Published online before print November 2, 2007
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Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA
1Correspondence: Dept. of Immunology and Infectious Diseases, Harvard School of Public Health, 651 Huntington Ave., Boston, MA 02115, USA. E-mail: fmartino{at}hsph.harvard.edu
ABSTRACT
The innate immune system in animals has been forged to detect microbes, coordinate symbiotic responses, and mount immune defenses against pathogens. Recently, innate immunity was shown to detect signals released by damaged cells or tissues such as uric acid or ATP. These danger signals were proposed to be important in promoting and regulating inflammation upon trauma or pathogen insults. The physiological relevance of these signals in the immune response and their mechanisms of action are still unclear. Recent findings suggest that some danger signals activate the NALP3 inflammasome, an innate immune complex that controls inflammatory caspases and IL-1 activation.
Key Words: inflammasome • autoinflammation • uric acid • gout
INTRODUCTION
Innate immunity constitutes the first line of defense that detects pathogens and orchestrates the immune response. For almost two decades, Charles Janeway and his colleagues have suggested that the innate immune system recognizes signatures of pathogens collectively called pathogen-associated molecular patterns (PAMPs). These PAMPs were shown to activate specific receptors referred to as pathogen recognition receptors (PRRs) [1
]. Three major families of PRRs have been identified: the Toll-like receptors (TLRs), the RIG-I-like receptors (RLRs), and the Nod-like receptors (NLRs) [2
]. TLRs are the best-characterized PRRs. TLRs are transmembrane proteins that recognize bacteria, viruses, fungi, and protozoa and may also detect some endogenous danger signals [3
] (Table 1
). RLRs include the cytosolic helicases RIG-I and MDA5, which sense viruses. NLRs, previously also called caterpillars, form the largest family of intracellular PRRs, with more than 20 members in humans [4
]. To date, NLRs have been shown to recognize bacteria, and several of them recognize danger signals. The biology of most mammalian NLRs is still ill-defined, yet some NLRs such as NOD2 and NALP3 are better characterized. NOD2 senses bacterial wall products and activates the transcription factor NF-
B, whereas NALP3 forms a caspase-1 activating molecular complex termed the inflammasome. Caspase-1 activation by the inflammasome promotes cytokine maturation, as in IL-1β [5
, 6
]. IL-1β, also known as the endogenous pyrogen, is a key player in the processes of inflammation and fever [7
]. Multiple stimuli, including pathogen recognition, activate NALP3 to trigger inflammation that is mainly mediated by IL-1β [8
, 9
]. Gain of function mutations in the NALP3 gene or deregulation in the inflammasome cause periodic fever syndromes in humans such as Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS), and chronic infantile neurological cutaneous, and articular (CINCA) syndrome, also known as neonatal-onset multisystem inflammatory disease (NOMID) [10
, 11
]. These hereditary autoinflammatory syndromes are characterized by an aberrant IL-1β overproduction that initiates periodic episodes of systemic inflammation in these patients [12
, 13
]. The fundamental role of IL-1β in this pathology was confirmed by clinical trials and case studies that demonstrated the efficacy of therapeutic strategies targeting the IL-1β cytokine [14
]. A decisive role for NALP3 in various pathologies related to inflammation could also be demonstrated in mice [15
, 16
]. Moreover, recent findings suggest that the NALP3 inflammasome detects endogenous danger signals such as uric acid and ATP released by damaged cells, suggesting that such signals may contribute to the pathology of autoimmune diseases and other inflammatory conditions.
