RESUMO
The synovium is a mesenchymal tissue composed mainly of fibroblasts, with a lining and sublining that surround the joints. In rheumatoid arthritis the synovial tissue undergoes marked hyperplasia, becomes inflamed and invasive, and destroys the joint1,2. It has recently been shown that a subset of fibroblasts in the sublining undergoes a major expansion in rheumatoid arthritis that is linked to disease activity3-5; however, the molecular mechanism by which these fibroblasts differentiate and expand is unknown. Here we identify a critical role for NOTCH3 signalling in the differentiation of perivascular and sublining fibroblasts that express CD90 (encoded by THY1). Using single-cell RNA sequencing and synovial tissue organoids, we found that NOTCH3 signalling drives both transcriptional and spatial gradients-emanating from vascular endothelial cells outwards-in fibroblasts. In active rheumatoid arthritis, NOTCH3 and Notch target genes are markedly upregulated in synovial fibroblasts. In mice, the genetic deletion of Notch3 or the blockade of NOTCH3 signalling attenuates inflammation and prevents joint damage in inflammatory arthritis. Our results indicate that synovial fibroblasts exhibit a positional identity that is regulated by endothelium-derived Notch signalling, and that this stromal crosstalk pathway underlies inflammation and pathology in inflammatory arthritis.
Assuntos
Artrite Reumatoide/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Receptor Notch3/metabolismo , Transdução de Sinais , Membrana Sinovial/patologia , Animais , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Células Endoteliais/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos , Receptor Notch3/antagonistas & inibidores , Receptor Notch3/deficiência , Receptor Notch3/genética , Antígenos Thy-1/metabolismoRESUMO
Glandular epithelia, including the mammary and prostate glands, are composed of basal cells (BCs) and luminal cells (LCs)1,2. Many glandular epithelia develop from multipotent basal stem cells (BSCs) that are replaced in adult life by distinct pools of unipotent stem cells1,3-8. However, adult unipotent BSCs can reactivate multipotency under regenerative conditions and upon oncogene expression3,9-13. This suggests that an active mechanism restricts BSC multipotency under normal physiological conditions, although the nature of this mechanism is unknown. Here we show that the ablation of LCs reactivates the multipotency of BSCs from multiple epithelia both in vivo in mice and in vitro in organoids. Bulk and single-cell RNA sequencing revealed that, after LC ablation, BSCs activate a hybrid basal and luminal cell differentiation program before giving rise to LCs-reminiscent of the genetic program that regulates multipotency during embryonic development7. By predicting ligand-receptor pairs from single-cell data14, we find that TNF-which is secreted by LCs-restricts BC multipotency under normal physiological conditions. By contrast, the Notch, Wnt and EGFR pathways were activated in BSCs and their progeny after LC ablation; blocking these pathways, or stimulating the TNF pathway, inhibited regeneration-induced BC multipotency. Our study demonstrates that heterotypic communication between LCs and BCs is essential to maintain lineage fidelity in glandular epithelial stem cells.
Assuntos
Comunicação Celular , Células Epiteliais/citologia , Células-Tronco Multipotentes/citologia , Animais , Linhagem da Célula , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Feminino , Homeostase , Humanos , Masculino , Glândulas Mamárias Animais/citologia , Camundongos , Células-Tronco Multipotentes/metabolismo , Organoides/citologia , Próstata/citologia , RNA Mensageiro/genética , RNA-Seq , Receptores Notch/metabolismo , Glândulas Salivares/citologia , Análise de Célula Única , Pele/citologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Wnt/metabolismoRESUMO
ZFP36L1 and ZFP36L2 are RNA-binding proteins (RBPs) that interact with AU-rich elements in the 3' untranslated region of mRNA, which leads to mRNA degradation and translational repression. Here we show that mice that lacked ZFP36L1 and ZFP36L2 during thymopoiesis developed a T cell acute lymphoblastic leukemia (T-ALL) dependent on the oncogenic transcription factor Notch1. Before the onset of T-ALL, thymic development was perturbed, with accumulation of cells that had passed through the beta-selection checkpoint without first expressing the T cell antigen receptor beta-chain (TCRbeta). Notch1 expression was higher in untransformed thymocytes in the absence of ZFP36L1 and ZFP36L2. Both RBPs interacted with evolutionarily conserved AU-rich elements in the 3' untranslated region of Notch1 and suppressed its expression. Our data establish a role for ZFP36L1 and ZFP36L2 during thymocyte development and in the prevention of malignant transformation.
