RESUMO
Systemic Lupus Erythematosus (SLE) is characterized by pathogenic autoantibodies against nucleic acid-containing antigens. Understanding which B-cell subsets give rise to these autoantibodies may reveal therapeutic approaches for SLE that spare protective responses. Mice lacking the tyrosine kinase Lyn, which limits B and myeloid cell activation, develop lupus-like autoimmune diseases characterized by increased autoreactive plasma cells (PCs). We used a fate-mapping strategy to determine the contribution of T-bet+ B cells, a subset thought to be pathogenic in lupus, to the accumulation of PCs and autoantibodies in Lyn-/- mice. Approximately, 50% of splenic PCs in Lyn-/- mice originated from T-bet+ cells, a significant increase compared to WT mice. In vitro, splenic PCs derived from T-bet+ B cells secreted both IgM and IgG anti-dsDNA antibodies. To determine the role of these cells in autoantibody production in vivo, we prevented T-bet+ B cells from differentiating into PCs or class switching in Lyn-/- mice. This resulted in a partial reduction in splenic PCs and anti-dsDNA IgM and complete abrogation of anti-dsDNA IgG. Thus, T-bet+ B cells make an important contribution to the autoreactive PC pool in Lyn-/- mice.
Assuntos
Lúpus Eritematoso Sistêmico , Plasmócitos , Animais , Camundongos , Autoanticorpos , Imunoglobulina G , Imunoglobulina M , Quinases da Família src/genéticaRESUMO
PI3K plays multiple roles throughout the life of a B cell. As such, its signaling is tightly regulated. The importance of this is illustrated by the fact that both loss- and gain-of-function mutations in PI3K can cause immunodeficiency in humans. PIK3IP1, also known as TrIP, is a transmembrane protein that has been shown to inhibit PI3K in T cells. Results from the ImmGen Consortium indicate that PIK3IP1 expression fluctuates throughout B cell development in a manner inversely correlated with PI3K activity; however, its role in B cells is poorly understood. In this study, we define the consequences of B cell-specific deletion of PIK3IP1. B cell development, basal Ig levels, and T-independent responses were unaffected by loss of PIK3IP1. However, there was a significant delay in the production of IgG during T-dependent responses, and secondary responses were impaired. This is likely due to a role for PIK3IP1 in the extrafollicular response because germinal center formation and affinity maturation were normal, and PIK3IP1 is not appreciably expressed in germinal center B cells. Consistent with a role early in the response, PIK3IP1 was downregulated at late time points after B cell activation, in a manner dependent on PI3K. Increased activation of the PI3K pathway was observed in PIK3IP1-deficient B cells in response to engagement of both the BCR and CD40 or strong cross-linking of CD40 alone. Taken together, these observations suggest that PIK3IP1 promotes extrafollicular responses by limiting PI3K signaling during initial interactions between B and T cells.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunidade Celular , Switching de Imunoglobulina , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas de Membrana/imunologia , Linfócitos T/imunologia , Animais , Linfócitos B/citologia , Antígenos CD40/genética , Antígenos CD40/imunologia , Centro Germinativo/citologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Linfócitos T/citologiaRESUMO
PURPOSE OF THE REVIEW: Systemic lupus erythematosus (SLE) is driven by nucleic acid-containing antigens that stimulate endosomal TLRs. We review new advances in our understanding of how TLR7 signaling in B cells drives autoimmunity. RECENT FINDINGS: Pathogenic B cell responses to TLR7 engagement are shaped by the disease-associated cytokine environment. TLR7, IFNγ, and IL-21 together promote the formation of autoreactive germinal centers and the ABC/DN2 B cell subset. BAFF and type 1 IFNs enhance autoantibody production from transitional B cells in concert with TLR7. TLR7 signaling components STAT1, BANK1, IRF5, SLC15A4, and CXorf21/TASL are associated genetically with SLE and important for lupus development in mice, while role of T-bet is controversial. Proper control of TLR7 trafficking by UNC93B1, syntenin-1, and αvß3 integrin is critical for preventing autoimmunity. A better understanding of TLR7 signaling has revealed potential new therapeutic approaches for SLE, several of which are being tested in animal models or clinical trials.
