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Of inflammasomes and pathogens – sensing of microbes by the inflammasome

2013; Springer Nature; Volume: 5; Issue: 6 Linguagem: Inglês

10.1002/emmm.201201771

1757-4684

Franz Bauernfeind, Veit Hornung,

Biomarkers in Disease Mechanisms

Review13 May 2013Open Access Of inflammasomes and pathogens – sensing of microbes by the inflammasome Franz Bauernfeind Franz Bauernfeind Institute for Clinical Chemistry and Pharmacology, Unit for Clinical Biochemistry, University Hospital, University of Bonn, Germany Department of Internal Medicine III, University Hospital, University of Bonn, Germany Search for more papers by this author Veit Hornung Corresponding Author Veit Hornung [email protected] Institute for Clinical Chemistry and Pharmacology, Unit for Clinical Biochemistry, University Hospital, University of Bonn, Germany Search for more papers by this author Franz Bauernfeind Franz Bauernfeind Institute for Clinical Chemistry and Pharmacology, Unit for Clinical Biochemistry, University Hospital, University of Bonn, Germany Department of Internal Medicine III, University Hospital, University of Bonn, Germany Search for more papers by this author Veit Hornung Corresponding Author Veit Hornung [email protected] Institute for Clinical Chemistry and Pharmacology, Unit for Clinical Biochemistry, University Hospital, University of Bonn, Germany Search for more papers by this author Author Information Franz Bauernfeind1,2 and Veit Hornung *,1 1Institute for Clinical Chemistry and Pharmacology, Unit for Clinical Biochemistry, University Hospital, University of Bonn, Germany 2Department of Internal Medicine III, University Hospital, University of Bonn, Germany *Tel: +49 228 28751200; Fax: +49 228 28751201 EMBO Mol Med (2013)5:814-826https://doi.org/10.1002/emmm.201201771 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Abstract Inflammasomes are signalling platforms that sense a diverse range of microbial products and also a number of stress and damage associated endogenous signals. Inflammasome complexes can be formed by members of the Nod-like receptor family or the PYHIN family member AIM2. Upon formation, inflammasomes trigger proteolysis of caspase-1, which subsequently leads to a potent inflammatory response through the maturation and secretion of IL-1 family cytokines, which can be accompanied by an inflammatory cell death termed pyroptosis. Here, we review the sensing mechanisms of the currently characterized inflammasome complexes and discuss how they are involved in the innate immune response against microbial pathogens. We especially highlight recent advances in the molecular understanding of how microbial patterns are detected and discriminated from endogenous compounds by inflammasome sensors. Further, we review how inflammasomes contribute to the anti microbial host defense by cytokine-dependent and cell autonomous mechanisms. This review is part of the review series on host-pathogen interactions. See more reviews from this series.. Introduction Physical barriers and immune defense systems have evolved to protect the host from microbial invasion. Acute inflammation is a response to infection or cellular disturbances by other means such as trauma. The initial inflammatory reaction limits harm to the body by directly impacting on microbial propagation and also by indicating the site of disturbed homeostasis to cells of the adaptive immune system. This immediate and innate immune effect is predominantly mediated via myeloid cells that sense conserved microbe associated molecular patterns (MAMPs) via a limited repertoire of germ-line encoded pattern recognition receptors (PRRs). MAMPs are mostly foreign structures and thus allow the specific sensing of invading organisms (Medzhitov, 2008). At the same time, some PRRs can also be triggered by endogenous substances that are formed or released during cell stress, perturbation of tissue homeostasis or metabolic imbalance. In analogy to the MAMP terminology, these signals are commonly referred to as damage associated molecular patterns (DAMPs). Activation of PRRs of the Toll-like receptor system (TLRs), RIG-I-like receptors (RLRs) or C-type lectin receptors (CLRs) initiates signalling cascades that result in pro-inflammatory