Assuntos
Proteínas Nucleares/deficiência , Leucemia-Linfoma Linfoblástico de Células T Precursoras/imunologia , Linfócitos T/imunologia , Timo/imunologia , Tristetraprolina/deficiência , Sequência de Aminoácidos , Animais , Fator 1 de Resposta a Butirato , Sequência Conservada , Humanos , Imunofenotipagem , Estimativa de Kaplan-Meier , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Receptor Notch1/genética , Receptor Notch1/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Alinhamento de Sequência , Timo/crescimento & desenvolvimento , Transcrição Gênica , Tristetraprolina/genética , Tristetraprolina/imunologiaRESUMO
Notch signaling is emerging as a critical regulator of T cell activation and function. However, there is no reliable cell surface indicator of Notch signaling across activated T cell subsets. In this study, we show that Notch signals induce upregulated expression of the Gcnt1 glycosyltransferase gene in T cells mediating graft-versus-host disease after allogeneic bone marrow transplantation in mice. To determine if Gcnt1-mediated O-glycosylation could be used as a Notch signaling reporter, we quantified the core-2 O-glycoform of CD43 in multiple T cell subsets during graft-versus-host disease. Pharmacological blockade of Delta-like Notch ligands abrogated core-2 O-glycosylation in a dose-dependent manner after allogeneic bone marrow transplantation, both in donor-derived CD4+ and CD8+ effector T cells and in Foxp3+ regulatory T cells. CD43 core-2 O-glycosylation depended on cell-intrinsic canonical Notch signals and identified CD4+ and CD8+ T cells with high cytokine-producing ability. Gcnt1-deficient T cells still drove lethal alloreactivity, showing that core-2 O-glycosylation predicted, but did not cause, Notch-dependent T cell pathogenicity. Using core-2 O-glycosylation as a marker of Notch signaling, we identified Ccl19-Cre+ fibroblastic stromal cells as critical sources of Delta-like ligands in graft-versus-host responses irrespective of conditioning intensity. Core-2 O-glycosylation also reported Notch signaling in CD8+ T cell responses to dendritic cell immunization, Listeria infection, and viral infection. Thus, we uncovered a role for Notch in controlling core-2 O-glycosylation and identified a cell surface marker to quantify Notch signals in multiple immunological contexts. Our findings will help refine our understanding of the regulation, cellular source, and timing of Notch signals in T cell immunity.
Assuntos
Transplante de Medula Óssea/efeitos adversos , Linfócitos T CD8-Positivos/metabolismo , Doença Enxerto-Hospedeiro/imunologia , N-Acetilglucosaminiltransferases/metabolismo , Receptores Notch/metabolismo , Animais , Biomarcadores/metabolismo , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Estudos de Viabilidade , Feminino , Citometria de Fluxo/métodos , Glicosilação/efeitos dos fármacos , Humanos , Leucossialina/metabolismo , Ligantes , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Sensibilidade e Especificidade , Sialomucinas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Células Estromais/imunologia , Células Estromais/metabolismo , Transplante Homólogo/efeitos adversos , Regulação para CimaRESUMO
Human NK cells develop in tonsils through discrete NK cell developmental intermediates (NKDIs), yet the mechanistic regulation of this process is unclear. We demonstrate that Notch activation in human tonsil-derived stage 3 (CD34-CD117+CD94-NKp80-) and 4A (CD34-CD117+/-CD94+NKp80-) NKDIs promoted non-NK innate lymphoid cell differentiation at the expense of NK cell differentiation. In contrast, stage 4B (CD34-CD117+/-CD94+NKp80+) NKDIs were NK cell lineage committed despite Notch activation. Interestingly, whereas NK cell functional maturation from stage 3 and 4A NKDIs was independent of Notch activation, the latter was required for high NKp80 expression and a stage 4B-like phenotype by the NKDI-derived NK cells. The Notch-dependent effects required simultaneous engagement with OP9 stromal cells and were also stage-specific, with NOTCH1 and NOTCH2 receptors regulating stage 3 NKDIs and NOTCH1 primarily regulating stage 4A NKDIs. These data establish stage-specific and stromal-dependent roles for Notch in regulating human NK cell developmental plasticity and maturation.
