RESUMO
Tumor necrosis factor receptor-associated factor 5 (TRAF5) is an intracellular protein that binds to the cytoplasmic portion of tumor necrosis factor receptors and mediates the activation of downstream nuclear factor-kappa B (NF-κB), interferon regulatory factor 3, and mitogen activated protein kinase signaling pathways. Compared with other TRAF proteins, TRAF5 is largely unknown in teleosts. In the present study, a TRAF5 homologue (HgTRAF5) from the hybrid grouper (Epinephelus fuscoguttatusâ × Epinephelus lanceolatusâ) was cloned and characterized. The open reading frame of HgTRAF5 consists of 1743 nucleotides encoding a 581 amino acid protein with a predicted molecular mass of 64.90 kDa. Similar to its mammalian counterpart, HgTRAF5 contains an N-terminal RING finger domain, a zinc finger domain, and a C-terminal TRAF domain, including a coiled-coil domain and a MATH domain. HgTRAF5 shares 99.83% identity with giant grouper (Epinephelus lanceolatus) TRAF5. Quantitative real-time PCR analysis indicated that HgTRAF5 mRNA was broadly expressed in all examined tissues. The expression of HgTRAF5 increased after Singapore grouper iridovirus (SGIV) infection in grouper spleen (GS) cells. Intracellular localization analysis demonstrated that the full-length HgTRAF5 protein mainly distributed in the cytoplasm. HgTRAF5 overexpression also promoted SGIV replication during viral infection in vitro. HgTRAF5 significantly promoted the activities of interferon-ß, interferon-sensitive response element, and NF-κB. Taken together, these results are important for a better understanding of the function of TRAF5 in fish and reveal its involvement in the host response to immune challenge by SGIV.
Assuntos
Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Fator 5 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/imunologia , Sequência de Aminoácidos , Animais , Bass , Infecções por Vírus de DNA/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Ranavirus/fisiologia , Alinhamento de Sequência/veterinária , Fator 5 Associado a Receptor de TNF/químicaRESUMO
Conserved immune cell signaling in fish was recently highlighted by the identification of various immune cell signaling molecules. Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins are critical adaptor molecules in immune cell signaling and contain E3 ubiquitin ligase activity. Here, we report the first crystal structure of the TRAF5 TRAF domain from the black rockcod (Notothenia coriiceps; ncTRAF5). Our structure revealed both similarities and differences with mammalian TRAF5. Structural and biochemical analyses indicated that ncTRAF5 forms a functional trimer unit in solution, with a structural flexibility that might be critical for imparting resistance to cold temperature-induced stress. We also found conserved surface residues on ncTRAF5 that might be critical binding hot spots for interaction with various receptors.
Assuntos
Doenças dos Peixes/imunologia , Imunidade Inata/genética , Perciformes/genética , Perciformes/imunologia , Fator 5 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Alinhamento de Sequência/veterinária , Transdução de Sinais , Fator 5 Associado a Receptor de TNF/químicaRESUMO
Nile tilapia (Oreochromis niloticus) is a pivotal economic fish that has been plagued by Streptococcus infections. Tumor necrosis factor receptor-associated factor 5 (TRAF5) is a crucial adaptor molecule, which can trigger downstream signaling cascades involved in immune pathway. In this study, Nile tilapia TRAF5 coding sequence (named OnTRAF5) was obtained, which contained typical functional domains, such as RING, zinc finger, coiled-coil and MATH domain. Different from other TRAF molecules, OnTRAF5 had shown relatively low identify with its homolog, and it was clustered into other teleost TRAF5 proteins. qRT-PCR was used to analysis the expression level of OnTRAF5 in gill, skin, muscle, head kidney, heart, intestine, thymus, liver, spleen and brain, In healthy Nile tilapia, the expression level of OnTRAF5 in intestine, gill and spleen were significantly higher than other tissues. While under Streptococcus agalactiae infection, the expression level of OnTRAF5 was improved significantly in all detected organs. Additionally, over-expression WT OnTRAF5 activated NF-κB, deletion of RING or zinc finger caused the activity impaired. In conclusion, OnTRAF5 participate in anti-bacteria immune response and is crucial for the signaling transduction.
