© 2006 Nature Publishing Group
Gout-associated uric acid crystals activate the
Fabio Martinon1, Virginie Pe ´trilli1, Annick Mayor1, Aubry Tardivel1& Ju ¨rg Tschopp1
Development of the acute and chronic inflammatory responses
known as gout and pseudogout are associated with the deposition
ofmonosodium urate(MSU) orcalcium pyrophosphate dihydrate
(CPPD) crystals, respectively, in joints and periarticular tissues.
Although MSU crystals were first identified as the aetiological
agent of gout in the eighteenth century1and more recently as a
‘danger signal’released from dying cells2, little is known about the
molecular mechanisms underlying MSU- or CPPD-induced
inflammation. Here we show that MSU and CPPD engage the
caspase-1-activating NALP3 (also called cryopyrin) inflamma-
some, resulting in the production of active interleukin (IL)-1b
and IL-18. Macrophages from mice deficient in various compo-
nents of the inflammasome such as caspase-1, ASC and NALP3 are
defective in crystal-induced IL-1b activation. Moreover, an
impaired neutrophil influx is found in an in vivo model of
crystal-induced peritonitis in inflammasome-deficient mice or
mice deficient in the IL-1b receptor (IL-1R). These findings
provide insight into the molecular processes underlying the
inflammatory conditions of gout and pseudogout, and further
support a pivotal role of the inflammasome in several auto-
The notion of autoinflammatory diseases delineates a hetero-
geneous group of pathologies characterized by spontaneous periodic
inflammation and fever in the absence of infectious or autoimmune
arthritis, Still’s disease, Behc ¸et’s disease and the metabolic disorders
gout and pseudogout are examples of such inflammatory maladies.
clear evidence for a pivotal role of this cytokine in triggering auto-
inflammation4–8. IL-1b, also known as the endogenous pyrogen, is a
highly inflammatorycytokine whose production istightlycontrolled
by at least three distinct steps9. The first step involves the production
of the pro-IL-1b protein (p35); this is followed by cleavage of the
precursor pro-IL-1b to produce the active IL-1b protein (p17), and
finally IL-1b is released into the extracellular environment. The
middle step, processing of pro-IL-1b, involves the activation of a
caspase-1-activating complex, the best characterized being the
Upon activation, the inflammasome is formed by a member of
the NALP protein family, such as NALP1, NALP2 or NALP3, and the
adaptor protein ASC that connects the NALPs with caspase-1
(ref. 12). Signals and mechanisms leading to inflammasome acti-
vation are still poorly understood. Muramyl dipeptide (MDP), a
degradation product of the bacterial cell wall component peptido-
glycan and contaminant of crude lipopolysaccharide (LPS), was
recently shown to activate a NALP3 inflammasome13through the
leucine-rich repeat domain of NALP3, suggesting that NALPs,
like Toll-like receptors (TLRs), are fundamental for microbial
detection14. However, the inflammasome is also proficient in sensing
stress or endogenous danger signals, such as extracellular ATP or
hypotonic stress10,11,15. Recently, MSU crystals were identified as a
danger signal formed after release of uric acid from dying cells2. This
observation, and the well-known role of uric acid crystals in gouty
arthritis16, prompted us to investigate whether MSU crystals could
activate the inflammasome.
Cells from the differentiated monocytic cell line THP1 were
incubated with MSU crystals. Maturation of IL-1b was indeed
detected after stimulation with as little as 10mgml21of the crystals
(Fig. 1a). The caspase-1 dependency of the pro-IL-1b cleavage was
confirmed by addition of the caspase-1 inhibitor zYVAD-fmk, which
completely blocked MSU-induced IL-1b activation (Fig. 1a). CPPD,
another type of pathogenic crystal involved in calcium pyrophos-
phate deposition disease, also known as pseudogout, was as active as
MSU (Fig. 1b). Crystal-induced IL-1b processing was specific for
pathogenic agents, as the non-inflammatory allopurinol ordiamond
crystals and particulate elements such as zymosan and aluminium
powder failed to induce pro-IL-1b processing (Fig. 1c), despite their
similar size and/or chemical composition. Compared to the known
activators of the inflammasome (that is, crude LPS, ATP), MSU and
CPPD were more active11,13(Fig. 1c). This superior potency was
particularly evident when analysing processing of pro-IL-18, the
second known substrate of caspase-1 (Fig. 1c). Previously, we
demonstrated that the inflammatory caspases are cleaved and
released along with active IL-1b after activation of the inflamma-
some13. This was also observed when cells were treated with MSU
As shown in Fig. 1d, a strong response to both pathogenic crystals
was also elicited in primary cells.