Assuntos
Diferenciação Celular/imunologia , Células Matadoras Naturais/fisiologia , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Plasticidade Celular/imunologia , Células Cultivadas , Humanos , Imunidade Inata , Lectinas Tipo C/metabolismo , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Cultura Primária de Células , Receptores de Células Matadoras Naturais/metabolismo , Transdução de Sinais/imunologiaRESUMO
Graft-versus-host disease (GVHD) is the most serious complication of allogeneic hematopoietic cell transplantation. Notch signals delivered during the first 48 h after transplantation drive proinflammatory cytokine production in conventional T cells (Tconv) and inhibit the expansion of regulatory T cells (Tregs). Short-term Notch inhibition induces long-term GVHD protection. However, it remains unknown whether Notch blockade blunts GVHD through its effects on Tconv, Tregs, or both and what early Notch-regulated molecular events occur in alloantigen-specific T cells. To address these questions, we engineered T cell grafts to achieve selective Notch blockade in Tconv versus Tregs and evaluated their capacity to trigger GVHD in mice. Notch blockade in Tconv was essential for GVHD protection as GVHD severity was similar in the recipients of wild-type Tconv combined with Notch-deprived versus wild-type Tregs. To identify the impact of Notch signaling on the earliest steps of T cell activation in vivo, we established a new acute GVHD model mediated by clonal alloantigen-specific 4C CD4+ Tconv. Notch-deprived 4C T cells had preserved early steps of activation, IL-2 production, proliferation, and Th cell polarization. In contrast, Notch inhibition dampened IFN-γ and IL-17 production, diminished mTORC1 and ERK1/2 activation, and impaired transcription of a subset of Myc-regulated genes. The distinct Notch-regulated signature had minimal overlap with known Notch targets in T cell leukemia and developing T cells, highlighting the specific impact of Notch signaling in mature T cells. Our findings uncover a unique molecular program associated with the pathogenic effects of Notch in T cells at the earliest stages of GVHD.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Doença Enxerto-Hospedeiro/imunologia , Isoantígenos/imunologia , Receptores Notch/imunologia , Animais , Transplante de Medula Óssea/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Transplante Homólogo/efeitos adversosRESUMO
Prevailing dogma holds that cell-cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.
Assuntos
Anticorpos/uso terapêutico , Transdiferenciação Celular , Pulmão/citologia , Pulmão/metabolismo , Receptores Notch/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Asma/tratamento farmacológico , Asma/metabolismo , Asma/patologia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Rastreamento de Células , Transdiferenciação Celular/efeitos dos fármacos , Cílios/metabolismo , Modelos Animais de Doenças , Feminino , Células Caliciformes/citologia , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/patologia , Homeostase/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteína Jagged-2 , Ligantes , Pulmão/efeitos dos fármacos , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Serrate-Jagged , Transdução de Sinais/efeitos dos fármacosRESUMO
Lateral meningocele syndrome (LMS), a genetic disorder characterized by meningoceles and skeletal abnormalities, is associated with NOTCH3 mutations. We created a mouse model of LMS (Notch3tm1.1Ecan ) by introducing a tandem termination codon in the Notch3 locus upstream of the proline (P), glutamic acid (E), serine (S) and threonine (T) domain. Microcomputed tomography demonstrated that Notch3tm1.1Ecan mice exhibit osteopenia. The cancellous bone osteopenia was no longer observed after the intraperitoneal administration of antibodies directed to the negative regulatory region (NRR) of Notch3. The anti-Notch3 NRR antibody suppressed the expression of Hes1, Hey1, and Hey2 (Notch target genes), and decreased Tnfsf11 (receptor activator of NF Kappa B ligand) messenger RNA in Notch3tm1.1Ecan osteoblast (OB) cultures. Bone marrow-derived macrophages (BMMs) from Notch3tm1.1Ecan mutants exhibited enhanced osteoclastogenesis in culture, and this was increased in cocultures with Notch3tm1.1Ecan OB. Osteoclastogenesis was suppressed by anti-Notch3 NRR antibodies in Notch3tm1.1Ecan OB/BMM cocultures. In conclusion, the cancellous bone osteopenia of Notch3tm1.1Ecan mutants is reversed by anti-Notch3 NRR antibodies.
