Isoliquiritigenin inhibits NLRP3 inflammasome activation with CAPS mutations by suppressing caspase-1 activation and mutated NLRP3 aggregation.

The nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome contributes to the development of inflammatory diseases. Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory disease caused by NLRP3 gene mutations that results in excessive IL-1ß production. We previously identified isoliquiritigenin (ILG), a component of Glycyrrhiza uralensis extracts, as a potent inhibitor of the NLRP3 inflammasome. Here, we aimed to investigate whether ILG inhibits the activation of NLRP3 inflammasome caused by NLRP3 gene mutations. We demonstrated that ILG significantly inhibited NLRP3 inflammasome-mediated lactate dehydrogenase (LDH) release and IL-1ß production in two CAPS model THP-1 cell lines, NLRP3-D303N and NLRP3-L353P, in a dose-dependent manner. Interestingly, the NLRP3 inhibitor MCC950 inhibited LDH release and IL-1ß production in NLRP3-D303N cells, but not in NLRP3-L353P cells. Western blotting and caspase-1 activity assays showed that ILG, as well as caspase inhibitors, including Z-VAD and YVAD, suppressed caspase-1 activation. Notably, ILG prevented cryo-sensitive foci formation of NLRP3 without affecting the levels of intracellular Ca2+. We concluded that ILG effectively prevents the constitutive activation of the inflammasome associated with NLRP3 gene mutations by inhibiting the aggregation of cryo-sensitive mutated NLRP3.

Activation of the inflammasome drives peritoneal deterioration in a mouse model of peritoneal fibrosis.

During peritoneal dialysis (PD), the peritoneum is exposed to a bioincompatible dialysate, deteriorating the tissue and limiting the long-term effectiveness of PD. Peritoneal fibrosis is triggered by chronic inflammation induced by a variety of stimuli, including peritonitis. Exposure to PD fluid alters peritoneal macrophages phenotype. Inflammasome activation triggers chronic inflammation. First, it was determined whether inflammasome activation causes peritoneal deterioration. In the in vivo experiments, the increased expression of the inflammasome components, caspase-1 activity, and concomitant overproduction of IL-1ß and IL-18 were observed in a mouse model of peritoneal fibrosis. ASC-positive and F4/80-positive cells colocalized in the subperitoneal mesothelial cell layer. These macrophages expressed high CD44 levels indicating that the CD44-positive macrophages contribute to developing peritoneal deterioration. Furthermore, intravital imaging of the peritoneal microvasculature demonstrated that the circulating CD44-positive leukocytes may contribute to peritoneal fibrosis. Bone marrow transplantation in ASC-deficient mice suppressed inflammasome activation, thereby attenuating peritoneal fibrosis in a high glucose-based PD solution-injected mouse model. Our results suggest inflammasome activation in CD44-positive macrophages may be involved in developing peritoneal fibrosis. The inflammasome-derived pro-inflammatory cytokines might therefore serve as new biomarkers for developing encapsulating peritoneal sclerosis.

NLRP3 inflammasome-driven IL-1ß and IL-18 contribute to lipopolysaccharide-induced septic cardiomyopathy.

