NLR‐containing inflammasomes: Central mediators of host defense and inflammation

Since its discovery of 25 years ago, the basic mechanisms through which IL-1 drives inflammation and contributes to the pathogenesis of inflammatory diseases have been well documented (reviewed in 1). The underlying mechanisms responsible for IL-1 production, however, have only been uncovered relatively recently. A major breakthrough in this regard was the purification in 2002 by Tschopp and colleagues of the inflammasome 2, a large multiprotein complex that assembles in cells and leads to the activation of the proteolytic enzyme caspase-1. To date, four inflammasome complexes have been partially characterized, containing NLRP1 3, NLRP3 4, IPAF/NLRC4 5 or AIM2 6-9 (Fig. 1). The NLR proteins (officially called the nucleotide-binding domain and leucine-rich repeat (LRR)-containing receptors, although almost exclusively called the NOD (nucleotide-binding oligomerization domain)-like receptors by the research community) are a family of intracellular immune receptors with more than 20 members currently known in humans. The largest group of these are characterized by the presence of an amino terminal pyrin domain (PYD), caspase activation and recruitment domain (CARD) or baculovirus inhibitory repeat domain followed by a nucleotide-binding domain, and LRRs at the C- terminus. Architecture of characterized inflammasomes. The NLRP1 inflammasome contains an N-terminal PYD, which interacts with the adapter molecule ASC, an ATP-binding NACHT domain, an LRR domain and a C-terminal CARD that interacts with caspase-5. The adapter molecule ASC binds to caspase-1. To date, muramyl dipeptide and lethal factor of Bacillus anthracis are the only characterized ligands of the NRLP1 inflammasome. NLRP3 contains an N-terminal PYD, which interacts with ASC. NLRP3's NACHT domain can bind to ATP and the LRR bind to a yet to be defined ligand. NLRC4 (IPAF) can directly associate with caspase-1 via its N-terminal CARD. NAIP5 can also interact with NLRC4 and bacterial flagellin can activate the NLRC4 inflammasome. The AIM2 inflammasome directly binds dsDNA via the HIN200 domain and ASC via the PYD. Upon activation, the NLR are thought to oligomerize via homotypic interactions between NACHT domains thereby clustering PYD domains, which recruit an adapter molecule called apoptosis-associated speck-like protein containing a CARD (ASC). ASC assembly facilitates the recruitment of procaspase-1. Procaspase-1 clustering, in turn, leads to autocleavage and generation of active caspase-1, which cleaves and activates the pro-inflammatory cytokines IL-1β and IL-18. In this Viewpoint series, leading experts studying NLR have been commissioned by the editorial team at the European Journal of Immunology to describe recent developments related to these important host sensors. A special Viewpoint from Charles Dinarello recounts the critical discoveries in the field of IL-1 biology and NLR inflammasomes, celebrating the 25th anniversary of the cloning of IL-1 10. The following 11 Viewpoint articles detail the discovery and characterization of inflammasome complexes, describing their role in host defense and sterile inflammation and address the role of the NLR in adjuvanticity, auto-inflammatory diseases, as well as the pathogenesis of other inflammasome-mediated diseases. These Viewpoints highlight the impact of the NLR on our understanding of the basic mechanisms of host defense and reveal how the progress made over the past decade has reshaped our understanding of inflammatory disease pathogenesis. The pro-inflammatory cytokine IL-1β is among the arsenal of defense measures deployed by the innate immune system to combat invading microbes. Its pro-inflammatory activity is regulated at the level of expression, processing and secretion 11, although additional control occurs via antagonism by IL-1 receptor antagonist (IL-1RA). Several classes of innate immune receptors including the TLR and the C-type lectin Dectin-1, induce pro-IL-1 expression 12, 13; however, caspase-1 activation is only mediated by the NLR or AIM2 inflammasomes. In the Viewpoint by Nunez and colleagues, the role of the NLR in regulating caspase-1 activity and IL-1β production during microbial infection is described 14. NLRP1 has been shown to form an inflammasome in response to muramyl dipeptide, as well as to anthrax lethal toxin in mice. NLRP3 inflammasomes form in cells infected with bacteria (Staphlococcus aureus, Listeria monocytogenes, Escherichia coli, Mycobacterium marinarum and