RESUMO
Tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) functions downstream of multiple TNF receptors and receptors that induce interferon-α (IFN-α), IFN-ß, and IFN-λ production, including Toll-like receptor 3 (TLR3), which is deficient in some patients with herpes simplex virus-1 encephalitis (HSE). Mice lacking TRAF3 die in the neonatal period, preventing direct investigation of the role of TRAF3 in immune responses and host defenses in vivo. Here, we report autosomal dominant, human TRAF3 deficiency in a young adult with a history of HSE in childhood. The TRAF3 mutant allele is loss-of-expression, loss-of-function, dominant-negative and associated with impaired, but not abolished, TRAF3-dependent responses upon stimulation of both TNF receptors and receptors that induce IFN production. TRAF3 deficiency is associated with a clinical phenotype limited to HSE resulting from the impairment of TLR3-dependent induction of IFN. Thus, TLR3-mediated immunity against primary infection by HSV-1 in the central nervous system is critically dependent on TRAF3.
Assuntos
Encefalite por Herpes Simples/imunologia , Fator 3 Associado a Receptor de TNF/fisiologia , Receptor 3 Toll-Like/fisiologia , Células Cultivadas , Suscetibilidade a Doenças , Humanos , Interferons/fisiologia , Mutação , Receptores do Fator de Necrose Tumoral/fisiologia , Fator 3 Associado a Receptor de TNF/genéticaRESUMO
In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of toll-like receptor 3 (TLR3) immunity are prone to HSV-1 encephalitis (HSE). We tested the hypothesis that the pathogenesis of HSE involves non-haematopoietic CNS-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of interferon-ß (IFN-ß) and/or IFN-λ1 in response to stimulation by the dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-ß and IFN-λ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele showed that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was rescued further by treatment with exogenous IFN-α or IFN-ß ( IFN-α/ß) but not IFN-λ1. Thus, impaired TLR3- and UNC-93B-dependent IFN-α/ß intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3-pathway deficiencies.
Assuntos
Sistema Nervoso Central/patologia , Herpesvirus Humano 1/imunologia , Células-Tronco Pluripotentes Induzidas/citologia , Receptor 3 Toll-Like/deficiência , Astrócitos/imunologia , Astrócitos/virologia , Biomarcadores , Diferenciação Celular , Linhagem da Célula , Separação Celular , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/virologia , Criança , Suscetibilidade a Doenças , Encefalite por Herpes Simples/imunologia , Encefalite por Herpes Simples/metabolismo , Encefalite por Herpes Simples/patologia , Encefalite por Herpes Simples/virologia , Herpesvirus Humano 1/patogenicidade , Humanos , Imunidade Inata , Células-Tronco Pluripotentes Induzidas/virologia , Interferons/imunologia , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Células-Tronco Neurais/imunologia , Células-Tronco Neurais/virologia , Neurônios/imunologia , Neurônios/patologia , Neurônios/virologia , Oligodendroglia/imunologia , Oligodendroglia/patologia , Oligodendroglia/virologia , Receptor 3 Toll-Like/genéticaRESUMO
High-throughput genomic data reveal thousands of gene variants per patient, and it is often difficult to determine which of these variants underlies disease in a given individual. However, at the population level, there may be some degree of phenotypic homogeneity, with alterations of specific physiological pathways underlying the pathogenesis of a particular disease. We describe here the human gene connectome (HGC) as a unique approach for human mendelian genetic research, facilitating the interpretation of abundant genetic data from patients with the same disease, and guiding subsequent experimental investigations. We first defined the set of the shortest plausible biological distances, routes, and degrees of separation between all pairs of human genes by applying a shortest distance algorithm to the full human gene network. We then designed a hypothesis-driven application of the HGC, in which we generated a Toll-like receptor 3-specific connectome useful for the genetic dissection of inborn errors of Toll-like receptor 3 immunity. In addition, we developed a functional genomic alignment approach from the HGC. In functional genomic alignment, the genes are clustered according to biological distance (rather than the traditional molecular evolutionary genetic distance), as estimated from the HGC. Finally, we compared the HGC with three state-of-the-art methods: String, FunCoup, and HumanNet. We demonstrated that the existing methods are more suitable for polygenic studies, whereas HGC approaches are more suitable for monogenic studies. The HGC and functional genomic alignment data and computer programs are freely available to noncommercial users from http://lab.rockefeller.edu/casanova/HGC and should facilitate the genome-wide selection of disease-causing candidate alleles for experimental validation.