gene expression. Additionally, PRR engagement sets off cascades that culminate in the proteolytical activation of inflammatory caspases. Hereby, a major inflammatory pathway is the cleavage and activation of caspase-1, which is initiated upon the formation of large multiprotein signalling platforms, the so-called inflammasomes. Activated caspase-1 proteolytically cleaves the cytokine precursors of interleukin-1β (IL-1β) and interleukin-18 (IL-18) to initiate a pro-inflammatory and antimicrobial response (Bauernfeind et al, 2011a). Various sensor proteins have been identified that can trigger the formation of inflammasome platforms. These inflammasome-forming PRRs, except for the DNA sensor AIM2, belong to the Nod-like receptor (NLR) family. NLRs are cytosolic PRRs with a tripartite domain architecture comprising of C-terminal leucine-rich repeats (LRRs) that are thought to sense microbial molecules or endogenous stress mediators; a central NACHT nucleoside triphosphatase domain that mediates NLR oligomerization and formation of the core structure of the inflammasome; and an N-terminal effector domain required for signal transduction. The latter can consist of a pyrin domain (PYD), a caspase recruitment domain (CARD) or a baculovirus inhibitor of apoptosis protein repeat (BIR) domain (Ting et al, 2008). Several NLRP proteins (NLR subfamily with N-terminal PYD domain) and the protein NLRC4 (NLR family CARD domain-containing protein 4) have been shown to form an inflammasome upon stimulation with the respective activator. Hereby, oligomerization of the inflammasome sensors NLRP1, NLRP3, NLRP6 or AIM2 (HIN200 protein family) allows interaction of the respective N-terminal PYD with the PYD of the protein ASC (Apoptosis associated speck-like containing a CARD domain) (Bauernfeind et al, 2011a; Elinav et al, 2011). ASC itself then recruits pro-caspase-1 via CARD-CARD interactions. According to its bipartite structure, composed of a PYD and CARD, ASC is often referred to as a bridging or adapter protein. The interaction of NLRPs, ASC and caspase-1 is based on homotypic interactions, which are common for death fold domains of the same subfamily, usually forming dimers or multimers. On the other hand, interactions across death domain subfamilies (e.g. CARD with PYD) are uncommon, even though somewhat surprising given their overall structural similarities. An overview of inflammasome proteins is depicted in Fig 1. Figure 1. Assembled inflammasomes. The nucleotide binding domain and LRR containing (NLR) family proteins NLRP1, NLRP3, NLRC4, NLRP6, NLRP12 and the pyhin protein AIM2 recruit and activate pro-caspase-1 indirectly through the bridging protein apoptosis-associated speck-like protein containing a CARD (ASC). Download figure Download PowerPoint Inflammasomes sense microbes in the cytosol to initiate an innate response Phagocytic cells of the innate immune system, e.g. macrophages, respond to microbial infection using several mechanistically diverse signalling cascades. TLRs or CLRs, for example, sense extracellular or endolysosomal MAMPs and subsequently orchestrate immune responses by the transcriptional induction of anti-microbial effector molecules or mechanisms. At the same time, PRR activation can also activate effector mechanisms that function in the absence of de novo protein expression. For example, TLR4 ligation leads to rearrangement of the cytoskeleton and subsequently enhanced phagocytosis (Blander and Medzhitov, 2004). On the other hand, activated inflammasomes lead to proximity-induced autoproteolytic cleavage of the pro-enzyme caspase-1, resulting in its activation. Its thus generated subunits (p10 and p20) build tetramers to form the active cysteine protease, which converts the inactive IL-1β precursor to the C-terminal and active fragment. The pro-inflammatory cytokine IL-1β is an acute phase response mediator and promotes inflammation, vasodilation, hyperthermia and extravasation of immune cells, and is further involved in generation of Th17 cells (Dinarello, 2009). Notably, pro-IL-1β is expressed only at limiting amounts in resting cells and needs to be induced via the TLR signalling axis (Dinarello, 2009). The second well characterized caspase-1 substrate IL-18 is also expressed as a pro-cytokine but underlies