Assuntos
Imunidade Adaptativa/genética , Ciclídeos/genética , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fator 5 Associado a Receptor de TNF/genética , Sequência de Aminoácidos , Animais , Ciclídeos/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Alinhamento de Sequência/veterinária , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/veterinária , Streptococcus agalactiae/fisiologia , Fator 5 Associado a Receptor de TNF/química , Fator 5 Associado a Receptor de TNF/imunologiaRESUMO
Tumor necrosis factor receptor-associated factor 5 (TRAF5) is a key adapter molecule that participates in numerous signaling pathways. The function of TRAF5 in fish is largely unknown. In the present study, a TRAF5 cDNA sequence (EcTRAF5) was identified in grouper (Epinephelus coioides). Similar to its mammalian counterpart, EcTRAF5 contained an N-terminal RING finger domain, a zinc finger domain, a C-terminal TRAF domain, including a coiled-coil domain and a MATH domain. The EcTRAF5 protein shared relatively low sequence identity with that of other species, but clustered with TRAF5 sequences from other fish. Real-time PCR analysis revealed that EcTRAF5 mRNA was broadly expressed in numerous tissues, with relatively high expression in skin, hindgut, and head kidney. Additionally, the expression of EcTRAF5 was up-regulated in gills and head kidney after infection with Cryptocaryon irritans. Intracellular localization analysis demonstrated that the full-length EcTRAF5 protein was uniformly distributed in the cytoplasm; while a deletion mutant of the coiled-coil domain of EcTRAF5 was observed uniformly distributed in the cytoplasm and the nucleus. After exogenous expression in HEK293T cells, TRAF5 significantly activated NF-κB. The deletion of the EcTRAF5 RING domain or of the zinc finger domain dramatically impaired its ability to activate NF-κB, implying that the RING domain and the zinc finger domain are required for EcTRAF5 signaling.
Assuntos
Bass/genética , Bass/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fator 5 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/imunologia , Sequência de Aminoácidos , Animais , Cilióforos/fisiologia , Infecções por Cilióforos/imunologia , Infecções por Cilióforos/veterinária , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Alinhamento de Sequência/veterinária , Transdução de Sinais , Fator 5 Associado a Receptor de TNF/químicaRESUMO
Tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 constitutively bind to glycoprotein 130 kDa (gp130) and inhibit IL-6-driven activation of signal transducer and activator of transcription 3 (STAT3) in CD4+ T cells, which limits the differentiation of pro-inflammatory IL-17-producing helper T cells that require IL-6-receptor (IL-6R) signals for their development. However, it is not known how the interaction between TRAF and gp130 negatively regulates STAT3 activity in the IL-6R complex. We hypothesized that TRAF proteins associated with gp130 might limit the activation of Janus kinase that is needed for the activation of STAT3. To test this, we transfected HEK293T cells to express gp130 and TRAF2 or TRAF5 together with two chimeric JAK1 proteins combined with either the N-terminal or the C-terminal protein fragment of firefly luciferase. Using this luciferase fragment complementation system, we found that the recovery of luciferase enzyme activity was coincident with proximal JAK1-JAK1 interaction and phosphorylation of JAK1 in the IL-6R complex and that the expression of TRAF protein significantly inhibited the recovery of luciferase activity. The binding of TRAF to gp130 via the C-terminal TRAF domain was essential for the inhibition. In accordance with this, upon stimulation of endogenous gp130 with a complex of IL-6 and IL-6R, Traf5-/- CD4+ T cells displayed significantly higher amounts of phosphorylated JAK1 than did their wild-type counterparts. Therefore, our results demonstrate that gp130-associated TRAF2 and TRAF5 inhibit the interaction between two JAK proteins in the IL-6R complex that is essential for initiating the JAK-STAT signaling pathway.