Sepsis is a life-threatening syndrome, and its associated mortality is increased when cardiac dysfunction and damage (septic cardiomyopathy [SCM]) occur. Although inflammation is involved in the pathophysiology of SCM, the mechanism of how inflammation induces SCM in vivo has remained obscure. NLRP3 inflammasome is a critical component of the innate immune system that activates caspase-1 (Casp1) and causes the maturation of IL-1ß and IL-18 as well as the processing of gasdermin D (GSDMD). Here, we investigated the role of the NLRP3 inflammasome in a murine model of lipopolysaccharide (LPS)-induced SCM. LPS injection induced cardiac dysfunction, damage, and lethality, which was significantly prevented in NLRP3-/- mice, compared to wild-type (WT) mice. LPS injection upregulated mRNA levels of inflammatory cytokines (Il6, Tnfa, and Ifng) in the heart, liver, and spleen of WT mice, and this upregulation was prevented in NLRP3-/- mice. LPS injection increased plasma levels of inflammatory cytokines (IL-1ß, IL-18, and TNF-α) in WT mice, and this increase was markedly inhibited in NLRP3-/- mice. LPS-induced SCM was also prevented in Casp1/11-/- mice, but not in Casp11mt, IL-1ß-/-, IL-1α-/-, or GSDMD-/- mice. Notably, LPS-induced SCM was apparently prevented in IL-1ß-/- mice transduced with adeno-associated virus vector expressing IL-18 binding protein (IL-18BP). Furthermore, splenectomy, irradiation, or macrophage depletion alleviated LPS-induced SCM. Our findings demonstrate that the cross-regulation of NLRP3 inflammasome-driven IL-1ß and IL-18 contributes to the pathophysiology of SCM and provide new insights into the mechanism underlying the pathogenesis of SCM.

Caspase-11 deficiency attenuates neutrophil recruitment into the atherosclerotic lesion in apolipoprotein E-deficient mice.

Caspase-11 is an inflammatory caspase that triggers an inflammatory response by regulating non-canonical NLRP3 inflammasome activation. Although the deficiency of both caspase-11 and caspase-1, another inflammatory caspase that functions as an executor of the inflammasome, prevents the development of atherosclerosis, the effect of caspase-11 deficiency alone on the development of atherosclerosis has not been fully evaluated. In the present study, we found that caspase-11 deficiency prevented the formation of the necrotic core, whereas it did not affect the development of atherosclerosis in Apoe-deficient mice. Notably, the infiltration of neutrophils into atherosclerotic lesions was attenuated by caspase-11 deficiency. RNA-seq analysis of stage-dependent expression of atherosclerotic lesions revealed that both upregulations of caspase-11 and neutrophil migration are common features of advanced atherosclerotic lesions. Furthermore, similar expression profiles were observed in unstable human plaque. These data suggest that caspase-11 regulates neutrophil recruitment and plaque destabilization in advanced atherosclerotic lesions.

NLRP3 Inflammasome Activation in Lung Vascular Endothelial Cells Contributes to Intestinal Ischemia/Reperfusion-Induced Acute Lung Injury.

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-1ß, 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-1ß 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-1ß is a novel potential target for treating and preventing this disorder.

NLRP3 Inflammasome as a Common Denominator of Atherosclerosis and Abdominal Aortic Aneurysm.

Atherosclerosis and abdominal aortic aneurysm (AAA) are multifactorial diseases characterized by inflammatory cell infiltration, matrix degradation, and thrombosis in the arterial wall. Although there are some differences between atherosclerosis and AAA, inflammation is a prominent common feature of these disorders. The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a cytosolic multiprotein complex that activates caspase-1 and regulates the release of proinflammatory cytokines interleukin (IL)-1ß and IL-18, as well as the induction of lytic cell death, termed pyroptosis, thereby leading to inflammation. Previous experimental and clinical studies have demonstrated that inflammation in atherosclerosis and AAA is mediated primarily through the NLRP3 inflammasome. Furthermore, recent results of the Canakinumab Anti-inflammatory Thrombosis and Outcome Study (CANTOS) showed that IL-1ß inhibition reduces systemic inflammation and prevents atherothrombotic events; this supports the concept that the NLRP3 inflammasome is a promising therapeutic target for cardiovascular diseases, including atherosclerosis and AAA. This review summarizes current knowledge with a focus on the role of the NLRP3 inflammasome in atherosclerosis and AAA, and discusses the prospects of NLRP3 inflammasome-targeted therapy.

Cutting Edge: G Protein Subunit ß 1 Negatively Regulates NLRP3 Inflammasome Activation.

