RESUMO
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid progenitor cells that dampen overwhelming adaptive immune responses through multiple mechanisms and are recognized as an attractive novel immune intervention therapy for counteracting the destructive effects of graft- versus -host disease (GVHD) developing after allogeneic bone marrow transplantation (BMT). MDSCs can be produced in great numbers for cellular therapy, but they present a mixture of subsets whose functions in GVHD prevention are undefined. Here, we generated MDSCs in vitro from murine BM cells in the presence of GM-CSF and defined the integrin CD11c as a marker to subdivide MDSCs into two functional subgroups: CD11b+CD11c+ and CD11b+CD11c- MDSCs. Isolated CD11b+CD11c+ and CD11b+CD11c- MDSCs both inhibited alloantigen-stimulated T-cell proliferation in vitro, although CD11b+CD11c+ MDSCs were more efficient and expressed higher levels of different immunosuppressive molecules. Likewise, expression of surface markers such as MHC class II, CD80, CD86, or PD-L1 further delineated both subsets. Most importantly, only the adoptive transfer of CD11b+CD11c+ MDSCs into a single MHC class I-disparate allogeneic BMT model prevented GVHD development and strongly decreased disease-induced mortality, while CD11b+CD11c- MDSCs were totally ineffective. Surprisingly, allogeneic T-cell homing and expansion in lymphatic and GVHD target organs were not affected by cotransplanted CD11b+CD11c+ MDSCs indicating a clear contradiction between in vitro and in vivo functions of MDSCs. However, CD11b+CD11c+ MDSCs shifted immune responses towards type 2 immunity reflected by increased Th2-specific cytokine expression of allogeneic T cells. Induction of type 2 immunity was mandatory for GVHD prevention, since CD11b+CD11c+ MDSCs were ineffective if recipients were reconstituted with STAT6-deficient T cells unable to differentiate into Th2 cells. Most importantly, the beneficial graft- versus -tumor (GVT) effect was maintained in the presence of CD11b+CD11c+ MDSCs since syngeneic tumor cells were efficiently eradicated. Strong differences in the transcriptomic landscape of both subpopulations underlined their functional differences. Defining CD11b+CD11c+ MDSCs as the subset of in vitro-generated MDSCs able to inhibit GVHD development might help to increase efficiency of MDSC therapy and to further delineate relevant target molecules and signaling pathways responsible for GVHD prevention.
Assuntos
Antígenos CD11/análise , Antígeno CD11b/análise , Doença Enxerto-Hospedeiro/prevenção & controle , Células Supressoras Mieloides/imunologia , Aloenxertos , Animais , Transplante de Medula Óssea/efeitos adversos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Ontologia Genética , Efeito Enxerto vs Tumor , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Imunidade Celular , Separação Imunomagnética , Camundongos , Células Supressoras Mieloides/química , Células Supressoras Mieloides/classificação , Células Supressoras Mieloides/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Quimera por Radiação , Subpopulações de Linfócitos T/imunologia , TranscriptomaRESUMO
Slit2 exerts antitumor effects in various cancers; however, the underlying mechanism, especially its role in regulating the immune, especially in the bone marrow niche, system is still unknown. Elucidating the behavior of macrophages in tumor progression can potentially improve immunotherapy. Using a spontaneous mammary tumor virus promoter-polyoma middle T antigen (PyMT) breast cancer mouse model, we observed that Slit2 increased the abundance of antitumor M1 macrophage in the bone marrow upon differentiation in vitro. Moreover, myeloablated PyMT mice injected with Slit2-treated bone marrow allografts showed a marked reduction in tumor growth, with enhanced recruitment of M1 macrophage in their tumor stroma. Mechanistic studies revealed that Slit2 significantly enhanced glycolysis and reduced fatty acid oxidation in bone marrow-derived macrophages (BMDMs). Slit2 treatment also altered mitochondrial respiration metabolites in macrophages isolated from healthy human blood that were treated with plasma from breast cancer patients. Overall, this study, for the first time, shows that Slit2 increases BMDM polarization toward antitumor phenotype by modulating immune-metabolism. Furthermore, this study provides evidence that soluble Slit2 could be developed as novel therapeutic strategy to enhance antitumor immune response.