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As cytoplasmic protein complexes that are pivotal for innate immunity, inflammasomes act primarily through the detection of pathogen- or danger-associated molecular patterns. Nucleotide oligomerisation domain-like receptor family and caspase activation recruitment domain-containing protein 4 (NLRC4) inflammasomes identify and eliminate intracellular pathogens, a process contingent on the ligand-recognition capabilities of neuronal apoptosis inhibitory proteins (NAIPs). Upon detection of specific molecules indicative of intracellular infection, NAIPs discern distinct pathogenic components and subsequently transmit signals to NLRC4, thus initiating their activation and triggering an inflammatory response. However, the mechanisms underlying NLRC4 inflammasome remain unclear. In this study, we elucidated the critical role of ATG16L2 in activating the NLRC4 inflammasome. ATG16L2-deficient macrophages exhibited reduced NLRC4 inflammasome activation, characterised by decreased oligomerisation of apoptosis-associated speck-like protein containing a CARD and attenuated cleavage of Pro-caspase-1, Pro-IL-1β and gasdermin D. Co-immunoprecipitation assays revealed an interaction between ATG16L2 and NAIPs. Furthermore, ATG16L2 enhanced the association between NAIPs and NLRC4 by binding to NAIPs. For ATG16L2-knockout mice infected with Salmonella typhimurium, pathogen clearance and survival rates markedly decreased. Collectively, our findings suggest that ATG16L2 is a significant modulator of the innate immune system, influencing the activity of the NLRC4 inflammasome and the host's defensive response to intracellular pathogens., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

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., (© 2024. Springer Nature Limited.)

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that uses two distinct type III secretion systems (T3SSs), termed Salmonella pathogenicity island (SPI)-1 and SPI-2, to deliver virulence factors into the host cell. The SPI-1 T3SS enables Salmonella to invade host cells, while the SPI-2 T3SS facilitates Salmonella's intracellular survival. In mice, a family of cytosolic immune sensors, including NAIP1, NAIP2, and NAIP5/6, recognizes the SPI-1 T3SS needle, inner rod, and flagellin proteins, respectively. Ligand recognition triggers assembly of the NAIP/NLRC4 inflammasome, which mediates caspase-1 activation, IL-1 family cytokine secretion, and pyroptosis of infected cells. In contrast to mice, humans encode a single NAIP that broadly recognizes all three ligands. The role of NAIP/NLRC4 or other inflammasomes during Salmonella infection of human macrophages is unclear. We find that although the NAIP/NLRC4 inflammasome is essential for detecting T3SS ligands in human macrophages, it is partially required for responses to infection, as Salmonella also activated the NLRP3 and CASP4/5 inflammasomes. Importantly, we demonstrate that combinatorial NAIP/NLRC4 and NLRP3 inflammasome activation restricts Salmonella replication in human macrophages. In contrast to SPI-1, the SPI-2 T3SS inner rod is not sensed by human or murine NAIPs, which is thought to allow Salmonella to evade host recognition and replicate intracellularly. Intriguingly, we find that human NAIP detects the SPI-2 T3SS needle protein. Critically, in the absence of both flagellin and the SPI-1 T3SS, the NAIP/NLRC4 inflammasome still controlled intracellular Salmonella burden. These findings reveal that recognition of Salmonella SPI-1 and SPI-2 T3SSs and engagement of both the NAIP/NLRC4 and NLRP3 inflammasomes control Salmonella infection in human macrophages., Competing Interests: The authors have declared that no competing interests exist.

Inflammasomes are cytosolic multi-protein complexes that detect infection or cellular damage and activate the Caspase-1 (CASP1) protease. The NAIP5/NLRC4 inflammasome detects bacterial flagellin and is essential for resistance to the flagellated intracellular bacterium Legionella pneumophila. The effectors required downstream of NAIP5/NLRC4 to restrict bacterial replication remain unclear. Upon NAIP5/NLRC4 activation, CASP1 cleaves and activates the pore-forming protein Gasdermin-D (GSDMD) and the effector caspase-7 (CASP7). However, Casp1-/- (and Casp1/11-/-) mice are only partially susceptible to L. pneumophila and do not phenocopy Nlrc4-/-mice, because NAIP5/NLRC4 also activates CASP8 for restriction of L. pneumophila infection. Here we show that CASP8 promotes the activation of CASP7 and that Casp7/1/11-/- and Casp8/1/11-/- mice recapitulate the full susceptibility of Nlrc4-/- mice. Gsdmd-/- mice exhibit only mild susceptibility to L. pneumophila, but Gsdmd-/-Casp7-/- mice are as susceptible as the Nlrc4-/- mice. These results demonstrate that GSDMD and CASP7 are the key substrates downstream of NAIP5/NLRC4/CASP1/8 required for resistance to L. pneumophila., Competing Interests: At the time of writing, Russell Vance was a scientific advisory board member of Metchnikoff Therapeutics, Inc. Metchnikoff Therapeutics did not fund research in his lab, or research that directly related to the research in this article. The authors have declared that no other competing interests exist.

