RESUMO
We studied the effect of an experimental synthetic organoselenium compound 2,6-dipyridinium- 9-selenabicyclo[3.3.1]nonane dibromide (974zh) on the cell composition of the red bone marrow and peripheral blood in white mice. The study drug co-administered with Yersinia pestis EV vaccine strain (103 CFU) potentiated maturation and migration of mature neutrophils from the bone marrow into the circulation. Reducing the dose of the live vaccine and the anti-inflammatory properties of the study drug made it possible to reduce the allergic reaction during the vaccination process.
Assuntos
Linfopoese/efeitos dos fármacos , Compostos Organosselênicos/farmacologia , Vacinação , Vacinas Atenuadas/farmacologia , Yersinia pestis/imunologia , Animais , Animais não Endogâmicos , Contagem de Células Sanguíneas , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/imunologia , Células Sanguíneas/patologia , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Camundongos , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/uso terapêuticoRESUMO
Natural killer (NK) cells are important components of the innate immune defense against infections and cancers. Signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is essential for NK cell maturation and NK cell-dependent tumor surveillance. Two alternatively spliced isoforms of STAT1 exist: a full-length STAT1α and a C-terminally truncated STAT1ß isoform. Aberrant splicing is frequently observed in cancer cells and several anti-cancer drugs interfere with the cellular splicing machinery. To investigate whether NK cell-mediated tumor surveillance is affected by a switch in STAT1 splicing, we made use of knock-in mice expressing either only the STAT1α (Stat1α/α) or the STAT1ß (Stat1ß/ß ) isoform. NK cells from Stat1α/α mice matured normally and controlled transplanted tumor cells as efficiently as NK cells from wild-type mice. In contrast, NK cells from Stat1ß/ß mice showed impaired maturation and effector functions, albeit less severe than NK cells from mice that completely lack STAT1 (Stat1-/- ). Mechanistically, we show that NK cell maturation requires the presence of STAT1α in the niche rather than in NK cells themselves and that NK cell maturation depends on IFNγ signaling under homeostatic conditions. The impaired NK cell maturation in Stat1ß/ß mice was paralleled by decreased IL-15 receptor alpha (IL-15Rα) surface levels on dendritic cells, macrophages and monocytes. Treatment of Stat1ß/ß mice with exogenous IL-15/IL-15Rα complexes rescued NK cell maturation but not their effector functions. Collectively, our findings provide evidence that STAT1 isoforms are not functionally redundant in regulating NK cell activity and that the absence of STAT1α severely impairs, but does not abolish, NK cell-dependent tumor surveillance.
Assuntos
Células Matadoras Naturais/citologia , Linfopoese/fisiologia , Fator de Transcrição STAT1/imunologia , Animais , Transplante de Medula Óssea , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Vigilância Imunológica/efeitos dos fármacos , Vigilância Imunológica/imunologia , Fator Gênico 3 Estimulado por Interferon/deficiência , Fator Gênico 3 Estimulado por Interferon/genética , Fator Gênico 3 Estimulado por Interferon/imunologia , Interleucina-15/farmacologia , Subunidade alfa de Receptor de Interleucina-15 , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Depleção Linfocítica , Tecido Linfoide/citologia , Linfoma/imunologia , Linfoma/patologia , Linfopoese/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Receptores de Interferon/deficiência , Fator de Transcrição STAT1/deficiência , Fator de Transcrição STAT1/genética , Organismos Livres de Patógenos Específicos , Baço/citologia , Receptor de Interferon gamaRESUMO
IL-9-producing CD4+ (Th9) cells are a subset of CD4+ T-helper cells that are endowed with powerful antitumor capacity. Both IL-4 and TGF-ß have been reported to be indispensable for Th9 cell-priming and differentiation. Here we show, by contrast, that Th9 cell development can occur in the absence of TGF-ß signaling. When TGF-ß was replaced by IL-1ß, the combination of IL-1ß and IL-4 efficiently promoted IL-9-producing T cells (Th9IL-4+IL-1ß). Th9IL-4+ IL-1ß cells are phenotypically distinct T cells compared to classic Th9 cells (Th9IL-4+TGF-ß) and other Th cells, and are enriched for IL-1 and NF-κB gene signatures. Inhibition of NF-κB but not TGF-ß-signaling negates IL-9 production by Th9IL-4+IL-1ß cells. Furthermore, when compared with classic Th9IL-4+TGF-ß cells, Th9IL-4+IL-1ß cells are less exhausted, exhibit cytotoxic T effector gene signature and tumor killing function, and exert a superior antitumor response in a mouse melanoma model. Our study thus describes an alternative pathway for Th9 cell differentiation and provides a potential avenue for antitumor therapies.
