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Keywords:

  • Danger signals;
  • Inflammasome;
  • Inflammation;
  • Oxidative stress

Abstract

  1. Top of page
  2. Abstract
  3. Redox signaling and oxidative stress
  4. ROS is required for NLRP3/NALP3 Inflammasome activation
  5. How do NLRP3/NALP3 activators promote ROS generation?
  6. How does ROS trigger NLRP3/NALP3 activation?
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

Inflammasomes are innate immune signaling pathways that sense pathogens and injury to direct the proteolytic maturation of inflammatory cytokines such as IL-1β and IL-18. Among inflammasomes, the NLRP3/NALP3 inflammasome is the most studied. However, little is known on the molecular mechanisms that mediate its assembly and activation. Recent findings suggest that ROS are produced by NLRP3/NALP3 activators and are essential secondary messengers signaling NLRP3/NALP3 inflammasome activation.


Redox signaling and oxidative stress

  1. Top of page
  2. Abstract
  3. Redox signaling and oxidative stress
  4. ROS is required for NLRP3/NALP3 Inflammasome activation
  5. How do NLRP3/NALP3 activators promote ROS generation?
  6. How does ROS trigger NLRP3/NALP3 activation?
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

ROS are free radicals that contain the oxygen atom and include hydrogen peroxide (H2O2), superoxide anion (equation image) and hydroxyl radical (OHċ). These molecules are highly reactive (oxidizing/electron-capturing) due to the presence of unpaired valence shell electron. ROS mainly originate as a byproduct of oxygen metabolism in the electron transport chain within the mitochondria (Fig. 1). ROS are also generated by the activity of cellular enzymes such as NADPH oxidases, xanthine oxidoreductases, lipoxygenases and cyclooxygenases 1. Cellular production of ROS regulates several important physiological responses, such as oxygen sensing, angiogenesis, control of vascular tone, and regulation of cell growth, differentiation and migration. While ROS is also important for cell signaling (a phenomenon known as redox signaling), sustained ROS production can cause cellular damage. To cope with this stress, several enzymes displaying anti-oxidant activities, including thioredoxin (TRX), superoxide dismutases, glutathione peroxidases and catalase, are involved in neutralizing ROS. The imbalance between the formation of ROS and the ability to detoxify these oxidizing radicals can produce a cellular state known as oxidative stress 2. ROS-mediated oxidative stress plays an important role in pathological processes such as aging, hypertension, atherosclerosis, cancer, ischemia, neurodegenerative diseases and diabetes 1.

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Figure 1. Examples of ROS generating pathways. (A) During respiration 1–2% of the oxygen is partially reduced to equation image, which can be converted H2O2 and OHċ. The major sites of for production in the mitochondrial respiratory chain are at complexes I and III. Complex I accepts electrons from NADH; these electrons move down an electrochemical gradient through ubiquinone (Q cycle) to complex III, from complex III to cytochrome c (C) and from C to complex IV that use the electrons to reduce molecular oxygen to water. The mechanisms involved in generation of equation image by complex I are poorly understood. Complex III generate equation image by auto-oxidation of ubisemiquinone generated during the Q cycle, (IM, inner mitochondrial membrane). (B) NADPH oxidases such as the NOX2 complex transport electrons across biological membranes to reduce oxygen to superoxide. The activation of NOX2 occurs through a complex series of protein/protein interactions. Phosphorylation of p47phox leads to a conformational change allowing its interaction with p22phox. The localization of p47phox to the membrane brings p67phox into contact with NOX2 and also brings the small subunit p40phox to the complex. Finally, the GTPase Rac interacts with NOX2. Once assembled, the complex is active and generates superoxide by transferring an electron from NADPH in the cytosol to oxygen on the luminal or extracellular space.

