From allergy to schistosomes: role of Fc receptors and adhesion molecules in eosinophil effector function

The dual function of eosinophils has been evidenced in protective immunity against parasites as well as in pathological manifestations during allergic disorders. We have demonstrated that a new class of IgE receptors, FcepsilonRII/CD23, was involved in the functional duality of eosinophils and other proinflammatory cells. More recently, we have shown that FcepsilonRI, the high affinity IgE receptor thought to be only expressed by basophils and mast cells, was involved in eosinophil-mediated cytotoxicity against schistosomes as well as in mediator release. These results favour the view that both IgE and its receptors have been primarily associated to a protective immune response, rather than to pathology. Not only IgE receptors but also members belonging to the family of adhesion molecules can participate as co-receptors in eosinophil effector function. The inhibitory role of monoclonal antibodies to LewisX (LeX, CD15) or to selectins in eosinophil-mediated cytotoxicity towards schistosomes and the detection of LeX and 'selectin-like' molecules on schistosomula surface indicate a double interaction mediated by selectins and their carbohydrate ligands between eosinophils and schistosomula. These results suggest new functions for these adhesion molecules, previously known to be involved mainly in cell infiltration.

Positive and negative roles of the trans-acting T cell factor-1 for the acquisition of distinct Ly-49 MHC class I receptors by NK cells.

Members of the Ly-49 gene family code for class I MHC-specific receptors that regulate NK cell function. Due to a combinatorial distribution of Ly-49 receptors, NK cells display considerable clonal heterogeneity. The acquisition of one Ly-49 receptor, Ly-49A is strictly dependent on the transcriptional trans-acting factor T cell-specific factor-1 (TCF-1). Indeed, TCF-1 binds to two sites in the Ly-49a promoter and regulates its activity, suggesting that the Ly-49a gene is a direct TCF-1 target. TCF-1 deficiency resulted in the altered usage of additional Ly-49 receptors. We show in this study, using TCF-1 beta(2)-microglobulin double-deficient mice, that these repertoire alterations are not due to Ly-49/MHC class I interactions. Our findings rather suggest a TCF-1-dependent, cell autonomous effect on the acquisition of multiple Ly-49 receptors. Besides reduced receptor usage (Ly-49A and D), we also observed no effect (Ly-49C) and significantly expanded (Ly-49G and I) receptor usage in the absence of TCF-1. These effects did not in all cases correlate with the presence of TCF binding sites in the respective proximal promoter. Therefore, besides TCF-1 binding to the proximal promoter, Ly-49 acquisition may also be regulated by TCF-1 binding to more distant cis-acting elements and/or by regulating the expression of additional trans-acting factors. Consistent with the observed differential, positive or negative role of TCF-1 for Ly-49 receptor acquisition, reporter gene assays revealed the presence of an inducing as well as a repressing TCF site in certain proximal Ly-49 promoters. These findings reveal an important role of TCF-1 for the formation of the NK cell receptor repertoire.

Fast subplasma membrane Ca2+ transients control exo-endocytosis of synaptic-like microvesicles in astrocytes

