Summary The NACHT, LRR and PYD domains containing protein (NALP3) inflammasome is a key regulator of interleukin‐1β (IL‐1β) secretion. As there is strong evidence for a pro‐inflammatory role of IL‐1β in rheumatoid arthritis (RA) and in murine models of arthritis, we explored the expression of the different components of the NALP3 inflammasome as well as other nucleotide oligomerization domain (NOD)‐like receptors (NLRs) in synovium obtained from patients with RA. The expression of NLRs was also studied in fibroblast lines derived from joint tissue. By immunohistology, NALP3 and apoptosis‐associated speck‐like protein containing a CARD domain (ASC) were expressed in myeloid and endothelial cells and B cells. T cells expressed ASC but lacked NALP3. In synovial fibroblast lines, NALP3 expression was not detected at the RNA and protein levels and stimulation with known NALP3 agonists failed to induce IL‐1β secretion. Interestingly, we were unable to distinguish RA from osteoarthritis synovial samples on the basis of their basal level of RNA expression of known NLR proteins, though RA samples contained higher levels of caspase‐1 assayed by enzyme‐linked immunsorbent assay. These results indicate that myeloid and endothelial cells are the principal sources of inflammasome‐mediated IL‐1β production in the synovium, and that synovial fibroblasts are unable to activate caspase‐1 because they lack NALP3. The NALP3 inflammasome activity does not account for the difference in level of inflammation between RA and osteoarthritis.
Abstract Because IL-1β plays an important role in inflammation in human and murine arthritis, we investigated the contribution of the inflammasome components ASC, NALP-3, IPAF, and caspase-1 to inflammatory arthritis. We first studied the phenotype of ASC-deficient and wild-type mice during Ag-induced arthritis (AIA). ASC−/− mice showed reduced severity of AIA, decreased levels of synovial IL-1β, and diminished serum amyloid A levels. In contrast, mice deficient in NALP-3, IPAF, or caspase-1 did not show any alteration of joint inflammation, thus indicating that ASC associated effects on AIA are independent of the classical NALP-3 or IPAF inflammasomes. Because ASC is a ubiquitous cytoplasmic protein that has been implicated in multiple cellular processes, we explored other pathways through which ASC may modulate inflammation. Ag-specific proliferation of lymph node and spleen cells from ASC-deficient mice was significantly decreased in vitro, as was the production of IFN-γ, whereas IL-10 production was enhanced. TCR ligation by anti-CD3 Abs in the presence or absence of anti-CD28 Abs induced a reduction in T cell proliferation in ASC−/− T cells compared with wild-type ones. In vivo lymph node cell proliferation was also significantly decreased in ASC−/− mice, but no effects on apoptosis were observed either in vitro or in vivo in these mice. In conclusion, these results strongly suggest that ASC modulates joint inflammation in AIA through its effects on cell-mediated immune responses but not via its implication in inflammasome formation.
Objectives: We tested the effects of the three forms of basic calcium phosphate (BCP) crystals (octacalcium phosphate (OCP), carbonate-substituted apatite (CA) and hydroxyapatite (HA)) on monocytes and macrophages on IL-1β secretion. The requirement for the NALP3 inflammasome and TLR2 and TLR4 receptors in this acute response was analyzed.
Basic calcium phosphate (BCP) crystals are associated with severe osteoarthritis and acute periarticular inflammation. Three main forms of BCP crystals have been identified from pathological tissues: octacalcium phosphate, carbonate-substituted apatite, and hydroxyapatite. We investigated the proinflammatory effects of these BCP crystals in vitro with special regard to the involvement of the NLRP3-inflammasome in THP-1 cells, primary human monocytes and macrophages, and mouse bone marrow-derived macrophages (BMDM). THP-1 cells stimulated with BCP crystals produced IL-1β in a dose-dependent manner. Similarly, primary human cells and BMDM from wild-type mice also produced high concentrations of IL-1β after crystal stimulation. THP-1 cells transfected with short hairpin RNA against the components of the NLRP3 inflammasome and mouse BMDM from mice deficient for NLRP3, apoptosis-associated speck-like protein, or caspase-1 did not produce IL-1β after BCP crystal stimulation. BCP crystals induced macrophage apoptosis/necrosis as demonstrated by MTT and flow cytometric analysis. Collectively, these results demonstrate that BCP crystals induce IL-1β secretion through activating the NLRP3 inflammasome. Furthermore, we speculate that IL-1 blockade could be a novel strategy to inhibit BCP-induced inflammation in human disease.