Assuntos
Anormalidades Múltiplas/genética , Anormalidades Múltiplas/terapia , Anticorpos/uso terapêutico , Meningocele/genética , Meningocele/terapia , Receptor Notch3/imunologia , Animais , Osso e Ossos/anormalidades , Feminino , Predisposição Genética para Doença , Macrófagos/fisiologia , Masculino , Camundongos , Mutação , Osteoblastos/fisiologia , Microtomografia por Raio-XRESUMO
BACKGROUND: Transplantation is the treatment of choice for many patients with end-stage organ disease. Despite advances in immunosuppression, long-term outcomes remain suboptimal, hampered by drug toxicity and immune-mediated injury, the leading cause of late graft loss. The development of therapies that promote regulation while suppressing effector immunity is imperative to improve graft survival and minimize conventional immunosuppression. Notch signaling is a highly conserved pathway pivotal to T-cell differentiation and function, rendering it a target of interest in efforts to manipulate T cell-mediated immunity. METHODS: We investigated the pattern of Notch-1 expression in effector and regulatory T cells (Tregs) in both murine and human recipients of a solid-organ transplant. Using a selective human anti-Notch-1 antibody (aNotch-1), we examined the effect of Notch-1 receptor inhibition in full major histocompatibility complex-mismatch murine cardiac and lung transplant models, and in a humanized skin transplant model. On the basis of our findings, we further used a genetic approach to investigate the effect of selective Notch-1 inhibition in Tregs. RESULTS: We observed an increased proportion of Tregs expressing surface and intracellular (activated) Notch-1 in comparison with conventional T cells, both in mice with transplants and in the peripheral blood of patients with transplants. In the murine cardiac transplant model, peritransplant administration of aNotch-1 (days 0, 2, 4, 6, 8, and 10) significantly prolonged allograft survival in comparison with immunoglobulin G-treated controls. Similarly, aNotch-1 treatment improved both histological and functional outcomes in the murine lung transplant model. The use of aNotch-1 resulted in a reduced proportion of both splenic and intragraft conventional T cells, while increasing the proportion of Tregs. Furthermore, Tregs isolated from aNotch-1-treated mice showed enhanced suppressive function on a per-cell basis, confirmed with selective Notch-1 deletion in Tregs (Foxp3EGFPCreNotch1fl/fl). Notch-1 blockade inhibited the mammalian target of rapamycin pathway and increased the phosphorylation of STAT5 (signal transducer and activator of transcription 5) in murine Tregs. Notch-1low Tregs isolated from human peripheral blood exhibited more potent suppressive capacity than Notch-1high Tregs. Last, the combination of aNotch-1 with costimulation blockade induced long-term tolerance in a cardiac transplant model, and this tolerance was dependent on CTLA-4 (cytotoxic T-lymphocyte-associated antigen-4) signaling. CONCLUSIONS: Our data reveal a promising, clinically relevant approach for immune modulation in transplantation by selectively targeting Notch-1.
Assuntos
Rejeição de Enxerto/metabolismo , Receptor Notch1/antagonistas & inibidores , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Anticorpos Bloqueadores/farmacologia , Células Cultivadas , Regulação da Expressão Gênica , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/mortalidade , Humanos , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transplante de Órgãos , Receptor Notch1/genética , Receptor Notch1/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Análise de SobrevidaRESUMO
Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic hematopoietic cell transplantation (allo-HCT) and remains an area of unmet clinical need with few treatment options available. Notch blockade prevents acute GVHD in multiple mouse models, but the impact of Notch signaling on cGVHD remains unknown. Using genetic and antibody-mediated strategies of Notch inhibition, we investigated the role of Notch signaling in complementary mouse cGVHD models that mimic several aspects of human cGVHD in search of candidate therapeutics. In the B10.D2âBALB/c model of sclerodermatous cGVHD, Delta-like ligand 4 (Dll4)-driven Notch signaling was essential for disease development. Antibody-mediated Dll4 inhibition conferred maximum benefits when pursued early in a preventative fashion, with anti-Dll1 enhancing early protection. Notch-deficient alloantigen-specific T cells showed no early defects in proliferation or helper polarization in vivo but subsequently exhibited markedly decreased cytokine secretion and enhanced accumulation of FoxP3+ regulatory T cells. In the B6âB10.BR major histocompatibility complex-mismatched model with multi-organ system cGVHD and prominent bronchiolitis obliterans (BO), but not skin manifestations, absence of Notch signaling in T cells provided long-lasting disease protection that was replicated by systemic targeting of Dll1, Dll4, or both Notch ligands, even during established disease. Notch inhibition decreased target organ damage and germinal center formation. Moreover, decreased BO-cGVHD was observed upon inactivation of Notch1 and/or Notch2 in T cells. Systemic targeting of Notch2 alone was safe and conferred therapeutic benefits. Altogether, Notch ligands and receptors regulate key pathogenic steps in cGVHD and emerge as novel druggable targets to prevent or treat different forms of cGVHD.
