RESUMEN
Senescent cells accumulate during ageing in various tissues and contribute to organismal ageing. However, factors that are involved in the induction of senescence in vivo are still not well understood. SNEV(P) (rp19/) (PSO) (4) is a multifaceted protein, known to be involved in DNA damage repair and senescence, albeit only in vitro. In this study, we used heterozygous SNEV(+/-) mice (SNEV-knockout results in early embryonic lethality) and wild-type littermate controls as a model to elucidate the role of SNEV(P) (rp19/) (PSO) (4) in DNA damage repair and senescence in vivo. We performed PUVA treatment as model system for potently inducing cellular senescence, consisting of 8-methoxypsoralen in combination with UVA on mouse skin to induce DNA damage and premature skin ageing. We show that SNEV(P) (rp19/) (PSO) (4) expression decreases during organismal ageing, while p16, a marker of ageing in vivo, increases. In response to PUVA treatment, we observed in the skin of both SNEV(P) (rp19/) (PSO) (4) and wild-type mice an increase in γ-H2AX levels, a DNA damage marker. In old SNEV(P) (rp19/) (PSO) (4) mice, this increase is accompanied by reduced epidermis thickening and increase in p16 and collagenase levels. Thus, the DNA damage response occurring in the mouse skin upon PUVA treatment is dependent on SNEV(P) (rp19/) (PSO) (4) expression and lower levels of SNEV(P) (rp19/) (PSO) (4) , as in old SNEV(+/-) mice, result in increase in cellular senescence and acceleration of premature skin ageing.
Asunto(s)
Colagenasas/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Terapia PUVA/métodos , Factores de Empalme de ARN/genética , Envejecimiento de la Piel/fisiología , Piel/metabolismo , Envejecimiento Prematuro , Animales , Senescencia Celular , Colágeno/metabolismo , Daño del ADN , Epidermis/metabolismo , Femenino , Genotipo , Heterocigoto , Histonas/metabolismo , Masculino , Metoxaleno/química , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Empalme de ARN/metabolismoRESUMEN
BACKGROUND: Acute graft-versus-host disease (aGVHD) poses a major limitation for broader therapeutic application of allogeneic hematopoietic cell transplantation (allo-HCT). Early diagnosis of aGVHD remains difficult and is based on clinical symptoms and histopathological evaluation of tissue biopsies. Thus, current aGVHD diagnosis is limited to patients with established disease manifestation. Therefore, for improved disease prevention it is important to develop predictive assays to identify patients at risk of developing aGVHD. Here we address whether insights into the timing of the aGVHD initiation and effector phases could allow for the detection of migrating alloreactive T cells before clinical aGVHD onset to permit for efficient therapeutic intervention. METHODS: Murine major histocompatibility complex (MHC) mismatched and minor histocompatibility antigen (miHAg) mismatched allo-HCT models were employed to assess the spatiotemporal distribution of donor T cells with flow cytometry and in vivo bioluminescence imaging (BLI). Daily flow cytometry analysis of peripheral blood mononuclear cells allowed us to identify migrating alloreactive T cells based on homing receptor expression profiles. RESULTS: We identified a time period of 2 weeks of massive alloreactive donor T cell migration in the blood after miHAg mismatch allo-HCT before clinical aGVHD symptoms appeared. Alloreactive T cells upregulated α4ß7 integrin and P-selectin ligand during this migration phase. Consequently, targeted preemptive treatment with rapamycin, starting at the earliest detection time of alloreactive donor T cells in the peripheral blood, prevented lethal aGVHD. CONCLUSIONS: Based on this data we propose a critical time frame prior to the onset of aGVHD symptoms to identify alloreactive T cells in the peripheral blood for timely and effective therapeutic intervention.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/cirugía , Trasplante de Células Madre Hematopoyéticas/métodos , Subgrupos de Linfocitos T/trasplante , Enfermedad Aguda , Animales , Incompatibilidad de Grupos Sanguíneos/inmunología , Femenino , Predicción , Enfermedad Injerto contra Huésped/inmunología , Antígenos HLA/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Subgrupos de Linfocitos T/inmunología , Factores de Tiempo , Trasplante Homólogo , Resultado del TratamientoRESUMEN
