RESUMO
Hematopoietic stem cells (HSCs) are the most primitive cells in the hematopoietic system and are under tight regulation for self-renewal and differentiation. Notch signals are essential for the emergence of definitive hematopoiesis in mouse embryos and are critical regulators of lymphoid lineage fate determination. However, it remains unclear how Notch regulates the balance between HSC self-renewal and differentiation in the adult bone marrow (BM). Here we report a novel mechanism that prevents HSCs from undergoing premature lymphoid differentiation in BM. Using a series of in vivo mouse models and functional HSC assays, we show that leukemia/lymphoma related factor (LRF) is necessary for HSC maintenance by functioning as an erythroid-specific repressor of Delta-like 4 (Dll4) expression. Lrf deletion in erythroblasts promoted up-regulation of Dll4 in erythroblasts, sensitizing HSCs to T-cell instructive signals in the BM. Our study reveals novel cross-talk between HSCs and erythroblasts, and sheds a new light on the regulatory mechanisms regulating the balance between HSC self-renewal and differentiation.
Assuntos
Proteínas de Ligação a DNA/genética , Eritroblastos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Proteínas de Ligação ao Cálcio , Diferenciação Celular/genética , Proliferação de Células , Microambiente Celular/genética , Proteínas de Ligação a DNA/metabolismo , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Receptor Notch1/genética , Receptor Notch1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Linfócitos T/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transcriptoma/genéticaRESUMO
The thymus is the site of T cell development. Several stromal and hematopoietic cell types are necessary for the proper function of thymic selection and eventually peripheral immunity. Thymic epithelial cells (TECs) are essential for T cell lineage commitment, expansion, and maturation in the thymus. We were interested in developing an in vivo model in which exogenous gene expression could be transiently induced in embryonic TEC (Tet-On system). To this end, we have generated a bacterial artificial chromosome (BAC) transgenic mouse line in which the reverse tetracycline-dependent transactivator (rtTA) is expressed under the control of the Foxn1 promoter, a transcriptional factor indispensable for TEC development. To analyze the expression pattern and efficiency of this novel mouse model, we crossed the Foxn1-rtTA founder with a Tet-Responsive Element (TRE)-LacZ GFP mouse reporter to obtain a double transgenic mouse. In the presence of doxycycline, rtTA can interact with TRE and induce the expression of GFP and LacZ. In this double transgenic mouse, we observed that GFP expression was high, inducible and limited to TEC in fetal thymus. In contrast, in adult thymus, when TEC development and maturation is completed, GFP was barely detectable. Therefore, Foxn1-rtTA represents a new and efficient transgenic mouse model to induce genes of interest specifically in fetal thymic epithelium.
Assuntos
Células Epiteliais/metabolismo , Fatores de Transcrição Forkhead/genética , Expressão Gênica , Camundongos Transgênicos , Modelos Animais , Timo/embriologia , Animais , Células Cultivadas , Cromossomos Artificiais Bacterianos , Epitélio/embriologia , Epitélio/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Regiões Promotoras Genéticas , Timo/metabolismoRESUMO
The adult mammalian forebrain contains neural stem/progenitor cells (NSCs) that generate neurons throughout life. As in other somatic stem cell systems, NSCs are proposed to be predominantly quiescent and proliferate only sporadically to produce more committed progeny. However, quiescence has recently been shown not to be an essential criterion for stem cells. It is not known whether NSCs show differences in molecular dependence based on their proliferation state. The subventricular zone (SVZ) of the adult mouse brain has a remarkable capacity for repair by activation of NSCs. The molecular interplay controlling adult NSCs during neurogenesis or regeneration is not clear but resolving these interactions is critical in order to understand brain homeostasis and repair. Using conditional genetics and fate mapping, we show that Notch signaling is essential for neurogenesis in the SVZ. By mosaic analysis, we uncovered a surprising difference in Notch dependence between active neurogenic and regenerative NSCs. While both active and regenerative NSCs depend upon canonical Notch signaling, Notch1-deletion results in a selective loss of active NSCs (aNSCs). In sharp contrast, quiescent NSCs (qNSCs) remain after Notch1 ablation until induced during regeneration or aging, whereupon they become Notch1-dependent and fail to fully reinstate neurogenesis. Our results suggest that Notch1 is a key component of the adult SVZ niche, promoting maintenance of aNSCs, and that this function is compensated in qNSCs. Therefore, we confirm the importance of Notch signaling for maintaining NSCs and neurogenesis in the adult SVZ and reveal that NSCs display a selective reliance on Notch1 that may be dictated by mitotic state.