The NLRP3 inflammasome has important roles in the pathogenesis of various inflammatory diseases. However, the regulatory mechanisms of the NLRP3 inflammasome are not fully understood. In this study, we attempted to identify molecules that interact with NLRP3 upon its activation. We identified G protein subunit ß 1 (GNB1), a downstream molecule of G protein-coupled receptors (GPCRs), which regulates the NLRP3 inflammasome activation. GNB1 was physically associated with NLRP3 via the pyrin domain of NLRP3. Activation of the NLRP3 inflammasome was enhanced in GNB1-knockdown or GNB1-deficient murine macrophages, although a lack of GNB1 did not affect activation of the AIM2 inflammasome. ASC oligomerization induced by NLRP3 was enhanced by GNB1 deficiency. Conversely, NLRP3-dependent ASC oligomerization was inhibited by the overexpression of GNB1. This study indicates that GNB1 negatively regulates NLRP3 inflammasome activation by suppressing NLRP3-dependent ASC oligomerization, and it provides a regulatory mechanism of the NLRP3 inflammasome.

Inflammasome-Independent and Atypical Processing of IL-1ß Contributes to Acid Aspiration-Induced Acute Lung Injury.

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-1ß 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-1ß-/- mice, but not NLRP3-/- mice. In vitro experiments revealed that severe acidic stress (pH 1.75) induced the processing of pro-IL-1ß into its 18-kDa mature form (p18-IL-1ß), which was different from the caspase-1-processed 17-kDa form (p17-IL-1ß), in human THP-1 macrophages and primary murine macrophages. Deficiency of NLRP3 and caspase-1 had no effect on acidic stress-produced IL-1ß. The production of IL-1ß 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-1ß 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-1ß, we identified D109 as a novel cleavage site of pro-IL-1ß 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-1ß 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-1ß production and inflammation independent of NLRP3 inflammasome and provide new insights into the therapeutic strategies for aspiration pneumonitis and ALI.

Deletion of inflammasome adaptor protein ASC enhances functional recovery after spinal cord injury in mice.

BACKGROUND: Research has revealed the crucial roles of inflammasomes in various central nervous system disorders. However, the role of inflammasomes in secondary damage following spinal cord injury (SCI) remains incompletely understood. METHODS: Here, we investigated the role of apoptosis-associated speck-like protein (ASC), an adaptor protein for inflammasome formation, after contusion SCI in ASC homozygous knockout (ASC-/-) mice. Contusion SCI was induced using a force of 60 kdyn, and recovery of open-field locomotor performance was evaluated using the nine-point Basso Mouse Scale (BMS). Bone marrow transplantation (BMT) was performed to create mice chimeric for ASC expression in bone marrow cells. RESULTS: Western blot analysis revealed that protein expression of NLRP3, ASC, Caspase-1, and IL-ß were increased in injured spinal cords compared with sham-control spinal cords at 1 day post injury (dpi). Double immunostaining showed that ASC expression was co-localized to cellular constituents of the spinal cord, including NeuN+ neurons, CD11b+ microglia/macrophages, GFAP+ astrocytes, and MOG+ oligodendrocytes. ASC-/- mice had significantly better locomotor function assessed by BMS than wild-type (WT) mice. ASC-/- mice also had significantly reduced levels of Nlrp3, Casp1, IL1b, Il-6, Tnfa, Cxcl1, and Ly6g mRNA compared with WT mice. BMT (WT→ASC-/-) mice had significantly better BMS scores than BMT (WT→WT) mice. BMT (ASC-/-→WT) mice also had significantly better BMS scores than BMT (WT→WT) mice. However, the statistical significance was limited to time points between 7 and 21 dpi. CONCLUSIONS: These results suggest that ASC-dependent inflammasome formation, especially in resident cells of the spinal cord, plays a pivotal role in the progression of secondary damage following SCI.

Endothelial Dysfunction Accelerates Impairment of Mitochondrial Function in Ageing Kidneys via Inflammasome Activation.

