RESUMEN
BackgroundSome clinical features of severe COVID-19 represent blood vessel damage induced by activation of host immune responses initiated by the coronavirus SARS-CoV-2. We hypothesized autoantibodies against angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor expressed on vascular endothelium, are generated during COVID-19 and are of mechanistic importance.MethodsIn an opportunity sample of 118 COVID-19 inpatients, autoantibodies recognizing ACE2 were detected by ELISA. Binding properties of anti-ACE2 IgM were analyzed via biolayer interferometry. Effects of anti-ACE2 IgM on complement activation and endothelial function were demonstrated in a tissue-engineered pulmonary microvessel model.ResultsAnti-ACE2 IgM (not IgG) autoantibodies were associated with severe COVID-19 and found in 18/66 (27.2%) patients with severe disease compared with 2/52 (3.8%) of patients with moderate disease (OR 9.38, 95% CI 2.38-42.0; P = 0.0009). Anti-ACE2 IgM autoantibodies were rare (2/50) in non-COVID-19 ventilated patients with acute respiratory distress syndrome. Unexpectedly, ACE2-reactive IgM autoantibodies in COVID-19 did not undergo class-switching to IgG and had apparent KD values of 5.6-21.7 nM, indicating they are T cell independent. Anti-ACE2 IgMs activated complement and initiated complement-binding and functional changes in endothelial cells in microvessels, suggesting they contribute to the angiocentric pathology of COVID-19.ConclusionWe identify anti-ACE2 IgM as a mechanism-based biomarker strongly associated with severe clinical outcomes in SARS-CoV-2 infection, which has therapeutic implications.FUNDINGBill & Melinda Gates Foundation, Gates Philanthropy Partners, Donald B. and Dorothy L. Stabler Foundation, and Jerome L. Greene Foundation; NIH R01 AR073208, R01 AR069569, Institutional Research and Academic Career Development Award (5K12GM123914-03), National Heart, Lung, and Blood Institute R21HL145216, and Division of Intramural Research, National Institute of Allergy and Infectious Diseases; National Science Foundation Graduate Research Fellowship (DGE1746891).
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Autoanticuerpos , Células Endoteliales , Humanos , Inmunoglobulina M , SARS-CoV-2RESUMEN
V(D)J recombination is initiated by the recombination-activating gene protein (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with gene transcription and with epigenetic marks characteristic of active chromatin, including histone H3 trimethylated at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 induces conformational changes in RAG-1, allosterically stimulating substrate binding and catalysis. To better understand the path of allostery from the RAG-2 PHD finger to RAG-1, here we employed phylogenetic substitution. We observed that a chimeric RAG-2 protein in which the mouse PHD finger is replaced by the corresponding domain from the shark Chiloscyllium punctatum binds H3K4me3 but fails to transmit an allosteric signal, indicating that binding of H3K4me3 by RAG-2 is insufficient to support recombination. By substituting residues in the C. punctatum PHD with the corresponding residues in the mouse PHD and testing for rescue of allostery, we demonstrate that H3K4me3 binding and transmission of an allosteric signal to RAG-1 are separable functions of the RAG-2 PHD finger.
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Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Regulación Alostérica/genética , Regulación Alostérica/fisiología , Animales , Sitios de Unión , Cromatina/metabolismo , Histonas/fisiología , Lisina/metabolismo , Metilación , Ratones , Filogenia , Unión Proteica , Recombinasas/metabolismo , Tiburones/metabolismo , Especificidad por Sustrato , Recombinación V(D)J/genética , Recombinación V(D)J/fisiología , VDJ Recombinasas/metabolismoRESUMEN
T and B cells are the two known lineages of adaptive immune cells. Here, we describe a previously unknown lymphocyte that is a dual expresser (DE) of TCR and BCR and key lineage markers of both B and T cells. In type 1 diabetes (T1D), DEs are predominated by one clonotype that encodes a potent CD4 T cell autoantigen in its antigen binding site. Molecular dynamics simulations revealed that this peptide has an optimal binding register for diabetogenic HLA-DQ8. In concordance, a synthetic version of the peptide forms stable DQ8 complexes and potently stimulates autoreactive CD4 T cells from T1D patients, but not healthy controls. Moreover, mAbs bearing this clonotype are autoreactive against CD4 T cells and inhibit insulin tetramer binding to CD4 T cells. Thus, compartmentalization of adaptive immune cells into T and B cells is not absolute, and violators of this paradigm are likely key drivers of autoimmune diseases.