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Production of reactive ROS is crucial to the regulation of innate immune responses. In plants for example, pathogen recognition generates ROS in an NADPH oxidase-dependent manner to cause the oxidative burst leading to the hypersensitive response 3. Similarly, during inflammation and immune responses in vertebrates, activated phagocytic cells such as neutrophils generate a ROS-dependent respiratory burst that directs toxicity towards invading microbes 4. ROS is also involved in signaling injury to the immune system. Beyond its antiseptic function, release of ROS (H2O2) by damaged tissues can form a decreasing concentration gradient that directs leukocytes recruitment at the site of tissue injury, demonstrating that ROS can orchestrate inflammatory responses in tissues 5. Redox signaling is also important in the signaling pathways engaged by various inflammatory conditions. ROS production by the PRR, TLR, regulates activation of redox-regulated transcription factors (NF-κB and AP-1) and cytokines production 6, 7. Recently, ROS has been proposed to play an important role in the activation of the NLRP3/NALP3 inflammasome 8, 9.

ROS is required for NLRP3/NALP3 Inflammasome activation

  1. Top of page
  2. Abstract
  3. Redox signaling and oxidative stress
  4. ROS is required for NLRP3/NALP3 Inflammasome activation
  5. How do NLRP3/NALP3 activators promote ROS generation?
  6. How does ROS trigger NLRP3/NALP3 activation?
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

The inflammasome is a cytosolic molecular complex that once activated has an enzymatic activity mediated by the recruitment and activation of caspase-1. The inflammasome senses pathogens and stress or danger signals to promote the maturation of cytokines such as IL-1β. The release of active IL-1β engages IL-1 receptor-harboring cells and promotes inflammatory responses 10. Although the activation of caspase-1 and the maturation of IL-1β is common to virtually all kinds of inflammasomes, the scaffolding unit involved in sensing pathogens or danger signals may vary 9. The NLR protein NLRP3/NALP3 forms the most studied inflammasome. Upon activation, NLRP3/NALP3 recruits the adaptor ASC and the enzyme caspase-1 to form the NLRP3/NALP3 inflammasome. No NLRP3/NALP3 activators have been shown to directly interact and activate NLRP3/NALP3, suggesting that NLRP3/NALP3 may sense these signals indirectly. Interestingly, most identified NLRP3/NALP3 activators also trigger ROS production. Moreover, the use of antioxidants has been shown to inhibit NLRP3/NALP3 inflammasome activation, suggesting that redox signaling or oxidative stress is involved in NLRP3/NALP3 activation.

Extracellular ATP is an inflammatory signal that has been implicated in innate immunity in both plants and animals 11. In mammals, extracellular ATP binds to P2X7 receptors and activates the NLRP3/NALP3 inflammasome 12. Treatment of macrophages with ATP results in the rapid production of ROS and the use of the broad spectrum NADPH oxidase inhibitor, diphenyleneiodonium (DPI) inhibits ATP-mediated caspase-1 activation 13, 14. The NLRP3/NALP3 activating particulate elements uric acid crystals, alum and particulate metals have been shown to induce ROS production 15–18. Similarly, ROS is detectable quickly upon exposure of macrophages to silica or asbestos 16, 19–21 Other NLRP3/NALP3 activators, such as the toxin nigericin, UV light and skin sensitizers (e.g. dinitrochlorobenzene) activate a cellular redox imbalance required for inflammasome formation 14, 22, 23. ROS has also been implicated in NLRP3/NALP3 activation by the malaria pathogenic crystal, hemozoin, the influenza virus and the yeast Candida albicans24–26.

How do NLRP3/NALP3 activators promote ROS generation?

  1. Top of page
  2. Abstract
  3. Redox signaling and oxidative stress
  4. ROS is required for NLRP3/NALP3 Inflammasome activation
  5. How do NLRP3/NALP3 activators promote ROS generation?
  6. How does ROS trigger NLRP3/NALP3 activation?
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

Various pathways have been proposed to mediate ROS production by NLRP3/NALP3 activators; however, the general picture of how NLRP3/NALP3 activators trigger ROS is still unclear. Potassium efflux and decrease in cytosolic potassium concentration are the most striking features associated with NLRP3/NALP3 activators 27. Interestingly, potassium efflux has been linked to ROS production at the membrane in plants 28. Moreover, potassium efflux has been shown to trigger ROS production in human granulocytes 29. It is therefore tempting to speculate that potassium efflux by NLRP3/NALP3 activators could be involved in ROS generation.