Assuntos
Interleucina-1beta/farmacologia , Interleucina-4/farmacologia , Linfopoese/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Animais , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Interleucina-1/genética , Interleucina-1/imunologia , Interleucina-9 , Linfopoese/imunologia , Melanoma Experimental/imunologia , Camundongos , NF-kappa B/genética , NF-kappa B/imunologia , Fenótipo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , TranscriptomaRESUMO
OBJECTIVE: Signaling lymphocytic activation molecule family member 1 (SLAMF1) homophilic interactions promote immunoglobulin production and T cell-B cell cross-talk. SLAMF1 is overexpressed on T and B cells in patients with systemic lupus erythematosus (SLE). This study was undertaken to determine the role of SLAMF1 monoclonal antibody (mAb) in modulating T cell-B cell interaction and B cell activation. METHODS: Anti-IgM-prestimulated naive or total B cells from either healthy donors or patients with SLE were cocultured with autologous T cells under CD3/CD28 stimulation, in the presence or absence of the SLAMF1 mAb. Naive B cells were stimulated with anti-IgM and CD40L in the presence of the SLAMF1 antibody. Cytokine production by CD4+ T cells and B cells was examined by flow cytometry and/or quantitative polymerase chain reaction. Plasmablast formation and T cell and B cell conjugates were assessed by flow cytometry. IgG and antinuclear antibody production was determined by enzyme-linked immunosorbent assay. RESULTS: SLAMF1 ligation in a human peripheral blood T cell-B cell culture system reduced the following in both healthy controls and patients with SLE: conjugate formation, interleukin-6 (IL-6) production by B cells, IL-21 and IL-17A production by T cells, and Ig and autoantibody production. Whereas the SLAMF1 mAb directly affected the function of isolated peripheral B cells by decreasing IL-6 and Ig production in vitro, it did not affect cytokine production by isolated T cells stimulated in vitro. CONCLUSION: The SLAMF1 antibody inhibits T cell-B cell interaction and suppresses B cell cytokine production and differentiation, thereby acting as a potential therapeutic tool in the treatment of patients with SLE.
Assuntos
Linfócitos B/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Plasmócitos/imunologia , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Adulto , Linfócitos B/efeitos dos fármacos , Estudos de Casos e Controles , Técnicas de Cocultura , Feminino , Humanos , Interleucina-17/imunologia , Interleucina-6/imunologia , Interleucinas/imunologia , Linfopoese/efeitos dos fármacos , Linfopoese/imunologia , Masculino , Pessoa de Meia-Idade , Plasmócitos/citologia , Plasmócitos/efeitos dos fármacos , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Células Th17/imunologiaRESUMO
Glucocorticoids are produced and released by the adrenal gland and become elevated in response to stress. Although glucocorticoids are well known for their immunosuppressive effects, less is known about their effects on B cells. ABCB1 is an efflux pump expressed in both cancer and normal cells, modulating the gradient of various metabolites, including hydrocortisone. Our goal was to evaluate the effect of this glucocorticoid on murine B cell differentiation and whether sensitivity to hydrocortisone could be related to ABCB1 activity in vivo. C57BL/6 mice received one or three consecutive i.p. injections of hydrocortisone (70, 140 and 200 mg/kg/day). ABCB1 activity was evaluated via the rhodamine-123 transport and inhibited by cyclosporin A in hydrocortisone-treated and control mice. Cells from bone marrow, spleen and blood were counted, incubated with antibodies and analyzed by flow cytometry. A single hydrocortisone injection did not alter the number of bone marrow subsets. Conversely, three daily injections were able to reduce the cell number of most bone marrow subsets, excepting c-kit-sca-1+ and mature B cells. This treatment reduced marginal zone, follicular and transitional B cells, though splenic subsets were more resistant than bone marrow B cells. Recirculating follicular B cells in the blood were resistant to hydrocortisone. With the exception of follicular B cells, all subpopulations exhibited ABCB1 activity. However, hydrocortisone treatment did not affect ABCB1 activity in most subsets analyzed. Results suggest that hydrocortisone is able to regulate B cell lymphopoiesis although ABCB1 activity is not related to the susceptibility to that glucocorticoid in B cell subsets.