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Table 1. Examples of Danger Signals
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THE CONCEPT OF IMMUNE DANGER IN MAMMALS
Historically, immunologists predicted that a major function of the immune system was to differentiate self from nonself and to respond to self with tolerance and to mount an immune response against nonself. Innate immunity, for example, detects various microbial (nonself) molecules via the TLRs, providing the costimulation that is required to mount an immune response, a phenomenon referred to adjuvanticity. More recently, Polly Matzinger and colleagues [17 , 18 ], in order to account for some unsolved questions of the self from nonself model, put forward an alternative hypothesis, in which the immune system responds with tolerance to most antigens, and what triggers an immune response is the presentation of an antigen in the context of a danger signal rather than the foreignness of the antigen. Despite the fact that these models were occasionally presented as different and opposing views of how the immune system works, it is becoming more and more clear that both the self from nonself model and the danger model are important pathways of our immunity and that the combination of the two types of signals determines the quality and extent of the activation of the innate immune system.
NLRS AND DANGER: LESSONS FROM THE PLANT IMMUNE SYSTEM
Although the danger hypothesis was first proposed in mammals, evidence for such mechanisms in innate immunity was initially better documented in plants. Plant immunity relies exclusively on innate immunity that, like the mammalian innate immune system, was divided into two types. The first, known as PAMP-triggered immunity or nonhost resistance, discriminates self from nonself by recognizing common pathogen-associated molecular patterns (PAMPs) [19 ]. The second type, also called effector-triggered immunity, is more specific and is the result of an exclusive evolutionary adaptation between a precise pathogen and its host. This effector-triggered immunity mainly detects pathogen-driven modifications, stress, or danger signals in the host-infected cell. This type of immune surveillance resembles the danger model of immunity proposed by Matzinger [20 ]. Interestingly, PAMP-sensors in plants use transmembrane pathogen recognition receptors that resemble mammalian TLRs [21 ], whereas danger signal sensors in plants are mainly formed by a large family of hundreds of NLR-like proteins [22 ], further suggesting that convergent evolution has resulted in analogous mechanisms in both plant and mammalian innate immunity and highlighting the possible role of NLRs as danger signal sensors. In line with this hypothesis, it is worth noting that plant NLRs and mammalian NLRs seem to be regulated in a similar fashion. Both plant NLR genes involved in the effector-triggered immunity and mammalian NLRs have the same modular organization [23 ]. Furthermore, various mammalian NLRs bind SGT1 and HSP90 [24 , 25 ], two proteins whose plant orthologs were previously shown to interact with plant NLRs [26 ]. The combined action of HSP90 and SGT1 is required to modulate plant NLR accumulation and signaling competence. Likewise, in mammals, the activity of SGT1 is essential for NALP3 and NOD1 activation [24 , 25 ]. SGT1 depletion affects HSP90 binding to NALP3 and low-dose incubations with HSP90 inhibitors reduce SGT1 interaction with NALP3 and block its activation. This suggests that an HSP90-SGT1 complex keeps the inflammasome inactive but competent for activation. These findings further highlight some similarities between the mammalian danger signal sensor NALP3 and danger signal sensors in plants.
URIC ACID IS A DANGER SIGNAL DETECTED BY NALP3
Various observations over the years by several groups have suggested that cellular debris or components from the cytoplasm can act as danger signals that activate the immune system [27 28 29 ]. Some examples of such identified molecules and signals are listed in Table 1 . In a seminal paper, the laboratory of Kenneth Rock purified ultraviolet irradiated BALB/c 3T3 cells to get a low molecular fraction that could activate the immune system in an in vivo model of adjuvanticity [30 ]. Biochemical characterization of that fraction revealed that the nature of that danger signal was uric acid. In their study, the authors proposed that extracellular uric acid could nucleate and form monosodium urate (MSU) crystals, a well-known inflammatory mediator involved in the pathology of gout.