Some NLRP3/NALP3 activators such as uric acid crystals, alum, asbestos and silica are large particulate elements that can induce the so-called frustrated phagocytosis at the cell surface. Frustrated phagocytosis has been associated with ROS production; however, the mechanism by which this occurs is unclear 30, 31. Frustrated phagocytosis may not be the only mechanism used by macrophages to sense pathogenic particles. Evidence has demonstrated that uric acid crystals can be phagocytosed. Ultrastructure studies of uric acid crystal-containing phagolysosomes show a disrupted membrane and possibly release of part of their content in the cytoplasm 32. In line with these early observations, silica crystals and alum trigger damage and rupture of the lysosome 33, as described by the companion Viewpoint article 34. Importantly, the release of cathepsin B by damaged lysosomes has been proposed to mediate inflammasome activation 33. It is unclear whether this mechanism works in parallel to the ionic imbalance and oxidative stress pathway. It is also possible that lysosomal damage and cathepsin B release act upstream of ROS production. In line with this model, cathepsin B has been shown to promote ROS production in hepatocytes and neurones 35, 36.

Multiple lines of evidence suggest that ROS poduction by NLRP3/NALP3 activators involves NADPH-oxidases (NOX). NOX are a family of transmembrane enzymes that generate ROS by carrying electrons across biological membranes from a cytosolic electron donor (such as NADPH) to an electron acceptor (oxygen) in the extracellular or luminal space 37. The observation that NOX inhibitors such as DPI or (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate inhibits inflammasome activation by virtually all NLRP3/NALP3 activators identified so far suggests that NOX are involved in ROS production. This has also been demonstrated in vivo. Indeed, DPI inhibits caspase-1 mediated IL-18 activation in mice undergoing physical stress 38.

NOX inhibitors may have additional targets. DPI can exert inhibitory effect on mitochondrial ROS production in addition to NOX 39. However, in line with the possibility that NOX are involved in inflammasome activation, extracellular ATP has been shown to trigger translocation of cytosolic NOX components (p47phox, p40phox, p67phox and p21rac) onto membrane-bound NOX2, forming an active macromolecular complex 14. Indeed, NOX2 deficiency impairs ATP-mediated ROS production by macrophages, suggesting that NOX2 may be involved in ATP-mediated NLRP3/NALP3 activation 40. On the contrary, NOX2-deficient macrophages have no defect in inflammasome activation upon stimulation with other NLRP3/NALP3 agonists including uric acid crystals and silica 33 while knockdown of p22phox, in the monocytic cell line THP1 impairs inflammasome activation by hemozoin, silica uric acid crystal and asbestos 16, 41. Because p22phox deficiency affects several NOX including NOX1, NOX2, NOX3 and NOX4 37, it is possible that multiple NOX mediate ROS production to trigger NLRP3/NALP3 inflammasome assembly.

Overall, many studies using anti-oxidants support a model in which ROS production by NLRP3/NALP3 agonists drive inflammasome assembly. However, the mechanisms of production and the nature of ROS involved in inflammasome activation are unknown. Future work should focus on characterizing how frustrated phagocytosis, cathepsin B, potassium efflux and NOX may synergize and contribute to ROS production and inflammasome activation.

How does ROS trigger NLRP3/NALP3 activation?

  1. Top of page
  2. Abstract
  3. Redox signaling and oxidative stress
  4. ROS is required for NLRP3/NALP3 Inflammasome activation
  5. How do NLRP3/NALP3 activators promote ROS generation?
  6. How does ROS trigger NLRP3/NALP3 activation?
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

ROS production by H2O2 activates the inflammasome 16, 42, furthermore, knockdown of TRX, a cellular anti-oxidant protein, enhances IL-1β activation by silica, uric acid crystal and asbestos 41. These findings suggest that oxidative stress could be sufficient to trigger NLRP3/NALP3 activation and lead to interrogate how NLRP3/NALP3 senses ROS. ROS may either directly trigger inflammasome assembly or be indirectly sensed through cytoplasmic proteins that modulate inflammasome activity. ATP-mediated ROS production has been shown to stimulate the PI3K pathway, and pharmacological inhibition of PI3K inhibits ATP-mediated caspase-1 activation suggesting that PI3K may be involved in inflammasome activation downstream of ROS 13.

