RESUMEN
BACKGROUND: Urticarial lesions are observed in both cutaneous and systemic disorders. Familial forms of urticarial syndromes are rare and can be encountered in systemic autoinflammatory diseases. OBJECTIVE: We sought to investigate a large family with dominantly inherited chronic urticarial lesions associated with hypercytokinemia. METHODS: We performed a genetic linkage analysis in 14 patients from a 5-generation family, as well as whole-exome sequencing, cytokine profiling, and transcriptomic analyses on samples from 2 patients. The identified candidate protein was studied after in vitro expression of the corresponding normal and mutated recombinant proteins. An unsupervised proteomic approach was used to unveil the associated protein network. RESULTS: The disease phenotype of the most affected family members is characterized by chronic urticarial flares associated with extremely high plasma levels of proinflammatory (IL-1ß, IL-6, and TNF-α) and anti-inflammatory (IL-10 and IL-1 receptor antagonist [IL-1RA]) cytokines, with no secondary organ dysfunction, no susceptibility to infections, no fever, and normal C-reactive protein levels. Monocyte transcriptomic analyses identified an immunotolerant profile in the most affected patient. The affected family members carried a loss-of-function mutation in RNF213 that encodes mysterin, a protein with a poorly known physiologic role. We identified the deubiquitinase CYLD, a major regulator of inflammation, as an RNF213 partner and showed that CYLD expression is inhibited by wild-type but not mutant RNF213. CONCLUSION: We identified a new entity characterized by chronic urticarial lesions associated with a clinically blunted hypercytokinemia. This disease, which is due to loss of function of RNF213, reveals mysterin's key role in the complex molecular network of innate immunity.
Asunto(s)
Síndrome de Liberación de Citoquinas , Proteómica , HumanosRESUMEN
OBJECTIVE: To identify the molecular basis of a systemic autoinflammatory disorder (SAID) evocative of TNF receptor-associated periodic syndrome (TRAPS). METHODS: (i) Deep next generation sequencing (NGS) through a SAID gene panel; (ii) variant allele distribution in peripheral blood subpopulations; (iii) in silico analyses of mosaic variants using TNF receptor superfamily 1A (TNFRSF1A) crystal structure; (iv) review of the very rare TNFRSF1A mosaic variants reported previously. RESULTS: In a 36-year-old man suffering from recurrent fever for 12 years, high-depth NGS revealed a TNFRSF1A mosaic variant, c.176G>A p.(Cys59Tyr), which Sanger sequencing failed to detect. This mosaic variant displayed a variant allele fraction of 14% in whole blood; it affects both myeloid and lymphoid lineages. p.(Cys59Tyr), a recurrent germline pathogenic variant, affects a crucial cysteine located in the first cysteine-rich domain (CRD1) and involved in a disulphide bridge. Introduction of a tyrosine at this position is expected to disrupt the CRD1 structure. Review of the three previously reported TNFRSF1A mosaic variants revealed that they are all located in a small region of CRD2 and that germinal cells can be affected. CONCLUSION: This study expands the localization of TNFRSF1A mosaic variants to the CRD1 domain. Noticeably, residues involved in germline TNFRSF1A mutational hot spots can also be involved in post-zygotic mutational events. Including our study, only four patients have been thus far reported with TNFRSF1A mosaicism, highlighting the need for a high-depth NGS-based approach to avoid the misdiagnosis of TRAPS. Genetic counselling has to consider the potential occurrence of TNFRSF1A mosaic variants in germinal cells.
