The NAIP-NLRC4 inflammasome in innate immune detection of bacterial flagellin and type III secretion apparatus.

Yue Zhao Feng Shao

The NAIP–NLRC4 inflammasome in innate immune detection of bacterial flagellin and type III secretion apparatus

Authors’ address Yue Zhao1, Feng Shao1 1 National Institute of Biological Sciences, Beijing, China.

Summary: Bacterial flagella and type III secretion system (T3SS) are evolutionarily related molecular transport machineries. Flagella mediate bacterial motility; the T3SS delivers virulence effectors to block host defenses. The inflammasome is a cytosolic multi-protein complex that activates caspase-1. Active caspase-1 triggers interleukin-1b (IL-1b)/IL18 maturation and macrophage pyroptotic death to mount an inflammatory response. Central to the inflammasome is a pattern recognition receptor that activates caspase-1 either directly or through an adapter protein. Studies in the past 10 years have established a NAIP–NLRC4 inflammasome, in which NAIPs are cytosolic receptors for bacterial flagellin and T3SS rod/needle proteins, while NLRC4 acts as an adapter for caspase-1 activation. Given the wide presence of flagella and the T3SS in bacteria, the NAIP–NLRC4 inflammasome plays a critical role in anti-bacteria defenses. Here, we review the discovery of the NAIP– NLRC4 inflammasome and further discuss recent advances related to its biochemical mechanism and biological function as well as its connection to human autoinflammatory disease.

Correspondence to: Feng Shao National Institute of Biological Sciences #7 Science Park Road Zhongguancun Life Science Park Beijing 102206, China Tel.: +86 010 80726688 8560 e-mail: [email protected] Acknowledgements We thank M. Shi for helping with the artwork. Work in the authors’ laboratory was supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute and the Beijing Scholar Program to F. S. The work was also supported by the National Basic Research Program of China 973 Programs (2012CB518700 and 2014CB849602), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB08020202), and the China National Science Foundation Program for Distinguished Young Scholars (31225002) to F. S. The authors have no conflicts of interest to declare.

This article is part of a series of reviews covering Inflammasomes appearing in Volume 265 of Immunological Reviews.

Immunological Reviews 2015 Vol. 265: 85–102

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Immunological Reviews 0105-2896

Keywords: pattern recognition, innate immunity, NOD-like receptor, NLRs, inflammasome, flagellin

Bacterial flagellin and type III secretion system The flagellum is a whip-like filament appended to the bacterial surface, providing the major force for bacterial motility. The flagellum polymer is built from repeating flagellin monomers that stack helically. Monomeric flagellin (30–60 kDa, dependent upon the taxa of the bacterium) is secreted from the bacterium through the flagellar export system (Fig. 1A). Structurally, the carboxyl (C) terminus of the flagellin folds back to the amino terminus (N); the whole molecule contains four distinct globular domains, D0, D1, D2, and D3, shaped into a ‘boomerang’ (1) (Fig. 1B). About 40 amino acids from each terminus of the flagellin molecule constitute the D0 domain. The D1 domain contains about 100 residues from the N-terminus and 50 residues from the C-terminus. Through intermolecular contacts between the D1 domains of two adjacent monomers, flagellins pile up longitudinally to

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

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Zhao & Shao The NAIP–NLRC4 inflammasome

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Fig. 1. Comparisons of a flagellum and the T3SS: their overall architecture and structural regions recognized by the NAIP–NLRC4 inflammasome. (A) Schematic models of the flagellum and the T3SS needle structure. Both systems contain structurally similar basal bodies (pink) spanning the bacterial inner and outer membranes. The flagellum filament, formed by polymerization of monomeric flagellin, is connected to the base through the hook structure. The T3SS needle is connected to the base through the inner rod ring located within the periplasmic space. (B) Ribbon diagram of structures of flagellin, the T3SS rod protein, and the T3SS needle protein. The D0 domain in flagellin (PDB ID: 1UCU) is recognized by the NAIP5/6 receptor. The T3SS rod protein (structure obtained by homology modeling) and the T3SS needle protein (PrgI from S. typhimurium, PDB ID: 2JOW) are recognized by mouse NAIP2 and mouse NAIP1/hNAIP, respectively. The flagellin D0 domain and the T3SS rod and needle proteins all adopt similar helix hairpin structures with surface-exposed hydrophobic residues (highlighted) that are essential for recognition by the NAIP.

form one profilament unit. A total of 11 profilaments assemble to form a flagellum filament, which is a hollow tube of approximately 15 lm in length with external and inner diameters of approximately 240 A and 20 A, respectively (2). The D0 and adjacent D1 domains from the profilaments make up the inner and outer cores of the hollow filament; the D2 and D3 domains, which come from the central segment of the flagellin polypeptide, form projections on the outer surface of the filament (1, 2). The D0 and D1 domains both assume primarily a-helical structures. The D2 and D3 domains are composed largely of b-sheets (Fig. 1B). The D0 and D1 domains are crucial for assembly of the helical filamentous structure and are therefore highly conserved among different species of bacteria, whereas the other two domains exhibit high sequence diversity. The flagellum filament is anchored into the bacterial membrane by a basal body, which is a rotary motor powered by proton motive force. A flexible hook complex bridges the filament and the basal body (3) (Fig. 1A). The filament, the hook, and the basal body together constitute the complete flagellum machinery, which, through rotation of the filament, enables the bacterium to swim. Flagella-mediated motility is important for the bacterium in the search for nutrients and for the avoidance of harmful substances in the environment. The flagellum is also vital in facilitating both

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bacterial adhesion and invasion of host cells (4). For enteric bacteria, flagellum-mediated motility is advantageous for coping with rapid epithelial cell renewal, tight intercellular junctions, and the viscous mucus present in gastrointestinal tissue. For example, the spiral-shaped Campylobacter jejuni, a common cause of gastroenteritis, and Helicobater pylori, the leading causative agent for chronic gastritis and gastric ulcers, both bear polar-positioned flagella; mutations disrupting flagella render both bacteria incapable of establishing a replication niche in the gastrointestinal tract (5). Other bacterial pathogens such as Pseudomonas aeruginosa (6) and Vibrio cholera (7) also rely on an intact flagellum for colonization of their respective hosts. Supporting the notion of the critical function of the flagellum, flagellated bacteria are the primary cause of a number of serious skin, soft tissue, pulmonary, and urinary tract infections. Flagellin, due to its wide presence in diverse bacterial species and extreme abundance in each bacterial cell, is a major target of host immune system. Flagellin is highly antigenic; variations in its antigenicity (H antigen) lead to the generation of serotype-specific antibodies in hosts. Flagellin has been heavily explored for use as vaccine antigens and adjuvants. The innate immune system in mammals has evolved to detect flagellin through germline-encoded pattern recognition receptors (PRRs). In the extracellular space, flagellin is © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


recognized by Toll-like receptor 5 (TLR5), a plasma membrane-localized PRR (8). Similar to other TLR family members, the extracellular leucine-rich repeat (LRR) domain in TLR5 directly binds to flagellin. TLR5 then transmits the activation signal to the nuclear factor jB (NF-jB) signaling cascade, which upregulates the transcription of many proinflammatory cytokines and chemokines. This serves as an important host defense mechanism. It is known that insects and plants have also evolved mechanisms for the perception of flagellin (9, 10). In plants, flagellin detection is mediated by the flagellinsensitive 2 (FLS2) protein, an LRR domain-containing receptor kinase (9). Flagellin binding activates the serine/threonine kinase activity of FLS2, resulting in a phosphorylation cascade of the mitogen-activated protein kinase (MAPK) pathway and subsequent transcriptional induction of defense genes (11). TLR5 and FLS2-like receptors, though effective in detecting extracellular flagellin, are powerless to detect flagellin that has reached the host cytosol, a situation that often occurs during infection. Recent studies have defined a new innate immune detection pathway for cytosolic flagellin; this new detection pathway is a primary focus of this review. Many Gram-negative bacterial pathogens harbor a conserved type III secretion system (T3SS) that injects virulence effector proteins into host cells. Structurally, the T3SS is composed of a base, a needle, and a translocon located at the tip of the needle (12). The base contains two rings that are located in the bacterial inner and outer membrane; a rod structure between the two rings further connects the base to the needle. The needle is hollow and serves as a tunnel through which the effectors are delivered into the host cytosol via the translocon complex. The translocon consists of pore-forming translocator proteins inserted into the host cell membrane. Despite the absence of a translocon in the flagellum, the base of the T3SS structurally resembles the flagellar basal body, and the needle is seen as being analogous to the flagellar hook (Fig. 1A). Many of the structural proteins in the T3SS and flagellum systems bear significant sequence homology; the two systems display a highly similar three-dimensional architecture. Indeed, the T3SS is believed to have evolved from the flagellum. The conduits of the T3SS directly connect the bacteria and the host, culminating in the high-efficiency transport of bacterial proteins into host cytosol (with a small portion leaked to the extracellular media) (13). T3SS-mediated secretion is a highly ordered event; the inner rod and needle proteins are secreted prior to the secretion of the translocator and subsequent effectors. This order is a prerequisite for the assembly of a complete and functional T3SS. The T3SS does not have © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