Assuntos
Células-Tronco Adultas/fisiologia , Ventrículos Laterais/citologia , Neurogênese/fisiologia , Receptor Notch1/metabolismo , Nicho de Células-Tronco/fisiologia , Células-Tronco Adultas/efeitos dos fármacos , Animais , Proteínas de Transporte/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Diferenciação Celular/genética , Citarabina/farmacologia , Antagonistas de Estrogênios/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Imunossupressores/farmacologia , Proteínas de Filamentos Intermediários/genética , Ventrículos Laterais/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nestina , Neurogênese/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas/genética , Proteínas/metabolismo , RNA não Traduzido , Receptor Notch1/deficiência , Receptores de Estrogênio/genética , Nicho de Células-Tronco/genética , Tamoxifeno/farmacologia , Fatores de TempoRESUMO
Notch1 (N1) receptor signaling is essential and sufficient for T cell development, and recently developed in vitro culture systems point to members of the Delta family as being the physiological N1 ligands. We explored the ability of Delta1 (DL1) and DL4 to induce T cell lineage commitment and/or maturation in vitro and in vivo from bone marrow (BM) precursors conditionally gene targeted for N1 and/or N2. In vitro DL1 can trigger T cell lineage commitment via either N1 or N2. N1- or N2-mediated T cell lineage commitment can also occur in the spleen after short-term BM transplantation. However, N2-DL1-mediated signaling does not allow further T cell maturation beyond the CD25(+) stage due to a lack of T cell receptor beta expression. In contrast to DL1, DL4 induces and supports T cell commitment and maturation in vitro and in vivo exclusively via specific interaction with N1. Moreover, comparative binding studies show preferential interaction of DL4 with N1, whereas binding of DL1 to N1 is weak. Interestingly, preferential N1-DL4 binding reflects reduced dependence of this interaction on Lunatic fringe, a glycosyl transferase that generally enhances the avidity of Notch receptors for Delta ligands. Collectively, our results establish a hierarchy of Notch-Delta interactions in which N1-DL4 exhibits the greatest capacity to induce and support T cell development.
Assuntos
Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Células-Tronco Hematopoéticas/citologia , Proteínas de Membrana/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/citologia , Animais , Primers do DNA , Citometria de Fluxo , Glicosiltransferases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Transgênicos , Ligação Proteica , Receptor Notch2/metabolismo , Retroviridae , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Estromais , TransfecçãoRESUMO
Introduction: People with DS are highly predisposed to Alzheimer's disease (AD) and demonstrate very similar clinical and pathological features. Ts65Dn mice are widely used and serve as the best-characterized animal model of DS. Methods: We undertook studies to characterize age-related changes for AD-relevant markers linked to Aß, Tau, and phospho-Tau, axonal structure, inflammation, and behavior. Results: We found age related changes in both Ts65Dn and 2N mice. Relative to 2N mice, Ts65Dn mice showed consistent increases in Aß40, insoluble phospho-Tau, and neurofilament light protein. These changes were correlated with deficits in learning and memory. Discussion: These data have implications for planning future experiments aimed at preventing disease-related phenotypes and biomarkers. Interventions should be planned to address specific manifestations using treatments and treatment durations adequate to engage targets to prevent the emergence of phenotypes.
RESUMO
Amyloid-ß (Aß) deposition is an initiating factor in Alzheimer's disease (AD). Microglia are the brain immune cells that surround and phagocytose Aß plaques, but their phagocytic capacity declines in AD. This is in agreement with studies that associate AD risk loci with genes regulating the phagocytic function of immune cells. Immunotherapies are currently pursued as strategies against AD and there are increased efforts to understand the role of the immune system in ameliorating AD pathology. Here, we evaluated the effect of the Aß targeting ACI-24 vaccine in reducing AD pathology in an amyloidosis mouse model. ACI-24 vaccination elicited a robust and sustained antibody response in APPPS1 mice with an accompanying reduction of Aß plaque load, Aß plaque-associated ApoE and dystrophic neurites as compared to non-vaccinated controls. Furthermore, an increased number of NLRP3-positive plaque-associated microglia was observed following ACI-24 vaccination. In contrast to this local microglial activation at Aß plaques, we observed a more ramified morphology of Aß plaque-distant microglia compared to non-vaccinated controls. Accordingly, bulk transcriptomic analysis revealed a trend towards the reduced expression of several disease-associated microglia (DAM) signatures that is in line with the reduced Aß plaque load triggered by ACI-24 vaccination. Our study demonstrates that administration of the Aß targeting vaccine ACI-24 reduces AD pathology, suggesting its use as a safe and cost-effective AD therapeutic intervention.