Background Auto-inflammatory diseases (AID) are characterized by recurrent or chronic febrile episodes due to a defect in the regulation of the inflammatory response linked to the innate immunity. For some AID, a monogenic origin has been established recently. The genetic defect is related or is suspected to be related to a molecular complex called inflammasome, which is involved in the regulation of IL1b, a cytokine with a central role in innate immunity. The inflammasome activates the proinflammatory caspase-1, which then cleaves the pro-IL-1b to IL-1b. PFAPA syndrome is characterized by recurrent fever associated with aphtosis, pharyngitis, and cervical adenitis with a spontaneous resolution in most cases until adolescence. There is no clear aetiology found up to now and recently a familial predominance has been shown suggesting a genetic cause.
To determine the mechanisms involved in inflammatory responses to octacalcium phosphate (OCP) crystals in vivo.OCP crystal-induced inflammation was monitored using a peritoneal model of inflammation in mice with different deficiencies affecting interleukin-1 (IL-1) secretion (IL-1α(-/-) , IL-1β(-/-) , ASC(-/-) , and NLRP3(-/-) mice) or in mice pretreated with IL-1 inhibitors (anakinra [recombinant IL-1 receptor antagonist] and anti-IL-1β). The production of IL-1α, IL-1β, and myeloid-related protein 8 (MRP-8)-MRP-14 complex was determined by enzyme-linked immunosorbent assay. Peritoneal neutrophil recruitment and cell viability were determined by flow cytometry. Depletion of mast cells or resident macrophages was performed by pretreatment with compound 48/80 or clodronate liposomes, respectively.OCP crystals induced peritoneal inflammation, as demonstrated by neutrophil recruitment and up-modulation of IL-1α, IL-1β, and MRP-8-MRP-14 complex, to levels comparable with those induced by monosodium urate monohydrate crystals. This OCP crystal-induced inflammation was both IL-1α- and IL-1β-dependent, as shown by the inhibitory effects of anakinra and anti-IL-1β antibody treatment. Accordingly, OCP crystal stimulation resulted in milder inflammation in IL-1α(-/-) and IL-1β(-/-) mice. Interestingly, ASC(-/-) and NLRP3(-/-) mice did not show any alteration in their inflammation status in response to OCP crystals. Depletion of the resident macrophage population resulted in a significant decrease in crystal-induced neutrophil infiltration and proinflammatory cytokine production in vivo, whereas mast cell depletion had no effect. Finally, OCP crystals induced apoptosis/necrosis of peritoneal cells in vivo.These data indicate that macrophages, rather than mast cells, are important for initiating and driving OCP crystal-induced inflammation. Additionally, OCP crystals induce IL-1-dependent peritoneal inflammation without requiring the NLRP3 inflammasome.
Summary :
The purpose of this study was to investigate the role of the inflammasome in human and experimental murine models (such as ΑΙΑ and K/BxN) of rheumatoid arthritis (RA)RA, affecting 1% of the population is the most frequent inflammatory disease characterized by synovial hyperplasia and cartilage and bone erosion, leading to joint destruction. In general, women are 3 times more affected by RA suggesting a role of estrogen in this disease. The inflammasome is a multiproteic complex triggering the activation of caspase-1 leading to the activation of IL-1 β, an important pro-inflammatory cytokine implicated in arthritis. The inflammasome has been implicated in several inflammatory diseases and particularly in gout.
To highlight a possible role of the inflammasome in murine arthritis, we obtained ASC, caspase-1 and NALP3 +/+ and -/- littermate mice to perform ΑΙΑ and K/BxN arthritis. NALP3 -/- and caspase-1 -/- mice were as arthritic as wild type littermate mice in both ΑΙΑ and K/BxN models implicating that the NALP3 inflammasome is not involved in experimental arthritis. By contrast, ΑΙΑ severity was significantly diminished in ASC- deficient male and female mice, and in the K/BxN model, in ASC-deficient female mice. These results were supported by histological scoring and acute phase protein serum amyloid A (SAA) levels that were equivalent between NALP+/+ and NALP3-/- mice and diminished in ASC -/- mice.
In ΑΙΑ and K/BxN murine experimental models, we observed a sexdependent phenotype. We studied the role of estradiol in both the ALA and the K/BxN models. Castrated female or male ASC -/- mice that received estradiol had a decreased arthritis severity. This implies a protective role of estrogen in the absence of ASC.
In the ΑΙΑ model, proliferation assay were performed using splenocytes from mBSA- immunized ASC +/+ and -/- mice. The mBSA-induced proliferation was significantly lower in ASC-/- splenocytes. Moreover the CD3-specific proliferation of purified splenic Τ cells was significantly lower in ASC-/- cells. Finally, Τ cells from ASC-/- mice produced significantly decreased levels of IFN-gamma associated with increased levels of IL-10. These results imply a possible role of ASC in the TCR-signaling pathway and Τ cell cytokine production.