Recently Jürg Tschopp's laboratory identified TXNIP/VDUP1 as an essential protein that may directly activate NLRP3/NALP3 upon oxidative stress 42. The authors of this study suggest that, in resting cells, TXNIP/VDUP1 interacts with TRX and is therefore unable to activate NLRP3/NALP3. Upon oxidative stress TXNIP is released from oxidized TRX and in turn directly binds the leucine-rich region of NLRP3/NALP3 leading to inflammasome assembly 42. Consistent with this finding, TXNIP/VDUP1-deficient macrophages treated with extracellular ATP or uric acid crystals have decreased caspase-1 and IL-1β processing 42. This finding provides support for a model in which TXNIP/VDUP1 and NLRP3/NALP3 set up a surveillance of cellular stress, preparing to drive inflammation in case of excessive stress or danger signals.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Redox signaling and oxidative stress
  4. ROS is required for NLRP3/NALP3 Inflammasome activation
  5. How do NLRP3/NALP3 activators promote ROS generation?
  6. How does ROS trigger NLRP3/NALP3 activation?
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

In contradiction with classical PRR, rather than directly recognizing pathogenic elements, NLRP3/NALP3 seems to detect oxidative stress produced by pathogenic insults 43 (Fig. 2). This model shares similarities with the guard model in plants. Pathogen-mediated changes in plant cellular physiology trigger activation of the R genes, a family of innate immune sensors that cope with infections and share structural similarities with NLRP3/NALP3 44. Although the mechanisms involved in inflammasome activation by oxidative stress are still unclear, ROS is emerging as the central and common element regulating NLRP3/NALP3 activation. A fine-tuning of the events underlining inflammasome activation and inflammation responses by oxidative stress is likely key to proper immunity and tissue repair. In line with the role of ROS in activating NLRP3/NALP3, inhibition of ROS production in M2 polarized macrophages dampens inflammasome activation 45. On the other hand, prolonged oxidative stress can dampen inflammatory mediators 12 including inhibition of caspase-1 by reversible oxidation and glutathionylation of redox-sensitive cysteine residues 46, suggesting that beyond its role in activating NLRP3/NALP3, oxidative stress may be part of a regulatory loop negatively regulating IL-1β activation.

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Figure 2. Model of NLRP3/NALP3 inflammasome assembly and activation. Multiple NLRP3/NALP3 inflammasome activators such as extracellular ATP and particulate elements trigger ROS production. Possible pathways involved in ROS production include potassium efflux, frustrated phagocytosis, phagolysosomes disruption, Cathepsin B release and NOX activation. Oxidative stress triggers inflammasome-activating signals such as PI3K and TXNIP release from oxidized TRX. Binding of TXNIP to NLRP3/NALP3 promotes assembly and oligomerization of the inflammasome. The recruitment of the adaptor ASC and the enzyme caspase-1 to the inflammasome are crucial for its proIL-1β cleaving activity.

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Oxidative stress and tissue injury are major hallmarks of numerous pathologies ranging from diabetes to neurodegenerative disorders. Most of theses pathologies have an inflammatory component 47. Dissecting the possible involvement of the inflammasome in such pathologies and identifying how oxidative stress regulates NLRP3/NALP3 activation and IL-1β activity will likely shed some new light on these pathologies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Redox signaling and oxidative stress
  4. ROS is required for NLRP3/NALP3 Inflammasome activation
  5. How do NLRP3/NALP3 activators promote ROS generation?
  6. How does ROS trigger NLRP3/NALP3 activation?
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

The author is supported by a Human Frontier Science Program long-term fellowship.

Conflict of interest: The author declares no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Redox signaling and oxidative stress
  4. ROS is required for NLRP3/NALP3 Inflammasome activation
  5. How do NLRP3/NALP3 activators promote ROS generation?
  6. How does ROS trigger NLRP3/NALP3 activation?
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Redox signaling and oxidative stress
  4. ROS is required for NLRP3/NALP3 Inflammasome activation
  5. How do NLRP3/NALP3 activators promote ROS generation?
  6. How does ROS trigger NLRP3/NALP3 activation?
  7. Concluding remarks
  8. Acknowledgements
  9. References
  10. Supporting Information

See accompanying Viewpoint: http://dx.doi.org/10.1002/eji.200940185

Please note: Wiley Blackwell 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.