Chronic kidney disease is a common problem in the elderly and is associated with increased mortality. We have reported on the role of nitric oxide, which is generated from endothelial nitric oxide synthase (eNOS), in the progression of aged kidneys. To elucidate the role of endothelial dysfunction and the lack of an eNOS-NO pathway in ageing kidneys, we conducted experiments using eNOS and ASC-deficient mice. C57B/6 J mice (wild type (WT)), eNOS knockout (eNOS KO), and ASC knockout (ASC KO) mice were used in the present study. Then, eNOS/ASC double-knockout (eNOS/ASC DKO) mice were generated by crossing eNOS KO and ASC KO mice. These mice were sacrificed at 17-19 months old. The Masson positive area and the KIM-1 positive area tended to increase in eNOS KO mice, compared with WT mice, but not eNOS/ASC DKO mice. The COX-positive area was significantly reduced in eNOS KO mice, compared with WT and eNOS/ASC DKO mice. To determine whether inflammasomes were activated in infiltrating macrophages, the double staining of IL-18 and F4/80 was performed. IL-18 and F4/80 were found to be co-localised in the tubulointerstitial areas. Inflammasomes play a pivotal role in inflammaging in ageing kidneys. Furthermore, inflammasome activation may accelerate cellular senescence via mitochondrial dysfunction. The importance of endothelial function as a regulatory mechanism suggests that protection of endothelial function may be a potential therapeutic target.

Crucial role of NLRP3 inflammasome in a murine model of Kawasaki disease.

Kawasaki disease (KD) is a systemic febrile syndrome during childhood that is characterized by coronary arteritis. The etiopathogenesis of KD remains to be elucidated. NLRP3 inflammasome is a large multiprotein complex that plays a key role in IL-1ß-driven sterile inflammatory diseases. In the present study, we investigated the role of NLRP3 inflammasome in a murine model of KD induced by Candida albicans water-soluble fraction (CAWS) and found that NLRP3 inflammasome is required for the development of CAWS-induced vasculitis. CAWS administration induced IL-1ß production, caspase-1 activation, leukocyte infiltration, and fibrotic changes in the aortic root and coronary arteries, which were significantly inhibited by a deficiency of IL-1ß, NLRP3, and ASC. In vitro experiments showed that among cardiac resident cells, macrophages, but not endothelial cells or fibroblasts, expressed Dectin-2, but did not produce IL-1ß in response to CAWS. In contrast, CAWS induced caspase-1 activation and IL-1ß production in bone marrow-derived dendritic cells (BMDCs), which were inhibited by a specific caspase-1 inhibitor and a deficiency of NLRP3, ASC, and caspase-1. CAWS induced NLRP3 and pro-IL-1ß expression through a Dectin-2/Syk/JNK/NF-κB pathway, and caspase-1 activation and cleavage of pro-IL-1ß through Dectin-2/Syk/JNK-mediated mitochondrial ROS generation, indicating that CAWS induces the priming and activation of NLRP3 inflammasome in BMDCs. These findings provide new insights into the pathogenesis of KD vasculitis, and suggest that NLRP3 inflammasome may be a potential therapeutic target for KD.

Glucose regulates hypoxia-induced NLRP3 inflammasome activation in macrophages.