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 a multiprotein 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., (Copyright © 2017, American Association for the Advancement of Science.)

Myeloid cells assemble inflammasomes in response to infection or cell damage; cytosolic sensors activate pro-caspase-1, indirectly for the most part, via the adaptors ASC and NLRC4. This leads to secretion of proinflammatory cytokines and pyroptosis. To explore complex formation under physiological conditions, we generated an alpaca single domain antibody, VHHASC, which specifically recognizes the CARD of human ASC via its type II interface. VHHASC not only impairs ASC(CARD) interactions in vitro, but also inhibits inflammasome activation in response to NLRP3, AIM2, and NAIP triggers when expressed in living cells, highlighting a role of ASC in all three types of inflammasomes. VHHASC leaves the Pyrin domain of ASC functional and stabilizes a filamentous intermediate of inflammasome activation. Incorporation of VHHASC-EGFP into these structures allowed the visualization of endogenous ASC(PYD) filaments for the first time. These data revealed that cross-linking of ASC(PYD) filaments via ASC(CARD) mediates the assembly of ASC foci., (© 2016 Schmidt et al.)

Biochemical studies suggest that the NAIP family of NLR proteins are cytosolic innate receptors that directly recognize bacterial ligands and trigger NLRC4 inflammasome activation. In this study, we generated Naip5(-/-), Naip1(-/-), and Naip2(-/-) mice and showed that bone marrow macrophages derived from these knockout mice are specifically deficient in detecting bacterial flagellin, the type III secretion system needle, and the rod protein, respectively. Naip1(-/-), Naip2(-/-), and Naip5(-/-) mice also resist lethal inflammasome activation by the corresponding ligand. Furthermore, infections performed in the Naip-deficient macrophages have helped to define the major signal in Legionella pneumophila, Salmonella Typhimurium and Shigella flexneri that is detected by the NAIP/NLRC4 inflammasome. Using an engineered S. Typhimurium infection model, we demonstrate the critical role of NAIPs in clearing bacterial infection and protecting mice from bacterial virulence-induced lethality. These results provide definitive genetic evidence for the important physiological function of NAIPs in antibacterial defense and inflammatory damage-induced lethality in mice., (© 2016 Zhao et al.)

Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1β secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Inflammasomes are cytosolic protein complexes that serve as platforms for the recruitment and activation of the pro-inflammatory CASPASE-1 protease. CASPASE-1 activation leads to processing and maturation of the cytokines interleukin-1β and interleukin-18 and a lytic form of cell death termed pyroptosis. Inflammasome assembly is initiated by cytosolic sensors in response to microbial infections. Many of these sensors, including NLRP1 (NLR family, pyrin domain containing 1), are described to form an inflammasome, but until recently, the mechanism of inflammasome activation and its physiological functions in host defense have remained unclear. In the last few years, important advances in our understanding of NLRP1 biology have been achieved. In this review, we discuss the activation of NLRP1 by various stimuli, including Bacillus anthracis lethal toxin, Toxoplasma gondii, muramyl dipeptide, and host intracellular ATP depletion. The role NLRP1 plays in pathogen recognition and resistance during infection is also discussed, as is the regulation of NLRP1 by host and viral proteins. We conclude by discussing the unexpected differences in the mechanism of NLRP1 inflammasome activation, as compared to the activation of other inflammasomes, such as the NAIP (NLR family, apoptosis inhibitory protein)/NLRC4 inflammasomes., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A major function of a subfamily of NLR (nucleotide-binding domain, leucine-rich repeat containing, or NOD-like receptor) proteins is in inflammasome activation, which has been implicated in a multitude of disease models and human diseases. This work will highlight key progress in understanding the mechanisms that activate the best-studied NLRs (NLRP3, NLRC4, NAIP, and NLRP1) and in uncovering inflammasome NLRs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The innate immune system of mammals encodes several families of immune detector proteins that monitor the cytosol for signs of pathogen invasion. One important but poorly understood family of cytosolic immunosurveillance proteins is the NLR (nucleotide-binding domain, leucine-rich repeat containing) proteins. Recent work has demonstrated that one subfamily of NLRs, the NAIPs (NLR family, apoptosis inhibitory proteins), are activated by specific interaction with bacterial ligands, such as flagellin. NAIP activation leads to assembly of a large multiprotein complex called the inflammasome, which initiates innate immune responses by activation of the Caspase-1 protease. NAIPs therefore appear to detect pathogen molecules via a simple and direct receptor-ligand mechanism. Interestingly, other NLR family members appear to detect pathogens indirectly, perhaps by responding to host cell "stress" caused by the pathogen. Thus, the NLR family may have evolved surprisingly diverse mechanisms for detecting pathogens., (Copyright © 2012 WILEY Periodicals, Inc.)