Gout is an autoinflammatory disease that is characterized by arthropathies generated by the inflammatory reaction to MSU microcrystals in the joints and periarticular tissues [31 , 32 ]. MSU stimulates the caspase-1-activating NALP3 inflammasome to produce active IL-1β [31 , 32 ]. Macrophages from mice deficient in components of the inflammasome, such as caspase-1 and NALP3, have a reduced crystal-induced IL-1β activation. Moreover, in a model of MSU crystal-induced peritonitis in mice, impaired inflammation is found in inflammasome-deficient mice or mice deficient in the IL-1 receptor (IL-1R), suggesting that in gout as well as in the above-mentioned hereditary autoinflammatory diseases caused by mutation in NALP3, the inflammatory cascade is initiated by overproduction of IL-1β [24 , 33 , 34 ]. The importance of IL-1 in the pathology of gout is also suggested by promising preliminary studies in humans. Two pilot open-labeled studies using inhibitors of IL-1β to treat a small number of patients with documented acute gouty attacks that could not tolerate or had failed standard anti-inflammatory therapies revealed a very rapid and efficient response in those patients to IL-1 blockade, suggesting that targeting IL-1 or the inflammasome could be an effective therapeutic alternative in gout [35 , 36 ].
Although the role of uric acid and MSU in the pathology of gout is well defined, the in vivo contribution of uric acid as a danger signal in an immune reaction remains to be fully addressed. A follow-up study by Rock and colleagues showed that elimination of uric acid reduced the generation of cytotoxic T cells to an antigen in transplanted syngeneic cells and the proliferation of autoreactive T cells in a transgenic diabetes model, supporting the idea that uric acid maybe important in vivo [37 ]. The role of the inflammasome in these models remains to be addressed.
DETECTING EXTRACELLULAR ATP BY NALP3
Extracellular ATP is a well-characterized danger signal that activates NALP3 and caspase-1 [38 ]. ATP is most likely released from cells as a consequence of cell damage or nonapoptotic cell death. Other cellular stresses may release ATP by regulated nonlytic mechanisms in endothelial, as well as epithelial cells, for example. In these cells, ATP release is triggered by mechanical stimuli as diverse as fluid shear stress [39 , 40 ], compression [41 ], hydrostatic pressure changes [42 ], hypotonic shock [43 ], and stretch [44 ]. It is also worth noting that certain secretory organelles store large amounts of ATP that are possibly released and may act as danger signals [45 46 47 48 ]. Interestingly, adrenal medullary chromaffin granules, which may be released upon physical or psychological stress, have concentrations of ATP around 100 mM, whereas platelet-dense granules contain concentrations of ATP that can reach 500 mM, which is 100 times more than the cytosolic concentration.
Exposure of cells to extracellular ATP has been known for years to activate caspase-1 [49 ], and several studies have demonstrated the requirement of P2X7 receptors for ATP-induced caspase-1 activation and subsequent IL-1β maturation [50 ]. More recently, another type of channel, the pannexin-1 channel, activated by P2X7 activation, was shown to be required for ATP-induced caspase-1 activation [51 52 53 ]. ASC is an adaptor protein that is essential for the recruitment of caspase-1 to the NALP-based inflammasome. The generation of ASC-deficient mice demonstrated that ATP-mediated caspase-1 activation requires ASC and was therefore probably dependent on the activation of a NALP protein [54 ]. This assumption was confirmed in studies using NALP3-deficient mice [33 , 55 , 56 ]. These findings clearly show that extracellular ATP can act as a danger signal to activate a NALP3 inflammasome and promote caspase-1 activation and IL-1β maturation. Yet the physiological relevance of extracellular ATP-mediated NALP3 inflammasome activation remains to be addressed in vivo, as the concentration of ATP (2 to 5 mM) required in vitro seems relatively high, taking in account that in vivo, most of the extracellular ATP may be rapidly hydrolyzed by ectonucleotidases [57 ]. Identifying pathologies and immune responses that may depend on extracellular ATP activation of the inflammasome would therefore be a very important step toward the understanding of the physiological function of this danger signal.
