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The spreading of pathology within and between brain areas is a hallmark of neurodegenerative disorders. In patients with Alzheimer’s disease, deposition of amyloid-β is accompanied by activation of the innate immune system and involves inflammasome-dependent formation of ASC specks in microglia. ASC specks released by microglia bind rapidly to amyloid-β and increase the formation of amyloid-β oligomers and aggregates, acting as an inflammation-driven cross-seed for amyloid-β pathology. Here we show that intrahippocampal injection of ASC specks resulted in spreading of amyloid-β pathology in transgenic double-mutant APP Swe PSEN1 dE9 mice. By contrast, homogenates from brains of APP Swe PSEN1 dE9 mice failed to induce seeding and spreading of amyloid-β pathology in ASC-deficient APP Swe PSEN1 dE9 mice. Moreover, co-application of an anti-ASC antibody blocked the increase in amyloid-β pathology in APP Swe PSEN1 dE9 mice. These findings support the concept that inflammasome activation is connected to seeding and spreading of amyloid-β pathology in patients with Alzheimer’s disease. Deposition and spreading of amyloid-β pathology in mice requires binding to microglia-released ASC specks. ASC specks bind to amyloid-β Innate immune activation in Alzheimer's disease involves the inflammasome-dependent formation of specks of adapter protein ASC (an apoptosis-associated speck-like protein containing a caspase recruitment domain) in microglial cells. Here it is shown that ASC specks released by microglia bind to amyloid-β and increase amyloid-β oligomer and aggregate formation, acting as an inflammation-driven cross-seed for amyloid-β pathology.

Phosphorylation of the NOD-like receptor NLRC4 is essential for activation of the NLRC4 inflammasome complex in response to bacterial stimuli. NLRC4 phosphorylation in innate immunity The NOD-like receptor NLRC4 acts as an inflammasome receptor and is an important part of the innate immune system. Vishva Dixit and colleagues now show that NLRC4 phosphorylation is essential for activation of the NLRC4 inflammasome complex in response to bacterial stimuli. Biochemical purification of the NLRC4-phosphorylating activity and a screen of kinase inhibitors suggest that PKCδ is the NLRC4 kinase involved. NLRC4 is a cytosolic member of the NOD-like receptor family that is expressed in innate immune cells. It senses indirectly bacterial flagellin and type III secretion systems, and responds by assembling an inflammasome complex that promotes caspase-1 activation and pyroptosis 1 , 2 , 3 , 4 , 5 , 6 . Here we use knock-in mice expressing NLRC4 with a carboxy-terminal 3×Flag tag to identify phosphorylation of NLRC4 on a single, evolutionarily conserved residue, Ser 533, following infection of macrophages with Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium ). Western blotting with a NLRC4 phospho-Ser 533 antibody confirmed that this post-translational modification occurs only in the presence of stimuli known to engage NLRC4 and not the related protein NLRP3 or AIM2. Nlrc4 −/− macrophages reconstituted with NLRC4 mutant S533A, unlike those reconstituted with wild-type NLRC4, did not activate caspase-1 and pyroptosis in response to S. typhimurium , indicating that S533 phosphorylation is critical for NLRC4 inflammasome function. Conversely, phosphomimetic NLRC4 S533D caused rapid macrophage pyroptosis without infection. Biochemical purification of the NLRC4-phosphorylating activity and a screen of kinase inhibitors identified PRKCD (PKCδ) as a candidate NLRC4 kinase. Recombinant PKCδ phosphorylated NLRC4 S533 in vitro , immunodepletion of PKCδ from macrophage lysates blocked NLRC4 S533 phosphorylation in vitro , and Prkcd −/− macrophages exhibited greatly attenuated caspase-1 activation and IL-1β secretion specifically in response to S. typhimurium . Phosphorylation-defective NLRC4 S533A failed to recruit procaspase-1 and did not assemble inflammasome specks 7 during S. typhimurium infection, so phosphorylation of NLRC4 S533 probably drives conformational changes necessary for NLRC4 inflammasome activity and host innate immunity.

