RESUMEN
The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.
Asunto(s)
Síndromes de Inmunodeficiencia , Proteínas del Tejido Nervioso , Ubiquitinas , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Femenino , Masculino , FN-kappa B/metabolismo , Ubiquitina-Proteína Ligasas/genética , Inflamación/inmunología , Inflamación/genética , Linfocitos B/inmunología , Mutación con Pérdida de Función , Fibroblastos/metabolismo , Fibroblastos/inmunología , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Ratones , AlelosRESUMEN
Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by homozygous or compound heterozygous gain-of-function mutations in MEFV, which encodes pyrin, an inflammasome protein. Heterozygous carrier frequencies for multiple MEFV mutations are high in several Mediterranean populations, suggesting that they confer selective advantage. Among 2,313 Turkish people, we found extended haplotype homozygosity flanking FMF-associated mutations, indicating evolutionarily recent positive selection of FMF-associated mutations. Two pathogenic pyrin variants independently arose >1,800 years ago. Mutant pyrin interacts less avidly with Yersinia pestis virulence factor YopM than with wild-type human pyrin, thereby attenuating YopM-induced interleukin (IL)-1ß suppression. Relative to healthy controls, leukocytes from patients with FMF harboring homozygous or compound heterozygous mutations and from asymptomatic heterozygous carriers released heightened IL-1ß specifically in response to Y. pestis. Y. pestis-infected MefvM680I/M680I FMF knock-in mice exhibited IL-1-dependent increased survival relative to wild-type knock-in mice. Thus, FMF mutations that were positively selected in Mediterranean populations confer heightened resistance to Y. pestis.
Asunto(s)
Resistencia a la Enfermedad/genética , Fiebre Mediterránea Familiar/genética , Peste , Pirina/genética , Selección Genética/genética , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Resistencia a la Enfermedad/inmunología , Haplotipos , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Peste/inmunología , Peste/metabolismo , Pirina/inmunología , Pirina/metabolismo , Turquía , Factores de Virulencia/inmunología , Factores de Virulencia/metabolismo , Yersinia pestisRESUMEN
Mutations in the genes encoding pyrin and mevalonate kinase (MVK) cause distinct interleukin-1ß (IL-1ß)-mediated autoinflammatory diseases: familial Mediterranean fever (FMF) and hyperimmunoglobulinemia D syndrome (HIDS). Pyrin forms an inflammasome when mutant or in response to bacterial modification of the GTPase RhoA. We found that RhoA activated the serine-threonine kinases PKN1 and PKN2 that bind and phosphorylate pyrin. Phosphorylated pyrin bound to 14-3-3 proteins, regulatory proteins that in turn blocked the pyrin inflammasome. The binding of 14-3-3 and PKN proteins to FMF-associated mutant pyrin was substantially decreased, and the constitutive IL-1ß release from peripheral blood mononuclear cells of patients with FMF or HIDS was attenuated by activation of PKN1 and PKN2. Defects in prenylation, seen in HIDS, led to RhoA inactivation and consequent pyrin inflammasome activation. These data suggest a previously unsuspected fundamental molecular connection between two seemingly distinct autoinflammatory disorders.
Asunto(s)
Fiebre Mediterránea Familiar/metabolismo , Inflamasomas/metabolismo , Deficiencia de Mevalonato Quinasa/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Pirina/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteínas 14-3-3/metabolismo , Adolescente , Adulto , Animales , Células Cultivadas , Niño , Femenino , Humanos , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Mutación/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteína Quinasa C/metabolismo , Pirina/genética , Transducción de Señal , Adulto Joven , Proteínas de Unión al GTP rho/genética , Proteína de Unión al GTP rhoARESUMEN
Deficiency in mevalonate kinase (MVK) causes systemic inflammation. However, the molecular mechanisms linking the mevalonate pathway to inflammation remain obscure. Geranylgeranyl pyrophosphate, a non-sterol intermediate of the mevalonate pathway, is the substrate for protein geranylgeranylation, a protein post-translational modification that is catalyzed by protein geranylgeranyl transferase I (GGTase I). Pyrin is an innate immune sensor that forms an active inflammasome in response to bacterial toxins. Mutations in MEFV (encoding human PYRIN) result in autoinflammatory familial Mediterranean fever syndrome. We found that protein geranylgeranylation enabled Toll-like receptor (TLR)-induced activation of phosphatidylinositol-3-OH kinase (PI(3)K) by promoting the interaction between the small GTPase Kras and the PI(3)K catalytic subunit p110δ. Macrophages that were deficient in GGTase I or p110δ exhibited constitutive release of interleukin 1ß that was dependent on MEFV but independent of the NLRP3, AIM2 and NLRC4 inflammasomes. In the absence of protein geranylgeranylation, compromised PI(3)K activity allows an unchecked TLR-induced inflammatory responses and constitutive activation of the Pyrin inflammasome.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Fiebre Mediterránea Familiar/metabolismo , Inflamasomas/metabolismo , Macrófagos/fisiología , Mutación/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Pirina/genética , Transferasas Alquil y Aril/genética , Animales , Células Cultivadas , Fiebre Mediterránea Familiar/genética , Humanos , Inmunidad Innata , Interleucina-1beta/metabolismo , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfatos de Poliisoprenilo/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal , Receptores Toll-Like/metabolismoRESUMEN
RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.
