RESUMEN
The bone marrow (BM) microenvironment (niche) plays important roles in supporting normal/abnormal haematopoiesis. We investigated the interaction between leukaemic mesenchymal niche and haematopoietic stem and progenitor cells (HSPCs) using the model of Fanconi anaemia (FA), a genetic disorder characterized by BM failure and leukaemia. Healthy donor HSPCs co-cultured on mesenchymal stromal cells (MSCs) derived from FA patients with acute myeloid leukaemia (AML) exhibited higher human engraftment and myeloid expansion in Non-obese diabetic severe combined immunodeficiency IL-2γ-/- /SGM3 recipients. Untargeted metabolomics analysis revealed the progressively elevated prostaglandins (PGs) in the MSCs of FA patients with myelodysplastic syndromes (MDS) and AML. Reduced secretion of PGs subsequent to inflammatory cyclooxygenase 2 (COX2) inhibition ameliorated HSPC/myeloid expansion. Transcriptome analysis demonstrated dysregulation of genes involved in the NR4A family of transcription factors (TFs) and WNT/ß-catenin signalling pathway in FA-AML-MSC-co-cultured-CD34+ cells. COX2 inhibition led to significantly decreased NR4A TFs and WNT signalling genes expression. Mechanistically, NR4A1 and NR4A2 synergistically activate the CTNNB1 gene promoter . Knocking down CTNNB1 or NR4A1 in AML-MSC-co-cultured-CD34+ cells increased leukaemia-reactive T-effector cells production and rescued anti-leukaemia immunity. Together, these findings suggest that specific interactions between leukaemic mesenchymal niche and HSPCs orchestrate a novel COX2/PG-NR4A/WNT signalling axis, connecting inflammation, cellular metabolism and cancer immunity.
Asunto(s)
Ciclooxigenasa 2/inmunología , Células Madre Hematopoyéticas/inmunología , Leucemia Mieloide Aguda/inmunología , Células Madre Mesenquimatosas/inmunología , Proteínas de Neoplasias/inmunología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Vía de Señalización Wnt/inmunología , Animales , Femenino , Células Madre Hematopoyéticas/patología , Humanos , Leucemia Mieloide Aguda/patología , Masculino , Células Madre Mesenquimatosas/patología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCIDAsunto(s)
Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/terapia , Autoinmunidad/inmunología , Inmunoterapia , Terapia Molecular Dirigida , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/terapia , Prolactina/antagonistas & inhibidores , Humanos , Inmunoterapia/métodos , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/fisiología , Prolactina/farmacología , Prolactina/fisiología , Proteínas de Dominio T Box/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Células Th17/inmunologíaRESUMEN
In Candida albicans-infected resident peritoneal macrophages, activation of group IVA cytosolic phospholipase A2(cPLA2α) by calcium- and mitogen-activated protein kinases triggers the rapid production of prostaglandins I2 and E2 through cyclooxygenase (COX)-1 and regulates gene expression by increasing cAMP. InC. albicans-infected cPLA2α(-/-)or COX-1(-/-)macrophages, expression ofI l10,Nr4a2, and Ptgs2 was lower, and expression ofTnfα was higher, than in wild type macrophages. Expression was reconstituted with 8-bromo-cAMP, the PKA activator 6-benzoyl-cAMP, and agonists for prostaglandin receptors IP, EP2, and EP4 in infected but not uninfected cPLA2α(-/-)or COX-1(-/-)macrophages. InC. albicans-infected cPLA2α(+/+)macrophages, COX-2 expression was blocked by IP, EP2, and EP4 receptor antagonists, indicating a role for both prostaglandin I2 and E2 Activation of ERKs and p38, but not JNKs, by C. albicansacted synergistically with prostaglandins to induce expression of Il10,Nr4a2, and Ptgs2. Tnfα expression required activation of ERKs and p38 but was suppressed by cAMP. Results using cAMP analogues that activate PKA or Epacs suggested that cAMP regulates gene expression through PKA. However, phosphorylation of cAMP-response element-binding protein (CREB), the cAMP-regulated transcription factor involved inIl10,Nr4a2,Ptgs2, andTnfα expression, was not mediated by cAMP/PKA because it was similar inC. albicans-infected wild type and cPLA2α(-/-)or COX-1(-/-)macrophages. CREB phosphorylation was blocked by p38 inhibitors and induced by the p38 activator anisomycin but not by the PKA activator 6-benzoyl-cAMP. Therefore, MAPK activation inC. albicans-infected macrophages plays a dual role by promoting the cPLA2α/prostaglandin/cAMP/PKA pathway and CREB phosphorylation that coordinately regulate immediate early gene expression.