Assuntos
Alelos , Genes/genética , Genômica/métodos , Fenótipo , Transdução de Sinais/genética , Software , Análise por Conglomerados , Genes/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Modelos Genéticos , Receptor 3 Toll-Like/genéticaRESUMO
TLR3 is a receptor for dsRNA, which is generated during most viral infections. However, other cellular processes may also produce dsRNA and there are other receptors for dsRNA. The role of TLR3 in protective immunity to viruses has been investigated in mice and humans with genetically impaired TLR3 responses. TLR3-deficient mice responded to experimental challenge with 16 different viruses in various ways. They were susceptible to eight viruses, normally resistant to three other viruses, and their survival rates were higher than those of wild-type mice following infection with four other viruses. Conflicting results were obtained for the other virus tested. These data are difficult to understand in terms of a simple pattern based on virus structure or tissue tropism. Surprisingly, the known human patients with inborn errors of the TLR3 pathway have remained healthy or developed encephalitis in the course of natural primary infection with HSV-1. These patients display no clear susceptibility to other infections, including viral infections, such as other forms of viral encephalitis and other HSV-1 diseases in particular. This restricted susceptibility to viruses seems to result from impaired TLR3-dependent IFN-α/ß production by central nervous system (CNS)-resident non-hematopoietic cells infected with HSV-1. These studies neatly illustrate the value of combining genetic studies of experimental infections in mice and natural infections in humans, to elucidate the biological function of host molecules in protective immunity.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/imunologia , Encefalite/imunologia , Herpesvirus Humano 1/imunologia , RNA de Cadeia Dupla/imunologia , Receptor 3 Toll-Like/imunologia , Viroses/imunologia , Animais , Encefalite/etiologia , Predisposição Genética para Doença , Humanos , Imunidade Inata/genética , Camundongos , Camundongos Knockout , Polimorfismo Genético , Transdução de Sinais/genética , Receptor 3 Toll-Like/genética , Viroses/complicaçõesRESUMO
Childhood herpes simplex virus-1 (HSV-1) encephalitis (HSE) may result from single-gene inborn errors of TLR3 immunity. TLR3-dependent induction of IFN-α/ß or IFN-λ is crucial for protective immunity against primary HSV-1 infection in the central nervous system (CNS). We describe here two unrelated children with HSE carrying different heterozygous mutations (D50A and G159A) in TBK1, the gene encoding TANK-binding kinase 1, a kinase at the crossroads of multiple IFN-inducing signaling pathways. Both mutant TBK1 alleles are loss-of-function but through different mechanisms: protein instability (D50A) or a loss of kinase activity (G159A). Both are also associated with an autosomal-dominant (AD) trait but by different mechanisms: haplotype insufficiency (D50A) or negative dominance (G159A). A defect in polyinosinic-polycytidylic acid-induced TLR3 responses can be detected in fibroblasts heterozygous for G159A but not for D50A TBK1. Nevertheless, viral replication and cell death rates caused by two TLR3-dependent viruses (HSV-1 and vesicular stomatitis virus) were high in fibroblasts from both patients, and particularly so in G159A TBK1 fibroblasts. These phenotypes were rescued equally well by IFN-α2b. Moreover, the IFN responses to the TLR3-independent agonists and viruses tested were maintained in both patients' peripheral blood mononuclear cells and fibroblasts. The narrow, partial cellular phenotype thus accounts for the clinical phenotype of these patients being limited to HSE. These data identify AD partial TBK1 deficiency as a new genetic etiology of childhood HSE, indicating that TBK1 is essential for the TLR3- and IFN-dependent control of HSV-1 in the CNS.
