RESUMEN
Regulation of innate immune responses is essential for maintenance of immune homeostasis and development of an appropriate immunity against microbial infection. We show here that miR-3614-5p, product of the TRIM25 host gene, is induced by type I interferon (IFN-I) in several human non-immune and immune cell types, in particular in primary myeloid cells. Studies in HeLa cells showed that miR-3614-5p represses both p110 and p150 ADAR1 and reduces constitutive and IFN-induced A-to-I RNA editing. In line with this, activation of innate sensors and expression of IFN-ß and the pro-inflammatory IL-6 are promoted. MiR-3614-5p directly targets ADAR1 transcripts by binding to one specific site in the 3'UTR. Moreover, we could show that endogenous miR-3614-5p is associated with Ago2 and targets ADAR1 in IFN-stimulated cells. Overall, we propose that, by reducing ADAR1, IFN-I-induced miR-3614-5p contributes to lowering the activation threshold of innate sensors. Our findings provide new insights into the role of miR-3614-5p, placing it as a potential fine tuner of dsRNA metabolism, cell homeostasis and innate immunity.
Asunto(s)
Adenosina Desaminasa/metabolismo , Inmunidad Innata , Interferón Tipo I , MicroARNs , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Anticuerpos , Antivirales , Células HeLa , Humanos , MicroARNs/genética , Isoformas de Proteínas , ARN BicatenarioRESUMEN
Human USP18 is an interferon (IFN)-stimulated gene product and a negative regulator of type I IFN (IFN-I) signaling. It also removes covalently linked ISG15 from proteins, in a process called deISGylation. In turn, ISG15 prevents USP18 from being degraded by the proteasome. Autosomal recessive complete USP18 deficiency is life-threatening in infancy owing to uncontrolled IFN-I-mediated autoinflammation. We report three Moroccan siblings with autoinflammation and mycobacterial disease who are homozygous for a new USP18 variant. We demonstrate that the mutant USP18 (p.I60N) is normally stabilized by ISG15 and efficient for deISGylation but interacts poorly with the receptor-anchoring STAT2 and is impaired in negative regulation of IFN-I signaling. We also show that IFN-γ-dependent induction of IL-12 and IL-23 is reduced owing to IFN-I-mediated impairment of myeloid cells to produce both cytokines. Thus, insufficient negative regulation of IFN-I signaling by USP18-I60N underlies a specific type I interferonopathy, which impairs IL-12 and IL-23 production by myeloid cells, thereby explaining predisposition to mycobacterial disease.
Asunto(s)
Ubiquitina Tiolesterasa , Ubiquitinas , Citocinas/metabolismo , Humanos , Inflamación/genética , Interleucina-12 , Interleucina-23 , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
USP18 is an isopeptidase that cleaves the ubiquitin-like ISG15 from conjugates and is also an essential negative feedback regulator of type I interferon signaling. We and others reported that USP18 protein is stabilized by ISG15 and targeted for degradation by SKP2 (S-phase kinase associated protein 2), the substrate-recognition subunit of the SCFSKP2 ubiquitin E3 ligase complex, which operates in cell cycle progression. Here, we have analyzed how, under non stimulated conditions, USP18, ISG15 and SKP2 communicate with each other, by enforcing or silencing their expression. We found that USP18 and SKP2 interact and that free ISG15 abrogates the complex, liberating USP18 from degradation and concomitantly driving SKP2 to degradation and/or ISGylation. These data reveal a dynamic interplay where the substrate USP18 stabilizes SKP2, both exogenous and endogenous. Consistent with this we show that silencing of baseline USP18 slows down progression of HeLa S3 cells towards S phase. Our findings point to USP18 and ISG15 as unexpected new SKP2 regulators, which aid in cell cycle progression at homeostasis.
