5.3 Microglia inflammasome signaling drives Alzheimer's disease phenotypes
Nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome activation in microglia is a major stress-induced innate immune mechanism that drives AD pathology.90,91 The inflammasome is a multiprotein complex consisting of NLRP3, the adapter molecular apoptosis-associated spec-like protein (ASC) and the cysteine protease caspase 1. Of particular importance, the NLRP3 inflammasome is activated through mechanisms of "sterile inflammation," which is an immunological reaction in the absence of a pathogenic insult. There are a number of endogenous molecules released following stress that act as danger-associated molecular patterns (DAMPs), including HMGB1, ATP or S100b that act upon their respective pattern recognition receptors (PPRs) such as P2X7 or RAGE, leading to the assembly of the NLRP3 inflammasome complex. In addition, a second signal is required to fully initiate the inflammasome's activity. Microbial-associated molecular patterns (MAMPS) or other DAMPs will signal through the toll-like receptors (TLRs) subsequently activating NF-κB and stimulating pro-IL-1β transcription. Caspase 1 activation in the inflammasome complex will then cleave pro-IL-1β into the active form initiating the inflammatory response.92,93
In the context of AD, it is important to note that fibrillar Aβ can act as an endogenous ligand to stimulate NLRP3 activity.94 Indeed, when transgenic APP/PS1 mice were crossed with a Nlrp3−/− or Casp1−/− line, there was reduced brain caspase 1 and IL-1β activation as well as enhanced Aβ clearance, likely due to the increased phagocytic state in microglia.95 In a more recent study, a NLRP3 inhibitor, JC-124, was given to CRND8 APP transgenic mice. The JC-124 inhibitor reduces NLRP3 inflammasome assembly and concurrently reduced Aβ deposition in the brain. Interestingly, the reduced Aβ was at least partially due to reduced β-cleavage of APP and/or reduced microglia activation, but not astrocytosis.96 Finally, Aβ deposition was also shown to induce ASC speck formation in the microglia, stimulating NLRP3 activity further driving Aβ pathology in a feed-forward loop.97 Clearly, NLRP3 activation is an important causative mechanism for the stress-induced neuroinflammatory development of AD.
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Pathophysiology of Neonatal Bacterial Meningitis
Tatiana Barichello, in Fetal and Neonatal Physiology (Fifth Edition), 2017
NLRP3 Inflammasome–Sensing Bacteria
The Nod-like receptor protein-3 (NLRP3) inflammasome is composed of NOD-like receptor 3, procaspase 1, and the adaptor protein apoptosis-associated speck-like protein comprising a caspase recruitment domain (ASC).10 The NLRP3 inflammasome oligomerization is activated by a large number of stimuli, such as bacterial toxins, bacterial compounds, and DAMPs. NLRP3 and absent in melanoma 2 can be activated by the exotoxin pneumolysin and bacterial DNA in pneumococcal infection.99 S. agalactiae activates NLRP3 through the expression of β-hemolysin, an important virulence factor. NLRP3 has been implicated in the responses to several microorganisms, such as Staphylococcus aureus (hemolysin O), L. monocytogenes (listeriolysin O), Klebsiella pneumoniae, and E. coli.100 L. monocytogenes is also sensed by absent in melanoma 2 and NLR family, CARD domain–containing 4 (NLRC-4), which, with NLRP3, orchestrate a strong caspase 1 activation and proinflammatory response.101 On activation, NLRP3 oligomerizes with ASC and procaspase 1. Procaspase 1 is converted to activated caspase 1, which subsequently matures and secretes the proinflammatory cytokines IL-1β and IL-18 in their mature forms.102
The NLPR3 inflammasome is responsible for the production of important proinflammatory cytokines that are identified in the CSF of patients with bacterial meningitis. In patients with bacterial meningitis, increased levels of IL-1β and IL-18 in the CSF are related to unfavorable disease outcomes.103 In the murine model of pneumococcal meningitis, NLRP3 induced caspase 1 activation. Additionally, NLRP3 and ASC inhibition were associated with reduced clinical and histologic disease severity and decreased brain inflammation.104 In another experimental study, NLRP3- and ASC-deficient mice displayed a decrease in systemic inflammatory responses. However, the same mice showed an increase in cerebral neutrophil infiltration and cerebral hemorrhages compared with wild-type mice.103 Mice lacking NLRP3, ASC, or caspase 1 were more susceptible to S. agalactiae infection than were wild-type mice.105 The challenge lies in identifying when the immune response is essential to eliminate the microorganism versus when its activation results in damage to the host.