Astrocytes are the most abundant glial cell type in the brain. Although not apposite for long-range rapid electrical communication, astrocytes share with neurons the capacity of chemical signaling via Ca(2+)-dependent transmitter exocytosis. Despite this recent finding, little is known about the specific properties of regulated secretion and vesicle recycling in astrocytes. Important differences may exist with the neuronal exocytosis, starting from the fact that stimulus-secretion coupling in astrocytes is voltage independent, mediated by G-protein-coupled receptors and the release of Ca(2+) from internal stores. Elucidating the spatiotemporal properties of astrocytic exo-endocytosis is, therefore, of primary importance for understanding the mode of communication of these cells and their role in brain signaling. We here take advantage of fluorescent tools recently developed for studying recycling of glutamatergic vesicles at synapses (Voglmaier et al., 2006; Balaji and Ryan, 2007); we combine epifluorescence and total internal reflection fluorescence imaging to investigate with unprecedented temporal and spatial resolution, the stimulus-secretion coupling underlying exo-endocytosis of glutamatergic synaptic-like microvesicles (SLMVs) in astrocytes. Our main findings indicate that (1) exo-endocytosis in astrocytes proceeds with a time course on the millisecond time scale (tau(exocytosis) = 0.24 +/- 0.017 s; tau(endocytosis) = 0.26 +/- 0.03 s) and (2) exocytosis is controlled by local Ca(2+) microdomains. We identified submicrometer cytosolic compartments delimited by endoplasmic reticulum tubuli reaching beneath the plasma membrane and containing SLMVs at which fast (time-to-peak, approximately 50 ms) Ca(2+) events occurred in precise spatial-temporal correlation with exocytic fusion events. Overall, the above characteristics of transmitter exocytosis from astrocytes support a role of this process in fast synaptic modulation.

Tumor necrosis-like weak inducer of apoptosis as a proinflammatory cytokine in human adipocyte cells: up-regulation in severe obesity is mediated by inflammation but not hypoxia.

CONTEXT Adipose tissue hypoxia and endoplasmic reticulum (ER) stress may link the presence of chronic inflammation and macrophage infiltration in severely obese subjects. We previously reported the up-regulation of TNF-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) axis in adipose tissue of severely obese type 2 diabetic subjects. OBJECTIVES The objective of the study was to examine TWEAK and Fn14 adipose tissue expression in obesity, severe obesity, and type 2 diabetes in relation to hypoxia and ER stress. DESIGN In the obesity study, 19 lean, 28 overweight, and 15 obese nondiabetic subjects were studied. In the severe obesity study, 23 severely obese and 35 control subjects were studied. In the type 2 diabetes study, 11 type 2 diabetic and 36 control subjects were studied. The expression levels of the following genes were analyzed in paired samples of sc and visceral adipose tissue: Fn14, TWEAK, VISFATIN, HYOU1, FIAF, HIF-1a, VEGF, GLUT-1, GRP78, and XBP-1. The effect of hypoxia, inflammation, and ER stress on the expression of TWEAK and Fn14 was examined in human adipocyte and macrophage cell lines. RESULTS Up-regulation of TWEAK/Fn14 and hypoxia and ER stress surrogate gene expression was observed in sc and visceral adipose tissue only in our severely obese cohort. Hypoxia modulates TWEAK or Fn14 expression in neither adipocytes nor macrophages. On the contrary, inflammation up-regulated TWEAK in macrophages and Fn14 expression in adipocytes. Moreover, TWEAK had a proinflammatory effect in adipocytes mediated by the nuclear factor-kappaB and ERK but not JNK signaling pathways. CONCLUSIONS Our data suggest that TWEAK acts as a pro-inflammatory cytokine in the adipose tissue and that inflammation, but not hypoxia, may be behind its up-regulation in severe obesity.

Probing the interactions of NK cell receptors with ligand expressed in trans and cis.

Certain receptors on natural killer (NK) cells, which are specific for MHC class I (MHC-I) molecules, do not only interact with ligand expressed on opposing cell membranes (in trans) but also interact with those on the same cell membrane (in cis). Cis interactions have been demonstrated for only a small number of cell surface receptors. However, this has not been tested systematically, raising the possibility that additional receptors may be able to bind ligand expressed in cis. Here we describe a number of approaches to evaluate trans and cis binding of the Ly49A NK cell receptor to its H-2D(d) ligand. These procedures should facilitate the investigation of cis/trans interactions of other receptor-ligand pairs and simplify the analysis of NK cell receptor variants.

Identification of CARDIAK, a RIP-like kinase that associates with caspase-1.