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Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Adolescente , Adulto , Autoantígenos/inmunología , Niño , Preescolar , Diabetes Mellitus Tipo 1/metabolismo , Epítopos/inmunología , Femenino , Células HEK293 , Antígenos HLA-DQ/inmunología , Antígenos HLA-DQ/ultraestructura , Humanos , Activación de Linfocitos/inmunología , Linfocitos/inmunología , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Simulación de Dinámica Molecular , Péptidos , Unión Proteica/inmunologíaRESUMEN
BACKGROUND: Rheumatoid arthritis (RA) is a systemic autoimmune disease associated with immune dysregulation and increased risk of infections. The presence of autoantibodies and immunoglobulin abnormalities indicates B-cell and antibody-secreting cell (ASC) dysfunction. We hypothesize that soluble factors associated with B-cell and ASC activity are decreased in RA patients and that this is linked to higher susceptibility to infections. METHODS: Using the Johns Hopkins Arthritis Cohort and Biorepository, we contrasted serum protein levels of soluble factors involved in B-cell activation (CD40, CD40L) and B-cell/ASC homing (CXCL10, CXCL11, and CXCL13) or survival (BAFF, APRIL, TACI, and BCMA) in 10 healthy subjects and 23 adult RA patients (aged 24-65 years). We subdivided RA patients into those with (n = 17) and those without infections (n = 6) within a 2-year period. In order to reduce the effect of RA treatment, we only included patients receiving methotrexate monotherapy or no RA treatments at baseline. Soluble serum protein levels of B-cell/ASC factors were quantified by multiplex immunoassays. RESULTS: We identified that (1) serum levels of soluble BCMA, APRIL, CD40, and CD40L were significantly decreased in RA patients relative to healthy individuals; (2) serum soluble BCMA, predominantly released by ASC, correlated with serum concentrations of class-switched immunoglobulins, IgG and IgA; and (3) RA patients with a history of infections had significantly lower soluble BCMA levels compared with healthy donors and with RA patients without infections. CONCLUSIONS: Our study using soluble factors linked to B-cell/ASC activation and survival suggests that there is a paucity of ASC in a subset of RA patients and that this may be linked to altered antibody production and increased risk of infections. Further delineating the link between ASC and infection susceptibility in RA may optimize disease management and provide novel insights into disease pathogenesis that are susceptible to intervention.
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Células Productoras de Anticuerpos/inmunología , Artritis Reumatoide/diagnóstico , Linfocitos B/inmunología , Infecciones/diagnóstico , Adolescente , Adulto , Anciano , Artritis Reumatoide/epidemiología , Autoanticuerpos/sangre , Antígeno de Maduración de Linfocitos B/sangre , Biomarcadores/sangre , Antígenos CD40/sangre , Supervivencia Celular , Femenino , Humanos , Infecciones/epidemiología , Activación de Linfocitos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Riesgo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/sangre , Estados Unidos/epidemiología , Adulto JovenRESUMEN
The mechanisms that drive T cell aging are not understood. We report that children and adult telomerase mutation carriers with short telomere length (TL) develop a T cell immunodeficiency that can manifest in the absence of bone marrow failure and causes life-threatening opportunistic infections. Mutation carriers shared T cell-aging phenotypes seen in adults 5 decades older, including depleted naive T cells, increased apoptosis, and restricted T cell repertoire. T cell receptor excision circles (TRECs) were also undetectable or low, suggesting that newborn screening may identify individuals with germline telomere maintenance defects. Telomerase-null mice with short TL showed defects throughout T cell development, including increased apoptosis of stimulated thymocytes, their intrathymic precursors, in addition to depleted hematopoietic reserves. When we examined the transcriptional programs of T cells from telomerase mutation carriers, we found they diverged from older adults with normal TL. Short telomere T cells upregulated DNA damage and intrinsic apoptosis pathways, while older adult T cells upregulated extrinsic apoptosis pathways and programmed cell death 1 (PD-1) expression. T cells from mice with short TL also showed an active DNA-damage response, in contrast with old WT mice, despite their shared propensity to apoptosis. Our data suggest there are TL-dependent and TL-independent mechanisms that differentially contribute to distinct molecular programs of T cell apoptosis with aging.