Asunto(s)
Ciclo Celular/genética , Citocinas/genética , Proteínas Quinasas Asociadas a Fase-S/genética , Ubiquitina Tiolesterasa/genética , Ubiquitinas/genética , Células HeLa , Humanos , Inmunidad Innata/genética , Transducción de Señal , Ubiquitina/genéticaRESUMEN
Homozygous mutations in EVER genes cause epidermodysplasia verruciformis (EV), characterized by an immune defect and the development of skin cancers associated with ß-human papillomavirus (HPV) infections. The effects of EVER protein loss on the keratinocyte immune response remain unknown. We show here that EVER2 plays a critical role in the interplay between the NF-κB and JNK/AP-1 signaling pathways. EVER2-deficient cells overproduce IL-6 following the upregulation of JNK activation. They respond poorly to phorbol ester and TNF via the NF-κB pathway. They have lower levels of IKKα subunit, potentially accounting for impairments of p100 processing and the alternative NF-κB pathway. The loss of EVER2 is associated with an unusual TRAF protein profile. We demonstrate that EVER2 deficiency sustains TRAF2 ubiquitination and decreases the pool of TRAF2 available in the detergent-soluble fraction of the cell. Finally, we demonstrate that EVER2 loss induces constitutive PKCα-dependent c-jun phosphorylation and facilitates activation of the HPV5 long control region through a JNK-dependent pathway. These findings indicate that defects of the EVER2 gene may create an environment conducive to HPV replication and the persistence of lesions with the potential to develop into skin cancer.
Asunto(s)
Resistencia a la Enfermedad/genética , Queratinocitos/metabolismo , Proteínas de la Membrana/deficiencia , FN-kappa B/metabolismo , Infecciones por Papillomavirus/metabolismo , Transducción de Señal/fisiología , Western Blotting , Cartilla de ADN/genética , Humanos , Quinasa I-kappa B/metabolismo , Inmunoprecipitación , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Fluorescente , Infecciones por Papillomavirus/genética , Fosforilación , Proteína Quinasa C-alfa/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor 2 Asociado a Receptor de TNF/metabolismoRESUMEN
Human papillomaviruses (HPV) cause a variety of mucosal and skin lesions ranging from benign proliferations to invasive carcinomas. The clinical manifestations of infection are determined by host-related factors that define the natural anti-HPV barrier. Key elements of this barrier are the EVER1 and EVER2 proteins, as deficiency in either one of the EVER proteins leads to Epidermodysplasia Verruciformis (EV), a genodermatosis associated with HPV-induced skin carcinoma. Although EVERs have been shown to regulate zinc homeostasis in keratinocytes, their expression and function in other cell types that may participate to the anti-HPV barrier remain to be investigated. In this work, we demonstrate that EVER genes are expressed in different tissues, and most notably in lymphocytes. Interestingly, in contrast to the skin, where EVER2 transcripts are hardly detectable, EVER genes are both abundantly expressed in murine and human T cells. Activation of CD4+ and CD8+ T cells via the TCR triggers a rapid and profound decrease in EVER expression, accompanied by an accumulation of free Zn(2+) ions. Thus, EVER proteins may be involved in the regulation of cellular zinc homeostasis in lymphocytes. Consistent with this hypothesis, we show that the concentration of Zn(2+) ions is elevated in lymphoblastoid cells or primary T cells from EVER2-deficient patients. Interestingly, we also show that Zn(2+) excess blocks T-cell activation and proliferation. Therefore, EVER proteins appear as key components of the activation-dependent regulation of Zn(2+) concentration in T cells. However, the impact of EVER-deficiency in T cells on EV pathogenesis remains to be elucidated.