Asunto(s)
Caspasa 8/metabolismo , Enfermedades Autoinflamatorias Hereditarias/metabolismo , Mutación , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Caspasa 3/metabolismo , Femenino , Enfermedades Autoinflamatorias Hereditarias/genética , Enfermedades Autoinflamatorias Hereditarias/patología , Humanos , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linaje , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genéticaRESUMEN
OBJECTIVES: To study the molecular pathogenesis of PAPA (pyogenic arthritis, pyoderma gangrenosum and acne) syndrome, a debilitating hereditary autoinflammatory disease caused by dominant mutation in PSTPIP1. METHODS: Gene knock-out and knock-in mice were generated to develop an animal model. THP1 and retrovirally transduced U937 human myeloid leukaemia cell lines, peripheral blood mononuclear cells, small interfering RNA (siRNA) knock-down, site-directed mutagenesis, cytokine immunoassays, coimmunoprecipitation and immunoblotting were used to study inflammasome activation. Cytokine levels in the skin were evaluated by immunohistochemistry. Responsiveness to Janus kinase (JAK) inhibitors was evaluated ex vivo with peripheral blood mononuclear cells and in vivo in five treatment-refractory PAPA patients. RESULTS: The knock-in mouse model of PAPA did not recapitulate the human disease. In a human myeloid cell line model, PAPA-associated PSTPIP1 mutations activated the pyrin inflammasome, but not the NLRP3, NLRC4 or AIM2 inflammasomes. Pyrin inflammasome activation was independent of the canonical pathway of pyrin serine dephosphorylation and was blocked by the p.W232A PSTPIP1 mutation, which disrupts pyrin-PSTPIP1 interaction. IFN-γ priming of monocytes from PAPA patients led to IL-18 release in a pyrin-dependent manner. IFN-γ was abundant in the inflamed dermis of PAPA patients, but not patients with idiopathic pyoderma gangrenosum. Ex vivo JAK inhibitor treatment attenuated IFN-γ-mediated pyrin induction and IL-18 release. In 5/5 PAPA patients, the addition of JAK inhibitor therapy to IL-1 inhibition was associated with clinical improvement. CONCLUSION: PAPA-associated PSTPIP1 mutations trigger a pyrin-IL-18-IFN-γ positive feedback loop that drives PAPA disease activity and is a target for JAK inhibition.
