RESUMEN
The B-cell CLL/lymphoma 11B gene (BCL11B) plays a crucial role in T-cell development, but its role in T-cell malignancies is still unclear. To study its role in the development of T-cell neoplasms, we generated an inducible BCL11B knockout in a murine T cell leukemia/lymphoma model. Mice, bearing human oncogenes TAL BHLH Transcription Factor 1 (TAL1; SCL) or LIM Domain Only 1 (LMO1), responsible for T-cell acute lymphoblastic leukemia (T-ALL) development, were crossed with BCL11B floxed and with CRE-ER/lox mice. The mice with a single oncogene BCL11Bflox/floxCREtg/tgTAL1tg or BCL11Bflox/floxCREtg/tgLMO1tg were healthy, bred normally, and were used to maintain the mice in culture. When crossed with each other, >90% of the double transgenic mice BCL11Bflox/floxCREtg/tgTAL1tgLMO1tg, within 3 to 6 months after birth, spontaneously developed T-cell leukemia/lymphoma. Upon administration of synthetic estrogen (tamoxifen), which binds to the estrogen receptor and activates the Cre recombinase, the BCL11B gene was knocked out by excision of its fourth exon from the genome. The mouse model of inducible BCL11B knockout we generated can be used to study the role of this gene in cancer development and the potential therapeutic effect of BCL11B inhibition in T-cell leukemia and lymphoma.
Asunto(s)
Leucemia de Células T , Factores de Transcripción , Animales , Modelos Animales de Enfermedad , Proteínas con Dominio LIM/genética , Leucemia de Células T/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas Represoras/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
T cell activation is an energy-demanding process fueled by increased glucose consumption and accompanied by upregulation of the insulin receptor (INSR). In this article, we report that silencing the INSR in inducible knockdown rats impairs selective T cell functions but not thymocyte development. Glucose transport and glycolysis in activated CD4+ T cells were compromised in the absence of the INSR, which was associated with alterations in intracellular signaling pathways. The observed metabolic defects coincided with reduced cytokine production, proliferation, and migration, as well as increased apoptosis of CD4+ T cells. The cytotoxicity of CD8+ T cells in response to alloantigens was also diminished under these conditions, whereas the frequency and suppressive capacity of regulatory T cells were unaffected. The observed impairments proved to be decisive in vivo because silencing of the INSR attenuated clinical symptoms in animal models of acute graft-versus-host disease and multiple sclerosis. Taken together, our results suggest that upregulation of the INSR on T cells following activation is required for efficient adaptive immunity.
Asunto(s)
Inmunidad Adaptativa , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/fisiología , Receptor de Insulina/deficiencia , Receptor de Insulina/fisiología , Timocitos/fisiología , Animales , Apoptosis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular , Citocinas/biosíntesis , Citocinas/inmunología , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Glucólisis/inmunología , Enfermedad Injerto contra Huésped/inmunología , Activación de Linfocitos/inmunología , Ratas , Receptor de Insulina/genética , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Timocitos/inmunologíaRESUMEN
Background Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex three-dimensional morphology of podocytes depends on the actin cytoskeleton, we studied the role in podocytes of the actin bundling protein palladin, which is highly expressed therein.Methods We knocked down palladin in cultured podocytes by siRNA transfection or in zebrafish embryos by morpholino injection and studied the effects by immunofluorescence and live imaging. We also investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld-/- mice) by immunofluorescence and ultrastructural analysis and kidney biopsy specimens from patients by immunostaining for palladin.Results Compared with control-treated podocytes, palladin-knockdown podocytes had reduced actin filament staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin and α-actinin-4. Furthermore, palladin-knockdown podocytes were more susceptible to disruption of the actin cytoskeleton with cytochalasin D, latrunculin A, or jasplakinolide and showed altered migration dynamics. In zebrafish embryos, palladin knockdown compromised the morphology and dynamics of epithelial cells at an early developmental stage. Compared with PodoPalld+/+ controls, PodoPalld-/- mice developed glomeruli with a disturbed morphology, an enlarged subpodocyte space, mild effacement, and significantly reduced expression of nephrin and vinculin. Furthermore, nephrotoxic serum injection led to significantly higher levels of proteinuria in PodoPalld-/- mice than in controls. Kidney biopsy specimens from patients with diabetic nephropathy and FSGS showed downregulation of palladin in podocytes as well.Conclusions Palladin has an important role in podocyte function in vitro and in vivo.
