RESUMEN
Loss of interleukin (IL)-7 or the IL-7 receptor alpha (IL-7Ralpha, CD127) results in severe immunodeficiencies in mice and humans. To more precisely identify signals governing IL-7 function in vivo, we have disrupted the IL-7Ralpha Y449XXM motif in mice by knock-in mutagenesis (IL-7Ralpha(449F)). Thymic precursors were reduced in number in IL-7Ralpha(449F) mice, but in marked contrast to IL-7Ralpha(-/-) knockout mice, thymocytes and peripheral T cells developed normally. Strikingly, Listeria infection revealed that CD4 and CD8 T cells had different requirements for IL-7Ralpha signals. CD4 T cells failed to mount a primary response, but despite normal CD8 primary responses, maintenance of CD8 memory was impaired in IL-7Ralpha(449F) mice. Furthermore, we show that Bcl-2 is IL-7Ralpha Y449 independent and insufficient for IL-7-mediated maintenance of CD8 memory.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica/genética , Receptores de Interleucina-7/genética , Animales , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Mutagénesis Sitio-Dirigida , Transducción de Señal/genéticaRESUMEN
BACKGROUND: Gastrointestinal complications are an important problem of antithrombotic therapy. Proton-pump inhibitors (PPIs) are believed to decrease the risk of such complications, though no randomized trial has proved this in patients receiving dual antiplatelet therapy. Recently, concerns have been raised about the potential for PPIs to blunt the efficacy of clopidogrel. METHODS: We randomly assigned patients with an indication for dual antiplatelet therapy to receive clopidogrel in combination with either omeprazole or placebo, in addition to aspirin. The primary gastrointestinal end point was a composite of overt or occult bleeding, symptomatic gastroduodenal ulcers or erosions, obstruction, or perforation. The primary cardiovascular end point was a composite of death from cardiovascular causes, nonfatal myocardial infarction, revascularization, or stroke. The trial was terminated prematurely when the sponsor lost financing. RESULTS: We planned to enroll about 5000 patients; a total of 3873 were randomly assigned and 3761 were included in analyses. In all, 51 patients had a gastrointestinal event; the event rate was 1.1% with omeprazole and 2.9% with placebo at 180 days (hazard ratio with omeprazole, 0.34, 95% confidence interval [CI], 0.18 to 0.63; P<0.001). The rate of overt upper gastrointestinal bleeding was also reduced with omeprazole as compared with placebo (hazard ratio, 0.13; 95% CI, 0.03 to 0.56; P = 0.001). A total of 109 patients had a cardiovascular event, with event rates of 4.9% with omeprazole and 5.7% with placebo (hazard ratio with omeprazole, 0.99; 95% CI, 0.68 to 1.44; P = 0.96); high-risk subgroups did not show significant heterogeneity. The two groups did not differ significantly in the rate of serious adverse events, though the risk of diarrhea was increased with omeprazole. CONCLUSIONS: Among patients receiving aspirin and clopidogrel, prophylactic use of a PPI reduced the rate of upper gastrointestinal bleeding. There was no apparent cardiovascular interaction between clopidogrel and omeprazole, but our results do not rule out a clinically meaningful difference in cardiovascular events due to use of a PPI. (Funded by Cogentus Pharmaceuticals; ClinicalTrials.gov number, NCT00557921.).
Asunto(s)
Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Hemorragia Gastrointestinal/prevención & control , Omeprazol/uso terapéutico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Inhibidores de la Bomba de Protones/uso terapéutico , Ticlopidina/análogos & derivados , Anciano , Aspirina/efectos adversos , Aspirina/uso terapéutico , Enfermedades Cardiovasculares/epidemiología , Clopidogrel , Interacciones Farmacológicas , Quimioterapia Combinada , Femenino , Hemorragia Gastrointestinal/inducido químicamente , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Omeprazol/efectos adversos , Inhibidores de Agregación Plaquetaria/efectos adversos , Inhibidores de la Bomba de Protones/efectos adversos , Ticlopidina/efectos adversos , Ticlopidina/uso terapéuticoRESUMEN
The development of a permissive small animal model for the study of human immunodeficiency virus type (HIV)-1 pathogenesis and the testing of antiviral strategies has been hampered by the inability of HIV-1 to infect primary rodent cells productively. In this study, we explored transgenic rats expressing the HIV-1 receptor complex as a susceptible host. Rats transgenic for human CD4 (hCD4) and the human chemokine receptor CCR5 (hCCR5) were generated that express the transgenes in CD4(+) T lymphocytes, macrophages, and microglia. In ex vivo cultures, CD4(+) T lymphocytes, macrophages, and microglia from hCD4/hCCR5 transgenic rats were highly susceptible to infection by HIV-1 R5 viruses leading to expression of abundant levels of early HIV-1 gene products comparable to those found in human reference cultures. Primary rat macrophages and microglia, but not lymphocytes, from double-transgenic rats could be productively infected by various recombinant and primary R5 strains of HIV-1. Moreover, after systemic challenge with HIV-1, lymphatic organs from hCD4/hCCR5 transgenic rats contained episomal 2-long terminal repeat (LTR) circles, integrated provirus, and early viral gene products, demonstrating susceptibility to HIV-1 in vivo. Transgenic rats also displayed a low-level plasma viremia early in infection. Thus, transgenic rats expressing the appropriate human receptor complex are promising candidates for a small animal model of HIV-1 infection.
