Inhibition of 7-dehydrocholesterol reductase prevents hepatic ferroptosis under an active state of sterol synthesis.

Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl ß-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.

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.

Gasdermin D regulates soluble fms-like tyrosine kinase 1 release in macrophages.

Preeclampsia (PE) is a serious complication, and soluble fms-like tyrosine kinase (sFLT1) released from the placenta is one of the causes of PE pathology. Trophoblasts are the primary source of sFLT1; however, monocytes/macrophages exist enough in the placenta can also secrete sFLT1. Sterile inflammatory responses, especially NLRP3 inflammasome and its downstream gasdermin D (GSDMD)-regulated pyroptosis, may be involved in the development of PE pathology. In this study, we investigated whether human monocyte/macrophage cell line THP-1 cells secrete sFLT1 depending on the NLRP3 inflammasome and GSDMD. To differentiate THP-1 monocytes into macrophages, treatment with phorbol 12-myristate 13-acetate (PMA) induced sFLT1 with interleukin (IL)- 1ß, but did not induce cell lytic death. IL-1ß secretion induced by PMA inhibited by deletion of NLRP3 and inhibitors of NLRP3 and caspase-1, but deletion of NLRP3 and these inhibitors did not affect sFLT1 secretion in THP-1 cells. Both gene deletion and inhibition of GSDMD dramatically decreased IL-1ß and sFLT1 secretion from THP-1 cells. Treatment with CA074-ME (a cathepsin B inhibitor) also reduced the secretion of both sFLT1 and IL-1ß in THP-1 cells. In conclusion, THP-1 macrophages release sFLT1 in a GSDMD-dependent manner, but not in the NLRP3 inflammasome-dependent manner, and this sFLT1 release may be associated with the non-lytic role of GSDMD. In addition, sFLT1 levels induced by PMA are associated with lysosomal cathepsin B in THP-1 macrophages. We suggest that sFLT1 synthesis regulated by GSDMD are involved in the pathology of PE.

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-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.

A Myb enhancer-guided analysis of basophil and mast cell differentiation.

The transcription factor MYB is a crucial regulator of hematopoietic stem and progenitor cells. However, the nature of lineage-specific enhancer usage of the Myb gene is largely unknown. We identify the Myb -68 enhancer, a regulatory element which marks basophils and mast cells. Using the Myb -68 enhancer activity, we show a population of granulocyte-macrophage progenitors with higher potential to differentiate into basophils and mast cells. Single cell RNA-seq demonstrates the differentiation trajectory is continuous from progenitors to mature basophils in vivo, characterizes bone marrow cells with a gene signature of mast cells, and identifies LILRB4 as a surface marker of basophil maturation. Together, our study leads to a better understanding of how MYB expression is regulated in a lineage-associated manner, and also shows how a combination of lineage-related reporter mice and single-cell transcriptomics can overcome the rarity of target cells and enhance our understanding of gene expression programs that control cell differentiation in vivo.

dsDNA-induced AIM2 pyroptosis halts aberrant inflammation during rhabdomyolysis-induced acute kidney injury.

Rhabdomyolysis is a severe condition that commonly leads to acute kidney injury (AKI). While double-stranded DNA (dsDNA) released from injured muscle can be involved in its pathogenesis, the exact mechanism of how dsDNA contributes to rhabdomyolysis-induced AKI (RIAKI) remains obscure. A dsDNA sensor, absent in melanoma 2 (AIM2), forms an inflammasome and induces gasdermin D (GSDMD) cleavage resulting in inflammatory cell death known as pyroptosis. In this study using a mouse model of RIAKI, we found that Aim2-deficiency led to massive macrophage accumulation resulting in delayed functional recovery and perpetuating fibrosis in the kidney. While Aim2-deficiency compromised RIAKI-induced kidney macrophage pyroptosis, it unexpectedly accelerated aberrant inflammation as demonstrated by CXCR3+CD206+ macrophage accumulation and activation of TBK1-IRF3/NF-κB. Kidney macrophages with intact AIM2 underwent swift pyroptosis without IL-1ß release in response to dsDNA. On the other hand, dsDNA-induced Aim2-deficient macrophages escaped from swift pyroptotic elimination and instead engaged STING-TBK1-IRF3/NF-κB signalling, leading to aggravated inflammatory phenotypes. Collectively, these findings shed light on a hitherto unknown immunoregulatory function of macrophage pyroptosis. dsDNA-induced rapid macrophage cell death potentially serves as an anti-inflammatory program and determines the healing process of RIAKI.

Cryo-sensitive aggregation triggers NLRP3 inflammasome assembly in cryopyrin-associated periodic syndrome.

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-1ß. 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-1ß 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.

NLRP3 inflammasome is involved in testicular inflammation induced by lipopolysaccharide in mice.