Asunto(s)
Actinas/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Podocitos/metabolismo , Animales , Citoesqueleto , Femenino , Adhesiones Focales , Expresión Génica , Silenciador del Gen , Humanos , Glomérulos Renales/patología , Masculino , Ratones Noqueados , Proteínas de Microfilamentos/metabolismo , Morfolinos/farmacología , Podocitos/patología , ARN Mensajero/metabolismo , Vinculina/genética , Vinculina/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
T-cell lymphopenia is a major risk factor for autoimmunity. Here we describe congenic Lewis (LEW) rats with a loss-of-function mutation in the Gimap5 gene, leading to a 92% reduction in peripheral T-cell numbers. Gimap5-deficient LEW rats developed eosinophilic autoimmune gastroenteritis accompanied by a 40-fold increase in IgE serum levels. This phenotype was ameliorated by antibiotic treatment, indicating a critical role of the microbial flora in the development of inflammatory bowel disease. Interestingly, Gimap5-deficient LEW rats showed strongly aggravated experimental autoimmune encephalomyelitis (EAE) after immunization with guinea pig myelin basic protein. This phenotype, however, persisted after antibiosis, confirming that the enhanced CNS autoimmune response in T-cell lymphopenic Gimap5-deficient LEW rats was unrelated to the composition of the microbial flora. Rather, it seems that it was caused by the 7-fold increase in the percentage of activated T cells producing IL-17 and IFN-γ, and the skewed T-cell receptor (TCR) repertoire, both of which were the result of T-cell lymphopenia and not affected by antibiosis. This notion was supported by the observation that adoptive T-cell transfer corrected the TCR repertoire and improved EAE. Collectively, our findings confirm a critical albeit differential role of T-cell lymphopenia in the susceptibility to organ-specific autoimmune responses.-Fischer, H. J., Witte, A.-K., Walter, L., Gröne, H.-J., van den Brandt, J., Reichardt, H. M. Distinct roles of T-cell lymphopenia and the microbial flora for gastrointestinal and CNS autoimmunity.
Asunto(s)
Enfermedades Autoinmunes/microbiología , Sistema Nervioso Central/microbiología , Proteínas de Unión al GTP/metabolismo , Tracto Gastrointestinal/inmunología , Linfopenia , Linfocitos T/fisiología , Traslado Adoptivo , Animales , Enfermedades Autoinmunes/metabolismo , Sistema Nervioso Central/inmunología , Enfermedades del Sistema Nervioso Central/genética , Enfermedades del Sistema Nervioso Central/metabolismo , Proteínas de Unión al GTP/genética , Gastroenteritis/genética , Gastroenteritis/metabolismo , Gastroenteritis/microbiología , Tracto Gastrointestinal/microbiología , Regulación de la Expresión Génica/inmunología , Mutación Puntual , RatasRESUMEN
Brain-derived neurotrophic factor (BDNF) promotes neuronal survival, regeneration, and plasticity. Emerging evidence also indicates an essential role for BDNF outside the nervous system, for instance in immune cells. We therefore investigated the impact of BDNF on T cells using BDNF knockout (KO) mice and conditional KO mice lacking BDNF specifically in this lymphoid subset. In both settings, we observed diminished T-cell cellularity in peripheral lymphoid organs and an increase in CD4(+) CD44(+) memory T cells. Analysis of thymocyte development revealed diminished total thymocyte numbers, accompanied by a significant increase in CD4/CD8 double-negative (DN) thymocytes due to a partial block in the transition from the DN3 to the DN4 stage. This was neither due to increased thymocyte apoptosis nor defects in the expression of the TCR-ß chain or the pre-TCR. In contrast, pERK but not pAKT levels were diminished in DN3 BDNF-deficient thymocytes. BDNF deficiency in T cells did not result in gross deficits in peripheral acute immune responses nor in changes of the homeostatic proliferation of peripheral T cells. Taken together, our data reveal a critical autocrine and/or paracrine role of T-cell-derived BDNF in thymocyte maturation involving ERK-mediated TCR signaling pathways.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/deficiencia , Factor Neurotrófico Derivado del Encéfalo/genética , Diferenciación Celular , Femenino , Memoria Inmunológica , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Linfopoyesis , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/inmunologíaRESUMEN