100% fidelity; the rod/needle proteins can be inadvertently translocated into the cytosol of the host. The T3SS is a critical virulence mechanism due to the powerful activities of the injected effectors (14, 15). One well-known function of the T3SS is to mediate bacterial adhesion and invasion of host cells. For example, extracellular pathogens such as enteropathogenic and enterohemorrhagic E. coli (EPEC and EHEC, respectively) establish an intimate contact with host cell to initiate infection; in this process, the bacteria deliver a T3SS-translocated intimin receptor (Tir) into host cell membrane. Tir then acts as a receptor for the bacterial outer membrane protein intimin. The Tir-intimin interaction triggers a signaling cascade, which, together with activities of other T3SS effectors, stimulates host actin polymerization and pedestal formation (16). This process causes the development of the attaching and effacing (A/E) lesion on the intestinal epithelial surface, thereby providing the bacteria a special replication niche. Salmonella and Shigella, the major causes of typhoid fever and dysentery, respectively, rely on a group of T3SS effectors to invade host cells. The effectors function to drive host actin polymerization, either directly or indirectly, by modulating the activities of host Rho GTPases (17). Many bacteria replicate within the host cytosol. Taking Salmonella as an example, following successful invasion, the bacterium resides in a Salmonella-containing vacuole (SCV) that can avoid phagolysosomal fusion. The biogenesis and maintenance of the SCV require a cadre of T3SS effectors that function to interfere with host membrane trafficking. The SCV is a niche for Salmonella to replicate and spread in the host (18). Other bacteria are instead adapted to live freely in the host cytosol. For example, Shigella and some pathogenic Burkholderia spp. escape from the endosome shortly after entry into host cells; this process also requires the activities of secreted T3SS effectors. These examples highlight the general function of T3SS effectors in the intracellular life cycle of bacterial pathogens. A third important virulence activity of the T3SS is to block host innate immunity. The MAPK and NF-jB signaling pathways are crucial for combating microbial infections and therefore are often targeted by various bacterial pathogens. For example, the Yersinia T3SS effector YopJ acetylates critical residues in the activation loops of MAPK kinase and IKKb to shut down the MAPK and NF-jB signaling, respectively (19). The Shigella OspF effector has a unique MAPK phosphothreonine lyase activity that inactivates MAPK through an elimination modification (20). The EPEC T3SS effector NleE methylates a zinc-finger cysteine of TAB 2/3 in the

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NF-jB pathway (21) and the NleB effector GlcNAcylates a conserved arginine in the death domains of the TNFR receptor complex (22); both of the enzymatic reactions contribute to bacterial suppression of host inflammatory responses. From the perspective of the host, how does the innate immune system deal with the virulence activity of the T3SS? Plants have evolved so-called ‘effector-triggered immunity’ (23), in which a specific PRR detects the virulence activity of a T3SS effector and triggers a defense response for restricting bacterial growth. Different from the situation in plants, which encode hundreds of PRR proteins, the number of PRRs in mammals is limited and cannot match the large number of bacterial effectors that a mammalian organism may encounter. Recent studies have revealed that the mammalian innate immune system has evolved to recognize conserved structural components in the T3SS, i.e. the rod and needle proteins (discussed in detail below). In this way, mammals can rely on a limited number of PRRs to detect as many as possible bacterial pathogens. Pattern recognition, the inflammasome, and NLR proteins The concept of pattern recognition in innate immune detection of pathogen-associated molecular patterns (PAMPs) was first proposed by Dr. Charles Janeway in 1989 (24). Since then, several PRR families have been identified (25). TLRs

and C-type lectin receptors (CLRs) are two families of membrane-anchored PRRs that have been shown to detect extracellular PAMPs. Besides flagellin mentioned above, TLRs can recognize bacterial cell wall components such as lipopolysaccharide (LPS), peptidoglycan (PGN), and lipoprotein, as well as pathogen-derived nuclear acids through extracellular LRR domains. In addition to inducing NF-jB activation, activation of certain TLRs can also stimulate interferon regulatory factor (IRF) signaling to upregulate proinflammatory gene transcription (26). Other families of PRRs act in the cytoplasmic compartment to detect intracellular PAMPs or threat signals. These include the RIG-I like receptors (RLRs) that recognize double-strand RNA and the newly identified cyclic GMP-AMP synthase (cGAS) that senses double-stranded DNA (dsDNA) and generates a cyclic di-nucleotide second messenger cGAMP (27). RLRs and cGAS play dominant roles in antiviral defenses by activating interferon and inflammatory cytokine transcription. Several cytoplasmic PRRs are known to function in inflammasome-based innate immunity. The concept of the inflammasome, borrowed from the apoptosome in apoptosis research, refers to a multi-protein complex for caspase-1 activation (Fig. 2); these complexes are present mainly in macrophage and dendritic cells (28). Caspase-1 activation leads to a lytic inflammatory cell death called pyroptosis as well as to the concurrent processing/maturation of IL-1b

Fig. 2. Inflammasomes mediated by pyrin domain (PYD)-containing PRRs. Cytosolic PRR proteins AIM2, PYRIN, and NLRP3 in the NLRP subfamily of NLRs all contain an N-terminal PYD. AIM2 recognizes the dsDNA through the C-terminal HIN200 domain. PYRIN indirectly senses inactivating modifications of host Rho GTPases by various bacterial toxins or infections. NLRP3 can detect a variety of microbial or dangle signals such as LPS plus extracellular ATP through an unknown mechanism. All three PRR sensors, upon stimulation, form an inflammasome complex with the ASC adapter through homotypical PYD interactions to induce caspase-1 activation, IL-1b/IL-18 maturation, and macrophage pyroptosis.

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and IL-18. Similar to the requirement of APAF-1 (apoptotic protease-activating factor 1) for apoptosome assembly, assembly of the inflammasome is mediated by a PRR in response to a microbial or a danger signal. Caspase-1 belongs to the inflammatory caspase family that also includes caspase-11 in mice, caspase-4 and caspase-5 in human, as well as caspase-12, all of which bear an N-terminal caspase activation and recruitment domain (CARD) in addition to the protease domain. Caspase-11 is activated upon Gram-negative bacterial infection and critically mediates LPS-induced endotoxic shock in mice. To distinguish it from the caspase-1 inflammasome, a non-canonical inflammasome for caspase-11 activation has been proposed (29). Recently, our group showed that caspase-11 as well as caspase-4/5 directly recognize cytosolic LPS and become activated as a result of the binding-induced oligomerization (30). Thus, these inflammatory caspases represent a unique type of PRR that combines the sensory and effector functions within a single polypeptide. The canonical inflammasome often requires or involves a cytosolic adapter called ASC. ASC contains an N-terminal Pyrin domain (PYD) and a C-terminal CARD. Both the PYD and the CARD, as members of the death domain (DD) superfamily, are protein interaction modules of approximately 90 amino acids in size that are structured into a compact globular bundle of six anti-parallel helices (31). The PYD in ASC can homotypically interact with the upstream PRRs, as most of these also contain a PYD; the CARD in ASC promotes caspase-1 activation through CARD– CARD interaction (Fig. 2). A hallmark of inflammasome activation is the massive aggregation of ASC, forming a large speck structure in stimulated cells. Key to inflammasome assembly is the PRR sensor that confers ligand specificity. Three types of PRRs are known to mediate inflammasome assembly: the absent in melanoma 2 (AIM2)-like receptor (ALR), PYRIN, and the nucleotide-binding domain (NBD) LRR (NLR) family (Fig. 2). AIM2 is a receptor for cytosolic dsDNA of bacterial or viral origin (32). Upon dsDNA binding to the C-terminal HIN200 domain in AIM2 (33), the Nterminal PYD in AIM2 specifically interacts with the PYD in ASC, leading to caspase-1 activation (Fig. 2). AIM2 inflammasome activation is negatively regulated by the p202 protein that contains two HIN200 domains but lacks the PYD, which has implications in lupus pathogenesis (34, 35). Human also has three AIM2-like proteins, of which IFI16 has been suggested to activate caspase-1 in the nucleus in response to herpes virus infection (36). PYRIN is a unique PRR that contains an N-terminal PYD; PYRIN can stimulate © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

