Mammalian NLR proteins - Nature

Immunology and Cell Biology (2007) 85, 495–502 & 2007 Australasian Society for Immunology Inc. All rights reserved 0818-9641/07 $30.00 www.nature.com/icb

REVIEW

Mammalian NLR proteins; discriminating foe from friend Maria Kaparakis1, Dana J Philpott2 and Richard L Ferrero1 Eukaryotic organisms of the plant and animal kingdoms have developed evolutionarily conserved systems of defence against microbial pathogens. These systems depend on the specific recognition of microbial products or structures by molecules of the host innate immune system. The first mammalian molecules shown to be involved in innate immune recognition of, and defence against, microbial pathogens were the Toll-like receptors (TLRs). These proteins are predominantly but not exclusively located in the transmembrane region of host cells. Interestingly, mammalian hosts were subsequently found to also harbour cytosolic proteins with analogous structures and functions to plant defence molecules. The members of this protein family exhibit a tripartite domain structure and are characterized by a central nucleotide-binding oligomerization domain (NOD). Moreover, in common with TLRs, most NOD proteins possess a C-terminal leucine-rich repeat (LRR) domain, which is required for the sensing of microbial products and structures. Recently, the name ‘nucleotide-binding domain and LRR’ (NLR) was coined to describe this family of proteins. It is now clear that NLR proteins play key roles in the cytoplasmic recognition of whole bacteria or their products. Moreover, it has been demonstrated in animal studies that NLRs are important for host defence against bacterial infection. This review will particularly focus on two subfamilies of NLR proteins, the NODs and ‘NALPs’, which specifically recognize bacterial products, including cell wall peptidoglycan and flagellin. We will discuss the downstream signalling events and host cell responses to NLR recognition of such products, as well as the strategies that bacterial pathogens employ to trigger NLR signalling in host cells. Cytosolic recognition of microbial factors by NLR proteins appears to be one mechanism whereby the innate immune system is able to discriminate between pathogenic bacteria (‘foe’) and commensal (‘friendly’) members of the host microflora. Immunology and Cell Biology (2007) 85, 495–502; doi:10.1038/sj.icb.7100105; published online 7 August 2007 Keywords: NODs; NLR; NALP; innate immunity; bacterial pathogens; peptidoglycan

Multicellular organisms have developed a diverse array of strategies to mount an initial defence against infection by microbial pathogens. These strategies depend on recognition of conserved microbial structures or products by pathogen recognition molecules (PRMs) that are associated with the membranes or cytosol of host cells. This review will focus on PRMs that belong to the nucleotide-binding oligomerization domain (NOD) protein family. NOD proteins have the peculiarity of being located within the cytosolic compartment of host cells. We will discuss the roles of NOD family members in innate immune recognition of bacterial pathogens and the strategies that bacteria use to induce NOD-dependent inflammatory responses in mammalian hosts. NLR PROTEIN BIOLOGY AND EVOLUTION NOD family proteins have been evolutionarily conserved and play diverse roles in apoptosis, inflammation and host defence among members of the plant and animal kingdoms.1 These multidomain proteins share a central NOD or NACHT (NAIP, CIITA, HET-E and TP-1 protein) domain that is important for protein

self-oligomerization.2,3 In contrast, the N-terminal regions of NOD family proteins are much more diverse, acting as effector domains in downstream signalling events through homophilic and heterophilic protein interactions.2 The N-terminal regions of NOD proteins generally consist of either pyrin (PYD), baculovirus inhibitor-ofapoptosis repeat (BIR), TOLL/interleukin-1 receptor (TIR) or caspase activation recruitment domains (CARDs). In the case of NOD family members that function as PRMs, detection of microbial products occurs via a C-terminal leucine-rich repeat (LRR) region, which is shared with the TOLL-like receptor (TLR) group of innate immune molecules. Examples of NOD family proteins include plant disease-resistance proteins (or R proteins), mammalian NOD-LRR proteins and apoptosis-regulatory proteins, such as apoptotic protease activating factor 1 (APAF-1) in mammals and ICE protease activating factor (IPAF) in Caenorhabditis elegans.2 Mammalian NOD-LRR proteins have recently been re-named and grouped into the ‘nucleotide-binding domain and LRR’ (NLR) family. The current list of NLR family members can be found on the website of the Human Gene Nomenclature Committee