Asunto(s)
Antígenos CD4 , Modelos Animales de Enfermedad , Infecciones por VIH , VIH-1 , Receptores CCR5 , Animales , Animales Modificados Genéticamente , Antígenos CD4/genética , Antígenos CD4/inmunología , VIH-1/fisiología , Humanos , Macrófagos/inmunología , Ratas , Receptores CCR5/genética , Receptores CCR5/inmunología , Replicación ViralRESUMEN
Shp1, encoded by the gene Ptpn6, is a protein tyrosine phosphatase that transduces inhibitory signals downstream of immunoreceptors in many immune cell types. Blocking Shp1 activity represents an exciting potential immunotherapeutic strategy for the treatment of cancer, as Shp1 inhibition would be predicted to unleash both innate and adaptive immunity against tumor cells. Antibodies blocking the interaction between CD47 on tumor cells and SIRPα on macrophages enhance macrophage phagocytosis, show efficacy in preclinical tumor models, and are being evaluated in the clinic. Here we found that Shp1 bound to phosphorylated peptide sequences derived from SIRPα and transduced the anti-phagocytic signal, as Shp1 loss in mouse bone marrow-derived macrophages increased phagocytosis of tumor cells in vitro. We also generated a novel mouse model to evaluate the impact of global, inducible Ptpn6 deletion on anti-tumor immunity. We found that inducible Shp1 loss drove an inflammatory disease in mice that was phenotypically similar to that seen when Ptpn6 is knocked out from birth. This indicates that acute perturbation of Shp1 in vivo could drive hyperactivation of immune cells, which could be therapeutically beneficial, though at the risk of potential toxicity. In this model, we found that Shp1 loss led to robust anti-tumor immunity against two immune-rich syngeneic tumor models that are moderately inflamed though not responsive to checkpoint inhibitors, MC38 and E0771. Shp1 loss did not promote anti-tumor activity in the non-inflamed B16F10 model. The observed activity in MC38 and E0771 tumors was likely due to effects of both innate and adaptive immune cells. Following Shp1 deletion, we observed increases in intratumoral myeloid cells in both models, which was more striking in E0771 tumors. E0771 tumors also contained an increased ratio of effector to regulatory T cells following Shp1 loss. This was not observed for MC38 tumors, though we did find increased levels of IFNγ, a cytokine produced by effector T cells, in these tumors. Overall, our preclinical data suggested that targeting Shp1 may be an attractive therapeutic strategy for boosting the immune response to cancer via a mechanism involving both innate and adaptive leukocytes.
