RESUMO
The coordination of nutrient and energy availability with cell growth and division is essential for proper immune cell development and function. By using a chemical mutagenesis strategy in mice, we identified a pedigree that has a complete block in B cell development at the pre-B cell stage resulting from a deletion in the Fnip1 gene. Enforced expression of an immunoglobulin transgene failed to rescue B cell development. Whereas essential pre-B cell signaling molecules were activated normally in Fnip1-null pre-B cells, the metabolic regulators AMPK and mTOR were dysregulated, resulting in excessive cell growth and enhanced sensitivity to apoptosis in response to metabolic stress (pre-B cell receptor crosslinking, oncogene activation). These results indicate that Folliculin-interacting protein 1 (Fnip1) is vital for B cell development and metabolic homeostasis and reveal a metabolic checkpoint that may ensure that pre-B cells have sufficient metabolic capacity to support division, while limiting lymphomagenesis caused by deregulated growth.
Assuntos
Linfócitos B/citologia , Linfócitos B/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Diferenciação Celular/genética , Estrona/genética , Estrona/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Apoptose/genética , Divisão Celular/genética , Hematopoese/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Camundongos , Camundongos Transgênicos , Células Precursoras de Linfócitos B/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Folliculin interacting protein 1 (Fnip1) is a cytoplasmic protein originally discovered through its interaction with the master metabolic sensor 5' AMP-activated protein kinase (AMPK) and Folliculin, a protein mutated in individuals with Birt-Hogg-Dubé Syndrome. In response to low energy, AMPK stimulates catabolic pathways such as autophagy to enhance energy production while inhibiting anabolic pathways regulated by the mechanistic target of rapamycin complex 1 (mTORC1). We previously found that constitutive disruption of Fnip1 in mice resulted in a lack of peripheral B cells because of a block in B cell development at the pre-B cell stage. Both AMPK and mTORC1 were activated in Fnip1-deficient B cell progenitors. In this study, we found inappropriate mTOR localization at the lysosome under nutrient-depleted conditions. Ex vivo lysine or arginine depletion resulted in increased apoptosis. Genetic inhibition of AMPK, inhibition of mTORC1, or restoration of cell viability with a Bcl-xL transgene failed to rescue B cell development in Fnip1-deficient mice. Fnip1-deficient B cell progenitors exhibited increased nuclear localization of transcription factor binding to IgHM enhancer 3 (TFE3) in developing B cells, which correlated with an increased expression of TFE3-target genes, increased lysosome numbers and function, and increased autophagic flux. These results indicate that Fnip1 modulates autophagy and energy response pathways in part through the regulation of AMPK, mTORC1, and TFE3 in B cell progenitors.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Linfócitos B/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Transporte/metabolismo , Homeostase , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that coordinates nutrient and growth factor availability with cellular growth, division, and differentiation. Studies examining the roles of mTOR signaling in immune function revealed critical roles for mTOR in regulating T cell differentiation and function. However, few studies have investigated the roles of mTOR in early B cell development. In this study, we found that mTOR is highly activated during the pro- and pre-B stages of mouse B cell development. Conditional disruption of the mTOR coactivating protein Raptor in developing mouse B cells resulted in a developmental block at the pre-B cell stage, with a corresponding lack of peripheral B cells and loss of Ag-specific Ab production. Pre-B cell survival and proliferation were significantly reduced in Raptor-deficient mice. Forced expression of a transgenic BCR or a BclxL transgene on Raptor-deficient B cells failed to rescue B cell development, suggesting that pre-BCR signaling and B cell survival are impaired in a BclxL-independent manner. Raptor-deficient pre-B cells exhibited significant decreases in oxidative phosphorylation and glycolysis, indicating that loss of mTOR signaling in B cells significantly impairs cellular metabolic capacity. Treatment of mice with rapamycin, an allosteric inhibitor of mTOR, recapitulated the early B cell developmental block. Collectively, our data reveal a previously uncharacterized role for mTOR signaling in early B cell development, survival, and metabolism.