Asunto(s)
Cisteína , Enfermedades Autoinflamatorias Hereditarias , Masculino , Humanos , Adulto , Cisteína/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Fiebre/genética , Enfermedades Autoinflamatorias Hereditarias/genética , Enfermedades Autoinflamatorias Hereditarias/diagnóstico , MutaciónRESUMEN
BACKGROUND: NLRP3-associated autoinflammatory diseases (NLRP3-AIDs) include conditions of various severities, due to germline or somatic mosaic NLRP3 mutations. OBJECTIVE: To identify mosaic- versus germline-specific NLRP3 mutations' characteristics, we reinterpreted all the mutations reported in NLRP3-AIDs and performed an in-depth study of 3 novel patients. METHODS: The pathogenicity of all reported mosaic/germline mutations was reassessed according to international recommendations and their location on the NLRP3 3-dimensional structure. Deep-targeted sequencing and NLRP3-inflammasome-activation assays were used to identify the disease-causing mutation in 3 patients. RESULTS: We identified, in 3 patients, mosaic mutations affecting the same NLRP3 amino acid (Glu569). This residue belongs to 1 of the 2 mosaic mutational hot spots that face each other in the core of the NLRP3 ATPase domain. The review of the 90 NLRP3 mutations identified in 277 patients revealed that those hot spots account for 68.5% of patients (37 of 54) with mosaic mutations. Glu569 is affected in 22% of the patients (12 of 54) with mosaic mutations and in 0.4% of patients (1 of 223) with germline mutations. Only 8 of 90 mutations were found in mosaic and germinal states. All of the germline mutations were associated with a severe phenotype. These data suggest that mutations found only in mosaic state could be incompatible with life if present in germinal state. None of the 5 most frequent germline mutations was identified in mosaic state. Mutations found only in germinal state could, therefore, be asymptomatic in mosaic state. CONCLUSIONS: The phenotypic spectrum of NLRP3-AIDs appears to be related to the germinal/mosaic status and localization of the underlying mutations.
Asunto(s)
Enfermedades Autoinmunes/genética , Inflamasomas/metabolismo , Inflamación/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Preescolar , Cristalografía por Rayos X , Femenino , Mutación de Línea Germinal/genética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inflamasomas/genética , Masculino , Persona de Mediana Edad , Proteína con Dominio Pirina 3 de la Familia NLR/química , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Fenotipo , Conformación Proteica , Índice de Severidad de la Enfermedad , Células THP-1RESUMEN
OBJECTIVE: Atherosclerosis is an inflammatory disease, where activated immunocompetent cells, including dendritic cells (DCs) and T cells are abundant in plaques. Low-density lipoprotein modified either by oxidation (oxLDL) or by human group X-secreted phospholipase A2 (LDLx) and heat shock proteins (HSP), especially HSP60 and 90, have been implicated in atherosclerosis. We previously reported that Annexin A5 inhibits inflammatory effects of phospholipids, decreases vascular inflammation and improves vascular function in apolipoprotein E(-/-) mice. Here, we focus on the LDLx effects on human DCs and T cells. APPROACH AND RESULTS: Human DCs were differentiated from peripheral blood monocytes, stimulated by oxLDL or LDLx. Naive autologous T cells were cocultured with pretreated DCs. oxLDL and LDLx, in contrast to LDL, induced DC-activation and T-cell proliferation. T cells exposed to LDLx-treated DCs produced interferon-γ, interleukin (IL)-17 but not IL-4 and IL-10. Annexin A5 abrogated LDLx effects on DCs and T cells and increased production of transforming growth factor-ß and IL-10. Furthermore, IL-10 producing T cells suppressed primary T-cell activation via soluble IL-10, transforming growth factor-ß, and cell-cell contact. Lentiviral-mediated shRNA knock-down HSP60 and 90 in DCs attenuated the effect of LDLx on DCs and subsequent T-cell proliferation. Experiments on DC and T cells derived from carotid atherosclerotic plaques gave similar results. CONCLUSIONS: Our data show that modified forms of LDL such as LDLx but not native LDL activate human T cells through DCs. HSP60 and 90 contribute to such T-cell activation. Annexin A5 promotes induction of regulatory T cells and is potentially interesting as a therapeutic agent.
