INTRODUCTION:

A quick immune response defines life and death for the host. To detect infection quickly, the immune system relies on innate immunity, the first line of defense against microbial infection that engages adaptive immunity ¹. Several families of innate immune receptors have been described ^2,3. These receptor families include Toll-like receptors (TLRs), retinoic acid-inducible gene I-like receptors (RLRs), and nucleotide-binding oligomerization domain-like receptors (NLRs). TLRs are membrane bound receptors located either in the plasma membrane or on vesicles of the endocytic pathway ⁴. TLRs are widely studied and detect microbial pathogens that include viruses, bacteria, protozoa, and fungi. TLR recognition of pathogen-associated molecular patterns is done via extracellular leucine-rich repeat motifs that transmit signals through the cytoplasmic Toll interleukin (IL)-1 receptor (TIR) domain ⁵. RLRs are distinct from the TLR pathway and recognize viral RNA present within the cytoplasm ³. RLRs proteins have a RNA-binding helicase domain and two amino (N)-terminal caspase recruitment domains (CARDs) for propagation to the interferon-regulatory factor and NF-κB signaling pathways ^6,7. NLRs are a family of intracellular sensors that have key roles in innate immunity and inflammation ^8-11. NLRs are defined by an architecture that contains a N-terminus, a middle nucleotide binding and oligomerization domain, and a leucine-rich repeat domain that is variable in the repeats composition and number.

A number of NLR family members can form multiprotein complexes, called inflammasomes, and are capable of activating the cysteine protease caspase-1 in response to a wide range of stimuli including both microbial and self-molecules. NLRs induce the recruitment of the adaptor molecule ASC (apoptosis associated speck-like protein containing a CARD), leading to the processing and activation of pro-IL-1β and IL-18 through caspase-1. NLRs are mostly cytosolic and structurally related to the disease resistant protein family (R protein) ¹². In this review we will discuss mechanisms of inflammasome activation according to the environment in which each cell type is located.

The Inflammasome

The term inflammasome was coined to describe a complex of proteins that activates caspase-1 and the cytokine IL-1β. Inflammasome is derived from the word inflammation and the suffix “some”, which is used to define molecular complexes¹⁰. This term was also chosen to highlight functional similarities with another platform that triggers apoptosis—the apoptosome¹³. The inflammasome is a multiprotein oligomer consisting of caspase 1, PYCARD, NALP and sometimes caspase 5 (also known as caspase 11 or ICH-3). It is expressed in myeloid cells and is a component of the innate immune system. The exact composition of an inflammasome depends on the activator which initiates inflammasome assembly, e.g. dsRNA will trigger one inflammasome composition whereas asbestos will assemble a different variant. The inflammasome promotes the maturation of inflammatory cytokines Interleukin 1β (IL-1β) and Interleukin 18 (IL-18).¹The inflammasome is responsible for activation of inflammatory processes,² and has been shown to induce cell pyroptosis, a process of programmed cell death distinct from apoptosis.³ In humans the NLR family is composed of 22 members¹⁰. They are divided into subfamilies according to their N-terminal effector domains. The effector domains lead to inflammatory caspase or NF-κB activation. In humans, 14 NLR members share a pyrin effector domain. Five other members are part of the NOD subfamily. CIITA, NLRC4/IPAF and the BIR-containing NAIP form the remaining NLR members. NLR molecules have been shown to form a complex with caspase-1 and the adaptor molecule ASC termed the inflammasome. The central effector molecule of the inflammasome is the cysteine protease caspase-1 that, upon activation cleaves cytosolic pro-IL-1β, pro-IL-18 to their active forms. Recent studies have shown that the NLRP1, NLRP3, and NLRC4 inflammasomes have a clear role in host defense ¹³.