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Células Precursoras de Linfócitos B/fisiologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Glicólise/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Fosforilação/efeitos dos fármacos , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos B/metabolismo , Proteína Regulatória Associada a mTOR , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/deficiência , Fatores de Transcrição , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Mammalian skeletal muscle is broadly characterized by the presence of two distinct categories of muscle fibers called type I "red" slow twitch and type II "white" fast twitch, which display marked differences in contraction strength, metabolic strategies, and susceptibility to fatigue. The relative representation of each fiber type can have major influences on susceptibility to obesity, diabetes, and muscular dystrophies. However, the molecular factors controlling fiber type specification remain incompletely defined. In this study, we describe the control of fiber type specification and susceptibility to metabolic disease by folliculin interacting protein-1 (Fnip1). Using Fnip1 null mice, we found that loss of Fnip1 increased the representation of type I fibers characterized by increased myoglobin, slow twitch markers [myosin heavy chain 7 (MyH7), succinate dehydrogenase, troponin I 1, troponin C1, troponin T1], capillary density, and mitochondria number. Cultured Fnip1-null muscle fibers had higher oxidative capacity, and isolated Fnip1-null skeletal muscles were more resistant to postcontraction fatigue relative to WT skeletal muscles. Biochemical analyses revealed increased activation of the metabolic sensor AMP kinase (AMPK), and increased expression of the AMPK-target and transcriptional coactivator PGC1α in Fnip1 null skeletal muscle. Genetic disruption of PGC1α rescued normal levels of type I fiber markers MyH7 and myoglobin in Fnip1-null mice. Remarkably, loss of Fnip1 profoundly mitigated muscle damage in a murine model of Duchenne muscular dystrophy. These results indicate that Fnip1 controls skeletal muscle fiber type specification and warrant further study to determine whether inhibition of Fnip1 has therapeutic potential in muscular dystrophy diseases.
Assuntos
Proteínas de Transporte/fisiologia , Fibras Musculares de Contração Rápida/patologia , Fibras Musculares de Contração Rápida/fisiologia , Fibras Musculares de Contração Lenta/patologia , Fibras Musculares de Contração Lenta/fisiologia , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteínas de Transporte/genética , Modelos Animais de Doenças , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Complexos Multiproteicos/metabolismo , Contração Muscular/fisiologia , Fadiga Muscular/fisiologia , Distrofia Muscular de Duchenne/genética , Mioglobina/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Folliculin-interacting protein 1 (Fnip1) is an adaptor protein that physically interacts with AMPK, an energy-sensing kinase that stimulates mitochondrial biogenesis and autophagy in response to low ATP, while turning off energy consumption mediated by mammalian target of rapamycin. Previous studies with Fnip1-null mice revealed that Fnip1 is essential for pre-B-cell development. Here we report a critical role of Fnip1 in invariant natural killer T (iNKT) cell development. Thymic iNKT development in Fnip1(-/-) mice was arrested at stage 2 (NK1.1(-)CD44(+)) but development of CD4, CD8, γδ T-cell, and NK cell lineages proceeded normally. Enforced expression of a Vα14Jα18 iNKT TCR transgene or loss of the proapoptotic protein Bim did not rescue iNKT cell maturation in Fnip1(-/-) mice. Whereas most known essential transcription factors for iNKT cell development were represented normally, Fnip1(-/-) iNKT cells failed to down-regulate Promyelocytic leukemia zinc finger compared with their WT counterparts. Moreover, Fnip1(-/-) iNKT cells contained hyperactive mTOR and reduced mitochondrial number despite lower ATP levels, resulting in increased sensitivity to apoptosis. These results indicate that Fnip1 is vital for iNKT cell development by maintaining metabolic homeostasis in response to metabolic stress.