Asunto(s)
Acné Vulgar , Artritis Infecciosa , Modelos Animales de Enfermedad , Inflamasomas , Interferón gamma , Piodermia Gangrenosa , Piodermia Gangrenosa/genética , Humanos , Animales , Ratones , Acné Vulgar/inmunología , Inflamasomas/metabolismo , Inflamasomas/inmunología , Interferón gamma/metabolismo , Inhibidores de las Cinasas Janus/uso terapéutico , Inhibidores de las Cinasas Janus/farmacología , Ratones Noqueados , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Retroalimentación Fisiológica , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Pirina/genética , Mutación , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Técnicas de Sustitución del Gen , Interleucina-18/metabolismo , Células THP-1RESUMEN
BACKGROUND: Familial Mediterranean fever (FMF), caused by mutations in the pyrin-encoding MEFV gene, is characterized by uncontrolled caspase-1 activation and IL-1ß secretion. A similar mechanism drives inflammation in cryopyrin-associated periodic fever syndrome (CAPS) caused by mutations in NLRP3. CAPS and FMF, however, result in largely different clinical manifestations, pointing to additional, autoinflammatory pathways involved in FMF. Another hallmark of FMF is extraordinarily high expression of S100A8 and S100A9. These alarmins are ligands of Toll-like receptor 4 and amplifiers of inflammation. However, the relevance of this inflammatory pathway for the pathogenesis of FMF is unknown. OBJECTIVE: This study investigated whether mutations in pyrin result in specific secretion of S100A8/A9 alarmins through gasdermin D pores' amplifying FMF pathology. METHODS: S100A8/A9 levels in FMF patients were quantified by enzyme-linked immunosorbent assay. In vitro models with knockout cell lines and specific protein inhibitors were used to unravel the S100A8/A9 secretion mechanism. The impact of S100A8/A9 to the pathophysiology of FMF was analyzed with FMF (MEFVV726A/V726A) and S100A9-/- mouse models. Pyrin-S100A8/A9 interaction was investigated by coimmunoprecipitation, immunofluorescence, and enzyme-linked immunosorbent assay studies. RESULTS: The S100A8/A9 complexes directly interacted with pyrin. Knocking out pyrin, caspase-1, or gasdermin D inhibited the secretion of these S100 alarmins. Inflammatory S100A8/A9 dimers were inactivated by tetramer formation. Blocking this inactivation by targeted S100A9 deletion in a murine FMF model demonstrated the relevance of this novel autoinflammatory pathway in FMF. CONCLUSION: This is the first proof that members of the S100 alarmin family are released in a pyrin/caspase-1/gasdermin D-dependent pathway and directly drive autoinflammation in vivo.
Asunto(s)
Síndromes Periódicos Asociados a Criopirina , Fiebre Mediterránea Familiar , Animales , Ratones , Alarminas , Calgranulina A/genética , Caspasas/metabolismo , Síndromes Periódicos Asociados a Criopirina/genética , Fiebre Mediterránea Familiar/genética , Gasderminas , Inflamación , Pirina/genéticaRESUMEN
BACKGROUND: Autoinflammatory diseases, a diverse group of inherited conditions characterized by excessive innate immune activation, have limited therapeutic options. Neuroimmune circuits of the inflammatory reflex control innate immune overactivation and can be stimulated to treat disease using the acetylcholinesterase inhibitor galantamine. METHODS: We tested the efficacy of galantamine in a rodent model of the prototypical autoinflammatory disease familial Mediterranean fever (FMF). Multiple chronic disease markers were evaluated in animals that received long-term galantamine treatment compared to vehicle. RESULTS: Long-term treatment with galantamine attenuated the associated splenomegaly and anemia which are characteristic features of this disease. Further, treatment reduced inflammatory cell infiltration into affected organs and a subcutaneous air pouch. CONCLUSIONS: These findings suggest that galantamine attenuates chronic inflammation in this mouse model of FMF. Further research is warranted to explore the therapeutic potential of galantamine in FMF and other autoinflammatory diseases.
Asunto(s)
Fiebre Mediterránea Familiar , Ratones , Animales , Fiebre Mediterránea Familiar/tratamiento farmacológico , Galantamina/farmacología , Galantamina/uso terapéutico , Acetilcolinesterasa/uso terapéutico , Modelos Animales de Enfermedad , Inflamación/tratamiento farmacológicoRESUMEN
