CARD8 is a negative regulator for NLRP3 inflammasome, but mutant NLRP3 in cryopyrin-associated periodic syndromes escapes the restriction
© Ito et al.; licensee BioMed Central Ltd. 2014
Received: 1 October 2013
Accepted: 4 February 2014
Published: 12 February 2014
NLRP3 plays a role in sensing various pathogen components or stresses in the innate immune system. Once activated, NLRP3 associates with apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and procaspase-1 to form a large protein complex termed inflammasome. Although some investigators have proposed a model of NLRP3-inflammasome containing an adaptor protein caspase recruitment domain-containing protein 8 (CARD8), the role of this molecule remains obscure. This study aimed to clarify the interaction between CARD8 and wild-type NLRP3 as well as mutant forms of NLRP3 linked with cryopyrin-associated periodic syndromes (CAPS).
In here HEK293 expression system, cells were transfected with the cDNAs for inflammasome components. Also used were peripheral blood mononuclear cells (PBMCs) and human monocyte-derived macrophages (HMDMs) from healthy volunteers. The interaction of CARD8 and NLRP3 was studied by immunoprecipitation. The effect of CARD8 expression on IL-1β secretion was assessed by ELISA. CARD8 knockdown experiments were carried out by transfection of the specific siRNA into HMDMs.
In HEK293 cells, CARD8 interacted with wild-type NLRP3, but not with CAPS-associated mutant NLRP3. CARD8 significantly reduced IL-1β secretion from cells transfected with wild-type NLRP3, but not if they were transfected with mutant NLRP3. In addition, association of endogenously expressed CARD8 with NLRP3 was confirmed in resting PBMCs, and CARD8 knockdown resulted in higher amount of IL-1β secretion from HMDMs.
Until specific stimuli activate NLRP3, CARD8 holds NLRP3, and is supposed to prevent activation by subtle stimuli. However, CAPS-associated mutant NLRP3 is unable to bind with CARD8, which might be relevant to the pathogenesis of CAPS.
The NLR (nucleotide binding oligomerization domain-like receptor) protein family plays a role as an intracellular sensor for pathogens and cell injury by detecting conserved structures, such as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [1, 2]. NLRP3, a member of the NLR, is a cytoplasmic protein expressed predominantly in monocytes and macrophages, and forms a caspase-1 activating multiprotein complex termed inflammasome with other proteins, including an adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)) and procaspase-1 [3, 4]. The NLRP3 is composed of three domains referred to as pyrin domain (PYD) at the N-terminus, leucine-rich repeats (LRRs) at the C-terminus, and nucleotide binding-oligomerization domain (NOD) in the middle. When PAMPs or DAMPs are recognized by the LRRs, NLRP3 oligomerizes by self-association through the NOD, and binds with ASC via their PYD domains [5, 6]. ASC and procaspase-1 bind with each other through their CARD [7, 8], which leads to the assembly and autocleavage of the procaspase-1 to produce active caspase-1. Then caspase-1 processes proIL-1β to mature IL-1β, which is released into the extracellular space .
Mutations in the cold-induced autoinflammatory syndrome 1 (CIAS1) gene encoding NLRP3 result in cryopyrin-associated periodic syndromes (CAPS) characterized by recurrent episodes of systemic inflammatory attacks in the absence of infection or autoimmune diseases . CAPS include three clinical entities: familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS) and chronic infantile neurologic cutaneous articular syndrome (CINCA). The mildest form, FCAS, presents with a cold-induced urticarial rash, fever and arthralgia. CINCA, the most severe form of CAPS, includes neonatal-onset high fever, aseptic meningitis, sensory hearing loss, papilledema, arthritis with bone overgrowth and secondary amyloidosis. MWS is the intermediate phenotype, and overlapping cases between FCAS and MWS, or MWS and CINCA exist as well. More than 50 CAPS-associated missense mutations have been reported and, of note, most of them are clustered in the NOD domain of NLRP3 . As for the mechanism by which those mutations activate NLRP3 leading to autoinflammation, Lee et al. have recently noticed the role of the calcium-sensing receptor that regulates NLRP3 inflammasome through Ca2+ and cAMP , but other mechanisms might be contributed as well.