Assuntos
Receptor gp130 de Citocina/metabolismo , Interleucina-6/metabolismo , Janus Quinase 1/metabolismo , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 5 Associado a Receptor de TNF/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Receptor gp130 de Citocina/imunologia , Interleucina-6/imunologia , Janus Quinase 1/imunologia , Camundongos , Fosforilação , Ligação Proteica , Fator 2 Associado a Receptor de TNF/imunologia , Fator 5 Associado a Receptor de TNF/imunologiaRESUMO
Upon viral infection, the cytoplasmic viral sensor retinoic acid-inducible gene-I (RIG-I) recognizes viral RNA to activate antiviral signaling to induce type I interferon (IFN). RIG-I-like receptors (RLRs) activate antiviral signaling in a tissue-specific manner. The molecular mechanism underlying antiviral signaling in the respiratory system remains unclear. We studied antiviral signaling in the lower respiratory tract (LRT), which is the site of many harmful viral infections. Epithelial cells of the LRT can be roughly divided into two groups: bronchial epithelial cells (BECs) and pulmonary alveolar epithelial cells (AECs). These two cell types exhibit different phenotypes; therefore, we hypothesized that these cells may play different roles in antiviral innate immunity. We found that BECs exhibited higher antiviral activity than AECs. TNF receptor-associated factor 3 (TRAF3) has been shown to be a crucial molecule in RLR signaling. The expression levels of TRAF3 and TRAF5, which have conserved domains that are nearly identical, in the LRT were examined. We found that the bronchus exhibited the highest expression levels of TRAF3 and TRAF5 in the LRT. These findings suggest the importance of the bronchus in antiviral innate immunity in the LRT and indicate that TRAF3 and TRAF5 may contribute to RLR signaling.
Assuntos
RNA Helicases DEAD-box/genética , Células Epiteliais/efeitos dos fármacos , Fator 3 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/genética , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/imunologia , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/imunologia , Células Epiteliais/citologia , Células Epiteliais/imunologia , Esôfago/química , Esôfago/efeitos dos fármacos , Esôfago/imunologia , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Interferon beta/biossíntese , Interferon beta/imunologia , Laringe/química , Laringe/efeitos dos fármacos , Laringe/imunologia , Leucócitos Mononucleares/química , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Especificidade de Órgãos , Poli I-C/farmacologia , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/imunologia , Receptores Imunológicos , Transdução de Sinais , Fator 3 Associado a Receptor de TNF/imunologia , Fator 5 Associado a Receptor de TNF/imunologiaRESUMO
Interleukin 17 (IL-17) is important in infection and autoimmunity; how it signals remains poorly understood. In this study, we identified the ubiquitin-specific protease USP25 as a negative regulator of IL-17-mediated signaling and inflammation. Overexpression of USP25 inhibited IL-17-triggered signaling, whereas USP25 deficiency resulted in more phosphorylation of the inhibitor IκBα and kinase Jnk and higher expression of chemokines and cytokines, as well as a prolonged half-life for chemokine CXCL1-encoding mRNA after treatment with IL-17. Consistent with that, Usp25(-/-) mice showed greater sensitivity to IL-17-dependent inflammation and autoimmunity in vivo. Mechanistically, stimulation with IL-17 induced the association of USP25 with the adaptors TRAF5 and TRAF6, and USP25 induced removal of Lys63-linked ubiquitination in TRAF5 and TRAF6 mediated by the adaptor Act1. Thus, our results demonstrate that USP25 is a deubiquitinating enzyme (DUB) that negatively regulates IL-17-triggered signaling.
Assuntos
Inflamação/genética , Interleucina-17/genética , Transdução de Sinais/genética , Ubiquitina Tiolesterase/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Quimiocina CXCL1/genética , Quimiocina CXCL1/imunologia , Deleção de Genes , Expressão Gênica , Regulação da Expressão Gênica/imunologia , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Inflamação/imunologia , Inflamação/patologia , Interleucina-17/imunologia , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/imunologia , Camundongos , Camundongos Knockout , Fosforilação , Transdução de Sinais/imunologia , Fator 5 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/imunologia , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/imunologia , Ubiquitina Tiolesterase/deficiência , Ubiquitina Tiolesterase/imunologia , UbiquitinaçãoRESUMO
A large and diverse group of receptors utilizes the family of cytoplasmic signaling proteins known as tumor necrosis factor receptor (TNFR)-associated factors (TRAFs). In recent years, there has been a resurgence of interest and exploration of the roles played by TRAF3 and TRAF5 in cellular regulation, particularly in cells of the immune system, the cell types of focus in this review. This work has revealed that TRAF3 and TRAF5 can play diverse roles for different receptors even in the same cell type, as well as distinct roles in different cell types. Evidence indicates that TRAF3 and TRAF5 play important roles beyond the TNFR-superfamily (SF) and viral mimics of its members, mediating certain innate immune receptor and cytokine receptor signals, and most recently, signals delivered by the T-cell receptor (TCR) signaling complex. Additionally, much research has demonstrated the importance of TRAF3-mediated cellular regulation via its cytoplasmic interactions with additional signaling proteins. In particular, we discuss below evidence for the participation by TRAF3 in a number of the regulatory post-translational modifications involving ubiquitin that are important in various signaling pathways.