Asunto(s)
Candida albicans/fisiología , Ciclooxigenasa 1/inmunología , Regulación de la Expresión Génica , Fosfolipasas A2 Grupo IV/inmunología , Interacciones Huésped-Patógeno , Macrófagos Peritoneales/inmunología , Proteínas de la Membrana/inmunología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/inmunología , Ciclooxigenasa 1/deficiencia , Ciclooxigenasa 1/genética , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/inmunología , Dinoprostona/biosíntesis , Epoprostenol/biosíntesis , Fosfolipasas A2 Grupo IV/deficiencia , Fosfolipasas A2 Grupo IV/genética , Interleucina-10/genética , Interleucina-10/inmunología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/microbiología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/inmunología , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/inmunología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/antagonistas & inhibidores , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/inmunología , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/inmunologíaRESUMEN
Development of acute experimental autoimmune encephalomyelitis (EAE) depends on Th17 cells expressing the nuclear factor NR4A2. However, in mice lacking NR4A2 in T cells, a late-onset disease is still inducible, despite a great reduction in acute inflammation. We here reveal that development of this late onset disease depends on cytotoxic T-cell-like CD4(+) T cells expressing the T-box transcription factor Eomesodermin (Eomes). T-cell-specific deletion of the Eomes gene remarkably ameliorates the late-onset EAE. Strikingly, similar Eomes(+) CD4(+) T cells are increased in the peripheral blood and cerebrospinal fluid from patients in a progressive state of multiple sclerosis. Collective data indicate an involvement of granzyme B and protease-activated receptor-1 in the neuroinflammation mediated by Eomes(+) CD4(+) T cells.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Granzimas/inmunología , Esclerosis Múltiple Crónica Progresiva/inmunología , Receptor PAR-1/inmunología , Proteínas de Dominio T Box/inmunología , Células Th17/inmunología , Adulto , Animales , Encefalomielitis Autoinmune Experimental/genética , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/inmunología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Proteínas de Dominio T Box/genética , Linfocitos T Colaboradores-Inductores/inmunología , Adulto JovenRESUMEN
The nuclear receptor subfamily 4 (NR4A) is composed of 3 related proteins sharing a DNA binding domain (DBD) and a ligand-binding domain (LBD). The nuclear receptor related 1 protein (Nurr1 or NR4A2) plays a key role in the maintenance of the dopaminergic system. Dopamine dysfunctions associated with the Nurr1 gene include Parkinson's disease, schizophrenia and manic depression among others. Furthermore, recent evidence indicates that Nurr1 is also expressed in other brain areas such as the hippocampus and plays critical roles for learning and memory. The other members of the family are nerve growth factor IB (Nur77 or NR4A1) and neuron-derived orphan receptor 1 (NOR1 or NR4A3). To help investigate the precise functional roles of Nurr1 in dopaminergic and other brain region-related neuronal dysfunctions antibodies devoid of cross-reactivities against Nur77 and NOR1 were needed. Since the proteins are more divergent in their LBDs than in their DNA binding domains immunization with purified LBDs should yield antibodies specific for Nurr1 with minimal reactivities against Nur77 and/or NOR1. Although anti-Nurr1 antibodies were successfully generated these showed significant immunoreactivity against the other members of the family. Affinity chromatography over immobilized Protein A followed by pre-adsorption against immobilized Nur77 and NOR1 LBDs yielded Nurr1 specific antibodies free of cross-reactivity. Here, we selectively target antibodies against a specific member of a highly conserved family of proteins by immunizing animals with their most divergent regions followed by removing cross reactive antibodies by pre-adsorption. The goal of the protocol is to increase polyclonal antibodies specificity through pre-adsorption against cross-reactive antigens.