only minimal transcriptional regulation. The caspase-1 cleaved C-terminal and secretable part of IL-18 promotes, together with IL-12, the production of IFN-γ in Th1, natural killer (NK) and cytotoxic T cells (Dinarello, 2009). Apart from its processive function, caspase-1 is required for an unconventional protein secretion pathway that is crucial for the release of IL-1β and various other target proteins (Keller et al, 2008). In addition, activity of caspase-1 in myeloid cells results in a special type of cell death, known as pyroptosis. Pyroptotic cell death is programmed, caspase-1 dependent and pro-inflammatory. In contrast, apoptosis is not inflammatory, does not require caspase-1 yet is dependent on the effector caspases caspase-3, caspase-6 and caspase-7 (Bergsbaken et al, 2009; Kuida et al, 1995). Apoptotic cell death further results in regulated degradation and clearance of cellular contents (orchestrated disassembly of the cell). However, during pyroptosis cellular contents are released to the extracellular space and can induce inflammation (Bergsbaken et al, 2009). Thus, pyroptosis shares features of apoptosis and necrosis. In contrast to the former assumption that pyroptosis may be a pro-microbial mechanism employed by bacteria to destroy host phagocytes via induction of suicide in host cells, current data suggest, that pyroptosis rather functions as a host defense mechanism used to clear intracellular pathogens (Miao et al, 2010a). AIM2 inflammasome The PYHIN family protein AIM2 is the only inflammasome sensor that does not belong to the NLR family, nevertheless some structural features are shared. AIM2 is characterized by the presence of an N-terminal PYD and a C-terminal HIN200 DNA-binding domain. Since AIM2 lacks a CARD, it essentially requires, similar to NLRPs, the bridging protein ASC to recruit caspase-1. Unlike other members of the PYHIN family, AIM2 is preferentially localized in the cytosol and operates as a direct intracellular sensor for cytosolic DNA (Fernandes-Alnemri et al, 2009; Hornung et al, 2009). So far, no substantial prerequisites for its ligand DNA have been described (e.g. sequence motifs or nucleotide modifications), beside the DNA needs to be double stranded and of more than 80 bp in length to accomplish sufficient AIM2 inflammasome formation to allow caspase-1 cleavage (Jin et al, 2012). Since AIM2 does not contain a NACHT domain, which could facilitate its multimerization, it was already initially speculated that the dsDNA sensed by AIM2 could provide the matrix for oligomerization (Bauernfeind et al, 2011a). This hypothesis is now tightened by the recently published crystal structure of the HIN domain of AIM2 in complex with its dsDNA ligand. Here, it was shown that non-sequence-specific DNA recognition is accomplished through electrostatic attraction between the positively charged HIN domain residues and the negatively charged dsDNA sugar-phosphate backbone. Upon binding, the pyrin domain is liberated from an intramolecular autoinhibitory complex (PYD/HIN domain complex). This facilitates the assembly of inflammasomes along the DNA staircase, whereby a HIN domain spans a spacing of 7–8 bp on each side of the dsDNA (Jin et al, 2012). As such, it is not surprising that transfected double stranded DNA (dsDNA) of either viral, procaryotic or mammalian origin was shown to bind to and activate AIM2 (Hornung et al, 2009). The fact that AIM2 activation does not require a certain sequence motif also implies that unintentional AIM2 activation by endogenous DNA is only prevented by AIM2's cytosolic compartmentalization, where dsDNA is not present under physiological conditions. Although it was demonstrated in the past that endogenous DNA could accumulate or access cytosolic compartments when improperly degraded or insufficiently cleared from the extracellular space (Kawane et al, 2006), AIM2 activation through endogenous DNA has yet not been demonstrated in a physiological disease model. However, several microbial invaders can gain access to the cytosol of phagocytic cells and release foreign DNA to trigger AIM2 and caspase-1 activation. This has been shown for the DNA viruses vaccinia virus (VACV) and murine cytomegalovirus (MCMV) (Rathinam et al, 2010) as well as upon infection with bacterial