SummaryHost- and pathogen-associated cytoplasmic double-stranded DNA triggers the activation of a NALP3 (also known as cryopyrin and NLRP3)-independent inflammasome 1, 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 2, 3, 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 4, 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.

Pyroptosis is a caspase-1-dependent inflammatory form of cell death. The adapter protein ASC binds directly to caspase-1 and is critical for caspase-1 activation in response to a broad range of stimuli. To elucidate the mechanism of activation of caspase-1 by ASC and its exact role in macrophage pyroptosis, we performed time-lapse confocal bioimaging analysis on human THP-1 macrophages stably expressing an ASC-GFP fusion protein. We show that stimulation of these cells with several proinflammatory stimuli trigger the formation of a large supramolecular assembly of ASC, termed here pyroptosome. Only one distinct pyroptosome in each stimulated cell is formed, which rapidly recruits and activates caspase-1 resulting in pyroptosis and the release of the intracellular proinflammatory cytokines. The pyroptosome is largely composed of oligomerized ASC dimers. Dimerization of ASC is driven by subphysiological concentrations of potassium as in vitro incubation of purified recombinant ASC in the presence of subphysiological concentrations of potassium induces the assembly of a functional pyroptosome. Furthermore, stimulation of potassium efflux in THP-1 cells with potassium-depleting agents induces formation of the pyroptosome, while increasing potassium concentrations in the culture medium or pharmacological inhibition of this efflux inhibits its assembly. Our results establish that macrophage pyroptosis is mediated by a unique pyroptosome, distinct from the inflammasome.

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

Activation of the inflammasome generates the pro-inflammatory cytokines interleukin-1␤ 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-B 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-B inhibitors revealed that the synthetic IB kinase-␤ inhibitor Bay 11-7082 and structurally related vinyl sulfone compounds selectively inhibit NLRP3 inflammasome activity in macrophages independent of their inhibitory effect on NF-B 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.

Mutations in cryopyrin and pyrin proteins are responsible for several autoinflammatory disorders in humans, suggesting that these proteins play important roles in regulating inflammation. Using a HEK293 cell-based reconstitution system that stably expresses ASC and procaspase-1 we demonstrated that neither cryopyrin nor pyrin or their corresponding disease-associated mutants could significantly activate NF-jB in this system. However, both cryopyrin and two disease-associated cryopyrin mutants induced ASC oligomerization and ASC-dependent caspase-1 activation, with the disease-associated mutants being more potent than the wild-type (WT) cryopyrin, because of increased selfoligomerization. Contrary to the proposed anti-inflammatory activity of WT pyrin, our results demonstrated that pyrin, like cryopyrin, can also assemble an inflammasome complex with ASC and procaspase-1 leading to ASC oligomerization, caspase-1 activation and interleukin-1b processing. Thus, we propose that pyrin could function as a proinflammatory molecule.

The molecular mechanism by which mutations in the cytoskeleton-organizing protein PSTPIP1 cause the autoinflammatory PAPA syndrome is still elusive. Here, we demonstrate that PSTPIP1 requires the familial Mediterranean fever protein pyrin to assemble the ASC pyroptosome, a molecular platform that recruits and activates caspase-1. We provide evidence that pyrin is a cytosolic receptor for PSTPIP1. Pyrin exists as a homotrimer in an autoinhibited state due to intramolecular interactions between its pyrin domain (PYD) and B-box. Ligation by PSTPIP1, which is also a homotrimer, activates pyrin by unmasking its PYD, thereby allowing it to interact with ASC and facilitate ASC oligomerization into an active ASC pyroptosome. Because of their high binding affinity to pyrin's B-box, PAPA-associated PSTPIP1 mutants were found to be more effective than WT PSTPIP1 in inducing pyrin activation. Therefore, constitutive ligation and activation of pyrin by mutant PSTPIP1 proteins explain the autoinflammatory phenotype seen in PAPA syndrome.

NALP proteins, also known as NLRPs, belong to the CATERPILLER protein family involved, like Toll-like receptors, in the recognition of microbial molecules and the subsequent activation of inflammatory and immune responses. Current advances in the function of NALPs support the recently proposed model of a disease continuum bridging autoimmune and autoinflammatory disorders. Among these diseases, hereditary periodic fevers (HPFs) are Mendelian disorders associated with sequence variations in very few genes; these variations are mostly missense mutations whose deleterious effect, which is particularly difficult to assess, is often questionable. The growing number of identified sporadic cases of periodic fever syndrome, together with the lack of discriminatory clinical criteria, has greatly hampered the identification of new disease-causing genes, a step that is, however, essential for appropriate management of these disorders. Using a candidate gene approach, we identified nonambiguous mutations in NALP12 (i.e., nonsense and splice site) in two families with periodic fever syndromes. As shown by means of functional studies, these two NALP12 mutations have a deleterious effect on NF-B signaling. Overall, these data identify a group of HPFs defined by molecular defects in NALP12, opening up new ways to manage these disorders. The identification of these first NALP12 mutations in patients with autoinflammatory disorder also clearly demonstrates the crucial role of NALP12 in inflammatory signaling pathways, thereby assigning a precise function to this particular member of an emerging family of proteins whose putative biological properties are currently inferred essentially through in vitro means.genetics ͉ Mendelian disorder ͉ NLRP ͉ autoinflammatory disorder ͉ CATERPILLER

Sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular events in humans with type 2 diabetes (T2D); however, the underlying mechanism remains unclear. Activation of the NLR family, pyrin domain-containing 3 (NLRP3) inflammasome and subsequent interleukin (IL)-1β release induces atherosclerosis and heart failure. Here we show the effect of SGLT2 inhibitor empagliflozin on NLRP3 inflammasome activity. Patients with T2D and high cardiovascular risk receive SGLT2 inhibitor or sulfonylurea for 30 days, with NLRP3 inflammasome activation analyzed in macrophages. While the SGLT2 inhibitor's glucoselowering capacity is similar to sulfonylurea, it shows a greater reduction in IL-1β secretion compared to sulfonylurea accompanied by increased serum β-hydroxybutyrate (BHB) and decreased serum insulin. Ex vivo experiments with macrophages verify the inhibitory effects of high BHB and low insulin levels on NLRP3 inflammasome activation. In conclusion, SGLT2 inhibitor attenuates NLRP3 inflammasome activation, which might help to explain its cardioprotective effects.