PROBLEM: Systemic inflammation induced by infection, which is associated with testicular inflammation, predisposes males to subfertility. Recently, the nucleotide-binding oligomerization domain, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome was identified as a key mediator of inflammation, and excessive activation of the NLRP3 inflammasome was shown to contribute to the pathogenesis of a wide variety of diseases. However, the mechanisms underlying infectious inflammation in the testis remain unclear. We investigated the effect of lipopolysaccharide (LPS)-induced systemic inflammation on the role of the NLRP3 inflammasome in murine testes. METHOD OF STUDY: We performed in vivo and in vitro studies using an LPS-induced model of NLRP3 inflammasome activation and testicular inflammation. RESULTS: Intraperitoneal administration of LPS significantly impaired sperm motility in the epididymis of wild type (WT) and NLRP3-knockout (KO) mice. LPS administration stimulated interleukin (IL)-1ß production and secretion in the testes of WT mice, and these adverse effects were improved in the testes of NLRP3-KO mice. LPS administration also stimulated neutrophil infiltration as well as its chemoattractant C-C motif chemokine ligand 2 (CCL2) in WT testes, which were suppressed in NLRP3-KO testes. In in vitro cell culture, treatment with LPS and NLRP3 inflammasome activation significantly induced IL-1ß and CCL2 secretion from WT but not NLRP3-KO testicular cells. CONCLUSIONS: Taken together, our results suggest that testicular cells have the potential to secrete IL-1ß and CCL2 in an NLRP3 inflammasome-dependent manner and that these cytokines from the testis may further exacerbate testicular function, resulting in subfertility during infectious diseases.

Elevated S100A9 in preeclampsia induces soluble endoglin and IL-1ß secretion and hypertension via the NLRP3 inflammasome.

OBJECTIVES: Maternal systemic and placental inflammatory responses participate in the pathogenesis of hypertensive disorders of pregnancy including preeclampsia, a pregnancy-specific syndrome, although the role of inflammation remains unclear. The NLRP3 inflammasome has been implicated in the control of sterile inflammation involved in preeclampsia. In the present study, we hypothesized that S100A9, as major alarmin, are associated with the pathogenesis of preeclampsia and induction of a preeclampsia-like phenotype in pregnant mice. METHODS: Plasma were taken from normal pregnant women and preeclampsia patients. Human placental tissues, trophoblast cell line Sw.71 cells, and human umbilical vein endothelial cells (HUVEC) were treated with S100A9 with or without inhibitors associated with NLRP3 inflammasome. Pregnant mice were administered S100A9. RESULTS: S100A9 was elevated in plasma and released from placentas of preeclampsia patients. S100A9 activated the NLRP3 inflammasome, resulting in IL-1ß secretion, by human placental tissues and trophoblasts. In addition, secretion of soluble endoglin, a main contributor to the pathogenesis of preeclampsia, is regulated via S100A9-stimulated NLRP3 inflammasome activation in the human placenta and HUVECs. S100A9 administration significantly elevated maternal blood pressure and neutrophil accumulation within the placentas of pregnant mice, and both were significantly decreased in Nlrp3-knock out pregnant mice. CONCLUSION: The results of this study demonstrated that S100A9 acts as a danger signal to activate the NLRP3 inflammasome in the placenta, associating with hypertension during pregnancy.

ß-hydroxybutyrate suppresses NLRP3 inflammasome-mediated placental inflammation and lipopolysaccharide-induced fetal absorption.

The immune system contributes to the regulation of pregnancy, and the disruption of well-controlled immune functions leads to pregnancy complications. Recently, the nucleotide-binding oligomerization domain, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome mechanisms [(a protein complex of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1)] have been reported to play roles in controlling placental inflammation involved in pregnancy pathologies. The ketone body ß-hydroxybutyrate (BHB) can suppress NLRP3 inflammasome activation and improve various inflammatory diseases. Therefore, we hypothesized that BHB could suppress activation of the NLRP3 inflammasome in the placenta, resulting in the improvement of pregnancy complications. In human placental tissue culture, treatment with BHB suppressed the secretion levels of inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and IL-8, but did not affect the mRNA expression levels of NLRP3 inflammasome-associated factors. Treatment with BHB reduced IL-1ß secretion and the amount of mature IL-1ß protein induced by lipopolysaccharide (LPS) stimulation in the placenta. In human trophoblast cells, BHB reduced ASC and activated-caspase-1 expression, resulting in the inhibition of IL-1ß secretion. To investigate the effect of BHB during pregnancy, we used an animal model of LPS (100 µg/kg intraperitoneally [i.p.] on gestational day 14)-induced pregnancy complications. Administration of BHB (100 mg/kg i.p.) clearly suppressed the absorption rate and IL-1ß production in the placenta induced by LPS in pregnant mice. Moreover, LPS-induced pregnancy abnormalities were improved in NLRP3-deficient mice. These findings suggest that BHB play a role in reducing placental inflammation and pregnancy complications via inhibition of NLRP3 inflammasome activation.

Calciprotein Particles Induce IL-1ß/α-Mediated Inflammation through NLRP3 Inflammasome-Dependent and -Independent Mechanisms.