Asunto(s)
Anticuerpos/inmunología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Anticuerpos/química , Especificidad de Anticuerpos , Cromatografía de Afinidad , Reacciones Cruzadas , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/inmunología , Proteína Estafilocócica A/química , Proteína Estafilocócica A/inmunologíaRESUMEN
Regulatory T (T reg) cells are central mediators of immune suppression. As such, T reg cells are characterized by a distinct pattern of gene expression, which includes up-regulation of immunosuppressive genes and silencing of inflammatory cytokine genes. Although an increasing number of transcription factors that regulate T reg cells have been identified, the mechanisms by which the T reg cell-specific transcriptional program is maintained and executed remain largely unknown. The Nr4a family of nuclear orphan receptors, which we recently identified as essential for the development of T reg cells, is highly expressed in mature T reg cells as well, suggesting that Nr4a factors play important roles even beyond T reg cell development. Here, we showed that deletion of Nr4a genes specifically in T reg cells caused fatal systemic immunopathology. Nr4a-deficient T reg cells exhibited global alteration of the expression of genes which specify the T reg cell lineage, including reduction of Foxp3 and Ikzf4. Furthermore, Nr4a deficiency abrogated T reg cell suppressive activities and accelerated conversion to cells with Th2 and follicular helper T (Tfh) effector-like characteristics, with heightened expression of Th2 and Tfh cytokine genes. These findings demonstrate that Nr4a factors play crucial roles in mature T reg cells by directly controlling a genetic program indispensable for T reg cell maintenance and function.
Asunto(s)
Receptores de Esteroides/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica , Interleucina-4/genética , Interleucina-4/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/inmunología , Receptores de Hormona Tiroidea/metabolismo , Células Th2/inmunología , Transcripción GenéticaRESUMEN
Follicular T helper (Tfh) cells promote germinal center (GC) reaction and high-affinity antibody production. The molecular mechanisms that regulate development and function of Tfh cells are not fully understood. Here we report that ligand-independent nuclear receptors of the Nr4a family are highly expressed in Tfh cells. In a well-established adoptive transfer model, enforced expression of Nr4a receptors reduces helper T cell expansion but apparently increased the T cell capacity to promote the GC response. On the other hand, deletion of all Nr4a receptors in T cells did not significantly affect expansion or differentiation of Tfh cells or the development of GC reaction. These findings suggest that Nr4a receptors may promote but are not necessary for Tfh development or function in vivo.
Asunto(s)
Proteínas de Unión al ADN/inmunología , Proteínas del Tejido Nervioso/inmunología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Receptores de Esteroides/inmunología , Receptores de Hormona Tiroidea/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Diferenciación Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Centro Germinal , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , ARN Mensajero/metabolismo , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
The functional roles of the orphan nuclear receptor, Nurr1, have been extensively studied and well established in the development and survival of midbrain dopamine neurons. As Nurr1 and other NR4A members are widely expressed in the brain in overlapping and distinct manners, it has been an open question whether Nurr1 has important function(s) in other brain areas. Recent studies suggest that up-regulation of Nurr1 expression is critical for cognitive functions and/or long-term memory in forebrain areas including hippocampal formation. Questions remain about the association between Nurr1 expression and Alzheimer's disease (AD) brain pathology. Here, using our newly developed Nurr1-selective antibody, we report that Nurr1 protein is prominently expressed in brain areas with Aß accumulation, that is, the subiculum and the frontal cortex, in the 5XFAD mouse and that Nurr1 is highly co-expressed with Aß at early stages. Furthermore, the number of Nurr1-expressing cells significantly declines in the 5XFAD mouse in an age-dependent manner, accompanied by increased plaque deposition. Thus, our findings suggest that altered expression of Nurr1 is associated with AD progression. Using our newly developed Nurr1-selective antibody, we show that Nurr1 protein is prominently expressed in brain areas accumulating amyloid-beta (Aß) in the transgenic mouse model of Alzheimer's disease (AD) and that Nurr1 is highly co-expressed with Aß at early stages (upper panel). Furthermore, in the AD brain the number of Nurr1-expressing cells significantly declines in an age-dependent manner concomitant with increased Aß accumulation (lower diagram) highlighting a possible Nurr1 involvement in AD pathology.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/fisiología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/fisiología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Técnica del Anticuerpo Fluorescente Directa , Hipocampo/patología , Técnicas para Inmunoenzimas , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/biosíntesis , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
PURPOSE OF REVIEW: To understand chronic inflammatory diseases such as atherosclerosis, we require in-depth knowledge on immune-cell differentiation, function of specific immune-cell subsets and endothelial cell-mediated extravasation. In this review, we summarize a number of very recent observations on the pivotal function of NR4A nuclear receptors in immunity and atherosclerosis. RECENT FINDINGS: NR4A nuclear receptors are involved in negative selection of thymocytes, Treg differentiation and the development of Ly6C monocytes. Nur77 and Nurr1 attenuate atherosclerosis in mice whereas NOR-1 aggravates vascular lesion formation. SUMMARY: These exciting, novel insights on the function of NR4A nuclear receptors in immunity, vascular cells and atherosclerosis will initiate a plethora of studies to understand the underlying molecular mechanisms, which will culminate in the identification of novel NR4A targets to modulate chronic inflammatory disease.