Asunto(s)
Anexina A5/metabolismo , Comunicación Celular , Chaperonina 60/metabolismo , Células Dendríticas/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Lipoproteínas LDL/metabolismo , Activación de Linfocitos , Proteínas Mitocondriales/metabolismo , Subgrupos de Linfocitos T/metabolismo , Enfermedades de las Arterias Carótidas/inmunología , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Chaperonina 60/genética , Técnicas de Cocultivo , Citocinas/metabolismo , Células Dendríticas/inmunología , Fosfolipasas A2 Grupo X/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Humanos , Mediadores de Inflamación/metabolismo , Proteínas Mitocondriales/genética , Placa Aterosclerótica , Interferencia de ARN , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , TransfecciónRESUMEN
OBJECTIVE: Several secreted phospholipases A2 (sPLA2s), including group IIA, III, V, and X, have been linked to the development of atherosclerosis, which led to the clinical testing of A-002 (varespladib), a broad sPLA2 inhibitor for the treatment of coronary artery disease. Group X sPLA2 (PLA2G10) has the most potent hydrolyzing activity toward phosphatidylcholine and is believed to play a proatherogenic role. METHODS AND RESULTS: Here, we show that Ldlr(-/-) mice reconstituted with bone marrow from mouse group X-deficient mice (Pla2g10(-/-)) unexpectedly display a doubling of plaque size compared with Pla2g10(+/+) chimeric mice. Macrophages of Pla2g10(-/-) mice are more susceptible to apoptosis in vitro, which is associated with a 4-fold increase of plaque necrotic core in vivo. In addition, chimeric Pla2g10(-/-) mice show exaggerated T lymphocyte (Th)1 immune response, associated with enhanced T-cell infiltration in atherosclerotic plaques. Interestingly, overexpression of human PLA2G10 in murine bone marrow cells leads to significant reduction of Th1 response and to 50% reduction of lesion size. CONCLUSIONS: PLA2G10 expression in bone marrow cells controls a proatherogenic Th1 response and limits the development of atherosclerosis. The results may provide an explanation for the recently reported inefficacy of A-002 (varespladib) to treat patients with coronary artery disease. Indeed, A-002 is a nonselective sPLA2 inhibitor that inhibits both proatherogenic (groups IIA and V) and antiatherogenic (group X) sPLA2s. Our results suggest that selective targeting of individual sPLA2 enzymes may be a better strategy to treat cardiovascular diseases.
Asunto(s)
Aorta Torácica/enzimología , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Fosfolipasas A2 Grupo X/metabolismo , Receptores de LDL/deficiencia , Inmunidad Adaptativa , Animales , Aorta Torácica/inmunología , Aorta Torácica/patología , Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/patología , Apoptosis , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Trasplante de Médula Ósea , Células Cultivadas , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Fosfolipasas A2 Grupo X/deficiencia , Fosfolipasas A2 Grupo X/genética , Humanos , Macrófagos/inmunología , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Necrosis , Placa Aterosclerótica , Receptores de LDL/genética , Células TH1/inmunología , Factores de TiempoRESUMEN
BACKGROUND: Our previous work showed that dietary oxidized linoleic acid given, as a single fatty acid, to LDL receptor knockout mice decreased weight gain as compared to control mice. Other studies have also reported that animals fed oils heated for 24 h or greater showed reduced weight gain. These observations, while important, have limited significance since fried foods in the typical human diet do not contain the extreme levels of oxidized lipids used in these studies. The main goal of this study was to investigate the effects of a diet containing soybean oil heated for 3 h on weight gain and fat pad mass in mice. Additionally, because PPARγ and UCP-1 mediate adipocyte differentiation and thermogenesis, respectively, the effect of this diet on these proteins was also examined. FINDINGS: Four to six week old male C57BL/6 J mice were randomly divided into three groups and given either a low fat diet with heated soybean oil (HSO) or unheated soybean oil (USO) or pair fed for 16 weeks. Weight and food intake were monitored and fat pads were harvested upon the study's termination. Mice consuming the HSO diet had significantly increased fat pad mass but gained less weight as compared to mice in the USO group despite a similar caloric intake and similar levels of PPARγ and UCP1. CONCLUSION: This is the first study to show that a diet containing soybean oil heated for a short time increases fat mass despite a decreased weight gain in C57BL/6 J mice. The subsequent metabolic consequences of this increased fat mass merits further investigation.
Asunto(s)
Tejido Adiposo/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Dieta con Restricción de Grasas , Grasas de la Dieta/administración & dosificación , Aceite de Soja/farmacología , Tejido Adiposo/metabolismo , Animales , Composición Corporal/efectos de los fármacos , Culinaria , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Energía/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Calor , Humanos , Canales Iónicos/genética , Canales Iónicos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Aceite de Soja/química , Proteína Desacopladora 1 , Aumento de Peso/efectos de los fármacosRESUMEN
A20 haploinsufficiency (HA20) is an autoinflammatory disease caused by heterozygous loss-of-function variations in TNFAIP3, the gene encoding the A20 protein. Diagnosis of HA20 is challenging due to its heterogeneous clinical presentation and the lack of pathognomonic symptoms. While the pathogenic effect of TNFAIP3 truncating variations is clearly established, that of missense variations is difficult to determine. Herein, we identified a novel TNFAIP3 variation, p.(Leu236Pro), located in the A20 ovarian tumor (OTU) domain and demonstrated its pathogenicity. In the patients' primary cells, we observed reduced A20 levels. Protein destabilization was predicted in silico for A20_Leu236Pro and enhanced proteasomal degradation was confirmed in vitro through a flow cytometry-based functional assay. By applying this approach to the study of another missense variant, A20_Leu275Pro, for which no functional characterization has been performed to date, we showed that this variant also undergoes enhanced proteasomal degradation. Moreover, we showed a disrupted ability of A20_Leu236Pro to inhibit the NF-κB pathway and to deubiquitinate its substrate TRAF6. Structural modeling revealed that two residues involved in OTU pathogenic missense variations (i.e. Glu192Lys and Cys243Tyr) establish common interactions with Leu236. Interpretation of newly identified missense variations is challenging, requiring, as illustrated here, functional demonstration of their pathogenicity. Together with functional studies, in silico structure analysis is a valuable approach that allowed us (i) to provide a mechanistic explanation for the haploinsufficiency resulting from missense variations and (ii) to unveil a region within the OTU domain critical for A20 function.