Glucocorticoids (GCs) repress lymphocyte function by controlling gene expression. In this study, we investigated Ag-specific effector T cells and provide evidence that GCs also modulate these cells' cytoskeletal architecture by nongenomic mechanisms. Following GC treatment, effector T cells rapidly lose their polarized morphology, which impedes both their migratory capacity and their interaction with APCs. The cytoskeleton rearrangements are preceded by an activation of ezrin-radixin-moesin proteins, which transiently increases the cellular rigidity but seems to occur independently of altered tyrosine phosphorylation. Phospholipase C activity is critically involved in mediating these nongenomic effects, because its inhibition prevents both T cell depolarization and ezrin-radixin-moesin phosphorylation after GC exposure. GC administration in vivo induced similar morphological changes in effector T cells as observed in vitro, suggesting that the above process plays a role in modulating inflammatory diseases. Taken together, our findings identify a novel mechanism through which GCs rapidly repress T cell function independently of gene transcription.
Asunto(s)
Células Presentadoras de Antígenos/inmunología , Inhibición de Migración Celular/inmunología , Proteínas del Citoesqueleto/fisiología , Dexametasona/farmacología , Proteínas de la Membrana/fisiología , Proteínas de Microfilamentos/fisiología , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Inhibición de Migración Celular/efectos de los fármacos , Polaridad Celular/efectos de los fármacos , Polaridad Celular/inmunología , Células Cultivadas , Ratas , Ratas Endogámicas Lew , Ratas Transgénicas , Subgrupos de Linfocitos T/ultraestructuraRESUMEN
Glucocorticoids (GCs) are the standard therapy for treating multiple sclerosis (MS) patients suffering from an acute relapse. One of the main mechanisms of GC action is held to be the induction of T cell apoptosis leading to reduced lymphocyte infiltration into the CNS, yet our analysis of experimental autoimmune encephalomyelitis (EAE) in three different strains of genetically manipulated mice has revealed that the induction of T cell apoptosis is not essential for the therapeutic efficacy of GCs. Instead, we identified the redirection of T cell migration in response to chemokines as a new therapeutic principle of GC action. GCs inhibited the migration of T cells towards CCL19 while they enhanced their responsiveness towards CXCL12. Importantly, blocking CXCR4 signaling in vivo by applying Plerixafor(®) strongly impaired the capacity of GCs to interfere with EAE, as revealed by an aggravated disease course, more pronounced CNS infiltration and a more dispersed distribution of the infiltrating T cells throughout the parenchyma. Our observation that T cells lacking the GC receptor were refractory to CXCL12 further underscores the importance of this pathway for the treatment of EAE by GCs. Importantly, methylprednisolone pulse therapy strongly increased the capacity of peripheral blood T cells from MS patients of different subtypes to migrate towards CXCL12. This indicates that modulation of T cell migration is an important mechanistic principle responsible for the efficacy of high-dose GC therapy not only of EAE but also of MS.
