RESUMEN
The paracaspase mucosa-associated lymphoid tissue lymphoma translocation protein-1 (MALT1) regulates nuclear-factor-kappa-B (NF-κB) activation downstream of surface receptors with immunoreceptor tyrosine-based activation motifs (ITAMs), such as the B-cell or T-cell receptor and has thus emerged as a therapeutic target for autoimmune diseases. However, recent reports demonstrate the development of lethal autoimmune inflammation due to the excessive production of interferon gamma (IFN-É£) and defective differentiation of regulatory T-cells in genetically modified mice deficient in MALT1 paracaspase activity. To address this issue, we explored the effects of pharmacological MALT1 inhibition on the balance between T-effector and regulatory T-cells. Here we demonstrate that allosteric inhibition of MALT1 suppressed Th1, Th17 and Th1/Th17 effector responses, and inhibited T-cell dependent B-cell proliferation and antibody production. Allosteric MALT1 inhibition did not interfere with the suppressive function of human T-regulatory cells, although it impaired de novo differentiation of regulatory T-cells from naïve T-cells. Treatment with an allosteric MALT1 inhibitor alleviated the cytokine storm, including IFN-É£, in a mouse model of acute T-cell activation, and long-term treatment did not lead to an increase in IFN-É£ producing CD4 cells or tissue inflammation. Together, our data demonstrate that the effects of allosteric inhibition of MALT1 differ from those seen in mice with proteolytically inactive MALT1, and thus we believe that MALT1 is a viable target for B and T-cell driven autoimmune diseases.
Asunto(s)
Linfocitos B/efectos de los fármacos , Células Dendríticas/efectos de los fármacos , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Linfocitos T Citotóxicos/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Células Cultivadas , Células Dendríticas/inmunología , Femenino , Transferencia Resonante de Energía de Fluorescencia , Voluntarios Sanos , Humanos , Inyecciones Intraperitoneales , Interferón gamma/inmunología , Interferón gamma/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Noqueados , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/inmunología , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Fenotiazinas/farmacología , Cultivo Primario de Células , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Células TH1/efectos de los fármacos , Células TH1/inmunología , Células TH1/metabolismoRESUMEN
Chronic obstructive pulmonary disease (COPD) is a serious global health problem characterized by chronic airway inflammation, progressive airflow limitation and destruction of lung parenchyma. Remodeling of the bronchial airways in COPD includes changes in both the bronchial epithelium and the subepithelial extracellular matrix (ECM). To explore the impact of an aberrant ECM on epithelial cell phenotype in COPD we developed a new ex vivo model, in which normal human bronchial epithelial (NHBE) cells repopulate and differentiate on decellularized human bronchial scaffolds derived from COPD patients and healthy individuals. By using transcriptomics, we show that bronchial ECM from COPD patients induces differential gene expression in primary NHBE cells when compared to normal bronchial ECM. The gene expression profile indicated altered activity of upstream mediators associated with COPD pathophysiology, including hepatocyte growth factor, transforming growth factor beta 1 and platelet-derived growth factor B, which suggests that COPD-related changes in the bronchial ECM contribute to the defective regenerative ability in the airways of COPD patients.
Asunto(s)
Matriz Extracelular/genética , Pulmón/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/genética , Transcriptoma/genética , Bronquios/metabolismo , Bronquios/patología , Factor de Crecimiento del Tejido Conjuntivo/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Matriz Extracelular/patología , Regulación de la Expresión Génica/genética , Factor de Crecimiento de Hepatocito/genética , Humanos , Pulmón/patología , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-sis/genética , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/patología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Factor de Crecimiento Transformador beta1/genéticaRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0173115.].