Members of the tumor necrosis factor receptor (TNFR) superfamily have an important role in the induction of cellular signals resulting in cell growth, differentiation and death. TNFR-1 recruits and assembles a signaling complex containing a number of death domain (DD)-containing proteins, including the adaptor protein TRADD and the serine/threonine kinase RIP, which mediates TNF-induced NF-kappa B activation. RIP also recruits caspase-2 to the TNFR-1 signaling complex via the adaptor protein RAIDD, which contains a DD and a caspase-recruiting domain (CARD). Here, we have identified a RIP-like kinase, termed CARDIAK (for CARD-containing interleukin (IL)-1 beta converting enzyme (ICE) associated kinase), which contains a serine/threonine kinase domain and a carboxy-terminal CARD. Overexpression of CARDIAK induced the activation of both NF-kappa B and Jun N-terminal kinase (JNK). CARDIAK interacted with the TNFR-associated factors TRAF-1 and TRAF-2, and a dominant-negative form of TRAF-2 inhibited CARDIAK-induced NF-kappa B activation. Interestingly, CARDIAK specifically interacted with the CARD of caspase-1 (previously known as ICE), and this interaction correlated with the processing of pro-caspase-1 and the formation of the active p20 subunit of caspase-1. Together, these data suggest that CARDIAK may be involved in NF-kappa B/JNK signaling and in the generation of the proinflammatory cytokine IL-1 beta through activation of caspase-1.

Exendin-(9-39) is an inverse agonist of the murine glucagon-like peptide-1 receptor: implications for basal intracellular cyclic adenosine 3',5'-monophosphate levels and beta-cell glucose competence.

The effect of exendin-(9-39), a described antagonist of the glucagon-like peptide-1 (GLP-1) receptor, was evaluated on the formation of cAMP- and glucose-stimulated insulin secretion (GSIS) by the conditionally immortalized murine betaTC-Tet cells. These cells have a basal intracellular cAMP level that can be increased by GLP-1 with an EC50 of approximately 1 nM and can be decreased dose dependently by exendin-(9-39). This latter effect was receptor dependent, as a beta-cell line not expressing the GLP-1 receptor was not affected by exendin-(9-39). It was also not due to the endogenous production of GLP-1, because this effect was observed in the absence of detectable preproglucagon messenger RNA levels and radioimmunoassayable GLP-1. Importantly, GSIS was shown to be sensitive to this basal level of cAMP, as perifusion of betaTC-Tet cells in the presence of exendin-(9-39) strongly reduced insulin secretion. This reduction of GSIS, however, was observed only with growth-arrested, not proliferating, betaTC-Tet cells; it was also seen with nontransformed mouse beta-cells perifused in similar conditions. These data therefore demonstrated that 1) exendin-(9-39) is an inverse agonist of the murine GLP-1 receptor; 2) the decreased basal cAMP levels induced by this peptide inhibit the secretory response of betaTC-Tet cells and mouse pancreatic islets to glucose; 3) as this effect was observed only with growth-arrested cells, this indicates that the mechanism by which cAMP leads to potentiation of insulin secretion is different in proliferating and growth-arrested cells; and 4) the presence of the GLP-1 receptor, even in the absence of bound peptide, is important for maintaining elevated intracellular cAMP levels and, therefore, the glucose competence of the beta-cells.

The N-terminal DNA-binding 'zinc finger' of the oestrogen and glucocorticoid receptors determines target gene specificity.

Steroid hormone receptors activate specific gene transcription by binding as hormone-receptor complexes to short DNA enhancer-like elements termed hormone response elements (HREs). We have shown previously that a highly conserved 66 amino acid region of the oestrogen (ER) and glucocorticoid (GR) receptors, which corresponds to part of the receptor DNA binding domain (region C) is responsible for determining the specificity of target gene activation. This region contains two sub-regions (CI and CII) analogous to the 'zinc-fingers' of the transcription factor TFIIIA. We show here that CI and CII appear to be separate domains both involved in DNA binding. Furthermore, using chimaeric ERs in which either the first (N-terminal) (CI) or second (CII) 'zinc finger' region has been exchanged with that of the GR, indicates that it is the first 'zinc finger' which largely determines target gene specificity. We suggest that receptor recognition of the HRE is analogous to that of the helix-turn-helix DNA binding motif in that the receptor binds to DNA as a dimer with the first 'zinc finger' lying in the major groove recognizing one half of the palindromic HRE, and that protein-DNA interaction is stabilized through non-specific DNA binding and dimer interactions contributed by the second 'zinc finger'.