Neisseria gonnorrhoeae), viruses (Sendai, influenza and adenovirus) and fungi (Candida albicans), as well as in response to products from these and other pathogens. IPAF senses flagellin and was considered a devoted flagellin sensor; however, a recent study suggests that IPAF can mediate caspase-1 activation independent of flagellin 15. The role of the NLR inflammasomes is not confined to sensing microbial infection; Cassel and Sutterwala describe how NLRP3 responds to endogenous molecules released from damaged or dying cells 16. These include nucleic acids, ATP and uric acid crystals. The ability of NLRP3 to sense these endogenous molecules has revealed an important role for NLRP3 and IL-1 in a variety of metabolic disorders including gout. An important open question, which currently drives inflammasome research and arises from the identification of the diverse activators of NLR, is to determine how inflammasome complexes are activated. While a direct ligand–receptor interaction has been shown for activation of the AIM2 inflammasome by cytosolic dsDNA 6, 7, 9, such a mechanism is unlikely for NLRP3 in light of the diverse physico-chemical nature of its activating stimuli. There are divergent opinions regarding this matter and a number of models have been proposed. Two Viewpoints address two of these models. In the first, Martinon describes the importance of ROS in mediating the upstream events for NLRP3 activation 17. The link between ROS and NLRP3 activation appears to involve thioredoxin-interacting protein (TXNIP) 18, which may act as an upstream activating ligand for NLRP3. The importance of ROS is questioned in the Viewpoints by Dinarello 10 and Hornung and Latz 19. Indeed an alternative model is put forward in the latter Viewpoint, instead of functioning as a sensor for ROS, NLRP3 activation is proposed to sense perturbations in membrane integrity and thus senses membrane disruption 19. In this model, membrane disruption drives NLRP3 activation in response to particulate or crystalline activators such as silica. Inefficient digestion of phagocytosed material leads to phagosomal destabilization and rupture 20, 21. Lysosomes contain a plethora of proteolytic enzymes, many of which are activated by acidification of lysosomal pathways. The lysosomal proteinase cathepsin B is released into the cytosolic compartment and data from Latz and colleagues suggest that cathepsin B activity is important to trigger NLRP3 activation 20, 21. Whether cathepsin B acts directly or indirectly at the level of NLRP3 is unclear at present. It is possible, of course, that the ROS and cathepsin B models are not mutually exclusive and direct or indirect crosstalk may occur. A focus on the IL-1 pathway as the signature response activated by the NLR proteins has led to important insights in defense and disease; however IL-1 and IL-18 production are not the only responses triggered by these proteins. Another response induced is an inflammatory form of cell death called pyroptosis. Pyroptosis differs from apoptosis in that it is an inflammatory process stimulated by a wide range of microbes and pathological events (including stroke, cardiovascular disease and cancer). Kroemer and colleagues outline the mechanisms triggering pyroptosis and discuss the pathophysiological relevance of pyroptosis both to infection and disease pathogenesis 22. Growing evidence also indicates the involvement of other NLR in the immune response. Kanneganti and colleagues discuss NOD1 and NOD2, two well-characterized mediators of anti-microbial defenses, which discriminate between different peptidoclycan structures and activate transcription of immune response genes 23. They also discuss how NLRP12 and NLRX1 have emerged as negative regulators acting on TLR or RNA helicase signaling pathways, respectively. Unlike, NLRX1, NLRP12 has previously been shown to assemble ASC-containing inflammasomes in vitro suggesting that under certain circumstances NLRP12 could form an inflammasome to process IL-1 24. Understanding the role of the remaining uncharacterized NLR is an area ripe for further investigation with much potential for new discoveries. The potent immunomodulatory functions of NLRP3 are further highlighted in the final five Viewpoints, which discuss clinical aspects of NLRP3 function. Several related auto-inflammatory diseases, collectively called cryopyrin-associated periodic syndromes (CAPS), in which activating NLRP3 mutations result in dysregulated IL-1β production and inflammation are described by McDermott and colleagues 25. Therapeutic