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Apoptosis/inmunología , Trastornos del Crecimiento , Hipercalcemia , Síndromes de Inmunodeficiencia , Enfermedades Metabólicas , Mutación , Nefrocalcinosis , Telomerasa , Homeostasis del Telómero/inmunología , Adulto , Envejecimiento/genética , Envejecimiento/inmunología , Envejecimiento/patología , Animales , Apoptosis/genética , Daño del ADN/inmunología , Femenino , Trastornos del Crecimiento/complicaciones , Trastornos del Crecimiento/genética , Trastornos del Crecimiento/inmunología , Trastornos del Crecimiento/patología , Humanos , Hipercalcemia/complicaciones , Hipercalcemia/genética , Hipercalcemia/inmunología , Hipercalcemia/patología , Síndromes de Inmunodeficiencia/etiología , Síndromes de Inmunodeficiencia/genética , Masculino , Enfermedades Metabólicas/complicaciones , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/inmunología , Enfermedades Metabólicas/patología , Ratones , Ratones Noqueados , Nefrocalcinosis/complicaciones , Nefrocalcinosis/genética , Nefrocalcinosis/inmunología , Nefrocalcinosis/patología , Enfermedades de Inmunodeficiencia Primaria , Linfocitos T/inmunología , Linfocitos T/patología , Telomerasa/genética , Telomerasa/inmunologíaRESUMEN
Accessibility of antigen receptor loci to RAG is correlated with the presence of H3K4me3, which binds to a plant homeodomain (PHD) in the RAG-2 subunit and promotes V(D)J recombination. A point mutation in the PHD, W453A, eliminates binding of H3K4me3 and impairs recombination. The debilitating effect of the W453A mutation is ameliorated by second-site mutations that locate an inhibitory domain in the interval from residues 352 through 405 of RAG-2. Disruption of the inhibitory domain stimulates V(D)J recombination within extrachromosomal substrates and at endogenous antigen receptor loci. Association of RAG-1 and RAG-2 with chromatin at the IgH locus in B cell progenitors is dependent on recognition of H3K4me3 by the PHD. Strikingly, disruption of the inhibitory domain permits association of RAG with the IgH locus in the absence of H3K4me3 binding. Thus, the inhibitory domain acts as a gate that prohibits RAG from accessing the IgH locus unless RAG-2 is engaged by H3K4me3.
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Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , VDJ Recombinasas/metabolismo , Inmunidad Adaptativa , Regulación Alostérica , Sustitución de Aminoácidos , Animales , Línea Celular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Reordenamiento Génico de Cadena Pesada de Linfocito B , Genes de las Cadenas Pesadas de las Inmunoglobulinas , Células HEK293 , Código de Histonas , Humanos , Ratones , Modelos Inmunológicos , Células 3T3 NIH , Mutación Puntual , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/metabolismo , Dominios ProteicosRESUMEN
The stromal signals that promote B lymphopoiesis remain poorly understood. Hedgehog (Hh) signaling promotes B lymphopoiesis in a non-cell-autonomous fashion in vitro, and depletion of the Hh effector Smoothened (Smo) from stromal cells is associated with the loss of osteoblastoid markers. These observations suggested that Hh signaling in the osteoblastoid lineage promotes B lymphopoiesis in vivo. To test this, we employed a mouse model for conditional ablation of Smo in the osteoblastoid lineage. Depletion of Smo from osteoblastoid cells is associated with profound and selective reductions in the number and proportion of bone marrow B-lymphoid progenitors. Upon partial bone marrow ablation, mutant animals exhibit delayed repopulation of the B-lymphoid compartment after the early lymphoid progenitor stage. Primary osteoblasts from mutant mice are defective in supporting B lymphopoiesis in vitro, whereas hematopoietic progenitors from mutant mice exhibit normal differentiation. We conclude that efficient B lymphopoiesis in vivo is dependent on the maintenance of Hh signaling in the osteoblastoid lineage.