Assuntos
Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Metabolismo Energético/imunologia , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Síndrome de Birt-Hogg-Dubé/imunologia , Síndrome de Birt-Hogg-Dubé/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proteínas de Transporte/genética , Sobrevivência Celular/imunologia , Feminino , Homeostase/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/citologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Serina-Treonina Quinases TOR/imunologia , Serina-Treonina Quinases TOR/metabolismo , Timo/citologia , Timo/imunologiaRESUMO
Inborn errors of immunity (IEI) are a group of diseases in humans that typically present as increased susceptibility to infections, autoimmunity, hyperinflammation, allergy, and in some cases malignancy. Among newly identified genes linked to IEIs include 3 independent reports of 9 individuals from 7 independent kindreds with severe primary immunodeficiency disease (PID) and autoimmunity due to loss-of-function mutations in the NCKAP1L gene encoding Hematopoietic protein 1 (HEM1). HEM1 is a hematopoietic cell specific component of the WASp family verprolin homologous (WAVE) regulatory complex (WRC), which acts downstream of multiple immune receptors to stimulate actin nucleation and polymerization of filamentous actin (F-actin). The polymerization and branching of F-actin is critical for creating force-generating cytoskeletal structures which drive most active cellular processes including migration, adhesion, immune synapse formation, and phagocytosis. Branched actin networks at the cell cortex have also been implicated in acting as a barrier to regulate inappropriate vesicle (e.g. cytokine) secretion and spontaneous antigen receptor crosslinking. Given the importance of the actin cytoskeleton in most or all hematopoietic cells, it is not surprising that HEM1 deficient children present with a complex clinical picture that involves overlapping features of immunodeficiency and autoimmunity. In this review, we will provide an overview of what is known about the molecular and cellular functions of HEM1 and the WRC in immune and other cells. We will describe the common clinicopathological features and immunophenotypes of HEM1 deficiency in humans and provide detailed comparative descriptions of what has been learned about Hem1 disruption using constitutive and immune cell-specific mouse knockout models. Finally, we discuss future perspectives and important areas for investigation regarding HEM1 and the WRC.
Assuntos
Síndromes de Imunodeficiência , Humanos , Animais , Camundongos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologiaRESUMO
At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the alphabeta T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide-MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection). Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf-MEK-ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to 'weak' positive selecting signals but not in response to 'strong' negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.
Assuntos
Calcineurina/metabolismo , Diferenciação Celular , Sistema de Sinalização das MAP Quinases , Timo/citologia , Timo/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Calcineurina/deficiência , Calcineurina/genética , Células Cultivadas , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ligantes , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Timo/enzimologia , Quinases raf/metabolismoRESUMO
Appropriate B cell activation is essential for adaptive immunity. In contrast to the molecular mechanisms that regulate positive signaling in immune responses, the counterbalancing negative regulatory pathways remain insufficiently understood. The Src homology domain 3 (SH3)-containing adapter protein SH3 lymphocyte protein 2 (SLy2, also known as hematopoietic adapter-containing SH3 and sterile α-motif (SAM) domains 1; HACS1) is strongly up-regulated upon B cell activation and functions as an endogenous immunoinhibitor in vivo, but the underlying molecular mechanisms of SLy2 function have been elusive. We have generated transgenic mice overexpressing SLy2 in B and T cells and have studied the biological effects of elevated SLy2 levels in Jurkat and HeLa cells. Our results demonstrate that SLy2 induces Rac1-dependent membrane ruffle formation and regulates cell spreading and polarization and that the SLy2 SH3 domain is essential for these effects. Using immunoprecipitation and confocal microscopy, we provide evidence that the actin nucleation-promoting factor cortactin is an SH3 domain-directed interaction partner of SLy2. Consistent with an important role of SLy2 for actin cytoskeletal reorganization, we further show that SLy2-transgenic B cells are severely defective in cell spreading. Together, our findings extend our mechanistic understanding of the immunoinhibitory roles of SLy2 in vivo and suggest that the physiological up-regulation of SLy2 observed upon B cell activation functions to counteract excessive B cell spreading.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/biossíntese , Linfócitos B/metabolismo , Citoesqueleto/metabolismo , Ativação Linfocitária/fisiologia , Regulação para Cima/fisiologia , Actinas/genética , Actinas/imunologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Linfócitos B/imunologia , Citoesqueleto/genética , Citoesqueleto/imunologia , Células HeLa , Humanos , Células Jurkat , Camundongos , Camundongos Transgênicos , Neuropeptídeos/genética , Neuropeptídeos/imunologia , Neuropeptídeos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/imunologia , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Domínios de Homologia de srcRESUMO