Asunto(s)
Mutación Missense , FN-kappa B , Humanos , FN-kappa B/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genéticaRESUMEN
OBJECTIVE: To identify the molecular basis of a severe systemic autoinflammatory disorder (SAID) and define its main phenotypic features, and to functionally assess the sequence variations identified in LYN, a gene encoding a nonreceptor tyrosine kinase. METHODS: We used targeted next-generation sequencing and in vitro functional studies of Lyn phosphorylation state and Lyn-dependent NF-κB activity after expression of recombinant Lyn isoforms carrying different sequence variations. RESULTS: We identified a de novo LYN variation (p.Tyr508His) in a patient presenting since birth with recurrent fever, chronic urticaria, atopic dermatitis, arthralgia, increased inflammatory biomarkers, and elevated plasma cytokine levels. We studied the consequences on Lyn phosphorylation state of the p.Tyr508His variation and of the 2 LYN variations reported so far (p.Tyr508Phe and p.Tyr508*), and found that all 3 variations prevent phosphorylation of residue 508 and lead to autophosphorylation of Tyr397. Additionally, these 3 LYN variations activate the NF-κB pathway. These results show a gain-of-function effect of the variations involving Tyr508 on Lyn activity. CONCLUSION: This study demonstrates the pathogenicity of the first 3 LYN variations identified in SAID patients and delineates the phenotypic spectrum of a disease entity characterized by severe, early-onset, systemic inflammatory disease affecting neonates with no family history of SAID. All 3 LYN variations affect the same tyrosine residue located in the C-terminus of Lyn, thereby demonstrating the critical role of this residue in the proper regulation of Lyn activity in humans.
Asunto(s)
FN-kappa B , Familia-src Quinasas , Recién Nacido , Humanos , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo , FN-kappa B/metabolismo , Mutación con Ganancia de Función , Fosforilación , Proteínas Tirosina QuinasasRESUMEN
Secreted phospholipases A2 (sPLA2s) are present in atherosclerotic plaques and are now considered novel attractive therapeutic targets and potential biomarkers as they contribute to the development of atherosclerosis through lipoprotein-dependent and independent mechanisms. We have previously shown that hGX-sPLA2-phospholipolyzed LDL (LDL-X) induces proinflammatory responses in human umbilical endothelial cells (HUVECs); here we explore the molecular mechanisms involved. Global transcriptional gene expression profiling of the response of endothelial cells exposed to either LDL or LDL-X revealed that LDL-X activates multiple distinct cellular pathways including the unfolded protein response (UPR). Mechanistic insight showed that LDL-X activates UPR through calcium depletion of intracellular stores, which in turn disturbs cytoskeleton organization. Treatment of HUVECs and aortic endothelial cells (HAECs) with LDL-X led to activation of all 3 proximal initiators of UPR: eIF-2alpha, IRE1alpha, and ATF6. In parallel, we observed a sustained phosphorylation of the p38 pathway resulting in the phosphorylation of AP-1 downstream targets. This was accompanied by significant production of the proinflammatory cytokines IL-6 and IL-8. Our study demonstrates that phospholipolyzed LDL uses a range of molecular pathways including UPR to initiate endothelial cell perturbation and thus provides an LDL oxidation-independent mechanism for the initiation of vascular inflammation in atherosclerosis.