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Linfocitos B/metabolismo , Linaje de la Célula , Linfopoyesis , Osteoblastos/citología , Transducción de Señal , Receptor Smoothened/metabolismo , Animales , Médula Ósea/metabolismo , Proteínas Hedgehog/metabolismo , Ratones TransgénicosRESUMEN
V(D)J recombination is initiated by the recombination-activating gene (RAG) recombinase, consisting of RAG-1 and RAG-2 subunits. The susceptibility of gene segments to cleavage by RAG is associated with histone modifications characteristic of active chromatin, including trimethylation of histone H3 at lysine 4 (H3K4me3). Binding of H3K4me3 by a plant homeodomain (PHD) in RAG-2 stimulates substrate binding and catalysis, which are functions of RAG-1. This has suggested an allosteric mechanism in which information regarding occupancy of the RAG-2 PHD is transmitted to RAG-1. To determine whether the conformational distribution of RAG is altered by H3K4me3, we mapped changes in solvent accessibility of cysteine thiols by differential isotopic chemical footprinting. Binding of H3K4me3 to the RAG-2 PHD induces conformational changes in RAG-1 within a DNA-binding domain and in the ZnH2 domain, which acts as a scaffold for the catalytic center. Thus, engagement of H3K4me3 by the RAG-2 PHD is associated with dynamic conformational changes in RAG-1, consistent with allosteric control by active chromatin.
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Dominio Catalítico , Cromatina/metabolismo , Genes RAG-1/fisiología , Histonas/metabolismo , Proteínas de Plantas/química , VDJ Recombinasas/química , Regulación Alostérica , Sitios de Unión , Cisteína/metabolismo , ADN/metabolismo , Metilación de ADN , Lisina/metabolismo , Fenómenos Fisiológicos de las Plantas , Unión Proteica , Conformación Proteica , Recombinación V(D)JAsunto(s)
Inmunidad Innata , Interleucina-33/metabolismo , Degeneración Macular/inmunología , Animales , Femenino , Humanos , MasculinoRESUMEN
V(D)J recombination is initiated by a specialized transposase consisting of the subunits RAG-1 and RAG-2. The susceptibility of gene segments to DNA cleavage by the V(D)J recombinase is correlated with epigenetic modifications characteristic of active chromatin, including trimethylation of histone H3 on lysine 4 (H3K4me3). Engagement of H3K4me3 by a plant homeodomain (PHD) in RAG-2 promotes recombination in vivo and stimulates DNA cleavage by RAG in vitro. We now show that H3K4me3 acts allosterically at the PHD finger to relieve autoinhibition imposed by a separate domain within RAG-2. Disruption of this autoinhibitory domain was associated with constitutive increases in recombination frequency, DNA cleavage activity, substrate binding affinity, and catalytic rate, thus mimicking the stimulatory effects of H3K4me3. Our observations support a model in which allosteric control of RAG is enforced by an autoinhibitory domain whose action is relieved by engagement of active chromatin.