Assuntos
Imunidade Inata , NF-kappa B/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Transdução de Sinais/imunologia , Fator 3 Associado a Receptor de TNF/imunologia , Fator 5 Associado a Receptor de TNF/imunologia , Ubiquitina/imunologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Expressão Gênica/imunologia , Humanos , Camundongos , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/imunologia , Receptores do Fator de Necrose Tumoral/genética , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/metabolismo , Fator 5 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
Interleukin 17 (IL-17) is critical in the pathogenesis of inflammatory and autoimmune diseases. Here we report that Act1, the key adaptor for the IL-17 receptor (IL-7R), formed a complex with the inducible kinase IKKi after stimulation with IL-17. Through the use of IKKi-deficient mice, we found that IKKi was required for IL-17-induced expression of genes encoding inflammatory molecules in primary airway epithelial cells, neutrophilia and pulmonary inflammation. IKKi deficiency abolished IL-17-induced formation of the complex of Act1 and the adaptors TRAF2 and TRAF5, activation of mitogen-activated protein kinases (MAPKs) and mRNA stability, whereas the Act1-TRAF6-transcription factor NF-κB axis was retained. IKKi was required for IL-17-induced phosphorylation of Act1 on Ser311, adjacent to a putative TRAF-binding motif. Substitution of the serine at position 311 with alanine impaired the IL-17-mediated Act1-TRAF2-TRAF5 interaction and gene expression. Thus, IKKi is a kinase newly identified as modulating IL-17 signaling through its effect on Act1 phosphorylation and consequent function.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Quimiocina CXCL1/imunologia , Quinase I-kappa B , Neutrófilos/imunologia , Pneumonia/imunologia , Transdução de Sinais/imunologia , Células Th17/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Quinase I-kappa B/deficiência , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-17/farmacologia , Pulmão , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neutrófilos/metabolismo , Fosforilação , Pneumonia/genética , Pneumonia/metabolismo , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro , Receptores de Interleucina-17/imunologia , Fator 5 Associado a Receptor de TNF/imunologia , Fator 5 Associado a Receptor de TNF/metabolismo , Células Th17/metabolismoRESUMO
Interleukin 17 (IL-17) promotes the expression of chemokines and cytokines via the induction of gene transcription and post-transcriptional stabilization of mRNA. We show here that IL-17 enhanced the stability of chemokine CXCL1 mRNA and other mRNAs through a pathway that involved the adaptor Act1, the adaptors TRAF2 or TRAF5 and the splicing factor SF2 (also known as alternative splicing factor (ASF)). TRAF2 and TRAF5 were necessary for IL-17 to signal the stabilization of CXCL1 mRNA. Furthermore, IL-17 promoted the formation of complexes of TRAF5-TRAF2, Act1 and SF2 (ASF). Overexpression of SF2 (ASF) shortened the half-life of CXCL1 mRNA, whereas depletion of SF2 (ASF) prolonged it. SF2 (ASF) bound chemokine mRNA in unstimulated cells, whereas the SF2 (ASF)-mRNA interaction was much lower after stimulation with IL-17. Our findings define an IL-17-induced signaling pathway that links to the stabilization of selected mRNA species through Act1, TRAF2-TRAF5 and the RNA-binding protein SF2 (ASF).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quimiocina CXCL1/metabolismo , Inflamação/imunologia , Interleucina-17 , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/imunologia , Fator 5 Associado a Receptor de TNF/metabolismo , Células Th17/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Processamento Alternativo , Animais , Quimiocina CXCL1/genética , Quimiocina CXCL1/imunologia , Feminino , Meia-Vida , Células HeLa , Humanos , Inflamação/genética , Inflamação/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-17/farmacologia , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Processamento Pós-Transcricional do RNA , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Fatores de Processamento de Serina-Arginina , Fator 5 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/imunologia , Células Th17/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Gastrointestinal nematode (GIN) infections are the predominant cause of economic losses in sheep. Infections are controlled almost exclusively by the use of anthelmintics which has lead to the selection of drug resistant nematode strains. An alternative control approach would be the induction of protective immunity to these parasites. This study exploits an ovine microarray biased towards immune genes, an artificially induced immunity model and the use of pseudo-afferent lymphatic cannulation to sample immune cells draining from the intestine, to investigate possible mechanisms involved in the development of immunity. RESULTS: During the development of immunity to, and a subsequent challenge infection with Trichostrongylus colubriformis, the transcript levels of 2603 genes of cells trafficking in afferent intestinal lymph were significantly modulated (P < 0.05). Of these, 188 genes were modulated more than 1.3-fold and involved in immune function. Overall, there was a clear trend for down-regulation of many genes involved in immune functions including antigen presentation, caveolar-mediated endocytosis and protein ubiquitination. The transcript levels of TNF receptor associated factor 5 (TRAF5), hemopexin (HPX), cysteine dioxygenase (CDO1), the major histocompatability complex Class II protein (HLA-DMA), interleukin-18 binding protein (IL-18BP), ephrin A1 (EFNA1) and selenoprotein S (SELS) were modulated to the greatest degree. CONCLUSIONS: This report describes gene expression profiles of afferent lymph cells in sheep developing immunity to nematode infection. Results presented show a global down-regulation of the expression of immune genes which may be reflective of the natural temporal response to nematode infections in livestock.
Assuntos
Regulação para Baixo/imunologia , Gastroenteropatias/imunologia , Linfa/metabolismo , Tricostrongilose/imunologia , Trichostrongylus/imunologia , Animais , Cateterismo , Cisteína Dioxigenase/genética , Cisteína Dioxigenase/imunologia , Cisteína Dioxigenase/metabolismo , Gastroenteropatias/genética , Gastroenteropatias/parasitologia , Perfilação da Expressão Gênica , Hemopexina/genética , Hemopexina/imunologia , Hemopexina/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/imunologia , Intestinos/parasitologia , Intestinos/patologia , Linfa/citologia , Linfa/imunologia , Linfa/parasitologia , Análise Serial de Proteínas , Ovinos , Fator 5 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/imunologia , Fator 5 Associado a Receptor de TNF/metabolismo , Tricostrongilose/genética , Tricostrongilose/parasitologiaRESUMO
The CD40-CD40 ligand (CD40L) signaling axis plays an important role in immunological pathways. Consequently, this dyad is involved in chronic inflammatory diseases, including atherosclerosis. Inhibition of CD40L in apolipoprotein E (Apoe)-deficient (Apoe(-/-)) mice not only reduced atherosclerosis but also conferred a clinically favorable plaque phenotype that was low in inflammation and high in fibrosis. Blockade of CD40L may not be therapeutically feasible, as long-term inhibition will compromise systemic immune responses. Conceivably, more targeted intervention strategies in CD40 signaling will have less deleterious side effects. We report that deficiency in hematopoietic CD40 reduces atherosclerosis and induces features of plaque stability. To elucidate the role of CD40-tumor necrosis factor receptor-associated factor (TRAF) signaling in atherosclerosis, we examined disease progression in mice deficient in CD40 and its associated signaling intermediates. Absence of CD40-TRAF6 but not CD40-TRAF2/3/5 signaling abolishes atherosclerosis and confers plaque fibrosis in Apoe(-/-) mice. Mice with defective CD40-TRAF6 signaling display a reduced blood count of Ly6C(high) monocytes, an impaired recruitment of Ly6C(+) monocytes to the arterial wall, and polarization of macrophages toward an antiinflammatory regulatory M2 signature. These data unveil a role for CD40-TRAF6, but not CD40-TRAF2/3/5, interactions in atherosclerosis and establish that targeting specific components of the CD40-CD40L pathway harbors the potential to achieve therapeutic effects in atherosclerosis.