Ali Gharavi and colleagues report a genome-wide association analysis of IgA nephropathy in over 20,000 individuals of European and East Asian ancestry. They identify genome-wide significant signals at three new loci near VAV3 , CARD9 and ITGAM - ITGAX and correlations between genetic risk and pathogen diversity. We performed a genome-wide association study (GWAS) of IgA nephropathy (IgAN), the most common form of glomerulonephritis, with discovery and follow-up in 20,612 individuals of European and East Asian ancestry. We identified six new genome-wide significant associations, four in ITGAM - ITGAX , VAV3 and CARD9 and two new independent signals at HLA-DQB1 and DEFA . We replicated the nine previously reported signals, including known SNPs in the HLA-DQB1 and DEFA loci. The cumulative burden of risk alleles is strongly associated with age at disease onset. Most loci are either directly associated with risk of inflammatory bowel disease (IBD) or maintenance of the intestinal epithelial barrier and response to mucosal pathogens. The geospatial distribution of risk alleles is highly suggestive of multi-locus adaptation, and genetic risk correlates strongly with variation in local pathogens, particularly helminth diversity, suggesting a possible role for host–intestinal pathogen interactions in shaping the genetic landscape of IgAN.

CARD9 and CARD11 drive immune cell activation by nucleating Bcl10 polymerization, but are held in an autoinhibited state prior to stimulation. Here, we elucidate the structural basis for this autoinhibition by determining the structure of a region of CARD9 that includes an extensive interface between its caspase recruitment domain (CARD) and coiled-coil domain. We demonstrate, for both CARD9 and CARD11, that disruption of this interface leads to hyperactivation in cells and to the formation of Bcl10-templating filaments in vitro, illuminating the mechanism of action of numerous oncogenic mutations of CARD11. These structural insights enable us to characterize two similar, yet distinct, mechanisms by which autoinhibition is relieved in the course of canonical CARD9 or CARD11 activation. We also dissect the molecular determinants of helical template assembly by solving the structure of the CARD9 filament. Taken together, these findings delineate the structural mechanisms of inhibition and activation within this protein family. CARD9 and CARD11 propagate signaling by nucleating Bcl10 polymerization in immune cells and are both held in an autoinhibited state prior to activation. Here, the authors combine structural, biochemical, and cell-based approaches to reveal the structural basis for CARD9/11 autoinhibition and show that the two proteins are activated through similar but distinct mechanisms.

Inflammasomes are supramolecular complexes that form in the cytosol in response to pathogen-associated and damage-associated stimuli, as well as other danger signals that perturb cellular homoeostasis, resulting in host defence responses in the form of cytokine release and programmed cell death (pyroptosis). Inflammasome activity is closely associated with numerous human disorders, including rare genetic syndromes of autoinflammation, cardiovascular diseases, neurodegeneration and cancer. In recent years, a range of inflammasome components and their functions have been discovered, contributing to our knowledge of the overall machinery. Here, we review the latest advances in inflammasome biology from the perspective of structural and mechanistic studies. We focus on the most well-studied components of the canonical inflammasome — NAIP–NLRC4, NLRP3, NLRP1, CARD8 and caspase-1 — as well as caspase-4, caspase-5 and caspase-11 of the noncanonical inflammasome, and the inflammasome effectors GSDMD and NINJ1. These structural studies reveal important insights into how inflammasomes are assembled and regulated, and how they elicit the release of IL-1 family cytokines and induce membrane rupture in pyroptosis.This Review highlights new insights into the biology of inflammasomes from the perspective of structural and mechanistic studies, revealing how the supramolecular complexes that activate inflammatory caspases are assembled and regulated, to induce cytokine maturation and release, as well as pyroptotic cell death.

NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase−1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA (conserved in UNC5, PIDD, and ankyrins) subdomain for self-oligomerization, which in turn form the platform to recruit the inflammasome adaptor ASC (apoptosis-associated speck-like protein containing a CARD) or caspase-1, respectively. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked, UPAs surrounding the filaments. Through biochemical and cellular approaches, we demonstrate that the UPA itself reduces the threshold needed for NLRP1-CT and CARD8-CT filament formation and signalling. Structural analyses provide insights on the mode of ASC recruitment by NLRP1-CT and the contrasting direct recruitment of caspase-1 by CARD8-CT. We also discover that subunits in the central NLRP1 CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. Finally, we engineer and determine the structure of an ASC CARD –caspase-1 CARD octamer, which suggests that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. Together these structures capture the architecture and specificity of the active NLRP1 and CARD8 inflammasomes in addition to key heteromeric CARD-CARD interactions governing inflammasome signalling. Pathogen triggered N-terminal degradation of NLRP1 and CARD8 by the proteasome releases their C-terminal UPA-CARD fragments (CT) to form the inflammasome, which in turn activates caspase-1. Here, the authors present the cryo-EM structures of the NLRP1-CT and CARD8-CT helical filaments as well as the ASC − caspase-1 octamer structure, which together with in vitro and cell based assays provide further insights into the architecture and specificity of the active NLRP1 and CARD8 inflammasomes.

