Cells sense the presence of microbes or cell damage through the recognition of molecular or damage associated molecular patterns (PAMPS or DAMPS), such as lipopolysaccaride (LPS) or viral ssDNA. Recognition of PAMPS or DAMPS by pattern recognition receptors (PRRs) results in activation of NFkB and/or AP1 signaling pathways, resulting in proinflammatory cytokine and chemokine production and induction of the IRF transcription factors that mediate the type I interferon response
The NLR (NOD-like receptor) family of proteins share a number of common domains and many are involved in PAMP and DAMP sensing. All 22 members of the family share a NACHT domain which enables complex activation through ATP dependent oligomerization. The family can be divided into three subfamilies on the basis of NACHT domain phylogenetic analysis: NOD, IPAF and NLRP. The function of the individual proteins can be inferred from their domain organization, but most detailed functions are largely unknown. For example, NOD1 and 2 sense breakdown products of the bacterial cell wall. PAMP recognition results in oligomerization and recruitment of RIP2 through the CARD domain to form a signalasome which results in NFkB activation and inflammatory gene expression (2, 4).
NLRP proteins oligomerize directly or indirectly with caspase1 through the caspase recruitment domain (CARD) to form an inflammasome. Inflammasomes can be thought of as caspase activating complexes. There are a number of proinflammatory caspases (human caspase1, 4, and 5) that function in maturation of pro-cytokines, though caspase1 is the best understood. Caspase1 is auto activated in a signal dependent manner within the inflammasome and mediates processing of pro-cytokines such as IL-1β and IL-18 (2,4) (see figure
NLR inflammasome pathway) (1–5).
IL-1β is a proinflammatory mediator that is generated in response to injury or immune challenge. Secretion of IL-1β mediates recruitment of cells to the site of insult. This cytokine is also involved in regulation of sleep, appetite and body temperature. IL-1β activity is tightly regulated through inflammasome mediated control of maturation and release (2, 4).
The NLRP1 inflammasome was the first to be described. NLRP1 has a c-terminal CARD domain, therefore directly interacts with procaspase1, bypassing the need for an adaptor. This inflammasome also interacts with caspase5 which mediates IL-1β processing in human cells (2, 4).
The NLRP3 inflammasome is the best characterized NLRP containing inflammasome. It is composed of an NLRP3 scaffold upon which an ASC adaptor assembles and acts to recruit procaspase1. The NLRP3 inflammasome is activated by fungi, bacteria that produce pore forming toxins, and viruses. It can also be activated by endogenous indicators of cell stress. Examples of such indicators include, extracellular ATP, hyaluronan, fibrillar β amyloid peptide, signs of metabolic stress such as elevated glucose, and environmental irritants such as silica and asbestos (2, 4).
The NLRC4 inflammasome is activated by gram negative bacteria containing type III or type IV secretion systems, specifically Salmonella, Shigella, Legionella, Pseudomonas, and Yersinia. NLRC4 specifically recognizes flagellin, as bacterial strains deficient for flagellin can not induce caspase1 activation like their wild type counterparts. Flagellin protein alone has been shown to activate caspase1 in the absence of bacteria. An alternative flagellin independent mechanism of NLRC4 inflammasome activation has been observed, where caspase1 activation appears to be dependent on a rod protein in the type III secretion system that contains a flagellin-like motif (2, 4).
NLR independent inflammasomes have also been identified. The AIM2 inflammasome of the HIN-200 family of proteins recognizes cytosolic dsDNA. This was the first example of a non-NLR family member forming an inflammasome scaffold. The AIM2 inflammasome recognizes cytosolic DNA from different sources, including virus, bacteria, and host. This inflammasome has been proposed to contribute to the autoimmune response to DNA in Systemic lupus erythematosus (2, 4).
Inflammasome activation is regulated at two levels. Proinflammatory signals such as LPS or TNF induce proIL-1β and NLRP scaffold expression through NFkB signaling. This can be thought of as a priming step. Then presence of pathogen (or host) specific components activates inflammasome function and caspase1 activation. Inflammasomes can be negatively regulated through mechanisms of host and microbial origin. Host mechanisms are in place to control the level of immune activation. Pathogens have evolved a number of inflammasome inhibitors, such as the viral PYR proteins, and bacterial virulence factors that prevent caspase1 activation (2, 4).
