RESUMO
Purpose: Retinopathy of prematurity (ROP) results from postnatal hyperoxia exposure in premature infants and is characterized by aberrant neovascularization of retinal blood vessels. Epithelial membrane protein-2 (EMP2) regulates hypoxia-inducible factor (HIF)-induced vascular endothelial growth factor (VEGF) production in the ARPE-19 cell line and genetic knock-out of Emp2 in a murine oxygen-induced retinopathy (OIR) model attenuates neovascularization. We hypothesize that EMP2 blockade via intravitreal injection protects against neovascularization. Methods: Ex vivo choroid sprouting assay was performed, comparing media and human IgG controls versus anti-EMP2 antibody (Ab) treatment. In vivo, eyes from wild-type (WT) mice exposed to hyperoxia from postnatal (P) days 7 to 12 were treated with P12 intravitreal injections of control IgG or anti-EMP2 Abs. Neovascularization was assessed at P17 by flat mount imaging. Local and systemic effects of anti-EMP2 Ab treatment were assessed. Results: Choroid sprouts treated with 30 µg/mL of anti-EMP2 Ab demonstrated a 48% reduction in vessel growth compared to control IgG-treated sprouts. Compared to IgG-treated controls, WT OIR mice treated with 4 µg/g of intravitreal anti-EMP2 Ab demonstrated a 42% reduction in neovascularization. They demonstrated down-regulation of retinal gene expression in pathways related to vasculature development and up-regulation in genes related to fatty acid oxidation and tricarboxylic acid cycle respiratory electron transport, compared to controls. Anti-EMP2 Ab-treated OIR mice did not exhibit gross retinal histologic abnormalities, vision transduction abnormalities, or weight loss. Conclusions: Our results suggest that EMP2 blockade could be a local and specific treatment modality for retinal neovascularization in oxygen-induced retinopathies, without systemic adverse effects.
Assuntos
Oxigênio , Neovascularização Retiniana , Retinopatia da Prematuridade , Animais , Humanos , Camundongos , Animais Recém-Nascidos , Modelos Animais de Doenças , Hiperóxia/complicações , Injeções Intravítreas , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Oxigênio/toxicidade , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/prevenção & controle , Neovascularização Retiniana/patologia , Retinopatia da Prematuridade/tratamento farmacológico , Retinopatia da Prematuridade/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common pediatric hematological malignancy, with ETV6::RUNX1 being the most prevalent translocation whose exact pathogenesis remains unclear. IGF2BP1 (Insulin-like Growth Factor 2 Binding Protein 1) is an oncofetal RNA binding protein seen to be specifically overexpressed in ETV6::RUNX1 positive B-ALL. In this study, we have studied the mechanistic role of IGF2BP1 in leukemogenesis and its synergism with the ETV6::RUNX1 fusion protein. METHODS: Gene expression was analyzed from patient bone marrow RNA using Real Time RT-qPCR. Knockout cell lines were created using CRISPR-Cas9 based lentiviral vectors. RNA-Seq and RNA Immunoprecipitation sequencing (RIP-Seq) after IGF2BP1 pulldown were performed using the Illumina platform. Mouse experiments were done by retroviral overexpression of donor HSCs followed by lethal irradiation of recipients using a bone marrow transplant model. RESULTS: We observed specific overexpression of IGF2BP1 in ETV6::RUNX1 positive patients in an Indian cohort of pediatric ALL (n=167) with a positive correlation with prednisolone resistance. IGF2BP1 expression was essential for tumor cell survival in multiple ETV6::RUNX1 positive B-ALL cell lines. Integrated analysis of transcriptome sequencing after IGF2BP1 knockout and RIP-Seq after IGF2BP1 pulldown in Reh cell line revealed that IGF2BP1 targets encompass multiple pro-oncogenic signalling pathways including TNFα/NFκB and PI3K-Akt pathways. These pathways were also dysregulated in primary ETV6::RUNX1 positive B-ALL patient samples from our center as well as in public B-ALL patient datasets. IGF2BP1 showed binding and stabilization of the ETV6::RUNX1 fusion transcript itself. This positive feedback loop led to constitutive dysregulation of several oncogenic pathways. Enforced co-expression of ETV6::RUNX1 and IGF2BP1 in mouse bone marrow resulted in marrow hypercellularity which was characterized by multi-lineage progenitor expansion and strong Ki67 positivity. This pre-leukemic phenotype confirmed their synergism in-vivo. Clonal expansion of cells overexpressing both ETV6::RUNX1 and IGF2BP1 was clearly observed. These mice also developed splenomegaly indicating extramedullary hematopoiesis. CONCLUSION: Our data suggest a combined impact of the ETV6::RUNX1 fusion protein and RNA binding protein, IGF2BP1 in activating multiple oncogenic pathways in B-ALL which makes IGF2BP1 and these pathways as attractive therapeutic targets and biomarkers.
Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animais , Camundongos , Subunidade alfa 2 de Fator de Ligação ao Core , Camundongos Knockout , Fosfatidilinositol 3-Quinases , Variante 6 da Proteína do Fator de Translocação ETSRESUMO
Generation of chimeric antigen receptor (CAR) T cells from pluripotent stem cells (PSCs) will enable advances in cancer immunotherapy. Understanding how CARs affect T cell differentiation from PSCs is important for this effort. The recently described artificial thymic organoid (ATO) system supports in vitro differentiation of PSCs to T cells. Unexpectedly, PSCs transduced with a CD19-targeted CAR resulted in diversion of T cell differentiation to the innate lymphoid cell 2 (ILC2) lineage in ATOs. T cells and ILC2s are closely related lymphoid lineages with shared developmental and transcriptional programs. Mechanistically, we show that antigen-independent CAR signaling during lymphoid development enriched for ILC2-primed precursors at the expense of T cell precursors. We applied this understanding to modulate CAR signaling strength through expression level, structure, and presentation of cognate antigen to demonstrate that the T cell-versus-ILC lineage decision can be rationally controlled in either direction, providing a framework for achieving CAR-T cell development from PSCs.
Assuntos
Células-Tronco Pluripotentes , Linfócitos T , Imunidade Inata , Linfócitos/metabolismo , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Imunoterapia Adotiva/métodos , Antígenos CD19 , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Although metabolic pathways have been shown to control differentiation and activation in peripheral T cells, metabolic studies on thymic T cell development are still lacking, especially in human tissue. In this study, we use transcriptomics and extracellular flux analyses to investigate the metabolic profiles of primary thymic and in vitro-derived mouse and human thymocytes. Core metabolic pathways, specifically glycolysis and oxidative phosphorylation, undergo dramatic changes between the double-negative (DN), double-positive (DP), and mature single-positive (SP) stages in murine and human thymus. Remarkably, despite the absence of the complex multicellular thymic microenvironment, in vitro murine and human T cell development recapitulated the coordinated decrease in glycolytic and oxidative phosphorylation activity between the DN and DP stages seen in primary thymus. Moreover, by inducing in vitro T cell differentiation from Rag1-/- mouse bone marrow, we show that reduced metabolic activity at the DP stage is independent of TCR rearrangement. Thus, our findings suggest that highly conserved metabolic transitions are critical for thymic T cell development.
Assuntos
Diferenciação Celular , Metabolismo Energético , Linfócitos T/citologia , Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Animais , Evolução Biológica , Biomarcadores , Linhagem Celular , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfopoese , Metaboloma , Metabolômica/métodos , Camundongos , Organoides , Timócitos/imunologia , Técnicas de Cultura de TecidosRESUMO
Males and females exhibit striking differences in the prevalence of metabolic traits including hepatic steatosis, a key driver of cardiometabolic morbidity and mortality. RNA methylation is a widespread regulatory mechanism of transcript turnover. Here, we show that presence of the RNA modification N6-methyladenosine (m6A) triages lipogenic transcripts for degradation and guards against hepatic triglyceride accumulation. In male but not female mice, this protective checkpoint stalls under lipid-rich conditions. Loss of m6A control in male livers increases hepatic triglyceride stores, leading to a more 'feminized' hepatic lipid composition. Crucially, liver-specific deletion of the m6A complex protein Mettl14 from male and female mice significantly diminishes sex-specific differences in steatosis. We further surmise that the m6A installing machinery is subject to transcriptional control by the sex-responsive BCL6-STAT5 axis in response to dietary conditions. These data show that m6A is essential for precise and synchronized control of lipogenic enzyme activity and provide insights into the molecular basis for the existence of sex-specific differences in hepatic lipid traits.