1Department of Microbiology, Monash University, Melbourne, Victoria, Australia and 2Department of Immunology, University of Toronto, Toronto, Ontario, Canada Correspondence: Dr M Kaparakis, Department of Microbiology, Monash University, Building 53, Wellington Road, Clayton, Melbourne, Victoria 3800, Australia. E-mail: [email protected] Received 1 July 2007; accepted 2 July 2007; published online 7 August 2007

Mammalian NLR proteins M Kaparakis et al 496

(http://www.gene.ucl.ac.uk/nomenclature). Parallels are often drawn between the roles of mammalian NLRs and plant R proteins in innate immunity. Interestingly, however, it has been suggested that the functions of plant R proteins may actually be more analogous to those of the mammalian adaptive immune system.4 The numbers of NLR proteins across the plant and animal kingdoms vary widely. From bioinformatic studies, organisms such as Arabidopsis, rice and the purple sea urchin have been deduced to have 140, 4500 and 203 putative NLR proteins, respectively. In contrast, 22 are present in humans, and none in C. elegans or Drosophila melanogaster.1,5 It is noteworthy, however, that the gut appears to be a major site of NLR expression in both sea urchins and vertebrates.5 This suggests that the control of the microbial flora within the gut may have been a major selective pressure in the development of NLRs throughout evolution. Consistent with this suggestion, polymorphisms in an NLR gene of humans, NOD2, have been associated with deregulated immune responses to the gut microflora and the development of a form of chronic intestinal inflammation, known as Crohn’s disease (reviewed in Fritz et al.3). Furthermore, various NLR gene polymorphisms have now been linked to a range of inflammatory conditions affecting different tissues and organs. These conditions include Blau syndrome, Muckle–Wells syndrome, familial cold urticaria, gastric mucosa-associated lymphoid tissue lymphoma and asthma (for reviews see Fritz et al.3 and Martinon and Tschopp6). NLR RECOGNITION OF BACTERIAL PEPTIDOGLYCAN The NOD1 and NOD2 proteins were the first mammalian members of the NLR family to be reported to act as intracellular microbial sensors.7–10 Engagement of NLRs with their cognate agonists initiates a signalling cascade that ultimately results in the upregulation of nuclear factor-kB (NF-kB) and the production of pro-inflammatory cytokines (reviewed in Fritz et al.3). Unlike most PRMs, the NLRs are intracellular microbial sensors that recognize defined microbial products within the host cytoplasmic compartment. Nevertheless, the precise intracellular compartment of NLR–agonist interactions has yet to be elucidated. Both NOD1 and NOD2 sense peptidoglycan fragments released from the cell wall of bacteria; however, the motifs recognized by these receptors are different (reviewed in Inohara and Nunez2 and Fritz et al.3). NOD1 recognizes the disaccharide of N-acetyl glucosamine-Nacetyl muramic acid, linked to a tripeptide group of which the terminal amino acid is meso-diaminopimelate (mDAP)9 (Figure 1). The minimal component recognized by NOD1 is g-D-glutamyl-mesoDAP, which is not normally found in bacteria.10 Furthermore, mDAP is a component of the bacterial cell wall of most Gram-negative organisms, excluding Mycoplasma and some other species (reviewed in Chamaillard et al.11). Gram-positive bacteria usually have a lysine residue at the terminal position of their peptidoglycan and this aminoacid substitution inhibits recognition of the motif, rendering its peptidoglycan incapable of signalling via NOD1. Recognition of the tripeptide structure of peptidoglycan by human NOD1 is highly specific, as the presence of an additional amino acid linked to mDAP abrogates the sensing of this bacterial product.9 Moreover, recognition of peptidoglycan motifs has been shown to be hostspecific, as human NOD1 recognizes L-alanine-g-D-glutamatemeso-DAP whereas murine NOD1 is specific for recognition of the tetrapeptide structure L-alanine-D-glutamate-meso-DAP-D-alanine motif12 (Figure 1). NOD2 is responsible for the recognition of a broad range of bacterial pathogens as it recognizes muramyl dipeptide (MDP), Immunology and Cell Biology