Asunto(s)
Retículo Endoplásmico/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Lipoproteínas LDL/farmacología , Factor de Transcripción Activador 6/metabolismo , Western Blotting , Células Cultivadas , Endorribonucleasas/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Técnica del Anticuerpo Fluorescente , Silenciador del Gen , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipoproteínas LDL/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfolipasas A2 Secretoras , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Venas Umbilicales/citología , Respuesta de Proteína DesplegadaRESUMEN
OBJECTIVE: To study the association of PAF-acetyl hydrolase (PAFAH) activity with inflammation, oxidative stress, and atherosclerosis in hypercholesterolemic swine. METHODS AND RESULTS: Cholesterol-rich diet feeding of miniature pigs was associated with an increase in PAFAH activity and an increase of the PAFAH to PON1 ratio. PLA2G7 RNA (coding for PAFAH) expression was increased in blood monocytes and plaque macrophages. Increased PAFAH activity was associated with higher plasma lysophosphatidylcholine and correlated with oxidized LDL. In THP1 monocytes and macrophages and in human blood-derived macrophages, oxidized LDL induced PLA2G7 RNA expression. Atherogenic diet feeding induced the accumulation of macrophages and oxidized LDL in the arterial wall leading to atherosclerosis. PAFAH activity correlated positively with plaque size and TNFalpha expression in plaque macrophages. CONCLUSIONS: We demonstrated that an increase in PAFAH activity was associated with increased levels of lysophosphatidylcholine, oxidized LDL, and inflammation, resulting in accelerated atherosclerosis in hypercholesterolemic minipigs. The significant correlation between PLA2G7 RNA expression in plaque macrophages and plasma PAFAH activity suggests that the latter is a consequence, rather than a cause of macrophage accumulation. Our cell experiments suggest that oxidized LDL can induce PAFAH, resulting in accumulation of lysophosphatidylcholine that increases the inflammatory action of oxidized LDL.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa/metabolismo , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Hipercolesterolemia/complicaciones , Hipercolesterolemia/metabolismo , Inflamación/etiología , Inflamación/metabolismo , Metabolismo de los Lípidos , 1-Alquil-2-acetilglicerofosfocolina Esterasa/genética , Secuencia de Aminoácidos , Animales , Arildialquilfosfatasa/metabolismo , Aterosclerosis/genética , Aterosclerosis/patología , Vasos Coronarios/patología , Dieta Aterogénica , Expresión Génica , Humanos , Hipercolesterolemia/genética , Inflamación/genética , Macrófagos/metabolismo , Macrófagos/patología , Datos de Secuencia Molecular , Oxidación-Reducción , Estrés Oxidativo , ARN/genética , ARN/metabolismo , Porcinos , Porcinos EnanosRESUMEN
Chronic urticaria is a common skin disorder with heterogeneous causes. In the absence of physical triggers, chronic urticarial rash is called idiopathic or spontaneous. The objective of this study was to identify the molecular and cellular bases of a disease condition displayed by two unrelated patients aged over 60 years who presented for two decades with a chronic urticaria resistant to standard therapy that occurred in the context of systemic inflammation not triggered by cold. In both patients, a targeted sequencing approach using a next generation technology identified somatic mosaic mutations in NLRP3, a gene encoding a key inflammasome component. The study of several of both patients' cell types showed that, despite the late onset of the disease, NLRP3 mutations were not found to be restricted to myelomonocytic cells. Rather, the data obtained strongly suggested that the mutational event occurred very early, during embryonic development. As shown by functional studies, the identified mutations-an in-frame deletion and a recurrent NLRP3 missense mutation-have a gain-of-function effect on NLRP3-inflammasome activation. Consistently, a complete remission was obtained in both patients with anti-IL-1 receptor antagonists. This study unveils that in late-onset chronic urticaria, the search for autoinflammatory markers and somatic mosaic NLRP3 mutations may have important diagnostic and therapeutic consequences.
