Yoshiko Mizushina

Role of NLRP3 Inflammasomes for Rhabdomyolysis-induced Acute Kidney Injury

Rhabdomyolysis is one of the main causes of community-acquired acute kidney injury (AKI). Although inflammation is involved in the pathogenesis of rhabdomyolysis-induced AKI (RIAKI), little is known about the mechanism that triggers inflammation during RIAKI. Recent evidence has indicated that sterile inflammation triggered by tissue injury can be mediated through multiprotein complexes called the inflammasomes. Therefore, we investigated the role of NLRP3 inflammasomes in the pathogenesis of RIAKI using a glycerol-induced murine rhabdomyolysis model. Inflammasome-related molecules were upregulated in the kidney of RIAKI. Renal tubular injury and dysfunction preceded leukocyte infiltration into the kidney during the early phase of RIAKI, and they were markedly attenuated in mice deficient in NLRP3, ASC, caspase-1, and interleukin (IL)-1β compared with those in wild-type mice. No difference in leukocyte infiltration was observed between wild-type and NLRP3-deficient mice. Furthermore, NLRP3 deficiency strikingly suppressed the expression of renal injury markers and inflammatory cytokines and apoptosis of renal tubular cells. These results demonstrated that NLRP3 inflammasomes contribute to inflammation, apoptosis, and tissue injury during the early phase of RIAKI and provide new insights into the mechanism underlying the pathogenesis of RIAKI.

NLRP3 Protein Deficiency Exacerbates Hyperoxia-induced Lethality through Stat3 Protein Signaling Independent of Interleukin-1β

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

NLRP3 Deficiency Reduces Macrophage Interleukin-10 Production and Enhances the Susceptibility to Doxorubicin-induced Cardiotoxicity.

NLRP3 inflammasomes recognize non-microbial danger signals and induce release of proinflammatory cytokine interleukin (IL)-1β, leading to sterile inflammation in cardiovascular disease. Because sterile inflammation is involved in doxorubicin (Dox)-induced cardiotoxicity, we investigated the role of NLRP3 inflammasomes in Dox-induced cardiotoxicity. Cardiac dysfunction and injury were induced by low-dose Dox (15 mg/kg) administration in NLRP3-deficient (NLRP3−/−) mice but not in wild-type (WT) and IL-1β−/− mice, indicating that NLRP3 deficiency enhanced the susceptibility to Dox-induced cardiotoxicity independent of IL-1β. Although the hearts of WT and NLRP3−/− mice showed no significant difference in inflammatory cell infiltration, macrophages were the predominant inflammatory cells in the hearts, and cardiac IL-10 production was decreased in Dox-treated NLRP3−/− mice. Bone marrow transplantation experiments showed that bone marrow-derived cells contributed to the exacerbation of Dox-induced cardiotoxicity in NLRP3−/− mice. In vitro experiments revealed that NLRP3 deficiency decreased IL-10 production in macrophages. Furthermore, adeno-associated virus-mediated IL-10 overexpression restored the exacerbation of cardiotoxicity in the NLRP3−/− mice. These results demonstrated that NLRP3 regulates macrophage IL-10 production and contributes to the pathophysiology of Dox-induced cardiotoxicity, which is independent of IL-1β. Our findings identify a novel role of NLRP3 and provided new insights into the mechanisms underlying Dox-induced cardiotoxicity.

Interferon-Tau Attenuates Uptake of Nanoparticles and Secretion of Interleukin-1β in Macrophages

