Je-Wook Yu

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-1β 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.

The AIM2 inflammasome is critical for innate immunity to Francisella tularensis

Francisella tularensis, the causative agent of tularemia, infects host macrophages, which triggers production of the proinflammatory cytokines interleukin 1β (IL-1β) and IL-18. We elucidate here how host macrophages recognize F. tularensis and elicit this proinflammatory response. Using mice deficient in the DNA-sensing inflammasome component AIM2, we demonstrate here that AIM2 is required for sensing F. tularensis. AIM2-deficient mice were extremely susceptible to F. tularensis infection, with greater mortality and bacterial burden than that of wild-type mice. Caspase-1 activation, IL-1β secretion and cell death were absent in Aim2−/− macrophages in response to F. tularensis infection or the presence of cytoplasmic DNA. Our study identifies AIM2 as a crucial sensor of F. tularensis infection and provides genetic proof of its critical role in host innate immunity to intracellular pathogens.

Anti-inflammatory compounds parthenolide and Bay 11-7082 are direct inhibitors of the inflammasome.

Activation of the inflammasome generates the pro-inflammatory cytokines interleukin-1β and -18, which are important mediators of inflammation. Abnormal activation of the inflammasome leads to many inflammatory diseases, including gout, silicosis, neurodegeneration, and genetically inherited periodic fever syndromes. Therefore, identification of small molecule inhibitors that target the inflammasome is an important step toward developing effective therapeutics for the treatment of inflammation. Here, we show that the herbal NF-κB inhibitory compound parthenolide inhibits the activity of multiple inflammasomes in macrophages by directly inhibiting the protease activity of caspase-1. Additional investigations of other NF-κB inhibitors revealed that the synthetic IκB kinase-β inhibitor Bay 11-7082 and structurally related vinyl sulfone compounds selectively inhibit NLRP3 inflammasome activity in macrophages independent of their inhibitory effect on NF-κB activity. In vitro assays of the effect of parthenolide and Bay 11-7082 on the ATPase activity of NLRP3 demonstrated that both compounds inhibit the ATPase activity of NLRP3, suggesting that the inhibitory effect of these compounds on inflammasome activity could be mediated in part through their effect on the ATPase activity of NLRP3. Our results thus elucidate the molecular mechanism for the therapeutic anti-inflammatory activity of parthenolide and identify vinyl sulfones as a new class of potential therapeutics that target the NLRP3 inflammasome.

The Mitochondrial Antiviral Protein MAVS Associates with NLRP3 and Regulates Its Inflammasome Activity

NLRP3 assembles an inflammasome complex that activates caspase-1 upon sensing various danger signals derived from pathogenic infection, tissue damage, and environmental toxins. How NLRP3 senses these various stimuli is still poorly understood, but mitochondria and mitochondrial reactive oxygen species have been proposed to play a critical role in NLRP3 activation. In this article, we provide evidence that the mitochondrial antiviral signaling protein MAVS associates with NLRP3 and facilitates its oligomerization leading to caspase-1 activation. In reconstituted 293T cells, full-length MAVS promoted NLRP3-dependent caspase-1 activation, whereas a C-terminal transmembrane domain–truncated mutant of MAVS (MAVS-ΔTM) did not. MAVS, but not MAVS-ΔTM, interacted with NLRP3 and triggered the oligomerization of NLRP3, suggesting that mitochondrial localization of MAVS and intact MAVS signaling are essential for activating the NLRP3 inflammasome. Supporting this, activation of MAVS signaling by Sendai virus infection promoted NLRP3-dependent caspase-1 activation, whereas knocking down MAVS expression clearly attenuated the activation of NLRP3 inflammasome by Sendai virus in THP-1 and mouse macrophages. Taken together, our results suggest that MAVS facilitates the recruitment of NLRP3 to the mitochondria and may enhance its oligomerization and activation by bringing it in close proximity to mitochondrial reactive oxygen species.

Defective mitochondrial fission augments NLRP3 inflammasome activation.