Types

The generic structure of the inflammasome is comprised of a family member of the Nod-like receptors, either a NLRP/NALP or IPAF central protein, an adaptor protein, and a caspase recruitment domain (CARD) to facilitate caspase activation. Nod-like receptors are important for triggering innate immune responses to microbial pathogens. Nod-like receptors consist of a large family of intracellular pattern recognition receptors that contain a conserved nuclear oligodimerization domain, NOD. Nod-like receptors genes are highly conserved in many higher organisms. By binding to specific pathogens, different Nod-like receptors may allow the host to recognize a variety of intracellular pathogens.

The NLR (Nod-like receptors) Family

The NLRs are comprised of 22 human genes and many more mouse genes because of gene expansion since the last common ancestor. The NLR family is characterized by the presence of a central nucleotide-binding and oligomerization (NACHT) domain, which is commonly flanked by C-terminal leucine-rich repeats (LRRs) and N-terminal caspase recruitment (CARD) or pyrin (PYD) domains. LRRs are believed to function in ligand sensing and autoregulation, whereas CARD and PYD domains mediate homotypic protein-protein interactions for downstream signaling. The NACHT domain, which is the only domain common to all NLR family members, enables activation of the signaling complex via ATP-dependent oligomerization.

Phylogenetic analysis of NLR family NACHT domains reveals 3 distinct subfamilies within the NLR family:

* The NODs (NOD1-2, NOD3/ NLRC3, NOD4/NLRC5, NOD5/NLRX1, CIITA),

* The NLRPs (NLRP1-14, also called NALPs) and

* The IPAF subfamily, consisting of IPAF (NLRC4) and NAIP.

The phylogenetic relationships between subfamily members are also supported by similarities in domain structures. This is particularly clear for the NLRPs, which all contain PYD, NACHT, and LRR domains, with the exception of NLRP10, which lacks LRRs. The class II transactivator (CIITA) was the first NLR to be characterized, and is a key regulator of class II MHC genes that is mutated in bare lymphocyte syndrome ¹⁴. The transcriptional coactivator factor function of CIITA appears to be distinct among NLRs, as no other NLRs have been shown to exert transcriptional regulator activity or other nuclear functions. Other members of the NLR family are generally considered to perform cytoplasmic surveillance for PAMPs or DAMPs. NOD1 and NOD2 both recognize breakdown products of bacterial cell walls (mesodiaminopimelic acid and muramyl dipeptide [MDP], respectively) and, upon ligand sensing, oligomerize and recruit RIP2 via CARD-CARD interactions. Assembly of NOD1 and NOD2 signalosomes ultimately culminates in the activation of the NF-kB transcription factor, which drives proinflammatory gene regulation¹⁵. Mutations in NOD2 are associated with human inflammatory diseases such as Crohn’s disease and Blau syndrome^16-18. The functions of NOD3 and NOD4 await clarification. The function of NOD5 (NLRX1) is a matter of debate; recent reports position NOD5 within the mitochondrial matrix, or, alternatively, as recruited to the outer mitochondrial membrane, and propose functions in either suppressing MAVS-dependent antiviral pathways or promoting the generation of reactive oxygen species (ROS)^{19, 20, 21}.