Assuntos
Subpopulações de Linfócitos B/efeitos dos fármacos , Glucocorticoides/farmacologia , Hidrocortisona/farmacologia , Linfopoese/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Feminino , Citometria de Fluxo , Imunofenotipagem , Linfopoese/genética , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The liver is an immunological organ with a distinct immune cell profile. Although the composition and function of liver immune cells have been widely investigated, the mechanisms regulating the development and homeostasis of the specialized immune system, especially in humans, remain largely unknown. Herein, we address this question in humanized mice (hu-mice) that were constructed by transplantation of human fetal thymus and CD34+ hematopoietic stem/progenitor cells in immunodeficient mice with or without autologous human hepatocyte engraftment. Although the levels of human immune cell reconstitution in peripheral blood and spleen were comparable between hu-mice with and without human hepatocyte engraftment, the former group showed that human immune cell reconstitution in the liver was significantly improved. Notably, human immune cells, including Kupffer cells, dendritic cells and natural killer cells, were shown to be closely colocalized with human hepatocytes in the liver. Human hepatocytes engrafted in the mouse liver were found to produce IL-3, IL-15, GM-CSF, M-CSF, MCP-1, CXCL-1 and CXCL-10, which are known to be important for immune cell development, differentiation, tissue migration and retention, and have no or poor cross-reaction between humans and mice. Furthermore, human hepatocytes were able to support human immune cell survival and expansion in an in vitro co-culture assay. This study demonstrates an essential role for hepatocytes in the development and maintenance of the liver immune cell profile. The hu-mouse model with human autologous immune cell and hepatocyte reconstitution has potential for use in studies of the pathogenesis of liver immune disorders such as hepatotropic virus infections.
Assuntos
Hepatócitos/metabolismo , Sistema Imunitário/metabolismo , Fígado/imunologia , Animais , Anticorpos/farmacologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/transplante , Humanos , Fígado/citologia , Linfopoese/efeitos dos fármacos , Camundongos SCID , Timo/embriologia , Timo/transplanteRESUMO
The microenvironments of leukemia and cancer are critical for multiple stages of malignancies, and they are an attractive therapeutic target. While skeletal abnormalities are commonly seen in children with acute lymphoblastic leukemia (ALL) prior to initiating osteotoxic therapy, little is known about the alterations to the bone marrow microenvironment during leukemogenesis. Therefore, in this study, we focused on the development of precursor-B cell ALL (pre-B ALL) in an immunocompetent BCR-ABL1+ model. Here we show that hematopoiesis was perturbed, B lymphopoiesis was impaired, collagen production was reduced, and the number of osteoblastic cells was decreased in the bone marrow microenvironment. As previously found in children with ALL, the leukemia-bearing mice exhibited severe bone loss during leukemogenesis. Leukemia cells produced high levels of receptor activator of nuclear factor κB ligand (RANKL), sufficient to cause osteoclast-mediated bone resorption. In vivo administration of zoledronic acid rescued leukemia-induced bone loss, reduced disease burden and prolonged survival in leukemia-bearing mice. Taken together, we provide evidence that targeting leukemia-induced bone loss is a therapeutic strategy for pre-B ALL.
Assuntos
Medula Óssea/efeitos dos fármacos , Reabsorção Óssea/tratamento farmacológico , Osteoclastos/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Microambiente Tumoral/efeitos dos fármacos , Ácido Zoledrônico/uso terapêutico , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Reabsorção Óssea/metabolismo , Linhagem Celular , Células HEK293 , Hematopoese/efeitos dos fármacos , Humanos , Linfopoese/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Ligante RANK/metabolismoRESUMO
Stem memory T cells (Tscm) constitute the earliest developmental stage of memory T cells, displaying stem cell-like properties, such as self-renewal capacity. Their superior immune reconstitution potential has sparked interest in cancer immune therapy, vaccine development, and immune reconstitution, whereas their role in autoimmunity is largely unexplored. Here we show that autoreactive CD8+ Tscm specific for ß-cell antigens GAD65, insulin, and IGRP are present in patients with type 1 diabetes (T1D). In vitro, the generation of autoreactive Tscm from naive precursors required the presence of the homeostatic cytokine interleukin-7 (IL-7). IL-7 promotes glucose uptake via overexpression of GLUT1 and upregulation of the glycolytic enzyme hexokinase 2. Even though metabolism depends on glucose uptake, the subsequent oxidation of pyruvate in the mitochondria was necessary for Tscm generation from naive precursors. In patients with T1D, high expression of GLUT1 was a hallmark of circulating Tscm, and targeting glucose uptake via GLUT1 using the selective inhibitor WZB117 resulted in inhibition of Tscm generation and expansion. Our results suggest that autoreactive Tscm are present in patients with T1D and can be selectively targeted by inhibition of glucose metabolism.
