Neutrophils Facilitate Prolonged Inflammasome Response in the DAMP-Rich Inflammatory Milieu

Aberrant inflammasome activation contributes to various chronic inflammatory diseases; however, pyroptosis of inflammasome-active cells promptly terminates local inflammasome response. Molecular mechanisms underlying prolonged inflammasome signaling thus require further elucidation. Here, we report that neutrophil-specific resistance to pyroptosis and NLRP3 desensitization can facilitate sustained inflammasome response and interleukin-1β secretion. Unlike macrophages, inflammasome-activated neutrophils did not undergo pyroptosis, indicated by using in vitro cell-based assay and in vivo mouse model. Intriguingly, danger-associated molecular patterns (DAMP)-rich milieu in the inflammatory region significantly abrogated NLRP3-activating potential of macrophages, but not of neutrophils. This macrophage-specific NLRP3 desensitization was associated with DAMP-induced mitochondrial depolarization that was not observed in neutrophils due to a lack of SARM1 expression. Indeed, valinomycin-induced compulsory mitochondrial depolarization in neutrophils restored inflammasome-dependent cell death and ATP-induced NLRP3 desensitization in neutrophils. Alongside prolonged inflammasome-activating potential, neutrophils predominantly secreted interleukin-1β rather than other proinflammatory cytokines upon NLRP3 stimulation. Furthermore, inflammasome-activated neutrophils did not trigger efferocytosis-mediated M2 macrophage polarization essential for the initiation of inflammation resolution. Taken together, our results indicate that neutrophils can prolong inflammasome response via mitochondria-dependent resistance to NLRP3 desensitization and function as major interleukin-1β-secreting cells in DAMP-rich inflammatory region.ope

Non-invasive in vivo imaging of caspase-1 activation enables rapid and spatiotemporal detection of acute and chronic inflammatory disorders

Inflammasome plays a critical role in diverse inflammatory disorders, including cancers and Alzheimer's disease. It is induced by various pathogenic insults and activates caspase-1, a hallmark executor of inflammasome. Here, we developed an activatable fluorescence probe for visualization of active caspase-1. This caspase-1 probe is biocompatible, efficiently delivered into cells and tissues, and specifically emits fluorescence upon caspase-1 activation as assessed in in vitro and in vivo models of inflammatory conditions. We demonstrated efficient in vivo imaging of caspase-1 activation in early stages of various inflammatory conditions of mice models, including endotoxin shock, inflammatory bowel disorder, transplanted cancer, and Alzheimer's disease. Notably, the caspase-1 probe enables detection of neuroinflammation in vivo two months earlier than cognitive impairments occur in Alzheimer's disease model. We detected significant fluorescence emitted from inflamed sites, as well as their draining lymph nodes, by macroscopic imaging analysis within 30 min after systemic injection of the probe. This novel synthetic probe could be applied for efficient and rapid detection of caspase-1 activity in a spatiotemporal way by non-invasive imaging.ope

Zika Virus Impairs Host NLRP3-mediated Inflammasome Activation in an NS3-dependent Manner

Zika virus (ZIKV) is a mosquito-borne flavivirus associated with severe neurological disorders including Guillain-Barré syndrome and microcephaly. The host innate immune responses against ZIKV infection are essential for protection; however, ZIKV has evolved strategies to evade and antagonize antiviral responses via its nonstructural (NS) proteins. Here, we demonstrated that ZIKV infection unexpectedly inhibits NLRP3-dependent inflammasome activation in bone marrow-derived macrophages and mixed glial cells from mouse brain. ZIKV infection led to increased transcript levels of proinflammatory cytokines such as IL-1β and IL-6 via activating NF-κB signaling. However, ZIKV infection failed to trigger the secretion of active caspase-1 and IL-1β from macrophages and glial cells even in the presence of LPS priming or ATP costimulation. Intriguingly, ZIKV infection significantly attenuated NLRP3-dependent, but not absent in melanoma 2-dependent caspase-1 activation and IL-1β secretion from both cells. ZIKV infection further blocked apoptosis-associated speck-like protein containing a caspase recruitment domain oligomerization in LPS/ATP-stimulated macrophages. Interestingly, expression of ZIKV NS3 protein reduced NLRP3-mediated caspase-1 activation and IL-1β secretion in macrophages, whereas NS1 and NS5 proteins showed no effects. Furthermore, NLRP3 was found to be degraded by the overexpression of ZIKV NS3 in 293T cells. Collectively, these results indicate that ZIKV evades host NLRP3 inflammasome-mediated innate immune responses in macrophages and glial cells; this may facilitate ZIKV's ability to enhance the replication and dissemination in these cells.ope

Dysbiosis-induced IL-33 contributes to impaired antiviral immunity in the genital mucosa