The NLRP3 Inflammasome

The NLRP3 inflammasome is currently the most fully characterized inflammasome and consists of the NLRP3 scaffold, the ASC (PYCARD) adaptor, and caspase-1. NLRP3 is activated upon exposure to whole pathogens, as well as a number of structurally diverse PAMPs, DAMPs, and environmental irritants. Whole pathogens demonstrated to activate the NLRP3 inflammasome include the fungi Candida albicans and Saccharomyces cerevisiae that signal to the inflammasome via Syk²², bacteria that produce pore-forming toxins, including Listeria monocytogenes and Staphylococcus aureus²³, and viruses such as Sendai virus, adenovirus, and influenza virus^24,25. In some cases, the individual microbial components (PAMPs, virulence factors) that activate the inflammasome have been identified (for instance, the alpha-toxin)²⁶. The unexpected finding that the NLRP3 inflammasome can be activated by host-derived molecules forms part of an emerging literature supporting a model in which the innate immune system detects endogenous indicators of cellular danger or stress, a hypothesis with similarities to the ‘‘danger model’’ proposed for adaptive immune responses in place of the more simplistic self/nonself recognition model²⁷. A number of host-derived molecules indicative of injury activate the NLRP3 inflammasome, including extracellular ATP²⁸ and hyaluronan²⁹that are released by injured cells. Fibrillar amyloid-b peptide, the major component of Alzheimer’s disease brain plaques, also activates the NLRP3 inflammasome³⁰. The NLRP3 inflammasome also detects signs of metabolic stress, including elevated extracellular glucose³¹such as that occurring in metabolic syndrome, and monosodium urate (MSU) crystals that form as a consequence of hyperuricemia in the autoinflammatory disease gout³². Uric acid can also be released during cell injury, and uric acid-dependent pathways in this context also activate the inflammasome^33,34. Additionally, the NLRP3 inflammasome drives inflammation in response to a number of environmental irritants, including silica^35-37, asbestos^35,36, UVB irradiation³⁸, and skin irritants such as trinitrophenylchloride, trinitrochlorobenzene, and dinitrofluorobenzene^39,40. NLRP3 inflammasome activation in response to these insults has been linked to pathology associated with silicosis, asbestosis, sunburn, and contact hypersensitivity reactions, respectively.

The NLRP1 Inflammasome

The NLRP1/NALP1 inflammasome (also known as DEFCAP, NAC, CARD7 and CLR17.1) is formed as a reconstituted complex enriched for caspase-1, caspase-5, and the adaptor molecule ASC. The bacterial cell wall component muramyl dipeptide (MDP) strongly activates the inflammasome in-vitro, although direct binding of MDP to NLRP1/NALP1 was not demonstrated. The NLRP1 inflammasome was the first to be described. Human NLRP1 has three orthologs in mouse (Nlrp1a-c) that are highly polymorphic between inbred mouse strains⁴¹. Strain variation in the mouse Nlrp1b locus appears to underlie susceptibility to Bacillus anthracis lethal toxin (LeTx), as macrophages from susceptible, but not resistant, mouse strains activate caspase-1 after LeTx exposure⁴¹. The NLRP1 inflammasome can also be activated by MDP ⁴². As a consequence of domain structure differences between NLRP1 and NLRP3, the minimal components of the NLRP1 inflammasome are somewhat different to its NLRP3 counterpart. NLRP1 contains a C-terminal extension that harbors a CARD domain, which can interact directly with procaspase-1 and bypass the requirement for ASC, although ASC inclusion in the complex augmented human NLRP1 inflammasome activity ⁴². Unlike human NLRP1, murine NLRP1 orthologs lack functional PYD domains and are predicted to be unable to interact with ASC. Indeed, ASC is dispensable for caspase-1 activation by NLRP1b in mouse macrophages⁴³. In addition to caspase-1, NLRP1 also interacts with caspase-5, which may contribute to IL-1b processing in human cells⁴⁴. The exact mechanisms of NLRP1 activation remain obscure, but, as for NLRP3, K+ efflux appears to be essential^45,46. The adaptor molecule ASC was not required for NLRP1/NALP1 inflammasome activation although its presence did enhance caspase-1 activation. NLRP1/NALP1 also plays a role in anthrax immunity. Bacillus anthracis lethal toxin (LT) can induce caspase-1-dependent cell death of macrophages.