disaccharide-tripeptide and disaccharide-tetrapeptide CH2OH

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Figure 1 Peptidoglycan structures recognized by NOD1 and NOD2. (a) Murine and human NOD1 identify different tetrapeptide (TetraDAP) or tripeptide (TriDAP) structures, respectively, within the peptidoglycan degradation products from Gram-negative bacteria. (b) NOD2 senses muramyl dipeptide, a component of bacterial peptidoglycan common to both Gram-positive and -negative organisms.

a component common to both Gram-negative and Gram-positive bacterial peptidoglycan.8,13 Bacterial MDP can be released and is accessible to NOD2 during cell wall biosynthesis through the action of bacterial hydrolases, or after degradation of ingested bacteria by host lysozyme (reviewed in Meylan et al.14). The mechanism whereby NOD2 recognizes MDP is yet to be elucidated; it is unknown if NOD2 directly recognizes MDP or if another protein is required to interact with NOD2 and facilitate recognition of MDP (reviewed in Meylan et al.14). There is stereospecificity in the recognition of the NOD2 ligand, as MDP-LD, but not the MDP-LL or MDP-DD forms, is recognized by NOD2.13,15 Moreover, treatment of peptidoglycan with amidase, which cleaves between the sugar and peptide moieties of peptidoglycan, renders the peptidoglycan undetectable by NOD2.15 The immunostimulatory capacity of MDP is well characterized, as it is a key component of Freund’s complete adjuvant, and is capable of eliciting strong immunological responses in rodents to coadministered antigens.16 When Freund’s complete adjuvant is coadministered with emulsified antigen, the mycobacterial components within the adjuvant are able to signal to T lymphocytes. This results in the production of elevated levels of antigen-specific circulating antibodies.17 It is now clear that NOD2 agonists can synergize with TLR-stimulatory products, such as bacterial lipopolysaccharide (LPS) and lipoproteins, to regulate the onset of adaptive immune responses.18 Moreover, in mouse immunization studies, it was demonstrated that the stimulation of NOD1 alone induced the development of antigen-specific T-cell immunity with a Th2 profile, whereas the simultaneous stimulation of NOD1 and TLR molecules triggered Th1, Th2 and Th17 immune responses.19 Taken together, the data demonstrate the role of NLR proteins not only in innate immune detection of pathogens, but also in the development of adaptive immunity. DOWNSTREAM SIGNALLING EVENTS TO NLR RECOGNITION OF BACTERIAL PEPTIDOGLYCAN Recognition of peptidoglycan moieties by NOD1 or NOD2 induces a signalling cascade, ultimately resulting in NF-kB activation. NOD1and NOD2-dependent NF-kB activation is mediated by the recruitment and oligomerization of a member of the receptor-interacting protein kinase family,20 receptor-interacting protein (RIP)2.7,21,22 This protein is also known as RIP-like interacting CLARP kinase (RICK)23 or CARD-containing ICE-associated kinase (CARDIAK).24 RIP2