Missense mutations in the C-terminal B30.2 domain of pyrin cause familial Mediterranean fever (FMF), the most common Mendelian autoinflammatory disease. However, it remains controversial as to whether FMF is due to the loss of an inhibitor of inflammation or to the activity of a proinflammatory molecule. We generated both pyrin-deficient mice and "knockin" mice harboring mutant human B30.2 domains. Homozygous knockin, but not pyrin-deficient, mice exhibited spontaneous bone marrow-dependent inflammation similar to but more severe than human FMF. Caspase-1 was constitutively activated in knockin macrophages and active IL-1ß was secreted when stimulated with lipopolysaccharide alone, which is also observed in FMF patients. The inflammatory phenotype of knockin mice was completely ablated by crossing with IL-1 receptor-deficient or adaptor molecule ASC-deficient mice, but not NLRP3-deficient mice. Thus, our data provide evidence for an ASC-dependent NLRP3-independent inflammasome in which gain-of-function pyrin mutations cause autoinflammatory disease.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Proteínas Portadoras/inmunología , Proteínas del Citoesqueleto/genética , Mutación , Inmunidad Adaptativa , Animales , Enfermedades Autoinmunes/patología , Células Cultivadas , Femenino , Humanos , Inflamación/inmunología , Inflamación/patología , Interleucina-1beta/inmunología , Macrófagos/inmunología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Pirina , Receptores de Interleucina-1/deficiencia , Receptores de Interleucina-1/inmunologíaRESUMEN
The NLRP3 inflammasome is an intracellular innate immune sensor that is expressed in immune cells, including monocytes and macrophages. Activation of the NLRP3 inflammasome leads to IL-1ß secretion. Gain-of-function mutations of NLRP3 result in abnormal activation of the NLRP3 inflammasome, and cause the autosomal dominant systemic autoinflammatory disease spectrum, termed cryopyrin-associated periodic syndromes (CAPS). Here, we show that a missense mutation, p.Arg918Gln (c.2753G > A), of NLRP3 causes autosomal-dominant sensorineural hearing loss in two unrelated families. In family LMG446, hearing loss is accompanied by autoinflammatory signs and symptoms without serologic evidence of inflammation as part of an atypical CAPS phenotype and was reversed or improved by IL-1ß blockade therapy. In family LMG113, hearing loss segregates without any other target-organ manifestations of CAPS. This observation led us to explore the possibility that resident macrophage/monocyte-like cells in the cochlea can mediate local autoinflammation via activation of the NLRP3 inflammasome. The NLRP3 inflammasome can indeed be activated in resident macrophage/monocyte-like cells in the mouse cochlea, resulting in secretion of IL-1ß. This pathway could underlie treatable sensorineural hearing loss in DFNA34, CAPS, and possibly in a wide variety of hearing-loss disorders, such as sudden sensorineural hearing loss and Meniere's disease that are elicited by pathogens and processes that stimulate innate immune responses within the cochlea.
Asunto(s)
Pérdida Auditiva Sensorineural/genética , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Adulto , Animales , Secuencia de Bases , Proteínas Portadoras/metabolismo , Cóclea/metabolismo , Síndromes Periódicos Asociados a Criopirina/genética , Síndromes Periódicos Asociados a Criopirina/metabolismo , Sordera/genética , Familia , Femenino , Pérdida Auditiva , Pérdida Auditiva Sensorineural/metabolismo , Humanos , Inflamasomas/metabolismo , Inflamación/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/fisiología , Linaje , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/metabolismo , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Systemic autoinflammatory diseases are caused by mutations in genes that function in innate immunity. Here, we report an autoinflammatory disease caused by loss-of-function mutations in OTULIN (FAM105B), encoding a deubiquitinase with linear linkage specificity. We identified two missense and one frameshift mutations in one Pakistani and two Turkish families with four affected patients. Patients presented with neonatal-onset fever, neutrophilic dermatitis/panniculitis, and failure to thrive, but without obvious primary immunodeficiency. HEK293 cells transfected with mutated OTULIN had decreased enzyme activity relative to cells transfected with WT OTULIN, and showed a substantial defect in the linear deubiquitination of target molecules. Stimulated patients' fibroblasts and peripheral blood mononuclear cells showed evidence for increased signaling in the canonical NF-κB pathway and accumulated linear ubiquitin aggregates. Levels of proinflammatory cytokines were significantly increased in the supernatants of stimulated primary cells and serum samples. This discovery adds to the emerging spectrum of human diseases caused by defects in the ubiquitin pathway and suggests a role for targeted cytokine therapies.
