Explore the vast range of titles available.

Robust innate immune detection of rapidly evolving pathogens is critical for host defense. Nucleotide-binding domain leucine-rich repeat (NLR) proteins function as cytosolic innate immune sensors in plants and animals. However, the structural basis for ligand-induced NLR activation has so far remained unknown. NAIP5 (NLR family, apoptosis inhibitory protein 5) binds the bacterial protein flagellin and assembles with NLRC4 to form amultiprotein complex called an inflammasome. Here we report the cryo–electron microscopy structure of the assembled ~1.4-megadalton flagellin-NAIP5-NLRC4 inflammasome, revealing how a ligand activates an NLR. Six distinct NAIP5 domains contact multiple conserved regions of flagellin, prying NAIP5 into an open and active conformation. We show that innate immune recognition of multiple ligand surfaces is a generalizable strategy that limits pathogen evolution and immune escape.

The NLR family apoptosis inhibitory proteins (NAIPs) bind conserved bacterial ligands, such as the bacterial rod protein PrgJ, and recruit NLR family CARD-containing protein 4 (NLRC4) as the inflammasome adapter to activate innate immunity. We found that the PrgJ-NAIP2-NLRC4 inflammasome is assembled into multisubunit disk-like structures through a unidirectional adenosine triphosphatase polymerization, primed with a single PrgJ-activated NAIP2 per disk. Cryo–electron microscopy (cryo-EM) reconstruction at subnanometer resolution revealed a ∼90° hinge rotation accompanying NLRC4 activation. Unlike in the related heptameric Apaf-1 apoptosome, in which each subunit needs to be conformationally activated by its ligand before assembly, a single PrgJ-activated NAIP2 initiates NLRC4 polymerization in a domino-like reaction to promote the disk assembly. These insights reveal the mechanism of signal amplification in NAIP-NLRC4 inflammasomes.

Responding to stimuli, nucleotide-binding domain and leucine-rich repeat–containing proteins (NLRs) oligomerize into multiprotein complexes, termed inflammasomes, mediating innate immunity. Recognition of bacterial pathogens by NLR apoptosis inhibitory proteins (NAIPs) induces NLR family CARD domain–containing protein 4 (NLRC4) activation and formation of NAIP-NLRC4 inflammasomes. The wheel-like structure of a PrgJ-NAIP2-NLRC4 complex determined by cryogenic electron microscopy at 6.6 angstrom reveals that NLRC4 activation involves substantial structural reorganization that creates one oligomerization surface (catalytic surface). Once activated, NLRC4 uses this surface to catalyze the activation of an inactive NLRC4, self-propagating its active conformation to form the wheel-like architecture. NAIP proteins possess a catalytic surface matching the other oligomerization surface (receptor surface) of NLRC4 but not those of their own, ensuring that one NAIP is sufficient to initiate NLRC4 oligomerization.

The nucleotide-binding domain (NBD), leucine rich repeat (LRR) domain containing protein family (NLR family) apoptosis inhibitory proteins (NAIPs) are cytosolic receptors that play critical roles in the host defense against bacterial infection. NAIPs interact with conserved bacterial ligands and activate the NLR family caspase recruitment domain containing protein 4 (NLRC4) to initiate the NAIP—NLRC4 inflammasome pathway. Here we found the process of NAIP activation is completely different from NLRC4. Our cryo-EM structure of unliganded mouse NAIP5 adopts an unprecedented wide-open conformation, with the nucleating surface fully exposed and accessible to recruit inactive NLRC4. Upon ligand binding, the winged helix domain (WHD) of NAIP5 undergoes roughly 20° rotation to form a steric clash with the inactive NLRC4, which triggers the conformational change of NLRC4 from inactive to active state. We also show the rotation of WHD places the 17–18 loop at a position that directly bind the active NLRC4 and stabilize the NAIP5–NLRC4 complex. Overall, these data provide structural mechanisms of inactive NAIP5, the process of NAIP5 activation and NAIP-dependent NLRC4 activation. The cryo-EM structure of unliganded mouse NAIP5 reveals the mechanism for NAIP—NLRC4 inflammasome activation in which ligand binding drives a roughly 20° rotation in the WHD–HD2–LRR domains, resulting in the formation of a steric clash to activate NLRC4 and generating new interactions to stabilize the NAIP5–NLRC4 complex.

The human neuronal apoptosis inhibitory protein (NAIP) gene is no longer principally considered a member of the Inhibitor of Apoptosis Protein (IAP) family, as its domain structure and functions in innate immunity also warrant inclusion in the Nod-Like Receptor (NLR) superfamily. NAIP is located in a region of copy number variation, with one full length and four partly deleted copies in the reference human genome. We demonstrate that several of the NAIP paralogues are expressed, and that novel transcripts arise from both internal and upstream transcription start sites. Remarkably, two internal start sites initiate within Alu short interspersed element (SINE) retrotransposons, and a third novel transcription start site exists within the final intron of the GUSBP1 gene, upstream of only two NAIP copies. One Alu functions alone as a promoter in transient assays, while the other likely combines with upstream L1 sequences to form a composite promoter. The novel transcripts encode shortened open reading frames and we show that corresponding proteins are translated in a number of cell lines and primary tissues, in some cases above the level of full length NAIP. Interestingly, some NAIP isoforms lack their caspase-sequestering motifs, suggesting that they have novel functions. Moreover, given that human and mouse NAIP have previously been shown to employ endogenous retroviral long terminal repeats as promoters, exaptation of Alu repeats as additional promoters provides a fascinating illustration of regulatory innovations adopted by a single gene.