RESUMEN
BACKGROUND: BET proteins (BRD2, BRD3, BRDT and BRD4) belong to the family of bromodomain containing proteins, which form a class of transcriptional co-regulators. BET proteins bind to acetylated lysine residues in the histones of nucleosomal chromatin and function either as co-activators or co-repressors of gene expression. An imbalance between HAT and HDAC activities resulting in hyperacetylation of histones has been identified in COPD. We hypothesized that pan-BET inhibitor (JQ1) treatment of BET protein interactions with hyperacetylated sites in the chromatin will regulate excessive activation of pro-inflammatory genes in key inflammatory drivers of alveolar macrophages (AM) in COPD. METHODS AND FINDINGS: Transcriptome analysis of AM from COPD patients indicated up-regulation of macrophage M1 type genes upon LPS stimulation. Pan-BET inhibitor JQ1 treatment attenuated expression of multiple genes, including pro-inflammatory cytokines and regulators of innate and adaptive immune cells. We demonstrated for the first time that JQ1 differentially modulated LPS-induced cytokine release from AM or peripheral blood mononuclear cells (PBMC) of COPD patients compared to PBMC of healthy controls. Using the BET regulated gene signature, we identified a subset of COPD patients, which we propose to benefit from BET inhibition. CONCLUSIONS: This work demonstrates that the effects of pan-BET inhibition through JQ1 treatment of inflammatory cells differs between COPD patients and healthy controls, and the expression of BET protein regulated genes is altered in COPD. These findings provide evidence of histone hyperacetylation as a mechanism driving chronic inflammatory changes in COPD.
Asunto(s)
Ensamble y Desensamble de Cromatina , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Factores de Transcripción/metabolismo , Azepinas/farmacología , Estudios de Casos y Controles , Proteínas de Ciclo Celular , Células Cultivadas , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Citocinas/genética , Citocinas/metabolismo , Humanos , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Enfermedad Pulmonar Obstructiva Crónica/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Triazoles/farmacologíaRESUMEN
Mammalian cells are thought to protect themselves and their host organisms from DNA double strand breaks (DSBs) through universal mechanisms that restrain cellular proliferation until DNA is repaired. The Cyclin D3 protein drives G1-to-S cell cycle progression and is required for proliferation of immature T and B cells and of mature B cells during a T cell-dependent immune response. We demonstrate that mouse thymocytes and pre-B cells, but not mature B cells, repress Cyclin D3 protein levels in response to DSBs. This response requires the ATM protein kinase that is activated by DSBs. Cyclin D3 protein loss in thymocytes coincides with decreased association of Cyclin D3 mRNA with the HuR RNA binding protein that ATM regulates. HuR inactivation reduces basal Cyclin D3 protein levels without affecting Cyclin D3 mRNA levels, indicating that thymocytes repress Cyclin D3 expression via ATM-dependent inhibition of Cyclin D3 mRNA translation. In contrast, ATM-dependent transcriptional repression of the Cyclin D3 gene represses Cyclin D3 protein levels in pre-B cells. Retrovirus-driven Cyclin D3 expression is resistant to transcriptional repression by DSBs; this prevents pre-B cells from suppressing Cyclin D3 protein levels and from inhibiting DNA synthesis to the normal extent following DSBs. Our data indicate that immature B and T cells use lymphocyte lineage- and developmental stage-specific mechanisms to inhibit Cyclin D3 protein levels and thereby help prevent cellular proliferation in response to DSBs. We discuss the relevance of these cellular context-dependent DSB response mechanisms in restraining proliferation, maintaining genomic integrity, and suppressing malignant transformation of lymphocytes.