In order to provide direct evidence for the involvement of the
inflammasome in crystal-induced inflammation, we analysed peri-
toneal macrophages (PMFs) derived from mice deficient in various
key proteins of the inflammasome complex or other proinflamma-
tory pathways. Given the absence and/or rapid degradation of
pro-IL-1b in PMFs ex vivo, and because we failed to see any direct
induction ofthetranscription or translation ofpro-IL-1b byMSUor
CPPD, we stimulated TLR4 in PMFs with highly purified LPS to
findings in human monocytes, murine PMFs stimulated with MSU
or CPPD activated caspase-1 and secreted mature IL-1b (Fig. 2a).
Maturation was abolished in PMFs from caspase-1-deficient mice,
confirming the specificity of the activation. As expected, MyD88-
deficient PMFs did not produce mature IL-1b due to their defective
TLR signalling, resulting in a failure to produce pro-IL-1b after LPS
pre-stimulation (Fig. 2a). Nevertheless, MyD882/2PMFs still acti-
vated caspase-1 (Fig. 2a), further suggesting that this activation is
1Department of Biochemistry, University of Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland.
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© 2006 Nature Publishing Group
inflammasome13,15. ASC is a crucial adaptor protein required for the
recruitment of caspase-1 to the NALP platform of inflammasomes12.
ASC-deficient PMFs did not produce any mature IL-1b after
stimulation by MSU and CPPD crystals (Fig. 2b).
The human genome harbours a repertoire of 14 NALPs. It is
currently not clear how many of them form inflammasomes. NALP3
is expressed in both monocytes and macrophages and is well
and to drive inflammation in humans is well supported by its
implication in many hereditary autoinflammatory syndromes4. We
considered that the NALP3 inflammasome was possibly implicated
in crystal-induced caspase-1 activation and we therefore generated
NALP3-deficient mice (Supplementary Fig. 1 and V.P., F.M. and J.T.,
manuscript in preparation). Similar to PMFs from Asc2/2mice,
IL-1b release was impaired in NALP3-deficient PMFs upon MSU
and CPPD exposure (Fig. 2c). IL-1b induction by ATP, the other
known non-microbial stimulus of inflammasomes, was also depen-
inhibited ATP-driven inflammasome activation, it had no effect on
MSU-induced activation, indicating that the two inflammasome-
activating pathways act independently (Supplementary Fig. 2).
In addition to cytokines whose activity is dependent on caspase-1
activation, MSU and CPPD are known to induce release of other
cytokines such as TNF17,18, suggesting additional, inflammasome-
independent activities of the crystals. When assaying the release of
TNF, we realized that the production of TNF was relatively slow and
was preceded by the release of IL-1b19(Fig. 3a). It was therefore
possible that TNF secretion was initiated, at least in part, by the
released mature IL-1b. Indeed, blocking the maturation of IL-1b
with zYVAD-fmk considerably reduced the production of TNF
induced by MSU and CPPD, without affecting TNF production by
the TLR2 agonist zymosan (Fig. 3a). Similarly, IL-1ra, a natural
inhibitor of IL-1 signalling, significantly affected the production of
TNF and IL-6 by human monocytes (Fig. 3b). These results suggest
that the processing of IL-1b is a proximal event in the inflammatory
cascade initiated by pathogenic crystals, possibly explaining the
extraordinary success of IL-1ra in the treatment of some auto-
of autoinflammatory diseases, including familial Mediterranean
fever, acute gout and pseudogout episodes22. Pre-treatment with
intravenous colchicine before intra-articular MSU injections greatly
reduces inflammation23, suggesting that colchicine targets the initial
phase of inflammation. We therefore investigated the role of colchi-
cine in crystal-induced maturation of IL-1b. As shown in Fig. 3c,
blockedthe processing of IL-1b. In contrast, colchicine did not affect
IL-1b activation by extracellular ATP, indicating that the drug acts
upstream of inflammasome activation. Taken together, the above
Figure 1 | Monosodium urate (MSU) and calcium pyrophosphate dihydrate
(CPPD) crystals activate IL-1b cleavage and release. a–c, THP1 cells were
stimulated for 6h with the indicated amounts (per ml) of MSU crystals (a),
crystals, CPPD crystals, diamond crystals, aluminium particles, zymosan,
crude preparations of LPS, or 5mM of extracellular ATP as indicated (c).