Caspase recruitment domain-containing protein 8 (CARD8), also called tumor-up-regulated CARD-containing antagonist of caspase nine (TUCAN) or CARD inhibitor of NF-κB-activating ligands (Cardinal), is a member of the CARD family, and is composed of an N-terminal function to find (FIIND) domain and a C-terminal CARD domain. In a pioneering study by Razmara et al. , CARD8 was shown to interact physically with caspase-1 through the CARD-CARD homophilic interaction and to negatively regulate activation of caspase-1. On the other hand, Agostini et al.  showed an interaction between the FIIND domain of CARD8 and the NOD domain of NLRP3, and they proposed a model in which NLRP3 inflammasome consists of a complex of NLRP3, ASC, caspase-1 and CARD8. Subsequently, some review articles adopted the model of an NLRP3 inflammasome including CARD8 [14–16] while others excluded CARD8 [17–19]. Thus, the association of CARD8 protein with NLRP3 and its function in the inflammasome remain obscure.
Here, we show that CARD8 plays a role as a negative regulator of NLRP3 inflammasome through its binding with NLRP3. CAPS-associated mutant NLRP3 was, however, unable to bind to CARD8.
cDNA cloning of inflammasome components
The study protocols using human blood cells were approved by the Medical Research Ethics Committee for Genetic Research, Tokyo Medical and Dental University. Blood samples were obtained from healthy volunteers after obtaining written informed consent, and we did not use samples from patients in this study. RNA was isolated from peripheral blood leukocytes by the acid guanidinium thiocyanate-phenol-chloroform method . cDNA was synthesized from total RNA with SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) using random hexamer as a primer. The entire coding sequence of NLRP3, ASC, procaspse-1, proIL-1β and CARD8 (the T48 isoform, amino acids 1 to 431), truncated coding sequences of CARD8 (the FIIND domain, amino acids 1 to 346, or the CARD domain, amino acids 321 to 431) were amplified by PCR using modified PCR primers based on the cDNA sequences from GeneBank (NLRP3, AF410477; ASC, AB023416; procaspse-1, M87507; proIL-1β, BC008678; and CARD8, AF322184). The sequences were confirmed with an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The C-terminus Flag (−DYKDDDDK), GFP, and DsRed (Clontech, Palo Alto, CA, USA) tags were used to generate fusion proteins. cDNAs of proIL-1β, ASC and fluorescent fusion proteins were subcloned into the pTargeT vector (Promega, Mannheim, Germany). NLRP3, NLRP3-Flag, CARD8, CARD8-Flag, FIIND-Flag, CARD-Flag and procasapse-1 cDNAs were subcloned into the pcDNA3.1 vector (Invitrogen).
Polymorphism of CARD8
DNA samples were collected from healthy Japanese donors using Puregene (Gentra, Big Lake, MN, USA), and the CARD8 was amplified by PCR using the following primers: sense: 5′-GATGGAGTCGTAGGGGCCTGAG-3′ and antisense: 5′-CTCCCTCATCAGGGGCTTCACG-3′. c.30 T > A (rs2043211) variant was detected by sequencing.
Immunoprecipitation of transient transfectants
HEK293 cells were provided by the RIKEN BRC (Tsukuba, Japan) through the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) Japan. The cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum, and transfected with expression plasmids pcDNA or pTargeT using Lipofectamine LTX (Invitrogen) according to the manufacturer’s protocol. At 24 hours after transfection, cells were lysed with ice-cold lysis buffer (50 mM Tris-HCl, 150 mM KCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 8.0) and solubilized by a sonicator. The cell lysates were clarified by centrifugation for 10 minutes at 16,000 g and incubated for 60 minutes at 4°C with Anti-Flag M2 affinity gel (Sigma, St Louis, MO, USA). The gels were washed four times with lysis buffer and the final precipitates were subjected to Western blotting analysis using the following antibodies: anti-FLAG (Sigma), anti-CARD8 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-NLRP3 (nalpy3-b, Abcam, Cambridge, UK), anti-ASC (MBL, Nagoya, Japan), anti-caspase-1 (Santa Cruz Biotechnology), anti-GAPDH (Sigma), anti-IL-1β (Santa Cruz Biotechnology), and anti-cleaved IL-1β (Cell Signaling Technology, Beverly, MA, USA).
