渡邊 幸子

Abstract LPS interacts with TLR4, which play important roles in host-against-pathogen immune responses, by binding to MD-2 and inducing an inflammatory response. In this study, to our knowledge, we found a novel function of lipoteichoic acid (LTA), a TLR2 ligand, that involves suppression of TLR4-mediated signaling independently of TLR2 under serum-free conditions. LTA inhibited NF-κB activation induced by LPS or a synthetic lipid A in a noncompetitive manner in human embryonic kidney 293 cells expressing CD14, TLR4, and MD-2. This inhibition was abrogated by addition of serum or albumin. LTAs from different bacterial sources also inhibited NF-κB activation, although LTA from Enterococcus hirae had essentially no TLR2-mediated NF-κB activation. The TLR2 ligands tripalmitoyl-Cys-Ser-Lys-Lys-Lys-Lys (Pam3CSK4) and macrophage-activating lipopeptide-2 (MALP-2) did not affect the TLR4-mediated NF-κB activation. In bone marrow–derived macrophages from TLR2−/− mice, LTA inhibited LPS-induced IκB-α phosphorylation and production of TNF, CXCL1/KC, RANTES, and IFN-β without affecting cell surface expression of TLR4. LTA did not suppress IL-1β–induced NF-κB activation mediated through signaling pathways shared with TLRs. LTAs including E. hirae LTA, but not LPS, induced association of TLR4/MD-2 complexes, which was suppressed by serum. LTA also increased association of MD-2, but not TLR4 molecules. These results demonstrate that, under serum-free conditions, LTA induces association of MD-2 molecules to promote formation of an inactive TLR4/MD-2 complex dimer that in turn prevents TLR4-mediated signaling. The presence of LTA that poorly induces TLR2-mediated activation but inhibits TLR4 signaling provides insight into the role of Gram-positive bacteria in suppressing inflammation induced by Gram-negative bacteria in organs such as the intestines where serum is absent.

Excessive activation of Toll-like receptor (TLR) leads to sepsis. Inflammatory responses to various microbiological components are initiated via different TLR proteins, but all TLR signals are transmitted by TRAF6. We reported that TRAF6 associated with ubiquitinated IRAK-1 undergoes proteasome-mediated degradation, suggesting that IRAK-1 has a negative regulatory role in TLR signaling. Here, we investigated the minimal structural region of IRAK-1 needed for degradation of TRAF6. The IRAK-1 protein contains an N-terminal death domain (DD; amino acids 1-102), a serine/proline/threonine-rich ProST domain (amino acids 103-197), a central kinase domain with an activation loop (amino acids 198-522), and the C-terminal C1 and C2 domains (amino acids 523-712), which contain two and one putative TRAF6-binding (TB) sites, respectively. TRAF6 degradation was severely impaired by deletion of the DD or C1 domain, and a mutant (DC1) containing only the DD and C1 domains could induce TRAF6 degradation. IRAK-1 mutants lacking the N- or C-terminal amino acids of DD induced little degradation. Deletion or mutation of TB2 (amino acids 585-591) in the C1 domain also inhibited TRAF6 degradation. An IRAK-1 mutant possessing only DD and TB2 did not induce TRAF6 degradation, although a mutant in which a short spacer was inserted between DD and TB2 induced TRAF6 degradation, which and DC1-induced degradation were inhibited by proteasome inhibitors. All IRAK-1 mutants that induced TRAF6 degradation could be immunoprecipitated with TRAF6. Meanwhile, NF-κB activation was observed for all IRAK-1 mutants-including those that failed to induce degradation and was severely impaired only for a mutant carrying mutations in both TBs of C1. These results demonstrate that only DD and TB2 separated by an appropriate distance can induce TRAF6 degradation. Conformational analysis of this minimal structural unit may aid development of low molecular compounds that negatively regulate TLR signaling.

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

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-1β-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-1β-/- 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-1β, and provide novel insights into the link between inflammation and thrombosis.