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Neoplasias Mamárias Experimentais/terapia , Metaboloma/efeitos dos fármacos , Proteínas do Tecido Nervoso/fisiologia , Adulto , Idoso , Animais , Antígenos Transformantes de Poliomavirus/genética , Meios de Cultivo Condicionados , Feminino , Glicólise/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Receptores de Lipopolissacarídeos/análise , Macrófagos/imunologia , Macrófagos/metabolismo , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Vírus do Tumor Mamário do Camundongo/genética , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/farmacologia , Quimera por Radiação , Serina-Treonina Quinases TOR/fisiologia , Neoplasias de Mama Triplo Negativas/sangue , Neoplasias de Mama Triplo Negativas/química , Carga TumoralRESUMO
Ventricular remodeling following myocardial infarction (MI) is a major cause of heart failure, a condition prevalent in older individuals. Following MI, immune cells are mobilized to the myocardium from peripheral lymphoid organs and play an active role in orchestrating repair. While the effect of aging on mouse bone marrow (BM) has been studied, less is known about how aging affects human BM cells and their ability to regulate repair processes. In this study, we investigate the effect aging has on human BM cell responses post-MI using a humanized chimeric mouse model. BM samples were collected from middle aged (mean age 56.4 ± 0.97) and old (mean age 72.7 ± 0.59) patients undergoing cardiac surgery, CD34+/- cells were isolated, and NOD-scid-IL2rγnull (NSG) mice were reconstituted. Three months following reconstitution, the animals were examined at baseline or subjected to coronary artery ligation (MI). Younger patient cells exhibited greater repopulation capacity in the BM, blood, and spleen as well as greater lymphoid cell production. Following MI, CD34+ cell age impacted donor and host cellular responses. Mice reconstituted with younger CD34+ cells exhibited greater human CD45+ recruitment to the heart compared to mice reconstituted with old cells. Increased cellular responses were primarily driven by T-cell recruitment, and these changes corresponded with greater human IFNy levels and reduced mouse IL-1ß in the heart. Age-dependent changes in BM function led to significantly lower survival, increased infarct expansion, impaired host cell responses, and reduced function by 4w post-MI. In contrast, younger CD34+ cells helped to limit remodeling and preserve function post-MI.
Assuntos
Envelhecimento/metabolismo , Células da Medula Óssea/metabolismo , Infarto do Miocárdio/metabolismo , Neovascularização Fisiológica , Quimera por Radiação/metabolismo , Idoso , Animais , Antígenos CD34/metabolismo , Transplante de Medula Óssea/métodos , Estudos de Coortes , Vasos Coronários/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Remodelação VentricularRESUMO
NK cells are critical innate immune cells that target the tumor cells and cancer-initiating cells and clear viruses by producing cytokines and cytotoxic granules. However, the role of the purinergic receptor P2Y6 in the NK cells remains largely unknown. In this study, we discovered that the expression of P2Y6 was decreased upon the activation of the NK cells. Moreover, in the P2Y6-deficient mice, we found that the deficiency of P2Y6 promoted the development of the NK precursor cells into immature NK and mature NK cells. We also found that the P2Y6 deficiency increased, but the P2Y6 receptor agonist UDP or UDP analog 5-OMe-UDP decreased the production of IFN-γ in the activated NK cells. Furthermore, we demonstrated that the P2Y6-deficient NK cells exhibited stronger cytotoxicity in vitro and antimetastatic effects in vivo. Mechanistically, P2Y6 deletion promoted the expression of T-bet (encoded by Tbx21), with or without the stimulation of IL-15. In the absence of P2Y6, the levels of phospho-serine/threonine kinase and pS6 in the NK cells were significantly increased upon the stimulation of IL-15. Collectively, we demonstrated that the P2Y6 receptor acted as a negative regulator of the NK cell function and inhibited the maturation and antitumor activities of the NK cells. Therefore, inhibition of the P2Y6 receptor increases the antitumor activities of the NK cells, which may aid in the design of innovative strategies to improve NK cell-based cancer therapy.