Cleavage CARD6

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Figure 2 Pathogen and host-induced NLR signalling cascades. NOD1 simulation induces a signalling cascade via RICK and the IKK complex, leading ultimately to degradation of IkB, the activation of NF-kB and expression of pro-inflammatory chemokines. Induction of RICK activates the pro-caspase-1 molecule and facilitates the cleavage of pro-IL-1b to its active form. However, activation of RICK and the NOD1 regulators, SGT with HSP90 and CSN6 with COP9, can initiate the host cell to undergo pro-caspase-9-induced apoptosis. NOD2 activation is negatively regulated by erbin. Signalling via NOD2 results in the activation of IFN-b and NF-kB, via the two independent pathways of GRIM-19 and RICK, ultimately leading to the upregulation of chemokines and antimicrobial peptides. Induction of NALP via DAMPs leads to the removal of SGT1 and HSP60 from NALP, enabling the formation of the inflammasome with ASC and pro-caspase-1. The inflammasome is then capable of cleaving pro-IL-1b to generate the active form of IL-1b. DAMPs, danger-associated molecular patterns; IkB, inhibitory kB; IKKs, inhibitory kB kinase complex; RICK, RIP-like interacting CLARP kinase.

interacts with NOD proteins via its CARD region resulting in the development of a transient complex composed of NOD, RIP2 and the inhibitory kB (IkB) kinase complex (IKK)25,26 (Figure 2). Ultimately, this signalling cascade activates the IKK, which phosphorylates and degrades IkB proteins, enabling the migration of NF-kB into the nucleus to induce target gene transcription. This can lead to the generation of pro-inflammatory cytokines and chemoattractants, such as CXCL5 and CXCL8 (IL-8), which recruit neutrophils27 (Figure 2). Biochemical studies have revealed numerous potential interacting partners for NOD1 and/or NOD2 molecules. In certain cases, however, the data have arisen solely from overexpression studies, and thus the findings need to be analysed with a degree of circumspection. Nevertheless, protein–protein interactions involving NOD1/2 proteins have been proposed to regulate the functions of these proteins and to even promote host cell programmed cell death (Figure 2). Indeed, an early study on NOD1 reported the ability of this protein to induce apoptosis by binding to pro-caspase-9, leading to caspase-induced cell death.7 It was also found that NOD1 could interact with both RIP2 and pro-caspase-1 to enhance the processing of pro-IL-1b to its active form.28 More recently, NOD1 was shown to interact via its CARD

with the CSN6 component of the COP9 signalosome, a large multiprotein complex, resulting in apoptosis.29 As has been reported for mammalian TLRs, there also appears to be a regulatory mechanism in the NOD1 signalling cascade: NOD1 activation was shown to be positively regulated by SGT1, a highly conserved protein found in yeast, plants and animals. SGT1 is believed to play a role in the ubiquitination of regulatory proteins,30 and, combined with HSP90, is reported to be essential for inflammasome activity.31 The CARDcontaining protein CARD6 has also been reported to regulate NOD1 responses via interactions with NOD1 and RIP-2, resulting in the suppression of NF-kB responses in host cells.32 NOD2 appears to interact with RIP-2 as well as with transforming growth factor b-activated kinase 1 and GRIM-19, to mediate NF-kB activation in host cells.33,34 To date, the only inhibitor of NOD2 signalling is Erbin, a member of the PDZ domain-containing family, that is capable of inhibiting cytokine expression by host cells in response to MDP stimulation35,36 (Figure 2). In addition to the induction of pro-inflammatory cytokine production, NOD1/2 protein signalling can result in the generation of antimicrobial peptides by epithelial cells in response to bacterial Immunology and Cell Biology