Hematopoietic protein-1 (Hem-1) is a member of the actin-regulatory WASp family verprolin homolog (WAVE) complex. Loss-of-function variants in the NCKAP1L gene encoding Hem-1 were recently discovered to result in primary immunodeficiency disease (PID) in children, characterized by poor specific Ab responses, increased autoantibodies, and high mortality. However, the mechanisms of how Hem-1 deficiency results in PID are unclear. In this study, we utilized constitutive and B cell-specific Nckap1l-KO mice to dissect the importance of Hem-1 in B cell development and functions. B cell-specific disruption of Hem-1 resulted in reduced numbers of recirculating follicular (FO), marginal zone (MZ), and B1 B cells. B cell migration in response to CXCL12 and -13 were reduced. T-independent Ab responses were nearly abolished, resulting in failed protective immunity to Streptococcus pneumoniae challenge. In contrast, T-dependent IgM and IgG2c, memory B cell, and plasma cell responses were more robust relative to WT control mice. B cell-specific Hem-1-deficient mice had increased autoantibodies against multiple autoantigens, and this correlated with hyperresponsive BCR signaling and increased representation of CD11c+T-bet+ age-associated B cell (ABC cells) - alterations associated with autoimmune diseases. These results suggest that dysfunctional B cells may be part of a mechanism explaining why loss-of-function Hem-1 variants result in recurring infections and autoimmunity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Autoanticorpos , Doenças Autoimunes , Linfócitos B , Imunidade Humoral , Actinas , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Linfócitos B/imunologia , Camundongos , Camundongos KnockoutRESUMO
Hematopoietic protein-1 (Hem-1) is a hematopoietic cell-specific actin-regulatory protein. Loss-of-function (LOF) variants in the NCKAP1L gene encoding Hem-1 have recently been found to result in primary immunodeficiency disease (PID) in humans, characterized by recurring respiratory infections, asthma, and high mortality. However, the mechanisms of how Hem-1 variants result in PID are not known. In this study, we generated constitutive and myeloid cell-specific Nckap1l-KO mice to dissect the importance of Hem-1 in lung immunity. We found that Hem-1-deficient mice accumulated excessive surfactant and cell debris in airways (pulmonary alveolar proteinosis) due to impaired development of alveolar macrophages (AMs) and reduced expression of the AM differentiation factor Pparg. Residual Hem-1-deficient AMs shifted to a proinflammatory phenotype, and Hem-1-deficient neutrophils and monocytes failed to migrate normally. Myeloid cell-specific Hem-1-deficient mice exhibited increased morbidity following influenza A virus or Streptococcus pneumoniae challenge. These results provide potential mechanisms for how LOF variants in Hem-1 result in recurring respiratory diseases.
Assuntos
Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Diferenciação Celular/genética , Macrófagos Alveolares/imunologia , Proteinose Alveolar Pulmonar/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Diferenciação Celular/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Neutrófilos/imunologia , PPAR gama/metabolismo , Fagocitose/genética , Fagocitose/imunologia , Proteinose Alveolar Pulmonar/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Alterations in genes encoding transforming growth factor-beta-signaling components contribute to colon cancer in humans. Similarly, mice deficient in the transforming growth factor-beta signaling molecule, Smad3, develop colon cancer, but only after a bacterial trigger occurs, resulting in chronic inflammation. To determine whether Smad3-null lymphocytes contribute to increased cancer susceptibility, we crossed Smad3-null mice with mice deficient in both B and T lymphocytes (Rag2(-/-) mice). Helicobacter-infected Smad3/Rag2-double knockout (DKO) mice had more diffuse inflammation and increased incidence of adenocarcinoma compared with Helicobacter-infected Smad3(-/-) or Rag2(-/-) mice alone. Adoptive transfer of WT CD4(+)CD25(+) T-regulatory cells provided significant protection of Smad3/Rag2-DKO from bacterial-induced typhlocolitis, dysplasia, and tumor development, whereas Smad3(-/-) T-regulatory cells provided no protection. Immunohistochemistry, real-time reverse transcriptase-polymerase chain reaction, and Western blot analyses of colonic tissues from Smad3/Rag2-DKO mice 1 week after Helicobacter infection revealed an influx of macrophages, enhanced nuclear factor-kappaB activation, increased Bcl(XL)/Bcl-2 expression, increased c-Myc expression, accentuated epithelial cell proliferation, and up-regulated IFN-gamma, IL-1alpha, TNF-alpha, IL-1beta, and IL-6 transcription levels. These results suggest that the loss of Smad3 increases susceptibility to colon cancer by at least two mechanisms: deficient T-regulatory cell function, which leads to excessive inflammation after a bacterial trigger; and increased expression of proinflammatory cytokines, enhanced nuclear factor-kappaB activation, and increased expression of both pro-oncogenic and anti-apoptotic proteins that result in increased cell proliferation/survival of epithelial cells in colonic tissues.