Despite the fact that deregulated NLRP3 inflammasome activation contributes to the pathogenesis of chronic inflammatory or metabolic disorders, the underlying mechanism by which NLRP3 inflammasome signaling is initiated or potentiated remains poorly understood. Much attention is being paid to mitochondria as a regulator of NLRP3 inflammasome activation, but little is known about the role of mitochondrial dynamics for the inflammasome pathway. Here, we present evidence that aberrant mitochondrial elongation caused by the knockdown of dynamin-related protein 1 (Drp1) lead to a marked increase in NLRP3-dependent caspase-1 activation and interleukin-1-beta secretion in mouse bone marrow-derived macrophages. Conversely, carbonyl cyanide m-chlorophenyl hydrazone, a chemical inducer of mitochondrial fission, clearly attenuated NLRP3 inflammasome assembly and activation. Augmented activation of NLRP3 inflammasome by mitochondrial elongation is not resulted from the increased mitochondrial damages of Drp1-knockdown cells. Notably, enhanced extracellular signal-regulated kinase (ERK) signaling in Drp1-knockdown macrophages is implicated in the potentiation of NLRP3 inflammasome activation, possibly via mediating mitochondrial localization of NLRP3 to facilitate the assembly of NLRP3 inflammasome. Taken together, our results provide a molecular insight into the importance of mitochondrial dynamics in potentiating NLRP3 inflammasome activation, leading to aberrant inflammation.

Rotenone-induced Impairment of Mitochondrial Electron Transport Chain Confers a Selective Priming Signal for NLRP3 Inflammasome Activation

Background: Mitochondrial dysfunction is considered crucial for triggering NLRP3 inflammasome activation. Results: Rotenone-induced impairment of mitochondrial electron transport chain promotes NLRP3 inflammasome activation exclusively with ATP but not with other NLRP3-activating stimulators. Conclusion: High-grade mROS and hyperpolarization are essential for NLRP3 inflammasome activation upon rotenone-induced mitochondrial dysfunction. Significance: Mitochondrial impairment may selectively prime NLRP3 inflammasome activation, leading to many degenerative diseases. Mitochondrial dysfunction is considered crucial for NLRP3 inflammasome activation partly through its release of mitochondrial toxic products, such as mitochondrial reactive oxygen species (mROS)2 and mitochondrial DNA (mtDNA). Although previous studies have shown that classical NLRP3-activating stimulations lead to mROS generation and mtDNA release, it remains poorly understood whether and how mitochondrial damage-derived factors may contribute to NLRP3 inflammasome activation. Here, we demonstrate that impairment of the mitochondrial electron transport chain by rotenone primes NLRP3 inflammasome activation only upon costimulation with ATP and not with nigericin or alum. Rotenone-induced priming of NLRP3 in the presence of ATP triggered the formation of specklike NLRP3 or ASC aggregates and the association of NLRP3 with ASC, resulting in NLRP3-dependent caspase-1 activation. Mechanistically, rotenone confers a priming signal for NLRP3 inflammasome activation only in the context of aberrant high-grade, but not low-grade, mROS production and mitochondrial hyperpolarization. By contrast, rotenone/ATP-mediated mtDNA release and mitochondrial depolarization are likely to be merely an indication of mitochondrial damage rather than triggering factors for NLRP3 inflammasome activation. Our results provide a molecular insight into the selective contribution made by mitochondrial dysfunction to the NLRP3 inflammasome pathway.

Ribotoxic stress through p38 mitogen-activated protein kinase activates in vitro the human pyrin inflammasome

Background: Human pyrin is an important regulator of inflammation and is involved in the pathogenesis of familial Mediterranean fever (FMF). Results: Ribotoxic stress activates the human pyrin inflammasome. Conclusion: p38 MAPK signaling is required for assembly of the human pyrin inflammasome. Significance: Understanding the mechanism of activation of the pyrin inflammasome is crucial for the development of therapeutics to treat FMF. Human pyrin with gain-of-function mutations in its B30.2/SPRY domain causes the autoinflammatory disease familial Mediterranean fever by assembling an ASC-dependent inflammasome that activates caspase-1. Wild-type human pyrin can also form an inflammasome complex with ASC after engagement by autoinflammatory PSTPIP1 mutants. How the pyrin inflammasome is activated in the absence of disease-associated mutations is not yet known. We report here that ribotoxic stress triggers the assembly of the human pyrin inflammasome, leading to ASC oligomerization and caspase-1 activation in THP-1 macrophages and in a 293T cell line stably reconstituted with components of the pyrin inflammasome. Knockdown of pyrin and selective inhibition of p38 MAPK greatly attenuated caspase-1 activation by ribotoxic stress, whereas expression of the conditional mutant ΔMEKK3:ER* allowed the activation of caspase-1 without ribotoxic stress. Disruption of microtubules by colchicine also inhibited pyrin inflammasome activation by ribotoxic stress. Together, our results indicate that ribotoxic stress activates the human pyrin inflammasome through a mechanism that requires p38 MAPK signaling and microtubule stability.