Asunto(s)
Quimiocinas/metabolismo , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Glucocorticoides/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Linfocitos T/efectos de los fármacos , Adulto , Anciano , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Humanos , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Esclerosis Múltiple/fisiopatología , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Médula Espinal/fisiopatología , Linfocitos T/fisiologíaRESUMEN
We recently described that T cell specification in mice deficient in the Hedgehog (Hh) receptor Patched (Ptch) is blocked at the level of the common lymphoid progenitor in the bone marrow (BM). Adoptive transfer of wild-type BM in Ptch-deficient mice provides evidence that T cell development strictly depends on Ptch expression in the nonhematopoietic compartment. Transplantation experiments using BM deficient in the glucocorticoid receptor exclude any involvement of the stress hormone corticosterone in our model. Using cell-type-specific knockout mice, we show that T cell development is independent of T cell-intrinsic Ptch expression. Furthermore, Ptch expression by the thymus stroma is dispensable, as revealed by fetal thymus organ culture and thymus transplantation. In contrast, analysis of the earliest thymic progenitors in Ptch-deficient mice indicated that Ptch is required for the development or supply of thymic homing progenitors that give rise to earliest thymic progenitors. Collectively, our findings identified Ptch as an exclusive T cell-extrinsic factor necessary for proper development of T cells at their prethymic stage. This observation may be important for current considerations using Hh inhibitors upstream of Ptch in diseases accompanied by aberrant Hh signaling.
Asunto(s)
Diferenciación Celular/inmunología , Regulación del Desarrollo de la Expresión Génica/inmunología , Receptores de Superficie Celular/fisiología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Timo/inmunología , Timo/metabolismo , Animales , Diferenciación Celular/genética , Depleción Linfocítica , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Técnicas de Cultivo de Órganos , Receptores Patched , Receptor Patched-1 , Quimera por Radiación , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Células Madre/citología , Células Madre/inmunología , Células Madre/metabolismo , Células del Estroma/citología , Células del Estroma/inmunología , Células del Estroma/metabolismo , Subgrupos de Linfocitos T/metabolismo , Timo/citologíaRESUMEN
The activity of acid sphingomyelinase (aSMase) was previously reported to be involved in glucocorticoid-induced cell death (GICD) of T lymphocytes. This mechanism in turn is believed to contribute to the therapeutic efficacy of glucocorticoids (GCs) in the treatment of inflammatory diseases. In this study, we reassessed the role of aSMase in GICD by using aSMase knockout mice. The absence of aSMase largely abolished the partial protection that effector memory CD4(+) T cells in wild-type mice possess against GICD. Reduced IL-2 secretion by aSMase-deficient CD4(+) T cells suggested that a lack of this important survival factor might be the cause of these cells' enhanced susceptibility to GICD. Indeed, addition of IL-2 restored the protection against GICD, whereas neutralization of IL-2 abrogated the otherwise protective effect seen in wild-type effector memory CD4(+) T cells. The therapeutic implications of the altered sensitivity of aSMase-deficient T cells to GICD were assessed in models of inflammatory disorders; namely, experimental autoimmune encephalomyelitis and acute graft-versus-host disease. Surprisingly, GC treatment was equally efficient in both models in terms of ameliorating the diseases, regardless of the genotype of the T cells. Thus, our data reveal a hitherto unrecognized contribution of aSMase to the sensitivity of effector memory CD4(+) T cells to GICD and call into question the traditionally attributed importance of GICD of T cells to the treatment of inflammatory diseases by GCs.
