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.

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.

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.

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.

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.

Inflammasome Activation Aggravates Cutaneous Xanthomatosis and Atherosclerosis in ACAT1 (Acyl-CoA Cholesterol Acyltransferase 1) Deficiency in Bone Marrow.

Objective- ACAT1 (Acyl-CoA cholesterol acyltransferase 1) esterifies cellular free cholesterol, thereby converting macrophages to cholesteryl ester-laden foam cells in atherosclerotic lesions and cutaneous xanthoma. Paradoxically, however, loss of ACAT1 in bone marrow causes the aggravation of atherosclerosis and the development of severe cutaneous xanthoma in hyperlipidemic mice. Recently, it has been reported that cholesterol crystals activate NLRP3 (NACHT, LRR [leucine-rich repeats], and PYD [pyrin domain] domain-containing protein 3) inflammasomes, thereby contributing to the development of atherosclerosis. The present study aimed to clarify the role of NLRP3 inflammasomes in the worsening of atherosclerosis and cutaneous xanthoma induced by ACAT1 deficiency. Approach and Results- Ldlr-null mice were transplanted with bone marrow from WT (wild type) mice and mice lacking ACAT1, NLRP3, or both. After the 4 types of mice were fed high-cholesterol diets, we compared their atherosclerosis and skin lesions. The mice transplanted with Acat1-null bone marrow developed severe cutaneous xanthoma, which was filled with numerous macrophages and cholesterol clefts and had markedly increased expression of inflammatory cytokines, and increased atherosclerosis. Loss of NLRP3 completely reversed the cutaneous xanthoma, whereas it improved the atherosclerosis only partially. Acat1-null peritoneal macrophages showed enhanced expression of CHOP (C/EBP [CCAAT/enhancer binding protein] homologous protein) and TNF-α (tumor necrosis factor-α) but no evidence of inflammasome activation, after treatment with acetylated LDL (low-density lipoprotein). Conclusions- Elimination of ACAT1 in bone marrow-derived cells aggravates cutaneous xanthoma and atherosclerosis. The development of cutaneous xanthoma is induced mainly via the NLRP3 inflammasome activation.

Myeloid HMG-CoA (3-Hydroxy-3-Methylglutaryl-Coenzyme A) Reductase Determines Atherosclerosis by Modulating Migration of Macrophages.

Objective- Inhibition of HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) is atheroprotective primarily by decreasing plasma LDL (low-density lipoprotein)-cholesterol. However, it is unknown whether inhibition of HMGCR in myeloid cells contributes to this atheroprotection. We sought to determine the role of myeloid HMGCR in the development of atherosclerosis. Approach and Results- We generated mice with genetically reduced Hmgcr in myeloid cells ( Hmgcr m-/m-) using LysM (Cre) and compared various functions of their macrophages to those of Hmgcr fl/fl control mice. We further compared the extent of atherosclerosis in Hmgcr m-/ m- and Hmgcr fl/fl mice in the absence of Ldlr (LDL receptor). Hmgcr m-/ m- macrophages and granulocytes had significantly lower Hmgcr mRNA expression and cholesterol biosynthesis than Hmgcr fl/fl cells. In vitro, Hmgcr m-/ m- monocytes/macrophages had reduced ability to migrate, proliferate, and survive compared with Hmgcr fl/fl monocytes/macrophages. However, there was no difference in ability to adhere, phagocytose, store lipids, or polarize to M1 macrophages between the 2 types of macrophages. The amounts of plasma membrane-associated small GTPase proteins, such as RhoA (RAS homolog family member A), were increased in Hmgcr m-/ m- macrophages. In the setting of Ldlr deficiency, Hmgcr m-/ m- mice developed significantly smaller atherosclerotic lesions than Hmgcr fl/fl mice. However, there were no differences between the 2 types of mice either in plasma lipoprotein profiles or in the numbers of proliferating or apoptotic cells in the lesions in vivo. The in vivo migration of Hmgcr m-/ m- macrophages to the lesions was reduced compared with Hmgcr fl/fl macrophages. Conclusions- Genetic reduction of HMGCR in myeloid cells may exert atheroprotective effects primarily by decreasing the migratory activity of monocytes/macrophages to the lesions.

Saturated Fatty Acids Undergo Intracellular Crystallization and Activate the NLRP3 Inflammasome in Macrophages.