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Lung Defenses
James M. Stark MD, PhD, Gary A. Mueller MD, in Kendig & Chernick's Disorders of the Respiratory Tract in Children (Eighth Edition), 2012
Nod-Like Receptors
The nod-like receptor (NLR) family is a large family of intracellular receptors consisting of 23 reported members. They are located in the cytosol and regulate both inflammation and apoptosis (programmed cell death). These proteins are expressed in many cell types, including immune cells and epithelial cells.24,25 Their general structure includes a central nucleotide-binding oligomerization (NOD) domain, and a C-terminal leucine-rich repeat (LRR) domain. The N-terminal effector region varies between the different proteins, resulting in activation of diverse downstream signaling pathways.25 NOD1 and NOD2 (the best studied cytosolic NLR) are expressed in leukocytes and lung epithelial cells. NOD1 detects bacterial wall peptidoglycan containing meso-diaminopimelic acid (found primarily in the peptidoglycan cell wall of Gram-negative bacteria), whereas NOD2 recognizes muramyl dipeptide conserved in both Gram-positive and Gram-negative bacteria. These activate intracellular signaling cascades by activation of Rip2 kinase, leading to activation of NFκB and subsequent expression of pro-inflammatory cytokines, and reactive oxygen species production.25 The NLRP (NLR family, pyrin domain containing) consists of 14 members characterized by a PYD domain. At least three members of this family (NLRP1-3) form multiprotein complexes called inflammasomes.26 Inflammasomes consist of one or two NLRs, an adapter molecule ASC, and caspase 1. The inflammasomes respond to various microbial molecules and regulate caspase-1–mediated cell death and production of mature IL-1β and related cytokines (including IL-18) at a posttranslational level. NLRP3 is expressed in granulocytes, macrophages, monocytes, and dendritic cells. The NLRP3 inflammasome activators include microbial RNA, certain forms of DNA, bacterial pore-forming toxins, and MDP-1. This inflammasome mediates caspase-1–dependent processing of pro-IL-1β as well as pro-IL-18 to their mature forms and regulates caspase-1–dependent cell death. The NLR member NLRX1 (NLR family member X1) is the only NLR molecule localized in the mitochondrial membrane where it mediates production of reactive oxygen species upon bacterial infection.
In summary, the NLRs function as intracellular pattern recognition proteins involved in the recognition of conserved microbial components in addition to nonmicrobial signals such as silica and uric acid crystals. NLR signaling results in activation of NFκB, resulting in the induction of pro-inflammatory cytokines, chemokines, and antimicrobial molecules.24
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The Mucosal Microbiome
Jonathan Jacobs, Jonathan Braun, in Mucosal Immunology (Fourth Edition), 2015
Nod-Like Receptors
The Nod-like receptor (NLR) family consists of at least 23 proteins in humans with C-terminal leucine-rich repeats, a central nucleotide-binding oligomerization (NOD) domain, and N-terminal protein binding motifs such as a caspase activation and recruitment domain (CARD) (Franchi et al., 2009). The NLRs are cytoplasmic proteins that primarily recognize bacterial motifs. The best studied are NOD1, NOD2, and the NLR components of the inflammasome. NOD2 has particularly drawn attention because of its strong genetic link to Crohn's disease as well as a rare familial disease, Blau syndrome. NOD1 and NOD2 recognize moieties from peptidoglycan—meso-diaminopimelic acid and muramyl dipeptide (MDP), respectively—which are found on many bacterial and mycobacterial pathogens. NOD1 is expressed widely whereas NOD2 is expressed primarily in immune cells and epithelial cells. After microbial sensing, NOD1 and NOD2 translocate to the plasma membrane and recruit RIP-like interacting caspase-like apoptosis regulatory protein kinase (RICK, also known as Rip2), resulting in a proinflammatory response through NF-κB and mitogen-activated protein kinase activity (Ting et al., 2010). NOD1 and NOD2 are also able to induce the formation of autophagosomes by recruiting Atg16L1 to the plasma membrane, resulting in engulfment of invading bacteria (Travassos et al., 2010).