caspase-1 activation in vitro in an ASC-dependent manner (37, 38). Mutations in the PYRIN-encoding gene MEFV cause a hereditary autoinflammatory disease known as familial Mediterranean fever (FMF) (39, 40). Recently, we showed that PYRIN senses inactivating modifications of host Rho GTPases mediated by diverse bacterial toxins and infections (Fig. 2), including Rho glucosylation by Clostridium difficile cytotoxin TcdB and Rho deamidation by Burkholderia cenocepacia (41, 42). These processes lead to the formation of an ASC-containing inflammasome. Different from AIM2 and most other mammalian PRRs, PYRIN detects pathogen virulence activity rather than directly recognizing a PAMP, which appears to be similar to above discussed ‘effectortriggered immunity’ in plants. The NLR family constitutes the majority of PRRs that function in canonical inflammasome assembly. NLRs feature C-terminal LRR domains, a central nucleotide-binding oligomerization domain (NOD or NACHT), and a variable N-terminal protein–protein interaction module (CARD, PYD, or baculovirus inhibitor of apoptosis repeat [BIR] domains) (43). Based on the N-terminal domain structure, NLRs (of which there are more than 20 in human) can be divided into four subfamilies: NLRA with a transcriptional activation domain, NLRB with BIR domains, NLRC with a CARD, and the largest NLRP subfamily with a PYD. NLR proteins structurally resemble APAF-1 and plant disease resistance (R) proteins, which control mammalian apoptosis and plant innate immunity, respectively (44, 45). It is generally believed that the NLR family mainly functions in immunity as genetic mutations in several NLR genes are known to be associated with immunological diseases. For instance, NLRP1 variations confer risk for vitiligo and associated autoimmunity. Mutations in NLRP3 are associated with Muckle–Wells syndrome, familial cold urticaria, and neonatal onset multisystem inflammatory disease (46). The indication that NLR proteins may function to activate caspase-1 comes from an in vitro study in macrophage cellfree extracts (28). The inflammasome concept proposed in that study promoted further investigations of the inflammasome function of NLRs. NLRP3 was the first NLR shown to mediate the assembly of a physiological inflammasome; this was hinted at from the observation that the NLRP3 disease mutations caused caspase-1 activation and excessive IL-1b production (28, 47, 48). It is now well established that NLRP3 responds to a broad spectrum of agents, including the bacterial cell wall component MDP (muramyl dipeptide), certain viruses, extracellular ATP, toxins that can deplete intracellular potassium, monosodium urate crystals,

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aluminum adjuvants, and even UV radiation (49–52) (Fig. 2). However, how NLRP3 senses these diverse signals remains an important open question. Similar to AIM2 and PYRIN, the PYD in NLRP3 mediates the interaction with ASC, which then further activates caspase-1 (Fig. 2). The NLRP subfamily contains more than a dozen other members. PYD-mediated interaction with ASC has been observed for several other NLRPs (53–55), and there is evidence suggesting that some of those NLRPs may mediate inflammasome activation. However, in most such situations, a defined stimulus has been lacking. Moreover, certain NLRPs have been reported to perform inflammasome-independent functions. For instance, NLRP6 and NLRP12 have been suggested to negatively regulate the NF-jB signaling (56, 57). NLRC4 links the inflammasome to anti-bacteria defense by sensing cytosolic flagellin Following the identification and characterization of caspase1 as the IL-1b-converting enzyme in the early 1990s, there was a wave of effort to search for activators of caspase-1. The prevailing approach used at that time was based on the knowledge that apoptotic pro-caspase is activated by a CARD-containing protein through homotypical CARD interactions. As a result of the search efforts, NLRC4, originally named as Ipaf (ICE protease-activating factor), was cloned (58). NLRC4 belongs to the NLRC subfamily of NLRs. In overexpression assays, NLRC4 was shown to bind directly to caspase-1; deletion of its LRR domains resulted in caspase-1 processing, indicating a possible autoinhibitory function of the LRR (58). The physiological function of NLRC4 was not demonstrated until a study from the group of Vishva Dixit in 2004 (59). Previously, it was known that infection of mouse primary macrophages with S. typhimurium induced robust caspase-1-dependent IL-1b secretion and cell death (60). The Dixit study confirmed this observation and further observed that S. typhimurium-induced caspase-1 activation was diminished in Nlrc4 knockout mouse macrophages. Asc deletion could also block caspase-1 activation and IL-1b maturation, suggesting that NLRC4 functions together with ASC in sensing S. typhimurium infection. These results demonstrated a role of inflammasome activation in host defenses against bacterial infection. What is the bacterial signal that triggers NLRC4-dependent caspase-1 activation? A series of studies published in 2006 established that cytosolic flagellin is the responsible bacterial signal. Two studies from the research groups of Michele Swanson and Russell Vance (61, 62) focused on how Legionella pneumophila evades macrophage innate immu-

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nity. L. pneumophila is a motile Gram-negative bacterium with a single polar flagellum. L. pneumophila infects alveolar macrophages and causes a form of pneumonia known as Legionnaires’ disease. The bacterium relies on a Dot/Icm type IV secretion system (T4SS) to establish a replication niche called the Legionella-containing vacuole (LCV) (63). In permissive hosts such as macrophages from the A/J mice, the LCV prevents the bacteria from being sent to the lysosome for degradation. Infection of non-permissive or resistant host, such as macrophages from the C57BL/6J mice, induces rapid cell death, which in turn inhibits L. pneumophila intracellular replication. Both research groups found that L. pneumophila mutants deficient in flagellin expression were defective in triggering caspase 1-dependent proinflammatory death and therefore could not kill the resistant macrophages. Consistently, the flagellin-less L. pneumophila mutants showed more robust replication in C57BL/6J macrophages. Further genetic analyses suggested that L. pneumophila-induced caspase-1 activation was independent of flagella assembly and also TLR5 in the host, but required the T4SS system in the bacterium. The T4SS is similar to the T3SS in forming a large protein complex for effector injection into the cytosol of hosts. Different from the T3SS, the T4SS originates from a bacterial DNA conjugation system (64, 65). Flagellin possesses two conserved leucine residues at the C-terminus; this is a common feature of many type IV secretion substrates (66). Therefore, it is possible that erroneous translocation of L. pneumophila flagellin into the cytosol of macrophages via the T4SS induces caspase-1 activation. Supporting this notion, the two studies demonstrated that flagellin, when delivered into macrophage cytosol through the listeriolysin O (LLO) (a pore-forming toxin from Listeria monocytogenes), was sufficient to trigger caspase-1 activation and macrophage pyroptosis. Cytosolic flagellin-induced TLR5-independent caspase-1 activation was also revealed in two other studies using the S. typhimurium infection model (67, 68). The research group of Alan Aderem performed an unbiased bacterial genetic screen, while Gabriel Nunez and colleagues followed up on an educated guess; both studies concluded that flagellin is the bacterial trigger for S. typhimurium-induced caspase-1 activation, IL-1b production, and the killing of macrophages. S. typhimurium flagellin-induced caspase-1 activation requires a functional T3SS, consistent with the fact that flagellin is known to be a promiscuous substrate of Salmonella T3SS (69). Both studies further showed that Nlrc4 / macrophages were defective in S. typhimurium flagellin-induced caspase-1 activation (59). A subsequent study from the Nunez group © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