Phylogenetic analyses of conserved [neuronal apoptosis inhibitory protein (NAIP), MHC class II transcription activator (CIITA), incompatibility locus protein from Podospora anserina (HET-E), and telomerase-associated protein (TP1)] (NACHT) domains were used to reconstruct the evolutionary history of vertebrate NACHT-containing proteins. The results supported the hypothesis that NOD3 is basal to the other NACHT-containing proteins found in tetrapods. The latter formed two strongly supported clusters or subfamilies, here designated NALP and nucleotide-binding oligomerization domain (NOD). The presence of apparent bony fish orthologs of NOD3 and CIITA supported the hypothesis that the origin of these molecules predates the origin of tetrapods, and the presence of avian sequences in both NALP and NOD clusters supported the origin of these subfamilies before the bird-mammal divergence. However, the extensive diversification of the NALP subfamily seen in mammals evidently occurred within the mammalian lineage. Both NALP and NOD subfamilies include members with differential expression in the antigen-presenting cells of the immune system, and the phylogenetic analyses supported the hypothesis that this expression pattern has evolved independently more than once in each of these subfamilies.

Infection of macrophages by bacterial pathogens can trigger Toll‐like receptor (TLR) activation as well as Nod‐like receptors (NLRs) leading to inflammasome formation and cell death dependent on caspase‐1 (pyroptosis). Complicating the study of inflammasome activation is priming. Here, we develop a priming‐free NLRC4 inflammasome activation system to address the necessity and role of priming in pyroptotic cell death and damage‐associated molecular pattern (DAMP) release. We find pyroptosis is not dependent on priming and when priming is re‐introduced pyroptosis is unaffected. Cells undergoing unprimed pyroptosis appear to be independent of mitochondrial involvement and do not produce inflammatory cytokines, nitrous oxide (NO), or reactive oxygen species (ROS). Nevertheless, they undergo an explosive cell death releasing a chemotactic isoform of the DAMP high mobility group protein box 1 (HMGB1). Importantly, priming through surface TLRs but not endosomal TLRs during pyroptosis leads to the release of a new TLR4‐agonist cysteine redox isoform of HMGB1. These results show that pyroptosis is dominant to priming signals and indicates that metabolic changes triggered by priming can affect how cell death is perceived by the immune system. Pathogens activate innate immune receptors, triggering inflammasome formation and cell death (pyroptosis). Inflammosome activation is sufficient for release of the chemotactic protein HMGB1 and pyroptosis. Additional signalling events are required to convert HMGB1 into a TLR4 agonist.

Induction of an eicosanoid storm is shown to be an unexpected consequence of inflammasome activation in peritoneal macrophages, leading to vascular leakage and rapid death in mice. Eicosanoids mediate inflammasome function Inflammasomes are multiprotein complexes that initiate early cellular responses to cellular pathogens. The mechanisms of inflammasome activation have been the focus of intense research, but relatively little is known about what pathways are activated downstream of inflammasomes. This study shows that systemic activation of the inflammasome in vivo results in the rapid induction of potent signalling lipids called eicosanoids, which cause a catastrophic loss of fluid from the blood, contributing to the death of the animal within 30 minutes. When restricted to the site of infection, eicosanoids may have a beneficial role in host defence, for example by increasing local vascular permeability, allowing an influx of immune cells. Detection of microbial products by host inflammasomes is an important mechanism of innate immune surveillance. Inflammasomes activate the caspase-1 (CASP1) protease, which processes the cytokines interleukin (IL)-1β and IL-18, and initiates a lytic host cell death called pyroptosis 1 . To identify novel CASP1 functions in vivo , we devised a strategy for cytosolic delivery of bacterial flagellin, a specific ligand for the NAIP5 (NLR family, apoptosis inhibitory protein 5)/NLRC4 (NLR family, CARD-domain-containing 4) inflammasome 2 , 3 , 4 . Here we show that systemic inflammasome activation by flagellin leads to a loss of vascular fluid into the intestine and peritoneal cavity, resulting in rapid (less than 30 min) death in mice. This unexpected response depends on the inflammasome components NAIP5, NLRC4 and CASP1, but is independent of the production of IL-1β or IL-18. Instead, inflammasome activation results, within minutes, in an ‘eicosanoid storm’—a pathological release of signalling lipids, including prostaglandins and leukotrienes, that rapidly initiate inflammation and vascular fluid loss. Mice deficient in cyclooxygenase-1, a critical enzyme in prostaglandin biosynthesis, are resistant to these rapid pathological effects of systemic inflammasome activation by either flagellin or anthrax lethal toxin. Inflammasome-dependent biosynthesis of eicosanoids is mediated by the activation of cytosolic phospholipase A 2 in resident peritoneal macrophages, which are specifically primed for the production of eicosanoids by high expression of eicosanoid biosynthetic enzymes. Our results therefore identify eicosanoids as a previously unrecognized cell-type-specific signalling output of the inflammasome with marked physiological consequences in vivo .

The molecular components of inflammasomes and what they sense are poorly defined. Vance and colleagues now show the carboxy-terminal 35 amino acids of flagellin activate the inflammasome in a Naip5-dependent way. Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes and what they sense remain poorly defined. Here we demonstrate that 35 amino acids of the carboxyl terminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in the intracellular sensor Naip5. These mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Our data clarify the molecular basis for the cytosolic response to flagellin.