Asunto(s)
Dexametasona/toxicidad , Memoria Inmunológica , Esfingomielina Fosfodiesterasa/fisiología , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/inmunología , Animales , Apoptosis/genética , Apoptosis/inmunología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Muerte Celular/inmunología , Células Cultivadas , Dexametasona/antagonistas & inhibidores , Dexametasona/uso terapéutico , Enfermedad Injerto contra Huésped/enzimología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Humanos , Memoria Inmunológica/efectos de los fármacos , Memoria Inmunológica/genética , Interleucina-2/antagonistas & inhibidores , Interleucina-2/metabolismo , Coriomeningitis Linfocítica/enzimología , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Esfingomielina Fosfodiesterasa/deficiencia , Esfingomielina Fosfodiesterasa/genética , Subgrupos de Linfocitos T/efectos de los fármacosRESUMEN
Glucocorticoids (GCs) are widely used to treat acute relapses of multiple sclerosis (MS). In this study, we demonstrate that liposomal encapsulation augments the therapeutic potency of GCs as they ameliorate experimental autoimmune encephalomyelitis (EAE) to the same extent as free GC, but at strongly reduced dosage and application frequency. Importantly, this is accompanied by an altered mode of action. Unlike free GCs, which mainly target T lymphocytes during EAE therapy, liposomal GCs only marginally affect T cell apoptosis and function. In contrast, liposomal GCs efficiently repress proinflammatory macrophage functions and upregulate anti-inflammatory genes associated with the alternatively activated M2 phenotype. The GC receptor (GR) per se is indispensable for the therapeutic efficacy of liposomal GC. In contrast to free GCs, however, the individual deletion of the GR either in T cells or myeloid cells has little effect on the efficacy of liposomal GCs in the treatment of EAE. Only the combined deletion of the GR in both cellular compartments markedly compromises the therapeutic effect of liposomal GCs on disease progression. In conclusion, encapsulation of GC does not only enhance their efficacy in the treatment of EAE but also alters their target cell specificity and their mode of action compared with free GCs.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Glucocorticoides/administración & dosificación , Liposomas , Macrófagos/efectos de los fármacos , Animales , Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Glucocorticoides/deficiencia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Diabetes-prone BioBreeding (DP-BB) rats spontaneously develop type 1 diabetes mellitus (T1DM) on grounds of their MHC haplotype RT1(u) and a point mutation in the Gimap5 gene. In this study, we report that DP-BB rats exhibit an increasingly severe imbalance, in particular between Th17 and regulatory T (T(reg)) cells, within the first months of age. This can be assigned to an excess in effector T cells because neither the percentage nor the function of the T(reg) cells is compromised. Flow cytometric analysis of Vbeta segment usage and CDR3 spectratyping further suggest that the disturbed repertoire of peripheral T cells may also contribute to the development of T1DM in DP-BB rats. Importantly, expansion of T(reg) cells in vivo by means of a CD28 superagonistic Ab as well as adoptive transfer of T(reg) cells efficiently interferes with the development of T1DM in DP-BB rats, whereas treatment with conventional Th cells does not afford protection. Using a newly generated strain of enhanced GFP transgenic rats, we could further demonstrate that the transferred T(reg) cells persist in the recipient rats for several months and partially correct the imbalance between Th17 and T(reg) cells. Thus, our data support the hypothesis that unchecked effector T cell action and a disturbed T cell repertoire contribute to the development of T1DM in DP-BB rats, which may also have implications for a better understanding of the human disease.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Traslado Adoptivo , Animales , Línea Celular , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Femenino , Citometría de Flujo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/inmunología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Interleucina-17/inmunología , Interleucina-17/metabolismo , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Recuento de Linfocitos , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/patología , Masculino , Mutación , Ratas , Ratas Endogámicas BB , Ratas Transgénicas , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/trasplanteRESUMEN
Pancreatic ductal adenocarcinoma (PDAC), due to its genomic heterogeneity and lack of effective treatment, despite decades of intensive research, will become the second leading cause of cancer-related deaths by 2030. Step-wise acquisition of mutations, due to genomic instability, is considered to drive the development of PDAC; the KRAS mutation occurs in 95 to 100% of human PDAC, and is already detectable in early premalignant lesions designated as pancreatic intraepithelial neoplasia (PanIN). This mutation is possibly the key event leading to genomic instability and PDAC development. Our study aimed to investigate the role of the error-prone DNA double-strand breaks (DSBs) repair pathway, alt-EJ, in the presence of the KRAS G12D mutation in pancreatic cancer development. Our findings show that oncogenic KRAS contributes to increasing the expression of Polθ, Lig3, and Mre11, key components of alt-EJ in both mouse and human PDAC models. We further confirm increased catalytic activity of alt-EJ in a mouse and human model of PDAC bearing the KRAS G12D mutation. Subsequently, we focused on estimating the impact of alt-EJ inactivation by polymerase theta (Polθ) deletion on pancreatic cancer development, and survival in genetically engineered mouse models (GEMMs) and cancer patients. Here, we show that even though Polθ deficiency does not fully prevent the development of pancreatic cancer, it significantly delays the onset of PanIN formation, prolongs the overall survival of experimental mice, and correlates with the overall survival of pancreatic cancer patients in the TCGA database. Our study clearly demonstrates the role of alt-EJ in the development of PDAC, and alt-EJ may be an attractive therapeutic target for pancreatic cancer patients.