Assuntos
Diferenciação Celular/genética , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Melanoma/patologia , Receptores Purinérgicos P2/deficiência , Transdução de Sinais/genética , Neoplasias Cutâneas/patologia , Animais , Linhagem Celular Tumoral , Feminino , Técnicas de Inativação de Genes , Humanos , Interleucina-15/farmacologia , Neoplasias Pulmonares/genética , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quimera por Radiação/imunologia , Receptores Purinérgicos P2/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas com Domínio T/metabolismo , Carga Tumoral/genética , Carga Tumoral/imunologiaRESUMO
The generation of radiation chimeras allows researchers to substitute the hematopoietic system of a mouse with that of one or more donors. A suspension of hematopoietic stem cells (HSCs) is prepared from the bone marrow (BM) or the fetal liver (FL) of a donor mouse and adoptively transferred into an irradiated recipient. Within days, the donor's HSCs will engraft, and their progeny will quickly replace the blood cells of the recipient. This simple tool, together with the large availability of genetically modified mouse lines, can be harnessed to manipulate and study various aspects of blood cell biology in vivo. We present here protocols to generate three types of radiation chimera: (1) BM chimeras, which can assist in determining whether the origin of a genetically based phenotype is the hematopoietic or radio-resistant compartment and which are also conducive for studying the ecology of blood cells and for manipulating the environment hematopoietic cells live; (2) FL chimeras, which allow the study of hematopoietic systems from animals that carry genetic modifications incompatible with postnatal life; and (3) mixed BM chimeras, in which the hematopoietic system comprises blood cells of two different genotypes. Mixed BM chimeras can be used to identify genes that affect hematopoietic cell fitness and to establish whether secreted factors mediate a phenotype of interest. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Generation of bone marrow chimera Basic Protocol 2: Generation of fetal liver chimera Basic Protocol 3: Generation of mixed bone marrow chimera Support Protocol 1: Isolation of bone marrow cells Support Protocol 2: Cell counting by flow cytometry Support Protocol 3: Assessment of chimerism.
Assuntos
Medula Óssea , Quimera , Animais , Células-Tronco Hematopoéticas , Fígado , Camundongos , Quimera por RadiaçãoRESUMO
Myeloproliferative neoplasms (MPNs) are characterized by a pathologic expansion of myeloid lineages. Mutations in JAK2, CALR and MPL genes are known to be three prominent MPN disease drivers. Mutant CALR (mutCALR) is an oncoprotein that interacts with and activates the thrombopoietin receptor (MPL) and represents an attractive target for targeted therapy of CALR mutated MPN. We generated a transgenic murine model with conditional expression of the human mutant exon 9 (del52) from the murine endogenous Calr locus. These mice develop essential thrombocythemia like phenotype with marked thrombocytosis and megakaryocytosis. The disease exacerbates with age showing prominent signs of splenomegaly and anemia. The disease is transplantable and mutCALR stem cells show proliferative advantage when compared to wild type stem cells. Transcriptome profiling of hematopoietic stem cells revealed oncogenic and inflammatory gene expression signatures. To demonstrate the applicability of the transgenic animals for immunotherapy, we treated mice with monoclonal antibody raised against the human mutCALR. The antibody treatment lowered platelet and stem cell counts in mutant mice. Secretion of mutCALR did not constitute a significant antibody sink. This animal model not only recapitulates human MPN but also serves as a relevant model for testing immunotherapeutic strategies targeting epitopes of the human mutCALR.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Calreticulina/antagonistas & inibidores , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/metabolismo , Terapia de Alvo Molecular , Trombocitemia Essencial/terapia , Animais , Anticorpos Monoclonais/imunologia , Plaquetas/imunologia , Plaquetas/metabolismo , Calreticulina/genética , Calreticulina/imunologia , Calreticulina/fisiologia , Éxons/genética , Mutação da Fase de Leitura , Técnicas de Introdução de Genes , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Quimera por Radiação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Esplenomegalia/etiologia , Trombocitemia Essencial/sangue , Trombocitemia Essencial/complicações , Trombocitemia Essencial/genética , TranscriptomaRESUMO
Murine bone marrow (BM) chimeras are a versatile and valuable research tool in stem cell and immunology research. Engraftment of donor BM requires myeloablative conditioning of recipients. The most common method used for mice is ionizing radiation, and Cesium-137 gamma irradiators have been preferred. However, radioactive sources are being out-phased worldwide due to safety concerns, and are most commonly replaced by X-ray sources, creating a need to compare these sources regarding efficiency and potential side effects. Prior research has proven both methods capable of efficiently ablating BM cells and splenocytes in mice, but with moderate differences in resultant donor chimerism across tissues. Here, we compared Cesium-137 to 350 keV X-ray irradiation with respect to immune reconstitution, assaying complete, syngeneic BM chimeras and a mixed chimera model of autoimmune disease. Based on dose titration, we find that both gamma and X-ray irradiation can facilitate a near-complete donor chimerism. Mice subjected to 13 Gy Cesium-137 irradiation and reconstituted with syngeneic donor marrow were viable and displayed high donor chimerism, whereas X-ray irradiated mice all succumbed at 13 Gy. However, a similar degree of chimerism as that obtained following 13 Gy gamma irradiation could be achieved by 11 Gy X-ray irradiation, about 85% relative to the gamma dose. In the mixed chimera model of autoimmune disease, we found that a similar autoimmune phenotype could be achieved irrespective of irradiation source used. It is thus possible to compare data generated, regardless of the irradiation source, but every setup and application likely needs individual optimization.