Assuntos
Autoimunidade/imunologia , Linfócitos T CD8-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Células Progenitoras Linfoides/imunologia , Subpopulações de Linfócitos T/imunologia , Adolescente , Adulto , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Criança , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Glucose/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Glucose-6-Fosfatase/imunologia , Glutamato Descarboxilase/imunologia , Hexoquinase/metabolismo , Humanos , Hidroxibenzoatos/farmacologia , Memória Imunológica/imunologia , Técnicas In Vitro , Insulina/imunologia , Interleucina-7/imunologia , Linfopoese/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/metabolismo , Regulação para CimaRESUMO
Apart from the role of sex steroids in reproduction, sex steroids are also important regulators of the immune system. 17ß-estradiol (E2) represses T and B cell development, but augments B cell function, possibly explaining the different nature of immune responses in men and women. Both E2 and selective estrogen receptors modulators (SERM) act via estrogen receptors (ER). Activating functions (AF)-1 and 2 of the ER bind to coregulators and thus influence target gene transcription and subsequent cellular response to ER activation. The importance of ERαAF-1 and AF-2 in the immunomodulatory effects of E2/SERM has previously not been reported. Thus, detailed studies of T and B lymphopoiesis were performed in ovariectomized E2-, lasofoxifene- or raloxifene-treated mice lacking either AF-1 or AF-2 domains of ERα, and their wild-type littermate controls. Immune cell phenotypes were analyzed with flow cytometry. All E2 and SERM-mediated inhibitory effects on thymus cellularity and thymic T cell development were clearly dependent on both ERαAFs. Interestingly, divergent roles of ERαAF-1 and ERαAF-2 in E2 and SERM-mediated modulation of bone marrow B lymphopoiesis were found. In contrast to E2, effects of lasofoxifene on early B cells did not require functional ERαAF-2, while ERαAF-1 was indispensable. Raloxifene reduced early B cells partly independent of both ERαAF-1 and ERαAF-2. Results from this study increase the understanding of the impact of ER modulation on the immune system, which can be useful in the clarification of the molecular actions of SERMs and in the development of new SERM.
Assuntos
Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/genética , Linfopoese/genética , Ativação Transcricional/genética , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/fisiologia , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Feminino , Linfopoese/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Domínios Proteicos/genética , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Ativação Transcricional/efeitos dos fármacosRESUMO
BACKGROUND/OBJECTIVE: 17ß-estradiol (E2) has major effects on the immune system. It induces thymic atrophy, inhibits both T and B lymphopoiesis and stimulates antibody production treatment with E2 has protective effects on the skeleton but is associated with negative side effects in reproductive organs. A tissue-selective estrogen complex (TSEC) comprise of estrogens combined with a selective estrogen receptor modulator (SERM). TSEC therapy displays the bone-protective effects of estrogen, while the negative side effects on reproductive organs are blocked by the SERM. In a recent publication we showed that treatment with the TSEC E2+bazedoxifene (bza) potently inhibits experimental arthritis and associated osteoporosis. In order to elucidate immunological mechanisms involved in those effects, the aim of this study was to investigate how E2+bza treatment affects the healthy immune system. METHODS: Ovariectomized C57BL/6N mice were treated with vehicle, E2, bza or E2+bza. Weights of uterus and thymus were determined and fluorescence-activated cell sorting was used to analyze B cell populations in bone marrow and spleen. Immunoglobulin production from B cells in bone marrow and spleen were determined using ELISPOT. RESULTS: Addition of bza to E2-treatment totally antagonized the E2-mediated proliferative effect on uterus. On the contrary, addition of bza to E2-treatment did not block the E2-induced thymic atrophy or inhibition of B lymphopoiesis, and did not block the E2-induced increase in immunoglobulin secretion from bone marrow B cells. CONCLUSIONS: Addition of bza to E2-treatment blocks E2-induced uteroproliferation but does not alter E2-mediated effects on thymus, B lymphopoiesis or B cell function.