The IPAF Inflammasome

The NLRC4/Ipaf inflammasome (also known as CARD12, CLR2.1 and CLAN) is important for caspase-1 activation in response to Salmonella, Pseudomonas, Legionella, Listeria, Anaplasma and Shigella. NLRC4/IPAF responds to the presence of flagellin within the macrophage cytosol during infection by Salmonella, Legionella and Pseudomonas. Shigella, however, does not express flagellin, suggesting that NLRC4/IPAF may signal through a molecule independently of flagellin expression. For Legionella infection, Vance and colleagues have generated mice deficient in the intracellular sensor Naip5/Birc1e/NLRB1 and have elegantly demonstrated that these mice fail to activate the inflammasome in response to a segment of the carboxyl terminus of flagellin. The IPAF inflammasome is activated by gram-negative bacteria possessing type III or IV secretion systems, such as Salmonella typhimurium, Shigella flexneri, Legionella pneumophila, and Pseudomonas aeruginosa^47-52. As IPAF contains a CARD domain, it could be expected to interact directly with procaspase-1, which is indeed the case⁵³. Maximal caspase-1 activation in response to S. Typhimurium, S. flexneri, and P. aeruginosa requires the ASC adaptor^48,49,52. The role for ASC in the IPAF inflammasome remains unclear; it is presumed that these proteins do not interact directly as IPAF does not contain a PYD domain. It is possible that IPAF collaborates with a PYD-containing protein (such as an NLRP) for responses to these pathogens. ASC is dispensable for IPAF dependent caspase-1 activation in response to L. Pneumophila⁵⁴, but protection against this pathogen appears to require IPAF collaboration with another NLR family member, NAIP^55,56. IPAF-dependent caspase-1 activation is accompanied by rapid cell death⁵⁷. Interestingly, the pathways downstream of IPAF appear to be independently regulated; maximal caspase-1 activation by the IPAF inflammasome requires ASC, but IPAF-dependent cell death is independent of ASC upon macrophage infection with S. Flexneri^52,58 and P. aeruginosa⁵¹. The exact mechanisms directing IPAF inflammasome activation and the participation of ASC and NAIP in this process remain elusive. The LRR domain of IPAF is likely to autorepress in the absence of ligand, as removal of the LRR domain from IPAF results in a spontaneously active mutant⁵³. The IPAF inflammasome is activated by cytosolic flagellin⁵⁹. It is likely that other PAMPs modulate IPAF function, as P. aeruginosa appears to activate IPAF through flagellin-dependent and independent pathways^50,51, and nonflagellated bacteria such as S. flexneri trigger IPAF inflammasome activity⁵². IPAF activation depends upon virulence factor injection into the cytosol via bacterial type III and IV secretion systems^52,55,60. Cytosolic localization of flagellin by other means (liposomes, expression systems) is sufficient for IPAF-dependent caspase-1 activation, suggesting that the sole function of bacterial secretion systems in IPAF activation is cytoplasmic injection of bacterial components^61,62,63. Unlike NLRP3, IPAF inflammasome activity is not inhibited by high extracellular K+, suggesting that IPAF is not a sensor for ionic flux⁶⁴. Direct interaction between IPAF and an activating ligand has not been demonstrated, so it is possible that IPAF senses a common pathway induced by cytosolic PAMPs, analogous to the ROS pathway proposed for NLRP3. Signaling mechanisms that form the NLRC4/IPAF inflammasome has been recently expanded. Using Legionella as a model pathogen, have demonstrated that caspase-7 was activated downstream of the NLRC4/ IPAF inflammasome and required caspase-1 activation. Caspase-7 activation was mediated by flagellin and required a functional Naip5.