Determination of IL-1β secretion from HEK293 cells
Cells were plated on 12-well plates and transfected with cDNAs. At 24 hours after transfection, supernatants were replaced with fresh medium and cells were incubated for another 24 hours. Then, the second supernatants and pellets were subjected to ELISA and Western blotting, respectively. ELISA was performed using Human IL-1β/IL-1 F2 Immunoassay ELISA Kit (R&D Systems, Minneapolis, MN, USA). In some experiments, secreted IL-1β during the two-hour incubation in serum-free medium (Opti-MEM, Gibco, Carlsbad, CA, USA), with which supernatants were replaced at 24 hours after transfection, was detected by Western blotting as well.
Quantification of specks
HEK293 cells were plated on multi-well glass bottom dishes (Matsunami, Kishiwada, Japan) and transfected with the plasmids by lipofection. Twenty-four hours later, fluorescent images were obtained using a confocal laser scanning microscope FV500 (Olympus, Tokyo, Japan), and the percentage of speck-positive cells was calculated as the number of speck-positive cells divided by the total number of transfected cells.
Immunoprecipitation of endogenous proteins
Peripheral blood mononuclear cells (PBMCs, 1 × 107 cells/6 cm dish) from healthy volunteers were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, and primed with 25 ng/ml lipopolysaccharide (LPS, Escherichia coli 0111:B4, Sigma) for three hours. After the supernatant was replaced by fresh medium or Opti-MEM, cells were stimulated with 1.5 mM adenosine triphosphate (ATP) for one hour, then the supernatants were assayed for IL-1β ELISA or Western blotting, respectively. In addition, whole cell extracts were prepared with ice-cold W buffer (20 mM HEPES-KOH, 10 mM KCl, 1.5 mM MgCl2, 1 mM Na-EDTA, 1 mM Na-EGTA, 0.1 mM PMSF and protease inhibitor cocktail (Sigma), pH 7.5) and solubilized by a sonicator . The cell lysates were clarified by centrifugation for 10 minutes at 16,000 g and incubated overnight at 4°C with anti-NLRP3 antibody. The reaction mixtures were then incubated for 60 minutes at 4°C with protein G-Sepharose to precipitate the antigen-antibody complexes. The precipitates were washed five times with W buffer and subjected to Western blotting analysis.
RNAi knockdown of CARD8
Human monocytes were isolated from peripheral blood cells using RosetteSep (Stemcell Technologies, Vancouver, BC, Canada). Human monocyte-derived macrophages (HMDMs) were differentiated after incubation of the monocytes for seven days in RPMI-1640 medium supplemented with 10% human serum, 10% heat-inactivated fetal bovine serum and 50 ng/ml GM-CSF (PeproTech, Rocky Hill, NJ, USA). The human CARD8-specific siRNA (siCARD8-1; 5′-CCUCUUAUGCUUCUAAAGUCU-3′ and 5′-ACUUUAGAAGCAUAAGAGGAA-3′) was synthesized by Sigma. Another set of CARD8-specific siRNA (siCARD8-2; 5′-GAGCCUUUCUAUGCUGUCCUGGAAA-3′ and 5′-UUUCCAGGACAGCAUAGAAAGGCUC-3′) and negative control duplex were purchased from Invitrogen. HMDMs were transiently transfected with each siRNA (50 nM) using Lipofectamine RNAiMax (Invitrogen). At four days after transfection, knockdown efficiency was evaluated by real-time quantitative RT-PCR. Then the cells were primed with 10 ng/ml LPS for three hours followed by stimulation with 1.5 mM ATP for one hour, and IL-1β in the culture supernatants was measured by ELISA. In parallel, ATP-stimulated cells were labeled with a FAM-YVAD-fmk FLICA caspase-1 assay kit (Immunochemistry, Bloomington, IN, USA), and analyzed by flow cytometry to evaluate the activity of caspase-1.
Student’s unpaired t- tests were performed for statistical comparisons, and a P- value of less than 0.05 was considered significant.
Interaction of CARD8 with NLRP3
Next, to study the effect of ASC on the interaction of NLRP3 with CARD8, ASC expression plasmid was introduced to the cells. The interaction between CARD8 and NLRP3 was inhibited by the coexpression of ASC in a dose-dependent manner (lanes 3 to 6). When 100 ng or higher doses of ASC-expression plasmid were transfected, NLRP3 was coprecipitated with ASC rather than with CARD8 (lanes 5 to 7). Thus, the interaction of ASC with NLRP3 seemed to be stronger than that of CARD8 with NLRP3 in this condition.