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Cromatina/genética , Proteínas de Unión al ADN/genética , Histonas/genética , Recombinación V(D)J/genética , VDJ Recombinasas/genética , Animales , Sitios de Unión , Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Histonas/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Metilación , Ratones , Células 3T3 NIH , Unión Proteica , VDJ Recombinasas/metabolismoRESUMEN
BACKGROUND: Lymphocytes achieve diversity in antigen recognition in part by rearranging genomic DNA at loci encoding antibodies and cell surface receptors. The process, termed V(D)J recombination, juxtaposes modular coding sequences for antigen binding. Erroneous recombination events causing chromosomal translocations are recognized causes of lymphoid malignancies. Here we show a hybridization based method for sequence enrichment can be used to efficiently and selectively capture genomic DNA adjacent to V(D)J recombination breakpoints for massively parallel sequencing. The approach obviates the need for PCR amplification of recombined sequences. RESULTS: Using tailored informatics analyses to resolve alignment and assembly issues in these repetitive regions, we were able to detect numerous recombination events across a panel of cancer cell lines and primary lymphoid tumors, and an EBV transformed lymphoblast line. With reassembly, breakpoints could be defined to single base pair resolution. The observed events consist of canonical V(D)J or V-J rearrangements, non-canonical rearrangements, and putatively oncogenic reciprocal chromosome translocations. We validated non-canonical and chromosome translocation junctions by PCR and Sanger sequencing. The translocations involved the MYC and BCL-2 loci, and activation of these was consistent with histopathologic features of the respective B-cell tumors. We also show an impressive prevalence of novel erroneous V-V recombination events at sites not incorporated with other downstream coding segments. CONCLUSIONS: Our results demonstrate the ability of next generation sequencing to describe human V(D)J recombinase activity and provide a scalable means to chronicle off-target, unexpressed, and non-amplifiable recombinations occurring in the development of lymphoid cancers.
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Reordenamiento Génico , Leucemia Linfoide/genética , VDJ Recombinasas/fisiología , Linfocitos B/enzimología , Secuencia de Bases , Carcinogénesis/genética , Línea Celular Tumoral , Puntos de Rotura del Cromosoma , Mapeo Cromosómico , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Leucemia Linfoide/enzimología , Análisis de Secuencia de ADN , Translocación GenéticaRESUMEN
UNLABELLED: The DNA damage response (DDR) factors ataxia telangiectasia mutated (ATM) and p53 binding protein 1 (53BP1) function as tumor suppressors in humans and mice, but the significance of their mutual interaction to the suppression of oncogenic translocations in vivo has not been investigated. To address this question, the phenotypes of compound mutant mice lacking 53BP1 and ATM (Trp53bp1(-/-)/Atm(-/-)), relative to single mutants, were examined. These analyses revealed that loss of 53BP1 markedly decreased the latency of T-lineage lymphomas driven by RAG-dependent oncogenic translocations in Atm(-/-) mice (average survival, 14 and 23 weeks for Trp53bp1(-/-)/Atm(-/-) and Atm(-/-) mice, respectively). Mechanistically, 53BP1 deficiency aggravated the deleterious effect of ATM deficiency on nonhomologous end-joining (NHEJ)-mediated double-strand break repair. Analysis of V(D)J recombinase-mediated coding joints and signal joints in Trp53bp1(-/-)/Atm(-/-) primary thymocytes is, however, consistent with canonical NHEJ-mediated repair. Together, these findings indicate that the greater NHEJ defect in the double mutant mice resulted from decreased efficiency of rejoining rather than switching to an alternative NHEJ-mediated repair mechanism. Complementary analyses of irradiated primary cells indicated that defects in cell-cycle checkpoints subsequently function to amplify the NHEJ defect, resulting in more frequent chromosomal breaks and translocations in double mutant cells throughout the cell cycle. Finally, it was determined that 53BP1 is dispensable for the formation of RAG-mediated hybrid joints in Atm(-/-) thymocytes but is required to suppress large deletions in a subset of hybrid joints. IMPLICATIONS: The current study uncovers novel ATM-independent functions for 53BP1 in the suppression of oncogenic translocations and in radioprotection.