RESUMEN
Administration of the CD28 superagonistic antibody JJ316 is an efficient means to treat autoimmune diseases in rats, but the humanized antibody TGN1412 caused devastating side effects in healthy volunteers during a clinical trial. Here we show that JJ316 treatment of rats induced a dramatic redistribution of T lymphocytes from the periphery to the secondary lymphoid organs, resulting in severe T lymphopenia. Live imaging of secondary lymphoid organs revealed that JJ316 administration almost instantaneously (<2 minutes) arrested T cells in situ. This reduction in T cell motility was accompanied by profound cytoskeletal rearrangements and increased cell size. In addition, surface expression of lymphocyte function-associated antigen-1 was enhanced, endothelial differentiation sphingolipid G protein-coupled receptor 1 and L selectin levels were downregulated, and the cells lost their responsiveness to sphingosine 1-phosphate-directed migration. These proadhesive alterations were accompanied by signs of strong activation, including upregulation of CD25, CD69, CD134, and proinflammatory mediators. However, this did not lead to a cytokine storm similar to the clinical trial. While most of the early changes disappeared within 48 hours, we observed that CD4+CD25+FoxP3+ regulatory T cells experienced a second phase of activation, which resulted in massive cell enlargement, extensive polarization, and increased motility. These data suggest that CD28 superagonists elicit 2 qualitatively distinct waves of activation.
Asunto(s)
Anticuerpos/inmunología , Antígenos CD28/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos/administración & dosificación , Biomarcadores , Adhesión Celular/efectos de los fármacos , Adhesión Celular/inmunología , Movimiento Celular , Polaridad Celular/inmunología , Supervivencia Celular , Citocinas/inmunología , Citocinas/metabolismo , Citoesqueleto/inmunología , Activación de Linfocitos/efectos de los fármacos , Tejido Linfoide/inmunología , Linfopenia/inducido químicamente , Linfopenia/inmunología , Linfopenia/patología , Masculino , Ratas , Ratas Endogámicas Lew , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Currently, tools to generate loss-of-function mutations in rats are limited. Therefore, we have developed a lentiviral single-vector system for the temporal control of ubiquitous shRNA expression. Here, we report transgenic rats carrying an insulin receptor-specific shRNA transcribed from a regulatable promoter and identified by concomitant EGFP expression. In the absence of the inducer doxycycline (Dox), we observed no siRNA expression. However, Dox treatment at very low concentrations led to a rapid induction of the siRNA and ablation of INSR protein expression. As anticipated, blood glucose levels increased, whereas insulin signaling and glucose regulation were impaired. Importantly, this phenotype was reversible (i.e., discontinuation of Dox treatment led to INSR re-expression and remission of diabetes symptoms). The lentiviral system offers a simple tool for reversible gene ablation in the rat and can be used for other species that cannot be manipulated by conventional recombination techniques.