Distinct conformations of Ly49 natural killer cell receptors mediate MHC class I recognition in trans and cis.

Certain cell-surface receptors engage ligands expressed on juxtaposed cells and ligands on the same cell. The structural basis for trans versus cis binding is not known. Here, we showed that Ly49 natural killer (NK) cell receptors bound two MHC class I (MHC-I) molecules in trans when the two ligand-binding domains were backfolded onto the long stalk region. In contrast, dissociation of the ligand-binding domains from the stalk and their reorientation relative to the NK cell membrane allowed monovalent binding of MHC-I in cis. The distinct conformations (backfolded and extended) define the structural basis for cis-trans binding by Ly49 receptors and explain the divergent functional consequences of cis versus trans interactions. Further analyses identified specific stalk segments that were not required for MHC-I binding in trans but were essential for inhibitory receptor function. These data identify multiple distinct roles of stalk regions for receptor function.

Identificació i caracterització funcional dels complexes proteics dels receptors BRL1 i BRL3 en el teixit vascular d’Arabidopsis Thaliana

Els esteroids juguen papers clau en el creixement I el desenvolupament d’eucariotes multicel•lulars. En plantes, aquestes hormones, anomenades Brassinosteroides (BRs), estan involucrades en una gran varietat de processos biològics essencials per a les plantes. S’han descrit anteriorment dos receptors de BRs del tipus Leucine Rich Repeat Receptor Like Kinase LRR-RLK, BRASSINOSTEROID RECEPTOR LIKE 1 i 3 (BRL1 i BRL3 respectivalemt) que són homòlegs al receptor principal BRI1 i són necessaris pel desenvolupament vascular. Tot i que els principals components de la senyal ja han estat identificats pel seu homòleg més pròxim, el receptor BRI1, els complexes de BRL1 i BRL3 juntament amb els candidats co-receptors així com els components de la ruta de senyalització encara no han sigut identificats. Per tal d’entendre millor la funció molecular d’aquests receptors de BRs en la planta aquesta tesis doctoral planteja dues aproximacions: com a primera aproximació, vaig realitzar un estudi fenotípic del desenvolupament del teixit vascular a la planta model Arabidopsis thaliana (Arabidopsis). Disposant d'una amplia bateria de mutants de síntesis de la hormona i senyalització del receptor BRI1, vam analitzar quantitativament el seu patró vascular a la tija d'Arabidopsis. Vam establir els paràmetres en les plantes silvestres [Col-0 wild type, (WT)] i els vam analitzar a tots i cadascun dels mutants. Això conjuntament amb una col•laboració amb la Dr. Marta Ibañes, física de la Universitat de Barcelona que va construir un model matemàtic per simular la formació del patró vascular ens va permetre el•laborar una hipòtesis que vam demostrar experimentalment i va ser publicada a la revista PNAS. Posteriorment vam observar que les plantes knock-out d'aquests dos receptors BRL1 y BRL3 a diferència de BRI1, no tenien cap fenotip obvi en el teixit vascular de la planta adulta. Així, a continuació, per entendre quina necessitat té la planta de disposar de tres receptors tant altament homòlegs que poden percebre la mateixa hormona, vam utilitzar una aproximació bioquímica en col•laboració amb el Prof. de Vries de la Universitat de Wageningen (Holanda) per tal de purificar els complexes dels receptors in vivo i els seus interactors. Això ens ha permès entendre millor el paper funcional d'aquests receptors en la planta. Els resultats d’aquests experiments estan resumits en un article en preparació que aviat estarà en revisió.