Asunto(s)
Activación de Linfocitos , Proteínas de la Membrana/fisiología , Papillomaviridae/inmunología , Linfocitos T/inmunología , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Femenino , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la PolimerasaAsunto(s)
Luciferasas/genética , Luciferasas/metabolismo , Mapeo de Interacción de Proteínas/métodos , Animales , Benchmarking , Copépodos/enzimología , Bases de Datos de Proteínas , Prueba de Complementación Genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Luminiscencia , Unión Proteica , Mapeo de Interacción de Proteínas/normas , ProteómicaRESUMEN
Although the zeta-associated protein of 70 kDa (ZAP-70) is overexpressed in patients with chronic lymphocytic leukemia (CLL) displaying unmutated IGVH genes and poor prognosis, a previous microarray study from our group identified overexpression of LPL and ADAM29 genes among unmutated and mutated CLL, respectively. To assess the prognostic value of these genes, we quantified their expression by real-time quantitative polymerase chain reaction (PCR) in a cohort of 127 patients with CLL and correlated this with clinical outcome, IGVH mutational status, and ZAP-70 protein expression. IGVH mutational status, ZAP-70, and the LPL and ADAM29 mRNA ratios (L/A ratio) were predictive of event-free survival for the whole cohort and for patients with stage A disease. In patients in stage B and C, the L/A ratio was an independent prognostic factor, whereas ZAP-70 did not predict survival. Simultaneous usage of the L/A ratio and ZAP-70 expression allowed an almost perfect (99%) assessment of the IGVH status in the 80% of patients with concordant results (L/A+, ZAP-70+ or L/A-, ZAP-70-). LPL and ADAM29 gene expression could also be determined by a simple competitive multiplex reverse transcription PCR assay. Overall, quantification of LPL and ADAM29 gene expression is a strong prognostic indicator in CLL, providing better prognostic assessment than ZAP-70 in advanced stages of the disease.
Asunto(s)
Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/genética , Lipoproteína Lipasa/genética , Metaloendopeptidasas/genética , Proteínas ADAM , Secuencia de Bases , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , ADN Complementario/genética , ADN de Neoplasias/genética , Femenino , Expresión Génica , Genes de Inmunoglobulinas , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , Proteínas Tirosina Quinasas/genética , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Tirosina Quinasa ZAP-70RESUMEN
Low levels of B-cell-receptor (BCR) expression are the hallmark of tumoral B lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL). These cells also respond inadequately to stimulation through the BCR. This receptor consists of a surface immunoglobulin associated with a CD79a/CD79b heterodimer. We previously showed that the intracellular synthesis of BCR components, from transcription onward, is normal. Here, we investigated the glycosylation status and cellular localization of mu, CD79a, and CD79b chains in 10 CLL patients differing in surface immunoglobulin M (IgM) expression. We reported a severe impairment of the glycosylation and folding of mu and CD79a. These defects were associated with the retention of both chains in the endoplasmic reticulum and lower levels of surface IgM expression. In contrast, no clear impairment of glycosylation and folding was observed for CD79b. No sequence defects were identified for BCR components and for the chaperone proteins involved in BCR folding processes. These data show, for the first time, that lower levels of BCR surface expression observed in CLL are accounted for by an impaired glycosylation and folding of the mu and CD79a chains.
Asunto(s)
Antígenos CD/metabolismo , Inmunoglobulina M/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/fisiopatología , Receptores de Antígenos de Linfocitos B/metabolismo , Anciano , Antígenos CD/química , Antígenos CD/genética , Linfocitos B/metabolismo , Linfocitos B/ultraestructura , Antígenos CD79 , Dimerización , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Femenino , Regulación Leucémica de la Expresión Génica , Glicosilación , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Humanos , Inmunoglobulina M/química , Inmunoglobulina M/genética , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Agregación de Receptores , Receptores de Antígenos de Linfocitos B/química , Receptores de Antígenos de Linfocitos B/genéticaRESUMEN
Activation-induced cytidine deaminase (AID) is key to initiating somatic hypermutation (SHM) and class switch recombination (CSR), but its mode of action and regulation remains unclear. Since Pax-5 and Id-2 transcription factors play an opposing role in AID regulation, we have studied the expression of Pax-5, Id-2, and prdm-1 genes in 54 chronic lymphocytic leukemia (CLL) B cells. In 21 cases, presence of AID is constantly associated with high expression of the complete form of the Pax-5 gene (Pax-5a) and lower expression of the Id-2 and prdm-1 transcripts. In 33 cases, the absence of AID expression and CSR is associated with a reduction of Pax-5a and the appearance of a spliced form with a deletion in exon 8 (Pax-5/Delta-Ex8). Stimulation with CD40L+interleukin 4 (IL-4) induces CSR, the presence of AID transcripts, up-regulation of Pax-5a and down-regulation of Pax-5/Delta-Ex8, and Id-2 and prdm-1 transcripts. Pax-5a and Pax-5/Delta-Ex8 are translated into 2 isoforms of the B-cell-specific activator protein (BSAP) and both are able to bind the AID-promoter region. Overall, these results suggest that Pax-5/Delta-Ex8 could play an important role in the control of its own transcription and indirectly in AID expression and CSR.