Asunto(s)
Silenciador del Gen , Lentivirus/genética , ARN Viral/genética , Integración Viral , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/genética , Técnicas de Silenciamiento del Gen , Vectores Genéticos , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/administración & dosificación , Insulina/metabolismo , Ratas , Ratas Transgénicas , Transducción de SeñalRESUMEN
Proper and size selective blood filtration in the kidney depends on an intact morphology of podocyte foot processes. Effacement of interdigitating podocyte foot processes in the glomeruli causes a leaky filtration barrier resulting in proteinuria followed by the development of chronic kidney diseases. Since the function of the filtration barrier is depending on a proper actin cytoskeleton, we studied the role of the important actin-binding protein palladin for podocyte morphology. Podocyte-specific palladin knockout mice on a C57BL/6 genetic background (PodoPalldBL/6-/-) were back crossed to a 129 genetic background (PodoPalld129-/-) which is known to be more sensitive to kidney damage. Then we analyzed the morphological changes of glomeruli and podocytes as well as the expression of the palladin-binding partners Pdlim2, Lasp-1, Amotl1, ezrin and VASP in 6 and 12 months old mice. PodoPalld129-/- mice in 6 and 12 months showed a marked dilatation of the glomerular tuft and a reduced expression of the mesangial marker protein integrin α8 compared to controls of the same age. Furthermore, ultrastructural analysis showed significantly more podocytes with morphological deviations like an enlarged sub-podocyte space and regions with close contact to parietal epithelial cells. Moreover, PodoPalld129-/- of both age showed a severe effacement of podocyte foot processes, a significantly reduced expression of pLasp-1 and Pdlim2, and significantly reduced mRNA expression of Pdlim2 and VASP, three palladin-interacting proteins. Taken together, the results show that palladin is essential for proper podocyte morphology in mice with a 129 background.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/fisiología , Antecedentes Genéticos , Proteínas de Homeodominio/metabolismo , Proteínas con Dominio LIM/metabolismo , Proteínas de Microfilamentos/metabolismo , Podocitos/metabolismo , Citoesqueleto de Actina , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas del Citoesqueleto/genética , Proteínas de Homeodominio/genética , Riñón/metabolismo , Proteínas con Dominio LIM/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Fosforilación , Podocitos/citologíaRESUMEN
Autoimmune responses in the CNS can be induced by adoptive transfer of CD4(+) T effector cells after antigen-restimulation and expansion of clonal cell lines in vitro. However, pathogenic factors remain partially elusive due to the lack of appropriate methods to achieve gene inactivation. Here we describe a protocol for stable gene silencing in differentiated rat T cells by retroviral transfer of small hairpin RNAs. Through the combination of an expression cassette containing the green fluorescent protein with a puromycin selection cassette this allows for the generation of pure knockdown cell lines suitable for tracking in animals. Exemplified for the glucocorticoid receptor, we demonstrate that gene silencing renders T effector cells unresponsive to ligand-induced apoptosis and gene regulation without affecting their ability to induce EAE in rats. Interestingly, glucocorticoid administration remains effective in the treatment of EAE despite strongly diminished glucocorticoid receptor expression in antigen-specific T cells. This highlights an important role of other cell types and bystander T cells as targets of glucocorticoid therapy. Collectively, our approach provides a simple tool for stable and efficient gene silencing in T effector cells, which should help to better understand brain autoimmune pathophysiology.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/trasplante , Encefalomielitis Autoinmune Experimental/inmunología , Glucocorticoides/uso terapéutico , Receptores de Glucocorticoides/genética , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Silenciador del Gen , Glucocorticoides/farmacología , Interferencia de ARN/inmunología , Ratas , Ratas Endogámicas Lew , RetroviridaeRESUMEN
Rats are a reservoir of human- and livestock-associated methicillin-resistant Staphylococcus aureus (MRSA). However, the composition of the natural S. aureus population in wild and laboratory rats is largely unknown. Here, 144 nasal S. aureus isolates from free-living wild rats, captive wild rats and laboratory rats were genotyped and profiled for antibiotic resistances and human-specific virulence genes. The nasal S. aureus carriage rate was higher among wild rats (23.4%) than laboratory rats (12.3%). Free-living wild rats were primarily colonized with isolates of clonal complex (CC) 49 and CC130 and maintained these strains even in husbandry. Moreover, upon livestock contact, CC398 isolates were acquired. In contrast, laboratory rats were colonized with many different S.aureus lineages-many of which are commonly found in humans. Five captive wild rats were colonized with CC398-MRSA. Moreover, a single CC30-MRSA and two CC130-MRSA were detected in free-living or captive wild rats. Rat-derived S. aureus isolates rarely harbored the phage-carried immune evasion gene cluster or superantigen genes, suggesting long-term adaptation to their host. Taken together, our study revealed a natural S. aureus population in wild rats, as well as a colonization pressure on wild and laboratory rats by exposure to livestock- and human-associated S.aureus, respectively.