Assuntos
Doença de Alzheimer , Amiloidose , Camundongos , Animais , Microglia/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Camundongos Transgênicos , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/terapia , Doença de Alzheimer/metabolismo , Amiloidose/metabolismo , Placa Amiloide/metabolismo , Fenótipo , VacinaçãoRESUMO
It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4(-)CD8(-) thymocytes. Notch1 is progressively down-regulated at the CD4(+)CD8(+) and mature CD4(+) or CD8(+) thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1(+)CD25(-) cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117(+) early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.
Assuntos
Anticorpos Monoclonais/imunologia , Ativação Linfocitária/imunologia , Receptor Notch1/biossíntese , Receptor Notch2/biossíntese , Animais , Especificidade de Anticorpos , Diferenciação Celular , Membrana Celular/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Imunofluorescência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Receptor Notch1/imunologia , Receptor Notch2/imunologia , Transdução de Sinais/imunologia , Células-Tronco/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
Interactions between Notch1 receptors on lymphoid progenitors and Delta-like 4 (DL4) ligands on cortical thymic epithelial cells (cTEC) are essential for T cell lineage commitment, expansion, and maturation in the thymus. Using a novel mAb against DL4, we show that DL4 levels on cTEC are very high in the fetal and neonatal thymus when thymocyte expansion is maximal but decrease dramatically in the adult when steady-state homeostasis is attained. Analysis of mutant mouse strains where thymocyte development is blocked at different stages indicates that lymphostromal interactions ("thymus crosstalk") are required for DL4 down-regulation on cTEC. Reconstitution of thymocyte development in these mutant mice further suggests that maturation of thymocytes to the CD4(+)CD8(+) stage and concomitant expansion are needed to promote DL4 down-regulation on cTEC. Collectively, our data support a model where thymic crosstalk quantitatively regulates the rate of Notch1-dependent thymopoiesis by controlling DL4 expression levels on cTEC.
Assuntos
Comunicação Celular/imunologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Proteínas de Membrana/biossíntese , Timo/citologia , Timo/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Animais Recém-Nascidos , Proteínas de Ligação ao Cálcio , Comunicação Celular/genética , Regulação para Baixo/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linfopoese/genética , Linfopoese/imunologia , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Imunológicos , Ratos , Receptor Notch1/fisiologia , Células Estromais/citologia , Células Estromais/imunologia , Células Estromais/metabolismo , Timo/embriologia , Timo/metabolismoRESUMO
OBJECTIVE: Interleukin-17 (IL-17), a potent proinflammatory cytokine, has been implicated in allograft rejection. We analyzed the efficacy of an adenoviral vector expressing an IL-17 inhibitor in delaying acute allograft rejection in a rat model of heart transplantation, and the biological mechanisms underlying the protective effect. METHODS: We constructed an adenoviral vector expressing a soluble IL-17 receptor-immunoglobulin (IL-17R-Ig) fusion protein. IL-17R-Ig activity was assessed by inhibition of IL-17-induced IL-6 release in HeLa cells preincubated with the vector. Intracoronary vector administration was performed in F344 donor hearts that were placed as vascularized grafts into Lewis hosts. Inflammatory cells infiltrating the graft were analyzed by immunohistology. Cytokine transcripts in the graft were determined by real-time RT-PCR. RESULTS: IL-17R-Ig gene transfer resulted in prolonged allograft survival (16.1+/-3.1 days vs 10.3+/-2.5 days with control virus and 10.1+/-2.1 days with virus dilution buffer alone; p<0.001). IL-17R-Ig gene transfer reduced inflammatory cell infiltrates, especially monocytes/macrophages and CD4+ T cells (p<0.05). It also reduced intragraft cytokine transcripts for interferon-gamma and transforming growth factor-beta (p<0.05) and, to a lesser extent, IL-1beta and tumor necrosis factor-alpha (p=0.083). CONCLUSIONS: Local expression of soluble IL-17 receptor-immunoglobulin attenuates T helper type 1 (Th1) cytokine responses and leukocyte infiltration in rat cardiac allografts, thereby mediating prolonged graft survival. Intragraft IL-17 inhibition may be useful as an adjuvant therapy to systemic immunosuppression in heart transplantation.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Rejeição de Enxerto/prevenção & controle , Transplante de Coração , Receptores de Interleucina/fisiologia , Adenoviridae/genética , Animais , Linfócitos T CD4-Positivos/imunologia , Citocinas/biossíntese , Citocinas/genética , Vetores Genéticos , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew , Receptores de Interleucina/genética , Receptores de Interleucina-17 , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SolubilidadeRESUMO
Thymic T cell lineage commitment is dependent on Notch1 (N1) receptor-mediated signaling. Although the physiological ligands that interact with N1 expressed on thymic precursors are currently unknown, in vitro culture systems point to Delta-like 1 (DL1) and DL4 as prime candidates. Using DL1- and DL4-lacZ reporter knock-in mice and novel monoclonal antibodies to DL1 and DL4, we show that DL4 is expressed on thymic epithelial cells (TECs), whereas DL1 is not detected. The function of DL4 was further explored in vivo by generating mice in which DL4 could be specifically inactivated in TECs or in hematopoietic progenitors. Although loss of DL4 in hematopoietic progenitors did not perturb thymus development, inactivation of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus. These immature B cells were phenotypically indistinguishable from those developing in the thymus of conditional N1 mutant mice. Collectively, our results demonstrate that DL4 is the essential and nonredundant N1 ligand responsible for T cell lineage commitment. Moreover, they strongly suggest that N1-expressing thymic progenitors interact with DL4-expressing TECs to suppress B lineage potential and to induce the first steps of intrathymic T cell development.