Asunto(s)
Linaje de la Célula , Ciclina D3/genética , Roturas del ADN de Doble Cadena , Crecimiento y Desarrollo , Linfocitos/citología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Linfocitos B/citología , Linfocitos B/efectos de la radiación , Linaje de la Célula/genética , Linaje de la Célula/efectos de la radiación , Proliferación Celular/efectos de la radiación , Ciclina D3/metabolismo , ADN/biosíntesis , Roturas del ADN de Doble Cadena/efectos de la radiación , Regulación hacia Abajo/efectos de la radiación , Proteína 1 Similar a ELAV/metabolismo , Crecimiento y Desarrollo/genética , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Radiación Ionizante , Retroviridae/metabolismo , Transducción de Señal/efectos de la radiación , Especificidad por Sustrato/efectos de la radiación , Linfocitos T/citología , Linfocitos T/efectos de la radiación , Transcripción Genética/efectos de la radiaciónRESUMEN
The HuR RNA-binding protein posttranscriptionally controls expression of genes involved in cellular survival, proliferation, and differentiation. To determine roles of HuR in B cell development and function, we analyzed mice with B lineage-specific deletion of the HuR gene. These HuRΔ/Δ mice have reduced numbers of immature bone marrow and mature splenic B cells, with only the former rescued by p53 inactivation, indicating that HuR supports B lineage cells through developmental stage-specific mechanisms. Upon in vitro activation, HuRΔ/Δ B cells have a mild proliferation defect and impaired ability to produce mRNAs that encode IgH chains of secreted Abs, but no deficiencies in survival, isotype switching, or expression of germinal center (GC) markers. In contrast, HuRΔ/Δ mice have minimal serum titers of all Ab isotypes, decreased numbers of GC and plasma B cells, and few peritoneal B-1 B cells. Moreover, HuRΔ/Δ mice have severely decreased GCs, T follicular helper cells, and high-affinity Abs after immunization with a T cell-dependent Ag. This failure of HuRΔ/Δ mice to mount a T cell-dependent Ab response contrasts with the ability of HuRΔ/Δ B cells to become GC-like in vitro, indicating that HuR is essential for aspects of B cell activation unique to the in vivo environment. Consistent with this notion, we find in vitro stimulated HuRΔ/Δ B cells exhibit modestly reduced surface expression of costimulatory molecules whose expression is similarly decreased in humans with common variable immunodeficiency. HuRΔ/Δ mice provide a model to identify B cell-intrinsic factors that promote T cell-dependent immune responses in vivo.
Asunto(s)
Linfocitos B/inmunología , Proteína 1 Similar a ELAV/biosíntesis , Activación de Linfocitos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Linfocitos B/citología , Células de la Médula Ósea/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Proliferación Celular/genética , Proteína 1 Similar a ELAV/genética , Centro Germinal/inmunología , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/biosíntesis , Proteína p53 Supresora de Tumor/genéticaRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is a cancer of immature T cells that exhibits heterogeneity of oncogenic lesions, providing an obstacle for development of more effective and less toxic therapies. Inherited deficiency of ATM, a regulator of the cellular DNA damage response, predisposes young humans and mice to T-ALLs with clonal chromosome translocations. While acquired ATM mutation or deletion occurs in pediatric T-ALLs, the role of somatic ATM alterations in T-ALL pathogenesis remains unknown. We demonstrate here that somatic Atm inactivation in haematopoietic cells starting as these cells differentiate in utero predisposes mice to T-ALL at similar young ages and harboring analogous translocations as germline Atm-deficient mice. However, some T-ALLs from haematopoietic cell specific deletion of Atm were of more mature thymocytes, revealing that the developmental timing and celluar origin of Atm inactivation influences the phenotype of ATM-deficient T-ALLs. Although it has been hypothesized that ATM suppresses cancer by preventing deletion and inactivation of TP53, we find that Atm inhibits T-ALL independent of Tp53 deletion. Finally, we demonstrate that the Cyclin D3 protein that drives immature T cell proliferation is essential for transformation of Atm-deficient thymocytes. Our study establishes a pre-clinical model for pediatric T-ALLs with acquired ATM inactivation and identifies the cell cycle machinery as a therapeutic target for this aggressive childhood T-ALL subtype.