Although the intimate linkage between hypoxia and inflammation is well known, the mechanism underlying this linkage has not been fully understood. Nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome is an intracellular multiprotein complex that regulates interleukin-1ß (IL-1ß) secretion and pyroptosis, and is implicated in the pathogenesis of sterile inflammatory diseases. Here, we investigated the regulatory mechanism of NLRP3 inflammasome activation in response to hypoxia in macrophages. Severe hypoxia (0.1% O2 ) induced the processing of pro-IL-1ß, pro-caspase-1, and gasdermin D, as well as the release of IL-1ß and lactate dehydrogenase in lipopolysaccharide (LPS)-primed murine macrophages, indicating that hypoxia induces NLRP3 inflammasome-driven inflammation and pyroptosis. NLRP3 deficiency and a specific caspase-1 blockade inhibited hypoxia-induced IL-1ß release. Hypoxia-induced IL-1ß release and cell death were augmented under glucose deprivation, and an addition of glucose in the media negatively regulated hypoxia-induced IL-1ß release. Under hypoxia and glucose deprivation, hypoxia-induced glycolysis was not driven and subsequently, the intracellular adenosine triphosphates (ATPs) were depleted. Atomic absorption spectrometry analysis showed a reduction of intracellular K+ concentrations, indicating the K+ efflux occurring under hypoxia and glucose deprivation. Furthermore, hypoxia and glucose deprivation-induced IL-1ß release was significantly prevented by inhibition of K+ efflux and KATP channel blockers. In vivo experiments further revealed that IL-1ß production was increased in LPS-primed mice exposed to hypoxia (9.5% O2 ), which was prevented by a deficiency of NLRP3, an apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1. Our results demonstrate that NLRP3 inflammasome can sense intracellular energy crisis as a danger signal induced by hypoxia and glucose deprivation, and provide new insights into the mechanism underlying hypoxia-induced inflammation.

Iron overload as a risk factor for hepatic ischemia-reperfusion injury in liver transplantation: Potential role of ferroptosis.

Hepatic ischemia-reperfusion (I/R) injury is a major problem in liver transplantation (LT). Although hepatocyte cell death is the initial event in hepatic I/R injury, the underlying mechanism remains unclear. In the present study, we retrospectively analyzed the clinical data of 202 pediatric living donor LT and found that a high serum ferritin level, a marker of iron overload, of the donor is an independent risk factor for liver damage after LT. Since ferroptosis has been recently discovered as an iron-dependent cell death that is triggered by a loss of cellular redox homeostasis, we investigated the role of ferroptosis in a murine model of hepatic I/R injury, and found that liver damage, lipid peroxidation, and upregulation of the ferroptosis marker Ptgs2 were induced by I/R, and all of these manifestations were markedly prevented by the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) or α-tocopherol. Fer-1 also inhibited hepatic I/R-induced inflammatory responses. Furthermore, hepatic I/R injury was attenuated by iron chelation by deferoxamine and exacerbated by iron overload with a high iron diet. These findings demonstrate that iron overload is a novel risk factor for hepatic I/R injury in LT, and ferroptosis contributes to the pathogenesis of hepatic I/R injury.

Cigarette smoke extract induces ferroptosis in vascular smooth muscle cells.

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 (Z-VAD), caspase-1 (Z-YVAD), or necroptosis (necrostatin-1). CSE also upregulated IL-1ß, IL-6, TNF-α, 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.NEW & NOTEWORTHY Cigarette smoke extract (CSE)-induced cell death in rat vascular smooth muscle cells (VSMCs) was completely inhibited by specific ferroptosis inhibitors and an iron chelator. CSE also induced the upregulation of Ptgs2 mRNA, lipid peroxidation, and intracellular GSH depletion, which are key features of ferroptosis. CSE caused medial VSMC loss in ex vivo aortas. These findings demonstrate that ferroptosis is responsible for CSE-induced VSMC death.

ASC regulates platelet activation and contributes to thrombus formation independent of NLRP3 inflammasome.