Asunto(s)
Adenocarcinoma/enzimología , Neoplasias de la Mama/enzimología , Neoplasias del Colon/enzimología , Melanoma Experimental/enzimología , Proteína Tirosina Fosfatasa no Receptora Tipo 6/deficiencia , Neoplasias Cutáneas/enzimología , Macrófagos Asociados a Tumores/enzimología , Inmunidad Adaptativa , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Adenocarcinoma/terapia , Animales , Antígenos de Diferenciación/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Femenino , Humanos , Inmunidad Innata , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Receptores Inmunológicos/metabolismo , Transducción de Señal , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/terapia , Células THP-1 , Carga Tumoral , Microambiente Tumoral , Macrófagos Asociados a Tumores/inmunologíaRESUMEN
The protein tyrosine phosphatase SHP2 binds to phosphorylated signaling motifs on regulatory immunoreceptors including PD-1, but its functional role in tumor immunity is unclear. Using preclinical models, we show that RMC-4550, an allosteric inhibitor of SHP2, induces antitumor immunity, with effects equivalent to or greater than those resulting from checkpoint blockade. In the tumor microenvironment, inhibition of SHP2 modulated T-cell infiltrates similar to checkpoint blockade. In addition, RMC-4550 drove direct, selective depletion of protumorigenic M2 macrophages via attenuation of CSF1 receptor signaling and increased M1 macrophages via a mechanism independent of CD8+ T cells or IFNγ. These dramatic shifts in polarized macrophage populations in favor of antitumor immunity were not seen with checkpoint blockade. Consistent with a pleiotropic mechanism of action, RMC-4550 in combination with either checkpoint or CSF1R blockade caused additive antitumor activity with complete tumor regressions in some mice; tumors intrinsically sensitive to SHP2 inhibition or checkpoint blockade were particularly susceptible. Our preclinical findings demonstrate that SHP2 thus plays a multifaceted role in inducing immune suppression in the tumor microenvironment, through both targeted inhibition of RAS pathway-dependent tumor growth and liberation of antitumor immune responses. Furthermore, these data suggest that inhibition of SHP2 is a promising investigational therapeutic approach. SIGNIFICANCE: Inhibition of SHP2 causes direct and selective depletion of protumorigenic M2 macrophages and promotes antitumor immunity, highlighting an investigational therapeutic approach for some RAS pathway-driven cancers.
Asunto(s)
Neoplasias de la Mama/inmunología , Inmunosupresores/farmacología , Macrófagos/inmunología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Microambiente Tumoral/inmunología , Regulación Alostérica , Animales , Apoptosis , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/prevención & control , Proliferación Celular , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Transducción de Señal , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Cells derived from native rodents have limits at distinct steps of HIV replication. Rat primary CD4 T-cells, but not macrophages, display a profound transcriptional deficit that is ameliorated by transient trans-complementation with the human Tat-interacting protein Cyclin T1 (hCycT1). RESULTS: Here, we generated transgenic rats that selectively express hCycT1 in CD4 T-cells and macrophages. hCycT1 expression in rat T-cells boosted early HIV gene expression to levels approaching those in infected primary human T-cells. hCycT1 expression was necessary, but not sufficient, to enhance HIV transcription in T-cells from individual transgenic animals, indicating that endogenous cellular factors are critical co-regulators of HIV gene expression in rats. T-cells from hCD4/hCCR5/hCycT1-transgenic rats did not support productive infection of prototypic wild-type R5 HIV-1 strains ex vivo, suggesting one or more significant limitation in the late phase of the replication cycle in this primary rodent cell type. Remarkably, we identify a replication-competent HIV-1 GFP reporter strain (R7/3 YU-2 Env) that displays characteristics of a spreading, primarily cell-to-cell-mediated infection in primary T-cells from hCD4/hCCR5-transgenic rats. Moreover, the replication of this recombinant HIV-1 strain was significantly enhanced by hCycT1 transgenesis. The viral determinants of this so far unique replicative ability are currently unknown. CONCLUSION: Thus, hCycT1 expression is beneficial to de novo HIV infection in a transgenic rat model, but additional genetic manipulations of the host or virus are required to achieve full permissivity.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Ciclinas/metabolismo , VIH-1/fisiología , Transcripción Genética , Replicación Viral , Animales , Células Cultivadas , Ciclina T , VIH-1/crecimiento & desarrollo , Humanos , Macrófagos/virología , Ratas , Ratas TransgénicasRESUMEN
Oncogenic alterations in the RAS/RAF/MEK/ERK pathway drive the growth of a wide spectrum of cancers. While BRAF and MEK inhibitors are efficacious against BRAFV600E-driven cancers, effective targeted therapies are lacking for most cancers driven by other pathway alterations, including non-V600E oncogenic BRAF, RAS GTPase-activating protein (GAP) NF1 (neurofibromin 1) loss and oncogenic KRAS. Here, we show that targeting the SHP2 phosphatase (encoded by PTPN11) with RMC-4550, a small-molecule allosteric inhibitor, is effective in human cancer models bearing RAS-GTP-dependent oncogenic BRAF (for example, class 3 BRAF mutants), NF1 loss or nucleotide-cycling oncogenic RAS (for example, KRASG12C). SHP2 inhibitor treatment decreases oncogenic RAS/RAF/MEK/ERK signalling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. Our findings illuminate a critical function for SHP2 in promoting oncogenic RAS/MAPK pathway activation in cancers with RAS-GTP-dependent oncogenic BRAF, NF1 loss and nucleotide-cycling oncogenic KRAS. SHP2 inhibition is a promising molecular therapeutic strategy for patients with cancers bearing these oncogenic drivers.