Asunto(s)
Aterosclerosis/inmunología , Células Endoteliales/inmunología , Monocitos/inmunología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Linfocitos T Reguladores/inmunología , Animales , Aterosclerosis/patología , Diferenciación Celular/inmunología , Células Endoteliales/patología , Humanos , Ratones , Monocitos/patología , Linfocitos T Reguladores/patologíaRESUMEN
The biological function of the Nr4a subfamily of nuclear receptors is only partially understood. Here we show for the fist time that mast cell (MC) activation processes involve the regulation of Nr4a factors. Exposure of murine bone marrow-derived MCs (BMMCs) to live bacteria causes a robust and selective upregulation of all Nr4a members (Nr4a1-Nr4a3). In response to purified LPS, strong upregulation of Nr4a3, but not of Nr4a1 or Nr4a2 was seen. Nr4a3 expression was also induced after the activation of BMMCs by IgE receptor cross-linking. Moreover, Nr4a expression was induced in activated human MCs. As shown by Western blot analysis, Nr4a phosphorylation was induced by IgE receptor cross-linking and calcium ionophore stimulation of BMMCs and LAD2 cells, respectively. By using various inhibitors of signaling pathways, Nr4a3 induction in BMMCs was shown to be strongly dependent on Gö6976-sensitive kinases and partially dependent on the nuclear factor of activated T-cells (NFAT) pathway, while nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) inhibition failed to inhibit Nr4a3 expression in BMMCs. Together, these data reveal selective induction of Nr4a family members in activated MCs and implicate Nr4a family nuclear receptors in the regulation of MC function.
Asunto(s)
Mastocitos/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 3 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Transducción de Señal/inmunología , Animales , Western Blotting , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/inmunología , Humanos , Mastocitos/inmunología , Ratones , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Miembro 3 del Grupo A de la Subfamilia 4 de Receptores Nucleares/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Regulatory T cells (Tregs) have a central role in maintaining immune homoeostasis through various mechanisms. Although the Forkhead transcription factor Foxp3 defines the Treg cell lineage and functions, the molecular mechanisms of Foxp3 induction and maintenance remain elusive. Here we show that Foxp3 is one of the direct targets of Nr4a2. Nr4a2 binds to regulatory regions of Foxp3, where it mediates permissive histone modifications. Ectopic expression of Nr4a2 imparts Treg-like suppressive activity to naïve CD4(+) T cells by inducing Foxp3 and by repressing cytokine production, including interferon-γ and interleukin-2. Deletion of Nr4a2 in T cells attenuates induction of Tregs and causes aberrant induction of Th1, leading to the exacerbation of colitis. Nr4a2-deficeint Tregs are prone to lose Foxp3 expression and have attenuated suppressive ability both in vitro and in vivo. Thus, Nr4a2 has the ability to maintain T-cell homoeostasis by regulating induction, maintenance and suppressor functions of Tregs, and by repression of aberrant Th1 induction.