Assuntos
Adenocarcinoma/genética , Neoplasias do Colo/genética , Proteínas de Ligação a DNA/deficiência , Infecções por Helicobacter/complicações , Proteína Smad3/deficiência , Fator de Crescimento Transformador beta/metabolismo , Adenocarcinoma/imunologia , Adenocarcinoma/microbiologia , Animais , Western Blotting , Neoplasias do Colo/imunologia , Neoplasias do Colo/microbiologia , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Citometria de Fluxo , Infecções por Helicobacter/imunologia , Imuno-Histoquímica , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/patologia , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad3/genética , Linfócitos T Reguladores/imunologiaRESUMO
Accurate identification of lymph nodes in the mouse is critical for studies of tumor metastasis, and of regional immune responses following immunization. However, these small lymphatic organs are often difficult to identify in mice using standard dissection techniques, so that larger rats have been used to characterize rodent lymphatic drainage. We developed techniques injecting dye into the mouse footpad or tail, to label the lymphatic drainage of the hind leg and flank, pelvic viscera, prostate and mammary glands. While lymphatic drainage patterns were similar in mice and rats, the inguinal lymph nodes showed distinct differences in afferent and efferent drainage. These techniques allow accurate and rapid identification of lymph nodes and lymphatic drainage in normal as well as diseased mice.
Assuntos
Meios de Contraste/farmacocinética , Corantes Fluorescentes/farmacocinética , Linfonodos/anatomia & histologia , Linfonodos/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição TecidualRESUMO
The corepressor mSin3A is the core component of a chromatin-modifying complex that is recruited by multiple gene-specific transcriptional repressors. In order to understand the role of mSin3A during development, we generated constitutive germ line as well as conditional msin3A deletions. msin3A deletion in the developing mouse embryo results in lethality at the postimplantation stage, demonstrating that it is an essential gene. Blastocysts derived from preimplantation msin3A null embryos and mouse embryo fibroblasts (MEFs) lacking msin3A display a significant reduction in cell division. msin3A null MEFs also show mislocalization of the heterochromatin protein, HP1alpha, without alterations in global histone acetylation. Heterozygous msin3A(+/-) mice with a systemic twofold decrease in mSin3A protein develop splenomegaly as well as kidney disease indicative of a disruption of lymphocyte homeostasis. Conditional deletion of msin3A from developing T cells results in reduced thymic cellularity and a fivefold decrease in the number of cytotoxic (CD8) T cells, while helper (CD4) T cells are unaffected. We show that CD8 development is dependent on mSin3A at a step downstream of T-cell receptor signaling and that loss of mSin3A specifically decreases survival of double-positive and CD8 T cells. Thus, msin3A is a pleiotropic gene which, in addition to its role in cell cycle progression, is required for the development and homeostasis of cells in the lymphoid lineage.
Assuntos
Cromatina/metabolismo , Proteínas Repressoras/fisiologia , Linfócitos T/citologia , Animais , Apoptose , Blastocisto , Western Blotting , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Ciclo Celular , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Cromatina/química , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/metabolismo , Éxons , Fibroblastos/citologia , Fibroblastos/metabolismo , Citometria de Fluxo , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Glomerulonefrite Membranosa , Heterocromatina/metabolismo , Heterozigoto , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Modelos Genéticos , Recombinação Genética , Complexo Correpressor Histona Desacetilase e Sin3 , Esplenomegalia , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/citologia , Timo/citologia , Fatores de TempoRESUMO
Accumulating evidence suggests that intestinal microbial organisms may play an important role in triggering and sustaining inflammation in individuals afflicted with inflammatory bowel disease (IBD). Moreover, individuals with IBD are at increased risk for developing colorectal cancer, suggesting that chronic inflammation may initiate genetic or epigenetic changes associated with cancer development. We tested the hypothesis that bacteria may contribute to the development of colon cancer by synergizing with defective transforming growth factor-beta (TGF-beta) signaling, a pathway commonly mutated in human colon cancer. Although others have reported that mice deficient in the TGF-beta signaling molecule SMAD3 develop colon cancer, we found that SMAD3-deficient mice maintained free of the Gram-negative enterohepatic bacteria Helicobacter spp. for up to 9 months do not develop colon cancer. Furthermore, infection of SMAD3(-/-) mice with Helicobacter triggers colon cancer in 50% to 66% of the animals. Using real-time PCR, we found that Helicobacter organisms concentrate in the cecum, the preferred site of tumor development. Mucinous adenocarcinomas develop 5 to 30 weeks after infection and are preceded by an early inflammatory phase, consisting of increased proliferation of epithelial cells; increased numbers of cyclooxygenase-2-positive cells, CD4(+) T cells, macrophages; and increased MHC class II expression. Colonic tissue revealed increased transcripts for the oncogene c-myc and the proinflammatory cytokines interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IFN-gamma, and tumor necrosis factor-alpha, some of which have been implicated in colon cancer. These results suggest that bacteria may be important in triggering colorectal cancer, notably in the context of gene mutations in the TGF-beta signaling pathway, one of the most commonly affected cellular pathways in colorectal cancer in humans.
