RESUMO
The genome-wide association study (GWAS)-identified asthma susceptibility risk alleles on chromosome 17q21 increase the expression of ORMDL3 (ORMDL sphingolipid biosynthesis regulator 3) in lung tissue. Given the importance of epithelial integrity in asthma, we hypothesized that ORMDL3 directly impacted bronchial epithelial function. To determine whether and how ORMDL3 expression impacts the bronchial epithelium, in studies using both primary human bronchial epithelial cells and human bronchial epithelial cell line, 16HBE (16HBE14o-), we assessed the impact of ORMDL3 on autophagy. Studies included: autophagosome detection by electron microscopy, RFP-GFP-LC3B to assess autophagic activity, and Western blot analysis of autophagy-related proteins. Mechanistic assessments included immunoprecipitation assays, intracellular calcium mobilization assessments, and cell viability assays. Coexpression of ORMDL3 and autophagy-related genes was measured in primary human bronchial epithelial cells derived from 44 subjects. Overexpressing ORMDL3 demonstrated increased numbers of autophagosomes and increased levels of autophagy-related proteins LC3B, ATG3, ATG7, and ATG16L1. ORMDL3 overexpression promotes autophagy and subsequent cell death by impairing intracellular calcium mobilization through interacting with SERCA2. Strong correlation was observed between expression of ORMDL3 and autophagy-related genes in patient-derived bronchial epithelial cells. Increased ORMDL3 expression induces autophagy, possibly through interacting with SERCA2, thereby inhibiting intracellular calcium influx, and induces cell death, impairing bronchial epithelial function in asthma.
Assuntos
Asma , Proteínas de Membrana , Asma/genética , Asma/metabolismo , Asma/patologia , Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Cálcio/metabolismo , Epitélio/metabolismo , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
Rationale: Daily high-dose aspirin therapy benefits many patients with aspirin-exacerbated respiratory disease but provides no benefit for aspirin-tolerant patients with asthma. Type 2 inflammation characterizes aspirin-exacerbated respiratory disease.Objectives: To determine whether high-dose aspirin therapy changes biomarkers of type 2 inflammation in aspirin-exacerbated respiratory disease.Methods: Forty-two subjects with aspirin-exacerbated respiratory disease underwent an aspirin desensitization and were placed on high-dose aspirin (1,300 mg daily). Fifteen aspirin-tolerant subjects with asthma were also placed on high-dose aspirin. Biologic specimens and clinical parameters were collected at baseline and after 8 weeks on aspirin. Urinary eicosanoids, plasma tryptase and cytokine levels, platelet-leukocyte aggregates, and granulocyte transcripts were assessed.Measurements and Main Results: Eight weeks of high-dose aspirin decreased nasal symptoms and urinary prostaglandin E metabolite (P < 0.05) and increased urinary leukotriene E4 (P < 0.01) levels in subjects with aspirin-exacerbated respiratory disease, but not in those with aspirin-tolerant asthma. Urinary prostaglandin D2 and thromboxane metabolites decreased in both groups. Only in subjects with aspirin-exacerbated respiratory disease, exhaled nitric oxide (P < 0.05), plasma tryptase (P < 0.01), and blood eosinophil (P < 0.01) and basophil (P < 0.01) counts increased and plasma tryptase correlated with eosinophil counts (Pearson r = 0.514; P < 0.01) on aspirin. After correction for eosinophil counts, aspirin-induced changes in blood granulocyte transcripts did not differ between groups. Aspirin had no effect on platelet-leukocyte aggregates, platelet activation markers, or plasma cytokines in either group.Conclusions: High-dose aspirin therapy for 8 weeks paradoxically increases markers of type 2 inflammation in subjects with aspirin-exacerbated respiratory disease, despite reducing nasal symptoms. This effect of aspirin is unique to aspirin-exacerbated respiratory disease and not observed in subjects with aspirin-tolerant asthma.
Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Aspirina/efeitos adversos , Asma Induzida por Aspirina/tratamento farmacológico , Biomarcadores/urina , Hipersensibilidade a Drogas/complicações , Receptores de Leucotrienos/análise , Infecções Respiratórias/induzido quimicamente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anti-Inflamatórios não Esteroides/uso terapêutico , Aspirina/uso terapêutico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by asthma, recurrent nasal polyposis, and respiratory reactions on ingestion of COX-1 inhibitors. Increased numbers of platelet-leukocyte aggregates are present in the sinus tissue and blood of patients with AERD compared with that of aspirin-tolerant patients, and platelet activation can contribute to aspirin-induced reactions. OBJECTIVE: We sought to determine whether treatment with prasugrel, which inhibits platelet activation by blocking the type 12 purinergic (P2Y12) receptor, would attenuate the severity of sinonasal and respiratory symptoms induced during aspirin challenge in patients with AERD. METHODS: Forty patients with AERD completed a 10-week, double-blind, placebo-controlled crossover trial of prasugrel. All patients underwent oral aspirin challenges after 4 weeks of prasugrel and after 4 weeks of placebo. The primary outcome was a change in the provocative dose of aspirin that would elicit an increase in Total Nasal Symptom Score (TNSS) of 2 points. Changes in lung function, urinary eicosanoids, plasma tryptase, platelet-leukocyte aggregates, and platelet activation were also recorded. RESULTS: Prasugrel did not significantly change the mean increase in TNSS of 2 points (79 ± 15 for patients receiving placebo and 139 ± 32 for patients receiving prasugrel, P = .10), platelet-leukocyte aggregates, or increases in urinary leukotriene E4 and prostaglandin D2 metabolite levels during aspirin-induced reactions in the study population as a whole. Five subjects (responders) reacted to aspirin while receiving placebo but did not have any reaction to aspirin challenge after the prasugrel arm. In contrast to prasugrel nonresponders (35 subjects), the prasugrel responders had smaller reaction-induced increases in TNSS; did not have significant aspirin-induced increases in urinary leukotriene E4, prostaglandin D2 metabolite, or thromboxane B2 levels; and did not display increases in serum tryptase levels during aspirin reactions on the placebo arm, all of which were observed in the nonresponders. CONCLUSION: In the overall study population, prasugrel did not attenuate aspirin-induced symptoms, possibly because it failed to decrease the frequencies of platelet-adherent leukocytes or to diminish aspirin-induced mast cell activation. In a small subset of patients with AERD who had greater baseline platelet activation and milder upper respiratory symptoms during aspirin-induced reactions, P2Y12 receptor antagonism with prasugrel completely inhibited all aspirin-induced reaction symptoms, suggesting a contribution from P2Y12 receptor signaling in this subset.
Assuntos
Asma Induzida por Aspirina/tratamento farmacológico , Cloridrato de Prasugrel/administração & dosagem , Antagonistas do Receptor Purinérgico P2Y/administração & dosagem , Receptores Purinérgicos P2Y12/imunologia , Adulto , Asma Induzida por Aspirina/imunologia , Estudos Cross-Over , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
TREX1/DNASE III, the most abundant 3'-5' DNA exonuclease in mammalian cells, is tail-anchored on the endoplasmic reticulum (ER). Mutations at the N-terminus affecting TREX1 DNase activity are associated with autoimmune and inflammatory conditions such as Aicardi-Goutières syndrome (AGS). Mutations in the C-terminus of TREX1 cause loss of localization to the ER and dysregulation of oligosaccharyltransferase (OST) activity, and are associated with retinal vasculopathy with cerebral leukodystrophy (RVCL) and in some cases with systemic lupus erythematosus (SLE). Here we investigate mice with conditional expression of the most common RVCL mutation, V235fs, and another mouse expressing a conditional C-terminal mutation, D272fs, associated with a case of human SLE. Mice homozygous for either mutant allele express the encoded human TREX1 truncations without endogenous mouse TREX1, and both remain DNase active in tissues. The two mouse strains are similar phenotypically without major signs of retinal, cerebral or renal disease but exhibit striking elevations of autoantibodies in the serum. The broad range of autoantibodies is primarily against non-nuclear antigens, in sharp contrast to the predominantly DNA-related autoantibodies produced by a TREX1-D18N mouse that specifically lacks DNase activity. We also found that treatment with an OST inhibitor, aclacinomycin, rapidly suppressed autoantibody production in the TREX1 frame-shift mutant mice. Together, our study presents two new mouse models based on TREX1 frame-shift mutations with a unique set of serologic autoimmune-like phenotypes.