stimuli. The upregulation of the antimicrobial peptide, human b-defensin-2, in epithelial cells stimulated with the gastric pathogen, Helicobacter pylori, occurs via a NOD1-dependent mechanism37 (M Kaparakis, DJ Philpott, RL Ferrero; unpublished data). This finding is consistent with the increased numbers of H. pylori bacteria, which were present in the stomachs of Nod1-deficient mice that had been experimentally challenged with this bacterium.38 Similarly, it was suggested that the increased susceptibility of Nod2-deficient mice to Listeria infection was due to an impaired expression of intestinal antimicrobial peptides, known as cryptidins.39 Hence, NOD1 and NOD2 proteins appear to have a dual role in host defence via: first, the induction of chemokine production and the local recruitment of neutrophils; and second, the initiation of antimicrobial peptide synthesis. THE NALP SUBFAMILY OF NLRS NACHT-LRR-PYD-containing proteins, or NALPs, are characterized by the presence of an N-terminal PYD effector domain.40 NALPs are activated by a variety of danger-associated molecular patterns (DAMPs) and microbial products. These include double-stranded RNA from bacteria and viruses,41,42 toxins,43 ATP44 and MDP.45 This family of proteins even responded to host-derived danger signals, such as uric acid crystals released by necrotic cells.46 Once these NALPs are activated, their PYD domains associate with apoptosis associated speck-like protein (ASC), an adaptor molecule that subsequently interacts with the CARD of pro-caspase-1 and ultimately leads to the formation of a complex called the ‘inflammasome’.40 The inflammasome is a signalling platform scaffolded by proteins belonging to the NLR family, and is responsible for the processing and maturation of the proinflammatory cytokines, IL-1b and IL-18.6,40 To date, there are two known NALP inflammasome complexes: NALP1 and NALP2/NALP3. NALP3 is also known as cryopyrin and the PYD-containing APAF-1 like protein, or PYPAF1.47 It has been proposed that NALP3 activity is regulated by SGT1 and HSP90, and both these regulators maintain NALP3 inactive until it senses its agonist.31 The NALP1 inflammasome consists of the adaptor protein ASC, caspase-1 and caspase-5,40 whereas the NALP2/NALP3 inflammasome is comprised of NALP2, NALP3, CARDINAL, ASC and caspase-1.47 ASC is critical for caspase-1 activation and the secretion of IL-1b and IL-18 in response to several purified microbial components, intracellular bacteria, toxins, ATP and RNA.42,44,48–50 Mice deficient in ASC have defective IL-1b and IL-18 responses to Salmonella infection and are resistant to LPS-induced endotoxic shock, thus further supporting the role of ASC in the development of the inflammasome.50 THE ROLE OF CASPASE-1 IN NLR SIGNALLING Studies have suggested that proteins of the NLR family regulate the activation of caspase-1, a member of the evolutionarily conserved cysteine proteins known as caspases.6 Caspase-1 is responsible for the maturation of the pro-inflammatory cytokines IL-1b and IL-18, which play a key role in host defence against infection. Both IL-1b and IL-18 are synthesized as inactive cytoplasmic precursors and are proteolytically cleaved by caspase-1 to produce the biologically active mature forms.51 Although the intracellular signalling pathways leading to caspase-1 activation remain poorly defined, it has been shown that caspase-1 functions in unison with Pannexin-1 to interact with the inflammasome.52 Caspase-1 is known to play a key role in immune responses to bacterial pathogens. Indeed, mice lacking either caspase-1 or components of the inflammasome were more susceptible to oral Immunology and Cell Biology

Salmonella infection.53 Bacillus anthracis lethal toxin was shown to induce cell death of murine macrophages via caspase-1 signalling.54 Furthermore, the importance of caspase-1 during IL-1b- and IL-18induced inflammation has been confirmed in animal models of Shigella flexneri, Francisella tularensis and Listeria infection.49,55,56 BACTERIAL AND HOST MECHANISMS OF NLR INDUCTION Owing to the cytoplasmic nature of NLR proteins, bacterial products must find their way into the cytoplasmic compartment to induce an immunostimulatory response. Antigen-presenting cells such as macrophages and dendritic cells can obtain bacterial fragments from their extracellular milieu via phagocytosis or pinocytosis, respectively. In addition, it appears that bacteria can physically introduce their products, such as peptidoglycan, into the cytosolic compartment of non-phagocytic cells via various strategies, including invasion and the actions of bacterial secretion systems. Examples of bacteria that utilize these strategies to initiate NLR-dependent responses are summarized below and in Figure 3. Host cell invasion Both NOD1 and NOD2 proteins can be activated directly to mediate innate immunity by invasive bacteria, such as Shigella flexneri,26 enteroinvasive Escherichia coli,27 Streptococcus pneumoniae57 and Mycobacteria.58 Initial reports identifying the ability of Gram-negative organisms to initiate NOD1 responses in eukaryotic cells involved the examination of S. flexneri-induced inflammation. S. flexneri was shown to mediate NOD1-dependent NF-kB activation and c-jun Nterminal kinase signalling in epithelial cell lines.26,59 The requirement for bacterial products to be microinjected directly into host cells,59 together with the absence of NOD1 signalling to non-invasive S. flexneri bacteria,26 was sufficient evidence to conclude that NOD1 recognition was dependent on the location of bacterial components within the intracellular compartment. Consistent with these findings,