Commensal microbiota are well known to play an important role in antiviral immunity by providing immune inductive signals; however, the consequence of dysbiosis on antiviral immunity remains unclear. We demonstrate that dysbiosis caused by oral antibiotic treatment directly impairs antiviral immunity following viral infection of the vaginal mucosa. Antibiotic-treated mice succumbed to mucosal herpes simplex virus type 2 infection more rapidly than water-fed mice, and also showed delayed viral clearance at the site of infection. However, innate immune responses, including type I IFN and proinflammatory cytokine production at infection sites, as well as induction of virus-specific CD4 and CD8 T-cell responses in draining lymph nodes, were not impaired in antibiotic-treated mice. By screening the factors controlling antiviral immunity, we found that IL-33, an alarmin released in response to tissue damage, was secreted from vaginal epithelium after the depletion of commensal microbiota. This cytokine suppresses local antiviral immunity by blocking the migration of effector T cells to the vaginal tissue, thereby inhibiting the production of IFN-γ, a critical cytokine for antiviral defense, at local infection sites. These findings provide insight into the mechanisms of homeostasis maintained by commensal bacteria, and reveal a deleterious consequence of dysbiosis in antiviral immune defense.ope

Cobalt Chloride-induced Hypoxia Ameliorates NLRP3-Mediated Caspase-1 Activation in Mixed Glial Cultures

Hypoxia has been shown to promote inflammation, including the release of proinflammatory cytokines, but it is poorly investigated how hypoxia directly affects inflammasome signaling pathways. To explore whether hypoxic stress modulates inflammasome activity, we examined the effect of cobalt chloride (CoCl2)-induced hypoxia on caspase-1 activation in primary mixed glial cultures of the neonatal mouse brain. Unexpectedly, hypoxia induced by oxygen-glucose deprivation or CoCl2 treatment failed to activate caspase-1 in microglial BV-2 cells and primary mixed glial cultures. Of particular interest, CoCl2-induced hypoxic condition considerably inhibited NLRP3-dependent caspase-1 activation in mixed glial cells, but not in bone marrow-derived macrophages. CoCl2-mediated inhibition of NLRP3 inflammasome activity was also observed in the isolated brain microglial cells, but CoCl2 did not affect poly dA:dT-triggered AIM2 inflammasome activity in mixed glial cells. Our results collectively demonstrate that CoCl2-induced hypoxia may negatively regulate NLRP3 inflammasome signaling in brain glial cells, but its physiological significance remains to be determined.ope

AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA

Host- and pathogen-associated cytoplasmic double-stranded DNA triggers the activation of a NALP3 (also known as cryopyrin and NLRP3)-independent inflammasome, which activates caspase-1 leading to maturation of pro-interleukin-1beta and inflammation. The nature of the cytoplasmic-DNA-sensing inflammasome is currently unknown. Here we show that AIM2 (absent in melanoma 2), an interferon-inducible HIN-200 family member that contains an amino-terminal pyrin domain and a carboxy-terminal oligonucleotide/oligosaccharide-binding domain, senses cytoplasmic DNA by means of its oligonucleotide/oligosaccharide-binding domain and interacts with ASC (apoptosis-associated speck-like protein containing a CARD) through its pyrin domain to activate caspase-1. The interaction of AIM2 with ASC also leads to the formation of the ASC pyroptosome, which induces pyroptotic cell death in cells containing caspase-1. Knockdown of AIM2 by short interfering RNA reduced inflammasome/pyroptosome activation by cytoplasmic DNA in human and mouse macrophages, whereas stable expression of AIM2 in the non-responsive human embryonic kidney 293T cell line conferred responsiveness to cytoplasmic DNA. Our results show that cytoplasmic DNA triggers formation of the AIM2 inflammasome by inducing AIM2 oligomerization. This study identifies AIM2 as an important inflammasome component that senses potentially dangerous cytoplasmic DNA, leading to activation of the ASC pyroptosome and caspase-1.ope

Inhibition of apoptosis signal-regulating kinase 1 by nitric oxide through a thiol redox mechanism

Nitric oxide is an endogenous thiol-reactive molecule that modulates the functions of many regulatory proteins by a thiol-redox mechanism. NO has now been shown to inhibit the activation of apoptosis signal-regulating kinase 1 (ASK1) in murine fibrosarcoma L929 cells through such a mechanism. Exposure of L929 cells to interferon-gamma resulted in the endogenous production of NO and in inhibition of the activation of ASK1 by hydrogen peroxide. The interferon-gamma-induced inhibition of ASK1 activity was blocked by N(G)-nitro-l-arginine, an inhibitor of NO synthase. Furthermore, the NO donor S-nitro-N-acetyl-dl-penicillamine (SNAP) inhibited ASK1 activity in vitro, and this inhibition was reversed by thiol-reducing agents such as dithiothreitol and beta-mercaptoethanol. SNAP did not inhibit the kinase activities of MKK3, MKK6, or p38 in vitro. The inhibition of ASK1 by interferon-gamma was not changed by 1H- (1,2,4)oxadiazolo[4,3-alpha]quinoxalin-1-one, an inhibitor of guanylyl cyclase nor was it mimicked by 8-bromo-cyclic GMP. Site-directed mutagenesis revealed that replacement of cysteine 869 of ASK1 by serine rendered this protein resistant to the inhibitory effects both of interferon-gamma in intact cells and of SNAP in vitro. Co-immunoprecipitation data showed that NO production inhibited a binding of ASK1, but not ASK1(C869S), to MKK3 or MKK6. Moreover, interferon-gamma induced the S-nitrosylation of endogenous ASK1 in L929 cells. Together, these results suggest that NO mediates the interferon-gamma-induced inhibition of ASK1 in L929 cells through a thiolredox mechanism.ope