Asunto(s)
Linfocitos B/inmunología , Citosina Desaminasa/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Regulación Leucémica de la Expresión Génica/inmunología , Leucemia Linfocítica Crónica de Células B/inmunología , Factor de Transcripción PAX5/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Linfocitos B/metabolismo , Linfocitos B/patología , Secuencia de Bases , Ligando de CD40 , Citidina Desaminasa , Citosina Desaminasa/metabolismo , Femenino , Humanos , Proteína 2 Inhibidora de la Diferenciación/inmunología , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Persona de Mediana Edad , Factor de Transcripción PAX5/metabolismo , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Proteínas Represoras/inmunología , Proteínas Represoras/metabolismo , Eliminación de Secuencia/inmunología , Hipermutación Somática de Inmunoglobulina/inmunología , Factores de Transcripción/inmunología , Factores de Transcripción/metabolismo , Transcripción Genética/inmunología , Células Tumorales CultivadasRESUMEN
Base substitutions, deletions, and duplications are observed at the immunoglobulin locus in DNA sequences involved in class switch recombination (CSR). These mutations are dependent upon activation-induced cytidine deaminase (AID) and present all the characteristics of the ones observed during V gene somatic hypermutation, implying that they could be generated by the same mutational complex. It has been proposed, based on the V gene mutation pattern of patients with the cancer-prone xeroderma pigmentosum variant (XP-V) syndrome who are deficient in DNA polymerase eta (pol eta), that this enzyme could be responsible for a large part of the mutations occurring on A/T bases. Here we show, by analyzing switched memory B cells from two XP-V patients, that pol eta is also an A/T mutator during CSR, in both the switch region of tandem repeats as well as upstream of it, thus suggesting that the same error-prone translesional polymerases are involved, together with AID, in both processes.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Cambio de Clase de Inmunoglobulina , Hipermutación Somática de Inmunoglobulina , Adulto , Linfocitos B/inmunología , Secuencia de Bases , Citidina Desaminasa , Citosina Desaminasa/metabolismo , ADN/genética , Análisis Mutacional de ADN , Cartilla de ADN/genética , ADN Polimerasa Dirigida por ADN/deficiencia , ADN Polimerasa Dirigida por ADN/genética , Femenino , Humanos , Memoria Inmunológica , Intrones , Persona de Mediana Edad , Datos de Secuencia Molecular , Recombinación Genética , Xerodermia Pigmentosa/enzimología , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/inmunologíaRESUMEN
Chronic lymphocytic leukemia (CLL) results from the accumulation of small mature, slowly dividing, monoclonal B lymphocytes. The clinical course of this disease is heterogeneous, with some patients progressing rapidly with early death whilst others exhibit a more stable, possibly, non-progressing disease lasting many years. Despite progress in therapy, relapse invariably occurs and the disease remains uncurable. The clinical management of CLL is therefore challenging and considerable effort has been directed towards novel therapeutic strategies aimed at reducing the disease relapse rate. Recent insights into the role of dendritic cells as the pivotal antigen-presenting cells that initiate immune responses may provide the basis for generating more effective antitumor immune responses. Consequently, dendritic cells constitute an attractive approach in the context of CLL. However, understanding the relation between dendritic cells and the cellular immune response is crucial to elucidation of how to manipulate immune responses. After summarizing general properties of dendritic cells, this review focus on the approaches exploiting monocyte-derived dendritic cells in CLL, which should help design of novel treatment strategies in this disease.