OBJECTIVE: In the K/BxN mouse model of inflammatory arthritis, T cells carrying a transgenic T cell receptor initiate disease by helping B cells to produce arthritogenic anti-glucose-6-phosphate isomerase (anti-GPI) autoantibodies. We found that lethally- irradiated lymphocyte-deficient C57BL/6 (B6).g7 (I-A(g7) +) recombinase-activating gene-deficient (Rag(-/-)) mice reconstituted with K/BxN hematopoietic stem and progenitor cells exhibit arthritis by week 4. In contrast, healthy B6.g7 recipients of K/BxN hematopoietic stem and progenitor cells show only mild arthritis, with limited extent and duration. The objective of this study was to investigate the factors responsible for the attenuation of arthritis in B6.g7 recipients. METHODS: Antibody responses were measured by enzyme-linked immunosorbent assay. Fluorescence-activated cell sorting analyses were performed for testing chimerism, expression of markers of activation and suppression, tetramer binding, and intracellular cytokines in CD4+ T cells. Suppressive activity of CD4+ T cells was studied by adoptive transfer. RESULTS: Titers of anti-GPI antibodies in reconstituted B6.g7 mice were â¼60-fold lower than in reconstituted B6.g7 Rag(-/-) mice. Examination of chimerism in the reconstituted B6.g7 mice showed that B cells and myeloid cells in these mice were donor derived, but CD4+ T cells were primarily host derived and enriched for cells expressing the conventional regulatory markers CD25 and FoxP3. Notably, CD4+CD25-FoxP3- T cells expressed markers of suppressive function (CD73 and folate receptor 4), and delayed disease after adoptive transfer. Activation of donor-derived CD4+ T cells was reduced, and thymic deletion of these cells appeared increased. CONCLUSION: Despite myeloablation, host CD4+ T cells having a regulatory phenotype emerge in these mice and attenuate autoimmunity.
Asunto(s)
Artritis/inmunología , Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Células Madre Hematopoyéticas/inmunología , 5'-Nucleotidasa/metabolismo , Traslado Adoptivo , Animales , Artritis/etiología , Artritis/patología , Enfermedades Autoinmunes/etiología , Enfermedades Autoinmunes/patología , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Modelos Animales de Enfermedad , Factores de Transcripción Forkhead/metabolismo , Glucosa-6-Fosfato Isomerasa/genética , Glucosa-6-Fosfato Isomerasa/inmunología , Células Madre Hematopoyéticas/patología , Proteínas de Homeodominio/genética , Memoria Inmunológica/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Receptores de Superficie Celular/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Timo/citología , Timo/inmunología , Irradiación Corporal TotalRESUMEN
BACKGROUND & AIMS: Autoimmune pancreatitis (AIP) underlies 5%-11% of cases of chronic pancreatitis. An association between AIP and the human leukocyte antigen (HLA)-DRB1*0405/DQB1*0401 haplotype has been reported, but linkage disequilibrium has precluded the identification of predisposing HLA gene(s). We studied the role of single HLA genes in the development of AIP in transgenic mice. METHODS: CD4(+) T-cell-negative I-Abeta chain(-/-) (Ab0) mice develop AIP spontaneously, likely due to dysregulation of CD8(+) T- cell responses. We generated Ab0 nonobese diabetic (NOD) mice transgenic for HLA-DR*0405, leading to rescue of CD4(+) T cells; we compared their susceptibility to AIP with HLA-DQ8 or HLA-DR*0401 (single) transgenic, or HLA-DR*0405/DQ8 (double) transgenic mice. RESULTS: CD4(+) T-cell-competent HLA-DR*0405 transgenic Ab0 NOD mice develop AIP with high prevalence after sublethal irradiation and adoptive transfer of CD90(+) T cells, leading to complete pancreatic atrophy. HLA-DR*0405 transgenic mice can also develop unprovoked AIP, whereas HLA-DR*0401, HLA-DQ8, and HLA-DR*0405/DQ8 transgenic Ab0 NOD controls all remained normal, even after irradiation and adoptive transfer of CD90(+) T cells. Pancreas histology in HLA-DR*0405 transgenic mice was characterized by destructive infiltration of the exocrine tissue with CD4(+) and CD8(+) T cells, B cells, and macrophages. Mice with complete pancreatic atrophy lost weight, developed fat stools, and had reduced levels of serum lipase activity. CONCLUSIONS: Because HLA-DR*0405 expression fails to protect mice from AIP, the HLA-DRB1*0405 allele appears to be an important risk factor for AIP on the HLA-DRB1*0405/DQB1*0401 haplotype. This humanized mouse model should be useful for studying immunopathogenesis, diagnostic markers, and therapy of human AIP.