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Proteínas Cromosómicas no Histona/fisiología , Daño del ADN , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/fisiología , Rayos gamma , Estrés Fisiológico , Animales , Proteínas de la Ataxia Telangiectasia Mutada/deficiencia , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Células Cultivadas , Roturas del ADN de Doble Cadena/efectos de la radiación , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN por Unión de Extremidades/efectos de la radiación , Humanos , Linfoma/genética , Linfoma/metabolismo , Ratones , Ratones Noqueados , Modelos Animales , Estrés Fisiológico/genética , Timocitos/metabolismo , Timocitos/efectos de la radiación , Translocación Genética , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
The landscape of human phosphorylation networks has not been systematically explored, representing vast, unchartered territories within cellular signaling networks. Although a large number of in vivo phosphorylated residues have been identified by mass spectrometry (MS)-based approaches, assigning the upstream kinases to these residues requires biochemical analysis of kinase-substrate relationships (KSRs). Here, we developed a new strategy, called CEASAR, based on functional protein microarrays and bioinformatics to experimentally identify substrates for 289 unique kinases, resulting in 3656 high-quality KSRs. We then generated consensus phosphorylation motifs for each of the kinases and integrated this information, along with information about in vivo phosphorylation sites determined by MS, to construct a high-resolution map of phosphorylation networks that connects 230 kinases to 2591 in vivo phosphorylation sites in 652 substrates. The value of this data set is demonstrated through the discovery of a new role for PKA downstream of Btk (Bruton's tyrosine kinase) during B-cell receptor signaling. Overall, these studies provide global insights into kinase-mediated signaling pathways and promise to advance our understanding of cellular signaling processes in humans.
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Linfocitos B/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/genética , Agammaglobulinemia Tirosina Quinasa , Algoritmos , Secuencia de Aminoácidos , Linfocitos B/citología , Teorema de Bayes , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Humanos , Datos de Secuencia Molecular , Fosforilación , Análisis por Matrices de Proteínas , Mapas de Interacción de Proteínas , Proteínas Tirosina Quinasas/genética , Receptores de Antígenos de Linfocitos B/genética , Tirosina/metabolismoRESUMEN
The transcription factor Stat3 is an activator of systemic inflammatory genes. Two isoforms of Stat3 are generated by alternative splicing, Stat3α and Stat3ß. The ß isoform lacks the transactivation domain but retains other functions, including dimerization and DNA binding. Stat3ß-deficient mice exhibit elevated expression of systemic inflammatory genes and are hyperresponsive to lipopolysaccharide, suggesting that Stat3ß functions predominantly as a suppressor of systemic inflammation. To test whether Stat3ß deficiency would provoke pathologic effects associated with chronic inflammation, we asked whether selective removal of Stat3ß would exacerbate the development of atherosclerosis in apolipoprotein E-deficient mice. In apoE(-/-)Stat3ß(-/-) mice atherosclerotic plaque formation was significantly enhanced relative to apoE(-/-)Stat3ß(+/+) controls. The ability of Stat3ß deficiency to promote atherosclerosis was more pronounced in female mice, but could be unmasked in males by feeding a high fat diet. Infiltrating macrophages were not increased in aortas of apoE(-/-)Stat3ß(-/-) mice. In contrast, the proportion of pro-inflammatory TH17 cells was significantly elevated in aortic infiltrates from apoE(-/-)Stat3ß(-/-) mice, relative to paired apoE(-/-)Stat3ß(+/+) littermates. These observations indicate that Stat3ß can suppress pathologic sequelae associated with chronic inflammation. Our findings further suggest that in Stat3ß-deficient mice the unopposed action of Stat3α may enhance atherogenesis in part by promoting differentiation of TH17 cells.
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Apolipoproteínas E/deficiencia , Aterosclerosis/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Aorta/patología , Apolipoproteínas E/genética , Peso Corporal , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Colesterol/sangre , Citocinas/metabolismo , Dieta Alta en Grasa , Femenino , Macrófagos Peritoneales/metabolismo , Masculino , Ratones , Ratones Noqueados , Factor de Transcripción STAT3/genética , Triglicéridos/sangreRESUMEN
Invariant natural killer T cells have a distinct developmental pathway from conventional αß T cells. Here we demonstrate that the transcriptional repressor NKAP is required for invariant natural killer T cell but not conventional T cell development. In CD4-cre NKAP conditional knockout mice, invariant natural killer T cell development is blocked at the double-positive stage. This cell-intrinsic block is not due to decreased survival or failure to rearrange the invariant Vα14-Jα18 T cell receptor-α chain, but is rescued by overexpression of a rec-Vα14-Jα18 transgene at the double-positive stage, thus defining a role for NKAP in selection into the invariant natural killer T cell lineage. Importantly, deletion of the NKAP-associated protein histone deacetylase 3 causes a similar block in the invariant natural killer T cell development, indicating that NKAP and histone deacetylase 3 functionally interact to control invariant natural killer T cell development.