Asunto(s)
Células Dendríticas/trasplante , Inmunoterapia/métodos , Leucemia Linfocítica Crónica de Células B/terapia , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/trasplante , Linfocitos B/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/inmunología , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Linfocitos T/inmunologíaRESUMEN
Chronic lymphocytic leukemia (CLL) results from the accumulation of small mature, slowly dividing, monoclonal B lymphocytes. The clinical course of this disease is heterogeneous, with some patients progressing rapidly with early death whilst others exhibit a more stable, possibly, non-progressing disease lasting many years. Despite progress in therapy, relapse invariably occurs and the disease remains incurable. The clinical management of CLL is therefore challenging and considerable effort has been directed towards novel therapeutic strategies aimed at reducing the disease relapse rate. Recent insights into the role of dendritic cells as the pivotal antigen-presenting cells that initiate immune responses may provide the basis for generating more effective antitumor immune responses. Consequently, dendritic cells constitute an attractive approach in the context of CLL. However, understanding the relation between dendritic cells and the cellular immune response is crucial to elucidation of how to manipulate immune responses. After summarizing general properties of dendritic cells, this review focus on the approaches exploiting monocyte-derived dendritic cells in CLL, which should help design of novel treatment strategies in this disease.
Asunto(s)
Células Dendríticas/inmunología , Leucemia Linfocítica Crónica de Células B/terapia , Antígenos de Neoplasias/inmunología , Linfocitos B/inmunología , Linfocitos B/patología , Células Dendríticas/citología , Células Dendríticas/trasplante , Humanos , Inmunoterapia Adoptiva , Monocitos/citologíaRESUMEN
In B cells, somatic hypermutation (SHM) and class switch recombination (CSR) depend on the activation-induced cytidine deaminase (AID) gene product, although the precise mode of action of AID remains unknown. Because some chronic lymphocytic leukemia (CLL) B cells can undergo CSR without SHM, it constitutes a useful model to dissect AID function. In this work, we have studied AID expression, the presence of mutations in the preswitch mu DNA region, CSR, and the SHM in 65 CLL patients. Our results demonstrate that unmutated CLL B cells can constitutively express AID and that AID expression is associated with the presence of mutations in the preswitch region and in clonally related isotype-switched transcripts. They also demonstrate that in CLL without constitutive AID expression, AID induction on stimulation results in preswitch mutations and the CSR process. Our results show a dissociation between SHM and CSR in CLL and suggest that, in this disease, AID would require additional help for carrying out the SHM process.
Asunto(s)
Citidina Desaminasa/genética , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Recombinación Genética , Transcripción Genética , Linfocitos B/inmunología , Secuencia de Bases , Citidina Desaminasa/sangre , Cartilla de ADN , Humanos , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/inmunología , Reacción en Cadena de la Polimerasa , Edición de ARN/inmunología , Valores de ReferenciaRESUMEN
Idiotypic structures of immunoglobulins from malignant B cells constitute tumour-specific antigens, though the function of immunoglobulin-specific CD8+ T cells in disease control and rejection remains unclear. We have studied five cases of B chronic lymphocytic leukaemia patients affected with indolent (three patients) or aggressive (two patients) disease. We showed that CD8+ T cells with major histocompatibility complex class I-restricted cytotoxicity against autologous tumour B cells could be generated following repeated stimulations with idiotype-pulsed dendritic cells in vitro. CD8+ T-cell lines were able to upregulate CD69 expression and to release interferon (IFN)-gamma upon contact with the autologous B cells, though cytolytic activity was only substantiated for patients with indolent disease. The failure of cytolytic activity in patients with aggressive disease may be explained by a skewed maturation of memory CD8 cells.