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.

The C-type lectin receptor–Syk (spleen tyrosine kinase) adaptor CARD9 facilitates protective antifungal immunity within the central nervous system (CNS), as human deficiency in CARD9 causes susceptibility to fungus-specific, CNS-targeted infection. CARD9 promotes the recruitment of neutrophils to the fungus-infected CNS, which mediates fungal clearance. In the present study we investigated host and pathogen factors that promote protective neutrophil recruitment during invasion of the CNS by Candida albicans . The cytokine IL-1β served an essential function in CNS antifungal immunity by driving production of the chemokine CXCL1, which recruited neutrophils expressing the chemokine receptor CXCR2. Neutrophil-recruiting production of IL-1β and CXCL1 was induced in microglia by the fungus-secreted toxin Candidalysin, in a manner dependent on the kinase p38 and the transcription factor c-Fos. Notably, microglia relied on CARD9 for production of IL-1β, via both transcriptional regulation of Il1b and inflammasome activation, and of CXCL1 in the fungus-infected CNS. Microglia-specific Card9 deletion impaired the production of IL-1β and CXCL1 and neutrophil recruitment, and increased fungal proliferation in the CNS. Thus, an intricate network of host–pathogen interactions promotes antifungal immunity in the CNS; this is impaired in human deficiency in CARD9, which leads to fungal disease of the CNS. Innate immunity protects the central nervous system against fungal pathogens. Lionakis and colleagues identify Candidalysin, a Candida virulence factor that elicits microglial expression of the cytokine IL-1β and chemokine CXCL1 and facilitates neutrophil recruitment. Alteration of this pathway impairs antifungal responses.

Canonical inflammasomes are cytosolic supramolecular complexes that activate caspase-1 upon sensing extrinsic microbial invasions and intrinsic sterile stress signals. During inflammasome assembly, adaptor proteins ASC and NLRC4 recruit caspase-1 through homotypic caspase recruitment domain (CARD) interactions, leading to caspase-1 dimerization and activation. Activated caspase-1 processes proinflammatory cytokines and Gasdermin D to induce cytokine maturation and pyroptotic cell death. Here, we present cryo-electron microscopy (cryo-EM) structures of NLRC4 CARD and ASC CARD filaments mediated by conserved three types of asymmetric interactions (types I, II, and III). We find that the CARDs of these two adaptor proteins share a similar assembly pattern, which matches that of the caspase-1 CARD filament whose structure we defined previously. These data indicate a unified mechanism for downstream caspase-1 recruitment through CARD–CARD interactions by both adaptors. Using structure modeling, we further show that full-length NLRC4 assembles via two separate symmetries at its CARD and its nucleotide-binding domain (NBD), respectively.

Interleukin-1β (IL-1β) is a major cytokine that initiates and enhances inflammatory responses. Excessive IL-1β production is a characteristic of most chronic inflammatory diseases, including atherosclerosis, type 2 diabetes, and obesity, which affect a large proportion of the global population. The production of bioactive IL-1β is mediated by a caspase-1-activating complex known as an ‘inflammasome’. The NLRP3 inflammasome has been associated with several human inflammatory and autoimmune diseases and represents a potential therapeutic target for disrupting IL-1β production. We used molecular modeling guided by molecular dynamics simulations to design α-helical stapled peptides targeting the pyrin domain of the adaptor protein ASC to interrupt the development of its filament, which is crucial for NLRP3 inflammasome formation. The peptides were effectively internalized by human monocytic cells and efficiently suppressed the release of the inflammasome-regulated cytokines IL-1β and IL-18, following exogenous activation of the NLRP3 inflammasome. The peptides reduced ASC speck formation and caspase-1 processing thereby suppressing pro-IL-1β processing and release of active IL-1β. This is the first demonstration of the successful use of stapled peptides designed to target the adaptor protein ASC, and can be extended to other inflammatory pathways to disrupt excessive IL-1β production.