Inflammasomes are cytosolic protein complexes of a PRR with the adaptor protein ASC (apoptosis-associated speck-like protein containing CARD) and procaspase-1. The primary function of an inflammasome is to cleave caspase-1 into its active form, which can then activate the inflammatory cytokines interleukin (IL)-1β and IL-18 by proteolysis. The following NLRs have been documented to form inflammasomes: NLRP1, NLRP3, NLRP6, and NLRC4 (Franchi et al., 2012). NLRP1 is activated by MDP, although it remains unclear whether it binds it directly. NLRC4 recognizes flagellin and PrgJ-like proteins from Gram-negative bacteria in a manner that depends upon several other NLRs (Naip2, Naip5, and possibly others) and protein kinase C-theta (Franchi and Nunez, 2012). NLRP3 is distinct from NLRP1 and NLRC4 in that it can be activated by diverse microbial signals including pore-forming bacterial toxins, dsRNA, LPS, MDP, and lipopeptide as well as many nonmicrobial signals such as urea, silica, and aluminum (Leemans et al., 2011). This broad specificity suggests that it does not directly sense microbes. There is now growing evidence that NLRP3 is activated by reactive oxygen species generated by mitochondrial stress (Zhou et al., 2011). This is not surprising given the critical role of mitochondria as sensors of cell injury that can initiate pathways leading to apoptosis, angiogenesis, and inflammation (Galluzzi et al., 2012). NLRP6 has only recently been described to form an inflammasome (Elinav et al., 2011). It is found predominantly in epithelial cells, but its ligand remains unknown.
An additional inflammasome has also been described that incorporates a non-NLR PRR, absent in melanoma-2 (AIM2), which recognizes cytoplasmic double-stranded DNA (Hornung et al., 2009). This inflammasome was capable of activating caspase-1 in response to vaccinia virus. A second member of the AIM2-like family in humans has also been described, interferon inducible protein-16 (IFI16), that recognizes intracellular DNA and is involved in the response to herpes simplex virus (Unterholzner et al., 2010).
NLRs can also shape microbial composition, as was seen for some TLRs. NOD2 knockout mice were found to have an altered intestinal microbiome compared with controls (Petnicki-Ocwieja et al., 2009). Deficiency of NLRP6, or of ASC, resulted in an abnormal microbiota that conferred transmissible susceptibility to colitis and fatty liver disease (Elinav et al., 2011; Henao-Mejia et al., 2012). As with the TLRs, it is an open area of investigation whether the many remaining NLRs affect the microbiome.
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Innate Immunity to Toxoplasma gondii
Dana G. Mordue, Christopher A. Hunter, in Toxoplasma Gondii (Second Edition), 2014
24.7.6 NALP1 Inflammasome
NALP1 is a NOD-like receptor that is important for the activation of the NALP1 inflammasome. Recent studies have identified susceptibility alleles of NALP1 associated with congenital toxoplasmosis. Human monocytes depleted of NALP1 and infected with T. gondii were more likely to undergo cell death and less able to control replication of intracellular parasites. T. gondii infection also failed to increase expression of IL-1β and IL-18 protein and IL-12 from NALP1 knockdown monocytes. These results suggest that T. gondii infection of human monocytes activates the NALP1 inflammasome contributing to control of intracellular parasites (Witola et al., 2011). The refractoriness of the Lewis rat to toxoplasmosis independent of parasite genotype is intrinsic to bone marrow cells and is linked to a genetic locus (q24 region of rat c10 containing 86 putative genes) orthologous to a region in the human genome that contains NALP1 (Witola et al., 2011; Cavailles et al., 2006; Sergent et al., 2005). How T. gondii stimulates inflammasome activation from its location within its relatively sequestered PV is currently unknown.
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Cells of Innate Immunity: Mechanisms of Activation
T.J. Sellati, B. Sahay, in Pathobiology of Human Disease, 2014
Structural features
In general, each NLR contains three important domains that facilitate interaction with other proteins to form a multimeric complex known as an inflammasome. The domains include (i) an N-terminal effector binding region that consists of a CARD, PYD, or BIR domain, (ii) a NOD needed for nucleotide binding and self-oligomerization, and (iii) an LRR motif to detect conserved microbial patterns (Figure 6). CARD domains were originally associated with proteins involved in apoptosis and inflammation such as many of the caspases, including caspase-1. However, CARDs also can mediate caspase-independent interactions. The structure of PYD is homologous to that of CARD and promotes homophilic interactions with other PYD-containing proteins that are important for downstream signaling events. Both CARD and PYD are members of the death domain-fold superfamily, members of which are involved in both apoptosis and inflammation. BIR-containing proteins can be classified into two major groups, inhibitor of apoptosis proteins and neuronal apoptosis inhibitor proteins (NAIPs), both of which are NLR family members. Finally, NLRX represents a new subfamily with no strong homology to the others and only one member, NLRX1, which rather uniquely localizes to mitochondria. The presence of NLRX1 in the mitochondrial matrix amplifies NF-κB and JNK signaling by augmenting the production of mitochondrial reactive oxygen species (ROS).