DANGER SIGNALS TRIGGERED BY UVB AND SKIN IRRITANTS
The skin is the body’s first line of defense against external insult and serves as an effective barrier against ordinary environmental intrusions. As such, the skin is often damaged by various insults. Repeated exposure of the skin to irritant allergens, for example, induces a T cell-mediated immune response called contact hypersensitivity [58 ]. This response is divided into two phases. First, there is a sensitization phase that requires antigen uptake by skin-resident antigen presenting cells and their migration to draining lymph nodes where T cell priming occurs. Second, an elicitation phase occurs upon challenge with a corresponding hapten for the primed T cells. Interestingly, the sensitization phase requires activation of the immune system by the irritant effect. This innate immunity-related priming of the antigen depends on the presence of functional caspase-1, IL-1β, and IL-18 [59 , 60 ], suggesting potential involvement of the inflammasome. Indeed, the role of the inflammasome was confirmed in ASC- and NALP3-deficient mice that showed an impaired contact hypersensitivity response to the irritants, trinitrophenylchloride (TNP-Cl) [56 ], 2,4, 6-trinitrochlorobenzene (TNCB) and 2,4-dinitrofluorobenzene (DNFB) [61 ]. In these mice, transfer of primed T cells results in a normal contact hypersensitivity, suggesting that only the sensitization phase requires NALP3 and ASC. Interestingly, DNFB promotes the release of IL-1β in a caspase-1-dependent manner in primary keratinocytes, as well as in a dendritic cell line, suggesting that the inflammasome may either detect such compounds directly or, more likely, may detect some danger signals released or produced by these irritants [61 , 62 ]. Altogether, these findings suggest that the inflammasome can bridge danger signals triggered by the irritant effect of sensitizing chemicals with the activation of IL-1β and IL-18, thus promoting an efficient activation of the adaptive immune system.
Another skin insult, ultraviolet irradiation, was recently shown to activate the NALP3 inflammasome and promote IL-1β maturation in keratinocytes, suggesting that multiple danger signals in the skin may use the inflammasome to activate immunity, inflammation, and repair mechanisms [63 ].
CONCLUSIONS
The molecular nature of danger signals and the mechanisms underlining the activation of innate immune sensors by such signals is emerging [64 ]. As illustrated above, NALP3 and the inflammasome are probably important pathways, among others, possibly involved in the orchestration of both pathogen and danger immune recognition (Fig. 1 ). This is an emerging field of research with a plethora of open questions. For instance, the mechanism of NALP3 activation by danger signals is unknown. It has been suggested, for example, that disruption of the ionic environment by NALP3 activators may initiate a cellular alarm signal involved in the activation of the inflammasome [65 ]. Another important issue that remains open is the identification of additional danger signals and the possibility that other PRRs, such as other NALPs or other NLRs, may detect these signals. Finally, identifying the role and contribution of each of these danger signals in immune responses or inflammatory conditions in vivo is an important challenge that will indubitably shed more light on our understanding of pathologies such as trauma, sepsis, inflammation, and shock.
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Figure 1. Model of danger signals activation of the NALP3 inflammasome. Tissue injury leads to the formation and/or release of danger signals such as ATP or uric acid crystals that are recognized by the innate immune system. A number of these signals mediate a potassium efflux or other secondary intracellular danger signals that are required for inflammasome activation. The inflammasome preactivation complex includes NALP3, SGT1, and HSP90. Upon activation SGT1 and HSP90 are released, NALP3 oligomerizes to recruit the adaptor ASC and caspase-1. Activation of caspase-1 results in the processing and maturation of proIL-1β into its biologically active form, IL-1β. IL-1β will then trigger the IL-1R complex, leading to the activation of multiple cytokines involved in the inflammation cascade, including IL-8, TNF, or IL-17.
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ACKNOWLEDGEMENTS
I thank the organizers of the TSIS2007 meeting and all of the participants of the symposium on danger signals and NLRs for terrific discussions and inspiration. I would like to thank Jürg Tschopp and the members of his laboratory for discussions. F. M. is supported by a long-term fellowship from the Human Frontier Sciences Program.
Received June 8, 2007; revised September 13, 2007; accepted October 4, 2007.
REFERENCES
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