Asunto(s)
Ciclina D3/metabolismo , Silenciador del Gen , Células Madre Hematopoyéticas/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Susceptibilidad a Enfermedades , Eliminación de Gen , Inestabilidad Genómica , Heterocigoto , Ratones Endogámicos C57BL , Ratones Transgénicos , Timocitos/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
TP53 protects cells from transformation by responding to stresses including aneuploidy and DNA double-strand breaks (DSBs). TP53 induces apoptosis of lymphocytes with persistent DSBs at antigen receptor loci and other genomic loci to prevent these lesions from generating oncogenic translocations. Despite this critical function of TP53, germline Tp53(-/-) mice succumb to immature T-cell (thymic) lymphomas that exhibit aneuploidy and lack clonal translocations. However, Tp53(-/-) mice occasionally develop B lineage lymphomas and Tp53 deletion in pro-B cells causes lymphomas with oncogenic immunoglobulin (Ig) locus translocations. In addition, human lymphoid cancers with somatic TP53 inactivation often harbor oncogenic IG or T-cell receptor (TCR) locus translocations. To determine whether somatic Tp53 inactivation unmasks translocations or alters the frequency of B lineage tumors in mice, we generated and analyzed mice with conditional Tp53 deletion initiating in hematopoietic stem cells (HSCs) or in lineage-committed thymocytes. Median tumor-free survival of each strain was similar to the lifespan of Tp53(-/-) mice. Mice with HSC deletion of Tp53 predominantly succumbed to thymic lymphomas with clonal translocations not involving Tcr loci; however, these mice occasionally developed mature B-cell lymphomas that harbored clonal Ig translocations. Deletion of Tp53 in thymocytes caused thymic lymphomas with aneuploidy and/or clonal translocations, including oncogenic Tcr locus translocations. Our data demonstrate that the developmental stage of Tp53 inactivation affects karyotypes of lymphoid malignancies in mice where somatic deletion of Tp53 initiating in thymocytes is sufficient to cause thymic lymphomas with oncogenic translocations.
Asunto(s)
Silenciador del Gen , Predisposición Genética a la Enfermedad , Células Madre Hematopoyéticas/metabolismo , Linfoma de Células T/genética , Linfoma de Células T/patología , Timocitos/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Linaje de la Célula , Células Clonales , Eliminación de Gen , Células Madre Hematopoyéticas/patología , Humanos , Ratones , Timocitos/patologíaAsunto(s)
Daño del ADN , Código de Histonas , Animales , Histonas/genética , Histonas/metabolismo , Humanos , RatonesRESUMEN
Pyrethroid insecticides are one of the most commonly used residential and agricultural insecticides. Based on the increased use of pyrethroids and recent studies showing that pregnant women and children are exposed to pyrethroids, there are concerns over the potential for developmental neurotoxicity. However, there have been relatively few studies on the developmental neurotoxicity of pyrethroids. In this study, we sought to investigate the developmental toxicity of six common pyrethroids, three type I compounds (permethrin, resmethrin, and bifenthrin) and three type II compounds (deltamethrin, cypermethrin, and lambda-cyhalothrin), and to determine whether zebrafish embryos may be an appropriate model for studying the developmental neurotoxicity of pyrethroids. Exposure of zebrafish embryos to pyrethroids caused a dose-dependent increase in mortality and pericardial edema, with type II compounds being the most potent. At doses approaching the LC(50), permethrin and deltamethrin caused craniofacial abnormalities. These findings are consistent with mammalian studies demonstrating that pyrethroids are mildly teratogenic at very high doses. However, at lower doses, body axis curvature and spasms were observed, which were reminiscent of the classic syndromes observed with pyrethroid toxicity. Treatment with diazepam ameliorated the spasms, while treatment with the sodium channel antagonist MS-222 ameliorated both spasms and body curvature, suggesting that pyrethroid-induced neurotoxicity is similar in zebrafish and mammals. Taken in concert, these data suggest that zebrafish may be an appropriate alternative model to study the mechanism(s) responsible for the developmental neurotoxicity of pyrethroid insecticides and aid in identification of compounds that should be further tested in mammalian systems.