OBJECTIVE: Inflammation provoked by the imbalance of fatty acid composition, such as excess saturated fatty acids (SFAs), is implicated in the development of metabolic diseases. Recent investigations suggest the possible role of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3) inflammasome, which regulates IL-1ß (interleukin 1ß) release and leads to inflammation, in this process. Therefore, we investigated the underlying mechanism by which SFAs trigger NLRP3 inflammasome activation. APPROACH AND RESULTS: The treatment with SFAs, such as palmitic acid and stearic acid, promoted IL-1ß release in murine primary macrophages while treatment with oleic acid inhibited SFA-induced IL-1ß release in a dose-dependent manner. Analyses using polarized light microscopy revealed that intracellular crystallization was provoked in SFA-treated macrophages. As well as IL-1ß release, the intracellular crystallization and lysosomal dysfunction were inhibited in the presence of oleic acid. These results suggest that SFAs activate NLRP3 inflammasome through intracellular crystallization. Indeed, SFA-derived crystals activated NLRP3 inflammasome and subsequent IL-1ß release via lysosomal dysfunction. Excess SFAs also induced crystallization and IL-1ß release in vivo. Furthermore, SFA-derived crystals provoked acute inflammation, which was impaired in IL-1ß-deficient mice. CONCLUSIONS: These findings demonstrate that excess SFAs cause intracellular crystallization and subsequent lysosomal dysfunction, leading to the activation of the NLRP3 inflammasome, and provide novel insights into the pathogenesis of metabolic diseases.

Interaction of Neutrophils with Macrophages Promotes IL-1ß Maturation and Contributes to Hepatic Ischemia-Reperfusion Injury.

Accumulating evidence suggests that IL-1ß plays a pivotal role in the pathophysiology of hepatic ischemia-reperfusion (I/R) injury; however, the mechanism by which I/R triggers IL-1ß production in the liver remains unclear. Recent data have shown that neutrophils contribute to hepatic I/R injury independently of the inflammasomes regulating IL-1ß maturation. Thus, we investigated the role of neutrophils in IL-1ß maturation and tissue injury in a murine model of hepatic I/R. IL-1ß 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-1ß release and hepatic I/R injury. In vitro experiments revealed that neutrophil-derived proteinases process pro-IL-1ß derived from macrophages into its mature form independently of caspase-1. Furthermore, pharmacological inhibition of serine proteases attenuated IL-1ß release and hepatic I/R injury in vivo. Taken together, the interaction between neutrophils and macrophages promotes IL-1ß maturation and causes IL-1ß-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-1ß maturation in the liver.

ARIH2 Ubiquitinates NLRP3 and Negatively Regulates NLRP3 Inflammasome Activation in Macrophages.

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-1ß 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-1ß processing, and IL-1ß 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.

Serum Mac-2 binding protein glycosylation isomer predicts the activation of hepatic stellate cells after liver transplantation.

BACKGROUND AND AIM: Serum Mac-2 binding protein glycosylation isomer (M2BPGi) is a novel fibrosis marker for various chronic liver diseases. We investigated the ability of M2BPGi to predict liver fibrosis in liver transplant (LT) recipients. METHODS: A total of 116 liver biopsies were performed in 113 LT recipients. The serum level of M2BPGi was also measured on the same day. The median age at LT and liver biopsy was 1.1 and 11.8 years, respectively. Serum M2BPGi levels and liver fibrosis status using METAVIR fibrosis score were compared. Immunohistological evaluation by anti-α-smooth-muscle actin (αSMA) was performed, and the relationship between αSMA positive rate and serum M2BPGi levels was investigated. RESULTS: The median M2BPGi level was 0.78 (range, 0.22-9.50), and 65, 29, 16, 5, and 1 patient(s) had METAVIR fibrosis scores of F0, F1, F2, F3, and F4, respectively. In patients with F0 fibrosis, median M2BPGi level was 0.69 and was significantly lower than in patients with F1 (median 0.99, P < 0.01), F2 (median 1.00, P = 0.01), and F3 fibrosis (median 1.53, P < 0.01). Area-under-the-curve analysis of the ability of M2BPGi level to predict liver fibrosis grade were > F1: 0.716, > F2: 0.720, and > F3: 0.900. Three patients with acute cellular rejection showed high levels of M2BPGi, which decreased after the treatment. A positive correlation existed between M2BPGi levels and αSMA positive rate (r2  = 0.715, P < 0.01). CONCLUSION: Mac-2 binding protein glycosylation isomer is a novel liver fibrosis marker in LT recipients and is also increased in patients with acute liver injuries, especially acute cellular rejection, even when fibrosis is absent.

NLRP3 protein deficiency exacerbates hyperoxia-induced lethality through Stat3 protein signaling independent of interleukin-1ß.

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-1ß 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-1ß(-/-) mice, and there was no difference in IL-1ß 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-1ß production and contributes to the pathophysiology of HALI.