Assuntos
Adenosina/análogos & derivados , Metabolismo Energético , Regulação da Expressão Gênica , Característica Quantitativa Herdável , Transcrição Gênica , Adenosina/metabolismo , Animais , Feminino , Metabolismo dos Lipídeos , Masculino , Metilação , Camundongos , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Fator de Transcrição STAT5/metabolismo , Fatores Sexuais , Transdução de SinaisRESUMO
Post-transcriptional gene regulation, including that by RNA binding proteins (RBPs), has recently been described as an important mechanism in cancer. We had previously identified a set of RBPs that were highly dysregulated in B-cell acute lymphoblastic leukemia (B-ALL) with MLL translocations, which carry a poor prognosis. Here, we sought to functionally characterize these dysregulated RBP genes by performing a focused CRISPR dropout screen in B-ALL cell lines, finding dependencies on several genes including EIF3E, EPRS and USO1. Validating our findings, CRISPR/Cas9-mediated disruption of USO1 in MLL-translocated B-ALL cells reduced cell growth, promoted cell death, and altered the cell cycle. Transcriptomic analysis of USO1-deficient cells revealed alterations in pathways related to mTOR signaling, RNA metabolism, and targets of MYC. In addition, USO1-regulated genes from these experimental samples were significantly and concordantly correlated with USO1 expression in primary samples collected from B-ALL patients. Lastly, we found that loss of Uso1 inhibited colony formation of MLL-transformed in primary bone marrow cells from Cas9-EGFP mice. Together, our findings demonstrate an approach to performing focused sub-genomic CRISPR screens and highlight a putative RBP vulnerability in MLL-translocated B-ALL, thus identifying potential therapeutic targets in this disease.
Assuntos
Sistemas CRISPR-Cas , Proteínas da Matriz do Complexo de Golgi/fisiologia , Proteína de Leucina Linfoide-Mieloide/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas de Fusão Oncogênica/fisiologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas de Transporte Vesicular/fisiologia , Animais , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Regulação Leucêmica da Expressão Gênica , Genes Reporter , Predisposição Genética para Doença , Testes Genéticos , Proteínas da Matriz do Complexo de Golgi/genética , Homeostase , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Processamento Pós-Transcricional do RNA , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Transgenes , Translocação Genética , Ensaio Tumoral de Célula-Tronco , Proteínas de Transporte Vesicular/genéticaRESUMO
BACKGROUND AND AIMS: The host receptor for severe acute respiratory syndrome coronavirus 2, angiotensin-converting enzyme 2 (ACE2), is highly expressed in small bowel (SB). Our aim was to identify factors influencing intestinal ACE2 expression in Crohn's disease (CD), ulcerative colitis (UC), and non-inflammatory bowel disease (IBD) controls. METHODS: Using bulk RNA sequencing or microarray transcriptomics from tissue samples (4 SB and 2 colonic cohorts; n = 495; n = 387 UC; n = 94 non-IBD), we analyzed the relationship between ACE2 with demographics and disease activity and prognosis. We examined the outcome of anti-tumor necrosis factor and anti-interleukin-12/interleukin-23 treatment on SB and colonic ACE2 expression in 3 clinical trials. Univariate and multivariate regression models were fitted. RESULTS: ACE2 levels were consistently reduced in SB CD and elevated in colonic UC compared with non-IBD controls. Elevated SB ACE2 was also associated with demographic features (age and elevated body mass index) associated with poor coronavirus disease 2019 outcomes. Within CD, SB ACE2 was reduced in patients subsequently developing complicated disease. Within UC, colonic ACE2 was elevated in active disease and in patients subsequently requiring anti-tumor necrosis factor rescue therapy. SB and colonic ACE2 expression in active CD and UC were restored by anti-cytokine therapy, most notably in responders. CONCLUSIONS: Reduced SB but elevated colonic ACE2 levels in IBD are associated with inflammation and severe disease, but normalized after anti-cytokine therapy, suggesting compartmentalization of ACE2-related biology in SB and colonic inflammation. The restoration of ACE2 expression with anti-cytokine therapy might be important in the context of severe acute respiratory syndrome coronavirus 2 infection and potentially explain reports of reduced morbidity from coronavirus disease 2019 in IBD patients treated with anti-cytokines.