activation mechanism nrlp3

7/25/2019 Activation Mechanism NRLP3

1/4

Activation mechanisms

Signaling by ROS drives inflammasome activation

Fabio Martinon

Department of Immunology and Infectious Diseases, Harvard School of Public Health,

Boston, MA, USA DOI 10.1002/eji.200940168

Inflammasomes are innate immune

signaling pathways that sense patho-

gens and injury to direct the proteo-

lytic maturation of inflammatory

cytokines such as IL-1b and IL-18.

Among inflammasomes, the NLRP3/

NALP3 inflammasome is the most

studied. However, little is known onthe molecular mechanisms that

mediate its assembly and activation.

Recent findings suggest that ROS are

produced by NLRP3/NALP3 activators

and are essential secondary messen-

gers signaling NLRP3/NALP3 inflam-

masome activation.

Redox signaling and oxidativestress

ROS are free radicals that contain theoxygen atom and include hydrogen

peroxide (H2O2), superoxide anion

(O2) and hydroxyl radical (OH).

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, lipoxygenasesand 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 produc-

tion can cause cellular damage. To cope

with this stress, several enzymes display-

ing anti-oxidant activities, including

thioredoxin (TRX), superoxide dismu-

tases, glutathione peroxidases and cata-

lase, 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, athero-

sclerosis, cancer, ischemia, neurodegen-

erative diseases and diabetes [1].

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]. Simi-

larly, during inflammation and immuneresponses 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 responsesin 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-kB

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/NALP3Inflammasome activation

The inflammasome is a cytosolic mole-

cular complex that once activated has

an enzymatic activity mediated by the

recruitment and activation of caspase-1.

The inflammasome senses pathogensand stress or danger signals to promote

the maturation of cytokines such as

IL-1b. The release of active IL-1b

engages IL-1 receptor-harboring cells

and promotes inflammatory responses

[10]. Although the activation of

caspase-1 and the maturation of IL-1b

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 activa-tion, NLRP3/NALP3 recruits the adap-

tor 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 indir-

ectly. Interestingly, most identified

NLRP3/NALP3 activators also trigger

ROS production. Moreover, the use of

antioxidants has been shown to inhibit

NLRP3/NALP3 inflammasome activa-

tion, suggesting that redox signaling oroxidative 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, extra-

cellular 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

Eur. J. Immunol. 2010. 40: 595653FORUM16

&2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu


2/4

the broad spectrum NADPH oxidase

inhibitor, diphenyleneiodonium (DPI)

inhibits ATP-mediated caspase-1 acti-

vation [13, 14]. The NLRP3/NALP3

activating particulate elements uric acid

crystals, alum and particulate metals

have been shown to induce ROS

production [1518]. Similarly, ROS is

detectable quickly upon exposure of

macrophages to silica or asbestos

[16, 1921] Other NLRP3/NALP3 acti-

vators, such as the toxin nigericin, UV

light and skin sensitizers (e.g. dinitro-

chlorobenzene) activate a cellular redox

imbalance required for inflammasome

formation [14, 22, 23]. ROS has also

been implicated in NLRP3/NALP3 acti-

vation by the malaria pathogenic crys-tal, hemozoin, the influenza virus and

the yeast Candida albicans [2426].

How do NLRP3/NALP3 activatorspromote ROS generation?

Various pathways have been proposed

to mediate ROS production by NLRP3/

NALP3 activators; however, the general

picture of how NLRP3/NALP3 activa-

tors trigger ROS is still unclear. Potas-

sium efflux and decrease in cytosolicpotassium concentration are the most

striking features associated with

NLRP3/NALP3 activators [27]. Interest-

ingly, potassium efflux has been linked

to ROS production at the membrane in

plants [28]. Moreover, potassium efflux

has been shown to trigger ROS produc-

tion in human granulocytes [29]. It is

therefore tempting to speculate that

potassium efflux by NLRP3/NALP3 acti-

vators could be involved in ROS genera-

tion.