pathogens such as Francisella tularensis (Fernandes-Alnemri et al, 2010; Rathinam et al, 2010), Listeria monocytogenes (Kim et al, 2010) and certain Legionella pneumophila strains (Ge et al, 2012). It is widely accepted and experimentally proven that free cytosolic microbial DNA is required to activate AIM2. However, it is currently unknown for F. tularensis infection, if the DNA is released while the bacterium is still within the phagosome or when it has entered the cytosol. Francisella virulence is closely linked to its ability to replicate in the host cytosol. Hypercytotoxic mutants that are defective for membrane proteins that affect bacterial stability or susceptibility for lysis show a higher release of cytosolic DNA, resulting in enhanced AIM2 activation and pyroptosis (Peng et al, 2011). Legionella pneumophila, the causative agent of Legionnaires' disease, resides in a distinct vacuole structure called Legionella-containing vacuole (LCV), which functions to avoid the fusion with the lysosome. Efficient L. pneumophila replication in the host macrophage requires a bacterial secretion system to translocate the effector SdhA to prevent host cell death. SdhA is most likely involved in membrane trafficking and was recently shown to maintain the integrity of the LCV. Strains lacking SdhA are defective for intracellular replication due to host cell death by DNA initiated pyroptosis (Creasey & Isberg, 2012; Ge et al, 2012). L. monocytogenes, on the other hand, replicates in the cytosol after escaping the phagosome by employing the pore forming cytolysin listeriolysin O (LLO) (Dramsi & Cossart, 2002) and loss of cell wall integrity through cytosolic bacteriolysis results in the release of bacterial DNA into the macrophage cytosol (Sauer et al, 2010; Warren et al, 2010). Thus, it seems that AIM2 gets activated whenever foreign DNA is present in the cytosol of inflammasome competent cells. As a consequence, bacterial pathogens seem to avoid AIM2 recognition through maintenance of bacterial structural integrity and protection from cytosolic host factors in vacuolar niches. AIM2 deficiency increases the virulence of MCMV (Rathinam et al, 2010) and F. tularensis (Fernandes-Alnemri et al, 2010) in vivo. In a mouse model of MCMV infection, the AIM2 inflammasome mediates NK cell-dependent production of IFN-γ via IL-18 processing. These events were critical for the very early innate response to infection and are reflected by higher titers of MCMV in spleens of AIM2 deficient mice compared to littermate controls, however only for an early time point (Rathinam et al, 2010). Furthermore, AIM2 deficient mice are extremely susceptible to F. tularensis infections and display a higher bacterial burden in tissues, which is associated with greater mortality (Fernandes-Alnemri et al, 2010). The NLRC4 inflammasome NLRC4 (formerly known as IPAF, Card12) has been shown to form an inflammasome upon infection of macrophages with various gram-negative bacteria such as Salmonella typhimurium (Mariathasan et al, 2004), Legionella pneumophila (Zamboni et al, 2006), Shigella flexneri (Suzuki et al, 2007) and Pseudomonas aeruginosa (Miao et al, 2008). NLRC4 contains an N-terminal CARD for signal transduction, a central NACHT domain, and a C-terminal LRR domain and it was shown to associate directly and specifically with the CARD domain of pro-caspase-1 through CARD–CARD interactions in overexpression systems (Poyet et al, 2001). Nevertheless, ASC seems to be required for cytokine processing by caspase-1, whereas cell death initiated by NLRC4 signalling occurs independent of ASC (Aachoui et al, 2013; Broz et al, 2010). Early studies of NLRC4 inflammasome activation by gram-negative bacteria revealed that functional bacterial type III or type IV secretion systems seemed to be required for caspase-1 cleavage. Further experiments uncovered that S. typhimurium and L. pneumophila strains deficient for the protein flagellin, the main component of flagellum, were defective in their ability to activate NLRC4 (Miao et al, 2006; Zamboni et al, 2006). Flagellin independent NLRC4 activation by P. aeruginosa (Sutterwala et al, 2007) was later attributed to the sensing of compounds of the bacterial type III secretion (T3S) system (Miao et al, 2010b). T3S systems function