demonstrated that Nlrc4 was similarly required for flagellinstimulated caspase-1 activation in L. pneumophila infection (70). The important role of NLRC4 inflammasomes in sensing cytosolic flagellin and in anti-bacteria immune defense were also noted in studies of the extracellular pathogen Pseudomonas aeruginosa (71–73) as well as in studies of Gram-positive L. monocytogenes (74). These studies collectively indicated that the NLR-family member NLRC4 is likely a general innate immune sensor for cytosolic flagellin and mediates caspase-1 activation during bacterial infection. Flagellin-independent NLRC4 activation indicates a response to bacterial T3SS In the course of S. typhimurium infection, Miao et al. (68) observed ‘flagellin-independent but NLRC4-dependent IL-1b secretion’ when a high dose of bacteria was used. Other studies found that a flagellin-deficient mutant of P. aeruginosa and even the non-flagellated S. flexneri could still trigger NLRC4-dependent caspase-1 activation (72, 75). These results implied the existence of an additional bacterial signal that is detected by the NLRC4 inflammasome. The residual NLRC4-dependent IL-1b secretion induced by S. typhimurium fliCfljB mutants (devoid of both flagellin genes) was sensitive to mutation of prgJ, a gene encoding the inner rod protein of Salmonella pathogenicity island-1 (SPI1) T3SS (76). A requirement for the T3SS was similarly observed in studies of P. aeruginosa and S. flexneri (72, 75). As the absence of the inner rod disrupts the assembly of the T3SS, Miao et al. (76) then examined the biochemical activity of PrgJ. Transfection of macrophages with recombinant PrgJ protein or infection with PrgJ-expressing retrovirus was found to induce a significant (though not robust) inflammasome response. In the same assay, the rod protein of S. typhimurium SPI2 and the needle component of SPI1 appeared to be inert (76). Notably, the inflammasome activation region in flagellin (see below) and the rod protein share a similar secondary structural organization that is characterized by two long, parallel a helices (Fig. 1B). Mutation of the conserved hydrophobic surface residues in these a helices disrupted the activity of both flagellin and PrgJ in triggering NLRC4 inflammasome activation (76, 77). Like flagellin, the T3SS rod protein, as well as the needle protein, forms a hollow tube structure during the assembly of the corresponding secretion apparatus. In fact, the needle protein is highly similar to the rod both in size and in its three-dimensional structural arrangement (78). Unlike the rod protein that is erroneously secreted out of the bacteria by the T3SS, secretion of the © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

needle protein is essential for the assembly of the T3SS needle complex. Thus, it was somewhat counterintuitive at that time to note that the T3SS rod protein, but not the needle protein, was detected by the flagellin-sensing innate immune pathway. In the efforts to identify cytosolic receptors for flagellin and the T3SS rod protein (see below), both Russell Vance’s group and our group (79, 80) designed alternative experimental methods to deliver bacterial proteins into the cytosol of macrophages. Both groups confirmed the biochemical activity of the T3SS rod protein in activating the NLRC4 inflammasome. Our assay showed that the percentage of macrophage pyroptosis induced by the rod protein could be as high as 90% (80), suggesting a higher immune-stimulating activity in the rod protein than in the flagellin. We further discovered that the T3SS needle protein could also trigger NLRC4 inflammasome activation; this was found to be the case in both mouse cells and in human monocyte-derived macrophages (80, 81). Similar to flagellin and the rod protein, the activity of the needle protein was sensitive to mutations in the hydrophobic residues of its C-terminal a helix (Fig. 1B). Collectively, the NLRC4 inflammasome serves as effective cytosolic surveillance to detect bacteria that bears flagella or/and a T3SS. A role of NAIP5 in Legionella replication and flagellininduced NLRC4 activation For quite a few years after the identification of flagellin as the bacterial signal detected by the NLRC4 inflammasome, investigators had no idea how flagellin was recognized in the cytosol. A reasonable hypothesis or a presumed belief would be that NLRC4, the central NLR in this pathway, is the cytosolic flagellin receptor. However, no direct interactions between flagellin and NLRC4 have been reported. A final solution to this puzzle was hinted at from studies of L. pneumophila infection. As mentioned above, L. pneumophila replicates proficiently in permissive A/J macrophages, while macrophages from C57BL/6J and several other inbred strains restrict L. pneumophila infection (82). The genetic determinant for this differential response was mapped to Naip5 (also named Birc1e) (83, 84). NAIP5 is a unique NLRfamily member featuring three tandem BIR domains at its N-terminus. Mouse has seven NAIPs (NAIP1-7) encoded in the same genetic locus while humans generally possess one (hNAIP); the seven paralogs in mice likely originate from gene duplication events. The BIR domain is known as a caspase inhibitor in apoptosis, but it is now generally believed

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that NAIPs do not function in the inhibition of apoptosis. Compared with Naip5 from C57BL/6J mice, A/J micederived Naip5 has a number of polymorphisms that lead to 14 amino acid differences (83). A/J-mice Naip5 is also expressed at a much lower level than Naip5 in C57BL/6J mice (85). Transgenic expression of a restrictive Naip5 allele in a permissive host repressed L. pneumophila growth, suggesting the loss of NAIP function in A/J-derived macrophages. For many years, it was not clear how C57BL/6J-derived Naip5 confers macrophage resistance to L. pneumophila infection. The research group of Craig Roy (86) discovered that NAIP5 was capable of mediating L. pneumophila-induced caspase-1 activation in a reconstituted 293T cell infection system. Results obtained in this experimental system also showed that the A/J version of NAIP5 is intrinsically nonfunctional. However, and somewhat inconsistently, L. pneumophila infection of macrophages from A/J mice or B6.AChr13A/J mice (a C57BL/6 mouse homozygous for A/J chromosome 13 harboring the Naip5 gene) could still induce robust (though slightly attenuated) caspase-1 activation. The involvement of NAIP5 in L. pneumophila-induced caspase-1 activation or macrophage pyroptosis was also noted in the two studies that identified L. pneumophila flagellin as the trigger for caspase-1 activation (61, 62). All these studies observed consistent genetic results supporting the idea that flagellin-induced caspase-1 activation limits L. pneumophila growth only in the presence of the C57BL/6 allele of Naip5. However, the fact that the A/J allele of Naip5 was associated with evident caspase-1 activation prevented the investigators from realizing a critical role of NAIP5 in flagellin activation of the inflammasome. Meanwhile, there was also a report suggesting that macrophages harboring the A/J allele of Naip5 exhibit normal caspase-1 activation and IL-1b secretion in response to cytosolic flagellin or L. pneumophila infection (87). Those investigators even favored the idea that flagellin-induced NLRC4-mediated caspase-1 activation and the NAIP5 signaling were two parallel pathways that both restricted L. pneumophila growth. To clarify the physiological function of NAIP5 in L. pneumophila infection, the Vance group (77) generated Naip5 / mice in a pure C57BL/6 background. Using bone marrow macrophages derived from the knockout mice, it was demonstrated that Naip5, similar to Nlrc4, was genetically required for L. pneumophila-induced caspase-1 activation. To the contrary, P. aeruginosa and S. typhimurium infection-induced macrophage pyroptosis only partially required Naip5. To assess the function of NAIP5 in the absence of bacterial components, pore-forming toxins, and even in the absence

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of transfection reagents, Vance and colleagues established a retrovirus transduction assay to express flagellin in macrophages. The ability of flagellin to kill macrophages was determined by counting the percentage of flagellin-expressing cells that could be recovered from retroviral transduction. This assay confirmed that cytosolic flagellin can induce NLRC4-dependent caspase-1 activation and macrophage pyroptosis. Moreover, the smallest flagellin fragment capable of inducing NLRC4 activation was mapped to 35 residues of the C-terminus that contained three essential leucine residues (Fig. 1B). This 35-residue region is part of the D0 domain, distinct from the D1 domain that is sensed by TLR5 (88). When the retroviral transduction was performed with Naip5-deficient macrophages, the 35 residues from the C-terminus of flagellin strictly required Naip5 for induction of macrophage pyroptosis. Surprisingly, full-length flagellin killed Naip5 / macrophages just as efficiently as it killed wildtype cells. These intriguing results contradicted the genetic requirement of flagellin and Naip5 for L. pneumophilainduced caspase-1 inflammasome activation. To reconcile this apparent inconsistency, it was suggested that regions outside of the 35 residues in flagellin might trigger ‘Naip5independent but Nlrc4-dependent caspase-1 activation’, which was also proposed as a potential reason for the partial requirement of Naip5 in P. aeruginosa and S. typhimuriuminduced caspase-1 activation. Along this line, it was further suggested, based on retroviral lethality data, that an N-terminal region in flagellin can release the requirement of NAIP5 for NLRC4 activation (89). Looking back, we now know that Naip5-independent inflammasome activation in P. aeruginosa and S. typhimurium infections most likely results from the T3SS. The partial dependence on Naip5 observed with full-length flagellin is probably caused by the narrow assay window and the relatively high variation in the retroviral lethality assay. While Naip5 / macrophages have allowed for clarifying the role of Naip5 in restricting L. pneumophila infection to a certain degree, the observation of the partial requirement of Naip5 for P. aeruginosa and S. typhimurium-induced caspase-1 activation strengthened the historic misperception that NAIP5 is a host factor that is specific to L. pneumophila. Indeed, shortly after the discovery that the T3SS rod protein PrgJ is responsible for flagellin-independent NLRC4 activation in S. typhimurium infection (76), it was shown that PrgJ could activate the NLRC4 inflammasome in Naip5 / macrophages to a similar extent as in wildtype cells (89). This might be one reason why the susceptibility of the A/J allele of Naip5 has only been observed in L. pneumophila but not in © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