Asunto(s)
Animales Salvajes/microbiología , Infecciones Estafilocócicas/epidemiología , Staphylococcus aureus/aislamiento & purificación , Animales , Antibacterianos/farmacología , Coagulación Sanguínea , República Checa , Ecosistema , Alemania , Meticilina/farmacología , Epidemiología Molecular , Nariz/microbiología , Ratas Sprague-Dawley , Infecciones Estafilocócicas/veterinaria , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Factores de Virulencia/genéticaRESUMEN
Currently, gene disruption by homologous recombination in embryonic stem cells is only feasible in mice. To circumvent this problem, we silenced mineralocorticoid receptor (MR) expression by RNA interference in knockdown rats generated through lentiviral transgenesis. Analysis of the F1 progeny at 3 wk of age revealed strongly decreased MR levels. This was specific for the targeted gene and related to the abundance of the short interfering RNA. Reminiscent of MR knockout mice, the transgenic rats showed a reduced body weight, elevated serum aldosterone levels, increased plasma renin activity, and altered expression of MR target genes. Some of these effects correlated with the degree to which MR mRNA expression was reduced. Whereas disruption of the MR by gene targeting in mice leads to postnatal death, our strategy also allowed obtaining adult knockdown rats with defects in hormone and electrolyte homeostasis resembling pseudohypoaldosteronism. In conclusion, this is the first example of a human disease model based on RNA interference in rats.
Asunto(s)
Sistema Endocrino/efectos de los fármacos , Sistema Endocrino/fisiología , Homeostasis/efectos de los fármacos , Antagonistas de Receptores de Mineralocorticoides , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Factores de Edad , Aldosterona/sangre , Animales , Animales Modificados Genéticamente , Células Cultivadas , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Femenino , Vectores Genéticos , Células HeLa , Homeostasis/genética , Humanos , Lentivirus/genética , Seudohipoaldosteronismo/genética , Seudohipoaldosteronismo/patología , ARN Mensajero/metabolismo , Ratas , Receptores de Mineralocorticoides/genética , Equilibrio Hidroelectrolítico/genéticaRESUMEN
Currently, it is controversially discussed whether a relationship between obesity and cognition exists. We here analyzed a mouse model of obesity (leptin-deficient mice) to study the effects of obesity on the morphology of the hippocampus (a brain structure involved in mechanisms related to learning and memory) and on behavior. Mice aged 4 to 6 months were analyzed. At this age, the obese mice have nearly double the body weight as controls, but display smaller brains (brain volume is about 10% smaller) as control animals of the same age. Adult hippocampal neurogenesis, a process that is linked to learning and memory, might be disturbed in the obese mice and contribute to the smaller brain volume. Adult hippocampal neurogenesis was examined using specific markers for cell proliferation (phosphohistone H3), neuronal differentiation (doublecortin), and apoptosis (caspase 3). The number of phosphohistone H3 and doublecortin-positive cells was markedly reduced in leptin-deficient mice, but not the number of apoptotic cells, indicating that adult hippocampal neurogenesis on the level of cell proliferation was affected. In addition, dendritic spine densities of pyramidal neurons in the hippocampal area CA1 were analyzed using Golgi impregnation. However, no significant change in dendritic spine densities was noted in the obese mice. Moreover, the performance of the mice was analyzed in the open field as well as in the Morris water maze. In the open field test, obese mice showed reduced locomotor activity, but in the Morris water maze they showed similar performance compared with control animals.
RESUMEN
Administration of high-dose myelin antigen induces massive T cell apoptosis in experimental autoimmune encephalomyelitis (EAE) but the nature of the target cells remains elusive. Here we have used a cell line established in eGFP-transgenic Lewis rats to distinguish between pathogenic and bystander T cells in adoptive transfer EAE. Intravenous application of gpMBP strongly reduced the amount of encephalitogenic cells in spinal cord and spleen while the number of the other T cells remained constant. This could be attributed to their differential sensitivity to apoptosis. Thus, antigen therapy selectively targets pathogenic T cells and should therefore limit potential adverse effects.