to deliver multiple bacterial effectors that confer virulence into the host cell to disrupt certain host processes to allow bacterial replication, particularly by circumventing an innate immune response (Gong & Shao, 2012). The T3S system apparatus consists of roughly 20 proteins building a bacterial membrane spanning multi-ring core base with an inner rod and a protruding extracellular needle-like structure that can insert into host cell membranes and form a conduit for bacterial effector proteins. Noteworthy, flagellin also enters the host cell by a translocation-associated T3S system (Buttner, 2012). Therefore, flagellin-mediated stimulation of the inflammasome pathway by bacterial mutants harbouring genetic mutations that disrupt T3S systems is usually absent or significantly reduced (Miao et al, 2006). Mechanism of NLRC4/NAIP activation Together with the observation that even purified and transfected flagellin has the ability to activate NLRC4 (Franchi et al, 2006; Miao et al, 2006), these early studies suggested a model of direct sensing of the MAMP flagellin through NLRC4 after cytosolic delivery via T3S systems. However, several points challenged this assumption. (1) Direct interaction of NLRC4 and flagellin could not be shown (Franchi et al, 2006). (2) Infection with Shigella flexneri, an aflagellated bacterium, also induced NLRC4 inflammasomes whereby the presence of its T3S system was still required (Suzuki et al, 2007). (3) Wild type C57BL/6 macrophages restricted L. pneumophila replication, whereas A/J strains were highly permissive. The genomic locus accounting for this phenotype was mapped to a region containing NAIP proteins (NLR family, apoptosis inhibitory protein) (Kofoed & Vance, 2011) and NAIP5 was shown not only to restrict L. pneumophila replication but also to be required for caspase-1 activation and pyroptosis upon L. pneumophila infection (Lightfield et al, 2008). (4) The C-terminal portion spanning 35 amino acids of flagellin, forming an alpha helical domain was shown to rely on NAIP5, whereas full length flagellin did not require NAIP5 when massively overexpressed (Lightfield et al, 2008). Additionally, the P. aeruginosa mutant PAK ΔfliC, which is deficient in flagellin, is still capable of activating caspase-1 in an NLRC4-dependent manner without a requirement for NAIP5 (Sutterwala et al, 2007). (5) It could be shown that besides flagellin, a second bacterial protein that is present in many bacterial pathogens, the inner rod protein PrgJ of T3S systems is sensed by NLRC4 without the requirement for NAIP5 (Miao et al, 2010b). A sequence of recent studies has now clarified several of these issues, at least for the murine system (Kofoed & Vance, 2011; Zhao et al, 2011). Reconstitution of inflammasomes by overexpression of NLRC4, NAIPs and bacterial compounds in HEK cells followed by biochemical analysis led to a conclusive NLRC4 inflammasome model (Fig 2). Here, NLRC4 does not function as a receptor but distinct NAIP proteins bind to the MAMPs flagellin or PrgJ and associate with NLRC4. NAIP5 directly binds the C-terminal part of Salmonella flagellin and NAIP2 interacts with PrgJ-like proteins (PrgJ of S. typhimurium and BsaK of B. thailandensis) (Kofoed & Vance, 2011; Zhao et al, 2011). NAIP6 can also bind flagellin but its physiological role is unclear due to its expression at limiting amounts (Zhao et al, 2011). Ligand binding of NAIPs facilitates their interaction with NLRC4 and allows the latter to oligomerize and assemble an inflammasome. Reconstitution experiments with truncated dominant positive variants clearly demonstrated that NAIPs act upstream of NLRC4 and proposed that a 'folding back' of the LRR domain in the native state blocks the NBD domain responsible for oligomerization (Fig 2). Ligand binding could induce a conformational change of NAIPs and release the NBD from the autoinhibitory state (Gong & Shao, 2012; Kofoed & Vance, 2011) to allow assembly of an NLRC4/NAIP inflammasome, which also involves phosphorylation of the serine residue 553 of NLRC4 by kinases such as PKCδ (Qu et al, 2012). Figure 2. The NLRC4/NAIP inflammasome. NAIP proteins sense flagellin or structurally related subunits of T3S systems in the cytosol of host macrophages. Several flagellin species directly bind