Assuntos
Linfócitos B/efeitos dos fármacos , Estradiol/farmacologia , Estrogênios/farmacologia , Indóis/farmacologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Animais , Linfócitos B/citologia , Células da Medula Óssea/efeitos dos fármacos , Interações Medicamentosas , Feminino , Linfopoese/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Ovariectomia , Baço/citologia , Baço/efeitos dos fármacos , Timo/efeitos dos fármacos , Útero/efeitos dos fármacosRESUMO
B lymphopoiesis arrests precipitously in rabbits such that by 2-4 mo of age, before sexual maturity, little to no B lymphopoiesis occurs in the bone marrow (BM). Previously, we showed that in mice, adipocytes inhibit B lymphopoiesis in vitro by inducing inflammatory myeloid cells, which produce IL-1ß. In this study, we characterized rabbit BM after the arrest of B lymphopoiesis and found a dramatic increase in fat, increased CD11b+ myeloid cells, and upregulated expression of the inflammatory molecules, IL-1ß and S100A9, by the myeloid cells. We added BM fat, CD11b+ myeloid cells, and recombinant S100A9 to B lymphopoiesis cultures and found that they inhibited B lymphopoiesis and enhanced myelopoiesis. Unlike IL-1ß, which inhibits B lymphopoiesis by acting on early lymphoid progenitors, S100A9 inhibits B lymphopoiesis by acting on myeloid cells and promoting the release of inflammatory molecules, including IL-1ß. Many molecules produced by adipocytes activate the NLRP3 inflammasome, and the NLRP3 inhibitor, glibenclamide, restored B lymphopoiesis and minimized induction of myeloid cells induced by adipocyte-conditioned medium in vitro. We suggest that fat provides an inflammatory microenvironment in the BM and promotes/activates myeloid cells to produce inflammatory molecules such as IL-1ß and S100A9, which negatively regulate B lymphopoiesis.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Linfócitos B/fisiologia , Células da Medula Óssea/fisiologia , Calgranulina B/metabolismo , Microambiente Celular , Tecido Adiposo/patologia , Animais , Antígeno CD11b/metabolismo , Células Cultivadas , Glibureto/farmacologia , Interleucina-1beta/metabolismo , Linfopoese/efeitos dos fármacos , Camundongos , Mielopoese/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , CoelhosRESUMO
BACKGROUND: ELF2 (E74-like factor 2) also known as NERF (new Ets-related factor), a member of the Ets family of transcription factors, regulates genes important in B and T cell development, cell cycle progression, and angiogenesis. Conserved ELF2 isoforms, ELF2A, and ELF2B, arising from alternative promoter usage can exert opposing effects on target gene expression. ELF2A activates, whilst ELF2B represses, gene expression, and the balance of expression between these isoforms may be important in maintaining normal cellular function. METHODS: We compared the function of ELF2 isoforms ELF2A and ELF2B with other ELF subfamily proteins ELF1 and ELF4 in primary and cancer cell lines using proliferation, colony-forming, cell cycle, and apoptosis assays. We further examined the role of ELF2 isoforms in haemopoietic development using a Rag1 -/-murine bone marrow reconstitution model. RESULTS: ELF2B overexpression significantly reduced cell proliferation and clonogenic capacity, minimally disrupted cell cycle kinetics, and induced apoptosis. In contrast, ELF2A overexpression only marginally reduced clonogenic capacity with little effect on proliferation, cell cycle progression, or apoptosis. Deletion of the N-terminal 19 amino acids unique to ELF2B abrogated the antiproliferative and proapoptotic functions of ELF2B thereby confirming its crucial role. Mice expressing Elf2a or Elf2b in haemopoietic cells variously displayed perturbations in the pre-B cell stage and multiple stages of T cell development. Mature B cells, T cells, and myeloid cells in steady state were unaffected, suggesting that the main role of ELF2 is restricted to the early development of B and T cells and that compensatory mechanisms exist. No differences in B and T cell development were observed between ELF2 isoforms. CONCLUSIONS: We conclude that ELF2 isoforms are important regulators of cellular proliferation, cell cycle progression, and apoptosis. In respect to this, ELF2B acts in a dominant negative fashion compared to ELF2A and as a putative tumour suppressor gene. Given that these cellular processes are critical during haemopoiesis, we propose that the regulatory interplay between ELF2 isoforms contributes substantially to early B and T cell development.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/farmacologia , Linfopoese/efeitos dos fármacos , Fatores de Transcrição/farmacologia , Animais , Ciclo Celular , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Camundongos , Células Precursoras de Linfócitos B/efeitos dos fármacos , Isoformas de Proteínas , Linfócitos T/efeitos dos fármacosRESUMO
Hematopoietic stem cells (HSCs) have the capacity to self-renew and differentiate into hematopoietic cells and have been utilized to replace diseased bone marrow for patients with cancers and blood disorders. Although remarkable progress has been made in developing new tools to manipulate HSCs for clinic use, there is still no effective method to expand HSCs in vivo for quick repopulation of hematopoietic cells following sublethal irradiation. We have recently described a novel synthetic cytokine that is derived from the fusion of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4; named as GIFT4), and we have now discovered that GIFT4 fusokine promotes long-term hematopoietic regeneration in a B cell-dependent manner. We found that GIFT4 treatment triggered a robust expansion of endogenous bone marrow HSCs and multipotent progenitors in vivo. Delivery of GIFT4 protein together with B cells rescued lethally irradiated mice. Moreover, adoptive transfer of autologous or allogeneic GIFT4-treated B cells (GIFT4-B cells) enhanced long-term hematopoietic recovery in radiated mice and prevented the mice from irradiation-induced death. Our data suggest that GIFT4 as well as GIFT4-B cells could serve as means to augment HSC engraftment in the setting of bone marrow transplantation for patients with hematological malignancy.