Suppression of NLRP3 inflammasome by expression of CARD8
Suppression of NLRP3-speck formation by CARD8
Next, to investigate the role of CARD8, the number of speck-positive cells in the expression of different dosages of CARD8 was counted. When cells were transfected with NLRP3-GFP, ASC, procaspase-1 and proIL-1β, the percentage of NLRP3-speck positive cells was 27.1 ± 2.9% (mean ± SD). However, the number of speck positive cells was reduced by coexpression of CARD8 in a dose-dependent manner (Figure 3D). Transfection of ASC alone has been known to form speck [23, 24], but coexpression of CARD8 had no effect on the number of ASC-speck positive cells (data not shown). These results suggest that CARD8 negatively regulates formation of NLRP3 inflammasome, although the final product of the inflammasome does not contain CARD8. Therefore, we speculate that expression of CARD8 leads to a reduced amount of inflammasome formation, and this results in a reduced amount of IL-1β secretion by the cells than those without CARD8 expression.
No interaction between CARD8 and CAPS-associated NLRP3 mutants
Interaction of endogenous CARD8 and NLRP3
Enhanced ATP-induced IL-1β secretion by CARD8-knockdown
In the present study, we obtained four major findings: 1) in the HEK293 cell expression system, CARD8 interacted with wild-type NLRP3 but not with CAPS-associated mutant NLRP3; 2) CARD8 suppressed IL-1β secretion from the cells transfected with wild-type NLRP3, but not from the cells with CAPS-associated mutant NLRP3; 3) in PBMCs from healthy subjects, endogenous NLRP3 interacted with CARD8 in a resting state, but the partner changed to ASC after stimulation by LPS and ATP; and finally, 4) in HMDMs from healthy subjects, ATP-stimulated IL-1β secretion was increased by knockdown of CARD8.
Using HEK293T cells and calcium phosphate transfection methods, it has been reported that VSV-tagged CARD8 interacts with Flag-tagged truncated NLRP3 (ΔLRRs), but no interaction was detected between VSV-tagged CARD8 and Flag-tagged full-length NLRP3 . In our experiments, however, CARD8 interacted with full length NLRP3. Differences in tagging, cells, culture medium and transfection methods may produce different results. We were cautious, therefore, and experiments using Flag-tagged NLRP3 were repeated using Flag-tagged CARD8, with similar results. Furthermore, we confirmed the interaction between endogenous CARD8 and NLRP3 in normal PBMCs. In the transfection study with HEK293 cells, NLRP3 did interact with CARD8, but ASC prevented the interaction in a dose-dependent manner. Moreover, HEK293 cells transfected with every component of NLRP3 inflammasome spontaneously showed speck formation and produced IL-1β. These results suggest that gene transfer itself may induce conversion of NLRP3 to activated form, to which CARD8 does not bind. Consistent with this hypothesis, endogenous NLRP3 interacted with CARD8 in PBMCs only in a resting state; after stimulation with LPS and ATP, NLRP3 interacted not with CARD8 but with ASC. CARD8 may hold NLRP3 in an inactive form until the cells encounter stimuli over a threshold level.
Families of small proteins that are called CARD-only proteins (COPs) and PYD-only proteins (POPs) have recently been known to negatively regulate inflammasome and suppress spontaneous or unnecessary activation by playing a role as decoy partners [31–33]. Similar to COPs, the CARD domain of CARD8 is reported to interact with the CARD domain of procaspase-1; these CARD domains are highly homologous to each other, resulting in suppression of autocleavage of procaspase-1 . In the present study, IL-1β secretion was reduced in HEK293 cells transfected with every inflammasome component, by coexpression of the FIIND domain of CARD8, suggesting that this domain is also responsible for negative regulation of NLRP3 inflammasome by inhibiting NLRP3 oligomerization. Furthermore, CARD8-knockdown HMDMs secreted a significantly higher amount of IL-1β along with caspase-1 activation, suggesting that CARD8 plays a role as a negative regulator on the endogenous NLRP3 inflammasome as well. How much the suppressive effect of CARD8 on IL-1β secretion resulted from the interaction with NLRP3 independent of that with procaspase-1 remains to be elucidated. Analogous to this hypothesis, CARD8 has been recently reported to interact with the NOD domain of NOD2, which is one of the NLR family members, and inhibits nodosome assembly .