Asunto(s)
Biomarcadores de Tumor/genética , Guanosina Trifosfato/metabolismo , Mutación , Neoplasias/enzimología , Neoplasias/genética , Neurofibromina 1/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Ratones Endogámicos BALB C , Ratones Desnudos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Fenotipo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína SOS1/metabolismo , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas raf/metabolismoRESUMEN
The requirement for receptor components and the signalling effector, signal transducer and activator of transcription (STAT) 5A/5B, was assessed genetically in a lymphoma development model induced by interleukin-7 (IL-7). This growth factor for T- and B-cell progenitors and mature lymphocytes activates survival and proliferative pathways including Bcl-2, phosphatidylinositol-3 kinase and STAT5. Overexpression of IL-7 in vivo causes early mortality from lymphoma development. Mice overexpressing IL-7 that were heterozygous for the IL-7Ralpha subunit showed improved survival compared to wild-type mice. In addition, STAT5A/5B+/- compound heterozygous mice with one targeted allele each of STAT5A and STAT5B showed striking amelioration of IL-7-induced mortality and disease development. STAT5A/5B+/- compound heterozygous mice were otherwise normal in stem cell and lymphocyte development and cellularity. Lower STAT5 protein levels accompanied the reduction in STAT5A/5B copy number, which suggests that STAT5 haploinsufficiency is a modifier of IL-7 signal strength.
Asunto(s)
Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/genética , Interleucina-7/fisiología , Linfoma/genética , Proteínas de la Leche/análisis , Proteínas de la Leche/genética , Transactivadores/análisis , Transactivadores/genética , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Dosificación de Gen , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Haplotipos , Humanos , Interleucina-7/genética , Linfoma/inmunología , Linfoma/patología , Ratones , Receptores de Interleucina-7/fisiología , Factor de Transcripción STAT5 , Proteínas Supresoras de TumorRESUMEN
BACKGROUND: The COGENT (Clopidogrel and the Optimization of Gastrointestinal Events Trial) showed that proton-pump inhibitors (PPIs) safely reduced rates of gastrointestinal (GI) events in patients requiring dual antiplatelet therapy (DAPT). However, utilization of appropriate prophylactic PPI therapy remains suboptimal, especially with low-dose aspirin. OBJECTIVES: The authors investigated the safety and efficacy of PPI therapy in patients receiving DAPT in low- and high-dose aspirin subsets. METHODS: Randomized patients with available aspirin dosing information in COGENT (N = 3,752) were divided into "low-dose" (≤ 100 mg) and "high-dose" (>100 mg) aspirin groups. The primary GI and cardiovascular endpoints were composite upper GI events and major adverse cardiac events, respectively. All events were adjudicated by independent, blinded gastroenterologists and cardiologists. RESULTS: Median duration of follow-up was 110 days. Low-dose aspirin users (n = 2,480; 66.1%) were more likely to be older, female, and have higher rates of peripheral artery disease, prior stroke, and hypertension, whereas high-dose aspirin users (n = 1,272; 33.9%) had higher rates of hyperlipidemia, smoking, a history of percutaneous coronary intervention, and were more than twice as likely to be enrolled from sites within the United States (80.4% vs. 39.8%). High-dose aspirin was associated with similar 180-day Kaplan-Meier estimates of adjudicated composite GI events (1.7% vs. 2.1%; adjusted hazard ratio: 0.88; 95% confidence interval: 0.46 to 1.66) and major adverse cardiac events (4.8% vs. 5.5%; adjusted hazard ratio: 0.73; 95% confidence interval: 0.48 to 1.11) compared with low-dose aspirin. Randomization to PPI therapy reduced 180-day Kaplan-Meier estimates of the primary GI endpoint in low-dose (1.2% vs. 3.1%) and high-dose aspirin subsets (0.9% vs. 2.6%; p for interaction = 0.80), and did not adversely affect the primary cardiovascular endpoint in either group. CONCLUSIONS: Gastroprotection with PPI therapy should be utilized in appropriately selected patients with coronary artery disease requiring DAPT, even if the patients are on low-dose aspirin. (Clopidogrel and the Optimization of Gastrointestinal Events Trial [COGENT]; NCT00557921).