Asunto(s)
Alelos , Endopeptidasas/genética , Fibroblastos/patología , Enfermedades Autoinflamatorias Hereditarias/genética , Leucocitos Mononucleares/patología , Mutación , Edad de Inicio , Niño , Preescolar , Consanguinidad , Citocinas/genética , Citocinas/inmunología , Dermatitis/fisiopatología , Endopeptidasas/deficiencia , Endopeptidasas/inmunología , Insuficiencia de Crecimiento/fisiopatología , Femenino , Fiebre/fisiopatología , Fibroblastos/enzimología , Fibroblastos/inmunología , Regulación de la Expresión Génica , Células HEK293 , Enfermedades Autoinflamatorias Hereditarias/diagnóstico , Enfermedades Autoinflamatorias Hereditarias/enzimología , Enfermedades Autoinflamatorias Hereditarias/patología , Humanos , Leucocitos Mononucleares/enzimología , Leucocitos Mononucleares/inmunología , Masculino , FN-kappa B/genética , FN-kappa B/inmunología , Paniculitis/fisiopatología , Linaje , Transducción de Señal , Ubiquitina/genética , Ubiquitina/inmunologíaRESUMEN
Mutations in the gene encoding NLRP3 cause a spectrum of autoinflammatory diseases known as cryopyrin-associated periodic syndromes (CAPS). NLRP3 is a key component of one of several distinct cytoplasmic multiprotein complexes (inflammasomes) that mediate the maturation of the proinflammatory cytokine interleukin-1ß (IL-1ß) by activating caspase-1. Although several models for inflammasome activation, such as K(+) efflux, generation of reactive oxygen species and lysosomal destabilization, have been proposed, the precise molecular mechanism of NLRP3 inflammasome activation, as well as the mechanism by which CAPS-associated mutations activate NLRP3, remain to be elucidated. Here we show that the murine calcium-sensing receptor (CASR) activates the NLRP3 inflammasome, mediated by increased intracellular Ca(2+) and decreased cellular cyclic AMP (cAMP). Ca(2+) or other CASR agonists activate the NLRP3 inflammasome in the absence of exogenous ATP, whereas knockdown of CASR reduces inflammasome activation in response to known NLRP3 activators. CASR activates the NLRP3 inflammasome through phospholipase C, which catalyses inositol-1,4,5-trisphosphate production and thereby induces release of Ca(2+) from endoplasmic reticulum stores. The increased cytoplasmic Ca(2+) promotes the assembly of inflammasome components, and intracellular Ca(2+) is required for spontaneous inflammasome activity in cells from patients with CAPS. CASR stimulation also results in reduced intracellular cAMP, which independently activates the NLRP3 inflammasome. cAMP binds to NLRP3 directly to inhibit inflammasome assembly, and downregulation of cAMP relieves this inhibition. The binding affinity of cAMP for CAPS-associated mutant NLRP3 is substantially lower than for wild-type NLRP3, and the uncontrolled mature IL-1ß production from CAPS patients' peripheral blood mononuclear cells is attenuated by increasing cAMP. Taken together, these findings indicate that Ca(2+) and cAMP are two key molecular regulators of the NLRP3 inflammasome that have critical roles in the molecular pathogenesis of CAPS.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Proteínas Portadoras/metabolismo , AMP Cíclico/metabolismo , Inflamasomas/metabolismo , Receptores Sensibles al Calcio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Proteínas Portadoras/genética , Síndromes Periódicos Asociados a Criopirina/etiología , Síndromes Periódicos Asociados a Criopirina/genética , Síndromes Periódicos Asociados a Criopirina/metabolismo , Retículo Endoplásmico/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Interleucina-1beta/biosíntesis , Interleucina-1beta/metabolismo , Leucocitos Mononucleares/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Unión Proteica , Fosfolipasas de Tipo C/metabolismoRESUMEN
BACKGROUND: We observed a syndrome of intermittent fevers, early-onset lacunar strokes and other neurovascular manifestations, livedoid rash, hepatosplenomegaly, and systemic vasculopathy in three unrelated patients. We suspected a genetic cause because the disorder presented in early childhood. METHODS: We performed whole-exome sequencing in the initial three patients and their unaffected parents and candidate-gene sequencing in three patients with a similar phenotype, as well as two young siblings with polyarteritis nodosa and one patient with small-vessel vasculitis. Enzyme assays, immunoblotting, immunohistochemical testing, flow cytometry, and cytokine profiling were performed on samples from the patients. To study protein function, we used morpholino-mediated knockdowns in zebrafish and short hairpin RNA knockdowns in U937 cells cultured with human dermal endothelial cells. RESULTS: All nine patients carried recessively inherited mutations in CECR1 (cat eye syndrome chromosome region, candidate 1), encoding adenosine deaminase 2 (ADA2), that were predicted to be deleterious; these mutations were rare or absent in healthy controls. Six patients were compound heterozygous for eight CECR1 mutations, whereas the three patients with polyarteritis nodosa or small-vessel vasculitis were homozygous for the p.Gly47Arg mutation. Patients had a marked reduction in the levels of ADA2 and ADA2-specific enzyme activity in the blood. Skin, liver, and brain biopsies revealed vasculopathic changes characterized by compromised endothelial integrity, endothelial cellular activation, and inflammation. Knockdown of a zebrafish ADA2 homologue caused intracranial hemorrhages and neutropenia - phenotypes that were prevented by coinjection with nonmutated (but not with mutated) human CECR1. Monocytes from patients induced damage in cocultured endothelial-cell layers. CONCLUSIONS: Loss-of-function mutations in CECR1 were associated with a spectrum of vascular and inflammatory phenotypes, ranging from early-onset recurrent stroke to systemic vasculopathy or vasculitis. (Funded by the National Institutes of Health Intramural Research Programs and others.).
