivo. Taken together, the interaction between neutrophils and macrophages promotes IL-1β maturation and causes IL-1β--driven inflammation in the I/R liver. Both neutrophils and macrophages are indispensable in this process. These findings suggest that neutrophil-macrophage interaction is a therapeutic target for hepatic I/R injury and may also provide new insights into the inflammasome-independent mechanism of IL-1β maturation in the liver. [ABSTRACT FROM AUTHOR]

Supplemental oxygen inhalation is frequently used to treat severe respiratory failure; however, prolonged exposure to hyperoxia causes hyperoxic acute lung injury (HALI), which induces acute respiratory distress syndrome and leads to high mortality rates. Recent investigations suggest the possible role of NLRP3 inflammasomes, which regulate IL-1β production and lead to inflammatory responses, in the pathophysiology of HALI; however, their role is not fully understood. In this study, we investigated the role of NLRP3 inflammasomes in mice with HALI. Under hyperoxic conditions, NLRP3-/- mice died at a higher rate compared with wild-type and IL-1β-/- mice, and there was no difference in IL-1β production in their lungs. Under hyperoxic conditions, the lungs of NLRP3-/- mice exhibited reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, as well as increased and decreased expression of MMP-9 and Bcl-2, respectively. NLRP3-/- mice exhibited diminished expression and activation of Stat3, which regulates MMP-9 and Bcl-2, in addition to increased numbers of apoptotic alveolar epithelial cells. In vitro experiments revealed that alveolar macrophages and neutrophils promoted Stat3 activation in alveolar epithelial cells. Furthermore, NLRP3 deficiency impaired the migration of neutrophils and chemokine expression by macrophages. These findings demonstrate that NLRP3 regulates Stat3 signaling in alveolar epithelial cells by affecting macrophage and neutrophil function independent of IL-1β production and contributes to the pathophysiology of HALI. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]