Assuntos
Neoplasias do Colo/genética , Neoplasias do Colo/microbiologia , Infecções por Helicobacter/complicações , Proteína Smad3/genética , Animais , Ceco/microbiologia , Proliferação de Células , Citocinas/biossíntese , DNA Bacteriano/análise , Feminino , Predisposição Genética para Doença , Inflamação , Masculino , Camundongos , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-myc , Fatores de Risco , Transdução de Sinais , Proteína Smad3/fisiologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Noroviruses are a leading cause of gastroenteritis in humans and it was recently revealed that noroviruses can infect B cells. We demonstrate that murine norovirus (MNV) infection can significantly impair B cell development in the bone marrow in a signal transducer and activator of transcription 1 (STAT1) dependent, but interferon signaling independent manner. We also show that MNV replication is more pronounced in the absence of STAT1 in ex vivo cultured B cells. Interestingly, using bone marrow transplantation studies, we found that impaired B cell development requires Stat1-/- hematopoietic cells and Stat1-/- stromal cells, and that the presence of wild-type hematopoietic or stromal cells was sufficient to restore normal development of Stat1-/- B cells. These results suggest that B cells normally restrain norovirus replication in a cell autonomous manner, and that wild-type STAT1 is required to protect B cell development during infection.
Assuntos
Linfócitos B/metabolismo , Medula Óssea/metabolismo , Infecções por Caliciviridae/metabolismo , Gastroenterite/metabolismo , Norovirus/fisiologia , Fator de Transcrição STAT1/deficiência , Animais , Linfócitos B/virologia , Medula Óssea/virologia , Infecções por Caliciviridae/virologia , Feminino , Gastroenterite/virologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Norovirus/genética , Fator de Transcrição STAT1/genética , Replicação ViralRESUMO
Birt-Hogg-Dube' Syndrome (BHDS) is a rare genetic disorder in humans characterized by skin hamartomas, lung cysts, pneumothorax, and increased risk of renal tumors. BHDS is caused by mutations in the BHD gene, which encodes for Folliculin, a cytoplasmic adapter protein that binds to Folliculin interacting proteins-1 and -2 (Fnip1, Fnip2) as well as the master energy sensor AMP kinase (AMPK). Whereas kidney-specific deletion of the Bhd gene in mice is known to result in polycystic kidney disease (PKD) and renal cell carcinoma, the roles of Fnip1 in renal cell development and function are unclear. In this study, we utilized mice with constitutive deletion of the Fnip1 gene to show that the loss of Fnip1 is sufficient to result in renal cyst formation, which was characterized by decreased AMPK activation, increased mTOR activation, and metabolic hyperactivation. Using RNAseq, we found that Fnip1 disruption resulted in many cellular and molecular changes previously implicated in the development of PKD in humans, including alterations in the expression of ion and amino acid transporters, increased cell adhesion, and increased inflammation. Loss of Fnip1 synergized with Tsc1 loss to hyperactivate mTOR, increase Erk activation, and greatly accelerate the development of PKD. Our results collectively define roles for Fnip1 in regulating kidney development and function, and provide a model for how loss of Fnip1 contributes to PKD and perhaps renal cell carcinoma.