Role of the cannabinoid system in the effects induced by nicotine on anxiety-like behaviour in mice

Rationale: Acute behavioural effects and motivational responses induced by nicotine can be modulated by the endocannabinoid system supporting the existence of a physiological interaction between these two systems. Objectives: The present study was designed to examine the possible involvement of the cannabinoid system in the anxiolytic- and anxiogenic-like responses induced by nicotine in mice. Methods: Animals were only exposed once to nicotine. The acute administration of low (0.05, sc) or high (0.8 mg/kg, sc) doses of nicotine produced opposite effects in the elevated plus-maze, i.e., anxiolytic- and anxiogenic-like responses, respectively. The effects of the pretreatment with the CB1 cannabinoid receptor antagonist, rimonabant (0.25, 0.5 and 1 mg/kg, ip), and the cannabinoid agonist, 9-tetrahydrocannabinol (0.1 mg/kg, ip), were evaluated on the anxiolytic- and anxiogenic-like responses induced by nicotine. Results: Rimonabant completely abolished nicotine-induced anxiolytic-like effects and increased the anxiogenic-like responses of nicotine, suggesting an involvement of CB1 receptors in these behavioural responses. On the other hand, 9-tetrahydrocannabinol failed to modify nicotine anxiolytic-like responses, but attenuated its anxiogenic-like effects. In addition the association of non-effective doses of 9-tetrahydrocannabinol and nicotine produced clear anxiolytic-like responses. Conclusions: These results demonstrate that the endogenous cannabinoid system is involved in the regulation of nicotine anxiety-like behaviour in mice, and provide new findings to support the use of cannabinoid antagonists in the treatment of tobacco addiction.

Glucagon-like peptide-1 protects beta-cells against apoptosis by increasing the activity of an IGF-2/IGF-1 receptor autocrine loop.

OBJECTIVE: The gluco-incretin hormones glucagon-like peptide (GLP)-1 and gastric inhibitory peptide (GIP) protect beta-cells against cytokine-induced apoptosis. Their action is initiated by binding to specific receptors that activate the cAMP signaling pathway, but the downstream events are not fully elucidated. Here we searched for mechanisms that may underlie this protective effect. RESEARCH DESIGN AND METHODS: We performed comparative transcriptomic analysis of islets from control and GipR(-/-);Glp-1-R(-/-) mice, which have increased sensitivity to cytokine-induced apoptosis. We found that IGF-1 receptor expression was markedly reduced in the mutant islets. Because the IGF-1 receptor signaling pathway is known for its antiapoptotic effect, we explored the relationship between gluco-incretin action, IGF-1 receptor expression and signaling, and apoptosis. RESULTS: We found that GLP-1 robustly stimulated IGF-1 receptor expression and Akt phosphorylation and that increased Akt phosphorylation was dependent on IGF-1 but not insulin receptor expression. We demonstrated that GLP-1-induced Akt phosphorylation required active secretion, indicating the presence of an autocrine activation mechanism; we showed that activation of IGF-1 receptor signaling was dependent on the secretion of IGF-2. We demonstrated, both in MIN6 cell line and primary beta-cells, that reducing IGF-1 receptor or IGF-2 expression or neutralizing secreted IGF-2 suppressed GLP-1-induced protection against apoptosis. CONCLUSIONS: An IGF-2/IGF-1 receptor autocrine loop operates in beta-cells. GLP-1 increases its activity by augmenting IGF-1 receptor expression and by stimulating secretion; this mechanism is required for GLP-1-induced protection against apoptosis. These findings may lead to novel ways of preventing beta-cell loss in the pathogenesis of diabetes.