Assuntos
Lúpus Eritematoso Sistêmico , Receptor 7 Toll-Like , Animais , Autoimunidade , Linfócitos B/metabolismo , Humanos , Fatores Reguladores de Interferon , Camundongos , Transdução de Sinais , Receptor 7 Toll-Like/metabolismoRESUMO
B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR) or hyperactivation above maximum (for example, self-reactive BCR) thresholds of signalling strength causes negative selection. In â¼25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), which mimics constitutively active pre-BCR signalling. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival. We tested the hypothesis that targeted hyperactivation--above a maximum threshold--will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR-ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patient-derived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6 (ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1), we demonstrated that pharmacological hyperactivation of SYK and engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.
Assuntos
Linfócitos B/metabolismo , Linfócitos B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Transdução de Sinais , Motivos de Aminoácidos/genética , Animais , Antígenos CD/metabolismo , Linfócitos B/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Proteínas de Fusão bcr-abl/genética , Deleção de Genes , Humanos , Inositol Polifosfato 5-Fosfatases , Peptídeos e Proteínas de Sinalização Intracelular/agonistas , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/metabolismo , Células Precursoras de Linfócitos B/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos B/deficiência , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase Syk , Tirosina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Central tolerance checkpoints are critical for the elimination of autoreactive B cells and the prevention of autoimmunity. When autoreactive B cells encounter their Ag at the immature B cell stage, BCR cross-linking induces receptor editing, followed by apoptosis if edited cells remain autoreactive. Although the transcription factor Foxo1 is known to promote receptor editing, the role of the related factor Foxo3 in central B cell tolerance is poorly understood. We find that BCR-stimulated immature B cells from Foxo3-deficient mice demonstrate reduced apoptosis compared with wild type cells. Despite this, Foxo3-/- mice do not develop increased autoantibodies. This suggests that the increased survival of Foxo3-/- immature B cells allows additional rounds of receptor editing, resulting in more cells "redeeming" themselves by becoming nonautoreactive. Indeed, increased Igλ usage and increased recombining sequence recombination among Igλ-expressing cells were observed in Foxo3-/- mice, indicative of increased receptor editing. We also observed that deletion of high-affinity autoreactive cells was intact in the absence of Foxo3 in the anti-hen egg lysozyme (HEL)/membrane-bound HEL model. However, Foxo3 levels in B cells from systemic lupus erythematosus (SLE) patients were inversely correlated with disease activity and reduced in patients with elevated anti-dsDNA Abs. Although this is likely due in part to increased B cell activation in these SLE patients, it is also possible that low-affinity B cells that remain autoreactive after editing may survive inappropriately in the absence of Foxo3 and become activated to secrete autoantibodies in the context of other SLE-associated defects.
Assuntos
Apoptose/imunologia , Linfócitos B/imunologia , Proteína Forkhead Box O3/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoimunidade/imunologia , Diferenciação Celular/imunologia , Feminino , Tolerância Imunológica/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Precursoras de Linfócitos B/imunologiaRESUMO
SLE is a chronic autoimmune disease caused by perturbations of the immune system. The clinical presentation is heterogeneous, largely because of the multiple genetic and environmental factors that contribute to disease initiation and progression. Over the last 60 years, there have been a number of significant leaps in our understanding of the immunological mechanisms driving disease processes. We now know that multiple leucocyte subsets, together with inflammatory cytokines, chemokines and regulatory mediators that are normally involved in host protection from invading pathogens, contribute to the inflammatory events leading to tissue destruction and organ failure. In this broad overview, we discuss the main pathways involved in SLE and highlight new findings. We describe the immunological changes that characterize this form of autoimmunity. The major leucocytes that are essential for disease progression are discussed, together with key mediators that propagate the immune response and drive the inflammatory response in SLE.