Assuntos
Diferenciação Celular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptor Notch1/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/citologia , Timo/citologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Citometria de Fluxo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Timo/imunologia , Timo/metabolismo , beta-GalactosidaseRESUMO
Although it is well established that early expression of TCRbeta transgenes in the thymus leads to efficient inhibition of both endogenous TCRbeta and TCRgamma rearrangement (also known as allelic and "isotypic" exclusion, respectively) the role of pTalpha in these processes remains controversial. Here, we have systematically re-evaluated this issue using three independent strains of TCRbeta-transgenic mice that differ widely in transgene expression levels, and a sensitive intracellular staining assay that detects endogenous TCRVbeta expression in individual immature thymocytes. In the absence of pTalpha, both allelic and isotypic exclusion were reversed in all three TCRbeta-transgenic strains, clearly demonstrating a general requirement for pre-TCR signaling in the inhibition of endogenous TCRbeta and TCRgamma rearrangement. Both allelic and isotypic exclusion were pTalpha dose dependent when transgenic TCRbeta levels were subphysiological. Moreover, pTalpha-dependent allelic and isotypic exclusion occurred in both alphabeta and gammadelta T cell lineages, indicating that pre-TCR signaling can potentially be functional in gammadelta precursors. Finally, levels of endogenous RAG1 and RAG2 were not down-regulated in TCRbeta-transgenic immature thymocytes undergoing allelic or isotypic exclusion. Collectively, our data reveal a critical but lineage-nonspecific role for pTalpha in mediating both allelic and isotypic exclusion in TCRbeta-transgenic mice.
Assuntos
Linhagem da Célula/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Glicoproteínas de Membrana/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Alelos , Animais , Western Blotting , Citometria de Fluxo , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Phosphoinositide-dependent kinase l (PDK1) phosphorylates and activates multiple AGC serine kinases, including protein kinase B (PKB), p70Ribosomal S6 kinase (S6K) and p90Ribosomal S6 kinase (RSK). PDK1 is required for thymocyte differentiation and proliferation, and herein, we explore the molecular basis for these essential functions of PDK1 in T lymphocyte development. A key finding is that PDK1 is required for the expression of key nutrient receptors in T cell progenitors: CD71 the transferrin receptor and CD98 a subunit of L-amino acid transporters. PDK1 is also essential for Notch-mediated trophic and proliferative responses in thymocytes. A PDK1 mutant PDK1 L155E, which supports activation of PKB but no other AGC kinases, can restore CD71 and CD98 expression in pre-T cells and restore thymocyte differentiation. However, PDK1 L155E is insufficient for thymocyte proliferation. The role of PDK1 in thymus development thus extends beyond its ability to regulate PKB. In addition, PDK1 phosphorylation of AGC kinases such as S6K and RSK is also necessary for thymocyte development.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Receptores Notch/metabolismo , Linfócitos T/citologia , Linfócitos T/enzimologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Alimentos , Camundongos , Proteínas Mutantes/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Serina-Treonina Quinases TORRESUMO
Negative selection eliminates thymocytes bearing autoreactive T cell receptors (TCR) via an apoptotic mechanism. We have cloned an inhibitor of NF-kappa B, I kappa BNS, which is rapidly expressed upon TCR-triggered but not dexamethasone- or gamma irradiation-stimulated thymocyte death. The predicted protein contains seven ankyrin repeats and is homologous to I kappa B family members. In class I and class II MHC-restricted TCR transgenic mice, transcription of I kappa BNS is stimulated by peptides that trigger negative selection but not by those inducing positive selection (i.e., survival) or nonselecting peptides. I kappa BNS blocks transcription from NF-kappa B reporters, alters NF-kappa B electrophoretic mobility shifts, and interacts with NF-kappa B proteins in thymic nuclear lysates following TCR stimulation. Retroviral transduction of I kappa BNS in fetal thymic organ culture enhances TCR-triggered cell death consistent with its function in selection.