Asunto(s)
Aspirina/administración & dosificación , Omeprazol/uso terapéutico , Inhibidores de Agregación Plaquetaria/administración & dosificación , Inhibidores de la Bomba de Protones/uso terapéutico , Ticlopidina/análogos & derivados , Anciano , Aspirina/efectos adversos , Clopidogrel , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Dispepsia/inducido químicamente , Dispepsia/prevención & control , Femenino , Hemorragia Gastrointestinal/inducido químicamente , Hemorragia Gastrointestinal/prevención & control , Humanos , Obstrucción Intestinal/inducido químicamente , Obstrucción Intestinal/prevención & control , Perforación Intestinal/inducido químicamente , Perforación Intestinal/prevención & control , Masculino , Persona de Mediana Edad , Infarto del Miocardio/epidemiología , Revascularización Miocárdica/estadística & datos numéricos , Dolor/inducido químicamente , Dolor/prevención & control , Úlcera Péptica/inducido químicamente , Úlcera Péptica/prevención & control , Inhibidores de Agregación Plaquetaria/efectos adversos , Estudios Prospectivos , Accidente Cerebrovascular/epidemiología , Ticlopidina/administración & dosificación , Ticlopidina/efectos adversosRESUMEN
BACKGROUND: Proton-pump inhibitors (PPIs) have been demonstrated to reduce rates of gastrointestinal events in patients requiring dual antiplatelet therapy (DAPT). Data are limited regarding the efficacy and safety of PPIs in high-risk cardiovascular subsets after acute coronary syndrome or percutaneous coronary intervention. METHODS: All patients enrolled in COGENT (Clopidogrel and the Optimization of Gastrointestinal Events Trial) were initiated on DAPT (with aspirin and clopidogrel) for various indications within the prior 21 days. These post hoc analyses of the COGENT trial evaluated the efficacy and safety of omeprazole compared with placebo in subsets of patients requiring DAPT for the 2 most frequent indications: 1) patients undergoing percutaneous coronary intervention (for any indication) within 14 days of randomization (n = 2676; 71.2%); and 2) patients presenting with acute coronary syndrome managed with or without percutaneous coronary intervention (n = 1573; 41.8%). Unadjusted Cox proportional hazards models were used to estimate effect sizes through final follow-up. RESULTS: Median follow-up duration was 110 days (interquartile range 55-167). In percutaneous coronary intervention-treated patients, omeprazole significantly reduced rates of composite gastrointestinal events at 180 days (1.2% vs 2.7%; hazard ratio [HR] 0.43; 95% confidence interval [CI], 0.22-0.85; P = .02) without increasing composite cardiovascular events (5.4% vs 6.3%; HR 1.00; 95% CI, 0.67-1.50; P = 1.00). Similarly, omeprazole lowered risk of the primary gastrointestinal endpoint at 180 days in patients presenting with acute coronary syndrome (1.1% vs 2.7%; HR 0.37; 95% CI, 0.13-1.01; P = .05) without a significant excess in cardiovascular events (5.6% vs 4.5%; HR 1.40; 95% CI, 0.77-2.53; P = .27). CONCLUSIONS: PPI therapy attenuates gastrointestinal bleeding risk without significant excess in major cardiovascular events in high-risk cardiovascular subsets, regardless of indication for DAPT. Future studies will be needed to clarify optimal gastroprotective strategies for higher-intensity and longer durations of DAPT.