Assuntos
Aterosclerose/imunologia , Aterosclerose/metabolismo , Antígenos CD40/metabolismo , Ligante de CD40/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Anti-Inflamatórios/imunologia , Apolipoproteínas E/deficiência , Aterosclerose/patologia , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Progressão da Doença , Inflamação/imunologia , Inflamação/patologia , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Leucócitos/patologia , Camundongos , Transdução de Sinais/imunologia , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/imunologia , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/imunologia , Fator 3 Associado a Receptor de TNF/metabolismo , Fator 5 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/imunologia , Fator 5 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/imunologiaRESUMO
The cytoplasmic signaling protein TNF receptor-associated factor 5 (TRAF5) has been implicated in several biological roles in T-lymphocyte responses. However, a clear connection between in vivo TRAF5 immune cell functions and specific signaling pathways has not been made. This study shows that TRAF5 associated strongly with the viral oncogenic CD40 mimic latent membrane protein 1 (LMP1), in contrast to weaker association with CD40, for which it has been shown to play a modest role. LMP1 uses specific TRAFs differently than CD40, resulting in amplified and dysregulated CD40-like activation of B lymphocytes. When the cytoplasmic domain of LMP1 is expressed as a transgenic replacement for CD40 in mouse B cells, the resulting mouse exhibits measures of B-cell hyperactivity such as splenomegaly, lymphadenopathy, elevated serum IL-6, elevated serum autoantibodies, and abnormal splenic architecture. Thus, in contrast to CD40, TRAF5 may have an important nonredundant role as a positive mediator of LMP1 signaling and functions in B cells. To test this hypothesis, mice were created that express mCD40LMP1 in place of CD40, and are either sufficient or deficient in TRAF5. Results revealed that TRAF5 plays a critical role in LMP1-mediated c-Jun kinase signaling and is required for much of the abnormal phenotype observed in mCD40LMP1 transgenic mice. This is the first report showing a major requirement for TRAF5 in signaling by a specific receptor both in vitro and in vivo, as well as playing an important role in biological function in B lymphocytes.
Assuntos
Antígenos CD40/imunologia , Transdução de Sinais/imunologia , Fator 5 Associado a Receptor de TNF/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Anticorpos Antinucleares/sangue , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Western Blotting , Antígenos CD40/genética , Antígenos CD40/metabolismo , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Interleucina-6/sangue , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , MAP Quinase Quinase 4/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Baço/citologia , Baço/imunologia , Baço/metabolismo , Fator 5 Associado a Receptor de TNF/genética , Fator 5 Associado a Receptor de TNF/metabolismo , Fatores de Tempo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismoRESUMO
Receptors belonging to the TNF-receptor (TNF-R) superfamily include important costimulatory molecules, many of which specifically affect T cell activation. TNF receptor-associated factors (TRAFs) are recruited to many TNF-R superfamily members and are important modulators of the proximal signaling events that occur at the time of receptor engagement and activation. TRAF5 has been shown to be a positive regulator of a number of these receptors that are involved in T cell costimulation. However, the potential importance of TRAF5 in cellular immune responses to infection or in T cell expansion and memory have not been studied. We report in this study that TRAF5 was required for optimal CD8(+) T cell responses following infection with Listeria monocytogenes expressing OVA (LM-OVA). TRAF5 was necessary for optimal T cell expansion following primary infection with LM-OVA, and its absence resulted in fewer memory CD8(+) T cells following LM-OVA infection, together with higher bacterial loads in the liver. The effect of TRAF5 on CD8(+) T cell expansion was T cell intrinsic and not due to effects of TRAF5 deficiency on APCs. Although their proliferative ability remained intact, CD8(+) T cells from TRAF5(-/-) mice were more sensitive to apoptosis and were unresponsive to the prosurvival effects of the TNF-R superfamily costimulator CD27. Collectively, these studies identify TRAF5 as an important positive signaling element that