In parallel the expression of the different inflammasome components were analyzed in biopsies from rheumatoid arthritis (RA) and osteoarthritis (OA) patiens. The expression of the 14 different NALPs, their effector protein ASC, and caspase-1 and -5 was readily measurable by RT-PCR in a similar proportion in RA and OA synovial samples, with the exception of NALP-5 and NALP-13, which weren't found in samples from either disease. The corresponding NALP1, -3, -12 and ASC proteins were expressed at similar levels in both OA and RA biopsies, as determined by immunohistochemistry and Western-blot analysis. By contrast, caspase-1 levels were significantly enhanced in RA synovial tissues compared to those from OA patients. NALP-1, -2, -3, -10, -12 and -14, as well as ASC, caspase-1, and -5 were detected in RNA from unstimulated and stimulated RA synoviocytes. In FLS, only ASC and caspase-1 were expressed at the protein level. NALP1, 3 and 12 were not detected. However, upon stimulation, no secreted IL-Ιβ was detectable in either RA or in OA synoviocytes culture medium.
Resume :
Le but de ce projet etait d'etudier le role de l'inflammasome dans des modeles experimentaux d'arthrite tels que les modeles ΑΙΑ et K/BxN ainsi que dans la polyarthrite humaine (RA). La polyarthrite est une maladie inflammatoire tres frequente avec 1 % de la population affectee et touche 3 fois plus les femmes que les hommes, suggerant un role des hormones sexuelles dans cette pathologie.
L'inflammasome est un complexe multiproteique qui permet l'activation de la caspase-1, une cysteine protease qui va ensuite cliver et activer rinterleukine-ΐβ (IL-Ιβ). L'inflammasome a ete implique ces dernieres annees dans de nombreuses maladies inflammatoires notamment dans la goutte.
Pour mettre en evidence un eventuel role de l'inflammasome dans l'arthrite experimentale nous avons obtenu des souris deficientes pour certains des composants de l'inflammasome tels que ASC, NALP3 et caspase-1. Les souris NALP3 deficientes et caspase-1 deficientes sont aussi arthritiques que les souris wild type correspondantes que ce soit dans le modele ΑΙΑ ou K/BxN. Par contre les souris mâles et femelles ASC-deficientes sont moins arthritiques que les souris +/+ correspondantes dans le modele ΑΙΑ. Dans le modele KRN, le meme phenotype (diminution de la severite de l'arthrite) est observe uniquement chez les femelles ASC-/- Ce phenotype est correle avec l'histologie ainsi qu'avec le dosage du serum amyloid A (SAA) qui reflete l'inflammation systemique et qui est diminue chez les souris ASC-deficientes.
Nous avons ensuite etudie le role de Γ estradiol (une des formes active des estrogenes) dans les modeles K/BxN et ΑΙΑ. Les souris castrees maies ou femelles deficientes pour ASC ayant recu de l'estradiol ont une arthrite moins severe ce qui implique que les estradiol ont un effet protecteur en l'absence de ASC.
Dans le modele ΑΙΑ, nous nous sommes aussi interesses a la reponse immune. Des tests de proliferation ont ete effectues sur des splenocytes en presence de mBSA (qui est l'antigene utilise dans le modele ΑΙΑ). Les splenocytes ASC -/- ont une proliferation qui est diminuee en presence de l'antigene. De plus la proliferation de cellules Τ spleniques purifiees en presence d'anti-CD3 est diminuee chez les cellules Τ ASC-/-. Ces resultats nous indiquent une eventuelle implication de ASC dans la signalisation par le recepteur des cellules T. En parallele l'expression des differents composants de l'inflammasome a ete analysee dans des biopsies de patients atteints de polyarthrite rhumatoide (RA) et d'arthrose (OA). L'expression des 14 differents NALPs, de l'adaptateur ASC, ainsi que des caspase-1 et -5 etait similaires dans les echantillons RA et OA, a l'exception de NALP5 et 13 qui n'etaient pas detectables. L'expression proteique de NALP1, 3, 12 et ASC effectuee par Western blot et immunohistochimie etait similaire dans les biopsies RA et OA. Par contre la quantite de la caspase-1 mesuree par ELISA etait augmentee de facon significative dans les extraits proteiques de biopsies RA.
NALP-1, -2. -3, -10, -12, and -14 ainsi que ASC, caspase-1 et -5 etaient exprimes de facon similaire par les synoviocytes RA non stimules et stimules. Dans les synoviocytes seuls ASC et caspase-1 etaient detectable au niveau proteique. NALP-1, -3 et -12 n'etait pas detectables. Cependant apres stimulation il n'y avait d'IL-Ιβ secrete que ce soit dans les surnageants de cultures de synoviocytes RA ou OA.