Background Type I interferons (IFNs), including IFN-alpha (IFNA) and IFN-beta (IFNB), have anti-inflammatory properties and are used to treat patients with autoimmune and inflammatory disorders. However, little is known of the role of IFN-tau (IFNT), a type I IFN produced by ruminant animals for inflammation. Because IFNB has recently been shown to inhibit nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome activation and subsequent secretion of the potent inflammatory cytokine interleukin (IL)-1β, we examined the effects of ruminant IFNT on NLRP3 inflammasome-mediated IL-1β secretion in human THP-1 macrophages. Methods and Results IFNT dose-dependently inhibited IL-1β secretion induced by nano-silica, a well-known activators of NLRP3 inflammasomes, in human macrophages primed with lipopolysaccharide (LPS, TLR4 agonist) and Pam3CSK4 (TLR1/2 agonist). IFNT also suppressed phagocytosis of nano-silica and reactive oxygen species (ROS) generation. Western blot analysis showed that IFNT inhibited both pro-IL-1β and mature IL-1β. In addition, real-time RT-PCR analysis showed that IFNT suppressed IL-1β mRNA expression induced by LPS and Pam3CSK4. Although nano-silica particles did not induce IL-10 secretion, IFNT induced IL-10 secretion in a dose-dependent manner. Furthermore, IFNT-suppressed IL-1β secretion was restored by anti-IL-10 neutralizing antibody. Conclusions Ruminant IFNT inhibits NLRP3 inflammasome-driven IL-1β secretion in human macrophages via multiple pathways, including the uptake of nano-silica particles, generation of ROS, and IL-10-mediated inhibition of pro-IL-1β induction. It may be a therapeutic alternative to IFNA and IFNB.

Oligomerized CARD16 promotes caspase‐1 assembly and IL‐1β processing

Increasing evidence indicates that caspase recruitment domain (CARD)‐mediated caspase‐1 (CASP1) assembly is an essential process for its activation and subsequent interleukin (IL)‐1β release, leading to the initiation of inflammation. Both CARD16 and CARD17 were previously reported as inhibitory homologs of CASP1; however, their molecular function remains unclear. Here, we identified that oligomerization activity allows CARD16 to function as a CASP1 activator. We investigated the molecular characteristics of CARD16 and CARD17 in transiently transfected HeLa cells. Although both CARD16 and CARD17 interacted with CASP1CARD, only CARD16 formed a homo‐oligomer. Oligomerized CARD16 formed a filament‐like structure with CASP1CARD and a speck with apoptosis‐associated speck‐like protein containing a CARD. A filament‐like structure formed by CARD16 promoted CASP1 filament assembly and IL‐1β release. In contrast, CARD17 did not form a homo‐oligomer or filaments and inhibited CASP1‐dependent IL‐1β release. Mutated CARD16D27G, mimicking the CARD17 amino acid sequence, formed a homo‐oligomer but failed to form a filament‐like structure. Consequently, CARD16D27G weakly promoted CASP1 filament assembly and subsequent IL‐1β release. These results suggest that oligomerized CARD16 promotes CARD‐mediated molecular assembly and CASP1 activation.

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

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.

Immunoproteasome subunit LMP7 Deficiency Improves Obesity and Metabolic Disorders

Inflammation plays an important role in the development of obesity and metabolic disorders; however, it has not been fully understood how inflammation occurs and is regulated in their pathogenesis. Low-molecular mass protein-7 (LMP7) is a proteolytic subunit of the immunoproteasome that shapes the repertoire of antigenic peptides on major histocompatibility complex class I molecule. In this study, we investigated the role of LMP7 in the development of obesity and metabolic disorders using LMP7-deficient mice. LMP7 deficiency conveyed resistant to obesity, and improved glucose intolerance and insulin sensitivity in mice fed with high-fat diet (HFD). LMP7 deficiency decreased pancreatic lipase expression, increased fecal lipid contents, and inhibited the increase of plasma triglyceride levels upon oral oil administration or HFD feeding. Using bone marrow-transferred chimeric mice, we found that LMP7 in both bone marrow- and non-bone marrow-derived cells contributes to the development of HFD-induced obesity. LMP7 deficiency decreased inflammatory responses such as macrophage infiltration and chemokine expression while it increased serum adiponection levels. These findings demonstrate a novel role for LMP7 and provide new insights into the mechanisms underlying inflammation in the pathophysiology of obesity and metabolic disorders.