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Células T Asesinas Naturales/citología , Proteínas Represoras/metabolismo , Animales , Supervivencia Celular , Eliminación de Gen , Reordenamiento Génico de la Cadena alfa de los Receptores de Antígenos de los Linfocitos T , Histona Desacetilasas/metabolismo , Ratones , Ratones Noqueados , Células T Asesinas Naturales/metabolismo , Especificidad de Órganos , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores Notch/metabolismo , Recombinación Genética/genética , Proteínas Represoras/deficiencia , Timocitos/citología , Timocitos/metabolismoRESUMEN
The role of hedgehog (Hh) signaling in B lymphopoiesis has remained unclear. We observed that the proliferation of pro-B cells in stromal cocultures was impaired by interruption of Hh signaling, prompting us to investigate whether the target of Hh antagonism was intrinsic or extrinsic to the B-lymphoid compartment. In the present study, using conditional deletion of the pathway activator gene Smo, we found that cell-autonomous Hh signaling is dispensable for B-cell development, B-lymphoid repopulation of the BM, and humoral immune function. In contrast, depletion of the Smo protein from stromal cells was associated with impaired generation of B-lymphoid cells from hematopoietic stem progenitor cells, whereas reciprocal removal of Smo from these cells had no effect on the production of B-cell progenitors. Depletion of Smo from stromal cells was associated with coordinate down-regulation of genes for which expression is associated with osteoblastoid identity and B-lymphopoietic activity. The results of the present study suggest that activity of the Hh pathway within stromal cells promotes B lymphopoiesis in a non-cell-autonomous fashion.
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Linfocitos B/citología , Proteínas Hedgehog/metabolismo , Células Madre Hematopoyéticas/citología , Linfopoyesis , Receptores Acoplados a Proteínas G/metabolismo , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Eliminación de Gen , Proteínas Hedgehog/antagonistas & inhibidores , Células Madre Hematopoyéticas/metabolismo , Inmunidad Humoral , Ratones , Ratones Endogámicos C57BL , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/metabolismo , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Receptor Smoothened , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
Constitutive activation of FLT3 by internal tandem duplication (ITD) is one of the most common molecular alterations in acute myeloid leukemia (AML). FLT3/ITD mutations have also been observed in myelodysplastic syndrome patients both before and during progression to AML. Previous work has shown that insertion of an FLT3/ITD mutation into the murine Flt3 gene induces a myeloproliferative neoplasm, but not progression to acute leukemia, suggesting that additional cooperating events are required. We therefore combined the FLT3/ITD mutation with a model of myelodysplastic syndrome involving transgenic expression of the Nup98-HoxD13 (NHD13) fusion gene. Mice expressing both the FLT3/ITD and NHD13 transgene developed AML with 100% penetrance and short latency. These leukemias were driven by mutant FLT3 expression and were susceptible to treatment with FLT3 tyrosine kinase inhibitors. We also observed a spontaneous loss of the wild-type Flt3 allele in these AMLs, further modeling the loss of the heterozygosity phenomenon that is seen in human AML with FLT3-activating mutations. Because resistance to FLT3 inhibitors remains an important clinical issue, this model may help identify new molecular targets in collaborative signaling pathways.