25-hydroxycholesterol contributes to cerebral inflammation of X-linked adrenoleukodystrophy through activation of the NLRP3 inflammasome.

X-linked adrenoleukodystrophy (X-ALD), caused by an ABCD1 mutation, is a progressive neurodegenerative disorder associated with the accumulation of very long-chain fatty acids (VLCFA). Cerebral inflammatory demyelination is the major feature of childhood cerebral ALD (CCALD), the most severe form of ALD, but its underlying mechanism remains poorly understood. Here, we identify the aberrant production of cholesterol 25-hydroxylase (CH25H) and 25-hydroxycholesterol (25-HC) in the cellular context of CCALD based on the analysis of ALD patient-derived induced pluripotent stem cells and ex vivo fibroblasts. Intriguingly, 25-HC, but not VLCFA, promotes robust NLRP3 inflammasome assembly and activation via potassium efflux-, mitochondrial reactive oxygen species (ROS)- and liver X receptor (LXR)-mediated pathways. Furthermore, stereotaxic injection of 25-HC into the corpus callosum of mouse brains induces microglial recruitment, interleukin-1β production, and oligodendrocyte cell death in an NLRP3 inflammasome-dependent manner. Collectively, our results indicate that 25-HC mediates the neuroinflammation of X-ALD via activation of the NLRP3 inflammasome.

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

Significance Protective mechanisms of commensal bacteria against viral infection are limited to how immune inductive signals are provided by commensal bacteria for enhancing immunity. Whether, or how, commensal bacteria might influence the effector arm of immune responses remains unknown. Here, we demonstrate that dysbiosis within the vaginal microbiota results in severe impairment of antiviral protection against herpes simplex virus type 2 infection. IL-33 released into the vaginal tract after antibiotic treatment blocks the ability of effector T cells to migrate into the vaginal tissue and secrete the antiviral cytokine, IFN-γ. Thus, our findings suggest a previously unstudied role of commensal bacteria in the effector phase of the antiviral immune response against genital herpes. 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.

Non-transcriptional regulation of NLRP3 inflammasome signaling by IL-4

Th2 cytokine IL‐4 has been previously shown to suppress the production of proinflammatory cytokines in monocytes. However, the underlying molecular mechanism by which IL‐4 signaling antagonizes proinflammatory responses is poorly characterized. In particular, whether IL‐4 can modulate inflammasome signaling remains unknown. Here, we provide evidence that IL‐4 suppresses NLRP3‐dependent caspase‐1 activation and the subsequent IL‐1β secretion but does not inhibit absent in melanoma 2 (AIM2)‐ or NLRC4 (NOD‐like receptor family, CARD domain‐containing 4)‐dependent caspase‐1 activation in THP‐1 and mouse bone marrow‐derived macrophages. Upon lipopolysaccharide (LPS) or LPS/ATP stimulation, IL‐4 markedly inhibited the assembly of NLRP3 inflammasome, including NLRP3‐dependent ASC (apoptosis‐associated speck‐like protein containing a caspase recruitment domain) oligomerization, NLRP3‐ASC interaction and NLRP3 speck‐like oligomeric structure formation. The negative regulation of NLRP3 inflammasome by IL‐4 was not due to the impaired mRNA or protein production of NLRP3 and proinflammatory cytokines. Supporting this observation, IL‐4 attenuated NLRP3 inflammasome activation even in reconstituted NLRP3‐expressing macrophages in which NLRP3 expression is not transcriptionally regulated by TLR‐NF‐κB signaling. Furthermore, the IL‐4‐mediated suppression of NLRP3 inflammasome was independent of STAT6‐dependent transcription and mitochondrial reactive oxygen species (ROS). Instead, IL‐4 inhibited subcellular redistribution of NLRP3 into mitochondria and microtubule polymerization upon NLRP3‐activating stimulation. Our results collectively suggest that IL‐4 could suppress NLRP3 inflammasome activation in a transcription‐independent manner, thus providing an endogenous regulatory machinery to prevent excessive inflammasome activation.