Asunto(s)
Enfermedades Autoinmunes/etiología , Genes MHC Clase II , Antígenos HLA-DR/genética , Pancreatitis Crónica/etiología , Traslado Adoptivo , Animales , Atrofia , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Femenino , Antígenos HLA-DR/fisiología , Cadenas HLA-DRB1 , Humanos , Lipasa/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Páncreas/patología , Pancreatitis Crónica/genética , Pancreatitis Crónica/patología , RiesgoRESUMEN
In humans and NOD mice, defects in immune tolerance result in the spontaneous development of type-1-diabetes. Recent studies have ascribed a breakdown in tolerance to dysfunction in regulatory T cells that is secondary to reduced IL-2 production by T cells having the NOD diabetes susceptibility region insulin-dependent diabetes 3 (Idd3). In this study, we demonstrate a peripheral tolerance defect in the dendritic cells of NOD mice that is independent of regulatory T cells. NOD CD8 T cells specific for islet Ags fail to undergo deletion in the pancreatic lymph nodes. Deletion was promoted by expression of the protective alleles of both Idd3 (Il2) and Idd5 in dendritic cells. We further identify a second tolerance defect that involves endogenous CD4 T cell expression of the disease-promoting NOD alleles of these genetic regions. Pervasive insulitis can be reduced by expression of the Idd3 and Idd5 protective alleles by either the Ag-presenting cell or lymphocytes.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Células Dendríticas/metabolismo , Diabetes Mellitus/genética , Autotolerancia/inmunología , Alelos , Animales , Autoantígenos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos , Supresión Clonal , Células Dendríticas/patología , Expresión Génica , Islotes Pancreáticos/inmunología , Ratones , Ratones Endogámicos NODRESUMEN
In acute graft-versus-host disease (aGVHD), donor T cells attack the recipient's gastrointestinal tract, liver, and skin. We hypothesized that blocking access to distinct lymphoid priming sites may alter the specific organ tropism and prevent aGVHD development. In support of this initial hypothesis, we found that different secondary lymphoid organs (SLOs) imprint distinct homing receptor phenotypes on evolving alloreactive effector T cells in vivo. Yet preventing T-cell entry to specific SLOs through blocking monoclonal antibodies, or SLO ablation, did not alter aGVHD pathophysiology. Moreover, transfer of alloreactive effector T cells into conditioned secondary recipients targeted the intestines and liver, irrespective of their initial priming site. Thus, we demonstrate redundancy of SLOs at different anatomical sites in aGVHD initiation. Only prevention of T-cell entry to all SLOs could completely abrogate the onset of aGVHD.
