RESUMO
B cells predominate in a quiescent state until an antigen is encountered, which results in rapid growth, proliferation and differentiation of the B cells. These distinct cell states are probably accompanied by differing metabolic needs, yet little is known about the metabolic control of B cell fate. Here we show that glycogen synthase kinase 3 (Gsk3) is a metabolic sensor that promotes the survival of naive recirculating B cells by restricting cell mass accumulation. In antigen-driven responses, Gsk3 was selectively required for regulation of B cell size, mitochondrial biogenesis, glycolysis and production of reactive oxygen species (ROS), in a manner mediated by the co-stimulatory receptor CD40. Gsk3 was required to prevent metabolic collapse and ROS-induced apoptosis after glucose became limiting, functioning in part by repressing growth dependent on the myelocytomatosis oncoprotein c-Myc. Notably, we found that Gsk3 was required for the generation and maintenance of germinal center B cells, which require high glycolytic activity to support growth and proliferation in a hypoxic microenvironment.
Assuntos
Linfócitos B/fisiologia , Centro Germinativo/imunologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Animais , Antígenos CD19/genética , Antígenos CD19/metabolismo , Apoptose/genética , Ligante de CD40/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Glicogênio Sintase Quinase 3 beta/genética , Glicólise , Interleucina-4/metabolismo , Camundongos , Camundongos Knockout , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Mature naive B cells expressing BCRs of the IgM and IgD isotypes respond to Ag in secondary lymphoid organs. However, the vast majority of B cells do not undergo productive Ag encounter and have finite life spans dependent on survival signals propagated by the BCR and the BAFFR. In this study, we show that the E3 ubiquitin ligase Fbw7 is required for the maintenance of mature B cell populations in mice. BCR stimulation of B cells induced substantial apoptosis along with proliferative and growth defects upon the loss of Fbw7. Analysis of B cell proteomes revealed aberrant signaling patterns, including lower Bcl2 and diminished NF-κB signaling. Further, excessive accumulation of Fbw7 substrate c-Myc, increased Bim expression, and loss of PI3K signaling mediated apoptosis downstream of BCR signaling. In accordance, strong prosurvival signals delivered through ectopic expression of BCL2 in B cells could largely rescue apoptotic cells in the absence of Fbw7. Overall, this study reveals an unexpected role for Fbw7 in the survival and fitness of mature B cells.
Assuntos
Linfócitos B/fisiologia , Proteína 7 com Repetições F-Box-WD/metabolismo , Animais , Apoptose/imunologia , Proteína 11 Semelhante a Bcl-2/metabolismo , Proliferação de Células/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Proteína 7 com Repetições F-Box-WD/genética , Feminino , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Ubiquitinação/genética , Ubiquitinação/imunologiaRESUMO
B cells contribute critically to an effective immune response by producing Ag-specific Abs. During the immune response to so-called "thymus-dependent Ags," activated B cells seek T cell help and form germinal centers. In contrast, thymus-independent Ags generally do not induce germinal center formation. In the germinal center, B cells undergo somatic hypermutation, affinity-based clonal expansion, and differentiation to produce plasma cells and memory B cells. Valuable insight into these processes has been gained by using model hapten-carrier complexes or SRBCs. SRBCs induce robust germinal center formation in mice. Therefore, this Ag is commonly used to study germinal center responses. In contrast to haptenated Ags, thus far it has been difficult to measure the titer of Ag-specific Abs or the expansion of Ag-specific B cells after immunization with SRBCs. We have developed new, simple methods to access these parameters, thus providing new tools to study germinal center and Ab responses.
Assuntos
Linfócitos B/fisiologia , Transfusão de Eritrócitos/métodos , Eritrócitos/imunologia , Centro Germinativo/imunologia , Imunidade Humoral , Técnicas Imunológicas/métodos , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Bovinos , Diferenciação Celular , Células Cultivadas , Feminino , Imunização , Memória Imunológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hipermutação Somática de ImunoglobulinaRESUMO
Regnase-1 is an emerging regulator of immune responses with essential roles in the posttranscriptional control of immune cell activation. Regnase-1 is expressed in B cells; however, its B cell-specific functions remain unknown. Here, we demonstrate that Regnase-1 prevents severe autoimmune pathology and show its essential role in maintaining B cell homeostasis. Using Cre driver mice for ablation of Regnase-1 at various stages of B cell development, we demonstrate that loss of Regnase-1 leads to aberrant B cell activation and differentiation, resulting in systemic autoimmunity and early morbidity. The basis of these findings was informed by gene expression data revealing a regulatory role for Regnase-1 in the suppression of a transcriptional program that promotes B cell activation, survival, and differentiation. Overall, our study shows that Regnase-1 exerts critical control of B cell activation, which is required for prevention of immunopathology.
Assuntos
Autoimunidade/genética , Linfócitos B/metabolismo , Homeostase/genética , Ativação Linfocitária/genética , Ribonucleases/genética , Animais , Diferenciação Celular/genética , Perfilação da Expressão Gênica/métodos , Camundongos Knockout , Camundongos Transgênicos , RNA-Seq/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ribonucleases/metabolismoRESUMO
The PI3K pathway is integral for the germinal center (GC) response. However, the contribution of protein kinase B (AKT) as a PI3K effector in GC B cells remains unknown. Here, we show that mice lacking the AKT1 and AKT2 isoforms in B cells failed to form GCs, which undermined affinity maturation and antibody production in response to immunization. Upon B-cell receptor stimulation, AKT1/2-deficient B cells showed poor survival, reduced proliferation, and impaired mitochondrial and metabolic fitness, which collectively halted GC development. By comparison, Foxo1 T24A mutant, which cannot be inactivated by AKT1/2 phosphorylation and is sequestered in the nucleus, significantly enhanced antibody class switch recombination via induction of activation-induced cytidine deaminase (AID) expression. By contrast, repression of FOXO1 activity by AKT1/2 promoted IRF4-driven plasma cell differentiation. Last, we show that T-cell help via CD40, but not enforced expression of Bcl2, rescued the defective GC response in AKT1/2-deficient animals by restoring proliferative expansion and energy production. Overall, our study provides mechanistic insights into the key role of AKT and downstream pathways on B cell fate decisions during the GC response.