Inflammasomes are large cytoplasmic complexes that sense microbial infections/danger molecules and induce caspase-1 activation-dependent cytokine production and macrophage inflammatory death. The inflammasome assembled by the NOD-like receptor (NLR) protein NLRC4 responds to bacterial flagellin and a conserved type III secretion system (TTSS) rod component. How the NLRC4 inflammasome detects the two bacterial products and the molecular mechanism of NLRC4 inflammasome activation are not understood. Here we show that NAIP5, a BIR-domain NLR protein required for Legionella pneumophila replication in mouse macrophages, is a universal component of the flagellin-NLRC4 pathway. NAIP5 directly and specifically interacted with flagellin, which determined the inflammasome-stimulation activities of different bacterial flagellins. NAIP5 engagement by flagellin promoted a physical NAIP5-NLRC4 association, rendering full reconstitution of a flagellin-responsive NLRC4 inflammasome in non-macrophage cells. The related NAIP2 functioned analogously to NAIP5, serving as a specific inflammasome receptor for TTSS rod proteins such as Salmonella PrgJ and Burkholderia BsaK. Genetic analysis of Chromobacterium violaceum infection revealed that the TTSS needle protein CprI can stimulate NLRC4 inflammasome activation in human macrophages. Similarly, CprI is specifically recognized by human NAIP, the sole NAIP family member in human. The finding that NAIP proteins are inflammasome receptors for bacterial flagellin and TTSS apparatus components further predicts that the remaining NAIP family members may recognize other unidentified microbial products to activate NLRC4 inflammasome-mediated innate immunity., (© 2011 Macmillan Publishers Limited. All rights reserved)

Inflammasomes are a family of cytosolic multiprotein complexes that initiate innate immune responses to pathogenic microbes by activating the caspase 1 protease. Although genetic data support a critical role for inflammasomes in immune defence and inflammatory diseases, the molecular basis by which individual inflammasomes respond to specific stimuli remains poorly understood. The inflammasome that contains the NLRC4 (NLR family, CARD domain containing 4) protein was previously shown to be activated in response to two distinct bacterial proteins, flagellin and PrgJ, a conserved component of pathogen-associated type III secretion systems. However, direct binding between NLRC4 and flagellin or PrgJ has never been demonstrated. A homologue of NLRC4, NAIP5 (NLR family, apoptosis inhibitory protein 5), has been implicated in activation of NLRC4 (refs 7-11), but is widely assumed to have only an auxiliary role, as NAIP5 is often dispensable for NLRC4 activation. However, Naip5 is a member of a small multigene family, raising the possibility of redundancy and functional specialization among Naip genes. Here we show in mice that different NAIP paralogues determine the specificity of the NLRC4 inflammasome for distinct bacterial ligands. In particular, we found that activation of endogenous NLRC4 by bacterial PrgJ requires NAIP2, a previously uncharacterized member of the NAIP gene family, whereas NAIP5 and NAIP6 activate NLRC4 specifically in response to bacterial flagellin. We dissected the biochemical mechanism underlying the requirement for NAIP proteins by use of a reconstituted NLRC4 inflammasome system. We found that NAIP proteins control ligand-dependent oligomerization of NLRC4 and that the NAIP2-NLRC4 complex physically associates with PrgJ but not flagellin, whereas NAIP5-NLRC4 associates with flagellin but not PrgJ. Our results identify NAIPs as immune sensor proteins and provide biochemical evidence for a simple receptor-ligand model for activation of the NAIP-NLRC4 inflammasomes., (© 2011 Macmillan Publishers Limited. All rights reserved)

Inflammasomes are cytosolic multiprotein complexes that assemble in response to infectious or noxious stimuli and activate the CASPASE-1 protease. The inflammasome containing the nucleotide binding domain-leucine-rich repeat (NBD-LRR) protein NLRC4 (interleukin-converting enzyme protease-activating factor [IPAF]) responds to the cytosolic presence of bacterial proteins such as flagellin or the inner rod component of bacterial type III secretion systems (e.g., Salmonella PrgJ). In some instances, such as infection with Legionella pneumophila, the activation of the NLRC4 inflammasome requires the presence of a second NBD-LRR protein, NAIP5. NAIP5 also is required for NLRC4 activation by the minimal C-terminal flagellin peptide, which is sufficient to activate NLRC4. However, NLRC4 activation is not always dependent upon NAIP5. In this report, we define the molecular requirements for NAIP5 in the activation of the NLRC4 inflammasome. We demonstrate that the N terminus of flagellin can relieve the requirement for NAIP5 during the activation of the NLRC4 inflammasome. We also demonstrate that NLRC4 responds to the Salmonella protein PrgJ independently of NAIP5. Our results indicate that NAIP5 regulates the apparent specificity of the NLRC4 inflammasome for distinct bacterial ligands.