We also examined interaction of CARD8 and CAPS-associated NLRP3 mutants, but no significant interaction was detected with any mutants tested. This may result from a change of the local structure by replacement of an amino acid in the NOD domain that is normally the binding site for CARD8. As a result, CARD8 did not reduce IL-1β secretion by inflammasome containing mutant NLRP3. These results suggest that CAPS-associated mutant NLRP3 escapes the CARD8 restriction, which might be responsible for unnecessary activation of NLRP3 inflammasome in the patients.
Our data support the model of NLRP3 inflammasome excluding CARD8. Until specific stimuli activate NLRP3, CARD8 holds NLRP3, and is supposed to prevent activation by subtle stimuli. Because CAPS-associated mutant NLRP3 is not protected by CARD8, it may form inflammasome without obvious stimulation.
Apoptosis-associated speck-like protein containing a CARD
Cryopyrin-associated periodic syndromes
Caspase recruitment domain
Caspase recruitment domain-containing protein 8
CARD inhibitor of NF-κB-activating ligands
Cold-induced autoinflammatory syndrome 1
Chronic infantile neurologic cutaneous articular syndrome
Damage-associated molecular patterns
Familial cold autoinflammatory syndrome
Function to find
Human monocyte-derived macrophages
Mean fluorescence intensity
Nucleotide binding oligomerization domain
Pathogen-associated molecular patterns
Peripheral blood mononuclear cells
Polymerase chain reaction
Tumor-upregulated CARD-containing antagonist of caspase nine
This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology in Japan (#24590681).
- Martinon F, Mayor A, Tschopp J: The inflammasomes: guardians of the body. Annu Rev Immunol. 2009, 27: 229-265. 10.1146/annurev.immunol.021908.132715.View ArticlePubMedGoogle Scholar
- Franchi L, Muñoz-Planillo R, Núñez G: Sensing and reacting to microbes through the inflammasomes. Nat Immunol. 2012, 13: 325-332. 10.1038/ni.2231.View ArticlePubMedPubMed CentralGoogle Scholar
- Agostini L, Martinon F, Burns K, McDermott MF, Hawkins PN, Tschopp J: NALP3 forms an IL-1β-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. Immunity. 2004, 20: 319-325. 10.1016/S1074-7613(04)00046-9.View ArticlePubMedGoogle Scholar
- Mariathasan S, Monack DM: Inflammasome adaptors and sensors: intracellular regulators of infection andinflammation. Nat Rev Immunol. 2007, 7: 31-40. 10.1038/nri1997.View ArticlePubMedGoogle Scholar
- Dowds TA, Masumoto J, Zhu L, Inohara N, Núñez G: Cryopyrin-induced interleukin 1β secretion in monocytic cells: enhanced activity of disease-associated mutants and requirement for ASC. J Biol Chem. 2004, 279: 21924-219248. 10.1074/jbc.M401178200.View ArticlePubMedGoogle Scholar
- Manji GA, Wang L, Geddes BJ, Brown M, Merriam S, Al-Garawi A, Mak S, Lora JM, Briskin M, Jurman M, Cao J, DiStefano PS, Bertin J: PYPAF1, a PYRIN-containing Apaf1-like protein that assembles with ASC and regulates activation of NF-κB. J Biol Chem. 2002, 277: 11570-11575. 10.1074/jbc.M112208200.View ArticlePubMedGoogle Scholar
- Srinivasula SM, Poyet JL, Razmara M, Datta P, Zhang Z, Alnemri ES: The PYRIN-CARD protein ASC is an activating adaptor for caspase-1. J Biol Chem. 2002, 277: 21119-21122. 10.1074/jbc.C200179200.View ArticlePubMedGoogle Scholar
- Martinon F, Burns K, Tschopp J: The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-β. Mol Cell. 2002, 10: 417-426. 10.1016/S1097-2765(02)00599-3.View ArticlePubMedGoogle Scholar