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NOD1/2 NAIP5/NALP3/IPAF Figure 3 Bacterial mechanisms of NLR induction. Bacterial pathogens utilize various virulence mechanisms to initiate the NLR signalling cascade in host cells. (a) H. pylori encoding a T4SS and (b) invasive organisms, such as Shigella and E. coli, can transfer their peptidoglycan into the cytoplasm of non-phagocytic cells and induce NOD1/2 responses. (c) Listeria utilizes the toxin LLO to facilitate its escape from the phagosome and be released into the cytoplasm where it can trigger the NOD1/2 signalling cascade. Similarly, bacterial pathogens within phagocytic cells, such as macrophages, trigger NLR signalling once (d) internalized within these cells or via mechanisms involving T4SS-dependent (e) translocation of flagella, such as in L. pneumophila, or (f) in the case of Salmonella, by a flagellinindependent/T3SS-dependent function. LLO, listeriolysin O; NLR, nucleotide-binding domain and LRR; T3SS, type III secretion system; T4SS, type IV secretion system.


overexpression of a dominant-negative form of NOD1 in HEK293 cells blocked the capacity of S. flexneri to induce NOD1-dependent NF-kB activation in cells.26 NOD1 was subsequently confirmed to be the PRM responsible for the detection of internalized Gram-negative bacterial peptidoglycan.9,10 Since S. flexneri was identified as initiating NOD1 signalling, other invasive pathogens have been reported to have similar NLR recognition and stimulation capacities. A NOD1-expressing human colon epithelial cell line, Caco2, was shown to produce an NF-kB response to enteroinvasive E. coli infection in a NOD1-dependent manner.27 Similarly, Streptococcus pneumoniae, the invasive organism responsible for pneumococcal infection, is capable of upregulating both Nod1 and Nod2 levels in C57BL/6 mouse lung and the bronchial epithelial cell line, BEAS-2B, suggesting a possible function of NOD1/2 proteins in host cell recognition of this organism.57 Although organisms produce peptidoglycan that is capable of being recognized by NOD1, it does not necessarily follow that their peptidoglycan is sufficiently immunostimulatory to elicit a NOD1-dependent response in vivo. For example, Chlamydia trachomatis and Chlamydia muridarum are both invasive obligate bacteria that produce the peptidoglycan motif required for NOD1 recognition.60 Both organisms, however, produce insufficient levels of peptidoglycan to stimulate NOD1-dependent responses, as Nod1deficient mice infected with Chlamydia develop an immune response equivalent to wild-type animals. A possible explanation for this finding is that other PRMs of the innate immune system may compensate for the absence of NOD1 during murine infections with Chlamydia.60 Surprisingly, this is not the case for all Chlamydia species, as Chlamydia pneumonia can induce responses in human endothelial cells that express NOD1.61 Moreover, overexpression of NOD1 or NOD2 in HEK293 cells amplifies the capacity of C. pneumonia to induce NF-kB activation, and small interference RNA silencing of NOD1 reduced their ability to produce IL-8 in response to stimulation with Chlamydia.61 Collectively, these findings suggest that C. pneumonia can trigger NOD1 responses when this molecule is overexpressed, whereas under more physiological conditions, the inverse appears to be true. More recently, Moraxella catarrhalis, which is associated with lower respiratory tract infections, was believed to be predominantly an extracellular pathogen that was incapable of signalling via NOD1. A recent study, however, has described the ability of this pathogen to be internalized by the bronchial epithelial cell line BEAS-2B, pneumocytes and small airway epithelial cells and to induce NOD1 expression. Small interference RNA silencing of NOD1 within these cells reduced M. catarrhalis-induced IL-8 production.62 Bacterial and host secretion systems Non-invasive organisms have developed virulence mechanisms enabling them to facilitate the introduction of peptidoglycan into the host cell cytoplasm and to initiate NLR signalling. To date, there are a limited number of reports identifying the ability of extracellular organisms to induce NOD1 responses.38,63 H. pylori was the first extracellular organism identified as being capable of physically delivering its peptidoglycan to human epithelial cells.38 The delivery of H. pylori peptidoglycan into host cells is mediated via a type IV secretion system (T4SS), encoded by the H. pylori pathogenicity island (cagPAI). The T4SS is essential for epithelial cell sensing of H. pylori peptidoglycan; H. pylori strains lacking a cagPAI induce significantly reduced levels of NF-kB activation and IL-8 secretion in vitro.38 The observed cagPAI dependency of NOD1 responses to this pathogen was further demonstrated in vivo, as increased susceptibility