Asunto(s)
Síndrome Coronario Agudo/tratamiento farmacológico , Aspirina/uso terapéutico , Hemorragia Gastrointestinal/prevención & control , Omeprazol/uso terapéutico , Intervención Coronaria Percutánea/métodos , Inhibidores de Agregación Plaquetaria/uso terapéutico , Inhibidores de la Bomba de Protones/uso terapéutico , Aspirina/administración & dosificación , Aspirina/efectos adversos , Clopidogrel , Método Doble Ciego , Quimioterapia Combinada , Humanos , Omeprazol/efectos adversos , Intervención Coronaria Percutánea/efectos adversos , Intervención Coronaria Percutánea/estadística & datos numéricos , Inhibidores de Agregación Plaquetaria/administración & dosificación , Inhibidores de Agregación Plaquetaria/efectos adversos , Inhibidores de la Bomba de Protones/efectos adversos , Ticlopidina/administración & dosificación , Ticlopidina/efectos adversos , Ticlopidina/análogos & derivados , Ticlopidina/uso terapéutico , Resultado del TratamientoRESUMEN
OBJECTIVE: STAT5A/5B-deficient mice are recognized to manifest defects in multiple cell types and tissues. In particular, the hematopoietic defects in these mice are widespread, affecting multiple lineages and multiple stages of development. Previous studies indicate that deficiencies intrinsic to hematopoietic cells contribute substantially to the observed defects. However, in light of the broad physiologic effects of STAT5 in the context of the organism outside the blood system, we wished to investigate the possibility of STAT5-dependent environmental influence of nonhematopoietic origin on hematopoietic development in these mice. MATERIALS AND METHODS: We transplanted wild-type bone marrow into STAT5A/5B-deficient mice to determine the effects of loss of STAT5 in nonhematopoietic tissue on hematopoietic development. RESULTS: We observed that transplantation of wild-type marrow completely corrects hematopoietic defects in STAT5A/5B-deficient recipient mice, including peripheral blood counts, bone marrow cellularity, and reductions in specific progenitor subsets. CONCLUSION: These results indicate that the important role of STAT5 in hematolymphoid development are mediated directly through effects on hematopoietic cells and not indirectly.
Asunto(s)
Trasplante de Médula Ósea , Proteínas de Unión al ADN/deficiencia , Proteínas de la Leche , Transactivadores/deficiencia , Animales , Células Sanguíneas/citología , Células de la Médula Ósea/citología , Proteínas de Unión al ADN/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Sistema Hematopoyético/crecimiento & desarrollo , Sistema Linfático/crecimiento & desarrollo , Ratones , Ratones Noqueados , Factor de Transcripción STAT5 , Transactivadores/genéticaRESUMEN
OBJECTIVE: Cytokines bind high-affinity receptors expressed on hematopoietic cells to initiate signaling cascades that regulate differentiation, proliferation, and survival. Previous studies have established a role for STAT5 in transducing survival signals for hematopoietic progenitor cells in response to cytokines. MATERIALS AND METHODS: To determine if constitutive expression of a member of the bcl-2 family of anti-apoptotic proteins could compensate for the loss of STAT5, we utilized combinatorial genetics to generate STAT5A/5B-deficient mice expressing a bcl-2 transgene. RESULTS: Although bcl-2 expression restored peripheral blood counts to normal in STAT5A/5B(-/-) mice, we noted a striking failure of this transgene to correct defects in hematopoietic stem and progenitor cells. CONCLUSION: These data imply important effects of STAT5 in modulating hematopoietic cells in addition to promoting survival per se.
Asunto(s)
Proteínas de Unión al ADN/deficiencia , Sistema Hematopoyético/efectos de los fármacos , Sistema Linfático/efectos de los fármacos , Proteínas de la Leche , Proteínas Proto-Oncogénicas c-bcl-2/farmacología , Transactivadores/deficiencia , Animales , Células Sanguíneas/citología , Células Sanguíneas/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Sistema Hematopoyético/crecimiento & desarrollo , Sistema Linfático/crecimiento & desarrollo , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/uso terapéutico , Factor de Transcripción STAT5 , Transactivadores/genética , Transgenes , Insuficiencia del TratamientoRESUMEN
Granulocyte colony-stimulating factor (G-CSF) is essential for the host response to bacterial infection by controlling neutrophil production in the bone marrow. The G-CSF receptor (G-CSFR) activates the Jak/STAT pathway, although little is understood about how these signals regulate basal and stress-induced granulopoiesis. We examined STAT3 function in granulocytes using a bone marrow conditional knockout mouse model. Our results show that STAT3 has a crucial role in emergency granulopoiesis and mature neutrophil function. STAT3-deficient mice have an aberrant response to G-CSF in vivo, characterized by failure to accumulate immature granulocytes and an increased ratio of mature to immature neutrophils in the bone marrow, peripheral blood, and spleen. Acute neutrophil mobilization is impaired in STAT3-deficient mice as judged by their failure to up-regulate circulating neutrophils following short-term G-CSF exposure. STAT3 also controls neutrophil chemotactic responses to natural ligands for CXCR2 and regulates the magnitude of chemoattractant-induced actin polymerization. These functions of STAT3 are independent of its principal target gene Socs3, which encodes a crucial feedback inhibitor of cytokine signaling. Our results demonstrate the existence of distinct STAT3 target pathways in neutrophils required for granulopoiesis and innate immunity.