The AIM2 (absent in melanoma 2) Inflammasome

The recent identification of the HIN-200 family member, AIM2, as a cytosolic double-stranded DNA (dsDNA) sensor that induces caspase-1-dependent IL-1b maturation is an important advance in the inflammasome field^65-68. It is the first identification of a non-NLR family member forming an inflammasome scaffold, and oligomerization of the complex is suggested to be mediated not by a central oligomerization domain within the inflammasome scaffold protein (as for the NLR NACHT domain) but by clustering upon multiple binding sites in the ligand, dsDNA, to which AIM2 binds via its C-terminal HIN domain^65-67. The AIM2 inflammasome is composed of AIM2, ASC, and caspase-1. AIM2 contains a PYD domain that, as for NLRP3, interacts with ASC via homotypic PYD-PYD interactions, allowing the ASC CARD domain to recruit procaspase-1 to the complex. As for other inflammasomes, upon autoactivation, caspase-1 directs proinflammatory cytokine maturation and secretion (such as IL-1b and IL-18). Ligand requirements for AIM2 are quite permissive, as cytosolic dsDNA from virus, bacteria, or the host itself can activate the AIM2 inflammasome^25,67. Further studies are required to firmly establish the physiological relevance of this pathway, but AIM2 is proposed to function in cytosolic surveillance for DNA viruses and may contribute to autoimmune responses against self- DNA in systemic lupus erythematosus.

Negative Regulation of Inflammasome Activation

The importance of the inflammasome in controlling infection is highlighted by microbial evolution of inflammasome inhibitors. These include viral PYD proteins and various bacterial virulence factors that inhibit caspase-1 activation. A number of host mechanisms also suppress inflammasome activation and presumably function to inhibit the extent of potentially dangerous immune activation. Most recently, mouse CD4+ effector and memory T cells were demonstrated to suppress NLRP1 and NLRP3, but not IPAF, inflammasome-mediated caspase-1 activity and IL-1b secretion. Inhibition is dependent on cell-cell contact and is mediated by signaling by specific TNF family ligands⁶⁹. Such an inhibitory pathway is likely to aid in ‘‘switching off’’ innate immune responses once the adaptive arm of the immune system is engaged. A number of CARD- and PYD-containing proteins have been proposed to suppress inflammasome activity by blocking inflammasome component recruitment. Such PYD-containing proteins include pyrin, POP1 (PYDC1), and POP2 (PYDC2). The biological relevance of POP1- and POP2-dependent inflammasome inhibition is difficult to assess, as these genes are only present in primates. Conversely, modulation of inflammasome activity by pyrin has clear physiological relevance, as human pyrin mutations are responsible for familial Mediterranean fever ; however, the underlying disease-driving mechanisms await clarification. Pryin may inhibit inflammasome activation by sequestering ASC⁷⁰, or, alternatively, pyrin may itself form an inflammasome⁷¹. Further evidence for an important regulatory function for pyrin comes from a pyrin-interacting protein, PSTPIP1, mutations in which are associated with elevated IL-1b in the autoinflammatory disease, pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA)^72,73. CARD-containing proteins, such as human COP, INCA, iceberg, and caspase-12, are suggested to suppress inflammasome activation by preventing caspase-1 recruitment. Caspase-12 is proposed to function as a decoy inhibitor of caspase-1⁷⁴, but caspase-12 is nonfunctional in most human populations⁷⁵. As for POPs 1 and 2, COP, INCA, and iceberg do not have non- primate orthologs. The evolution of a raft of potential inflammasome inhibitors in humans suggests strong selection pressure for control over inflammasome activation. The antiapoptotic proteins Bcl-2 and Bcl-xL inhibit the inflammasome. These proteins interact with NLRP1 and suppress NLRP1-dependent caspase-1 activation and IL-1b secretion⁷⁶, potentially by inhibiting ATP binding to the NLRP1 NACHT domain, a necessary step for NACHT domain oligomerization⁷⁷. Activated caspase-1 is secreted alongside mature IL-1b after inflammasome activation. It is possible that caspase-1 has extracellular functions; however, given its potent intracellular functions and the almost undetectable levels of activated caspase-1 within stimulated cells in most experimental settings, it is tempting to speculate that rapid caspase-1 release after activation represents an important regulatory mechanism to limit the activity of cytosolic caspase-1.