of Nod1-deficient mice to H. pylori infection was observed only during challenge with cagPAI-positive strains.38 In addition to the capacity of extracellular organisms to induce NOD1 responses via secretion systems, bacteria residing within intracellular compartments can also utilize secretion systems to signal through NLRs. The invasive organisms Legionella and Salmonella are capable of inhibiting phagosome–lysosome fusion and hence reside and replicate within a protected intracellular niche.64,65 While located within this vacuole, these bacteria utilize their secretion systems to transport bacterial components into host cells. This results in the initiation of pro-inflammatory signalling via CARD-containing proteins, such as IPAF (CARD12) and NAIP (also known as baculoviral IAP repeat-containing 1 (Birc1), or as Naip5 in mice).66 Recognition of bacterial flagellin by these proteins in host cells results in inflammasome activation via a TLR5-independent pathway. IPAF, a member of the NLR family that is related to APAF-1,67 is critical for IL-1b responses to Legionella pneumophila68,69 and Salmonella infection.70 Also, IPAF is responsible for controlling intracellular L. pneumophila replication within the phagosome compartment of macrophages.71 L. pneumophila encodes a T4SS that is capable of delivering flagellin into the cytosolic compartment. Recognition of L. pneumophila flagellin by Naip5 activates the inflammasome complex within host macrophages, leading to the production of cytokines.72 Naip5 recognition of L. pneumophila flagellin restricts the bacterial infection.66,72 Consistent with this finding, flagellin mutants of Legionella fail to induce caspase-1-mediated cell death and immunity.69 Similar to Legionella, Salmonella enterica serovar typhimurium does not replicate freely in the cytosol and induces rapid death of macrophages via a caspase-1-dependent mechanism.73 This process is dependent on IPAF and ASC50,68 and not NALP3.74 Salmonella strains lacking flagellin or expressing mutant flagellin are defective in activating caspase-1.70 It was shown that Salmonella requires a functional pathogenicity island type III secretion system (T3SS), but not the flagellar T3SS, to signal via IPAF in macrophages.68 The former is encoded within the Salmonella pathogenicity island-1.75 Another invasive pathogen, the opportunistic bacterium Listeria monocytogenes, which can escape phagosomes using the pore-forming toxin listeriolysin O (LLO), contains an auxillary protein secretion system (SecA2), which promotes the release of muramyl peptides during infection.76 This secretion system plays a central role in the virulence of this organism. It was demonstrated that wild-type L. monocytogenes are capable of invading and inducing IL-8 secretion by human endothelial cells in a NOD1-dependent manner, whereas non-invasive mutants are significantly impaired in their ability to induce IL-8 production.77 L. monocytogenes was also suggested to transport bacterial products from the phagosome into the cytoplasmic compartment, thus activating NOD2 and the production of an IFN-b response.78 The findings presented above demonstrate that bacterial secretion systems are a key mechanism for the introduction of bacterial components into the host cell and thus the elicitation of innate immune responses via CARD molecules. Further to these bacterial mechanisms, however, it is plausible that host molecules may accomplish similar functions. Indeed, the human intestinal apical transporter hPepT1 was reported to mediate the uptake of bacterial MDP into host cells, ultimately leading to NOD2-dependent production of IL-8.79 Thus, collectively, these studies identify the ability of secretion systems and membrane transporters to introduce into the host cytoplasm virulence determinants capable of initiating NLR signalling cascades. Immunology and Cell Biology