Assuntos
Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Interleucina-4 , Linfopoese/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Animais , Linfócitos B/metabolismo , Biomarcadores , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Imunofenotipagem , Interleucina-4/genética , Masculino , Camundongos , Modelos Animais , Fenótipo , Proteínas Recombinantes de Fusão/genéticaRESUMO
The homeostasis of peripheral B cell compartment requires lifelong B lymphopoiesis from hematopoietic stem cells (HSC). As a result, the B cell repertoire is susceptible to disruptions of hematopoiesis. Increasing evidence, primarily from rodent models, shows that the aryl hydrocarbon receptor (AHR) regulates hematopoiesis. To study the effects of persistent AHR activation on human B cell development, a potent AHR agonist and known environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was utilized. An in vitro B cell development model system was established by co-culturing human cord blood-derived HSCs with irradiated human primary bone marrow stromal cells. Using this in vitro model, we found that TCDD significantly suppressed the total number of hematopoietic stem and progenitor cells (HSPC) in a concentration-dependent manner. Cell death analysis demonstrated that the decrease in cell number was not due to cytotoxicity by TCDD. In addition, TCDD markedly decreased CD34 expression on HSPCs. Structure-activity relationship studies using dioxin congeners demonstrated a correlation between the relative AHR binding affinity and the magnitude of decrease in the number of HSPCs and CD34 expression, suggesting that AHR mediates the observed TCDD-elicited changes in HSPCs. Moreover, a significant reduction in lineage committed B cell-derived from HSCs was observed in the presence of TCDD, indicating impairment of human B cell development. Similar effects of TCDD were observed regardless of the use of stromal cells in cultures indicating a direct effect of TCDD on HSCs. Collectively, we demonstrate that AHR activation by TCDD on human HSCs impairs early stages of human B lymphopoiesis.
Assuntos
Linfócitos B/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Células-Tronco Hematopoéticas/efeitos dos fármacos , Linfopoese/efeitos dos fármacos , Dibenzodioxinas Policloradas/toxicidade , Receptores de Hidrocarboneto Arílico/agonistas , Linfócitos B/citologia , Células Cultivadas , Técnicas de Cocultura , Células-Tronco Hematopoéticas/citologia , HumanosRESUMO
Introducción. El caseinato de sodio, una sal de la caseína utilizada como agente proinflamatorio en ratones, es capaz de inducir granulopoyesis en vivo e incrementar la producción de citocinas esenciales en dicho evento.Objetivo. Evaluar si el caseinato de sodio es capaz de inducir un efecto biológico en células de origen linfoide y la producción de citocinas involucradas con este linaje.Materiales y métodos: Se utilizaron ratones hembra BALB/c de 8 a 12 semanas de edad. Los animales se inyectaron cuatro veces, con intervalos de 48 horas, por vía intraperitoneal con 1 ml de caseinato de sodio (10 % de SFB p/v). La población de linfocitos B y la incorporación de bromodesoxiuridina (BrdU) se analizaron mediante citometría de flujo. La detección de la interleucina 7 se evaluó mediante la técnica de ELISA.Resultados. Tras la inyección por vía intraperitoneal, el número de linfocitos B 220+ provenientes del bazo de ratones tratados con caseinato de sodio aumentó comparados con los que solo recibieron el vehículo como tratamiento (89,01±1,03 Vs. 75,66±2,08), así como la incorporación de BrdU en células B220+ (38,59±4,48 Vs. 11,82±1,04). Se evidenció, asimismo, el incremento en la concentración de la interleucina 7 (IL-7) en el suero de los ratones tratados con caseinato de sodio, comparados con los que solo recibieron el vehículo (62,1±17,5 Vs. 26,9±4,4 pg/ml).Conclusión. El caseinato de sodio fue capaz de aumentar el número de linfocitos B en bazo de ratones, así como inducir la producción de IL-7, citocina clave para la linfopoyesis B.