BACKGROUND: Platelets are critical mediators of vascular homeostasis and thrombosis, and also contribute to the development of inflammation. NLRP3 inflammasome is a cytosolic multi-protein complex that consists of NLRP3, ASC and caspase-1, and regulates IL-1ß-mediated inflammation. METHOD AND RESULTS: Using two mouse models of thrombosis (i.e., occlusion of the middle cerebral artery and inferior vena cava), we found that thrombus formation was significantly enhanced in ASC-deficient (ASC-/-) mice, compared to that in wild-type (WT) and IL-1ß-/- mice. ASC deficiency had no effects on blood coagulation parameters (i.e., prothrombin time [PT] and activated partial thromboplastin time [APTT]). Platelets from WT mice express ASC, but neither NLRP3 nor caspase-1. ASC deficiency significantly enhanced the expression of P-selectin and GPIIb/IIIa in response to a GPVI agonist (collagen-related peptide [CRP]), but not to thrombin, in platelets. CRP induced ASC speck formation in WT platelets. ASC deficiency also enhanced cytosolic Ca2+ elevation and phosphorylation of ERK1/2 and Akt in platelets. CONCLUSION: Our results demonstrate that ASC negatively regulates GPVI signaling in platelets and enhances thrombus formation, independent of NLRP3 inflammasome and IL-1ß, and provide novel insights into the link between inflammation and thrombosis.

Palmitic acid activates NLRP3 inflammasome and induces placental inflammation during pregnancy in mice.

Maternal obesity is one of the major risk factors for pregnancy complications and is associated with low-grade chronic systemic inflammation due to higher levels of pro-inflammatory cytokines such as interleukin (IL)-1ß. Pregnant women with obesity have abnormal lipid profiles, characterized by higher levels of free fatty acids, especially palmitic acid (PA). Previously, we reported that PA stimulated IL-1ß secretion via activation of NLRP3 inflammasome in human placental cells. These observations led us to hypothesize that higher levels of PA induce NLRP3 inflammasome activation and placental inflammation, resulting in pregnancy complications. However, the effects of PA on NLRP3 inflammasome during pregnancy in vivo remain unclear. Therefore, PA solutions were administered intravenously into pregnant mice on day 12 of gestation. Maternal body weight was significantly decreased and absorption rates were significantly higher in PA-injected mice. The administration of PA significantly increased IL-1ß protein and the mRNA expression of NLRP3 inflammasome components (NLRP3, ASC, and caspase-1) within the placenta. In murine placental cell culture, PA significantly stimulated IL-1ß secretion, and this secretion was suppressed by a specific NLRP3 inhibitor (MCC950). Simultaneously, the number of macrophages/monocytes and neutrophils, together with the mRNA expression of these chemokines increased significantly in the placentas of PA-treated mice. Treatment with PA induced ASC assembling and IL-1ß secretion in macrophages, and this PA-induced IL-1ß secretion was significantly suppressed in NLRP3-knockdown macrophages. These results indicate that transient higher levels of PA exposure in pregnant mice activates NLRP3 inflammasome and induces placental inflammation, resulting in the incidence of absorption.

Exogenous nanoparticles and endogenous crystalline molecules as danger signals for the NLRP3 inflammasomes.

Inflammasome mechanisms are involved as some of the pathways of sterile inflammation. Inflammasomes are large multiprotein complexes in the cytosol and are a key system for the production of the pivotal inflammatory cytokines, interleukin (IL)-1ß and IL-18, and inflammatory cell death called pyroptosis. Although a number of inflammasomes have been described, the nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) is the most extensively investigated inflammasome. Exogenous pathogen-associated molecular patterns released during infection and endogenous crystalline danger/damage-associated molecular patterns (DAMPs) are well-known activators of NLRP3 inflammasomes. In addition, nanoparticle-associated molecular patterns (NAMPs), which are mediated by synthetic materials, including nanomaterials and nanoparticles, are proposed to be new danger signals of NLRP3 inflammasomes. Importantly, NAMP- and DAMP-triggered inflammation, a defining characteristic in inflammatory diseases, is termed as sterile inflammation because it occurs in the absence of foreign pathogens. This review focuses on the role of inflammasomes in exogenous NAMP- and endogenous crystalline DAMP-mediated sterile inflammation. Moreover, many regulatory mechanisms have been identified to attenuate NLRP3 inflammasomes. Therefore, we also summarize endogenous negative regulators of NLRP3 inflammasome activation, particularly induced by NAMPs or crystalline DAMPs.