Assuntos
Proteínas de Transporte/genética , Cistos/genética , Deleção de Genes , Rim/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Transcrição Gênica/genética , Proteína 1 do Complexo Esclerose Tuberosa/genética , Animais , Proteínas de Transporte/metabolismo , Cistos/patologia , Ativação Enzimática/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Perfilação da Expressão Gênica , Genótipo , Rim/crescimento & desenvolvimento , Rim/patologia , Camundongos , Tamanho do Órgão/genética , Fosforilação Oxidativa , Proteína 1 do Complexo Esclerose Tuberosa/deficiênciaRESUMO
Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase originally discovered as the molecular target of the immunosuppressant rapamycin. mTOR forms two compositionally and functionally distinct complexes, mTORC1 and mTORC2, which are crucial for coordinating nutrient, energy, oxygen, and growth factor availability with cellular growth, proliferation, and survival. Recent studies have identified critical, non-redundant roles for mTORC1 and mTORC2 in controlling B cell development, differentiation, and functions, and have highlighted emerging roles of the Folliculin-Fnip protein complex in regulating mTOR and B cell development. In this review, we summarize the basic mechanisms of mTOR signaling; describe what is known about the roles of mTORC1, mTORC2, and the Folliculin/Fnip1 pathway in B cell development and functions; and briefly outline current clinical approaches for targeting mTOR in B cell neoplasms. We conclude by highlighting a few salient questions and future perspectives regarding mTOR in B lineage cells.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Proteínas de Transporte/metabolismo , Proliferação de Células , Estrona/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos , FosforilaçãoRESUMO
A major challenge in inflammatory bowel disease (IBD) is the integration of diverse IBD data sets to construct predictive models of IBD. We present a predictive model of the immune component of IBD that informs causal relationships among loci previously linked to IBD through genome-wide association studies (GWAS) using functional and regulatory annotations that relate to the cells, tissues, and pathophysiology of IBD. Our model consists of individual networks constructed using molecular data generated from intestinal samples isolated from three populations of patients with IBD at different stages of disease. We performed key driver analysis to identify genes predicted to modulate network regulatory states associated with IBD, prioritizing and prospectively validating 12 of the top key drivers experimentally. This validated key driver set not only introduces new regulators of processes central to IBD but also provides the integrated circuits of genetic, molecular, and clinical traits that can be directly queried to interrogate and refine the regulatory framework defining IBD.
Assuntos
Redes Reguladoras de Genes , Genes Reguladores , Genômica/métodos , Doenças Inflamatórias Intestinais/genética , Modelos Genéticos , Transferência Adotiva , Animais , Causalidade , Células Cultivadas , Colite/induzido quimicamente , Colite/genética , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Interferente Pequeno/genética , Subpopulações de Linfócitos T/transplante , TranscriptomaRESUMO
Tumor-draining lymph nodes (TDLNs) often enlarge in human cancer patients and in murine tumor models, due to lymphocyte accumulation and lymphatic sinus growth. B lymphocytes within TDLNs can drive lymph node hypertrophy in response to tumor growth, however little is known about the mechanisms directing the preferential accumulation of B lymphocytes relative to T cells in enlarging TDLNs. To define why B and T lymphocytes accumulate in TDLNs, we quantified lymphocyte proliferation, apoptosis, entry, and exit in TDLNs versus contralateral non-TDLNs (NTDLNs) in a footpad B16-F10 melanoma mouse model. B and T lymphocyte proliferation and apoptosis were increased as the TDLNs enlarged, although relative rates were similar to those of NTDLNs. TDLN entry of B and T lymphocytes via high endothelial venules was also modestly increased in enlarged TDLNs. Strikingly, the egress of B cells was strongly reduced in TDLNs versus NTDLNs, while T cell egress was modestly decreased, indicating that regulation of lymphocyte exit from TDLNs is a major mechanism of preferential B lymphocyte accumulation. Surface sphingosine-1-phosphate receptor 1 (S1PR1) which binds S1P and signals lymphocyte egress, exhibited greater downregulation in B relative to T lymphocytes, consistent with preferential retention of B lymphocytes in TDLNs. TDLN lymphocytes did not activate surface CD69 expression, indicating a CD69-independent mechanism of downregulation of S1PR1. B and T cell trafficking via afferent lymphatics to enter TDLNs also increased, suggesting a pathway for accumulation of tumor-educated lymphocytes in TDLNs. These mechanisms regulating TDLN hypertrophy could provide new targets to manipulate lymphocyte responses to cancer.