Distinct Features of Brain-Resident Macrophages: Microglia and Non-Parenchymal Brain Macrophages

Tissue-resident macrophages play an important role in maintaining tissue homeostasis and innate immune defense against invading microbial pathogens. Brain-resident macrophages can be classified into microglia in the brain parenchyma and non-parenchymal brain macrophages, also known as central nervous system-associated or border-associated macrophages, in the brain-circulation interface. Microglia and non-parenchymal brain macrophages, including meningeal, perivascular, and choroid plexus macrophages, are mostly produced during embryonic development, and maintained their population by self-renewal. Microglia have gained much attention for their dual roles in the maintenance of brain homeostasis and the induction of neuroinflammation. In particular, diverse phenotypes of microglia have been increasingly identified under pathological conditions. Single-cell phenotypic analysis revealed that microglia are highly heterogenous and plastic, thus it is difficult to define the status of microglia as M1/M2 or resting/activated state due to complex nature of microglia. Meanwhile, physiological function of non-parenchymal brain macrophages remain to be fully demonstrated. In this review, we have summarized the origin and signatures of brain-resident macrophages and discussed the unique features of microglia, particularly, their phenotypic polarization, diversity of subtypes, and inflammasome responses related to neurodegenerative diseases.ope

SGK1 inhibition in glia ameliorates pathologies and symptoms in Parkinson disease animal models

Astrocytes and microglia are brain-resident glia that can establish harmful inflammatory environments in disease contexts and thereby contribute to the progression of neuronal loss in neurodegenerative disorders. Correcting the diseased properties of glia is therefore an appealing strategy for treating brain diseases. Previous studies have shown that serum/ glucocorticoid related kinase 1 (SGK1) is upregulated in the brains of patients with various neurodegenerative disorders, suggesting its involvement in the pathogenesis of those diseases. In this study, we show that inhibiting glial SGK1 corrects the pro-inflammatory properties of glia by suppressing the intracellular NFκB-, NLRP3-inflammasome-, and CGAS-STING-mediated inflammatory pathways. Furthermore, SGK1 inhibition potentiated glial activity to scavenge glutamate toxicity and prevented glial cell senescence and mitochondrial damage, which have recently been reported as critical pathologic features of and therapeutic targets in Parkinson disease (PD) and Alzheimer disease (AD). Along with those anti-inflammatory/neurotrophic functions, silencing and pharmacological inhibition of SGK1 protected midbrain dopamine neurons from degeneration and cured pathologic synuclein alpha (SNCA) aggregation and PD-associated behavioral deficits in multiple in vitro and in vivo PD models. Collectively, these findings suggest that SGK1 inhibition could be a useful strategy for treating PD and other neurodegenerative disorders that share the common pathology of glia-mediated neuroinflammation.ope

Iron Chlorin e6 Scavenges Hydroxyl Radical and Protects Human Endothelial Cells against Hydrogen Peroxide Toxicity

Iron chlorin e6 (FeCe6) has recently been proposed to be potentially antimutagenic and antioxidative. However, the antioxidant property of FeCe6 has not been elucidated in detail. In this study, we investigated the ability of FeCe6 to scavenge hydroxyl radical and to protect biomolecules and mammalian cells from oxidative stress-mediated damage. In electron spin resonance (ESR) experiments, FeCe6 showed excellent hydroxyl radical scavenging activity, whereas its iron-deficient molecule, chlorin e6 (Ce6) showed little effect. FeCe6 also significantly reduced hydroxyl radical-induced thiobarbituric acid reactive substance (TBARS) formation and benzoate hydroxylation in a dose-dependent manner. The rate constant for reaction between FeCe6 and hydroxyl radical was measured as 8.5 x 10(10) M(-1) s(-1) by deoxyribose degradation method, and this value was much higher than that of most hydroxyl radical scavengers. Superoxide dismutase (SOD) activity of FeCe6 was also confirmed by ESR study and cytochrome c reduction assay, but its in vitro activity appeared to be less efficient in comparison with other well-known SOD mimics. In addition, FeCe6 appreciably diminished hydroxyl radical-induced DNA single-strand breakage and protein degradation in Fe-catalyzed and Cu-catalyzed Fenton systems, and it significantly protected human endothelial cells against hydrogen peroxide (H2O2) toxicity. These results suggest that FeCe6 is a novel hydroxyl radical scavenger and may be useful for preventing oxidative injury in biological systems.ope