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-1β (IL-1β) 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-1β, pro-caspase-1, and gasdermin D, as well as the release of IL-1β 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-1β release. Hypoxia-induced IL-1β release and cell death were augmented under glucose deprivation, and an addition of glucose in the media negatively regulated hypoxia-induced IL-1β 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-1β release was significantly prevented by inhibition of K+ efflux and KATP channel blockers. In vivo experiments further revealed that IL-1β 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.

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

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.

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-1β release, a key feature of pyroptosis, is absent, whereas IL-1α release is induced. Notably, unprocessed pro-IL-1β 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.

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-1β, 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.

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.

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-1β-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-1β 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-1β, 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-1β in response to CAWS. In contrast, CAWS induced caspase-1 activation and IL-1β 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-1β expression through a Dectin-2/Syk/JNK/NF-κB pathway, and caspase-1 activation and cleavage of pro-IL-1β 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.

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.

Long-term peritoneal dialysis (PD) therapy leads to peritoneal inflammation and fibrosis. However, the mechanism underlying PD-related peritoneal inflammation and fibrosis remains unclear. NLRP3 inflammasome regulates the caspase-1-dependent release of interleukin-1β and mediates inflammation in various diseases. Here, we investigated the role of NLRP3 inflammasome in a murine model of PD-related peritoneal fibrosis induced by methylglyoxal (MGO). Inflammasome-related proteins were upregulated in the peritoneum of MGO-treated mice. MGO induced parietal and visceral peritoneal fibrosis in wild-type mice, which was significantly reduced in mice deficient in NLRP3, ASC, and interleukin-1β (IL-1β). ASC deficiency reduced the expression of inflammatory cytokines and fibrotic factors, and the infiltration of macrophages. However, myeloid cell-specific ASC deficiency failed to inhibit MGO-induced peritoneal fibrosis. MGO caused hemorrhagic ascites, fibrin deposition, and plasminogen activator inhibitor-1 upregulation, but all of these manifestations were inhibited by ASC deficiency. Furthermore, in vitro experiments showed that MGO induced cell death via the generation of reactive oxygen species in vascular endothelial cells, which was inhibited by ASC deficiency. Our results showed that endothelial NLRP3 inflammasome contributes to PD-related peritoneal inflammation and fibrosis, and provide new insights into the mechanisms underlying the pathogenesis of this disorder.

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

Kawasaki disease (KD), which is the leading cause of pediatric heart disease, is characterized by coronary vasculitis and subsequent aneurysm formation. Although intravenous immunoglobulin therapy is effective for reducing aneurysm formation, a certain number of patients are resistant to this therapy. Because interleukin-10 (IL-10) was identified as a negative regulator of cardiac inflammation in a murine model of KD induced by Candida albicans water-soluble fraction (CAWS), we investigated the effect of IL-10 supplementation in CAWS-induced vasculitis. Mice were injected intramuscularly with adeno-associated virus (AAV) vector encoding IL-10, then treated with CAWS. The induction of AAV-mediated IL-10 (AAV-IL-10) significantly attenuated the vascular inflammation and fibrosis in the aortic root and coronary artery, resulting in the improvement of cardiac dysfunction and lethality. The predominant infiltrating inflammatory cells in the vascular walls were Dectin-2+CD11b+ macrophages. In vitro experiments revealed that granulocyte/macrophage colony-stimulating factor (GM-CSF) induced Dectin-2 expression in bone marrow-derived macrophages and enhanced the CAWS-induced production of tumor necrosis factor-α (TNF-α) and IL-6. IL-10 had no effect on the Dectin-2 expression but significantly inhibited the production of cytokines. IL-10 also inhibited CAWS-induced phosphorylation of ERK1/2, but not Syk. Furthermore, the induction of AAV-IL-10 prevented the expression of TNF-α and IL-6, but not GM-CSF and Dectin-2 at the early phase of CAWS-induced vasculitis. These findings demonstrate that AAV-IL-10 may have therapeutic application in the prevention of coronary vasculitis and aneurysm formation, and provide new insights into the mechanism underlying the pathogenesis of KD.