Assuntos
Autoimunidade/imunologia , Linfócitos B/imunologia , Citocinas/imunologia , Meio Ambiente , Inflamação/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Autoimunidade/genética , Predisposição Genética para Doença , Humanos , Inflamação/genética , Lúpus Eritematoso Sistêmico/genética , Tolerância a Antígenos Próprios/genética , Tolerância a Antígenos Próprios/imunologiaRESUMO
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by excess B- and T-cell activation, the development of autoantibodies against self-antigens including nuclear antigens, and immune complex deposition in target organs, which triggers an inflammatory response and tissue damage. The genetic and environmental factors that contribute to the development of SLE have been studied extensively in both humans and mouse models of the disease. One of the important genetic contributions to SLE development is an alteration in the expression of the transcription factor Ets1, which regulates the functional differentiation of lymphocytes. Here, we review the genetic, biochemical, and immunological studies that have linked low levels of Ets1 to aberrant lymphocyte differentiation and to the pathogenesis of SLE.
RESUMO
Tight control of B cell differentiation into plasma cells (PCs) is critical for proper immune responses and the prevention of autoimmunity. The Ets1 transcription factor acts in B cells to prevent PC differentiation. Ets1(-/-) mice accumulate PCs and produce autoantibodies. Ets1 expression is downregulated upon B cell activation through the BCR and TLRs and is maintained by the inhibitory signaling pathway mediated by Lyn, CD22 and SiglecG, and SHP-1. In the absence of these inhibitory components, Ets1 levels are reduced in B cells in a Btk-dependent manner. This leads to increased PCs, autoantibodies, and an autoimmune phenotype similar to that of Ets1(-/-) mice. Defects in inhibitory signaling molecules, including Lyn and Ets1, are associated with human lupus, although the effects are more subtle than the complete deficiency that occurs in knockout mice. In this study, we explore the effect of partial disruption of the Lyn/Ets1 pathway on B cell tolerance and find that Lyn(+/-)Ets1(+/-) mice demonstrate greater and earlier production of IgM, but not IgG, autoantibodies compared with Lyn(+/-) or Ets1(+/-) mice. We also show that Btk-dependent downregulation of Ets1 is important for normal PC homeostasis when inhibitory signaling is intact. Ets1 deficiency restores the decrease in steady state PCs and Ab levels observed in Btk(-/-) mice. Thus, depending on the balance of activating and inhibitory signals to Ets1, there is a continuum of effects on autoantibody production and PC maintenance. This ranges from full-blown autoimmunity with complete loss of Ets1-maintaining signals to reduced PC and Ab levels with impaired Ets1 downregulation.
Assuntos
Anticorpos/imunologia , Proteínas Tirosina Quinases/imunologia , Proteína Proto-Oncogênica c-ets-1/imunologia , Quinases da Família src/imunologia , Tirosina Quinase da Agamaglobulinemia , Animais , Anticorpos/metabolismo , Autoanticorpos/sangue , Autoanticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Ensaio de Imunoadsorção Enzimática , Epistasia Genética , Citometria de Fluxo , Expressão Gênica/imunologia , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasmócitos/imunologia , Plasmócitos/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Baço/imunologia , Baço/metabolismo , Esplenomegalia/genética , Esplenomegalia/imunologia , Esplenomegalia/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Systemic lupus erythematosus (SLE) is characterized by antibody-mediated chronic inflammation in the kidney, lung, skin, and other organs to cause inflammation and damage. Several inflammatory pathways are dysregulated in SLE, and understanding these pathways may improve diagnosis and treatment. In one such pathway, Axl tyrosine kinase receptor responds to Gas6 ligand to block inflammation in leukocytes. A soluble form of the Axl receptor ectodomain (sAxl) is elevated in serum from patients with SLE and lupus-prone mice. We hypothesized that sAxl in SLE serum originates from the surface of leukocytes and that the loss of leukocyte Axl contributes to the disease. We determined that macrophages and B cells are a source of sAxl in SLE and in lupus-prone mice. Shedding of the Axl ectodomain from the leukocytes of lupus-prone mice is mediated by the matrix metalloproteases ADAM10 and TACE (ADAM17). Loss of Axl from lupus-prone macrophages renders them unresponsive to Gas6-induced anti-inflammatory signaling in vitro. This phenotype is rescued by combined ADAM10/TACE inhibition. Mice with Axl-deficient macrophages develop worse disease than controls when challenged with anti-glomerular basement membrane (anti-GBM) sera in an induced model of nephritis. ADAM10 and TACE also mediate human SLE PBMC Axl cleavage. Collectively, these studies indicate that increased metalloprotease-mediated cleavage of leukocyte Axl may contribute to end organ disease in lupus. They further suggest dual ADAM10/TACE inhibition as a potential therapeutic modality in SLE.