to the C-terminal part of NAIP5 or NAIP6, whereas PrgJ binds to NAIP2. Ligand binding of NAIPs results in oligomerization with PKCδ-phosphorylated NLRC4 to recruit caspase-1. The presence of caspase-1 is required for various inflammasome effector mechanisms, however catalytic activity is only needed for processing of IL-1β and IL-18. Phosphorylation of NLRC4 by PKCδ requires the cytosolic presence of flagellin or T3SS recognition, yet the exact mechanistics of this process are currently unclear. Download figure Download PowerPoint Thus, distinct NAIP proteins seem to allow the NLRC4 inflammasome to differentiate between its bacterial ligands. Hereby, NAIPs that were previously believed to function as inhibitors of apoptosis, act as receptors. C57BL/6 mice express four of the seven murine NAIP genes identified (NAIP1,2,5 and 6), whereas humans seem to be equipped with only one functional NAIP protein. Human NAIP can detect neither flagellin nor inner rod proteins of T3S systems. Instead, it recognizes the needle subunits (CprI or homologs) of bacterial T3S systems of EHEC, B. thailandensis, P. aeruginosa, S. typhimurium and S. flexneri (Zhao et al, 2011). In this regard, further studies are required to precisely elucidate differences between human and murine NLRC4 inflammasomes. NLRC4 inflammasome in vivo Hosts may use detection of bacterial flagellin to discriminate commensal from pathogenic strains. For example, flagellin differs between bacterial species. While flagellin from E. coli fails to trigger NLRC4 activation, even if delivered to the host cytosol (Ren et al, 2006), H. pylori flagellin can activate the NLRC4 inflammasome. In line with this, the ability of flagellins from ten different bacterial pathogens to bind to NAIP5 correlates well with their inflammasome stimulating activity (Zhao et al, 2011). The bacterial T3S system delivering virulence factors is structurally related to the bacterial flagellin system. Not surprisingly, since T3S system delivered virulence factors can modulate host-signalling pathways for bacterial benefit, the mammalian system has developed strategies to detect T3S activity via NLRC4 (Miao et al, 2006, 2008). The NLRC4 sensing machinery has been shown to contribute to pathogen defense by several distinct mechanisms. Conventional inflammasome activation resulting in IL-1β and IL-18 release was studied in vivo for diverse bacterial strains. For example, caspase-1 induced release of IL-1β and IL-18 is essential for S. flexneri defense in mice (Sansonetti et al, 2000), whereas it is not clear to what extent this depends on NLRC4. However, IL-1β and IL-18 are only partially required for the NAIP/NLRC4 dependent defense against S. typhimurium, L. pneumophila and B. thailandensis in vivo (Miao et al, 2010a). In this regard, a novel cell intrinsic, cytokine independent signalling output of NLRC4 activation was recently characterized. Von Moltke et al. identified eicosanoids as previously unrecognized inflammasome effectors. Activation of the NLRC4/caspase-1 axis through FlaTox (a synthetic fusion protein of Legionella flagellin and anthrax lethal factor delivered through anthrax protective antigen) specifically resulted in a rapid induction of inflammatory lipid mediators, a so called "eicosanoid storm". Mice deficient in cyclooxygenase-1, the critical enzyme in prostaglandin biosynthesis, were resistant to eicosanoid caused inflammation and vascular fluid loss (von Moltke et al, 2012). The relevance of these results needs to be verified in more physiological models, especially since NLRC4 activation was achieved by a highly dosed and artificial ligand. Nevertheless, this study disclosed, that the realm of signalling outputs of inflammasomes might be much broader than previously thought. On the cell autonomous level, NLRC4 induced activation can contribute to antimicrobial defense by degradation of pathogens inside macrophages, which is promoted by the fusion of the LCV, an endoplasmic reticulum (ER) derived compartment resembling an immature autophagosome where L. pneumophila replicates, with the lysosome. This effect is possibly mediated by caspase-7 acting downstream of caspase-1 (Akhter et al, 2009). The fusion of the pathogen-enclosing compartment with the lysosome is facilitated by the