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Anti-Inflamatórios/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Doença de Crohn/tratamento farmacológico , Intestinos/efeitos dos fármacos , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Enzima de Conversão de Angiotensina 2/genética , Anti-Inflamatórios/efeitos adversos , COVID-19/enzimologia , COVID-19/imunologia , COVID-19/virologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Colite Ulcerativa/enzimologia , Colite Ulcerativa/genética , Colite Ulcerativa/imunologia , Doença de Crohn/enzimologia , Doença de Crohn/genética , Doença de Crohn/imunologia , Bases de Dados Genéticas , Feminino , Regulação Enzimológica da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Intestinos/enzimologia , Intestinos/imunologia , Masculino , Pessoa de Meia-Idade , América do Norte , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Virais/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/imunologia , Índice de Gravidade de Doença , Resultado do Tratamento , Inibidores do Fator de Necrose Tumoral/efeitos adversos , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
We report a serum-free, 3D murine artificial thymic organoid (M-ATO) system that mimics normal murine thymopoiesis with the production of all T cell stages, from early thymic progenitors to functional single-positive (CD8SP and CD4SP) TCRαß and TCRγδ cells. RNA sequencing aligns M-ATO-derived populations with phenotypically identical primary thymocytes. M-ATOs initiated with Rag1-/- marrow produce the same differentiation block as seen in the endogenous thymus, and Notch signaling patterns in M-ATOs mirror primary thymopoiesis. M-ATOs initiated with defined hematopoietic stem cells (HSCs) and lymphoid progenitors from marrow and thymus generate each of the downstream differentiation stages, allowing the kinetics of T cell differentiation to be tracked. Remarkably, single HSCs deposited into each M-ATO generate the complete trajectory of T cell differentiation, producing diverse TCR repertoires across clones that largely match endogenous thymus. M-ATOs represent a highly reproducible and efficient experimental platform for the interrogation of clonal thymopoiesis from HSCs.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Timo/fisiologia , Animais , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , CamundongosRESUMO
Neonatal life marks the apogee of murine thymic growth. Over the first few days after birth, growth slows and the murine thymus switches from fetal to adult morphology and function; little is known about the cues driving this dramatic transition. In this study, we show for the first time (to our knowledge) the critical role of vascular endothelial growth factor (VEGF) on thymic morphogenesis beyond its well-known role in angiogenesis. During a brief window a few days after birth, VEGF inhibition induced rapid and profound remodeling of the endothelial, mesenchymal and epithelial thymic stromal compartments, mimicking changes seen during early adult maturation. Rapid transcriptional changes were seen in each compartment after VEGF inhibition, including genes involved in migration, chemotaxis, and cell adhesion as well as induction of a proinflammatory and proadipogenic signature in endothelium, pericytes, and mesenchyme. Thymocyte numbers fell subsequent to the stromal changes. Expression patterns and functional blockade of the receptors VEGFR2 and NRP1 demonstrated that VEGF mediates its pleiotropic effects through distinct receptors on each microenvironmental compartment of the developing mouse thymus.