Assuntos
Doenças Autoimunes/imunologia , Células da Medula Óssea/efeitos da radiação , Transplante de Medula Óssea/métodos , Medula Óssea/efeitos da radiação , Radioisótopos de Césio , Raios gama , Quimera por Radiação/imunologia , Animais , Modelos Animais de Doenças , Feminino , Doença Enxerto-Hospedeiro/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Irradiação Corporal Total , Raios XRESUMO
This study was conducted to determine the dosage effect of c-Myc on hematopoiesis and its distinct role in mediating the Wnt/ß-catenin pathway in hematopoietic stem cell (HSC) and bone marrow niche cells. c-Myc haploinsufficiency led to ineffective hematopoiesis by inhibiting HSC self-renewal and quiescence and by promoting apoptosis. We have identified Nr4a1, Nr4a2, and Jmjd3, which are critical for the maintenance of HSC functions, as previously unrecognized downstream targets of c-Myc in HSCs. c-Myc directly binds to the promoter regions of Nr4a1, Nr4a2, and Jmjd3 and regulates their expression. Our results revealed that Nr4a1 and Nr4a2 mediates the function of c-Myc in regulating HSC quiescence, whereas all 3 genes contribute to the function of c-Myc in the maintenance of HSC survival. Adenomatous polyposis coli (Apc) is a negative regulator of the Wnt/ß-catenin pathway. We have provided the first evidence that Apc haploinsufficiency induces a blockage of erythroid lineage differentiation through promoting secretion of IL6 in bone marrow endothelial cells. We found that c-Myc haploinsufficiency failed to rescue defective function of Apc-deficient HSCs in vivo but it was sufficient to prevent the development of severe anemia in Apc-heterozygous mice and to significantly prolong the survival of those mice. Furthermore, we showed that c-Myc-mediated Apc loss induced IL6 secretion in endothelial cells, and c-Myc haploinsufficiency reversed the negative effect of Apc-deficient endothelial cells on erythroid cell differentiation. Our studies indicate that c-Myc has a context-dependent role in mediating the function of Apc in hematopoiesis.
Assuntos
Genes myc , Hematopoese/fisiologia , Proteínas Proto-Oncogênicas c-myb/fisiologia , Proteína da Polipose Adenomatosa do Colo/fisiologia , Anemia/genética , Anemia/prevenção & controle , Animais , Apoptose/fisiologia , Transplante de Medula Óssea , Autorrenovação Celular/fisiologia , Ensaio de Unidades Formadoras de Colônias , Células Endoteliais/patologia , Células Eritroides/patologia , Deleção de Genes , Genes APC , Haploinsuficiência , Hematopoese/genética , Células-Tronco Hematopoéticas , Interleucina-6/fisiologia , Histona Desmetilases com o Domínio Jumonji/fisiologia , Camundongos Mutantes , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , Poli I-C/farmacologia , Quimera por Radiação , Via de Sinalização Wnt/fisiologiaRESUMO
Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.
Assuntos
AMP Cíclico/fisiologia , Dexametasona/farmacologia , Dinoprostona/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Sistemas do Segundo Mensageiro/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Criança , Cromograninas/antagonistas & inibidores , Colforsina/farmacologia , AMP Cíclico/farmacologia , Dexametasona/administração & dosagem , Dinoprostona/administração & dosagem , Dinoprostona/antagonistas & inibidores , Dinoprostona/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gs de Proteínas de Ligação ao GTP/deficiência , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Modelos Animais , Terapia de Alvo Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Quimera por Radiação , Receptores de Glucocorticoides/biossíntese , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/fisiologia , Receptores de Prostaglandina E Subtipo EP4/biossíntese , Receptores de Prostaglandina E Subtipo EP4/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Neutrophil migration to the site of infection is an essential process for the control and clearance of microbial growth within the host. Identifying the molecular factors that mediate neutrophil chemotaxis is therefore critical for our understanding of disease pathogenesis and the mechanisms underlying protective immunity. Here, we describe a protocol that enables analysis of neutrophil recruitment from the blood into fungal-infected organs in vivo, using mixed bone marrow chimeras and flow cytometry. This method directly assesses the relative contribution of a receptor or intracellular molecule in controlling neutrophil chemotaxis during fungal infection and can be adapted to a variety of other non-fungal infection experimental settings.