Asunto(s)
Quimiotaxis/fisiología , Inmunidad Innata/fisiología , Mielopoyesis/fisiología , Neutrófilos/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/fisiología , Animales , Infecciones Bacterianas/genética , Infecciones Bacterianas/metabolismo , Médula Ósea/metabolismo , Diferenciación Celular/fisiología , Factor Estimulante de Colonias de Granulocitos/metabolismo , Ratones , Ratones Noqueados , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Factor de Transcripción STAT3/deficiencia , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas/metabolismoRESUMEN
To enter human cells, HIV-1 usually uses CD4 and 1 of 2 coreceptors: CCR5 and CXCR4. Interestingly, even though CCR5 is expressed on far fewer T cells than is CXCR4, many patients in early- and late-stage HIV disease maintain high levels of CCR5-tropic (R5) viruses. We hypothesized that such high R5 viral loads may be sustained because, relative to CXCR4-tropic (X4) HIV-1 infection, R5 HIV-1 infection of permissive CD4(+)CCR5(+)CXCR4(+) T cells results in the production of significantly more infectious virus particles per target cell. To investigate this possibility, we compared the levels of virus production per target cell after isogenic R5 and X4 HIV-1 infection of 2 in vitro primary human lymphocyte culture systems: T-cell receptor-stimulated blood-derived CD4(+) T cells and tonsil histoculture (which requires no exogenous stimulation for ex vivo infection). We provide evidence that R5 HIV-1 does indeed compensate for a small target cell population by producing, on average, 5 to 10 times more infectious virus per CCR5(+) target cell than X4 HIV-1. This replicative advantage may contribute to the predominance of R5 HIV-1 in vivo.
Asunto(s)
Linfocitos T CD4-Positivos/virología , VIH-1/fisiología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , VIH-1/patogenicidad , Humanos , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Virulencia , Replicación ViralRESUMEN
Infection of macrophages has been implicated as a critical event in the transmission and persistence of human immunodeficiency virus type 1 (HIV-1). Here, we explore whether primary X4 HIV-1 isolates can productively infect tissue macrophages that have terminally differentiated in vivo. Using immunohistochemistry, HIV-1 RNA in situ hybridization, and confocal immunofluorescence microscopy, we demonstrate that macrophages residing in human tonsil blocks can be productively infected ex vivo by primary X4 HIV-1 isolates. This challenges the model in which macrophage tropism is a key determinant of the selective transmission of R5 HIV-1 strains. Infection of tissue macrophages by X4 HIV-1 may be highly relevant in vivo and contribute to key events in HIV-1 pathogenesis.
Asunto(s)
VIH-1/crecimiento & desarrollo , Macrófagos/virología , Antígenos CD/análisis , Células Cultivadas , VIH-1/clasificación , VIH-1/aislamiento & purificación , Humanos , Macrófagos/inmunología , Microscopía Confocal , Monocitos/virologíaRESUMEN
The filoviruses Ebola Zaire virus and Marburg virus are believed to infect target cells through endocytic vesicles, but the details of this pathway are unknown. We used a pseudotyping strategy to investigate the cell biology of filovirus entry. We observed that specific inhibitors of the caveola system, including cholesterol-sequestering drugs and phorbol esters, inhibited the entry of filovirus pseudotypes into human cells. We also measured slower cell entry kinetics for both filovirus pseudotypes than for pseudotypes of vesicular stomatitis virus (VSV), which has been recognized to exploit the clathrin-mediated entry pathway. Finally, visualization by immunofluorescence and confocal microscopy revealed that the filovirus pseudotypes colocalized with the caveola protein marker caveolin-1 but that VSV pseudotypes did not. Collectively, these results provide evidence suggesting that filoviruses use caveolae to gain entry into cells.