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-1β (interleukin 1β) 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-1β release in murine primary macrophages while treatment with oleic acid inhibited SFA-induced IL-1β 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-1β 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-1β release via lysosomal dysfunction. Excess SFAs also induced crystallization and IL-1β release in vivo. Furthermore, SFA-derived crystals provoked acute inflammation, which was impaired in IL-1β-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.

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

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

Cardiac glycosides such as digoxin are Na+/K+-ATPase inhibitors that are widely used for the treatment of chronic heart failure and cardiac arrhythmias; however, recent epidemiological studies have suggested a relationship between digoxin treatment and increased mortality. We previously showed that nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes, which regulate caspase-1-dependent interleukin (IL)-1β release, mediate the sterile cardiovascular inflammation. Because the Na+/K+-ATPase is involved in inflammatory responses, we investigated the role of NLRP3 inflammasomes in the pathophysiology of cardiac glycoside-induced cardiac inflammation and dysfunction. The cardiac glycoside ouabain induced cardiac dysfunction and injury in wild-type mice primed with a low dose of lipopolysaccharide (LPS), although no cardiac dysfunction was observed in mice treated with either ouabain or LPS alone. Ouabain also induced cardiac inflammatory responses, such as macrophage infiltration and IL-1β release, when mice were primed with LPS. These cardiac manifestations were all significantly attenuated in mice deficient in IL-1β. Furthermore, deficiency of NLRP3 inflammasome components, NLRP3 and caspase-1, also attenuated ouabain-induced cardiac dysfunction and inflammation. In vitro experiments revealed that ouabain induced NLRP3 inflammasome activation as well as subsequent IL-1β release from macrophages, and this activation was mediated by K+ efflux. Our findings demonstrate that cardiac glycosides promote cardiac inflammation and dysfunction through NLRP3 inflammasomes and provide new insights into the mechanisms underlying the adverse effects of cardiac glycosides.

Aquaporin 3 (AQP3), a water/glycerol channel protein, is capable of transporting hydrogen peroxide (H2O2). Here, we show that AQP3-mediated intracellular H2O2 is involved in epidermal growth factor (EGF)-induced cell signaling and its dependent cell function in the EGF receptor (EGFR)-positive cancer cell lines A431 and H1666. AQP3 knockdown suppressed the transport into the cells of extracellular H2O2 produced in response to EGF in A431 and H1666 cells. EGF-induced Erk and Akt activation, which occurred through SHP2 and/or PTEN modulation, was impaired by AQP3 knockdown. Cell growth and migration induced by EGF stimulation were attenuated in AQP3 knockdown cells compared with those in control cells. Coincidentally, tumor growth of A431 cell xenografts in immunodeficient mice was decreased by AQP3 knockdown. Accordingly, a xenograft with AQP3 knockdown A431 cells significantly enhanced the survival of recipient mice compared with the transplantation with control cells. In addition, AQP3 associated with EGFR and NADPH oxidase 2, which we propose is linked to AQP3 producing a localized increase in intracellular H2O2 to function as a second messenger during EGFR cell signaling. Therefore, our findings suggest that AQP3 is required for EGF-EGFR cell signaling in cancer cells and is a therapeutic target for cancer progression.

Aquaporin (AQP) 9, a member of the transmembrane water channel family, is defined as a water/glycerol transporting protein. Some AQPs including AQP3 and AQP8 have been recently found to transport hydrogen peroxide (H2O2). Here we show that AQP9 facilitates the membrane transport of H2O2 in human and mice cells. Enforced expression of human AQP9 in Chinese hamster ovary-K1 potentiated the increase in cellular H2O2 after adding exogenous H2O2. In contrast, AQP9 knockdown by siRNA in human hepatoma HepG2 cells reduced the import of extracellular H2O2. In addition, the uptake of extracellular H2O2 was suppressed in erythrocytes and bone marrow-derived mast cells from AQP9 knockout mice compared with wild-type cells. Coincidentally, H2O2-induced cytotoxicity was attenuated by AQP9 deficiency in human and mice cells. Our findings implicate the involvement of AQP9 in H2O2 transport in human and mice cells.