Assuntos
Transplante de Medula Óssea , Rastreamento de Células , Quimiotaxia de Leucócito , Citometria de Fluxo , Fungos/patogenicidade , Micoses/microbiologia , Neutrófilos/microbiologia , Quimera por Radiação , Animais , Modelos Animais de Doenças , Feminino , Fungos/imunologia , Interações Hospedeiro-Patógeno , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Micoses/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Transdução de SinaisRESUMO
B-cell lymphoma 6 (BCL6) is a transcription repressor and proto-oncogene that plays a crucial role in the innate and adaptive immune system and lymphoid neoplasms. However, its role in myeloid malignancies remains unclear. Here, we explored the role of BCL6 in acute myeloid leukemia (AML). BCL6 was expressed at variable and often high levels in AML cell lines and primary AML samples. AMLs with higher levels of BCL6 were generally sensitive to treatment with BCL6 inhibitors, with the exception of those with monocytic differentiation. Gene expression profiling of AML cells treated with a BCL6 inhibitor revealed induction of BCL6-repressed target genes and transcriptional programs linked to DNA damage checkpoints and downregulation of stem cell genes. Ex vivo treatment of primary AML cells with BCL6 inhibitors induced apoptosis and decreased colony-forming capacity, which correlated with the levels of BCL6 expression. Importantly, inhibition or knockdown of BCL6 in primary AML cells resulted in a significant reduction of leukemia-initiating capacity in mice, suggesting ablation of leukemia repopulating cell functionality. In contrast, BCL6 knockout or inhibition did not suppress the function of normal hematopoietic stem cells. Treatment with cytarabine further induced BCL6 expression, and the levels of BCL6 induction were correlated with resistance to cytarabine. Treatment of AML patient-derived xenografts with BCL6 inhibitor plus cytarabine suggested enhanced antileukemia activity with this combination. Hence, pharmacologic inhibition of BCL6 might provide a novel therapeutic strategy for ablation of leukemia-repopulating cells and increased responsiveness to chemotherapy.
Assuntos
Leucemia Mieloide Aguda/patologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-6/fisiologia , Animais , Antineoplásicos/farmacologia , Apoptose , Autorrenovação Celular , Citarabina/uso terapêutico , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células-Tronco Hematopoéticas/citologia , Humanos , Indóis/farmacologia , Indóis/uso terapêutico , Leucemia Mieloide Aguda/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/citologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , RNA-Seq , Quimera por Radiação , Tiazolidinedionas/farmacologia , Tiazolidinedionas/uso terapêutico , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the γ-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry and found that the endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1-knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared with wild-type littermates. Moreover, Gabbr1-null hematopoietic stem cells had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1-null HSPCs were less proliferative under steady-state conditions and upon stress. Colony-forming unit assays demonstrated that almost all Gabbr1-null HSPCs were in a slow or noncycling state. In vitro differentiation of Gabbr1-null HSPCs in cocultures produced fewer overall cell numbers with significant defects in differentiation and expansion of the B-cell lineage. To determine whether a GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared with GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation.
Assuntos
Células-Tronco Hematopoéticas/citologia , Receptores de GABA-B/fisiologia , Animais , Linfócitos B/patologia , Baclofeno/análogos & derivados , Baclofeno/farmacologia , Medula Óssea/inervação , Medula Óssea/metabolismo , Transplante de Medula Óssea , Divisão Celular , Linhagem da Célula , Feminino , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Células Endoteliais da Veia Umbilical Humana/transplante , Humanos , Linfopenia/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Quimera por Radiação , Receptores de GABA-B/deficiência , Receptores de GABA-B/genética , Nicho de Células-TroncoRESUMO
Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.