Asunto(s)
Caveolas/virología , Filoviridae/fisiología , Caveolina 1 , Caveolinas/metabolismo , Células Cultivadas , Humanos , Ésteres del Forbol/farmacología , Replicación ViralRESUMEN
Human infection by Marburg (MBG) or Ebola (EBO) virus is associated with fatal haemorrhagic fevers. While these filoviruses may both incite disease as a result of explosive virus replication, we hypothesized that expression of individual viral gene products, such as the envelope glycoprotein (GP), may directly alter target cells and contribute to pathogenesis. We found that expression of EBO GP in 293T cells caused significant levels of cellular detachment in the absence of cell death or virus replication. This detachment was induced most potently by membrane-bound EBO GP, rather than the shed glycoprotein products (sGP or GP1), and was largely attributable to a domain within the extracellular region of GP2. Furthermore, detachment was blocked by the Ser/Thr kinase inhibitor 2-aminopurine, suggesting the importance of a phosphorylation-dependent signalling cascade in inducing detachment. Since MBG GP did not induce similar cellular detachment, MBG and EBO GP interact with target cells by distinct processes to elicit cellular dysregulation.
Asunto(s)
Adhesión Celular , Ebolavirus/patogenicidad , Marburgvirus/patogenicidad , Animales , Línea Celular , Chlorocebus aethiops , Electroforesis en Gel de Poliacrilamida , Productos del Gen env/genética , Productos del Gen env/fisiología , Cabras , Cobayas , Transducción de Señal , Células VeroRESUMEN
STAT5 has previously been reported to be dispensable for the maintenance of tolerance in vivo. However, in examining hemopoiesis in mice lacking both isoforms of STAT5, STAT5A, and STAT5B, we noted that a subset of these mice demonstrated dramatic alterations in several bone marrow progenitor populations concomitant with lymphocytic infiltration of the bone marrow. In addition, cellular infiltration affecting the colon, liver, and kidney was observed in these mice. Survival analysis revealed that STAT5A/5B(-/-) mice exhibited early death. The increased mortality and the pathology affecting multiple organs observed in these mice were abrogated on the recombination-activating gene 1(-/-) background. In light of the similarities between STAT5A/5B-deficient mice and mice unable to signal through the IL-2R, we hypothesized that the tolerizing role of STAT5A/5B was triggered via activation of the IL-2R. In agreement with this, we found that IL-2Rbeta chain-deficient mice exhibited similar hemopoietic abnormalities. Because IL-2 signaling is thought to contribute to tolerance through maintenance of a CD4(+)CD25(+) regulatory T cell population, we examined these cells and observed a numerical reduction in STAT5A/5B(-/-) mice along with a higher rate of apoptosis. These data provide strong evidence for a requirement for STAT5 in the maintenance of tolerance in vivo.
Asunto(s)
Enfermedades Autoinmunes/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Tolerancia Inmunológica/genética , Proteínas de la Leche , Transactivadores/deficiencia , Transactivadores/genética , Traslado Adoptivo , Animales , Apoptosis/genética , Apoptosis/inmunología , Enfermedades Autoinmunes/mortalidad , Enfermedades Autoinmunes/patología , Enfermedades Autoinmunes/prevención & control , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/patología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Movimiento Celular/genética , Movimiento Celular/inmunología , Colon/inmunología , Colon/patología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/patología , Proteínas de Homeodominio/genética , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/inmunología , Síndromes de Inmunodeficiencia/patología , Memoria Inmunológica/genética , Subunidad beta del Receptor de Interleucina-2 , Riñón/inmunología , Riñón/patología , Hígado/inmunología , Hígado/patología , Tejido Linfoide/inmunología , Tejido Linfoide/patología , Linfopenia/genética , Linfopenia/inmunología , Linfopenia/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Receptores de Interleucina/deficiencia , Receptores de Interleucina/genética , Receptores de Interleucina-2/biosíntesis , Factor de Transcripción STAT5 , Tasa de Supervivencia , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
The transcription factor signal transducers and activators of transcription 5 (STAT5) is activated by numerous cytokines that orchestrate blood cell development. Multilineage peripheral blood cytopenias were observed in adult mice lacking both isoforms of STAT5 (STAT5A and STAT5B) as well as accelerated rates of apoptosis in the bone marrow. Although the hematopoietic stem cell (HSC) population was preserved in a number of these mice, the post-HSC progenitor populations were diminished and a marked reduction in functional progenitors (spleen colony-forming units) was detected. Competitive bone marrow transplantation studies in vivo revealed a profound impairment of repopulation potential of STAT5-null HSCs, leading to complete lack of contribution to the myeloid, erythroid, and lymphoid lineages. These abnormalities were associated with heightened proliferation activity in the HSC fraction, suggesting the action of homeostatic mechanisms to maintain sufficient levels of diverse blood cell types for viability. Thus, STAT5 normally sustains the robust hematopoietic reserve that contributes to host viability through crucial survival effects on early progenitor cells.