Asunto(s)
Urticaria Crónica/genética , ADN/genética , Inflamasomas/genética , Mutación , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Anciano , Urticaria Crónica/metabolismo , Análisis Mutacional de ADN , Femenino , Humanos , Inflamasomas/metabolismo , Masculino , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
Aims: Oxidative stress and inflammation play a pathogenic role in atherosclerosis. Thioredoxin-1 (Trx-1) is an anti-oxidative, anti-inflammatory protein with atheroprotective effects. However, in vivo cleavage of Trx-1 generates a truncated pro-inflammatory protein, Trx-80, which compromises the therapeutic use of Trx-1. Here we analysed whether the thioredoxin-mimetic peptide (TxMP), CB3 might exert anti-oxidative, anti-inflammatory, and atheroprotective effects in ApoE2.Ki mice. Methods and results: We synthesized a small TxMP, Ac-Cys-Pro-Cys-amide, CB3 and characterized its antioxidant and anti-inflammatory effects on cultured peritoneal murine macrophages. CB3 significantly and dose-dependently reduced the level of reactive oxygen species in lipopolysaccharides (LPS)-activated macrophages. In addition, it efficiently lowered LPS-induced inflammatory process through NF-κB inhibition, as evidenced by the reduced secretion of monocyte chemoattractant protein-1, interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α by macrophages. Nevertheless, CB3 did not affect cholesterol accumulation in macrophages. A daily-administered dose of 10 µg/g body weight CB3 to ApoE2.Ki mice on high fat diet did not affect plasma of total cholesterol and triglycerides levels but significantly reduced the plasma levels of pro-inflammatory cytokines (IL-33 and TNF-α) and oxidative markers. In contrast, it significantly induced the plasma levels of anti-inflammatory proteins (adiponectin, IL-10). In addition, CB3 reduced the number of pro-inflammatory M1 macrophages in spleen and decreased the ratio of M1/M2 macrophages in atherosclerotic lesion areas. Finally, CB3 significantly reduced the surface area of aortic lesions. Conclusions: Our results clearly showed that similar to the full length Trx-1, CB3 exerts protective effects, by reducing inflammation and oxidative stress in macrophages and in ApoE2.Ki mice. The atheroprotective effect of CB3 opens promising therapeutic approaches for treatment of atherosclerosis.
Asunto(s)
Antiinflamatorios/farmacología , Antioxidantes/farmacología , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Dieta Alta en Grasa , Mediadores de Inflamación/metabolismo , Imitación Molecular , Oligopéptidos/farmacología , Estrés Oxidativo/efectos de los fármacos , Placa Aterosclerótica , Compuestos de Sulfhidrilo/farmacología , Animales , Antiinflamatorios/síntesis química , Antioxidantes/síntesis química , Aorta/efectos de los fármacos , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Oligopéptidos/síntesis química , Transducción de Señal , Compuestos de Sulfhidrilo/síntesis química , Tiorredoxinas/metabolismoRESUMEN
Circulating serum amyloid A (SAA) is increased in various inflammatory conditions. The human SAA protein family comprises the acute phase SAA1/SAA2, known to activate a large set of innate and adaptive immune cells, and the constitutive SAA4. The liver synthesis of SAA1/SAA2 is well-established but there is still an open debate on extrahepatic SAA expression especially in macrophages. We aimed to investigate the ability of human primary monocytes and monocyte-derived macrophages to express SAA1, SAA2 and SAA4 at both the transcriptional and protein levels, as previous studies almost exclusively dealt with monocytic cell lines. Monocytes and derived macrophages from healthy donors were stimulated under various conditions. In parallel with SAA, pro-inflammatory IL1A, IL1B and IL6 cytokine expression was assessed. While LPS alone was non-effective, a combined LPS/dexamethasone treatment induced SAA1 and to a lesser extent SAA2 transcription in human monocytes and macrophages. In contrast, as expected, pro-inflammatory cytokine expression was strongly induced following stimulation with LPS, an effect which was dampened in the presence of dexamethasone. Furthermore, in monocytes polarized towards a pro-inflammatory M1 phenotype, SAA expression in response to LPS/dexamethasone was potentiated; a result mainly seen for SAA1. However, a major discrepancy was observed between SAA mRNA and intracellular protein levels under the experimental conditions used. Our results demonstrate that human monocytes and macrophages can express SAA genes, mainly SAA1 in response to an inflammatory environment. While SAA is considered as a member of a large cytokine network, its expression in the monocytes-macrophages in response to LPS-dexamethasone is strikingly different from that observed for classic pro-inflammatory cytokines. As monocytes-macrophages are major players in chronic inflammatory diseases, it may be hypothesized that SAA production from macrophages may contribute to the local inflammatory microenvironment, especially when macrophages are compactly organized in granulomas as in sarcoidosis.