Assuntos
Envelhecimento/genética , Regulação da Expressão Gênica/genética , Hematopoese/genética , Sistema Hematopoético/fisiologia , Código das Histonas/genética , Histona Desmetilases/fisiologia , Animais , Senescência Celular/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Feminino , Predisposição Genética para Doença , Hematopoese Extramedular , Histona Desmetilases/deficiência , Histona Desmetilases/genética , Reconstituição Imune , Histona Desmetilases com o Domínio Jumonji/metabolismo , Leucemia Experimental/genética , Leucemia Experimental/virologia , Masculino , Camundongos , Camundongos Knockout , Vírus da Leucemia Murina de Moloney/fisiologia , Células Mieloides/patologia , Quimera por Radiação , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/metabolismo , Integração ViralRESUMO
T cells increase cholesterol biosynthesis upon activation to generate substrates for cellular growth and proliferation. The ubiquitously expressed liver X receptor ß (LXRß) encoded by the Nr1h2 gene is a critical regulator of cholesterol homeostasis in mammalian cells; however, its cell-intrinsic role in T cell biology remains poorly understood. We report that ablation of LXRß in T cells leads to spontaneous T cell activation and T lymphocytopenia. Unexpectedly, analysis of mixed bone marrow chimeric mice revealed a cell-autonomous survival defect that reduced the fitness of LXRß-deficient effector T cells, suggesting that the heightened immune activation in mice harboring LXRß-deficient T cells was due to impaired regulatory T (T reg) cell functionality. Indeed, we found that single-copy deletion of Nr1h2 in T reg cells disrupted activated T reg cell metabolism and fitness and resulted in early-onset fatal autoimmune disease. Our study demonstrated an indispensable requirement for T reg cell-intrinsic LXRß function in immune homeostasis and provides a basis for immunological therapies through targeting of this receptor.
Assuntos
Doenças Autoimunes/imunologia , Homeostase/imunologia , Receptores X do Fígado/fisiologia , Ativação Linfocitária/genética , Linfócitos T Reguladores/imunologia , T-Linfocitopenia Idiopática CD4-Positiva/imunologia , Animais , Doenças Autoimunes/genética , Células Cultivadas , Colesterol/metabolismo , Feminino , Fatores de Transcrição Forkhead/genética , Homeostase/genética , Receptores X do Fígado/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quimera por Radiação/imunologia , Transdução de Sinais/genética , Linfócitos T Reguladores/metabolismo , T-Linfocitopenia Idiopática CD4-Positiva/genéticaRESUMO
Mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) comprise the majority of mutations found in clonal hematopoiesis (CH), an age-related condition that was recently found to affect outcomes in patients undergoing hematopoietic stem cell transplant (HSCT). Recent studies have indicated that patients with CH have worse prognoses after HSCT, suggesting stress imposed by HSCT preconditioning agents may impact hematopoietic stem cell (HSC) dynamics in transplant recipients. In this study, we used a competitive transplantation mouse model to investigate how treatment with the common preconditioning agents 5-fluorouracil (5-FU) and busulfan (BU) affect the prevalence of Dnmt3a-/- HSCs and progenitor cells in competition with wild-type cells. We found that, though sufficient to deplete peripheral blood counts, 5-FU preconditioning did not significantly alter the frequency of Dnmt3a-null hematopoietic stem and progenitor cells (HSPCs) in mosaic mice. In contrast, mice treated with BU had a sevenfold decline in total bone marrow cells and an increase in Dnmt3a-null HSPCs that was detectable in peripheral blood. Indeed, even though all mosaic mice had a starting engraftment of â¼10%-40%, 85%-100% of HSPCs were Dnmt3a-null in four of seven mice after BU treatment, indicating these cells expand dramatically during recovery. Overall, these results suggest that individual preconditioning regimens have different effects on the expansion of Dnmt3a-mutant cells in patients with pre-existing CH. Thus, the presence of CH-associated mutants should be evaluated prior to selecting preconditioning regimens for HSCT.