bacteria that contain the T3SS. Other possible reasons may lie in the unique intracellular life style of L. pneumophila. Notably, studies of L. pneumophila infection performed in other mouse strains suggest that the sequence differences between the A/J and C57BL/6 alleles of Naip5 are of no functional significance (90). Thus, it is most likely that the lower expression of NAIP5 in A/J macrophages results in a lower level of caspase-1 activation that reaches the threshold of inflammasome activation required for limiting L. pneumophila growth. In retrospect, we now know that although Naip5 is only associated with restricting L. pneumophila replication, this does not necessarily mean that the biochemical function of NAIP5 is only involved in detecting L. pneumophila, a situation that would otherwise be difficult to imagine given the limited number of PRR genes in every sequenced mammalian genome. NAIPs are inflammasome receptors for bacterial flagellin and T3SS Flagellin and the T3SS rod protein are both detected by the NLRC4 inflammasome. As mentioned above, the rod protein and flagellin D0 domain adopt a similar secondary structural arrangement but share no evident sequence homology. Similar to the situation with flagellin, direct interactions between NLRC4 and the rod protein have not been reported in the literature to date. This suggests that other PRRs may serve as cytosolic receptors for the two NLRC4 inflammasome agonists. In 2011, both the Vance group and our group independently showed that NAIP5 and its paralog in mice (NAIP2) could specifically recognize flagellin and the T3SS rod protein, respectively, and that this recognition signaled NLRC4 for caspase-1 activation and downstream inflammasome responses (79, 80) (Fig. 3). The Vance study used flagellinless L. monocytogenes as a vehicle to deliver Legionella flagellin and PrgJ into the macrophage cytosol. They showed that short hairpin (shRNA) knockdown of Naip2 expression blocked PrgJ but not flagellin-induced pyroptosis and caspase-1 activation while genetic ablation of Naip5 only diminished the responses to flagellin. Co-expression of flagellin with NAIP5 or PrgJ with NAIP2 in 293T cells led to the loss of membrane integrity and cell death in the presence of NLRC4 and caspase-1. It was further noted from the transfection assays that NLRC4, which by itself ran as a monomer (approximately 120 kDa) on a native polyacrylamide gel, was mobility-shifted as a large oligomer (approximately 1000 kDa). The oligomer contained the co-expressed NAIP5/flagellin or NAIP2/PrgJ. Formation of the heterooligomeric NLRC4 complex in the reconstitution system © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

(proposed to be the NLRC4 inflammasome) required the specific pairings of flagellin with NAIP5 and PrgJ with NAIP2. Furthermore, truncated NLRC4 devoid of the LRR (DLRR) was found to be capable of activating caspase1-mediated cell death in the absence of NAIP5. In contrast, NAIP5 DLRR could not activate caspase-1 in the absence of NLRC4. On a native gel, NAIP5 exhibited a flagellin-dependent mobility shift in the absence of NLRC4, while the mobility change in NLRC4 only occurred in the presence of NAIP5. These observations confirmed the requirement of NAIP5 for NLRC4 sensing of flagellin and further suggested that NAIP5 acts upstream of NLRC4 (79). Our study employed fusion of flagellin or the T3SS rod protein with the anthrax lethal factor N-terminal domain (LFn) (80). This endocytosis-mediated delivery is highly efficient and avoids the complications associated with performing bacterial infection (91). Using the LFn-mediated delivery, it was observed that flagellins derived from several bacterial species including S. typhimurium and Y. enterocolitica all induced NLRC4-dependent caspase-1 activation, IL-1b release, and macrophage pyroptosis. All of these responses were found to be equally sensitive to shRNA knockdown of Naip5 expression. Meanwhile, knockdown of Naip5 did not affect PrgJ or other bacterial rod protein-induced NLRC4 inflammasome activation. These results suggested that NAIP5 was not dedicated to counteract L. pneumophila infection, but was rather an integral component of the flagellin-NLRC4 pathway. Similarly, knockdown of Naip2 markedly blocked PrgJ- but not flagellin-induced inflammasome activation. We were also able to reconstitute the NLRC4 pathway in 293T cells and found that NAIP5 and NAIP2 were capable of supporting flagellin and the rod protein-induced IL-1b processing, respectively. Similar to observations in the Vance study, flagellin expression promoted the physical association of NAIP5 with NLRC4; formation of a trimeric caspase-1-activating complex containing NLRC4, aNAIP, and the corresponding bacterial ligand was observed in the transfected 293T cells. This result promoted us to explore whether or not NAIPs are indeed receptors for the two bacterial molecules. In both yeast two-hybrid and co-immunoprecipitation assays, direct and specific binding between NAIP5 and flagellin as well as that between NAIP2 and PrgJ was readily detected. NLRC4 was not found to interact with either flagellin or PrgJ (80). These results provided the first direct experimental evidence that NLR proteins can function as receptors for microbial molecules in the inflammasome pathway. We also noticed that NAIP5 encoded by the A/J allele was fully functional in both the binding and the

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Fig. 3. Model of the NAIP–NLRC4 inflammasome in anti bacteria defenses. The NAIP–NLRC4 inflammasome can detect flagellin and the T3SS rod and needle proteins from both extracellular (Pseudomonas) and intracellular (Salmonella and Shigella) bacterial pathogens. Flagellin, the T3SS rod protein, and the T3SS needle protein are directly recognized by mouse NAIP5/6, mouse NAIP2, and mouse NAIP1/hNAIP, respectively. The specific ligand-receptor ligation stimulates a physical association of the NAIP with an adapter NLR protein, NLRC4. Both NAIP and NLRC4 exist in autoinhibitory conformational states in the absence of the bacterial agonists, and upon stimulation undergo co-oligomerization to form the inflammasome complex for caspase-1 activation. Active caspase-1 then processes IL-1b/IL-18 and induces macrophage pyroptosis, mounting an inflammatory response to restrict bacterial infection.

biochemical assays, supporting the notion that the loss of NAIP5 function in A/J macrophages probably results from the lower expression level (90). Both the Vance study and our study showed that knockdown of Naip2 expression diminished T3SS-dependent inflammasome activation during S. typhimurium and EPEC infections. Four of the seven mouse NAIPs (NAIP1, NAIP2, NAIP5, and NAIP6) were expressed in the C57BL/6 strain (84). Consistent with the fact that NAIP6 is the most homologous to NAIP5 (approximately 95% sequence identify), NAIP6 was also observed to recognize flagellin and found to be biochemically indistinguishable from NAIP5 (79, 80). The absence of a noticeable genetic function for Naip6 is probably due to its low expression levels in intact macrophages. Different from mouse macrophages, human U937 and THP1 monocyte-derived macrophages cells did not respond to cytosolic delivery of either flagellin or the T3SS rod protein (80). This result was intriguing, given that U937 and THP1 cells do express NLRC4 and downstream inflammasome components. Even more puzzling, infection of U937 cells with S. typhimurium induced robust caspase-1

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activation that was sensitive to genetic deletion of prgJ but not sensitive to deletion of flagellin. Experiments of profiling various bacterial strains and their genetic mutants identified Chrombacterium violaceum by virtue of its stimulation of NLRC4-dependent caspase-1 activation and pyroptosis in U937 cells. Importantly, deletion of neither flagellin-encoding genes nor cprJ encoding the T3SS rod protein had evident effects on C. violaceum-induced inflammasome activation. However, disruption of the entire cprJ-containing T3SS locus largely diminished inflammasome activation. This situation enabled systemic genetic analyses that identified the crpI gene (encoding the needle component) required for C. violaceum-induced inflammasome activation in U937 cells (80). Biochemically, CprI but not other proteins encoded by the T3SS locus was capable of triggering NLRC4-dependent caspase-1 activation in U937 cells. Importantly, needle proteins derived from other bacteria including S. typhimurium and S. flexneri showed a similar inflammasome-stimulation activity. Furthermore, hNAIP could specifically bind to the T3SS needle protein and support the reconstitution of the needle protein-induced NLRC4 © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