Some NLRP3/NALP3 activators suchas 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 asso-

ciated with ROS production; however,

the mechanism by which this occurs is

unclear [30, 31]. Frustrated phagocy-

tosis may not be the only mechanism

used by macrophages to sense patho-

genic particles. Evidence has demon-

strated that uric acid crystals can be

phagocytosed. Ultrastructure studies ofuric acid crystal-containing phagolyso-

somes 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 lysoso-

mal damage and cathepsin B release act

upstream of ROS production. In line

with this model, cathepsin B has beenshown 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 transmem-

brane 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-dicar-

boxylate inhibits inflammasome activa-

tion 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 linewith 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 activa-

tion [40]. On the contrary, NOX2-defi-cient macrophages have no defect in

inflammasome activation upon stimu-

lation 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

Figure 1. Examples of ROS generating path-

ways. (A) During respiration 12% of theoxygen is partially reduced to O2

, whichcan be converted H2O2 and OH. The majorsites of for production in the mitochondrialrespiratory chain are at complexes I and III.Complex I accepts electrons from NADH;these electrons move down an electrochemi-cal gradient through ubiquinone (Q cycle) tocomplex III, from complex III to cytochrome c(C) and from C to complex IV that use theelectrons to reduce molecular oxygen towater. The mechanisms involved ingeneration of O2

by complex I are poorlyunderstood. Complex III generate O2

byauto-oxidation of ubisemiquinone generatedduring the Q cycle, (IM, inner mitochondrial

membrane). (B) NADPH oxidases such as theNOX2 complex transport electrons acrossbiological membranes to reduce oxygen tosuperoxide. The activation of NOX2 occursthrough a complex series of protein/proteininteractions. Phosphorylation of p47phox leadsto a conformational change allowing itsinteraction with p22phox. The localization ofp47phox to the membrane brings p67phox intocontact with NOX2 and also brings the smallsubunit p40phox to the complex. Finally, theGTPase Rac interacts with NOX2. Onceassembled, the complex is active and gener-ates superoxide by transferring an electronfrom NADPH in the cytosol to oxygen on theluminal or extracellular space.

Eur. J. Immunol. 2010. 40: 595653 FORUM 617



3/4

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?

ROS production by H2O2 activates the

inflammasome [16, 42], furthermore,

knockdown of TRX, a cellular anti-

oxidant protein, enhances IL-1b activa-

tion 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 inflammasomeactivity. ATP-mediated ROS production

has been shown to stimulate the PI3K

pathway, and pharmacological inhibi-

tion of PI3K inhibits ATP-mediated

caspase-1 activation suggesting that

PI3K may be involved in inflammasome

activation downstream of ROS [13].

Recently Jurg Tschopps 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/VDUP1interacts 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 macro-

phages treated with extracellular ATP

or uric acid crystals have decreased

caspase-1 and IL-1b 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

In contradiction with classical PRR,

rather than directly recognizing patho-

genic 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 thatcope 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 inflamma-

tion responses by oxidative stress is

likely key to proper immunity and tissue

repair. In line with the role of ROS inactivating NLRP3/NALP3, inhibition of

ROS production in M2 polarized macro-

phages dampens inflammasome activa-

tion [45]. On the other hand, prolonged

oxidative stress can dampen inflamma-

tory 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-1b activation.Oxidative stress and tissue injury are

major hallmarks of numerous pathologies

ranging from diabetes to neurodegen-

erative disorders. Most of theses pathol-

ogies have an inflammatory component

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

Eur. J. Immunol. 2010. 40: 595653FORUM18



4/4

[47]. Dissecting the possible involvement

of the inflammasome in such pathologies

and identifying how oxidative stress

regulates NLRP3/NALP3 activation and

IL-1b activity will likely shed some new

light on these pathologies.

Acknowledgements: 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.

1 Droge, W.,Physiol. Rev.2002.82: 4795.

2 Valko, M.et al.,Int. J. Biochem. Cell Biol.

2007.39: 4484.

3 Ma, W. and Berkowitz, G. A., Cell

Microbiol.2007.9: 25712585.

4 Bogdan, C. et al., Curr. Opin. Immunol.

2000.12: 6476.

5 Niethammer, P. et al., Nature 2009.

459: 996999.

6 Ogier-Denis, E. et al., Semin. Immuno-

pathol.2008. 30: 291300.

7 Iriti, M. and Faoro, F., Mycopathologia

2007.164: 5764.

8 Martinon, F. et al., Annu. Rev. Immunol.

2009.27: 229265.