Caspase1 can also activate pyroptosis, an inflammatory form of cell death that is associated with infection with intracellular pathogens. Pyroptosis differs from apoptosis in that it is a more chaotic process involving plasma membrane rupture, water influx, osmotic lysis, and release of proinflammatory contents. DNA cleavage and nuclear condensation differ from that observed in apoptosis (2, 4).
Inflammasome pathways have been linked to human disease such as cryopyrin-associated periodic syndromes (CAPS), gout and type 2 diabetes where the NLRP3 inflammasome has been implicated. In fact, elevated IL-1β has been detected in clinical samples. An IL-1 receptor agonist has been developed for treatment and IL-1β antagonists are in development (2, 4).
Inflammasomes are established pathogen sensors, important for activating the innate immune response. Now, Elinav et al describes the NLRP6 inflammasome and its role in preventing inflammatory bowel disease by maintaining a healthy gut microbiota. The commensal organisms that reside in the gut function to out-compete pathogenic organisms, thereby preventing the outgrowth of pathogenic species. Mice deficient in NLRP6 produced lower levels of IL-18, a proinflammatory cytokine, and exhibited an altered fecal microbiota (see figure
The NLRP6 inflammasome maintains a healthy gut microbiota ) (6).
The NLRP6 deficient mice had increased levels of Bacterioidetes and TM7, two phyla that have been associated with IBD. This altered microbiota was transferrable to wild type mice through cross fostering and cohousing. These mice exhibited increased recruitment of inflammatory cells to the gut as well as aggravated colitis induced by dextran sodium sulfate (DSS) (6).
The altered microbiota was associated with increased levels of CCL5/RANTES which directly associated with the development of colitis, as CCL5/RANTES deficient mice did not develop the exacerbated form of DSS induced colitis. This data suggests that defects in the inflammasome pathway, effecting the expression or function of NLRP6, ASC, caspase1, and/or IL-18, may predispose individuals or initiate the development of IBD (6).
The connection between NLR inflammasomes and the stability of the microbiota sheds light on the connection between gut bacteria and the development of IBD. However a number of questions remain. How does the NLRP6 inflammasome control the stability of the microbiota? Multiple factors have been implicated in the development of IBD in humans, including genetic polymorphism in proteins, innate and adaptive immune response, ER stress, and autophagy. Do genetic polymorphisms in the NLRP6 inflammasome pathway predispose individuals to IBD (7)?
Autophagy acts upstream of inflammasome activation. LPS alone does not result in activation of inflammasomes, however genetic inhibition of the autophagy regulators, Atg16L1 and Atg7, results in LPS induced inflammasome activation.The mechanism and function of autophagy in the inflammasome pathway remains unclear. It has been suggested that inflammasomes may be degraded via autophagy. Do polymorphisms in Atg16L1 and Atg7 effect inflammasome function in the gut epithelia (8)?
Elinav and colleagues demonstrate inflammasome activation and subsequent IL-18 production; however it is not known how IL-18 alters the composition of the microbiota. It is known that IL-18 can induce IFN-γ production and that IFN-γ induces microbicidal activity in macrophages, but the exact mechanism for targeting certain bacterial species remains unknown. How the NLRP6 inflammasome is activated is currently unclear and requires further investigation. Is NLRP6 expression induced by NFkB in response to a motif that is common on pathogenic gut bacteria? Analysis of the activation of different inflammasome pathways in response to altered microbiota will help to answer these questions (8).
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- Elinav, Eran, Till Strowig, Jorge Henao-Mejia, and Richard A Flavell, (2011) Regulation of the Antimicrobial Response by NLR Proteins. Immunity 34, 665-679.
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- Elinav, Eran, et al., (2011) NLRP6 Inflammasome Regulates Colonic Microbial Ecology and Risk for Colitis. Cell 145, 745-757.
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- van Ooij, Christiaan, (2011) Immunology: NLRP6 keeps the bad bacteria at bay. Nat. Rev. Micro. advance online publication