enhances T cell expansion and pathogen containment by providing a survival advantage to responding Ag-specific CD8(+) T cells during infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Listeria monocytogenes/imunologia , Listeriose/imunologia , Ativação Linfocitária , Fator 5 Associado a Receptor de TNF/imunologia , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Apoptose/genética , Apoptose/imunologia , Sobrevivência Celular , Listeria monocytogenes/genética , Listeriose/genética , Fígado/imunologia , Fígado/microbiologia , Ativação Linfocitária/genética , Camundongos , Camundongos Knockout , Fator 5 Associado a Receptor de TNF/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologiaRESUMO
Activation of the noncanonical pathway through the interaction of lymphotoxin (LT)-alpha(1)beta(2) and LT-betaR is essential for the development of secondary lymphoid organs including lymph nodes (LN) and Peyer's patches (PP). Although TNFR-associated factor (TRAF) 2 and TRAF5 were identified as signal transducers for the LT-betaR, roles for TRAF2 and TRAF5 in the development of secondary lymphoid organs remain obscure. In this study, we show that PP but not mesenteric LN development is severely impaired in traf2(-/-) and traf2(-/-)traf5(-/-) mice. Development of VCAM-1(+) and ICAM-1(+) mesenchymal cells and expression of CXCL13, a crucial chemokine for the development of PP, are severely impaired in PP anlagen in the intestines of traf2(-/-) mice. Surprisingly, TNF-alpha stimulation potently up-regulates cxcl13 mRNA expression in wild-type murine embryonic fibroblasts, which is impaired in traf2(-/-) and relA(-/-) murine embryonic fibroblasts. Moreover, RelA is recruited to the promoter of cxcl13 gene upon TNF-alpha stimulation and PP development is impaired in TNFR type 1 (tnfr1)(-/-) mice. These results underscore a crucial role for the TNFR1-TRAF2-RelA-dependent canonical pathway in the development of PP through up-regulation of cxcl13 mRNA.
Assuntos
Quimiocinas CXC/imunologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Nódulos Linfáticos Agregados/embriologia , Fator 2 Associado a Receptor de TNF/imunologia , Fator 5 Associado a Receptor de TNF/imunologia , Fator de Transcrição RelA/imunologia , Animais , Quimiocina CXCL13 , Quimiocinas CXC/biossíntese , Fibroblastos/citologia , Fibroblastos/imunologia , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/imunologia , Intestino Delgado/citologia , Intestino Delgado/embriologia , Intestino Delgado/imunologia , Linfonodos/embriologia , Linfonodos/imunologia , Heterotrímero de Linfotoxina alfa1 e beta2/imunologia , Receptor beta de Linfotoxina/imunologia , Mesoderma/citologia , Mesoderma/imunologia , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Fator 2 Associado a Receptor de TNF/deficiência , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 5 Associado a Receptor de TNF/deficiência , Fator 5 Associado a Receptor de TNF/metabolismo , Fator de Transcrição RelA/deficiência , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Regulação para Cima/imunologia , Molécula 1 de Adesão de Célula Vascular/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Escherichia coli is associated with inflammation in the brain. To investigate whether astrocytes are involved in E. coil-induced inflammation, we assessed the levels of expression of proinflammatory mediators produced by E. coli-infected astrocytes. E. coli infection in primary human astrocytes and cell lines increased expression of the CXC chemokine IL-8/GRO-alpha, the CC chemokine MCP-1, TNF-alpha, and iNOS. E. coli infection activated p65/p50 heterodimeric NF-kappaB and concurrently decreased the signals of IkappaBalpha. Blocking the NF-kappaB signals by IkappaBalpha-superrepressor-containing retrovirus or antisense p50 oligonucleotide transfection resulted in down-regulation of expression of the proinflammatory mediators. Furthermore, superrepressors of IkappaBalpha, IkappaB kinase (IKK) or NF-kappaB inducing kinase (NIK) inhibited the up-regulated expression of the downstream target genes of NF-kappaB such as IL-8 and MCP-1, and superrepressors of TNF receptor-associated factor (TRAF)2 and TRAF5 also inhibited expression of the E. coli-induced target genes of NF-kappaB. These results indicate that proinflammatory mediators such as the CXC chemokine IL-8/GRO-alpha, the CC chemokine MCP-1, TNF-alpha, and iNOS can be expressed in E. coli-infected astrocytes via an NF-kappaB pathway, suggesting that these mediators may contribute to inflammation in the brain, including infiltration of inflammatory cells.