inflammasome activation in 293T cells. Thus, hNAIP functions analogously to NAIP2/5 in mice and serves as the receptor for the T3SS needle protein (80) (Fig. 3). In retrospect, the reason why the activity of the needle protein was identified in the C. violaceum infection study is that the CprI protein can leak into infected cytosol independently of the rod protein and a functional T3SS. We even observed that ectopic expression of CprI in L. pneumophila could trigger hNAIP and NLRC4-dependent caspase-1 activation in infected U937 cells (81). It is important to note here that the observation of inflammasome activation by the T3SS needle protein in human cells was only performed with U937/THP-1 monocytes and it is not unlikely that other human cells may respond to cytosolic flagellin. The needle protein could also be detected in mouse macrophages. Using LFn-mediated delivery, we found that T3SS needle proteins from various bacterial pathogens could activate the NLRC4 inflammasome in mouse macrophages; such activation specifically required NAIP1 (81). Thus, NAIP1 is the mouse ortholog of hNAIP; both proteins function similarly to NAIP2/5 and serve as receptors for the T3SS needle protein (Fig. 3). A similar conclusion was reached in a later study by Rayamajhi et al. (92). Our studies (80, 81) also profiled and compared the activities of the three ligands from different bacteria in biochemical and macrophage/dendritic cellular assays. Several interesting findings are worth noting here. First, flagellin and the T3SS rod and needle proteins from different bacteria all showed differential activity in triggering NLRC4 inflammasome activation. Some bacterial molecules even lacked evident activity, which, at least in the case of flagellin, correlated with an inability to bind the cognate NAIP receptor. Secondly, T3SS rod proteins generally showed a stronger activity than flagellin and the needle protein from the same species of bacteria. Thirdly, the relative expression levels of different NAIPs varied among different types of mouse macrophage and dendritic cells. Thus, the inflammasome-stimulation activity of a particular NAIP ligand is dependent on its own nature as well as on the cell type assayed. For a particular pathogen, infection-induced inflammasome activation results from a mixture of differential contributions from multiple NAIPs. For example, in the non-flagellated S. flexneri, the rod protein showed a relatively lower activity and the needle protein appeared to dominate infection-induced NLRC4 inflammasome activation, especially in bone marrow dendritic cells that expressed a relatively high level of NAIP1 (81). © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

The role of ASC in NAIP–NLRC4 inflammasome activation The inflammasome adapter ASC was originally purified and cloned from promyelocytic leukemia HL-60 cells; it aggregates and forms a speck-like structure following induction of apoptosis by retinoic acid (93). Overexpression studies suggested that ASC is an activator for caspase-1 autoprocessing resulting from a homotypical CARD–CARD interaction (94). The supramolecular assembly of ASC was termed the ‘pyroptosome’. Such pyroptosomes are platforms for mediating caspase-1 activation and inflammatory cell death (95). In the inflammasome pathway, AIM2, PYRIN, and NLRP3 of the NLRP subfamily of NLRs all contain a PYD and absolutely require ASC to induce caspase-1 activation and macrophage pyroptosis (41, 52) (Fig. 2). In contrast, the role of ASC in NLRC4 inflammasome activation is more complicated. Biochemically, NLRC4 can activate caspase-1 by itself as a result of CARD–CARD interaction. At the physiological level, deletion of Asc resulted in the absence of detectable caspase-1 autoprocessing and IL-1b maturation in S. typhimurium-infected mouse macrophages (59). However, Asc / macrophages still undergo pyroptosis following infection, although the extent of the cell death is less severe than that in similarly infected wildtype macrophages. This type of differential requirement of ASC has also been observed with other bacterial pathogens that trigger NLRC4 inflammasome activation (96). The promiscuous role observed for ASC is not specific to bacterial infections; purified flagellin or T3SS rod/needle proteins were also shown to require ASC for the induction of caspase-1 processing and IL-1b secretion, while the pyroptosis is only partially attenuated by Asc deficiency (79, 80). The role of ASC in the inflammasome pathway was clarified in a study from the research group of Denise Monack (97). By employing caspase-1 mutants lacking auto-cleavage sites, two functionally distinct inflammasome complexes were observed. One is called the ‘death inflammasome’. This complex is devoid of ASC and therefore caspase-1 is activated by an upstream inflammasome sensor such as NLRC4. Caspase-1 activation by the ‘death inflammasome’ is absent from autoprocessing, and this activation efficiently mediates pyroptosis but contributes little to cytokine processing. The other complex, microscopically characterized by the ASC speck structure, is formed when ASC co-aggregates with the inflammasome sensor. This ASC-containing inflammasome activates caspase-1 by inducing its autoprocessing, which contributes primarily to IL-1b/IL-18 processing and has an

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auxiliary role in pyroptosis. Formation of the ASC-containing inflammasome requires PYD-mediated dimerization of ASC, consistent with the presence of ASC dimers within the speck structure (95). The CARD of one ASC monomer is connected to the inflammasome sensor (like NLRC4), while the CARD of the other monomer is responsible for inducing caspase-1 autoprocessing. This model of ASC inflammasome assembly is supported by the critical role of CARD-mediated ASC-caspase-1 interaction in ASC speck formation and IL-1b processing (98). Recent high-resolution cryo-electron microscopy (EM) studies showed that the PYD in ASC formed a filamentous structure in vitro (99). Formation of the ASC filaments resulted in clustering of its CARD, which then nucleated the CARD of caspase-1, leading to proximityinduced activation of the caspase. It was also proposed in the EM study that the PYD in inflammasome sensors such as NLRP3 may nucleate the PYD of ASC to promote the formation of filaments. This mechanism for ASC-containing inflammasomes is appealing for understating PYD-containing inflammasome sensors, but fails to address how NLRC4-like PRRs that bear no PYDs induce the formation of the ASC filaments. Structural mechanism of the NAIP–NLRC4 inflammasome NLRC4, like other NLRs as well as APAF-1 in the apoptosome, is kept in an inactive state in the absence of stimuli and therefore cannot relay a signal to the ASC adapter and caspase-1. A crystal structure of NLRC4 lacking the N-terminal CARD (DCARD) was recently determined (100). The overall structure, comprising the NOD, a linking helical domain 2 (HD2), and the C-terminal LRR domain, is shaped into an inverted question mark. The NOD domain can be divided into the NBD, helical domain 1 (HD1), and the winged-helix domain (WHD). An ADP molecule, albeit not intentionally supplemented in the experiments, was identified in the solved crystal structure. This phenomenon was previously observed in the crystal structure of APAF-1 purified from insect cells (101). Also similar to APAF-1, NLRC4 DCARD adopted an autoinhibitory conformation. Detailed analyses of the structure suggest that the ADP-mediated interactions between the NBD and the WHD, as well as the structural contact of HD2 with the NBD, play critical roles in maintaining the closed inactive conformation of NLRC4 DCARD. The LRR domain also contacts the NBD and sterically prevents NBD oligomerization-induced NLRC4 activation. Point mutations or truncation deletions disrupting these interaction interfaces all resulted in constitutive or partially constitutive NLRC4 activation, as indicated by the

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appearance of NAIP-independent IL-1b processing in transfected 293T cells (100). A recent study reported that Ser-533, located in the HD2 in the NLRC4 structure, was phosphorylated upon challenge by S. typhimurium; this phosphorylation appeared to be required for NLRC4 inflammasome activation (102). The phosphorylation was mediated by PKCd. Pharmacological inhibition of PKCd kinase activity could block NLRC4-mediated caspase-1 activation induced by S. typhimurium or L. pneumophila infection. Interestingly, in the crystal structure of NLRC4 DCARD, Ser-533 was found to be already phosphorylated (100), suggesting that phosphorylation is at least not sufficient for NLRC4 activation. Another study showed that genetic ablation of PKCd had no effect on S. flexneri- and S. typhimurium-induced NLRC4-mediated caspase-1 activation (103). Thus, the exact function of Ser-533 phosphorylation in NLRC4 inflammasome activation appears complicated and will require clarification in future studies. Upon ligand stimulation, the NAIP receptor and NLRC4 form an active oligomeric ‘inflammasome’ complex, as demonstrated in non-macrophage reconstitution assays (79, 80). Recently, Halff et al. (104) purified the NAIP5–NLRC4 complex from 293T cells and performed preliminary structural analyses using negative-stain EM analysis. In the EM micrographs, the inflammasomes appeared as disk-shaped particles with a radius of approximately 28 nm. The LRR domains could be clearly located at the perimeter of the disk; the CARD of NLRC4 was exposed on one face of the disk, and served as a platform for activating caspase-1 or the ASC adapter. Each disk contained 11–12 symmetric protomers, but their identity (NLRC4 or NAIP5) could not be determined due to the low resolution of the EM model. The size of the NAIP–NLRC4 inflammasome particle is larger than that of the apoptosome that contains 5–8 protomers (105, 106). Two NAIP5–NLRC4 disks appear to be stacked into pairs, as revealed by the side view the EM tomogram. In another study (107), the stoichiometry of the ligand, NAIP, and NLRC4 constituents within similarly purified ‘inflammasome’ complexes was estimated to be around 2:2:5. In the inflammasome activation model proposed from the EM analyses, flagellin recognition induces conformational changes of NAIP5, which by itself cannot oligomerize but recruits and activates NLRC4. This results in the progressive incorporation of NLRC4 monomers into the disk-shaped inflammasome complex. Consistent with this model, both the Vance group and our group showed that the ATP-binding P-loop in the NBD of NLRC4 was required for the reconstitution of functional NAIP–NLRC4 complexes in 293T cells (79, 80). In contrast, the equivalent P-loop mutant of © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


