Inflammasomes are key signalling platforms that detect pathogenic microorganisms and sterile stressors, and that activate the highly pro-inflammatory cytokines interleukin-1β (IL-1β) and IL-18. In this Review, we discuss the complex regulatory mechanisms that facilitate a balanced but effective inflammasome-mediated immune response, and we highlight the similarities to another molecular signalling platform - the apoptosome - that monitors cellular health. Extracellular regulatory mechanisms are discussed, as well as the intracellular control of inflammasome assembly, for example, via ion fluxes, free radicals and autophagy.

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Activation and regulation of the inflammasomes

Inflammasomes are multiprotein signalling platforms that control the inflammatory response and coordinate antimicrobial host defences. They are assembled by pattern-recognition receptors following the detection of pathogenic microorganisms and danger signals in the cytosol of host cells, and they activate inflammatory caspases to produce cytokines and to induce pyroptotic cell death. The clinical importance of inflammasomes reaches beyond infectious disease, as dysregulated inflammasome activity is associated with numerous hereditary and acquired inflammatory disorders. In this Review, we discuss the recent developments in inflammasome research with a focus on the molecular mechanisms that govern inflammasome assembly, signalling and regulation.

Inflammasomes: mechanism of assembly, regulation and signalling

Mechanisms and Functions of Inflammasomes

Cell death is a fundamental biological phenomenon that is essential for the survival and development of an organism. Emerging evidence also indicates that cell death contributes to immune defense against infectious diseases. Pyroptosis is a form of inflammatory programmed cell death pathway activated by human and mouse caspase-1, human caspase-4 and caspase-5, or mouse caspase-11. These inflammatory caspases are used by the host to control bacterial, viral, fungal, or protozoan pathogens. Pyroptosis requires cleavage and activation of the pore-forming effector protein gasdermin D by inflammatory caspases. Physical rupture of the cell causes release of the pro-inflammatory cytokines IL-1β and IL-18, alarmins and endogenous danger-associated molecular patterns, signifying the inflammatory potential of pyroptosis. Here, we describe the central role of inflammatory caspases and pyroptosis in mediating immunity to infection and clearance of pathogens.

Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases.

BACKGROUND Pathogens cause agricultural devastation and huge economic losses. Up to 30% of our crops are lost before or after harvest to pathogens and pests, wasting water and human effort. Diseases and pests are major problems for sustainable agriculture in the face of population growth. Similarly, microbial infection remains a major cause of human mortality and morbidity, responsible for ~25% of deaths worldwide in 2012. We lack vaccines for several major infectious diseases, and antibiotic resistance is an ever- growing concern. Plant and animal innate immune systems respond to pathogen infection and regulate beneficial interactions with commensal and symbiotic microbes. Plants and animals use intracellular proteins of the nucleotide binding domain (NBD), leucine-rich repeat (NLR) superfamily to detect many kinds of pathogens. Plant and animal NLRs evolved from distinct derivatives of a common ancestral prokaryotic adenosine triphosphatase (ATPase): the NBD shared by APAF-1, plant NLR proteins, and CED-4 (NB-ARC) domain class and that shared by apoptosis inhibitory protein (NAIP), CIITA, HET-E, TP1 (NACHT) domain class, respectively. Animals and fungi can carry both NB-ARC and NACHT domain proteins, but NACHT domain proteins are absent from plants and several animal taxa, such as Drosophila and nematodes. Despite the vast evolutionary distance between plants and animals, we describe trans-kingdom principles of NLR activation. We propose that NLRs evolved for pathogen-sensing in diverse organisms because the flexible protein domain architecture surrounding the NB-ARC and NACHT domains facilitates evolution of “hair trigger” switches, into which a virtually limitless number of microbial detection platforms can be integrated. ADVANCES Structural biology is beginning to shed light on pre- and postactivation NLR architectures. Various detection and activation platforms have evolved in both plant and animal NLR surveillance systems. This spectrum ranges from direct NLR activation, through binding of microbial ligands, to indirect NLR activation after the modification of host cellular targets, or decoys of those targets, by microbial virulence factors. Homo- and heterotypic dimerization and oligomerization of NLRs add complexity to signaling responses and can enable signal amplification. NLR population genomics across the plant and animal kingdoms is increasing owing to application of new capture-based sequencing methods. A more complete catalog of NLR repertoires within and across species will provide an enhanced toolbox for exploiting NLRs to develop therapeutic interventions. OUTLOOK Despite breakthroughs in our molecular understanding of NLR activation, many important questions remain. Biochemical mechanisms of NLR activation remain obscure. Events downstream of plant NLR activation and outputs such as transcription of defense genes, changes in cell permeability, localized cell death, and systemic signaling remain opaque. We do not know whether activated plant NLRs oligomerize or, if they do, how this is achieved, given the diversity of subcellular sites of activation observed for various NLRs. It is not clear whether and how the different N-terminal domains of plant NLRs signal. We have increasing knowledge regarding how animal NLRs assemble and signal, although knowledge gaps remain. Therapeutic interventions in humans targeting NLRs remain on the horizon. Design of novel recognition capabilities and engineering of new or extended NLR functions to counter disease in animals and plants provides tantalizing future goals to address plant and animal health problems worldwide.

Intracellular innate immune surveillance devices in plants and animals

NOD-like receptor (NLR) proteins (Nlrps) are cytosolic sensors responsible for detection of pathogen and danger-associated molecular patterns through unknown mechanisms. Their activation in response to a wide range of intracellular danger signals leads to formation of the inflammasome, caspase-1 activation, rapid programmed cell death (pyroptosis) and maturation of IL-1β and IL-18. Anthrax lethal toxin (LT) induces the caspase-1-dependent pyroptosis of mouse and rat macrophages isolated from certain inbred rodent strains through activation of the NOD-like receptor (NLR) Nlrp1 inflammasome. Here we show that LT cleaves rat Nlrp1 and this cleavage is required for toxin-induced inflammasome activation, IL-1 β release, and macrophage pyroptosis. These results identify both a previously unrecognized mechanism of activation of an NLR and a new, physiologically relevant protein substrate of LT.

Anthrax lethal factor cleavage of Nlrp1 is required for activation of the inflammasome.

Anthrax lethal factor (LF) is the protease component of anthrax lethal toxin (LT). LT induces pyroptosis in macrophages of certain inbred mouse and rat strains, while macrophages from other inbred strains are resistant to the toxin. In rats, the sensitivity of macrophages to toxin-induced cell death is determined by the presence of an LF cleavage sequence in the inflammasome sensor Nlrp1. LF cleaves rat Nlrp1 of toxin-sensitive macrophages, activating caspase-1 and inducing cell death. Toxin-resistant macrophages, however, express Nlrp1 proteins which do not harbor the LF cleavage site. We report here that mouse Nlrp1b proteins are also cleaved by LF. In contrast to the situation in rats, sensitivity and resistance of Balb/cJ and NOD/LtJ macrophages does not correlate to the susceptibility of their Nlrp1b proteins to cleavage by LF, as both proteins are cleaved. Two LF cleavage sites, at residues 38 and 44, were identified in mouse Nlrp1b. Our results suggest that the resistance of NOD/LtJ macrophages to LT, and the inability of the Nlrp1b protein expressed in these cells to be activated by the toxin are likely due to polymorphisms other than those at the LF cleavage sites.

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Kristina A. Hellmich

Papers

Anthrax lethal factor cleavage of Nlrp1 is required for activation of the inflammasome.

Anthrax Lethal Factor Cleaves Mouse Nlrp1b in Both Toxin-Sensitive and Toxin-Resistant Macrophages