Assuntos
Timo/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Endotélio/metabolismo , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Pericitos/metabolismo , Timócitos/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The finding that transformed mouse B-1 and B-2 progenitors give rise to B-cell acute lymphoblastic leukemias (B-ALLs) with varied aggressiveness suggests that B-cell lineage might also be a factor in the initiation and progression of pediatric B-ALLs in humans. If this is the case, we hypothesized that human pediatric B-ALLs would share gene expression patterns with mouse B-1 or B-2 progenitors. We tested this premise by deriving a distinct 30-gene B-1 and B-2 progenitor signature that was applied to a microarray data set of human pediatric ALLs. Cluster analysis revealed that CRLF2, E2A-PBX1, ERG, and ETV6-RUNX1 leukemias were B-1-like, whereas BCR-ABL1, hyperdiploid, and MLL leukemias were B-2-like. Examination of the 30-gene signature in two independent data sets of pediatric ALLs supported this result. Our data suggest that common genetic subtypes of human ALL have their origin in the B-1 or B-2 lineage.
Assuntos
Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Proteínas de Neoplasias , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Transcriptoma , Animais , Criança , Humanos , Camundongos , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/classificação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismoRESUMO
Purpose: Retinopathy of prematurity (ROP) is a leading cause of childhood blindness. ROP occurs as a consequence of postnatal hyperoxia exposure in premature infants, resulting in vasoproliferation in the retina. The tetraspan protein epithelial membrane protein-2 (EMP2) is highly expressed in the retinal pigment epithelium (RPE) in adults, and it controls vascular endothelial growth factor (VEGF) production in the ARPE-19 cell line. We, therefore, hypothesized that Emp2 knockout (Emp2 KO) protects against neovascularization in murine oxygen-induced retinopathy (OIR). Methods: Eyes were obtained from wildtype (WT) and Emp2 KO mouse pups at P7, P12, P17, and P21 after normoxia or hyperoxia (P7-P12) exposure. Following hyperoxia exposure, RNA sequencing was performed using the retina/choroid layers obtained from WT and Emp2 KO at P17. Retinal sections from P7, P12, P17, and P21 were evaluated for Emp2, hypoxia-inducible factor 1α (Hif1α), and VEGF expression. Whole mount images were generated to assess vaso-obliteration at P12 and neovascularization at P17. Results: Emp2 KO OIR mice demonstrated a decrease in pathologic neovascularization at P17 compared with WT OIR mice through evaluation of retinal vascular whole mount images. This protection was accompanied by a decrease in Hif1α at P12 and VEGFA expression at P17 in Emp2 KO animals compared with the WT animals in OIR conditions. Collectively, our results suggest that EMP2 enhances the effects of neovascularization through modulation of angiogenic signaling. Conclusions: The protection of Emp2 KO mice against pathologic neovascularization through attenuation of HIF and VEGF upregulation in OIR suggests that hypoxia-induced upregulation of EMP2 expression in the neuroretina modulates HIF-mediated neuroretinal VEGF expression.
Assuntos
Glicoproteínas de Membrana/fisiologia , Neovascularização Retiniana/patologia , Retinopatia da Prematuridade/patologia , Fator A de Crescimento do Endotélio Vascular/fisiologia , Animais , Animais Recém-Nascidos , Linhagem Celular , Hiperóxia/fisiopatologia , Hipóxia/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/patologia , Oxigênio/toxicidade , Epitélio Pigmentado da Retina/metabolismo , Vasos Retinianos/patologia , Regulação para Cima/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Aging results in increased myelopoiesis, which is linked to the increased incidence of myeloid leukemias and production of myeloid-derived suppressor cells. Here, we examined the contribution of plasma cells (PCs) to age-related increases in myelopoiesis, as PCs exhibit immune regulatory function and sequester in bone marrow (BM). PC number was increased in old BM, and they exhibited high expression of genes encoding inflammatory cytokines and pathogen sensors. Antibody-mediated depletion of PCs from old mice reduced the number of myeloid-biased hematopoietic stem cells and mature myeloid cells to levels in young animals, but lymphopoiesis was not rejuvenated, indicating that redundant mechanisms inhibit that process. PCs also regulated the production of inflammatory factors from BM stromal cells, and disruption of the PC-stromal cell circuitry with inhibitors of the cytokines IL-1 and TNF-α attenuated myelopoiesis in old mice. Thus, the age-related increase in myelopoiesis is driven by an inflammatory network orchestrated by PCs.