Assuntos
Doença Enxerto-Hospedeiro/patologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas de Membrana/imunologia , Receptores Notch/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Bronquiolite Obliterante/etiologia , Bronquiolite Obliterante/imunologia , Bronquiolite Obliterante/patologia , Proteínas de Ligação ao Cálcio , Doença Crônica , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/imunologia , Isoantígenos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Linfócitos T/patologia , Transplante Homólogo/efeitos adversosRESUMO
B-cell receptor (BCR)-activated B cells contribute to pathogenesis in chronic graft-versus-host disease (cGVHD), a condition manifested by both B-cell autoreactivity and immune deficiency. We hypothesized that constitutive BCR activation precluded functional B-cell maturation in cGVHD. To address this, we examined BCR-NOTCH2 synergy because NOTCH has been shown to increase BCR responsiveness in normal mouse B cells. We conducted ex vivo activation and signaling assays of 30 primary samples from hematopoietic stem cell transplantation patients with and without cGVHD. Consistent with a molecular link between pathways, we found that BCR-NOTCH activation significantly increased the proximal BCR adapter protein BLNK. BCR-NOTCH activation also enabled persistent NOTCH2 surface expression, suggesting a positive feedback loop. Specific NOTCH2 blockade eliminated NOTCH-BCR activation and significantly altered NOTCH downstream targets and B-cell maturation/effector molecules. Examination of the molecular underpinnings of this "NOTCH2-BCR axis" in cGVHD revealed imbalanced expression of the transcription factors IRF4 and IRF8, each critical to B-cell differentiation and fate. All-trans retinoic acid (ATRA) increased IRF4 expression, restored the IRF4-to-IRF8 ratio, abrogated BCR-NOTCH hyperactivation, and reduced NOTCH2 expression in cGVHD B cells without compromising viability. ATRA-treated cGVHD B cells had elevated TLR9 and PAX5, but not BLIMP1 (a gene-expression pattern associated with mature follicular B cells) and also attained increased cytosine guanine dinucleotide responsiveness. Together, we reveal a mechanistic link between NOTCH2 activation and robust BCR responses to otherwise suboptimal amounts of surrogate antigen. Our findings suggest that peripheral B cells in cGVHD patients can be pharmacologically directed from hyperactivation toward maturity.
Assuntos
Linfócitos B/metabolismo , Doença Enxerto-Hospedeiro/metabolismo , Transplante de Células-Tronco Hematopoéticas , Proteínas de Neoplasias/metabolismo , Receptor Notch2/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Aloenxertos , Linfócitos B/patologia , Doença Crônica , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/patologia , Humanos , Fatores Reguladores de Interferon/biossíntese , Fatores Reguladores de Interferon/genética , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Receptor Notch2/genética , Receptores de Antígenos de Linfócitos B/genética , Tretinoína/farmacologiaRESUMO
OBJECTIVE: Endothelial upregulation of adhesion molecules serves to recruit leukocytes to inflammatory sites and appears to be promoted by NOTCH1; however, current models based on interactions between active NOTCH1 and NF-κB components cannot explain the transcriptional selectivity exerted by NOTCH1 in this context. APPROACH AND RESULTS: Observing that Cre/Lox-induced conditional mutations of endothelial Notch modulated inflammation in murine contact hypersensitivity, we found that IL (interleukin)-1ß stimulation induced rapid recruitment of RELA (v-rel avian reticuloendotheliosis viral oncogene homolog A) to genomic sites occupied by NOTCH1-RBPJ (recombination signal-binding protein for immunoglobulin kappa J region) and that NOTCH1 knockdown reduced histone H3K27 acetylation at a subset of NF-κB-directed inflammatory enhancers. CONCLUSIONS: Our findings reveal that NOTCH1 signaling supports the expression of a subset of inflammatory genes at the enhancer level and demonstrate how key signaling pathways converge on chromatin to coordinate the transition to an infla mmatory endothelial phenotype.