Assuntos
Linfócitos B/efeitos dos fármacos , Caseínas/farmacologia , Linfopoese/efeitos dos fármacos , Animais , Medula Óssea/efeitos dos fármacos , Caseínas/administração & dosagem , Caseínas/toxicidade , Divisão Celular , Feminino , Injeções Intraperitoneais , Interleucina-7/biossíntese , Interleucina-7/sangue , Interleucina-7/genética , Contagem de Linfócitos , Linfocinas/biossíntese , Linfocinas/genética , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Chronic lipopolysaccharide (LPS) exposure to mice reduces the lymphoid compartment and skews the hematopoietic cell compartment toward myeloid-cells, which is considered to be a direct effect of LPS on hematopoietic stem cells. However, the effect of chronic LPS exposure on stromal-cells, which compose the hematopoietic microenvironment, has not been elucidated. Here, we investigated early- and late-phase effects of repeated LPS exposure on stromal-cells. During the early phase, when mice were treated with 5 or 25 µg LPS three times at weekly intervals, the numbers of myeloid-progenitor (colony forming unit-granulocyte macrophage (CFU-GM)) cells and B lymphoid-progenitor (CFU-preB) cells in the bone-marrow (BM) rapidly decreased after each treatment. The number of CFU-GM cells recovered from the initial decrease and then increased to levels higher than pretreatment levels, whereas the number of CFU-preB cells remained lower than pretreatment levels. In the BM, expression of genes for positive-regulators of myelopoiesis including granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), and interleukin (IL)-6 and negative-regulators of B lymphopoiesis including tumor necrosis factor (TNF)-α was up-regulated, whereas expression of positive-regulators of B lymphopoiesis including stromal cell-derived factor (SDF)-1, IL-7, and stem cell factor (SCF) was down-regulated. During the late phase, the number of CFU-preB cells remained lower than pretreatment levels 70 d after the first treatments with 5 and 25 µg LPS, whereas the number of CFU-GM cells returned to pretreatment levels. IL-7 gene expression in the BM remained down-regulated, whereas gene-expression levels of SDF-1 and SCF were restored. Thus, chronic LPS exposure may impair stromal-cell function, resulting in prolonged suppression of B lymphopoiesis, which may appear to be senescence similar to the hematological phenotype.
Assuntos
Lipopolissacarídeos/farmacologia , Linfopoese/efeitos dos fármacos , Mielopoese/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Animais , Células Cultivadas , Citocinas/genética , Expressão Gênica/efeitos dos fármacos , Células Progenitoras de Granulócitos e Macrófagos/citologia , Contagem de Leucócitos , Linfopoese/fisiologia , Masculino , Camundongos Endogâmicos BALB C , Mielopoese/fisiologia , Células Precursoras de Linfócitos B/citologia , Células Estromais/metabolismoRESUMO
During hematopoiesis, the balance between proliferation, differentiation, and apoptosis is tightly regulated in order to maintain homeostasis. Failure in these processes can ultimately lead to uncontrolled proliferation and leukemia. Phosphatase and tensin homolog (PTEN) is one of the molecular pathways involved in this balance. By opposing PI3-kinases, PTEN inhibits proliferation and promotes differentiation and is thus considered a tumor suppressor. Indeed, PTEN is frequently mutated in many cancers, including leukemias. Loss of PTEN often leads to lymphoid cancers. However, little is known about the molecular events that regulate PTEN signaling during lymphopoiesis. In this study, we used zebrafish to address this. We report that N-myc downstream-regulated gene 1b (ndrg1b) rescues lymphoid differentiation after PTEN inhibition. We also show that a previously uncharacterized gene, fam49ab, inhibits T-cell differentiation, a phenotype that can be rescued by ndrg1b We propose that ndrg1b and fam49ab are 2 new modulators of PTEN signaling that control lymphoid differentiation in the zebrafish thymus.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfopoese , PTEN Fosfo-Hidrolase/metabolismo , Linfócitos T/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Hematopoese/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linfopoese/efeitos dos fármacos , Morfolinos/farmacologia , Família Multigênica , Mutação/genética , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Timócitos/efeitos dos fármacos , Timócitos/metabolismo , Timo/metabolismo , Peixe-Zebra/embriologiaRESUMO