Assuntos
Encefalite por Herpes Simples/genética , Encefalite por Herpes Simples/imunologia , Mutação , Proteínas Serina-Treonina Quinases/genética , Receptor 3 Toll-Like/imunologia , Animais , Morte Celular/imunologia , Células Cultivadas , Criança , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/virologia , Genes Dominantes , Herpesvirus Humano 1/patogenicidade , Humanos , Interferon beta/imunologia , Masculino , Camundongos , Poli I-C/farmacologia , Proteínas Serina-Treonina Quinases/imunologia , Vesiculovirus/patogenicidadeRESUMO
Herpes simplex encephalitis (HSE) is the most common sporadic viral encephalitis of childhood. Autosomal recessive (AR) UNC-93B and TLR3 deficiencies and autosomal dominant (AD) TLR3 and TRAF3 deficiencies underlie HSE in some children. We report here unrelated HSE children with AR or AD TRIF deficiency. The AR form of the disease was found to be due to a homozygous nonsense mutation that resulted in a complete absence of the TRIF protein. Both the TLR3- and the TRIF-dependent TLR4 signaling pathways were abolished. The AD form of disease was found to be due to a heterozygous missense mutation, resulting in a dysfunctional protein. In this form of the disease, the TLR3 signaling pathway was impaired, whereas the TRIF-dependent TLR4 pathway was unaffected. Both patients, however, showed reduced capacity to respond to stimulation of the DExD/H-box helicases pathway. To date, the TRIF-deficient patients with HSE described herein have suffered from no other infections. Moreover, as observed in patients with other genetic etiologies of HSE, clinical penetrance was found to be incomplete, as some HSV-1-infected TRIF-deficient relatives have not developed HSE. Our results provide what we believe to be the first description of human TRIF deficiency and a new genetic etiology for HSE. They suggest that the TRIF-dependent TLR4 and DExD/H-box helicase pathways are largely redundant in host defense. They further demonstrate the importance of TRIF for the TLR3-dependent production of antiviral IFNs in the CNS during primary infection with HSV-1 in childhood.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/deficiência , Encefalite por Herpes Simples/genética , Herpesvirus Humano 1 , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Sequência de Aminoácidos , Pré-Escolar , Códon sem Sentido , Consanguinidade , RNA Helicases DEAD-box/fisiologia , Feminino , Genes Dominantes , Genes Recessivos , Heterogeneidade Genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Lactente , Interferon-alfa/biossíntese , Interferon-alfa/genética , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Linhagem , Arábia Saudita , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Receptor 3 Toll-Like/fisiologia , Receptor 4 Toll-Like/fisiologiaRESUMO
Autosomal dominant TLR3 deficiency has been identified as a genetic etiology of childhood herpes simplex virus 1 (HSV-1) encephalitis (HSE). This defect is partial, as it results in impaired, but not abolished induction of IFN-ß and -λ in fibroblasts in response to TLR3 stimulation. The apparently normal resistance of these patients to other infections, viral illnesses in particular, may thus result from residual TLR3 responses. We report here an autosomal recessive form of complete TLR3 deficiency in a young man who developed HSE in childhood but remained normally resistant to other infections. This patient is compound heterozygous for two loss-of-function TLR3 alleles, resulting in an absence of response to TLR3 activation by polyinosinic-polycytidylic acid (poly(I:C)) and related agonists in his fibroblasts. Moreover, upon infection of the patient's fibroblasts with HSV-1, the impairment of IFN-ß and -λ production resulted in high levels of viral replication and cell death. In contrast, the patient's peripheral blood mononuclear cells responded normally to poly(I:C) and to all viruses tested, including HSV-1. Consistently, various TLR3-deficient leukocytes from the patient, including CD14(+) and/or CD16(+) monocytes, plasmacytoid dendritic cells, and in vitro derived monocyte-derived macrophages, responded normally to both poly(I:C) and HSV-1, with the induction of antiviral IFN production. These findings identify a new genetic etiology for childhood HSE, indicating that TLR3-mediated immunity is essential for protective immunity to HSV-1 in the central nervous system (CNS) during primary infection in childhood, in at least some patients. They also indicate that human TLR3 is largely redundant for responses to double-stranded RNA and HSV-1 in various leukocytes, probably accounting for the redundancy of TLR3 for host defense against viruses, including HSV-1, outside the CNS.