Assuntos
Envelhecimento/fisiologia , Medula Óssea/fisiologia , Células-Tronco Hematopoéticas/patologia , Inflamação/metabolismo , Mielopoese/fisiologia , Plasmócitos/fisiologia , Animais , Células Cultivadas , Humanos , Interleucina-1/metabolismo , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Inflammatory bowel diseases, which include Crohn's disease and ulcerative colitis, affect several million individuals worldwide. Crohn's disease and ulcerative colitis are complex diseases that are heterogeneous at the clinical, immunological, molecular, genetic, and microbial levels. Individual contributing factors have been the focus of extensive research. As part of the Integrative Human Microbiome Project (HMP2 or iHMP), we followed 132 subjects for one year each to generate integrated longitudinal molecular profiles of host and microbial activity during disease (up to 24 time points each; in total 2,965 stool, biopsy, and blood specimens). Here we present the results, which provide a comprehensive view of functional dysbiosis in the gut microbiome during inflammatory bowel disease activity. We demonstrate a characteristic increase in facultative anaerobes at the expense of obligate anaerobes, as well as molecular disruptions in microbial transcription (for example, among clostridia), metabolite pools (acylcarnitines, bile acids, and short-chain fatty acids), and levels of antibodies in host serum. Periods of disease activity were also marked by increases in temporal variability, with characteristic taxonomic, functional, and biochemical shifts. Finally, integrative analysis identified microbial, biochemical, and host factors central to this dysregulation. The study's infrastructure resources, results, and data, which are available through the Inflammatory Bowel Disease Multi'omics Database ( http://ibdmdb.org ), provide the most comprehensive description to date of host and microbial activities in inflammatory bowel diseases.
Assuntos
Microbioma Gastrointestinal/genética , Doenças Inflamatórias Intestinais/microbiologia , Animais , Fungos/patogenicidade , Microbioma Gastrointestinal/imunologia , Saúde , Humanos , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/terapia , Doenças Inflamatórias Intestinais/virologia , Filogenia , Especificidade da Espécie , Transcriptoma , Vírus/patogenicidadeRESUMO
Current models propose that reductions in the number of lymphoid-biased hematopoietic stem cells (Ly-HSCs) underlie age-related declines in lymphopoiesis. We show that Ly-HSCs do not decline in number with age. Old Ly-HSCs exhibit changes in gene expression and a myeloid-biased genetic profile, but we demonstrate that they retain normal lymphoid potential when removed from the old in vivo environment. Additional studies showing that interleukin-1 inhibits Ly-HSC lymphoid potential provide support for the hypothesis that increased production of inflammatory cytokines during aging underlies declines in lymphocyte production. These results indicate that current models proposing that lymphopoiesis declines with age due to loss of Ly-HSCs require revision and provide an additional perspective on why lymphocyte development in the elderly is attenuated.
Assuntos
Envelhecimento/patologia , Células-Tronco Hematopoéticas/citologia , Linfócitos/citologia , Envelhecimento/metabolismo , Animais , Citocinas/genética , Feminino , Expressão Gênica/fisiologia , Inflamação/genética , Inflamação/patologia , Linfopoese/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos AnimaisRESUMO
The ability to generate T cells from pluripotent stem cells (PSCs) has the potential to transform autologous T cell immunotherapy by facilitating universal, off-the-shelf cell products. However, differentiation of human PSCs into mature, conventional T cells has been challenging with existing methods. We report that a continuous 3D organoid system induced an orderly sequence of commitment and differentiation from PSC-derived embryonic mesoderm through hematopoietic specification and efficient terminal differentiation to naive CD3+CD8αß+ and CD3+CD4+ conventional T cells with a diverse T cell receptor (TCR) repertoire. Introduction of an MHC class I-restricted TCR in PSCs produced naive, antigen-specific CD8αß+ T cells that lacked endogenous TCR expression and showed anti-tumor efficacy in vitro and in vivo. Functional assays and RNA sequencing aligned PSC-derived T cells with primary naive CD8+ T cells. The PSC-artificial thymic organoid (ATO) system presented here is an efficient platform for generating functional, mature T cells from human PSCs.