Assuntos
Autoimunidade/genética , Autoimunidade/imunologia , Exodesoxirribonucleases/genética , Mutação da Fase de Leitura , Fosfoproteínas/genética , Aclarubicina/análogos & derivados , Aclarubicina/farmacologia , Substituição de Aminoácidos , Animais , Apoptose/genética , Apoptose/imunologia , Autoanticorpos/imunologia , Autoimunidade/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Ativação Enzimática , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Camundongos , Camundongos Transgênicos , Fenótipo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Retina/imunologia , Retina/metabolismo , Retina/patologia , Timócitos/imunologia , Timócitos/metabolismo , TranscriptomaRESUMO
Autosomal dominant retinal vasculopathy with cerebral leukodystrophy is a microvascular endotheliopathy with middle-age onset. In nine families, we identified heterozygous C-terminal frameshift mutations in TREX1, which encodes a 3'-5' exonuclease. These truncated proteins retain exonuclease activity but lose normal perinuclear localization. These data have implications for the maintenance of vascular integrity in the degenerative cerebral microangiopathies leading to stroke and dementias.
Assuntos
Encefalopatias/genética , Exodesoxirribonucleases/genética , Mutação , Fosfoproteínas/genética , Doenças Retinianas/genética , Sequência de Aminoácidos , Encefalopatias/enzimologia , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Genes Dominantes , Predisposição Genética para Doença , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Doenças Retinianas/enzimologia , Homologia de Sequência de Aminoácidos , TransfecçãoAssuntos
Asma/genética , Cromossomos Humanos Par 17/genética , Predisposição Genética para Doença/genética , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Ilhas de CpG , Metilação de DNA , Regulação da Expressão Gênica/genética , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasAssuntos
CADASIL/diagnóstico por imagem , CADASIL/epidemiologia , Vasculite Retiniana/diagnóstico por imagem , Vasculite Retiniana/epidemiologia , Adulto , CADASIL/genética , Feminino , Humanos , Leucoencefalopatias/diagnóstico por imagem , Leucoencefalopatias/epidemiologia , Leucoencefalopatias/genética , Masculino , Pessoa de Meia-Idade , Vasculite Retiniana/genéticaRESUMO
The molecular and clinical features of a complex disease can be influenced by other diseases affecting the same individual. Understanding disease-disease interactions is therefore crucial for revealing shared molecular mechanisms among diseases and designing effective treatments. Here we introduce Flow Centrality (FC), a network-based approach to identify the genes mediating the interaction between two diseases in a protein-protein interaction network. We focus on asthma and COPD, two chronic respiratory diseases that have been long hypothesized to share common genetic determinants and mechanisms. We show that FC highlights potential mediator genes between the two diseases, and observe similar outcomes when applying FC to 66 additional pairs of related diseases. Further, we perform in vitro perturbation experiments on a widely replicated asthma gene, GSDMB, showing that FC identifies candidate mediators of the interactions between GSDMB and COPD-associated genes. Our results indicate that FC predicts promising gene candidates for further study of disease-disease interactions.