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Modelos Animales de Enfermedad , Proteínas de Homeodominio/genética , Leucemia Mieloide Aguda/genética , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Fusión Oncogénica/genética , Tirosina Quinasa 3 Similar a fms/genética , Animales , Western Blotting , Citometría de Flujo , Técnicas de Sustitución del Gen , Humanos , Ratones , Ratones Transgénicos , Mutación , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
V(D)J gene segment recombination is linked to the cell cycle by the periodic phosphorylation and destruction of the RAG-2 protein at the G1-to-S cell cycle transition. To examine the function of this coupling, we constructed mice in which the phosphorylation site at threonine 490 of RAG-2 was mutated to alanine. The RAG-2(T490A) mutation uncoupled DNA cleavage from cell cycle and promoted aberrant recombination. Similar aberrant recombination products were observed in mice deficient in the Skp2 ubiquitin ligase subunit, which is required for periodic destruction of RAG-2. On a p53-deficient background, the RAG-2(T490A) mutation induced lymphoid malignancies characterized by clonal chromosomal translocations involving antigen receptor genes. Taken together, these observations provide a direct link between the periodic destruction of RAG-2 and lymphoid tumorigenesis. We infer that cell cycle control of the V(D)J recombinase limits the potential genomic damage that could otherwise result from RAG-mediated DNA cleavage.
Asunto(s)
Ciclo Celular , Proteínas de Unión al ADN/fisiología , Reordenamiento Génico , Inestabilidad Genómica , Linfoma no Hodgkin/genética , Receptores de Antígenos de Linfocitos T/genética , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Femenino , Técnicas de Sustitución del Gen , Genes p53 , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Quinasas Asociadas a Fase-S/genética , Organismos Libres de Patógenos Específicos , Linfocitos T/metabolismo , Translocación GenéticaRESUMEN
We have generated an FLT3/ITD knock-in mouse model in which mice with an FLT3/ITD mutation develop myeloproliferative disease (MPD) and a block in early B-lymphocyte development. To elucidate the role of FLT3/ITD signaling in B-cell development, we studied VDJ recombination in the pro-B cells of FLT3/ITD mice and discovered an increased frequency of DNA double strand breaks (DSBs) introduced by the VDJ recombinase. Early pro-B cells from FLT3/ITD mice were found to have a lower efficiency and decreased accuracy of DSB repair by nonhomologous end joining (NHEJ), which is required for rejoining DSBs during VDJ recombination. Reduced NHEJ repair probably results from reduced expression of Ku86, a key component of the classic DNA-PK-dependent NHEJ pathway. In compensation, early pro-B cells from FLT3/ITD cells mice show increased levels of the alternative, and highly error-prone, NHEJ pathway protein PARP1, explaining the increase in repair errors. These data suggest that, in early pro-B cells from FLT3/ITD mice, impairment of classic NHEJ decreases the ability of cells to complete postcleavage DSB ligation, resulting in failure to complete VDJ recombination and subsequent block of B-lymphocyte maturation. These findings might explain the poor prognosis of leukemia patients with constitutive activation of FLT3 signaling.
Asunto(s)
Linfocitos B/citología , Mutación/genética , Recombinación Genética , Tirosina Quinasa 3 Similar a fms/metabolismo , Animales , Antígenos Nucleares/metabolismo , Linfocitos B/efectos de los fármacos , Linfocitos B/enzimología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Autoantígeno Ku , Ratones , Ratones Endogámicos C57BL , Poli(ADP-Ribosa) Polimerasas/metabolismo , Células Precursoras de Linfocitos B/citología , Células Precursoras de Linfocitos B/efectos de los fármacos , Células Precursoras de Linfocitos B/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Recombinación Genética/efectos de los fármacos , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidoresRESUMEN
In developing lymphocytes, V(D)J recombination is subject to tight spatial and temporal regulation. An emerging body of evidence indicates that some of these constraints, particularly with respect to locus specificity and cell cycle phase, are enforced by regulatory cues that converge directly on the RAG proteins themselves. Active chromatin is bound by RAG-2 through a specific histone modification that may serve the recombinase as an allosteric activator as well as a docking site. RAG-1 possesses intrinsic histone ubiquitin ligase activity, suggesting that the recombinase not only responds to chromatin modification but is itself able to modify chromatin. The cyclin A/Cdk2 component of the cell cycle clock triggers periodic destruction of RAG-2, thereby restricting V(D)J recombination to the G0/G1 cell cycle phases. These examples illustrate that the RAG proteins, in addition to their direct actions on DNA, are able to detect and respond to intracellular signals, thereby coordinating recombinase activity with intracellular processes such as cell division and transcription.