Assuntos
Diferenciação Celular , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Linfócitos T/citologia , Animais , Células Cultivadas , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos NOD , Organoides/imunologia , Células-Tronco Pluripotentes/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologiaRESUMO
Microbial metabolites are an emerging class of mediators influencing CD4+ T-cell function. To advance the understanding of direct causal microbial factors contributing to Crohn's disease, we screened 139 predicted Crohn's disease-associated microbial metabolites for their bioactivity on human CD4+ T-cell functions induced by disease-associated T helper 17 (Th17) polarizing conditions. We observed 15 metabolites with CD4+ T-cell bioactivity, 3 previously reported, and 12 unprecedented. A deeper investigation of the microbe-derived metabolite, ascorbate, revealed its selective inhibition on activated human CD4+ effector T cells, including IL-17A-, IL-4-, and IFNγ-producing cells. Mechanistic assessment suggested the apoptosis of activated human CD4+ T cells associated with selective inhibition of energy metabolism. These findings suggest a substantial rate of relevant T-cell bioactivity among Crohn's disease-associated microbial metabolites, and evidence for novel modes of bioactivity, including targeting of T-cell energy metabolism.
Assuntos
Ácido Ascórbico/metabolismo , Doença de Crohn/microbiologia , Microbiota/imunologia , Células Th17/imunologia , Apoptose , Diferenciação Celular , Respiração Celular , Células Cultivadas , Doença de Crohn/imunologia , Metabolismo Energético , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-4/metabolismo , Ativação Linfocitária , Programas de RastreamentoRESUMO
Nuclear receptors regulate gene expression in response to environmental cues, but the molecular events governing the cell type specificity of nuclear receptors remain poorly understood. Here we outline a role for a long noncoding RNA (lncRNA) in modulating the cell type-specific actions of liver X receptors (LXRs), sterol-activated nuclear receptors that regulate the expression of genes involved in cholesterol homeostasis and that have been causally linked to the pathogenesis of atherosclerosis. We identify the lncRNA MeXis as an amplifier of LXR-dependent transcription of the gene Abca1, which is critical for regulation of cholesterol efflux. Mice lacking the MeXis gene show reduced Abca1 expression in a tissue-selective manner. Furthermore, loss of MeXis in mouse bone marrow cells alters chromosome architecture at the Abca1 locus, impairs cellular responses to cholesterol overload, and accelerates the development of atherosclerosis. Mechanistic studies reveal that MeXis interacts with and guides promoter binding of the transcriptional coactivator DDX17. The identification of MeXis as a lncRNA modulator of LXR-dependent gene expression expands understanding of the mechanisms underlying cell type-selective actions of nuclear receptors in physiology and disease.
Assuntos
Aterosclerose/genética , Colesterol/metabolismo , RNA Helicases DEAD-box/genética , Receptores X do Fígado/genética , RNA Longo não Codificante/genética , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Células da Medula Óssea/metabolismo , Colesterol/genética , Regulação da Expressão Gênica/genética , Humanos , Receptores X do Fígado/metabolismo , Macrófagos/metabolismo , Camundongos , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
The PU.1 transcription factor plays a critical role in the regulation of T cell development, so a report that it is dispensable for fetal thymopoiesis is puzzling. To understand this paradox, we examined the requirement for PU.1, encoded by Spi1, during fetal, neonatal, and adult thymopoiesis in a PU.1 hypomorphic mouse generated by deletion of the Spi1 14-kb upstream regulatory element and by analysis of patterns of gene expression in fetal and adult T cell progenitors. Our data demonstrate that the initiation of thymopoiesis during early gestation is less dependent on PU.1 compared with T cell differentiation in adults and that fetal T cell progenitors express lower levels of Spi1 compared with their adult counterparts. We also show that expression of the core network of T lineage transcription factors regulated by PU.1 differs in fetal and adult T cell progenitors. In particular, PU.1-regulated genes that promote T cell differentiation are differentially expressed in fetal versus adult early T lineage progenitors. These results indicate that the transcriptional differences between the fetal and adult T cell developmental programs are driven in part by differential levels of PU.1 expression and that this likely underlies the differences in the properties of fetal and adult T cell progenitors.