Asunto(s)
Adenosina Desaminasa/deficiencia , Adenosina Desaminasa/genética , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Péptidos y Proteínas de Señalización Intercelular/genética , Mutación , Accidente Cerebrovascular/genética , Enfermedades Vasculares/genética , Edad de Inicio , Animales , Modelos Animales de Enfermedad , Endotelio Vascular/patología , Femenino , Fiebre/genética , Humanos , Masculino , Linaje , Poliarteritis Nudosa/genética , Análisis de Secuencia de ADN , Piel/patología , Vasculitis/genética , Vasculitis/patología , Pez CebraRESUMEN
PGE2 is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. Nucleotide-binding domain, leucine-rich repeat-containing protein (NLR)P3 inflammasome plays an important role in host defense. Uncontrolled activation of the NLRP3 inflammasome, owing to mutations in the NLRP3 gene, causes cryopyrin-associated periodic syndromes. In this study, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through PGE2 receptor subtype 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A or exchange protein directly activated by cAMP. A specific agonist of EP4 mimicked, whereas its antagonist or EP4 knockdown reversed, PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2-mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. Protein kinase A or exchange protein directly activated by cAMP agonists did not mimic, and their antagonists did not reverse, PGE2-mediated NLRP3 inhibition. Additionally, constitutive IL-1ß secretion from LPS-primed PBMCs of cryopyrin-associated periodic fever syndromes patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or small interfering RNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator.
Asunto(s)
Proteínas Portadoras/metabolismo , AMP Cíclico/metabolismo , Dinoprostona/fisiología , Macrófagos/inmunología , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Inhibidores de Adenilato Ciclasa , Adenilil Ciclasas/biosíntesis , Proteínas Portadoras/genética , Línea Celular , Síndromes Periódicos Asociados a Criopirina/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/farmacología , Dinoprostona/farmacología , Activación Enzimática , Fosfolipasas A2 Grupo IV/antagonistas & inhibidores , Fosfolipasas A2 Grupo IV/genética , Humanos , Inflamasomas/inmunología , Inflamación/inmunología , Interleucina-1beta/metabolismo , Leucocitos Mononucleares/inmunología , Lipopolisacáridos , Macrófagos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Inhibidores de Fosfodiesterasa/farmacología , Cultivo Primario de Células , Interferencia de ARN , ARN Interferente Pequeño , Subtipo EP2 de Receptores de Prostaglandina E/genética , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/genéticaRESUMEN
Microbial agents can aggravate inflammatory diseases, such as multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). An example is pertussis toxin (PTX), a bacterial virulence factor commonly used as an adjuvant to promote EAE, but whose mechanism of action is unclear. We have reported that PTX triggers an IL-6-mediated signaling cascade that increases the number of leukocytes that patrol the vasculature by crawling on its luminal surface. In the present study, we examined this response in mice lacking either TLR4 or inflammasome components and using enzymatically active and inactive forms of PTX. Our results indicate that PTX, through its ADP-ribosyltransferase activity, induces two series of events upstream of IL-6: 1) the activation of TLR4 signaling in myeloid cells, leading to pro-IL-1ß synthesis; and 2) the formation of a pyrin-dependent inflammasome that cleaves pro-IL-1ß into its active form. In turn, IL-1ß stimulates nearby stromal cells to secrete IL-6, which is known to induce vascular changes required for leukocyte adhesion. Without pyrin, PTX does not induce neutrophil adhesion to cerebral capillaries and is less effective at inducing EAE in transgenic mice with encephalitogenic T lymphocytes. This study identifies the first microbial molecule that activates pyrin, a mechanism by which infections may influence MS and a potential therapeutic target for immune disorders.