Assuntos
Bussulfano/farmacologia , DNA (Citosina-5-)-Metiltransferases/deficiência , Hematopoese/genética , Fatores de Crescimento de Células Hematopoéticas/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Animais , Medula Óssea/patologia , Divisão Celular/efeitos dos fármacos , Linhagem da Célula , Células Clonais , DNA Metiltransferase 3A , Fluoruracila/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Camundongos , Quimera por RadiaçãoRESUMO
Tristetraprolin (TTP) is a mRNA binding protein that binds to adenylate-uridylate-rich elements within the 3' untranslated regions of certain transcripts, such as tumor necrosis factor (Tnf) mRNA, and increases their rate of decay. Modulation of TTP expression is implicated in inflammation; however, its role in acute lung inflammation remains unknown. Accordingly, we tested the role of TTP in lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. LPS-challenged TTP-knockout (TTPKO) mice, as well as myeloid cell-specific TTP-deficient (TTPmyeKO) mice, exhibited significant increases in lung injury, although these responses were more robust in the TTPKO. Mice with systemic overexpression of TTP (TTPΔARE) were protected from ALI, as indicated by significantly reduced neutrophilic infiltration, reduced levels of neutrophil chemoattractants, and histological parameters of ALI. Interestingly, while irradiated wild-type (WT) mice reconstituted with TTPKO hematopoietic progenitor cells (HPCs) showed exaggerated ALI, their reconstitution with the TTPΔARE HPCs mitigated ALI. The reconstitution of irradiated TTPΔARE mice with HPCs from either WT or TTPΔARE donors conferred significant protection against ALI. In contrast, irradiated TTPΔARE mice reconstituted with TTPKO HPCs had exaggerated ALI, but the response was milder as compared to WT recipients that received TTPKO HPCs. Finally, the reconstitution of irradiated TTPKO recipient mice with TTPΔARE HPCs did not confer any protection to the TTPKO mice. These data together suggest that non-HPCs-specific overexpression of TTP within the lungs protects against ALI via downregulation of neutrophil chemoattractants and reduction in neutrophilic infiltration.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Células Epiteliais Alveolares/metabolismo , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Tristetraprolina/fisiologia , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Transplante de Medula Óssea , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Quimiotaxia de Leucócito , Citocinas/fisiologia , Feminino , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos , Neutrófilos/imunologia , Quimera por Radiação , Tristetraprolina/biossíntese , Tristetraprolina/deficiência , Tristetraprolina/genética , Regulação para CimaRESUMO
Myeloid cells are crucial for the development of vascular inflammation. Low-density lipoprotein receptor-related protein 8 (LRP8) or Apolipoprotein E receptor 2 (ApoER2), is expressed by macrophages, endothelial cells and platelets and has been implicated in the development of cardiovascular diseases. Our aim was to evaluate the role of LRP8, in particular from immune cells, in the development of vascular inflammation. METHODS: LRP8+/+ and LRP8-/- mice (on B6;129S background) were infused with angiotensin II (AngII, 1 mg/kg/day for 7 to 28 day) using osmotic minipumps. Blood pressure was recorded using tail cuff measurements. Vascular reactivity was assessed in isolated aortic segments. Leukocyte activation and infiltration were assessed by flow cytometry of aortic tissue and intravital videomicroscopy imaging. Histological analysis of aortic sections was conducted using sirius red staining. RESULTS: AngII infusion worsened endothelial-dependent vascular relaxation and immune cells rolling and adherence to the carotid artery in both LRP8+/+ as well as LRP8-/- mice. However, only LRP8-/- mice demonstrated a drastically increased mortality rate in response to AngII due to aortic dissection. Bone marrow transplantation revealed that chimeras with LRP8 deficient myeloid cells phenocopied LRP8-/- mice. CONCLUSION: AngII-infused LRP8 deficient mice could be a useful animal model to study aortic dissection reflecting the lethality of this disease in humans.
Assuntos
Angiotensina II/toxicidade , Dissecção Aórtica/induzido quimicamente , Proteínas Relacionadas a Receptor de LDL/deficiência , Acetilcolina/farmacologia , Animais , Pressão Sanguínea , Transplante de Medula Óssea , Adesão Celular/efeitos dos fármacos , Quimiotaxia de Leucócito/efeitos dos fármacos , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Bombas de Infusão , Proteínas Relacionadas a Receptor de LDL/fisiologia , Masculino , Camundongos , Microscopia de Vídeo , Células Mieloides/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Quimera por Radiação , Vasodilatação/efeitos dos fármacosRESUMO
Erythropoietin (EPO) provides the major survival signal to maturing erythroid precursors (EPs) and is essential for terminal erythropoiesis. Nonetheless, progenitor cells can irreversibly commit to an erythroid fate well before EPO acts, risking inefficiency if these progenitors are unneeded to maintain red blood cell (RBC) counts. We identified a new modular organization of erythropoiesis and, for the first time, demonstrate that the pre-EPO module is coupled to late EPO-dependent erythropoiesis by megakaryocyte (Mk) signals. Disrupting megakaryocytic transforming growth factor ß1 (Tgfb1) disorganized hematopoiesis by expanding the pre-EPO pool of progenitor cells and consequently triggering significant apoptosis of EPO-dependent EPs. Similarly, pharmacologic blockade of TGFß signaling in normal mice boosted the pre-EPO module, leading to apoptosis of EPO-sensitive EPs. Subsequent treatment with low-dose EPO triggered robust RBC production in both models. This work reveals modular regulation of erythropoiesis and offers a new strategy for overcoming chronic anemias.