NLRC4/caspase-1 axis and thereby restricts Legionella replication (Amer et al, 2006). Pyroptosis provides another clearing mechanism. S. typhimurium strains modified to persistently express flagellin induced caspase-1 dependent pyroptotic cell death. Bacteria released from pyroptotic macrophages were exposed to and taken up by neutrophils and killed by their reactive oxygen species (ROS). This has been shown to be a caspase-1 dependent process that occurs independently of IL-1β and IL-18 in vivo (Miao et al, 2010a). NLRP1 inflammasome Although NLRP1 has been the first protein described to form an inflammasome with the minimal requirement of caspase-1, caspase-5 and ASC (Martinon et al, 2002), its mechanism of activation remains poorly understood. NLRP1 structurally differs from other NLRs in its additional C-terminal extension consisting of a domain with unknown function (function to find domain, FIIND) and a CARD domain. Human NLRP1 forms an ASC-dependent inflammasome, whereas mouse NLRP1 may activate caspase-1 in an ASC-independent manner (Hsu et al, 2008). The best-characterized elicitor of NLRP1 activation is anthrax lethal toxin (Boyden & Dietrich, 2006), the major virulence factor of Bacillus anthracis. Its protective antigen (PA) subunit allows the effector subunit lethal factor (LF) to enter the cell cytosol. LF activates caspase-1 and induces rapid cell death via NLRP1. Since inactive but structurally virtually identical mutants of LF fail to activate caspase-1, it is most likely, that LF does not directly bind to NLRP1 (Fink et al, 2008). Indeed, the endoprotease activity of LF is required to alert NLRP1 activation. LF has the capability of cleaving cytosolic substrates, a process that additionally requires Ca2+ flux and probably also proteasome activity (Fink et al, 2008). Thus, it was proposed that LF could be sensed by NLRP1 through the presence of cleaved host substrates rather than direct binding (Fink et al, 2008). Some evidence also suggests that NLRP1 undergoes autoproteolytic cleavage at a conserved motif within its FIIND (D'Osualdo et al, 2011) and that direct cleavage of a conserved motif in the FIIND by LF may present a necessary but not sufficient step for NLRP1 activation (Hellmich et al, 2012; Levinsohn et al, 2012). Nevertheless, the precise mechanism by which LT triggers the NLRP1 inflammasome remains unclear. Humans harbour only one NLRP1 gene, whereas three paralogues (Nlrp1a, b, c) are present in mice (Boyden & Dietrich, 2006). Moreover, the murine Nlrp1b gene is highly polymorphic and different mouse strain variants of Nlrp1b confer susceptibility to LF induced caspase-1 activation (Boyden & Dietrich, 2006). Nlrp1b activation by LT represents an essential host defense mechanism in the control of B. anthracis infection. Caspase-1 induced pyroptosis of infected macrophages permits self-elimination of affected cells and initiation of an antimicrobial neutrophilic reaction, which also involves IL-1β (Terra et al, 2010; Welkos et al, 1986, 1989). Recently, a hyperactive mutation in the murine Nlrp1a paralogue was identified to trigger a systemic caspase-1 dependent inflammatory response in vivo (Masters et al, 2012). IL-1β was critical for the multiorgan neutrophilic disease, whereas ASC was dispensable for the inflammatory phenotype. Certainly, the exact Nlrp1a ligand or activation signal needs yet to be elucidated, but studies in Nlrp1a deficient mice suggest a role for Nlrp1a in triggering pyroptosis of haematopoietic progenitor cells during periods of haematopoietic stress induced by chemotherapy or infection (Masters et al, 2012). Whether this observation also has implications for human immunology and haematopoiesis needs to be investigated. NLRP3 inflammasome NLRP3 (Nalp3, cryopyrin) forms an inflammasome with ASC and caspase-1 (Agostini et al, 2004) and has been the most extensively studied NLR member due to a wide array of activators of microbial and non-microbial origin (Fig 3). The NLRP3 inflammasome has been implicated in sensing a plethora of pathogenic bacteria including S. aureus and E. coli (Rathinam et al, 2012), viral pathogens such as Influenza A virus (Allen et al, 2009; Thomas et al, 2009) or vesicular stomatitis virus (Rajan et al, 2011) and the fungal pathogens Candida albicans (