- Sutterwala FS, Ogura Y, Szczepanik M, Lara-Tejero M, Lichtenberger GS, Grant EP, Bertin J, Coyle AJ, Galán JE, Askenase PW, Flavell RA: Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity. 2006, 24: 317-327. 10.1016/j.immuni.2006.02.004.View ArticlePubMedGoogle Scholar
- Neven B, Callebaut I, Prieur AM, Feldmann J, Bodemer C, Lepore L, Derfalvi B, Benjaponpitak S, Vesely R, Sauvain MJ, Oertle S, Allen R, Morgan G, Borkhardt A, Hill C, Gardner-Medwin J, Fischer A, de Saint Basile G: Molecular basis of the spectral expression of CIAS1 mutations associated with phagocytic cell-mediated autoinflammatory disorders CINCA/NOMID, MWS, and FCU. Blood. 2004, 103: 2809-2815. 10.1182/blood-2003-07-2531.View ArticlePubMedGoogle Scholar
- Aksentijevich I, Kastner DL: Genetics of monogenic autoinflammatory diseases: past successes, future challenges. Nat Rev Rheumatol. 2011, 7: 469-478. 10.1038/nrrheum.2011.94.View ArticlePubMedGoogle Scholar
- Lee GS, Subramanian N, Kim AI, Aksentijevich I, Goldbach-Mansky R, Sacks DB, Germain RN, Kastner DL, Chae JJ: The calcium-sensing receptor regulates the NLRP3 inflammasome through Ca2+ and cAMP. Nature. 2012, 492: 123-127. 10.1038/nature11588.View ArticlePubMedPubMed CentralGoogle Scholar
- Razmara M, Srinivasula SM, Wang L, Poyet JL, Geddes BJ, DiStefano PS, Bertin J, Alnemri ES: CARD-8 protein, a new CARD family member that regulates caspase-1 activation and apoptosis. J Biol Chem. 2002, 277: 13952-13958. 10.1074/jbc.M107811200.View ArticlePubMedGoogle Scholar
- Hoffman HM, Brydges SD: Genetic and molecular basis of inflammasome-mediated disease. J Biol Chem. 2011, 286: 10889-10896. 10.1074/jbc.R110.135491.View ArticlePubMedPubMed CentralGoogle Scholar
- Busso N, So A: Mechanisms of inflammation in gout. Arthritis Res Ther. 2010, 12: 206-10.1186/ar2952.View ArticlePubMedPubMed CentralGoogle Scholar
- Verma D, Lerm M, Blomgran Julinder R, Eriksson P, Söderkvist P, Särndahl E: Gene polymorphisms in the NALP3 inflammasome are associated with interleukin-1 production and severe inflammation: relation to common inflammatory diseases?. Arthritis Rheum. 2008, 58: 888-894. 10.1002/art.23286.View ArticlePubMedGoogle Scholar
- Rathinam VA, Vanaja SK, Fitzgerald KA: Regulation of inflammasome signaling. Nat Immunol. 2012, 13: 333-342. 10.1038/ni.2237.View ArticlePubMedPubMed CentralGoogle Scholar
- Martinon F: Dangerous liaisons: mitochondrial DNA meets the NLRP3 inflammasome. Immunity. 2012, 36: 313-315. 10.1016/j.immuni.2012.03.005.View ArticlePubMedGoogle Scholar
- Stutz A, Golenbock DT, Latz E: Inflammasomes: too big to miss. J Clin Invest. 2009, 119: 3502-3511. 10.1172/JCI40599.View ArticlePubMedPubMed CentralGoogle Scholar
- Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987, 162: 156-159.View ArticlePubMedGoogle Scholar
- Yu JW, Wu J, Zhang Z, Datta P, Ibrahimi I, Taniguchi S, Sagara J, Fernandes-Alnemri T, Alnemri ES: Cryopyrin and pyrin activate caspase-1, but not NF-κB, via ASC oligomerization. Cell Death Differ. 2006, 13: 236-249. 10.1038/sj.cdd.4401734.View ArticlePubMedGoogle Scholar
- Bryan NB, Dorfleutner A, Rojanasakul Y, Stehlik C: Activation of inflammasomes requires intracellular redistribution of the apoptotic speck-like protein containing a caspase recruitment domain. J Immunol. 2009, 182: 3173-3182. 10.4049/jimmunol.0802367.View ArticlePubMedPubMed CentralGoogle Scholar