Bacterial evasion of NLR detection There is mounting evidence that bacterial pathogens can modify their peptidoglycan motifs to evade detection and clearance by the host. Recently, it was shown that L. monocytogenes could evade the human innate immune system by modifying its peptidoglycan via N-deacetylation. Inhibiting the N-deacetylation process, through deletion of pdgA, results in impaired growth of L. monocytogenes within macrophages and in vivo, as well as an increased sensitivity of the mutant to lysozyme.80 A similar evasion strategy has been observed in H. pylori to escape detection by human NOD1. The H. pylori AmiA protein, which is essential for the morphological transition of H. pylori from the rod to coccoid form, mediates modifications of the cell wall peptidoglycan that leads to the accumulation of the N-acetyl-Dglucosaminyl-b(1,4)-N-acetylmuramyl-L-Ala-D-Glu (GM-dipeptide) motif and a decrease in the GM-tripeptide motif.81 Hence coccoid forms of H. pylori containing the GM-dipeptide motif are no longer detected by human NOD1, nor are they capable of inducing NF-kB activation or IL-8 secretion by gastric epithelial cells.81 CONCLUSIONS AND PERSPECTIVES More than 30 years before the discovery of NLR proteins, bacteriologists observed the release of peptidoglycan fragments by bacteria. It was suggested that this activity may have some significance for host–pathogen interactions.82,83 This hypothesis was later confirmed in the pioneering studies of Rosenthal, Goldman and others, who demonstrated the toxic effects of these soluble peptidoglycan fragments on host cells.84–88 The Gram-negative bacterial pathogens, Neisseria gonorrhoea and Bordetella pertussis, were subsequently shown to release tetra and tri forms of mDAP-containing fragments into the external milieu.84,86–88 We now know that these forms are specifically recognized by mouse and human forms of NOD1, respectively.9 Interestingly, much later reports described the IL-8inducing activities of cell-free supernatants of the pathogens, Campylobacter jejuni89 and Pseudomonas aeruginosa,90 on non-phagocytic cells. These activities appeared to be associated with low molecular weight, heat-stable fractions in the extracts. With hindsight, it is likely that these active fractions were enriched in peptidoglycan. The findings from recent studies on NOD1 signalling in epithelial cell responses to C. jejuni91 and P. aeruginosa,63 seem to concur with this suggestion. One of the future challenges in this field is to understand better the mechanisms whereby Gram-negative bacterial pathogens signal through cytosolic NLR proteins. Although some pathogens clearly are able to invade or use secretion systems to deliver NLR agonists into the cytoplasm of host cells, bacteria also seem to be able to induce NLR-dependent responses in the absence of metabolically active cells.89,90 This raises the possibility of the existence of alternative mechanisms for bacterial delivery of NLR agonists to host cells. It will be important to identify such mechanisms, as this may provide clues to the basis by which the host innate immune system is able to discriminate between pathogenic and commensal bacteria. A second important question to be resolved in the future is what, if any, benefit do bacteria derive from inducing pro-inflammatory signalling through NLR and related adaptor molecules. By addressing this question, it may also be possible to gain a clearer understanding of the roles of NLRs in normal mammalian cell functions. ACKNOWLEDGEMENTS The work in RLF’s laboratory is supported by research grants from The National Health and Medical Research Council (NHMRC), The CASS Foundation Limited and The ANZ Charitable Trust. DJP is funded by The Canadian Immunology and Cell Biology

Institutes of Health Research and is an International Research Scholar of the Howard Hughes Medical Institute.

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