Assuntos
Células Endoteliais/efeitos dos fármacos , Histonas/metabolismo , Inflamação/prevenção & controle , Interleucina-1beta/farmacologia , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/metabolismo , Acetilação , Animais , Apendicite/metabolismo , Apendicite/patologia , Células Cultivadas , Dermatite de Contato/genética , Dermatite de Contato/metabolismo , Dermatite de Contato/patologia , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Receptor Notch1/genética , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
The mechanisms underlying lymphocyte lineage stability and plasticity remain elusive. Recent work indicates that innate lymphoid cells (ILC) possess substantial plasticity. Whereas natural ILC2 (nILC2) produce type-2 cytokines, plastic inflammatory ILC2 (iILC2) can coproduce both type-2 cytokines and the ILC3-characteristic cytokine, IL-17. Mechanisms that elicit this lineage plasticity, and the importance in health and disease, remain unclear. In this study we show that iILC2 are potent inducers of airway inflammation in response to acute house dust mite challenge. We find that Notch signaling induces lineage plasticity of mature ILC2 and drives the conversion of nILC2 into iILC2. Acute blockade of Notch signaling abolished functional iILC2, but not nILC2, in vivo. Exposure of isolated nILC2 to Notch ligands induced Rorc expression and elicited dual IL-13/IL-17 production, converting nILC2 into iILC2. Together these results reveal a novel role for Notch signaling in eliciting ILC2 plasticity and driving the emergence of highly proinflammatory innate lymphocytes.
Assuntos
Imunidade Inata , Linfócitos/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais , Animais , Linhagem da Célula , Citocinas/imunologia , Inflamação/imunologia , Interleucina-13/imunologia , Interleucina-17/imunologia , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Pyroglyphidae/imunologiaRESUMO
OBJECTIVE: We tested the ability of Notch pathway receptors Notch1 and Notch2 to regulate stem and epithelial cell homoeostasis in mouse and human gastric antral tissue. DESIGN: Mice were treated with the pan-Notch inhibitor dibenzazepine (DBZ) or inhibitory antibodies targeting Notch1 and/or Notch2. Epithelial proliferation, apoptosis and cellular differentiation were measured by histological and molecular approaches. Organoids were established from mouse and human antral glands; growth and differentiation were measured after treatment with Notch inhibitors. RESULTS: Notch1 and Notch2 are the predominant Notch receptors expressed in mouse and human antral tissue and organoid cultures. Combined inhibition of Notch1 and Notch2 in adult mice led to decreased epithelial cell proliferation, including reduced proliferation of LGR5 stem cells, and increased apoptosis, similar to the response to global Notch inhibition with DBZ. Less pronounced effects were observed after inhibition of individual receptors. Notch pathway inhibition with DBZ or combined inhibition of Notch1 and Notch2 led to increased differentiation of all gastric antral lineages, with remodelling of cells to express secretory products normally associated with other regions of the GI tract, including intestine. Analysis of mouse and human organoids showed that Notch signalling through Notch1 and Notch2 is intrinsic to the epithelium and required for organoid growth. CONCLUSIONS: Notch signalling is required to maintain gastric antral stem cells. Notch1 and Notch2 are the primary Notch receptors regulating epithelial cell homoeostasis in mouse and human stomach.