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Immunologic Repercussions of Cell Death
Helen M. Beere, Douglas R. Green, in Kelley and Firestein's Textbook of Rheumatology (Tenth Edition), 2017
NLRP1b Inflammasome
NLRP1, the first NLR to be identified as an inflammasome mediator,154 is encoded by a single gene in humans but by three orthologs in the mouse, denoted Nlrp1a, Nlrp1b, and Nlrp1c.155 Unlike human NLRP, which contains a pyrin domain, the murine paralogs do not contain this domain and are composed of an NBD, an LRR region, a FIIND ("function to find" domain) and a C-terminal CARD. The Nlrp1b locus is most frequently associated with inflammasome formation and activation of caspase-1, although aspects of its regulation and function remain unclear. Muramyl dipeptide (MDP), a minimal bioactive peptidoglycan peptide common to both positive and negative bacteria, was initially identified as a ligand for NLRP1b-mediated inflammasome formation. However, whether NLRP1b-mediated inflammasome formation and caspase-1 processing requires pyrin domain–dependent association with ASC or instead occurs via a direct interaction between NLRP1 and caspase-1 via their respective CARD domains remains unclear.156 Conflicting studies, using cells lacking NLRP1 expression, report that MDP-mediated caspase-1 processing was instead dependent upon NLRP3, either alone or in cooperation with NOD2.157,158
Lethal toxin (LT), the major virulence factor of Bacillus anthracis, is a potent ligand for NLRP1b-dependent inflammasome formation and triggers IL-1β and IL-18 processing and extensive pyroptosis of macrophages. LT promotes the cleavage of NLRPb1,159,160 an event that is reportedly both necessary and sufficient161 for inflammasome assembly. Although autoproteolysis of NLRP3 also correlates with susceptibility to LT-induced inflammasome assembly, it is insufficient for pro-IL-1β activation.162-164 Additional parameters that contribute to the susceptibility of cells to LT, inflammasome formation, and pyroptosis include extra-cellular ATP release and reduced p38-mediated survival.165
The physiologic significance of NLRP1b inflammasome activity in response to LT is supported by the observed resistance to B. anthracis infection in mice expressing an LT-sensitive allele of NLRP1b.166 However, other studies have noted that resistance to B. anthracis and IL-1β production does not necessarily correlate with NLRP1b activation,155 suggesting the contribution of additional factors.
NLRP1a is also associated with inflammasome formation and the consequent secretion of IL-1β and IL-18 in an ASC-independent manner.167 Regulation of NLRP1a activity is effected at multiple levels, including induction of its expression via NF-κB promoter elements, activation by post-translational autoprocessing. and inhibition via LRR-mediated association with the anti-apoptotic BCL-2 family members, BCL-2 and BCL-XL. Interestingly, an activating mutation of NLRP1 and consequent predisposition to inflammasome formation, although associated with lethal systemic inflammation via IL-1β, also led to caspase-1 activity and extensive pyroptosis in the hematopoietic compartment independently of IL-1β. NLRP1a-mediated inflammasome formation and pyroptosis therefore may have additional roles independent of that in IL-1β–mediated inflammation.