- Masumoto J, Taniguchi S, Nakayama J, Shiohara M, Hidaka E, Katsuyama T, Murase S, Sagara J: Expression of apoptosis-associated speck-like protein containing a caspase recruitment domain, a pyrin N-terminal homology domain-containing protein, in normal human tissues. J Histochem Cytochem. 2001, 49: 1269-1275. 10.1177/002215540104901009.View ArticlePubMedGoogle Scholar
- Richards N, Schaner P, Diaz A, Stuckey J, Shelden E, Wadhwa A, Gumucio DL: Interaction between pyrin and the apoptotic speck protein (ASC) modulates ASC-induced apoptosis. J Biol Chem. 2001, 276: 39320-39329. 10.1074/jbc.M104730200.View ArticlePubMedGoogle Scholar
- Hoffman HM (ed): Infevers NLRP3 sequence variants.http://fmf.igh.cnrs.fr/ISSAID/infevers/search.php?n=4,
- Koike R, Kubota T, Hara Y, Ito S, Suzuki K, Yanagisawa K, Uchibori K, Miyasaka N: A case of Muckle-Wells syndrome caused by a novel H312P mutation in NALP3 (cryopyrin). Mod Rheumatol. 2007, 17: 496-499. 10.3109/s10165-007-0616-5.View ArticlePubMedGoogle Scholar
- Bagnall RD, Roberts RG, Mirza MM, Torigoe T, Prescott NJ, Mathew CG: Novel isoforms of the CARD8 (TUCAN) gene evade a nonsense mutation. Eur J Hum Genet. 2008, 16: 619-625. 10.1038/sj.ejhg.5201996.View ArticlePubMedGoogle Scholar
- Büning C, Schmidt HH, Molnár T, Drenth JP, Fiedler T, Gentz E, Todorov T, Baumgart DC, Sturm A, Nagy F, Lonovics J, de Jong DJ, Landt O, Kage A, Nickel R, Büttner J, Lochs H, Witt H: No association of the CARD8 (TUCAN) c.30 T > A (p.C10X) variant with Crohn’s disease: a study in 3 independent European cohorts. Inflamm Bowel Dis. 2008, 14: 332-337. 10.1002/ibd.20337.View ArticlePubMedGoogle Scholar
- Mariathasan S, Weiss DS, Newton K, McBride J, O’Rourke K, Roose-Girma M, Lee WP, Weinrauch Y, Monack DM, Dixit VM: Cryopyrin activates the inflammasome in response to toxins and ATP. Nature. 2006, 440: 228-232. 10.1038/nature04515.View ArticlePubMedGoogle Scholar
- Gattorno M, Tassi S, Carta S, Delfino L, Ferlito F, Pelagatti MA, D’Osualdo A, Buoncompagni A, Alpigiani MG, Alessio M, Martini A, Rubartelli A: Pattern of interleukin-1β secretion in response to lipopolysaccharide and ATP before and after interleukin-1 blockade in patients with CIAS1 mutations. Arthritis Rheum. 2007, 56: 3138-3148. 10.1002/art.22842.View ArticlePubMedGoogle Scholar
- Stehlik C, Dorfleutner A: COPs and POPs: modulators of inflammasome activity. J Immunol. 2007, 179: 7993-7998. 10.4049/jimmunol.179.12.7993.View ArticlePubMedPubMed CentralGoogle Scholar
- Lee SH, Stehlik C, Reed JC: COP, a caspase recruitment domain-containing protein and inhibitor of caspase-1 activation processing. J Biol Chem. 2001, 276: 34495-34500. 10.1074/jbc.M101415200.View ArticlePubMedGoogle Scholar
- Humke EW, Shriver SK, Starovasnik MA, Fairbrother WJ, Dixit VM: ICEBERG: a novel inhibitor of interleukin-1β generation. Cell. 2000, 103: 99-111. 10.1016/S0092-8674(00)00108-2.View ArticlePubMedGoogle Scholar
- von Kampen O, Lipinski S, Till A, Martin SJ, Nietfeld W, Lehrach H, Schreiber S, Rosenstiel P: Caspase recruitment domain-containing protein 8 (CARD8) negatively regulates NOD2-mediated signaling. J Biol Chem. 2010, 285: 19921-19926. 10.1074/jbc.M110.127480.View ArticlePubMedPubMed CentralGoogle Scholar
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