Assuntos
Células Epiteliais/fisiologia , Homeostase , Organoides/crescimento & desenvolvimento , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Células-Tronco/fisiologia , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Apoptose , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Dibenzazepinas/farmacologia , Células Epiteliais/efeitos dos fármacos , Feminino , Mucosa Gástrica/citologia , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Organoides/efeitos dos fármacos , Antro Pilórico , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/genética , Receptor Notch2/antagonistas & inibidores , Receptor Notch2/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Células-Tronco/efeitos dos fármacosRESUMO
The Notch signaling pathway is known to regulate stem cells and epithelial cell homeostasis in gastrointestinal tissues; however, Notch function in the corpus region of the stomach is poorly understood. In this study we examined the consequences of Notch inhibition and activation on cellular proliferation and differentiation and defined the specific Notch receptors functioning in the mouse and human corpus. Notch pathway activity was observed in the mouse corpus epithelium, and gene expression analysis revealed NOTCH1 and NOTCH2 to be the predominant Notch receptors in both mouse and human. Global Notch inhibition for 5 days reduced progenitor cell proliferation in the mouse corpus, as well as in organoids derived from mouse and human corpus tissue. Proliferation effects were mediated through both NOTCH1 and NOTCH2 receptors, as demonstrated by targeting each receptor alone or in combination with Notch receptor inhibitory antibodies. Analysis of differentiation by marker expression showed no change to the major cell lineages; however, there was a modest increase in the number of transitional cells coexpressing markers of mucous neck and chief cells. In contrast to reduced proliferation after pathway inhibition, Notch activation in the adult stomach resulted in increased proliferation coupled with reduced differentiation. These findings suggest that NOTCH1 and NOTCH2 signaling promotes progenitor cell proliferation in the mouse and human gastric corpus, which is consistent with previously defined roles for Notch in promoting stem and progenitor cell proliferation in the intestine and antral stomach. NEW & NOTEWORTHY: Here we demonstrate that the Notch signaling pathway is essential for proliferation of stem cells in the mouse and human gastric corpus. We identify NOTCH1 and NOTCH2 as the predominant Notch receptors expressed in both mouse and human corpus and show that both receptors are required for corpus stem cell proliferation. We show that chronic Notch activation in corpus stem cells induces hyperproliferation and tissue hypertrophy, suggesting that Notch may drive gastric tumorigenesis.
Assuntos
Proliferação de Células/fisiologia , Células Epiteliais/fisiologia , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Estômago/fisiologia , Animais , Feminino , Mucosa Gástrica/citologia , Genes Reporter , Humanos , Masculino , Camundongos , Organoides/citologia , Organoides/fisiologia , Receptor Notch1/genética , Receptor Notch2/genética , Transdução de Sinais/fisiologia , Células-Tronco , Tamoxifeno/farmacologiaRESUMO
Despite use of newer approaches, some patients being considered for autologous hematopoietic cell transplantation (HCT) may only mobilize limited numbers of hematopoietic progenitor cells (HPCs) into blood, precluding use of the procedure, or being placed at increased risk of complications due to slow hematopoietic reconstitution. Developing more efficacious HPC mobilization regimens and strategies may enhance the mobilization process and improve patient outcome. Although Notch signaling is not essential for homeostasis of adult hematopoietic stem cells (HSCs), Notch-ligand adhesive interaction maintains HSC quiescence and niche retention. Using Notch receptor blocking antibodies, we report that Notch2 blockade, but not Notch1 blockade, sensitizes hematopoietic stem cells and progenitors (HSPCs) to mobilization stimuli and leads to enhanced egress from marrow to the periphery. Notch2 blockade leads to transient myeloid progenitor expansion without affecting HSC homeostasis and self-renewal. We show that transient Notch2 blockade or Notch2-loss in mice lacking Notch2 receptor lead to decreased CXCR4 expression by HSC but increased cell cycling with CXCR4 transcription being directly regulated by the Notch transcriptional protein RBPJ. In addition, we found that Notch2-blocked or Notch2-deficient marrow HSPCs show an increased homing to the marrow, while mobilized Notch2-blocked, but not Notch2-deficient stem cells and progenitors, displayed a competitive repopulating advantage and enhanced hematopoietic reconstitution. These findings suggest that blocking Notch2 combined with the current clinical regimen may further enhance HPC mobilization and improve engraftment during HCT.
Assuntos
Antineoplásicos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Receptor Notch2/antagonistas & inibidores , Animais , Antineoplásicos/uso terapêutico , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Movimento Celular/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Transgênicos , Receptor Notch2/deficiência , Receptor Notch2/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Rejection remains a major clinical challenge limiting allograft survival after solid organ transplantation. Both cellular and humoral immunity contribute to this complication, with increased recognition of Ab-mediated damage during acute and chronic rejection. Using a mouse model of MHC-mismatched heart transplantation, we report markedly protective effects of Notch inhibition, dampening both T cell and Ab-driven rejection. T cell-specific pan-Notch blockade prolonged heart allograft survival and decreased IFN-γ and IL-4 production by alloreactive T cells, especially when combined with depletion of recipient CD8(+) T cells. These effects were associated with decreased infiltration by conventional T cells and an increased proportion of regulatory T cells in the graft. Transient administration of neutralizing Abs specific for delta-like (Dll)1/4 Notch ligands in the peritransplant period led to prolonged acceptance of allogeneic hearts, with superior outcome over Notch inhibition only in T cells. Systemic Dll1/4 inhibition decreased T cell cytokines and graft infiltration, germinal center B cell and plasmablast numbers, as well as production of donor-specific alloantibodies and complement deposition in the transplanted hearts. Dll1 or Dll4 inhibition alone provided partial protection. Thus, pathogenic signals delivered by Dll1/4 Notch ligands early after transplantation promote organ rejection through several complementary mechanisms. Transient interruption of these signals represents an attractive new therapeutic strategy to enhance long-term allograft survival.