Calciprotein particles (CPPs) are nanoparticles composed of calcium phosphate crystals and fetuin-A and have been implicated in diseases associated with inflammation. In the current study, we investigated the molecular mechanisms underlying CPP-induced inflammation in mice. CPPs predominantly upregulated IL-1ß and IL-1α and provided priming and activation signals for the NLRP3 inflammasome in murine macrophages. Pharmacological and genetic inhibition of the NLRP3 inflammasome revealed that CPPs induced the release of IL-1ß and IL-1α via NLRP3 inflammasome-dependent and -independent mechanisms, respectively. CPPs also induced necrotic cell death, but gasdermin D was dispensable for CPP-induced IL-1ß release and necrotic cell death. Although phagocytosis of CPPs was required for CPP-induced IL-1ß/α release and necrotic cell death, lysosomal dysfunction and K+ efflux were mainly involved in CPP-induced NLRP3 inflammasome activation and subsequent IL-1ß release but not in CPP-induced IL-1α release and necrotic cell death. In vivo experiments showed that CPP administration evoked acute inflammatory responses characterized by neutrophil accumulation via both IL-1ß and IL-1α. In particular, CPP-induced neutrophil inflammation was mediated predominantly through an IL-1α-induced CXCL1/CXCR2 signaling pathway. These results provide new insights into the mechanism underlying CPP-induced inflammation and suggest that targeting both IL-1ß and IL-1α is necessary to regulate the CPP-induced inflammatory response and to treat CPP-associated inflammatory disorders.

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.

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.

GSDME-Dependent Incomplete Pyroptosis Permits Selective IL-1α Release under Caspase-1 Inhibition.

Pyroptosis is a form of regulated cell death that is characterized by gasdermin processing and increased membrane permeability. Caspase-1 and caspase-11 have been considered to be essential for gasdermin D processing associated with inflammasome activation. In the present study, we found that NLRP3 inflammasome activation induces delayed necrotic cell death via ASC in caspase-1/11-deficient macrophages. Furthermore, ASC-mediated caspase-8 activation and subsequent gasdermin E processing are necessary for caspase-1-independent necrotic cell death. We define this necrotic cell death as incomplete pyroptosis because IL-1ß release, a key feature of pyroptosis, is absent, whereas IL-1α release is induced. Notably, unprocessed pro-IL-1ß forms a molecular complex to be retained inside pyroptotic cells. Moreover, incomplete pyroptosis accompanied by IL-1α release is observed under the pharmacological inhibition of caspase-1 with VX765. These findings suggest that caspase-1 inhibition during NLRP3 inflammasome activation modulates forms of cell death and permits the release of IL-1α from dying cells.

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.

Role of the NLRP3 Inflammasome in Preeclampsia.

Reproduction involves tightly regulated series of events and the immune system is involved in an array of reproductive processes. Disruption of well-controlled immune functions leads to infertility, placental inflammation, and numerous pregnancy complications, including preeclampsia (PE). Inflammasomes are involved in the process of pathogen clearance and sterile inflammation. They are large multi-protein complexes that are located in the cytosol and play key roles in the production of the pivotal inflammatory cytokines, interleukin (IL)-1ß and IL-18, and pyroptosis. The nucleotide-binding oligomerization domain, leucine-rich repeat-, and pyrin domain-containing 3 (NLRP3) inflammasome is a key mediator of sterile inflammation induced by various types of damage-associated molecular patterns (DAMPs). Recent evidence indicates that the NLRP3 inflammasome is involved in pregnancy dysfunction, including PE. Many DAMPs (uric acid, palmitic acid, high-mobility group box 1, advanced glycation end products, extracellular vesicles, cell-free DNA, and free fatty acids) are increased and associated with pregnancy complications, especially PE. This review focuses on the role of the NLRP3 inflammasome in the pathophysiology of PE.

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.

Ferroptosis driven by radical oxidation of n-6 polyunsaturated fatty acids mediates acetaminophen-induced acute liver failure.

Acetaminophen (APAP) overdose is a common cause of drug-induced acute liver failure. Although hepatocyte cell death is considered to be the critical event in APAP-induced hepatotoxicity, the underlying mechanism remains unclear. Ferroptosis is a newly discovered type of cell death that is caused by a loss of cellular redox homeostasis. As glutathione (GSH) depletion triggers APAP-induced hepatotoxicity, we investigated the role of ferroptosis in a murine model of APAP-induced acute liver failure. APAP-induced hepatotoxicity (evaluated in terms of ALT, AST, and the histopathological score), lipid peroxidation (4-HNE and MDA), and upregulation of the ferroptosis maker PTGS2 mRNA were markedly prevented by the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1). Fer-1 treatment also completely prevented mortality induced by high-dose APAP. Similarly, APAP-induced hepatotoxicity and lipid peroxidation were prevented by the iron chelator deferoxamine. Using mass spectrometry, we found that lipid peroxides derived from n-6 fatty acids, mainly arachidonic acid, were elevated by APAP, and that auto-oxidation is the predominant mechanism of APAP-derived lipid oxidation. APAP-induced hepatotoxicity was also prevented by genetic inhibition of acyl-CoA synthetase long-chain family member 4 or α-tocopherol supplementation. We found that ferroptosis is responsible for APAP-induced hepatocyte cell death. Our findings provide new insights into the mechanism of APAP-induced hepatotoxicity and suggest that ferroptosis is a potential therapeutic target for APAP-induced acute liver failure.