NAIP5 could still form an inflammasome complex with NLRC4 and such complexes could bind the flagellin ligand robustly (104). These findings indicate that ATP-binding by NAIP5 is not a prerequisite for NLRC4 inflammasome assembly. Notwithstanding, all of the above studies were performed in artificial in vitro systems. Man et al. (108) recently performed preliminary analyses of the inflammasome complex by combining confocal and superresolution microscopy imaging in S. typhimurium-infected macrophages. The results indicated that endogenous NLRC4 forms a ring structure within the ring-like structure of the ASC speck. Caspase-1 and other inflammasome effectors/substrates are recruited to the inside of the inflammasome ring and get activated. Despite this, precisely how flagellin induces NAIP–NLRC4 inflammasome assembly in a physiological context still requires further biochemical and structural investigations. NAIP recognition of the bacterial ligand initiates the assembly of the NLRC4 inflammasome. Given the high sequence identity among the seven NAIPs in mice (more than 80%), an important outstanding question is how NAIPs recognize their cognate ligands and what determines ligand specificity. To address this, the Vance group (107) generated a series of NAIP2–NAIP5 chimeras and analyzed their ability to recognize the ligands and form functional inflammasomes upon stimulation. These extensive analyses revealed that the LRR domains in the NAIP receptor were dispensable in the recognition of flagellin and the T3SS rod protein. Unpublished data from our group confirm this observation. This finding is unexpected, given the widely believed dogma that the LRR domain is the PAMP-binding region of NLRs. NAIP is monomeric on its own; binding to the corresponding ligand induces co-oligomerization of the NAIP receptor with NLRC4 (107). It was further suggested in the Vance study that NBD-associated helical domains in NAIPs, but not the BIR domain or the NBD, confer ligandbinding specificity. Despite these important observations, it is not clear whether the NBD-associated helical domains are sufficient for ligand binding or other structural regions are also involved. Future studies in this direction shall be aimed to reveal the structural basis for specific ligand binding by the NAIP receptors. Function and regulation of the NAIP–NLRC4 inflammasome It is now well established that the NAIP–NLRC4 inflammasome plays a general role in the innate detection of bacteria © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

that are flagellated and/or harbor a T3SS. In the most extensively studied L. pneumophila infection model, activation of the NAIP5–NLRC4 inflammasome limits bacterial growth in infected macrophages as well as in the lungs and lymph nodes of infected mice (61, 62, 70, 77, 86, 109). This function of the NAIP5–NLRC4 inflammasome contributes to development of a coordinated host response that protects the mice from lethal L. pneumophila infection (110). It has been proposed that NAIP5/NLRC4-mediated caspase-1 activation may antagonize the ability of L. pneumophila to remodel the LCV into an ER-like compartment that would otherwise undergo fusion with the lysosome (70, 111), but the underlying mechanism has not been defined. Flagellin activation of NLRC4 could also promote clearance of L. monocytogenes (74), P. aeruginosa (72), B. thailandensis, and S. typhimurium (72, 76, 109, 112, 113). These bacteria have a life style different from that of L. pneumophila; therefore, the NLRC4 inflammasome in macrophages infected with these bacteria likely does not function in the same manner as that proposed for controlling L. pneumophila growth. Using engineered S. typhimurium constitutively expressing flagellin or PrgJ, Miao et al. (76, 109) showed that NLRC4 inflammasome activation inhibited systemic bacterial infection in mouse intraperitoneal infection. This effect was independent of IL-1b and IL-18, despite that many cytokines were upregulated upon sensing of flagellin and S. typhimurium infection (109, 114). Instead, restriction of S. typhimurium infection was critically mediated by caspase-1-mediated pyroptosis. Consistent with the absence of ASC in the ‘death NLRC4 inflammasome’ (97), clearance of the flagellinexpressing S. typhimurium did not require ASC. It has been further suggested that caspase-1-induced pyroptosis releases the bacteria from infected macrophages, resulting in uptake by the neutrophil and consequently killing of the bacteria by reactive oxygen species. NLRC4 inflammasome activation can also clear B. thailandensis and L. pneumophila infection through a cytokine-independent mechanism (109). Thus, neutrophil-mediated killing may apply generally to bacteria that are detected by NAIPs. On the host side, Nlrc4 / mice succumbed to infection with the flagellin-expressing S. typhimurium. This effect was also mediated primarily by pyroptosis while IL-1b/18 only played a minor role (109). Neutrophil itself responded to S. typhimurium both in vitro and during peritoneal infection (115). The resulting NLRC4 inflammasome activation did not induce pyroptosis, but provided a major source of IL-1b in peritoneal S. typhimurium infection. This finding illustrates an unexpected mechanism

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for NLRC4 inflammasome-mediated anti bacterial defense, which involves non-macrophage cells (neutrophils). In S. typhimurium oral infection of streptomycin-treated mice, the bacteria invade and breach the intestine mucosal epithelium to cause colitis. In this process, the NAIP– NLRC4–caspase-1 axis plays an important role by limiting bacterial replication and systemic infection of other organs (112, 114, 116, 117). A recent study proposed an interesting mechanism for NAIP–NLRC4 inflammasome function in this model (113). At the early phase of infection, activation of the inflammasome resulted in expulsion of infected enterocytes into the lumen, thereby disrupting the intraepithelial replication niche of the bacterium. As a result, deficiency of the NLRC4 pathway increased bacterial loads in the epithelium and promoted colonization in lymph nodes. Notably, expelling of the infected epithelial cells did not require IL-1 and IL-18 signaling. This finding favors the idea that it is caspase-1-induced pyroptosis that plays the key role, but this notion is not supported by subsequent preliminary analyses (113). The discovery of NLRC4 inflammasome function in non-hematopoietic cells brings a new concept to the study of inflammasome-mediated anti-bacteria defenses. The concept was also supported by another recent study that demonstrated that NLRC4 expression in epithelial crypts could limit Citrobacter rodentium infection (118). High doses of oral S. typhimurium infection also caused death of the mice; in this process, the role of the NLRC4 inflammasome appeared to be dependent upon the genetic background of the mice. In BALB/c mice (treated with streptomycin), loss of the NLRC4 inflammasome yielded higher lethality (119). Different from what was observed in peritoneal infection (109), IL-1b-mediated neutrophil recruitment has been suggested to play a critical defense role in the S. typhimurium intestinal infection model. As for the possible source of IL-1b, it was observed that intestinal phagocytes constitutively expressed high levels of pro-IL1b. In vitro, these cells produced substantial amounts of mature IL1b as a result of S. typhimurium infection-induced NLRC4 inflammasome activation. In light of the observation that intestinal phagocytes are anergic to commensals, it was proposed that the NLRC4 inflammasome can discriminate between pathogenic and commensal bacteria by inducing IL-1b maturation (119). This idea is consistent with the fact that NAIPs not only recognize flagellin but also recognize the T3SS that is specific to pathogenic bacteria. Hyperactivation of the NLRC4 inflammasome can lead to lethality in mice. This has been observed with LFn-mediated cytosolic delivery of flagellin as well as with the antibiotic-