Assuntos
Anticorpos Neutralizantes/imunologia , Rejeição de Enxerto/imunologia , Transplante de Coração/métodos , Imunidade/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas de Membrana/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anticorpos Neutralizantes/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proteínas de Ligação ao Cálcio , Citometria de Fluxo , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/imunologia , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Notch/antagonistas & inibidores , Receptores Notch/imunologia , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Transplante HomólogoRESUMO
Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody-siRNA complexes provide a possible solution. However, initial reports of antibody-siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, we built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. We report that such coupling generates monomeric antibody-siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technology, we generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. We identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only observed using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. Our broad characterization of multiple parameters using therapeutic-grade conjugate technology provides a thorough assessment of this delivery technology, highlighting both examples of success as well as remaining challenges.
Assuntos
Anticorpos , RNA Interferente Pequeno/administração & dosagem , Animais , Anticorpos/genética , Anticorpos/imunologia , Anticorpos/metabolismo , Linhagem Celular , Endossomos/metabolismo , Camundongos , Neoplasias/genética , Engenharia de Proteínas , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismoRESUMO
UNLABELLED: Primary liver cancer encompasses both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). The Notch signaling pathway, known to be important for the proper development of liver architecture, is also a potential driver of primary liver cancer. However, with four known Notch receptors and several Notch ligands, it is not clear which Notch pathway members play the predominant role in liver cancer. To address this question, we utilized antibodies to specifically target Notch1, Notch2, Notch3, or jagged1 (Jag1) in a mouse model of primary liver cancer driven by v-akt murine thymoma viral oncogene homolog and neuroblastoma RAS viral oncogene homolog (NRas). We show that inhibition of Notch2 reduces tumor burden by eliminating highly malignant HCC- and CCA-like tumors. Inhibition of the Notch ligand, Jag1, had a similar effect, consistent with Jag1 acting in cooperation with Notch2. This effect was specific to Notch2, because Notch3 inhibition did not decrease tumor burden. Unexpectedly, Notch1 inhibition altered the relative proportion of tumor types, reducing HCC-like tumors but dramatically increasing CC-like tumors. Finally, we show that Notch2 and Jag1 are expressed in, and Notch2 signaling is activated in, a subset of human HCC samples. CONCLUSIONS: These findings underscore the distinct roles of different Notch receptors in the liver and suggest that inhibition of Notch2 signaling represents a novel therapeutic option in the treatment of liver cancer.
Assuntos
Neoplasias Hepáticas/tratamento farmacológico , Receptores Notch/antagonistas & inibidores , Animais , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Modelos Animais de Doenças , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/análise , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/análise , Proteína Jagged-1 , Proteínas de Membrana/análise , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Receptores Notch/análise , Receptores Notch/fisiologia , Proteínas Serrate-JaggedRESUMO
Notch is long recognized as a signaling molecule important for stem cell self-renewal and fate determination. Here, we reveal a novel adhesive role of Notch-ligand engagement in hematopoietic stem and progenitor cells (HSPCs). Using mice with conditional loss of O-fucosylglycans on Notch EGF-like repeats important for the binding of Notch ligands, we report that HSPCs with faulty ligand binding ability display enhanced cycling accompanied by increased egress from the marrow, a phenotype mainly attributed to their reduced adhesion to Notch ligand-expressing stromal cells and osteoblastic cells and their altered occupation in osteoblastic niches. Adhesion to Notch ligand-bearing osteoblastic or stromal cells inhibits wild type but not O-fucosylglycan-deficient HSPC cycling, independent of RBP-JK -mediated canonical Notch signaling. Furthermore, Notch-ligand neutralizing antibodies induce RBP-JK -independent HSPC egress and enhanced HSPC mobilization. We, therefore, conclude that Notch receptor-ligand engagement controls HSPC quiescence and retention in the marrow niche that is dependent on O-fucosylglycans on Notch.