9 Bryant, C. and Fitzgerald, K. A., Trends

Cell Biol. 2009. 19: 455464.

10 Martinon, F. et al., Mol. Cell 2002. 10:

417426.

11 Chivasa, S. et al., Proteomics 2010. 10:

235244.

12 Carta, S. et al., J. Leukoc. Biol. 2009:

17.

13 Cruz, C. M. et al., J. Biol. Chem. 2007.

282: 28712879.

14 Hewinson, J. et al., J. Immunol. 2008.

180: 84108420.

15 Eisenbarth, S. C. et al., Nature 2008.

453: 11221126.

16 Dostert, C. et al., Science 2008. 320:

674677.

17 Sautin, Y. Y. et al., Am. J. Physiol. Cell

Physiol.2007. 293: C584C596.

18 Caicedo, M. et al., J. Orthop. Res. 2009

27: 847854.

19 Cassel, S. L. et al., Proc. Natl. Acad. Sci.

USA2008. 105: 90359040.20 Fubini, B. and Hubbard, A., Free Radic.

Biol. Med.2003. 34: 15071516.

21 Simeonova, P. P. and Luster, M. I., Am.

J. Respir. Cell Mol. Biol. 1995. 12:

676683.

22 Feldmeyer, L. et al., Curr. Biol. 2007.

17: 11401145.

23 Jin, G.-H.et al.,Radiat. Environ. Biophys.

2007. 46: 6168.

24 Jaramillo, M. et al., J. Immunol. 2005.

174: 475484.

25 Gross, O. et al., Nature 2009 459:

433436.

26 Allen, I. C. et al., Immunity 2009 30:

556565.27 Petrilli, V. et al., Cell Death Differ.2007.

14: 15831589.

28 Bolwell, G. P., Curr. Opin. Plant Biol.

1999. 2: 287294.

29 Fay, A. J.et al.,Proc. Natl. Acad. Sci. USA

2006. 103: 1754817553.

30 ONeill, L. A. J., Science 2008. 320:

619620.

31 Bergstrand, H., Agents Actions Suppl.

1990. 30: 199211.

32 Hoffstein, S. and Weissmann, G.,

Arthritis Rheum. 1975. 18: 153165.

33 Hornung, V.et al., Nat. Immunol. 2008.

9: 847856.

34 Hornung, V. and Latz, E., Eur. J.Immunol2010. 40: 620623.

35 Windelborn, J. A. and Lipton, P., J.

Neurochem.2008. 106: 5669.

36 Li, Z. et al., Hepatology 2008. 47:

14951503.

37 Bedard, K. and Krause, K.-H., Physiol.

Rev.2007. 87: 245313.

38 Sekiyama, A. et al., Immunity2005. 22:

669677.

39 Aldieri, E.et al.,Curr. Drug Metab.2008.

9: 686696.

40 Moore, S. F. and Mackenzie, A. B., J.

Immunol. 2009. 183: 33023308.

41 Dostert, C. et al., PLoS ONE 2009. 4:

e6510.42 Zhou, R. et al., Nat. Immunol. 2010.

Published online: 20 December 2009

doi:10.1038/ni.1831.

43 Martinon, F., J. Leukoc. Biol. 2008. 83:

507511.

44 Schneider, D. S., Cell 2002. 109:

537540.

45 Pelegrin, P. and Surprenant, A., EMBO

J. 2009. 28: 21142127.

46 Meissner, F. et al., Nat. Immunol. 2008.

9: 866872.

47 Medzhitov, R., Nature 2008. 454:

428435.

Correspondence: Dr. Fabio Martinon,

Department of Immunology and Infectious

Diseases, Harvard School of Public Health,

651 Huntington Ave, Boston, MA 02115,

USA

Fax: 11-617-432-0084

e-mail: [email protected]

Received: 18/11/2009

Revised: 21/12/2009

Accepted: 8/1/2010

Key words:Danger signals Inflammasome

Inflammation

Oxidative stress

Abbreviations: DPI: diphenyleneiodonium

NOX: NADPH-oxidase TRX: thioredoxin

See accompanying Viewpoint:

http://dx.doi.org/10.1002/eji.200940185

Eur. J. Immunol. 2010. 40: 595653 FORUM 619


activation mechanism nrlp3

Documents