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triggered systemic spread of a multidrug-resistant E. coli pathobiont (120, 121). In the former case (121), systemic inflammasome activation stimulated eicosanoid biosynthesis in resident peritoneal macrophages. This resulted in pathological release of multiple signaling lipids including prostaglandins and leukotriene. The ‘eicosanoid storm’ then triggered vascular fluid loss into the intestine and peritoneal cavity; concurrently severe systematic inflammation ultimately killed the mice. Such a pathological effect requires NAIP5, NLRC4, and caspase-1, but is independent of IL-1b/ 18. Pharmacological inhibition of eicosanoid synthesis did not affect pyroptosis, suggesting a possible new pathway linking caspase-1 to eicosanoid biosynthesis (121). Lethal NLRC4 inflammasome activation was also observed in mice treated with antibiotics plus dextran sulfate sodium (to induce intestinal injury) (120). In this process, dramatic changes in the microbiota composition and consequently extraintestinal outgrowth of a multidrug-resistant E. coli O21:H+ strain were observed. The mice died of a sepsis-like symptom, which was primarily caused by the virulence activity of the O21:H+ E. coli strain. Genetic analyses suggested that IL-1b signaling downstream of the NAIP5– NLRC4 axis played a dominant role in protecting the mice from the sepsis-like disease (120). Bacterial pathogens have also developed strategies to evade NLRC4-mediated innate immune defenses. First, bacteria often shut down the expression of the ligands of NAIP receptors following successful engagement with or invasion of the host. For example, Yersinia spp. turn off flagellin expression when exposed to 37°C in the host, despite that the flagellin protein of Yersinia can biochemically activate the NAIP5–NLRC4 inflammasome (80, 122). S. typhimurium relies on its SPI1 T3SS to invade phagocytic and non-phagocytic cells. However, following successful invasion in systemic infection, S. typhimurium shuts down the expression of both flagellin and the SPI1 T3SS to avoid NAIPs-mediated immune detection (109). Secondly, flagellins from several bacteria, including EPEC, EHEC, B. thailandensis, and C. violaceum, are intrinsically inactive in binding to NAIP5/6 and inducing inflammasome activation (80). Differential inflammasome-stimulating activity has also been observed with T3SS rod and needle proteins from different bacterial pathogens (81). For example, S. typhimurium SPI2 T3SS, which is activated after host cell invasion, is critical for bacterial intracellular growth. However, the SPI2 rod protein is devoid of inflammasome-stimulating activity. Thus, certain bacterial ligands, pressured by the host surveillance mechanism, have evolved to avoid innate immune detection by © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


the NAIP receptors. The third bacterial strategy is to deploy virulence factors to block NAIP–NLRC4 inflammasome activation. For example, the P. aeruginosa T3SS effector ExoU is known to inhibit infection-induced NLRC4 activation (72). Mycobacterium tuberculosis Zmp1 blocked both NLRC4- and NLRP3-mediated caspase-1 activation (123). The Yersinia YopK protein prevented inflammasome detection of the T3SS by interacting with the translocon within the host cell membrane (124). Concluding remarks and perspectives Studies in the past 10 years have defined an NAIP–NLRC4– ASC inflammasome pathway that has a general role in the cytosolic detection of bacteria and bacterial infections (59, 61, 62, 67, 68, 76, 77, 79–81, 83, 84). We now have a complete biochemical framework of the NAIP–NLRC4 inflammasome, in which NAIP1/hNAIP, NAIP2, and NAIP5/6 sense bacterial T3SS needle protein, rod protein, and flagellin, respectively. In this scenario, the NAIP functions as a ligand-specific receptor that transmits the signal to another adapter NLR (NLRC4) for the induction of caspase-1 activation. Given the evolutionary connection between flagella and the T3SS, it makes perfect sense that hosts have evolved closely related NAIP receptors to recognize these two systems. From a biochemical perspective, the mechanism of the NAIP–NLRC4 inflammasome echoes that of APAF-1 apoptosome; both systems employ a CARD-containing NLR to activate a caspase and trigger cell death through CARD– CARD interactions. The inflammasome detects a bacterial molecule, while the apoptosome captures cytochrome-C released from the mitochondria. The mitochondrium is known to have originated from a bacterium, but the different biological nature of bacteria and the mitochondria determines that one pathway triggers inflammatory cell death while the other induces non-inflammatory apoptosis. With this in mind, it is interesting to note the structural similarities between the T3SS needle protein and the Bcl-2 family of proteins in apoptosis, both of which bear a conserved helix-turn-helix motif and form membrane pores for substance release (125). Different from the apoptosome, the (NAIP–NLRC4) inflammasome detects the pore directly rather than detecting the released substrate, likely owing to the large number of diverse bacterial effectors that are encountered in the life of an animal. While mammals contain less than 30 NLR proteins per species, plants have evolved hundreds of them. This diversity allows plants to specifically recognize a large number of pathogenic © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

virulence effectors through the ‘effector-triggered immunity’. The NAIP–NLRC4 innate immune axis involves two NLR proteins for the detection of a single PAMP; one is a receptor and the other is an adapter. This mode of action is also seen in plant immunity (126). For example, two Arabidopsis NLR proteins RPS4 (resistance to Pseudomonas syringae 4) and RRS1 (resistance to Ralstonia solanacearum 1) associate together to monitor the virulence activity of the AvrRps4 or PopP2 effectors. This highlights the conservation of the innate immunity between mammals and plants. The NAIP– NLRC4 inflammasome is the best understood inflammasome pathway in both biochemical mechanism and biological function. However, we are still in the early stages of depicting a complete mechanistic model for ligand-induced activation of a specific NAIP–NLRC4 complex (79, 80, 100, 104, 107). From a more practical perspective, the innate immunestimulation activity of flagellin and T3SS rod/needle proteins may help to better combat bacterial infections. In particular, flagellin is being explored as a promising vaccine adjuvant; the differential inflammasome-stimulation activities in different bacterial flagellins may allow for fine-tuning the design of the adjuvant. In light of the differential activities of T3SS needle/rod proteins, it is worth considering exploring needle/rod proteins as new candidates for vaccine adjutants. In addition, bacterial genetic mutants with deficient or engineered T3SS needle/rod proteins may increase the success rate in the development of anti-bacteria vaccines. While NLRC4 inflammasome activation is critically important for immune defenses against bacterial infection, excessive NLRC4 inflammasome activation can lead to severe disease in human. Two recent studies reported that NLRC4 mutations cause a rare autoinflammatory disease termed SCAN4 (syndrome of enterocolitis and autoinflammation associated with mutation in NLRC4) (127) or NLRC4-MAS (NLRC4 macrophage activation syndrome) (128). The patients developed periodic fever, diarrhea, splenomegaly, duodenal inflammation, anemia, and severe autoinflammation, leading to death of a 23-day-old infant. Characterization of monocytes derived from the patients suggested a spontaneous hyperactivation of the inflammasome. The disease mutants of NLRC4 (V341A and T337S) are gain-of-function, resulting from the disruption of the auto-inhibited state of the NLRC4 structure. Different from autoinflammatory diseases caused by mutations in other inflammasome scaffolds like NLRP3 and PYRIN (39, 40, 47, 48, 129), the NLRC4-MAS patients suffered from unexpected enterocolitis. It remains to be

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determined whether and how NLRC4 activation gave rise to such gastrointestinal symptoms and what types of cells (macrophages, epithelial cells, or others?) are primarily responsible for the gastrointestinal symptoms. Knocking-in the disease mutation into mice, ideally in a tissue-specific

manner, may help to address this important question. Such a mouse model should also be beneficial for the in vivo study of the immunological function of the NAIP–NLRC4 inflammasome.

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The type III secretion system apparatus determines the intracellular niche of bacterial pathogens.

Functional Activation of the Flagellar Type III Secretion Export Apparatus.

Type III secretion systems: the bacterial flagellum and the injectisome.

Innate immune sensing of bacterial modifications of Rho GTPases by the Pyrin inflammasome.

Assembly of the bacterial type III secretion machinery.

Edwardsiella tarda-Induced cytotoxicity depends on its type III secretion system and flagellin.

ExsB is required for correct assembly of the Pseudomonas aeruginosa type III secretion apparatus in the bacterial membrane and full virulence in vivo.

Immune complexes inhibit IL-1 secretion and inflammasome activation.

Hexokinase Is an Innate Immune Receptor for the Detection of Bacterial Peptidoglycan.

Inflammasome activation in response to the Yersinia type III secretion system requires hyperinjection of translocon proteins YopB and YopD.

Type III secretion system.

The bacterial alarmone (p)ppGpp activates the type III secretion system in Erwinia amylovora.

Burkholderia cenocepacia Lipopolysaccharide Modification and Flagellin Glycosylation Affect Virulence but Not Innate Immune Recognition in Plants.

Bacterial autolysins trim cell surface peptidoglycan to prevent detection by the Drosophila innate immune system.

Hypothetical protein CT398 (CdsZ) interacts with σ(54) (RpoN)-holoenzyme and the type III secretion export apparatus in Chlamydia trachomatis.

Bacterial Control of Pores Induced by the Type III Secretion System: Mind the Gap.

Identification of the flagellin glycosylation system in Burkholderia cenocepacia and the contribution of glycosylated flagellin to evasion of human innate immune responses.

Editorial: secretion of cytokines and chemokines by innate immune cells.

The inflammasome adaptor ASC contributes to multiple innate immune processes in the resolution of otitis media.

Bacterial lipids: powerful modifiers of the innate immune response.

Structure of a bacterial type III secretion system in contact with a host membrane in situ.

The bacterial type III secretion system as a target for developing new antibiotics.

A bacterial type III secretion-based protein delivery tool for broad applications in cell biology.

Bacterial Exotoxins and the Inflammasome.