Assuntos
Linfócitos B/citologia , Centro Germinativo/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Formação de Anticorpos , Linfócitos B/metabolismo , Diferenciação Celular/imunologia , Feminino , Proteína Forkhead Box O1/metabolismo , Centro Germinativo/citologia , Switching de Imunoglobulina , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinases/metabolismo , Isoformas de Proteínas , Receptores de Antígenos de Linfócitos B/genética , Linfócitos T/imunologiaRESUMO
Immune homeostasis in the gut associated lymphoid tissues (GALT) is critical to prevent the development of inadvertent pathologies. B cells as the producers of antibodies and cytokines plays an important role in maintaining the GALT homeostasis. However, the mechanism by which B cells specifically direct their responses towards non-self-antigens and become ignorant to self-antigens in the GALT is not known. Therefore, we developed a novel mouse model by expressing Duck Egg Lysozyme (DEL) in gut epithelial cells in presence of HEL reactive B cells. Notably, we observed a transient activation and rapid deletion of self-reactive B cells in Peyers Patches and Mesenteric lymph nodes upon self-antigen exposure. The survival of self-reactive B cells upon exposure to their self-antigen was partially rescued by blocking receptor editing but could be completely rescued by stronger survival signal like ectopic expression of BCL2. Importantly, rescuing the self-reactive B cells promoted production of auto-antibodies and gut inflammation. Mechanistically, we identify a specific activation of TGFß signaling in self-reactive B cells in the gut and a critical role of this pathway in maintaining peripheral tolerance. Collectively, our studies describe functional consequences and fate of self-reactive B cells in GALT and provide novel mechanistic insights governing self-tolerance of B cells in the gut.
Assuntos
Linfócitos B/imunologia , Trato Gastrointestinal/imunologia , Inflamação/prevenção & controle , Ativação Linfocitária , Animais , Autoantígenos/imunologia , Medula Óssea , Células Epiteliais/imunologia , Trato Gastrointestinal/patologia , Homeostase , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Muramidase/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Galectin-3 is a member of a large family of animal lectins. This protein is expressed abundantly by macrophages, but its function in this cell type is not well understood. We have studied the effect of galectin-3 gene targeting on phagocytosis, a major function of macrophages. Compared with wild-type macrophages, galectin-3-deficient (gal3-/-) cells exhibited reduced phagocytosis of IgG-opsonized erythrocytes and apoptotic thymocytes in vitro. In addition, gal3-/- mice showed attenuated phagocytic clearance of apoptotic thymocytes by peritoneal macrophages in vivo. These mice also exhibited reduced IgG-mediated phagocytosis of erythrocytes by Kupffer cells in a murine model of autoimmune hemolytic anemia. Additional experiments indicate that extracellular galectin-3 does not contribute appreciably to the phagocytosis-promoting function of this protein. Confocal microscopic analysis of macrophages containing phagocytosed erythrocytes revealed localization of galectin-3 in phagocytic cups and phagosomes. Furthermore, gal3-/- macrophages exhibited a lower degree of actin rearrangement upon Fcgamma receptor crosslinkage. These results indicate that galectin-3 contributes to macrophage phagocytosis through an intracellular mechanism. Thus, galectin-3 may play an important role in both innate and adaptive immunity by contributing to phagocytic clearance of microorganisms and apoptotic cells.
Assuntos
Galectina 3/fisiologia , Macrófagos Peritoneais/fisiologia , Fagocitose/fisiologia , Anemia Hemolítica Autoimune/imunologia , Anemia Hemolítica Autoimune/patologia , Anemia Hemolítica Autoimune/fisiopatologia , Animais , Apoptose , Eritrócitos/imunologia , Eritrócitos/fisiologia , Galectina 3/deficiência , Galectina 3/genética , Imunoglobulina G/metabolismo , Técnicas In Vitro , Células de Kupffer/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Opsonizantes/metabolismo , Receptores de IgG/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
Galectin-3 is a member of a beta-galactoside-binding animal lectin family. Previous in vitro studies have demonstrated that galectin-3 is involved in a number of activities; however, the roles of this lectin in physiological and pathological processes in vivo remain to be elucidated. Herein, we show, in a murine model of ovalbumin (OVA)-induced asthma that 1) peribronchial inflammatory cells expressed large amounts of galectin-3; 2) bronchoalveolar lavage fluid from OVA-challenged mice contained significantly higher levels of galectin-3 compared to control mice; and 3) macrophages in bronchoalveolar lavage fluid were the major cell type that contained galectin-3. We investigated the role of galectin-3 in the allergic airway response by comparing galectin-3-deficient (gal3(-/-)) mice and wild-type (gal3(+/+)) mice. OVA-sensitized gal3(-/-) mice developed fewer eosinophils and lower goblet cell metaplasia, after airway OVA challenge compared to similarly treated gal3(+/+) mice. In addition, the OVA-sensitized gal3(-/-) mice developed significantly less airway hyperresponsiveness after airway OVA challenge compared to gal3(+/+) mice. Finally, gal3(-/-) mice developed a lower Th2 response, but a higher Th1 response, suggesting that galectin-3 regulates the Th1/Th2 response. We conclude that galectin-3 may play an important role in the pathogenesis of asthma and inhibitors of this lectin may prove useful for treatment of this disease.