Assuntos
Asma/genética , Predisposição Genética para Doença/genética , Mapas de Interação de Proteínas/genética , Doença Pulmonar Obstrutiva Crônica/genética , Asma/complicações , Asma/metabolismo , Linhagem Celular , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Humanos , Proteínas de Neoplasias/genética , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/metabolismo , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Mutations in the three-prime repair exonuclease 1 (TREX1) gene have been associated with neurological diseases, including Retinal Vasculopathy with Cerebral Leukoencephalopathy (RVCL). However, the endogenous expression of TREX1 in human brain has not been studied. METHODS: We produced a rabbit polyclonal antibody (pAb) to TREX1 to characterize TREX1 by Western blotting (WB) of cell lysates from normal controls and subjects carrying an RVCL frame-shift mutation. Dual staining was performed to determine cell types expressing TREX1 in human brain tissue. TREX1 distribution in human brain was further evaluated by immunohistochemical analyses of formalin-fixed, paraffin-embedded samples from normal controls and patients with RVCL and ischemic stroke. RESULTS: After validating the specificity of our anti-TREX1 rabbit pAb, WB analysis was utilized to detect the endogenous wild-type and frame-shift mutant of TREX1 in cell lysates. Dual staining in human brain tissues from patients with RVCL and normal controls localized TREX1 to a subset of microglia and macrophages. Quantification of immunohistochemical staining of the cerebral cortex revealed that TREX1+ microglia were primarily in the gray matter of normal controls (22.7 ± 5.1% and 5.5 ± 1.9% of Iba1+ microglia in gray and white matter, respectively) and commonly in association with the microvasculature. In contrast, in subjects with RVCL, the TREX1+ microglia were predominantly located in the white matter of normal appearing cerebral cortex (11.8 ± 3.1% and 38.9 ± 5.8% of Iba1+ microglia in gray and white matter, respectively). The number of TREX1+ microglia was increased in ischemic brain lesions in central nervous system of RVCL and stroke patients. CONCLUSIONS: TREX1 is expressed by a subset of microglia in normal human brain, often in close proximity to the microvasculature, and increases in the setting of ischemic lesions. These findings suggest a role for TREX1+ microglia in vessel homeostasis and response to ischemic injury.
Assuntos
Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Encéfalo/metabolismo , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/fisiologia , Microglia/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Idoso , Animais , Anticorpos/metabolismo , Encéfalo/patologia , Exodesoxirribonucleases/genética , Feminino , Mutação da Fase de Leitura , Células HEK293 , Células HeLa , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Homeostase/fisiologia , Humanos , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/genética , Coelhos/imunologia , Doenças Retinianas/genética , Doenças Retinianas/patologia , Doenças Vasculares/genética , Doenças Vasculares/patologiaRESUMO
Cerebroretinal vasculopathy (CRV) and the related diseases hereditary endotheliopathy with retinopathy, neuropathy, and stroke (HERNS), hereditary vascular retinopathy (HVR) and hereditary systemic angiopathy (HSA) [subsequently combined as retinovasculopathy and cerebral leukodystrophy (RVCL)] are devastating autosomal-dominant disorders of early to middle-age onset presenting with a core constellation of neurologic and ophthalmologic findings. This family of diseases is linked by specific mutations targeting a core region of a gene. Frameshift mutations in the carboxyl-terminus of three prime exonuclease-1 (TREX1), the major mammalian 3' to 5' DNA exonuclease on chromosome 3p21.1-p21.3, result in a systemic vasculopathy that follows an approximately 5-year course leading to death secondary to progressive neurologic decline, with sometimes a more protracted course in HERNS. Neuropathological features include a fibrinoid vascular necrosis or thickened hyalinized vessels associated with white matter ischemia, necrosis and often striking dystrophic calcifications. Ultrastructural studies of the vessel walls often demonstrate unusual multilaminated basement membranes.
Assuntos
Exodesoxirribonucleases/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Fosfoproteínas/genética , Doenças Retinianas/genética , Doenças Retinianas/patologia , Doenças Vasculares/genética , Doenças Vasculares/patologia , Encéfalo/irrigação sanguínea , Encéfalo/ultraestrutura , Mutação da Fase de Leitura , Humanos , Rim/ultraestruturaRESUMO
Aicardi-Goutières syndrome (AGS), Systemic Lupus Erythematosus (SLE), Familial Chilblain Lupus (FCL) and Retinal Vasculopathy and Cerebral Leukodystrophy (RVCL) {a new term encompassing three independently described conditions with a common etiology--Cerebroretinal Vasculopathy (CRV), Hereditary Vascular Retinopathy (HVR) and Hereditary Endotheliopathy, Retinopathy and Nephropathy (HERNS)}--have previously been regarded as distinct entities. However, recent genetic analysis has demonstrated that each of these diseases maps to chromosome 3p21 and can be caused by mutations in TREX1, the major human 3'-5' exonuclease. In this review, we discuss the putative functions of TREX1 in relationship to the clinical, genetic and functional characteristics of each of these conditions.