intervention with anti-IL-1 based therapeutics is showing real clinical promise in some of these conditions. NLRP3 and IL-1 are also being linked to the pathology of more common diseases including gout and cancer. An emerging literature also implicates IL-1β in pancreatic islet failure in Type 2 diabetes mellitus (T2DM) (reviewed in 26). A pathogenic role for TXNIP in T2DM also exists (reviewed in 27). The recent link, therefore, between NLRP3 and TXNIP suggests that therapeutic approaches to target NLRP3 may hold promise for the treatment of T2DM as well as other metabolic diseases such as gout. Gene-targeted mice expressing homologous mutations in NLRP3 associated with CAPS have been generated and have greatly enhanced our understanding of NLRP3-associated auto-inflammation. Meng and Strober detail how mice carrying these hyperactivating NLRP3 mutations have abnormalities similar to those found in human auto-inflammatory syndromes 28. These mice have revealed a clear Th17 cell bias in the auto-inflammation observed. Given the well-established role of IL-17 in inducing neutrophil-dependent inflammation, such a bias may help explain why neutrophils are a dominant component of auto-inflammation in CAPS in humans. Treatment of mice bearing hyperactivating NLRP3 mutations with either IL-1RA or with anti-IL-17A antibody reduced inflammation 29, suggesting that similar combined approaches in humans with IL-1 and IL-17 based therapies may benefit patients with CAPS. Disease-associated single nucleotide polymorphisms (SNP) of clinical significance within the NLR family are not confined to NlRP3. Rodrigue-Gervais and Saleh describe how SNP influence susceptibility to complex diseases such as Crohn's and vitiligo 30. NOD2, in particular, is of immense interest to Crohn's disease where SNP have been strongly linked to the disease. While interfering with NLRP3 activation is clearly beneficial in recurrent and chronic inflammatory diseases, there are situations where enhancing NLRP3 activity may be beneficial. Two Viewpoints outline recent evidence indicating a role for NLRP3 in the adjuvanticity of alum 31, 32. Alum has been approved for use as an adjuvant in the US for a long time, yet its mechanism of action is largely unknown. A major breakthrough in this regard was the recent discovery that alum activates the NLRP3 inflammasome. Five simultaneous reports showed that alum-mediated caspase-1 activation in vitro via NLRP3 21, 33-36. Despite agreement on the involvement of NLRP3 in vitro, how this translates to in vivo responses is controversial. Ricciardi-Castagnoli and colleagues 31 discuss these diverging opinions. Lavelle and colleagues 32 extend this theme to the role of NLRP3 in the adjuvant effect of other particulate adjuvants, discussing the evidence implicating NLRP3-driven IL-1 production in a number of different infection models including influenza, mycobacteria and C. albicans. The importance of NLRP3 in these infections suggests that the use of alum in vaccine formulations designed to eradicate these diseases could prove useful. As should be evident from the views expressed in this series, the discovery of the NLR inflammasomes has had a large impact on our understanding of inflammation in general. The link between the NLR and disease pathogenesis has revealed this family of proteins, particularly NLRP3, to be exciting new targets for therapeutic intervention in the treatment of not only auto-inflammatory but also metabolic diseases and cancer. The clinical benefit already observed with IL-1RA provides proof of principal that interfering with NLR activity itself will be therapeutically useful 37-39. The domain architecture of the NLR, particularly the NACHT domain, suggests that rational design of small molecule inhibitors may be possible. As with any field, the rapidity with which new knowledge is attained has raised many more questions than answers and differences in opinions act to catalyze progress forward. The last 25 years of IL-1 research have been fruitful and ripe with novel discoveries of therapeutic significance, the next 25 will likely be just as exciting with the added benefit that they may provide long awaited relief for patients suffering from debilitating inflammatory diseases. The author acknowledges funding from NIH (AI-067497 and AI083713) and Eicke Latz for Fig. 1. Conflict of interest: The author declares no financial or commercial conflict of interest. See accompanying article: All articles in the Viewpoint series Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.