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Pathogen- and Microbial- Associated Molecular Patterns (PAMPs/MAMPs) and the Innate Immune Response in Crohn's Disease
Amy K. Schaefer, ... Catherine L. Grimes, in Immunity and Inflammation in Health and Disease, 2018
14.2.2 NOD2 as an Innate Immune Receptor
As a class of PRRs, NLR proteins are tasked with identifying MAMPs and regulating an appropriate immune response (Benko et al., 2008). NLR proteins share a tripartate domain structure that consists of N-terminal protein-protein interaction domain(s) (i.e., caspase activation and recruitment domains (CARDs) or pyrin domains), a central nucleotide-binding oligomerization domain (NOD or NBD), and a C-terminal leucine rich repeat (LRR) domain. NLRs are predominantly found in the cytosol, making them important intracellular detectors of MAMPs (Girardin et al., 2002; Strober et al., 2006). NOD2 is a 1040 amino acid cytosolic innate immune receptor found primarily in intestinal epithelial cells, Paneth cells, monocytes and dendritic cells (Berrebi et al., 2003; Gutierrez et al., 2002; Hisamatsu et al., 2003; Ogura et al., 2001b, 2003; Rosenthiel et al., 2003). It consists of two N-terminal CARD, a central NOD domain and a C-terminal LRR domain. The receptor recognizes bacterial cell wall fragments and initiates signaling cascades through the NF-κB and MAPK pathways that mature as the inflammatory response.
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Neuroimmune Signaling in Drug Actions and Addictions
Gyongyi Szabo, Dora Lippai, in International Review of Neurobiology, 2014
3.1 Pattern recognition receptors sense danger signals in the brain (TLRs and NLRs)
Pattern recognition receptors (PRRs), including the TLR and Nod-like receptor (NLR) families, sense evolutionary conserved molecular patterns as "danger" signals and in response, activate innate immunity and proinflammatory pathways. TLR and NLR receptors, including TLR2, TLR4, TLR6, TLR9, NLRC4, NLRP1, and NLRP3, are upregulated in the alcoholic brain (Lippai, Bala, Petrasek, et al., 2013).
TLRs are expressed on the cell surface (TLR1–TLR6) or in endosomes (TLR3, TLR7–TLR9) and upon activation induce production of proinflammatory cytokines and/or type-1 interferons. Recruitment of the MyD88 adapter molecule to TLRs results in downstream activation of IRAK1/4 and NF-κB activation leading to proinflammatory cytokine production. The TLRs MyD88 (TLR7/8 and 9) or TRIF (TLR3 and TLR4) and signal through IRF3 to induce type-1 interferons (Szabo et al., 2006). TLR4 detects bacterial lipopolysaccharide (LPS) and it can be activated by DAMPs that are induced by alcohol. TLR4 activation contributes to microglia accumulation, astrogliosis, and inflammatory cytokine TNFα, MCP-1, and pro-IL-1β production (Alfonso-Loeches et al., 2010; Fernandez-Lizarbe, Pascual, & Guerri, 2009) and is involved in the pathogenesis of alcohol-induced neuroinflammation and neurodegeneration (Alfonso-Loeches et al., 2010; Lippai, Bala, Petrasek, et al., 2013).
The multiprotein complex, inflammasome, consists of an NLR (NLRs: NLRP1, NLRP3, NLRC4); an adapter molecule, ASC; and procaspase-1 (Mankan, Kubarenko, & Hornung, 2012). Inflammatory stimuli, including DAMPs and PAMPs, upregulate the expression of the inflammasome components via TLRs (Mankan et al., 2012; Fig. 11.1). Alcohol upregulates and activates NLRP3/ASC inflammasome evidenced by caspase-1 activation and increased mature IL-1β levels in mouse brains (Lippai, Bala, Petrasek, et al., 2013). NLRP3 inflammasome activation requires two signals, a priming signal to increase expression of inflammasome components and target proteins, and a second signal that leads to inflammasome activation and subsequent IL-1β secretion (Gross, Thomas, Guarda, & Tschopp, 2011). During this process, procaspase-1 is converted to a proteolytic effector enzyme, caspase-1 which then cleaves pro-IL-1β to mature, secretable IL-1β (Mankan et al., 2012). Suggestive of a feed-forward loop, alcohol-induced neuroinflammation is prevented by recombinant IL-1ra by inhibiting inflammasome activation, IL-1β, and TNFα production in the brain (Lippai, Bala, Petrasek, et al., 2013). TLR activation via DAMPs and PAMPs leads to NF-κB activation (Zhang & Ghosh, 2001). NF-κB induces the transcription of TNFα, MCP1, and IL-1β (Thompson & Van Eldik, 2009; Zhang & Ghosh, 2001), all of which are increased in alcohol-induced neuroinflammation (Alfonso-Loeches et al., 2010; Lippai, Bala, Petrasek, et al., 2013; Qin &