Guidelines for evaluating myocardial cell death.

Cell death is a fundamental process in cardiac pathologies. Recent studies have revealed multiple forms of cell death, and several of them have been demonstrated to underlie adverse cardiac remodeling and heart failure. With the expansion in the area of myocardial cell death and increasing concerns over rigor and reproducibility, it is important and timely to set a guideline for the best practices of evaluating myocardial cell death. There are six major forms of regulated cell death observed in cardiac pathologies, namely apoptosis, necroptosis, mitochondrial-mediated necrosis, pyroptosis, ferroptosis, and autophagic cell death. In this article, we describe the best methods to identify, measure, and evaluate these modes of myocardial cell death. In addition, we discuss the limitations of currently practiced myocardial cell death mechanisms.

Role of TLR5 in inflammation and tissue damage after intestinal ischemia-reperfusion injury.

BACKGROUND: Intestinal ischemia/reperfusion (I/R) injury is a life-threatening complication that leads to inflammation and remote organ damage. However, the underlying mechanism is not yet fully understood. Toll-like receptor 5 (TLR5) is highly expressed in mucosa and recognizes flagellin, the main component of the bacterial flagella. Here, we investigated the role of TLR5 in inflammation and tissue damage after intestinal I/R injury using TLR5-deficient mice. METHODS AND RESULTS: Intestinal levels of TLR5 mRNA and flagellin protein were elevated in wild-type mice subjected to intestinal I/R. Although TLR5 deficiency had no effect on intestinal flagellin levels, it significantly attenuated intestinal injury and inflammatory responses after intestinal I/R. TLR5 deficiency also markedly improved survival in mice after intestinal I/R injury. In wild-type mice, intestinal I/R injury induced remote organ damage, particularly in the lung, which was attenuated by TLR5 deficiency. Furthermore, TLR5 deficiency prevented lung inflammatory responses and vascular permeability after intestinal I/R injury. CONCLUSION: These findings demonstrate a novel role of TLR5 and provide new insights into the mechanism underlying inflammation and tissue damage after intestinal I/R injury.

Ultrafast bone scintigraphy scan for detecting bone metastasis using a CZT whole-body gamma camera.

PURPOSE: To evaluate the feasibility of short whole-body bone scan acquisition times using a novel gamma camera with cadmium-zinc-telluride (CZT) semiconductor detectors. METHODS: We retrospectively enrolled 78 consecutive patients with prostate cancer who underwent bone scintigraphy using a whole-body gamma camera with CZT detectors. After acquisition of list-mode data with 180 s per bed position, anterior and posterior whole-body images were reconstructed using the first 5%, 10%, 25%, 50%, 75% and 100% of the list-mode data. Two experienced nuclear medicine physicians interpreted the images, and interrater agreement and the diagnostic value of the images were determined. Quantitative artificial neural network (ANN) values, bone scan indexes (BSI) and hotspot numbers (HsN) were also calculated by automated diagnostic software. RESULTS: Excellent interrater reliabilities of the visual assessments were obtained for the 100%, 75%, 50%, and 25% images (κ = 0.88, 0.88, 0.88 and 0.88, respectively). The 5% images also showed high diagnostic value (sensitivity 0.94, specificity 0.84 and accuracy 0.86). Intraclass correlation coefficients (ICC) between the 100% images and the reduced acquisition time images were evaluated in quantitative analyses, and excellent correlations were observed for ANN value in the 75% images (ICC 0.77), for BSI in all the reduced acquisition time images (75%, 50%, 25%, 10% and 5%; ICC 0.99, 0.99, 0.99, 0.96 and 0.75, respectively), and for HsN in the 75%, 50%, 25% and 10% images (ICC 0.99, 0.99, 0.98 and 0.90, respectively). CONCLUSION: Whole-body gamma cameras with CZT detectors have the potential to reduce image acquisition times and the dose of radioisotope injected for bone scans.