Asunto(s)
Movimiento Celular , Enfermedad Injerto contra Huésped/prevención & control , Tejido Linfoide/patología , Linfocitos T/citología , Animales , Reactividad Cruzada , Femenino , Trasplante de Células Madre Hematopoyéticas , Ganglios Linfáticos/patología , Ratones , Especificidad de Órganos , Ganglios Linfáticos Agregados/patología , Receptores Mensajeros de Linfocitos/metabolismo , Acondicionamiento Pretrasplante , Imagen de Cuerpo EnteroRESUMEN
Before the onset of autoimmune destruction, type 1 diabetic patients and an animal model, the nonobese diabetic (NOD) mouse, show morphological and functional abnormalities in target organs, which may act as inciting events for leukocyte infiltration. To better understand these abnormalities, but without the complications associated with lymphocytic infiltrates, we examined genes expressed in autoimmune target tissues of NOD/severe combined immunodeficient (scid) mice and of autoimmune-resistant C57BL/6/scid mice. Our results suggest that the NOD genetic background may predispose them to diabetic complications, including insulin resistance in the absence of high circulating glucose levels and without autoimmune destruction of their beta cells. Several of these genes lie within known type 1 and 2 diabetes loci. These data suggest that the NOD mouse may be a good candidate to study an interface between type 1 and type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Predisposición Genética a la Enfermedad , Ratones Endogámicos NOD , Animales , Glucemia/metabolismo , Vasos Sanguíneos/anomalías , Complicaciones de la Diabetes/genética , Diabetes Mellitus Tipo 1/etiología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/genética , Retículo Endoplásmico/metabolismo , Matriz Extracelular/metabolismo , Femenino , Prueba de Tolerancia a la Glucosa , Humanos , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Células Secretoras de Insulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Pliegue de ProteínaRESUMEN
TNF-alpha has been linked to the development of type 1 diabetes (T1D). We previously reported that neonatal treatment of nonobese diabetic (NOD) mice with TNF-alpha accelerated the onset of T1D, whereas TNF-alpha blockade in the same time period resulted in a complete absence of diabetes. The mechanisms by which TNF-alpha modulates development of T1D in NOD mice remain unclear. Here we tested the effects of TNF-alpha on the maturation of dendritic cells (DCs) in the NOD mouse. We found that neonatal treatment with TNF-alpha caused an increase in expression of maturation markers on CD11c(+)CD11b(+) DC subpopulations, whereas treatment with anti-TNF-alpha resulted in a decrease in expression of maturation markers in the CD11c(+)CD11b(+) subset. Moreover, neonatal treatment with TNF-alpha resulted in skewed development of a CD8alpha(+)CD11b(-)CD11c(+) DC subset such that TNF-alpha decreases the CD8alpha(+)CD11c(+) DC subset, increases the CD11c(+)CD11b(+) subset, and causes an increase in the expression of CD40 and CD54 on mature DCs capable of inducing immunity. Anti-TNF-alpha-treated mice had an increase in the CD8alpha(+)CD11c(+) DCs. Notably, adoptively transferred naïve CD4(+) T cells from BDC2.5 T cell receptor transgenic mice proliferated in the pancreatic lymph nodes in TNF-alpha-treated NOD mice but not in anti-TNF-alpha-treated mice. Finally, we show that anti-TNF-alpha-treated mice showed immunological tolerance to islet cell proteins. We conclude that TNF-alpha plays an important role in the initiation of T1D in the NOD mouse by regulating the maturation of DCs and, thus, the activation of islet-specific pancreatic lymph node T cells.
Asunto(s)
Células Dendríticas/citología , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/etiología , Factor de Necrosis Tumoral alfa/inmunología , Traslado Adoptivo , Animales , Anticuerpos/administración & dosificación , Anticuerpos/farmacología , Antígenos CD/análisis , Linfocitos T CD4-Positivos/trasplante , Diferenciación Celular , Células Dendríticas/inmunología , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Tipo 1/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Inmunofenotipificación , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos NOD , Páncreas/inmunología , Páncreas/patología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Progression to hyperglycemia in young nonobese diabetic (NOD) mice is blocked by the transplantation of hematopoietic cells mismatched at the major histocompatibility complex (MHC). Because the NOD MHC class II allele, I-A(g7), is the primary disease susceptibility gene, it is logical to conclude that MHC-mismatched hematopoietic grafts prevent diabetes by replacement of this susceptibility allele on critical hematolymphoid populations. In this report, transplantation of MHC-matched purified hematopoietic stem cells (HSCs) pre-vented diabetes development in NOD mice, demonstrating that alleles of non-MHC background genes expressed on hematopoietic cells are sufficient to disrupt the autoaggressive process. Nonmarrow ablative conditioning was 100% protective, further showing that elimination of NOD hematopoiesis, including T-cells, was not required for the graft to block diabetes pathogenesis. The current standard clinical practice of hematopoietic cell transplantation uses donor/recipient pairs that are matched at the MHC. In our view, the principles established here using an MHC-matched engineered hematopoietic graft in conjunction with nonmarrow ablative conditioning to successfully block autoimmune diabetes sufficiently reduces the morbidity of the allogeneic transplantation procedure such that a similar approach can be translated to the treatment of human autoimmune disorders.