Impaired T lymphopoiesis is associated with immunosuppression of the adaptive immune response and plays a role in the morbidity and mortality of patients and animal models of sepsis. Although previous studies examined several intrathymic mechanisms that negatively affect T lymphopoiesis, the extrathymic mechanisms remain poorly understood. Here, we report a dramatic decrease in the percentage of early T lineage progenitors (ETPs) in three models of sepsis in mice (cecal ligation and puncture, lipopolysaccharide continuous injection, and poly I:C continuous injection). However, septic mice did not show a decrease in the number of bone marrow (BM) precursor cells. Instead, the BM progenitors for ETPs expressed reduced mRNA levels of CC chemokine receptor (CCR) 7, CCR9 and P-selectin glycoprotein ligand 1, and exhibited impaired homing capacity in vitro and in vivo. Furthermore, RNA-Seq analysis and real-time PCR showed a marked downregulation of several lymphoid-related genes in hematopoietic stem and progenitor cells. Hematopoietic stem and progenitor cells differentiated into myeloid cells but failed to generate T lymphocytes in vitro and in vivo. Our results indicate that the depletion of ETPs in septic mice might be a consequence of an impaired migration of BM progenitors to the thymus, as well as a defect in lymphoid lineage commitment. Stem Cells 2016;34:2902-2915.
Assuntos
Linfopoese , Sepse/complicações , Timo/patologia , Animais , Atrofia , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Perfilação da Expressão Gênica , Hematopoese Extramedular/efeitos dos fármacos , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Contagem de Linfócitos , Linfopoese/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Mielopoese/efeitos dos fármacos , Poli I-C/farmacologia , Receptores de Quimiocinas/metabolismo , Sepse/genética , Sepse/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/patologia , Timo/efeitos dos fármacos , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismoRESUMO
Thymic stromal lymphopoietin (TSLP) and IL-7 are cytokines that signal via the IL-7 receptor alpha (IL-7Rα) to exert both overlapping and unique functions during early stages of mouse B-cell development. In human B lymphopoiesis, the requirement for IL-7Rα signaling is controversial and the roles of IL-7 and TSLP are less clear. Here, we evaluated human B-cell production using novel in vitro and xenograft models of human B-cell development that provide selective IL-7 and human TSLP (hTSLP) stimulation. We show that in vitro human B-cell production is almost completely blocked in the absence of IL-7Rα stimulation, and that either TSLP or IL-7 can provide a signal critical for the production and proliferation of human CD19(+) PAX5(+) pro-B cells. Analysis of primary human bone marrow stromal cells shows that they express both IL-7 and TSLP, providing an in vivo source of these cytokines. We further show that the in vivo production of human pro-B cells under the influence of mouse IL-7 in a xenograft scenario is reduced by anti-IL-7 neutralizing antibodies, and that this loss can be restored by hTSLP at physiological levels. These data establish the importance of IL-7Rα mediated signals for normal human B-cell production.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Citocinas/metabolismo , Interleucina-7/metabolismo , Linfopoese , Receptores de Interleucina-7/metabolismo , Transdução de Sinais , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Células Cultivadas , Citocinas/farmacologia , Expressão Gênica , Humanos , Interleucina-7/farmacologia , Linfopoese/efeitos dos fármacos , Linfopoese/imunologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Transgênicos , Células Precursoras de Linfócitos B/citologia , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfopoietina do Estroma do TimoRESUMO
A prolonged period of T-cell recovery is the major challenge in hematopoietic stem cell transplantation (HSCT). Thymic epithelial cells (TECs) are the major component of the thymic microenvironment for T-cell generation. However, TECs undergo degeneration over time. FOXN1 plays a critical role in TEC development and is required to maintain adult TECs for thymopoiesis. To investigate the potential application of FOXN1, we have cloned and expressed recombinant FOXN1 protein (rFOXN1) that was fused with cell-penetrating peptides. We show here that the rFOXN1 protein can translocate from the cell surface into the cytoplasm and nucleus. Administration of rFOXN1 into both congenic and allogeneic HSCT recipient mice increased the number of TECs, resulting in enhanced thymopoiesis that led to an increased number of functional T cells in the periphery. The increased number of TECs is due to the enhanced survival and proliferation of TECs. Our results suggest that rFOXN1 has the potential to be used in enhancing T-cell regeneration in patients following HSCT.