Asunto(s)
Inflamación/sangre , Proteína Amiloide A Sérica/genética , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Voluntarios Sanos , Humanos , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/patología , Interleucina-1alfa/sangre , Interleucina-1beta/sangre , Interleucina-6/sangre , Lipopolisacáridos/toxicidad , Hígado/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismoRESUMEN
OBJECTIVE: To determine the molecular and cellular bases of autoinflammatory syndromes in a multigenerational French family with Muckle-Wells syndrome and in a patient originating from Portugal with familial cold autoinflammatory syndrome. METHODS: Sequencing of NLRP3 exon 3 was performed in all accessible patients. Microsatellite and whole-genome single nucleotide polymorphism genotyping was used i) to test the intrafamilial segregation of the identified variant and ii) to look for a founder effect. Functional analyses included the study of i) apoptosis-associated speck-like protein containing a CARD (ASC) speck formation in HEK293T cells (stably expressing ASC-green fluorescent protein and pro-caspase 1-FLAG) transiently expressing the wild-type or mutated NLRP3 protein, ii) levels of IL-1ß secreted from transfected THP-1 cells, and iii) inflammasome-related gene expression and cytokine secretion from monocytes isolated from patients in crisis (probands from the two families), related patients out of crisis, and from controls. RESULTS: The same heterozygous mutation (c.1322C>T, p.A441V) located in the NACHT domain, segregating with the disease within the first family, was identified in the two families. This mutation was found to be associated with different core haplotypes. NLRP3-A441V led to increased ASC speck formation and high levels of secreted IL-1ß. Monocyte inflammasome-related gene expression and cytokine secretion, which were within the normal range in patients out of crisis, were found to be differentially regulated between the two probands, correlating with their phenotypic status. CONCLUSION: These molecular and cellular findings, which indicate a recurrent mutational event, clearly demonstrate the pathogenicity of the p.A441V missense mutation in NLRP3-associated autoinflammatory disease and point to the interest of studying patients' primary cells to assess disease activity.
RESUMEN
It has been proposed that apolipoprotein J (apo J) and paraoxonase-1 (PON1) correlate with the extent and severity of ischemic heart disease (IHD). This article compares apo J and PON1 serum concentrations, PON1 activity, and the apo J/PON1 ratio in 138 IHD patients (64 statins users and 74 statin nonusers) referred for angiography and possible percutaneous coronary intervention. The effect of statin treatment on apo J and PON1 concentrations, PON1 activity, and the degree of coronary artery stenosis were evaluated. In both groups, apo J levels were increased, whereas PON1 concentration and activity decreased. IHD patients on statins had significantly lower apo J concentration and higher PON1 concentration and activity. Patients on statins had less coronary artery stenosis. High apo J levels, low PON1 levels, low PON1 activity, and a high apo J/PON1 ratio were associated with IHD. Statin treatment reverses these changes, probably by multiple beneficial actions.
Asunto(s)
Arildialquilfosfatasa/sangre , Clusterina/sangre , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Isquemia Miocárdica/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Arildialquilfosfatasa/efectos de los fármacos , Clusterina/efectos de los fármacos , Angiografía Coronaria , Estenosis Coronaria/sangre , Estenosis Coronaria/diagnóstico por imagen , Estenosis Coronaria/tratamiento farmacológico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Isquemia Miocárdica/sangre , Isquemia Miocárdica/diagnóstico por imagen , Índice de Severidad de la EnfermedadRESUMEN
Photoaging and epithelial skin tumorigenesis are complex processes triggered mainly by UV radiation from chronic sun exposure. This leads to DNA damage and reactive oxygen species (ROS) production, which initiate an inflammatory response that alters cell structure and function. Changes in cell homeostasis and ROS production activate intracellular multiprotein platforms called inflammasomes. Inflammasomes nucleate around cytoplasmic receptors mainly of the NLR (nucleotide-binding domain and leucine-rich repeat) family and regulate caspase-1-dependant secretion of pro-inflammatory interleukin (IL)1ß and IL18 cytokines, and an inflammatory form of death named pyroptosis. NLRP1 inflammasomes have taken centre stage in skin biology, as mutations in NLRP1 underlie the genetic etiology of dermatological diseases and increase the susceptibility to skin cancer. Targeting inflammasome(s) might be an important approach to improve skin inflammation, photoaging and reduce the risk of epithelial skin tumorigenesis. In this context, we discuss the potential implication of NLRP1 and NLRP3 inflammasomes.