Assuntos
Linhagem da Célula/fisiologia , Feto/metabolismo , Feto/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Linfócitos T/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular/fisiologia , Expressão Gênica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Células Precursoras de Linfócitos T/fisiologiaRESUMO
Various mesenchymal cell types have been identified as critical components of the hematopoietic stem/progenitor cell (HSPC) niche. Although several groups have described the generation of mesenchyme from human pluripotent stem cells (hPSCs), the capacity of such cells to support hematopoiesis has not been reported. Here, we demonstrate that distinct mesenchymal subpopulations co-emerge from mesoderm during hPSC differentiation. Despite co-expression of common mesenchymal markers (CD73, CD105, CD90, and PDGFRß), a subset of cells defined as CD146hiCD73hi expressed genes associated with the HSPC niche and supported the maintenance of functional HSPCs ex vivo, while CD146loCD73lo cells supported differentiation. Stromal support of HSPCs was contact dependent and mediated in part through high JAG1 expression and low WNT signaling. Molecular profiling revealed significant transcriptional similarity between hPSC-derived CD146++ and primary human CD146++ perivascular cells. The derivation of functionally diverse types of mesenchyme from hPSCs opens potential avenues to model the HSPC niche and develop PSC-based therapies.
Assuntos
Diferenciação Celular/genética , Células-Tronco Mesenquimais/citologia , Mesoderma/citologia , Células-Tronco Pluripotentes/citologia , Antígeno CD146/genética , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Hematopoese/genética , Humanos , Células-Tronco Mesenquimais/metabolismo , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Células-Tronco Pluripotentes/metabolismo , Nicho de Células-Tronco/genética , Via de Sinalização Wnt/genéticaRESUMO
BACKGROUND: Space travel is associated with continuous low dose rate exposure to high linear energy transfer (LET) radiation. Pathophysiological manifestations after low dose radiation exposure are strongly influenced by non-cytocidal radiation effects, including changes in the microbiome and host gene expression. Although the importance of the gut microbiome in the maintenance of human health is well established, little is known about the role of radiation in altering the microbiome during deep-space travel. RESULTS: Using a mouse model for exposure to high LET radiation, we observed substantial changes in the composition and functional potential of the gut microbiome. These were accompanied by changes in the abundance of multiple metabolites, which were related to the enzymatic activity of the predicted metagenome by means of metabolic network modeling. There was a complex dynamic in microbial and metabolic composition at different radiation doses, suggestive of transient, dose-dependent interactions between microbial ecology and signals from the host's cellular damage repair processes. The observed radiation-induced changes in microbiota diversity and composition were analyzed at the functional level. A constitutive change in activity was found for several pathways dominated by microbiome-specific enzymatic reactions like carbohydrate digestion and absorption and lipopolysaccharide biosynthesis, while the activity in other radiation-responsive pathways like phosphatidylinositol signaling could be linked to dose-dependent changes in the abundance of specific taxa. CONCLUSIONS: The implication of microbiome-mediated pathophysiology after low dose ionizing radiation may be an unappreciated biologic hazard of space travel and deserves experimental validation. This study provides a conceptual and analytical basis of further investigations to increase our understanding of the chronic effects of space radiation on human health, and points to potential new targets for intervention in adverse radiation effects.