Asunto(s)
Encefalomielitis Autoinmune Experimental/metabolismo , Inflamasomas/inmunología , Interleucina-1beta/biosíntesis , Neutrófilos/efectos de los fármacos , Toxina del Pertussis/farmacología , Animales , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/inmunología , Interleucina-1beta/inmunología , Interleucina-6/metabolismo , Ratones , Esclerosis Múltiple/metabolismo , Células Mieloides , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Hyperzincemia and hypercalprotectinemia (Hz/Hc) is a distinct autoinflammatory entity involving extremely high serum concentrations of the proinflammatory alarmin myeloid-related protein (MRP) 8/14 (S100A8/S100A9 and calprotectin). OBJECTIVE: We sought to characterize the genetic cause and clinical spectrum of Hz/Hc. METHODS: Proline-serine-threonine phosphatase-interacting protein 1 (PSTPIP1) gene sequencing was performed in 14 patients with Hz/Hc, and their clinical phenotype was compared with that of 11 patients with pyogenic arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome. PSTPIP1-pyrin interactions were analyzed by means of immunoprecipitation and Western blotting. A structural model of the PSTPIP1 dimer was generated. Cytokine profiles were analyzed by using the multiplex immunoassay, and MRP8/14 serum concentrations were analyzed by using an ELISA. RESULTS: Thirteen patients were heterozygous for a missense mutation in the PSTPIP1 gene, resulting in a p.E250K mutation, and 1 carried a mutation resulting in p.E257K. Both mutations substantially alter the electrostatic potential of the PSTPIP1 dimer model in a region critical for protein-protein interaction. Patients with Hz/Hc have extremely high MRP8/14 concentrations (2045 ± 1300 µg/mL) compared with those with PAPA syndrome (116 ± 74 µg/mL) and have a distinct clinical phenotype. A specific cytokine profile is associated with Hz/Hc. Hz/Hc mutations altered protein binding of PSTPIP1, increasing interaction with pyrin through phosphorylation of PSTPIP1. CONCLUSION: Mutations resulting in charge reversal in the y-domain of PSTPIP1 (EâK) and increased interaction with pyrin cause a distinct autoinflammatory disorder defined by clinical and biochemical features not found in patients with PAPA syndrome, indicating a unique genotype-phenotype correlation for mutations in the PSTPIP1 gene. This is the first inborn autoinflammatory syndrome in which inflammation is driven by uncontrolled release of members of the alarmin family.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Complejo de Antígeno L1 de Leucocito/metabolismo , Errores Innatos del Metabolismo de los Metales/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Adolescente , Alarminas/genética , Alarminas/metabolismo , Calgranulina A/genética , Calgranulina A/metabolismo , Niño , Citocinas/metabolismo , Proteínas del Citoesqueleto/genética , Femenino , Genotipo , Humanos , Complejo de Antígeno L1 de Leucocito/genética , Masculino , Errores Innatos del Metabolismo de los Metales/genética , Mutación Missense/genética , Fenotipo , Fosforilación , Unión Proteica/genética , Mapas de Interacción de Proteínas/genética , Multimerización de Proteína , Pirina , Adulto JovenRESUMEN
OBJECTIVE: To analyse gene expression patterns and to define a specific gene expression signature in patients with the severe end of the spectrum of cryopyrin-associated periodic syndromes (CAPS). The molecular consequences of interleukin 1 inhibition were examined by comparing gene expression patterns in 16 CAPS patients before and after treatment with anakinra. METHODS: We collected peripheral blood mononuclear cells from 22 CAPS patients with active disease and from 14 healthy children. Transcripts that passed stringent filtering criteria (p values≤false discovery rate 1%) were considered as differentially expressed genes (DEG). A set of DEG was validated by quantitative reverse transcription PCR and functional studies with primary cells from CAPS patients and healthy controls. We used 17 CAPS and 66 non-CAPS patient samples to create a set of gene expression models that differentiates CAPS patients from controls and from patients with other autoinflammatory conditions. RESULTS: Many DEG include transcripts related to the regulation of innate and adaptive immune responses, oxidative stress, cell death, cell adhesion and motility. A set of gene expression-based models comprising the CAPS-specific gene expression signature correctly classified all 17 samples from an independent dataset. This classifier also correctly identified 15 of 16 post-anakinra CAPS samples despite the fact that these CAPS patients were in clinical remission. CONCLUSIONS: We identified a gene expression signature that clearly distinguished CAPS patients from controls. A number of DEG were in common with other systemic inflammatory diseases such as systemic onset juvenile idiopathic arthritis. The CAPS-specific gene expression classifiers also suggest incomplete suppression of inflammation at low doses of anakinra.