Activation

Inflammasome Activation by PAMPs

Inflammasomes respond to immunomodulatory pathogen-associated molecular patterns, PAMPs, mainly bacterial Peptidoglycan, PGNs and nucleic acids. PGNs are structural units of cell walls common to all bacteria⁷⁸. Degradation of PGNs leads to the release of several structural units including muramyl dipeptide, MDP. MDP is sensed in the cytosol by the NLR NOD2, which activates NF-κB. MDP also activates caspase-1 and IL-1β⁷⁹ via NALP3 in human monocytes, suggesting that NALP3 is an additional MDP sensor ^80,81. The strength of the immune response to MDP varies greatly and depends on the animal species and genetic background. Mice are much less sensitive than humans, guinea pigs, or rats to these PGN- derived peptides; moreover, C57BL/6 mice are less sensitive than BALB/c mice^82,83. Interestingly, both NF-κB and IL-1β activation are greatly enhanced by MDP in the presence of the protein synthesis inhibitor cycloheximide (CHX), demonstrating that a CHX-sensitive pathway may affect MDP internalization or its presentation to NLRs⁸⁴. Genetic studies in mice have shown that IL-1β activation by MDP requires both NOD2 and NALP3, suggesting that both these NLRs may cooperate either in-directly or directly as part of the same molecular complex⁸⁴. This finding is consistent with the observation that monocytes from Crohn’s disease patients who have functional mutations in the NOD2 gene fail to activate IL-1β upon MDP stimulation⁸⁵. Moreover, Muckle- Wells patients that harbor a gain-of-function mutation in the NALP3 gene overproduce IL-1β upon stimulation with MDP⁸⁰. Similarly, a probable gain-of-function mutation in NOD2 in the mouse leads to increased IL-1β production upon stimulation of macrophages with MDP⁸⁶. NOD2 has also been suggested to play a role, together with NALP1, in MDP-induced caspase-1 activation, further suggesting that multiple NLRs may cooperate and synergize to mount host defenses⁸⁷. TLR9 and the intracellular protein DAI have been identified as sensors for DNA resulting in the triggering of a type I IFN response^88,89. Similar to DAI, the inflammasome is capable of sensing cytosolic DNA⁹⁰, although this is unlikely to be direct. Infection of a monocytic cell line or mouse macrophages with adenoviruses and herpesviruses leads to activation of caspase-1 and IL-1β. NALP3- and ASC-deficient mice display reduced innate inflammatory responses to infection with adenovirus. Inflammasome activation also occurs as a result of transfected cytosolic bacterial, viral, and mammalian (host) DNA; however, in this case sensing is dependent on ASC only and not on NALP3. It is also independent of TLRs and IRFs⁹⁰. Studies have also identified RNA as an activator of NALP3⁹¹, although this study could not be confirmed by other groups.

Pore-Forming Bacterial Toxins Activate the NALP3 Inflammasome

Many bacterial pathogens produce toxins that contribute to virulence by modifying host responses. Bacterial toxins are generally either enzymes or pore-forming proteins⁹². Most of the bacterial toxins that activate NALP3 are pore-forming toxins. These toxins are released by bacteria in a soluble form and subsequently polymerize into a ring-like structure forming a pore in the membrane of the host cell. Pore formation triggers ionic imbalance; in particular, potassium efflux and calcium influxes are observed. These two processes are common danger signals and are frequently associated with NALP3 inflammasome activation⁹³. Among pore-forming toxins, α-toxin from Staphylococcus aureus and aerolysin from Aeromonas hydrophila are potent activators of the NALP3 inflammasome⁹⁴. Similarly, listeriolysin O (LLO), a toxin released by Listeria monocytogenesis, activates caspase-1 in an ASC and NALP3-dependent manner^95,96. Interestingly, ivanolysin O, a LLO-related cytolysin that exhibits quite similar function regarding the contribution to the escape of Listeria from the phagosome into the cytosol, is unable to restore caspase-1 activation in LLO deficient strains, suggesting that these toxins may engage specific signals beyond their ability to form pores⁹⁷. The release into the cytosol of flagellin during Listeria infection activates the IPAF inflammasome (but not the NALP3 inflammasome, see below), highlighting the ability of pathogens to engage specific and multiple inflammasomes⁹⁸.