Supernatants (SN) were analysed for the presence of mature IL-1b, IL-18
or caspase-1, and cell extracts (Cell) for the presence of pro-IL-1b and
pro-IL-18. WB, western blot. d, Human monocytes were stimulated with
50mgml21of the indicated crystals for 6h and analysed by western blot for
IL-1b activation or by ELISA for released caspase-1 and IL-1b. Values are
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© 2006 Nature Publishing Group
results indicate that crystals are proinflammatory by virtue of their
capacity to activate the NALP3 inflammasome.
Clinically, gout and pseudogout are associated with oedema and
erythema of the joints, with consequent severe pain, conditions that
are associated with strong infiltration of neutrophils in the intra-
articular and periarticular spaces. This marked neutrophil influx can
be reproduced experimentally in mice by intraperitoneal injection of
crystals24. We used this well-established model to investigate the in
vivo role of the inflammasome in crystal-induced inflammation.
MSU, CPPD or allopurinol crystals were injected and the peritoneal
recruitment of neutrophils was analysed 6h later. Both MSU and
CPPD crystals elicited a considerable increase in the recruitment of
type C57BL/6 mice(Fig. 4a). Although neutrophil influx was slightly
increased in BALB/c mice, both BALB/c and C57BL/6 strains
adequately reproduced crystal-induced inflammation (Fig. 4 and
data not shown). Importantly, when pathogenic crystals were
injected in mice deficient in caspase-1 or ASC, neutrophil influx
was markedly impaired (Fig. 4b, c), indicating a pivotal role of the
inflammasome and IL-1b in this process. In agreement with this
notion was the observation that IL-1R-deficient mice exhibited a
similarly reduced recruitment of neutrophils after MSU and CPPD
injection (Fig. 4d). In contrast, zymosan-induced neutrophil influx
was not affected by ASC or IL-1R deficiency.
Gout and pseudogout are two common causes of inflammatory
joint diseases. Despite differences underlying their pathogenesis,
their clinical presentation and treatment share many common
features. On the basis of our findings that pathogenic crystal-
mediated IL-1b maturation requiresthe inflammasome components
NALP3, ASC and caspase-1, we proposethat both aetiological agents
of gout and pseudogout (that is, MSU and CPPD) mediate inflam-
mation in an inflammasome-dependent manner. This notion is
further supported by clinical data demonstrating that colchicine, a
drug able to resolve the initial inflammatory phase of both gout and
pseudogout, blocks IL-1b maturation by MSU and CPPD. Combin-
assembly with the observation that colchicine blocks crystal-induced
likely that the drug acts at the level of crystal endocytosis and/or
presentation to the inflammasome25. Notably, MSU uptake was also
proposed to be partly dependent on the presence of TLR2 and TLR4
(ref. 26). The mechanism whereby endocytosed MSU and CPPD are
sensedbytheNALP3 inflammasome iscurrentlynotknown,noris it
clear whether the crystals directly interact with NALP3 or whether
sensing occurs via intermediary protein(s). Because inflammasome-
activating MSU crystals and inflammasome neutral allopurinol are
chemically and structurally similar and, additionally, are both inter-
nalized, inflammasomes must have the capacity to distinguish
between subtle differences in crystal surface charge or form.
Microbial components (pathogen-associated molecular patterns
Figure 3 | IL-1b maturation is an early event after MSU and CPPD
stimulation, and is blocked by colchicine. a, THP1 cells were stimulated
with MSU, CPPD or zymosan (Zym) for the indicated times in the presence
or absence of the caspase-1 inhibitor zYVAD-fmk. Supernatants were
analysed for TNF (grey bars) and IL-1b (black bars) production by ELISA.
in the presence of two concentrations of IL-1ra. TNF and IL-6 production
was monitored by ELISA. Values are ^s.e.m. c, THP1 cells were stimulated
with MSU, CPPD or ATP in the presence or absence of colchicine (Colch).
Maturation of IL-1b was analysed by western blot.