Asunto(s)
Diabetes Mellitus Tipo 1/prevención & control , Trasplante de Células Madre Hematopoyéticas , Animales , Linfocitos B/fisiología , Complejo Mayor de Histocompatibilidad , Ratones , Ratones Endogámicos NOD , Fenotipo , Subgrupos de Linfocitos T , Quimera por Trasplante , Acondicionamiento Pretrasplante , Trasplante HomólogoRESUMEN
Graft-versus-host disease (GVHD) is a major obstacle in allogeneic hematopoietic cell transplantation. Given the dynamic changes in immune cell subsets and tissue organization, which occur in GVHD, localization and timing of critical immunological events in vivo may reveal basic pathogenic mechanisms. To this end, we transplanted luciferase-labeled allogeneic splenocytes and monitored tissue distribution by in vivo bioluminescence imaging. High-resolution analyses showed initial proliferation of donor CD4+ T cells followed by CD8+ T cells in secondary lymphoid organs with subsequent homing to the intestines, liver, and skin. Transplantation of purified naive T cells caused GVHD that was initiated in secondary lymphoid organs followed by target organ manifestation in gut, liver, and skin. In contrast, transplanted CD4+ effector memory T (T(EM)) cells did not proliferate in secondary lymphoid organs in vivo and despite their in vitro alloreactivity in mixed leukocyte reaction (MLR) assays did not cause acute GVHD. These findings underline the potential of T-cell subsets with defined trafficking patterns for immune reconstitution without the risk of GVHD.
Asunto(s)
Quimiotaxis de Leucocito , Enfermedad Injerto contra Huésped/etiología , Subgrupos de Linfocitos T/fisiología , Animales , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/fisiología , Proliferación Celular , Enfermedad Injerto contra Huésped/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Luciferasas , Ratones , Ratones Endogámicos , Bazo/citología , Factores de Tiempo , Trasplante HomólogoRESUMEN
Stem cell biology is scientifically, clinically, and politically a current topic. The hematopoietic stem cell, the common ancestor of all types of blood cells, is one of the best-characterized stem cells in the body and the only stem cell that is clinically applied in the treatment of diseases such as breast cancer, leukemias, and congenital immunodeficiencies. Multicolor cell sorting enables the purification not only of hematopoietic stem cells, but also of their downstream progenitors such as common lymphoid progenitors and common myeloid progenitors. Recent genetic approaches including gene chip technology have been used to elucidate the gene expression profile of hematopoietic stem cells and other progenitors. Although the mechanisms that control self-renewal and lineage commitment of hematopoietic stem cells are still ambiguous, recent rapid advances in understanding the biological nature of hematopoietic stem and progenitor cells have broadened the potential application of these cells in the treatment of diseases.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Animales , Apoptosis , Enfermedades Autoinmunes/terapia , Diferenciación Celular , División Celular , Movimiento Celular , Separación Celular , Diabetes Mellitus Tipo 1/terapia , Femenino , Perfilación de la Expresión Génica , Hematopoyesis , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Neoplasias/terapiaRESUMEN
Purified hematopoietic stem cells (HSCs) were transplanted into NOD mice to test whether development of hyperglycemia could be prevented. Engraftment of major histocompatibility complex-mismatched HSCs was compared with bone marrow (BM) grafts. HSCs differed from BM because HSCs were more strongly resisted and HSC recipients retained significant levels of NOD T-cells, whereas BM recipients were full donor chimeras. Despite persistent NOD T-cells, all HSC chimeras were protected from hyperglycemia, and attenuation of islet lesions was observed. T-cell selection was altered in allogeneic HSC recipients as demonstrated by deletion of both donor and host superantigen-specific T-cells. Syngeneic and congenic hematopoietic cell transplants were also performed to differentiate the influence of the preparative regimen(s) versus the allografts. Unlike the allogeneic HSC transplantations, syngeneic or congenic grafts did not retard diabetes development. In a pilot study, overtly diabetic NOD mice were cured by co-transplantation of allogeneic HSCs and donor-matched islets. We conclude that allogeneic HSC transplants block allo- and autoimmunity, despite residual host T-cell presence. These data demonstrate for the first time that purified HSC grafts block development of autoimmune diabetes and illuminate how HSC grafts alter thymic and peripheral T-cell responses against auto- and alloantigens.