Anthrax Lethal Toxin Activates NALP1

Bacillus anthracis is the causative agent of anthrax and depends for its virulence on the secretion of factors that form functional toxins. Anthrax lethal toxin (LeTx) is one of the major toxins produced by B. anthracis and is believed to be responsible for causing death in systemic anthrax infections. Macrophages from inbred mice are either susceptible or resistant to cell death in response to LeTx. This trait difference has been mapped to a locus on chromosome 11 and is associated with a polymorphism in the nalp1b gene that impinges on caspase-1 activation⁹⁹. How LeTx activates NALP1 and the role of the inflammasome in anthrax pathology are still unknown. Murine NALP1b does not contain a PYD; hence, it is not clear whether it requires ASC or dimerization with another NALP for caspase-1 recruitment. On the other hand, NALP1b possesses a CARD and a region related to CARDINAL. It is therefore possible that this CARD-containing region is able to activate caspase-1 in an ASC-independent manner, as was suggested for human NALP1 in vitro¹⁰⁰. Activation of caspase-1 by LeTx requires binding, uptake, and endosome acidification to mediate translocation of lethal factor (a functional subunit of LeTx) into the host cell cytosol. Interestingly, catalytically active lethal factor activates caspase-1 by a mechanism involving proteasome activity and potassium efflux ^101-103.

IPAF Inflammasome Activation by Injected Virulence Factors

IPAF and NAIP5 have been involved in the detection of virulence factors from Gram-negative bacteria. Recognition of Salmonella typhimurium and Shigella flexneri activates the IPAF inflammasome that requires the ASC adaptor¹⁰⁴. The role of ASC in the IPAF inflammasome is still unclear, but ASC may stabilize or facilitate caspase-1 recruitment to IPAF. Alternatively, IPAF may cooperate with a yet to be defined NALP to activate caspase-1. In contrast to S. typhimurium and S. Flexneri, Legionella pneumophila requires two murine NLRs, NAIP5 and IPAF, for inflammasome formation, but does not require ASC^105-107. The role of NAIP5 in IPAF inflammasome activation is unknown. It has been suggested that defective NAIP5 signaling renders macrophages permissive to L. Pneumophila despite caspase-1 activation, suggesting that NAIP5 may have additional functions beyond its role in IPAF and caspase-1 activation¹⁰⁸. Unlike the seven NAIP genes found in the murine genome, humans only harbor one copy. Consistent with findings in the mouse, knockdown of NAIP or IPAF in human cell lines leads to enhanced susceptibility to L. pneumophila¹⁰⁹. IL-18 production and protection against Anaplasma phagocytophilum, a neutrophilic obligate bacteria that causes human anaplasmosis, relies on ASC and caspase-1 and partially on IPAF, but not on NALP3¹¹⁰. Similarly, Pseudonomas aeruginosa specifically activates the IPAF inflammasome^111-113. Most Gram-negative pathogens that activate IPAF require the type III secretion system (T3SS) or the type IV secretion system (T4SS) to inject into the host cell IPAF-activating virulence factors, mainly flagellin. These bacterial injection machines span the two membranes from the bacteria and the host cell membrane¹¹⁴. Polymers of flagellin form the flagella, a structure anchored to the bacterial cell wall that enables bacterial motility. Flagellin is a well-known activator of host innate immunity through its capacity to trigger TLR5 activation. In this context, flagellin is considered a PAMP as it is a vital, evolutionarily conserved element of mobile bacteria. In the context of IPAF activation, flagellin could be interpreted as a virulence factor, as flagella formation is not required and flagellin release in the cytosol is apparently the result of an active process dependent on T3SS or T4SS injection systems¹¹⁴. Interestingly, S. flexneri, a pathogen that does not have a flagellum and apparently does not express flagellin, still requires the T3SS for IPAF-induced caspase-1 activation, indicating that flagellin may not be the only T3SS virulence factor used by pathogens to activate IPAF inflammasomes¹¹⁵. How flagellin or other virulence factors engage IPAF inflammasomes as well as the precise function of ASC or NAIP in detecting these signals and activating caspase-1 are fascinating questions that need to be solved in future studies.