Desensitization and phosphorylation of the glucagon-like peptide-1 (GLP-1) receptor by GLP-1 and 4-phorbol 12-myristate 13-acetate.

Glucagon-like peptide-1 (GLP-1) stimulates glucose-induced insulin secretion by binding to a specific G protein-coupled receptor linked to activation of the adenylyl cyclase pathway. Here, using insulinoma cell lines, we studied homologous and heterologous desensitization of GLP-1-induced cAMP production. Preexposure of the cells to GLP-1 induced a decrease in GLP-1-mediated cAMP production, as assessed by a 3- to 5-fold rightward shift of the dose-response curve and an approximately 20 percent decrease in the maximal production of cAMP. Activation of protein kinase C by the phorbol ester phorbol 12-myristate 13-acetate (PMA) also induced desensitization of the GLP-1-mediated response, leading to a 6- to 9-fold shift in the EC50 and a 30% decrease in the maximal production of cAMP. Both forms of desensitization were additive, and the protein kinase C inhibitor RO-318220 inhibited PMA-induced desensitization, but not agonist-induced desensitization. GLP-1- and PMA-dependent desensitization correlated with receptor phosphorylation, and the levels of phosphorylation induced by the two agents were additive. Furthermore, PMA-induced, but not GLP-1-induced, phosphorylation was totally inhibited by RO-318220. Internalization of the GLP-1 receptor did not participate in the desensitization induced by PMA, as a mutant GLP-1 receptor lacking the last 20 amino acids of the cytoplasmic tail was found to be totally resistant to the internalization process, but was still desensitized after PMA preexposure. PMA and GLP-1 were not able to induce the phosphorylation of a receptor deletion mutant lacking the last 33 amino acids of the cytoplasmic tail, indicating that the phosphorylation sites were located within the deleted region. The cAMP production mediated by this deletion mutant was not desensitized by PMA and was only poorly desensitized by GLP-1. Together, our results indicate that the production of cAMP and, hence, the stimulation of insulin secretion induced by GLP-1 can be negatively modulated by homologous and heterologous desensitization, mechanisms that involve receptor phosphorylation.

Peroxisome proliferator-activated receptors A link between endocrinology and nutrition?

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily like the steroid, thyroid, or retinoid hormone receptors, which are ligand-activated transcription factors regulating gene expression. PPARs mediate the induction of the enzymes of the peroxisomal and microsomal fatty-acid oxidation pathways by hypolipidemic drugs such as clofibrate and are probably also involved in the gene expression of other lipid-metabolism-associated proteins that are controlled by fibrate hypolipidemic drugs. That PPARs play an important role in the regulation of lipid metabolism is reinforced by the discovery of their activation by physiologic concentrations of fatty acids. This observation raises the question of whether fatty acids are ligands of PPARs, which would imply that nutritional fatty acids can act like hormones.