Aquaporin-9 (AQP9), a water/glycerol channel protein, is expressed in several immune cells including neutrophils; however, its role in immune response remains unknown. Here we show the involvement of AQP9 in hapten-induced contact hypersensitivity (CHS), as a murine model of skin allergic contact dermatitis, using AQP9 knockout (AQP9(-/-)) mice. First, the CHS response to hapten dinitrofluorobenzene (DNFB) was impaired in AQP9(-/-) mice compared with wild-type (WT) mice. Adoptive transfer of sensitized AQP9(-/-) draining lymph node (dLN) cells into WT recipients resulted in a reduced CHS response, indicating impaired sensitization in AQP9(-/-) mice. Second, administration of WT neutrophils into AQP9(-/-) mice during sensitization rescued the impaired CHS response. Neutrophil recruitment to dLNs upon hapten application was attenuated by AQP9 deficiency. Coincidentally, AQP9(-/-) neutrophils showed a reduced CC-chemokine receptor 7 (CCR7) ligand-induced migration efficacy, which was attributed to the attenuated recruitment of neutrophils to dLNs. Furthermore, we found that neutrophil deficiency, observed in AQP9(-/-) or neutrophil-depleted mice, decreased IL-17A production by dLN cells, which might be responsible for T cell activation during a subsequent CHS response. Taken together, these findings suggest that AQP9 is required for the development of sensitization during cutaneous acquired immune responses via regulating neutrophil function.

Lipopolysaccharide (LPS) is recognized by CD14 with Toll-like receptor 4 (TLR4), and initiates 2 major pathways of TLR4 signaling, the MyD88-dependent and TRIF-dependent signaling pathways. The MyD88-dependent pathway induces inflammatory responses such as the production of TNF-α, IL-6, and IL-12 via the activation of NFκB and MAPK. The TRIF-dependent pathway induces the production of type-I IFN, and RANTES via the activation of IRF-3 and NFκB, and is also important for the induction of adaptive immune responses. CD14 plays a critical role in initiating the TRIF-dependent signaling pathway response to LPS, to support the internalization of LPS via endocytosis. Here, we clearly demonstrate that intracellular delivery of LPS by LPS-formulated liposomes (LPS-liposomes) initiate only TRIF-dependent signaling via clathrin-mediated endocytosis, independent of CD14. In fact, LPS-liposomes do not induce the production of TNF-α and IL-6 but induce RANTES production in peritoneal macrophages. Additionally, LPS-liposomes could induce adaptive immune responses effectively in CD14-deficient mice. Collectively, our results strongly suggest that LPS-liposomes are useful as a TRIF-dependent signaling-based immune adjuvant without inducing unnecessary inflammation.

Lipopolysaccharide (LPS) is responsible for many of the inflammatory responses and pathogenic effects of Gram-negative bacteria, however, it also induces protective immune responses. LPS induces the production of inflammatory cytokines such as TNF-α, IL-6, and IL-12 from dendritic cells (DCs) and macrophages. It is thought that IL-12 is required for one of the protective immune responses induced by LPS, the T helper 1 (Th1)-immune response, which include the production of IFN-γ from Th1cells and IgG2c class switching. Here, we clearly demonstrate that intracellular delivery of LPS by LPS-formulated liposomes (LPS-liposomes) does not induce the production of inflammatory cytokines from DCs, but enhances Th1-immune responses via type-I IFNs, independent of IL-12. Collectively, our results strongly suggest that LPS-liposomes can effectively induce Th1-immune responses without inducing unnecessary inflammation, and may be useful as an immune adjuvant to induce protective immunity.