Asunto(s)
Envejecimiento de la Piel/efectos de la radiación , Neoplasias Cutáneas/metabolismo , Rayos Ultravioleta/efectos adversos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Humanos , Inflamasomas/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas NLR , Proteínas de Neoplasias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
Inflammasomes are intracellular multiprotein signaling complexes, mainly present in myeloid cells. They commonly assemble around a cytoplasmic receptor of the nucleotide-binding leucine-rich repeat containing receptor (NLR) family, although other cytoplasmic receptors like pyrin have been shown to form inflammasomes. The nucleation of the multiprotein scaffolding platform occurs upon detection of a microbial, a danger or a homeostasis pattern by the receptor that will, most commonly, associate with the adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) through homotypic domain interactions resulting in recruitment of procaspase-1. This will lead to the autoproteolytic activation of caspase-1, which regulates the secretion of proinflammatory IL1ß and IL18 cytokines and pyroptosis, a caspase-1-mediated form of cell death. Pyroptosis occurs through cleavage of Gasdermin D, a membrane pore forming protein. Recently, non-canonical inflammasomes have been described, which directly sense intracellular pathogens through caspase-4 and -5 in humans, leading to pyroptosis. Inflammasomes are important in host defense; however, a deregulated activity is associated with a number of inflammatory, immune and metabolic disorders. Furthermore, mutations in inflammasome receptor coding genes are causal for an increasing number of rare autoinflammatory diseases. Biotherapies targeting the products of inflammasome activation as well as molecules that directly or indirectly inhibit inflammasome nucleation and activation are promising therapeutic areas. This review discusses recent advances in inflammasome biology, the molecular pathology of several inflammasomes, and current therapeutic approaches in autoinflammatory diseases and in selected common multifactorial inflammasome-mediated disorders.
Asunto(s)
Inflamasomas/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteína con Dominio Pirina 3 de la Familia NLR/fisiología , Pirina/fisiología , Animales , Humanos , Inflamación/genética , Inflamación/inmunologíaRESUMEN
Mutations in the nucleotide-binding oligomerization domain protein 12 (NLRP12) cause recurrent episodes of serosal inflammation. Here we show that NLRP12 efficiently sequesters HSP90 and promotes K48-linked ubiquitination and degradation of NOD2 in response to bacterial muramyl dipeptide (MDP). This interaction is mediated by the linker-region proximal to the nucleotide-binding domain of NLRP12. Consequently, the disease-causing NLRP12 R284X mutation fails to repress MDP-induced NF-κB and subsequent activity of the JAK/STAT signaling pathway. While NLRP12 deficiency renders septic mice highly susceptible towards MDP, a sustained sensing of MDP through NOD2 is observed among monocytes lacking NLRP12. This loss of tolerance in monocytes results in greater colonization resistance towards Citrobacter rodentium. Our data show that this is a consequence of NOD2-dependent accumulation of inflammatory mononuclear cells that correlates with induction of interferon-stimulated genes. Our study unveils a relevant process of tolerance towards the gut microbiota that is exploited by an attaching/effacing enteric pathogen.
Asunto(s)
Acetilmuramil-Alanil-Isoglutamina/metabolismo , Cápsulas Bacterianas/metabolismo , Citrobacter rodentium/inmunología , Infecciones por Enterobacteriaceae/inmunología , Proteínas HSP90 de Choque Térmico/metabolismo , Tolerancia Inmunológica/inmunología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Animales , Línea Celular , Infecciones por Enterobacteriaceae/microbiología , Microbioma Gastrointestinal/inmunología , Células HEK293 , Humanos , Inflamación/inmunología , Inflamación/microbiología , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Noqueados , FN-kappa B/metabolismo , UbiquitinaciónRESUMEN
Plasma Platelet-activating-Factor (PAF)-acetylhydrolase (PAF-AH also named lipoprotein-PLA(2) or PLA(2)G7 gene) is secreted by macrophages, it degrades PAF and oxidation products of phosphatidylcholine produced upon LDL oxidation and/or oxidative stress, and thus is considered as a potentially anti-inflammatory enzyme. Cloning of PAF-AH has sustained tremendous promises towards the use of PAF-AH recombinant protein in clinical situations. The reason for that stems from the numerous animal models of inflammation, atherosclerosis or sepsis, where raising the levels of circulating PAF-AH either through recombinant protein infusion or through the adenoviral gene transfer showed to be beneficial. Unfortunately, neither in human asthma nor in sepsis the recombinant PAF-AH showed sufficient efficacy. One of the most challenging questions nowadays is as to whether PAF-AH is pro- or anti-atherogenic in humans, as PAF-AH may possess a dual pro- and anti-inflammatory role, depending on the concentration and the availability of potential substrates. It is equally possible that the plasma level of PAF-AH is a diagnostic marker of ongoing atherosclerosis.