Assuntos
Células Precursoras Eritroides/citologia , Eritropoese/fisiologia , Megacariócitos/citologia , Fator de Crescimento Transformador beta1/fisiologia , Animais , Apoptose/efeitos dos fármacos , Medula Óssea/patologia , Células Precursoras Eritroides/metabolismo , Eritropoetina/farmacologia , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Imunofenotipagem , Células Progenitoras de Megacariócitos e Eritrócitos/citologia , Células Progenitoras de Megacariócitos e Eritrócitos/metabolismo , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Quimera por Radiação , Proteínas Recombinantes/farmacologia , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
BACKGROUND: The Philadelphia chromosome (Ph), which leads to the creation and expression of the fusion gene product BCR-ABL, underlines the pathogenesis of chronic myelogenous leukemia (CML) and a fraction of adult and pediatric acute B-lymphoblastic leukemia (B-ALL). The BCR-ABL tyrosine kinase inhibitors (TKIs) have shown a remarkable clinical activity in patients with CML, but their efficacy in treating Ph+ B-ALL is limited. Identifying additional therapeutic targets is important for the effective treatment of Ph+ B-ALL. METHODS: Activation of the JNK signaling pathway in human and mouse BCR-ABL+ B-ALL cells with or without dasatinib treatment was analyzed by Western blotting. JNK was inhibited either by RNA interference or chemical inhibitors, such as JNK-IN-8. The effect of JNK inhibition with or without BCR-ABL TKI dasatinib on BCR-ABL+ B-ALL cells was analyzed by the CellTiter-Glo® Luminescent Cell Viability Assay. The in vivo effects of JNK-IN-8 and dasatinib alone or in combination were tested using a BCR-ABL induced B-ALL mouse model. RESULTS: We found that the c-JUN N-terminal kinase (JNK) signaling pathway is abnormally activated in both human and mouse BCR-ABL+ B-ALL cells, but the BCR-ABL TKI does not inhibit JNK activation in these cells. Inhibition of JNK, either by RNAi-mediated downregulation or by JNK inhibitors, could significantly reduce viability of Ph+ B-ALL cells. JNK inhibition by RNAi-mediated downregulation or JNK inhibitors also showed a synergistic effect with the BCR-ABL TKI, dasatinib, in killing Ph+ B-ALL cells in vitro. Furthermore, a potent JNK inhibitor, JNK-IN-8, in combination with dasatinib markedly improved the survival of mice with BCR-ABL induced B-ALL, as compared to the treatment with dasatinib alone. CONCLUSIONS: Our findings indicate that simultaneously targeting both BCR-ABL and JNK kinase might serve as a promising therapeutic strategy for Ph+ B-ALL.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antracenos/administração & dosagem , Azepinas/administração & dosagem , Benzamidas/administração & dosagem , Linhagem Celular Tumoral , Dasatinibe/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Mesilato de Imatinib/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Proto-Oncogênicas c-myc/biossíntese , Piridinas/administração & dosagem , Pirimidinas/administração & dosagem , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Quimera por Radiação , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Triazóis/administração & dosagemRESUMO
Acute erythroleukemia (AEL or acute myeloid leukemia [AML]-M6) is a rare but aggressive hematologic malignancy. Previous studies showed that AEL leukemic cells often carry complex karyotypes and mutations in known AML-associated oncogenes. To better define the underlying molecular mechanisms driving the erythroid phenotype, we studied a series of 33 AEL samples representing 3 genetic AEL subgroups including TP53-mutated, epigenetic regulator-mutated (eg, DNMT3A, TET2, or IDH2), and undefined cases with low mutational burden. We established an erythroid vs myeloid transcriptome-based space in which, independently of the molecular subgroup, the majority of the AEL samples exhibited a unique mapping different from both non-M6 AML and myelodysplastic syndrome samples. Notably, >25% of AEL patients, including in the genetically undefined subgroup, showed aberrant expression of key transcriptional regulators, including SKI, ERG, and ETO2. Ectopic expression of these factors in murine erythroid progenitors blocked in vitro erythroid differentiation and led to immortalization associated with decreased chromatin accessibility at GATA1-binding sites and functional interference with GATA1 activity. In vivo models showed development of lethal erythroid, mixed erythroid/myeloid, or other malignancies depending on the cell population in which AEL-associated alterations were expressed. Collectively, our data indicate that AEL is a molecularly heterogeneous disease with an erythroid identity that results in part from the aberrant activity of key erythroid transcription factors in hematopoietic stem or progenitor cells.