Inflammasome Activation Requirements

Dendritic cells, macrophages and circulating monocytes must respond to microbial threats and are essential to host defense. Inflammasome activation in monocytes and dendritic cells and macrophages are different. Two fundamental differences exist for inflammasome activation in dendritic cells, macrophages and circulating monocytes. While monocytes have constitutively activated caspase-1, the activation of caspase-1 in dendritic cells and macrophages needs to be induced. Furthermore, monocytes are capable of releasing endogenous ATP¹¹⁶—an important molecule for inflammasome activation, whereas the lack of endogenous ATP by macrophages and dendritic cells renders these cells incapable of IL-1β secretion with one single stimulus¹¹⁷. Monocytes only need a single TLR stimulus for IL-1β secretion, whereas dendritic cells and macrophages need a double stimulation—TLR (e.g., LPS) and NLR (e.g., ATP) stimuli. The reason for this differential regulation is poorly understood. Current evidence suggests an adaptation of each cell type to their respective environment. Circulating monocytes function in a pathogen-free environment and must promptly respond to a microbial threat. Dendritic cells and macrophages are confined to a non-sterile environment and are constantly exposed to microbial pathogens. Therefore, a second checkpoint mechanism is necessary to avoid deleterious inflammation. The difference in inflammasome activation requirements does not seem to be restricted to macrophages, dendritic cells and monocytes. In contrast to monocytes and macrophages, in which low intracellular K+ triggers inflammasome activation, neurons and astrocytes activate caspase-1 by high intracellular K+ ¹¹⁸. In epithelial cells, which do not secrete high levels of IL-1β, caspase-1 enhances lipid metabolism and is required for optimal growth of the intracellular bacterium Chlamydiae¹¹⁹. Mast cells were identified as the main cell population responsible for IL-1β in the skin of cryopyrin-associated periodic syndrome patients. Unlike normal mast cells that required stimulation with pro-inflammatory stimuli for IL-1β production, resident mast cells from cryopyrin-associated periodic syndrome patients constitutively produced IL-1β via the NLRP3 inflammasome¹²⁰. Collectively, these findings demonstrate that each cell type commits the inflammasome to the immediate environment in which they are located. This differential requirement for inflammasome activation operates to prevent accidental or uncontrolled inflammation, which may have devastated consequences for the host. A better understanding of the inflammasome activation in cells other than macrophages and dendritic cells may have significant ramifications for the treatment of infectious diseases and inflammatory disorders.

CONCLUDING REMARKS

The remarkable progress in this field offers new hope for many patients. The NLRs, together with the TLRs, are now appreciated to be part of important sensing system that allows the host to mount an effective immune response for elimination of the microbe and for establishment of an effective adaptive immune response for long-lasting immunity. We can anticipate a new generation of IL-1 antagonists in the near future. A new IL-1β antibody is currently in Phase II trials for rheumatoid arthritis, and various inflammatory diseases, such as gout, are now successfully treated with an IL-1 inhibitor. The precise roles and needs for molecules such as IPAF and NAIP remain ill defined. The ligands of many other NLRs are unknown and their functions elusive. Although mutations in NOD2 and NALP3 genes provide a basis for susceptibility to Crohn’s disease and inflammatory diseases.

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Received on 08.03.2014 Modified on 01.05.2014

Research J. Pharm. and Tech. 7(6): June, 2014; Page 695-703