Figure 2 | The NALP3 inflammasome is required for the maturation of
IL-1b. a–c, Mouse macrophages from wild-type (WT), caspase-1 (Casp1)-
or MyD88-deficient mice (a), ASC-deficient mice or littermate controls (b),
and NALP3-deficient mice or littermate controls (c) were stimulated as
indicated in the presence of ultra-pure LPS (1mgml21, Alexis or Invivogen)
in order to inducethe synthesis of precursor pro-IL-1b. In c, ultra-pure LPS
was added 1h before stimulation. Supernatant (SN) or cell extracts (Cell)
were analysed by western blot as indicated.
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© 2006 Nature Publishing Group
(PAMPs)) provide signals that alert our immune system to danger
and promote the innate generation of immunity27. However, PAMPs
(non-self) are not the only triggers of innate immunity. Innate
immunity is able to recognize abnormal self or danger signals, such
as uric acid released by injured cells2,28. How these danger signals
are recognized by cells is mostly unknown, but based on our results
inflammasomes probably constitute some of the long-sought
proximal sensors for stress or danger signals designed to initiate
In addition to gouty inflammation, the NALP3 inflammasome is
also implicatedinother autoinflammatorydiseases. Specificgain-of-
function mutations in the NALP3 protein lead to three related
familial autoinflammatory diseases: Muckle–Wells syndrome,
familial cold autoinflammatory syndrome and chronic infantile
neurologic cutaneous and articular syndrome4,29. In patients with
these diseases, mutations in NALP3 lead to a constitutive processing
of IL-1b30. In the case of gout and pseudogout, aberrant NALP3
inflammasome activation is not genetic, but mediated by local
deposition of crystals. Importantly, inflammation in hereditary
periodic fevers patients with mutations in NALP3 can be markedly
improved by treatments designated to block IL-1b20,21. Owing to the
similarity between NALP3-mediated hereditary periodic fevers and
benefit gout and pseudogout patients. It is also reasonable to foresee
that further identification of additional inflammasome-activating
endogenous danger signals will probably shed some light on the
molecular aetiology of other autoinflammatory diseases such as
systemic onset juvenile idiopathic arthritis and Behc ¸et’s disease
that share similarity with hereditary periodic fevers, gout or
Primary human monocyte and THP1 preparation and stimulation. THP1
cells were stimulated for 3h with 0.5mM of PMA the day before stimulation, as
described10. This treatment increases the phagocytic properties of the cells and
induces a constitutive production of pro-IL-1b. Human monocytes were
purified as described previously30. All cells were stimulated in OptiMEM
medium as indicated. Human mature IL-1b was detected with a specific
antibody directed against the cleaved epitope (D116) from Cell Signaling.
Mouse macrophage preparation. Eight-to-twelve-week-old mice of indicated
genotypes were injected intraperitoneally with 4% thioglycollate solution, and
macrophageswerecollectedbyperitoneallavage3days later. Cells wereplatedat
the density of 7 £ 105cells in 12-well dishes and non-adherent cells were
removed after 3h. Cells were cultured in RPMI complemented with 10% FCS,
sodiumpyruvate, penicillin/streptomycinand L-glutamine. All cellswere stimu-
lated in OptiMEM medium.
crystals or0.2mgofzymosanin0.5-mlsterilePBS.After6h,mice werekilledby
CO2exposureandperitonealcavitieswere washedwith 10mlofPBS.Thelavage
fluids were analysed for PMN recruitment byFACSusing the neutrophil marker
Ly-6G (1A8, BD Biosciences).
Mice and reagents. NALP3 targeting vector (Supplementary Fig. 1) was
electroporated into C57BL/6 embryonic stem (ES) cells (Ozgene). Homologous
into C57BL/6 blastocysts. Offspring were backcrossed to C57BL/6 mice and
germline transmission was confirmed by PCR of tail genomic DNA. Additional
Received 19 November; accepted 12 December 2005.
Published online 11 January 2006.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank C. Mattmann for technical support and A. So,
H. Everett, E. Meylan, M. Thome and P. Schneider for discussions and critical
reading of the manuscript. We thank S. Mariathasan, V. M. Dixit, R. A. Flavell,
M. Kopf and S. Akira for the gift of various knockout mice. This work was
supported by grants from the Swiss National Science Foundation and the
Commission of Technology and Innovation (CTI). V.P. is supported by a
fellowship of the FRM (Fondation pour la Recherche Me ´dicale); A.T. by a NCCR
Author Information Reprints and permissions information is available at
npg.nature.com/reprintsandpermissions. The authors declare no competing
financial interests. Correspondence and requests for materials should be
addressed to J.T. (firstname.lastname@example.org).
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