Characterization of NALP2 and NALP3 IL-1β processing inflammasomes

Résumé II y a cinq ans, la découverte d'un nouveau domaine, le PYD domaine, lié aux domaines de la mort, a permis la description de la nouvelle famille des NALP protéines. L'analyse structurelle de cette famille de protéines révéla la présence de deux autres domaines, impliqués dans l'oligomerisation, NACHT, et la détection des ligands, Leucine rich repeats ou LRR. Cette architecture protéique est homologue à celle qui est décrite pour les NODs, les Tol1 récepteurs et tes protéines de résistance chez les plantes. Cette homologie suggère une possible implication des NALPs dans la régulation de l'immunité innée. Premièrement, nous avons décrit les composants minimaux qui permettent à l'inflammasomeNALP3 d'activer la caspase pro-inflammatoire, caspase-1. En comparaison à NALP1, NALP3 ne contient pas de FIIND domaine, ni de CARD domaine en C-terminus et n'interagit pas avec caspase-5. Nous avons découvert une protéine très homologue au C-terminus de NALP1, Cardinal, qui se lie au NACHT domaine de NALP2 et NALP3 par l'intermédiaire de son FIIND domaine. Cardinal possède la capacité d'interagir avec caspase-l, mais seul ASC semble être nécessaire à la maturation de la prointerleukine-1β suite à la stimulation de NALP3. Deuxièmement, notre étude s'est concentrée sur la nature du stimulus capable d'induire la formation et l'activation de l'inflammasome-NALP3. Nous avons démontré que l'ajout de muramyl dipeptide (MDP), produit à partir de la digestion enzymatique de peptidoglycaris bactériens, induit à la fois l'expression de la proIL-1β par la voie NOD2 et sa maturation en IL-1β active par la voie NALP3. Bien que le MDP active l'inflammasome-NALP3, il est incapable d'induire la sécrétion de l'IL-1β mature dans la lignée cellulaire THP1, comparé aux monocytes primaires humains. Cette différence pourrait être liée à l'absence, dans les THP1, de la protéine Filamin, qui est proposée d'interagir avec Cardinal. L'implication de NALP3 dans la maturation de l'IL-lb est confirmée suite à la découverte de mutations sur le gène CIAS1/NALP3/cryopyrin associées à trois maladies auto-inflammatoires : le syndrome de Muckle-Wells (MWS), l'urticaire familial au froid (FCU) et le syndrome CINCA/NOMID. Une élévation constitutive de la maturation et de la sécrétion de la proIL-1β en absence de stimulation MDP est détectée dans les macrophages des patients Muckle-Wells. En conclusion, nos études ont démontré que l'inflammasome-NALP3 doit être finement régulé pour éviter une activité incontrôlée qui représente la base moléculaire des symptômes associés aux syndromes auto-inflammatoires liés à NALP3. Summary Five years ago, the description of the NALP family originated from the discovery of a new death-domain fold family, the PYD domain. NALP contains aprotein-protein interaction domain (PYD), an oligomerization domain (NACHT) and a ligand-sensing domain, leucine rich repeats or LRR. This protein architecture shares similarity with receptors involved in immunity, such as NODS, Toll receptors (TLRs) and related plant resistance proteins, and points to an important role of NALPs in defense mechanisms. We first described the minimal complex involved in the pro-inflammatory Interleukin-1beta (IL-1β) cytokine maturation, called the inflammasome, which contains NALP3. In contrast to NALP1, NALP3, like other members of the NALP family, is devoid of C-terminal FIIND and CARD domains and does not interact with the pro-inflammatory caspase-5. Interestingly, a homolog of the C-terminal portion of NALP1 was found in the human genome and was named Cardinal. We found that NALP2 and NALP3 interact with the CARD-containing proteins Cardinal. Cardinal is able to bind to caspase-1 but is not required for IL-1β maturation through NALP3 activation, as demonstrated for the adaptor ASC. Secondly, our study focused on the stimuli involved in the activation of the NALP3 inflammasome. MDP was shown to induce the expression of proIL1β through NOD2 and then the maturation into active IL-1β by activation of the NALP3 inflammasome. However, in the monocytic THP1 cell line, secretion of IL-1β upon MDP stimulation seems to be independent of the inflammasome activation compared to human primary monocytes. This difference might be linked to a Cardinal-interacting protein, filamin. Until now, the role of Cardinal and filamin is still unknown and remains to be elucidated. Finally, mutations in the NALP3/cryopyrin/CIAS1 gene are associated with three autoinflammatory diseases: Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and CINCA. Constitutive, elevated IL-1β maturation and secretion, even in the absence of MDP stimulation, was observed in macrophages from Muckle-Wells patients and confirmed a key role for the NALP3 inflammasome in innate immunity In conclusion, our studies describes the formation of the NALP3 inflammasome and suggests that this complex has to be tightly regulated to avoid an increased deregulated inflammasome activity that is the molecular basis for the symptoms associated with NALP3-dependent autoinflammatory disorders.