Asunto(s)
Antirreumáticos/uso terapéutico , Síndromes Periódicos Asociados a Criopirina/genética , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Transcriptoma/genética , Adulto , Estudios de Casos y Controles , Niño , Síndromes Periódicos Asociados a Criopirina/tratamiento farmacológico , Perfilación de la Expresión Génica , Humanos , Análisis por Micromatrices , Modelos Genéticos , Índice de Severidad de la Enfermedad , Transcriptoma/efectos de los fármacosRESUMEN
TNF, acting through p55 tumor necrosis factor receptor 1 (TNFR1), contributes to the pathogenesis of many inflammatory diseases. TNFR-associated periodic syndrome (TRAPS, OMIM 142680) is an autosomal dominant autoinflammatory disorder characterized by prolonged attacks of fevers, peritonitis, and soft tissue inflammation. TRAPS is caused by missense mutations in the extracellular domain of TNFR1 that affect receptor folding and trafficking. These mutations lead to loss of normal function rather than gain of function, and thus the pathogenesis of TRAPS is an enigma. Here we show that mutant TNFR1 accumulates intracellularly in peripheral blood mononuclear cells of TRAPS patients and in multiple cell types from two independent lines of knockin mice harboring TRAPS-associated TNFR1 mutations. Mutant TNFR1 did not function as a surface receptor for TNF but rather enhanced activation of MAPKs and secretion of proinflammatory cytokines upon stimulation with LPS. Enhanced inflammation depended on autocrine TNF secretion and WT TNFR1 in mouse and human myeloid cells but not in fibroblasts. Heterozygous TNFR1-mutant mice were hypersensitive to LPS-induced septic shock, whereas homozygous TNFR1-mutant mice resembled TNFR1-deficient mice and were resistant to septic shock. Thus WT and mutant TNFR1 act in concert from distinct cellular locations to potentiate inflammation in TRAPS. These findings establish a mechanism of pathogenesis in autosomal dominant diseases where full expression of the disease phenotype depends on functional cooperation between WT and mutant proteins and also may explain partial responses of TRAPS patients to TNF blockade.
Asunto(s)
Enfermedades Autoinflamatorias Hereditarias/inmunología , Enfermedades Autoinflamatorias Hereditarias/metabolismo , Mutación , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Animales , Enfermedades Autoinflamatorias Hereditarias/genética , Humanos , Lipopolisacáridos/inmunología , MAP Quinasa Quinasa 4/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: Dominantly inherited PSTPIP1 mutations cause a spectrum of autoinflammatory manifestations epitomized by PAPA syndrome (pyogenic sterile arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome.). The connections between PSTPIP1 and PAPA syndrome are poorly understood, although evidence suggests involvement of pyrin inflammasome activation. Interleukin-18 (IL-18) is an inflammasome-activated cytokine associated with susceptibility to macrophage activation syndrome (MAS). This study was undertaken to investigate an association of IL-18 with PAPA syndrome. METHODS: Clinical and genetic data and serum samples were obtained from patients referred to institutions due to symptoms indicative of PAPA syndrome. Serum IL-18, IL-18 binding protein (IL-18BP), and CXCL9 levels were assessed by bead-based assay, and free IL-18 levels were assessed by enzyme-linked immunosorbent assay. RESULTS: The symptoms of PSTPIP1-positive patients with PAPA syndrome overlapped with those of mutation-negative patients with PAPA-like conditions, but mutation-positive patients had earlier onset and a greater proportion had a history of arthritis. We found uniform elevation of total serum IL-18 in treated PAPA syndrome patients at levels nearly as high as those seen in NLRC4-associated autoinflammation with infantile enterocolitis patients, and well above levels found in most familial Mediterranean fever patients. Serum IL-18 elevation in PAPA syndrome patients persisted despite fluctuations in disease activity. Levels of the soluble IL-18 antagonist IL-18BP were modestly elevated, and PAPA syndrome patients had detectable free IL-18. PAPA syndrome was rarely associated with elevation of CXCL9, an indicator of interferon-γ activity, but no PAPA syndrome patients had a history of MAS. CONCLUSION: PAPA syndrome is a refractory and often disabling monogenic autoinflammatory disease associated with chronic and unopposed elevation of serum IL-18 levels but not with risk of MAS. These findings affect our understanding of the diseases in which IL-18 is overproduced and suggest a link between pyrin inflammasome activation, IL-18, and autoinflammation, without susceptibility to MAS.