Assuntos
Proteína ADAM10/imunologia , Proteína ADAM17/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Proteínas Proto-Oncogênicas/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Proteína ADAM10/metabolismo , Proteína ADAM17/metabolismo , Adulto , Animais , Western Blotting , Linhagem Celular , Feminino , Expressão Gênica/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto Jovem , Receptor Tirosina Quinase AxlRESUMO
Signaling through the BCR can drive B cell activation and contribute to B cell differentiation into Ab-secreting plasma cells. The positive BCR signal is counterbalanced by a number of membrane-localized inhibitory receptors that limit B cell activation and plasma cell differentiation. Deficiencies in these negative signaling pathways may cause autoantibody generation and autoimmune disease in both animal models and human patients. We have previously shown that the transcription factor Ets1 can restrain B cell differentiation into plasma cells. In this study, we tested the roles of the BCR and inhibitory receptors in controlling the expression of Ets1 in mouse B cells. We found that Ets1 is downregulated in B cells by BCR or TLR signaling through a pathway dependent on PI3K, Btk, IKK2, and JNK. Deficiencies in inhibitory pathways, such as a loss of the tyrosine kinase Lyn, the phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP1) or membrane receptors CD22 and/or Siglec-G, result in enhanced BCR signaling and decreased Ets1 expression. Restoring Ets1 expression in Lyn- or SHP1-deficient B cells inhibits their enhanced plasma cell differentiation. Our findings indicate that downregulation of Ets1 occurs in response to B cell activation via either BCR or TLR signaling, thereby allowing B cell differentiation and that the maintenance of Ets1 expression is an important function of the inhibitory Lyn â CD22/SiglecG â SHP1 pathway in B cells.
Assuntos
Diferenciação Celular/imunologia , Plasmócitos/imunologia , Proteína Proto-Oncogênica c-ets-1/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Western Blotting , Diferenciação Celular/genética , Linhagem Celular Tumoral , Expressão Gênica/imunologia , Lectinas/deficiência , Lectinas/genética , Lectinas/imunologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/imunologia , Plasmócitos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Proteína Proto-Oncogênica c-ets-1/deficiência , Proteína Proto-Oncogênica c-ets-1/genética , Receptores de Antígenos de Linfócitos B/deficiência , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/deficiência , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Transdução de Sinais/genética , Quinases da Família src/deficiência , Quinases da Família src/genética , Quinases da Família src/imunologiaRESUMO
The autoimmune disease systemic lupus erythematosus is characterized by loss of tolerance to nuclear Ags and a heightened inflammatory environment, which together result in end organ damage. Lyn-deficient mice, a model of systemic lupus erythematosus, lack an inhibitor of B-cell and myeloid cell activation. This results in B-cell hyper-responsiveness, plasma cell accumulation, autoantibodies, and glomerulonephritis (GN). IL-21 is associated with autoimmunity in mice and humans and promotes B-cell differentiation and class switching. Here, we explore the role of IL-21 in the autoimmune phenotypes of lyn(-/-) mice. We find that IL-21 mRNA is reduced in the spleens of lyn(-/-) IL-6(-/-) and lyn(-/-) Btk(lo) mice, neither of which produce pathogenic autoantibodies or develop significant GN. While IL-21 is dispensable for plasma cell accumulation and IgM autoantibodies in lyn(-/-) mice, it is required for anti-DNA IgG antibodies and some aspects of T-cell activation. Surprisingly, GN still develops in lyn(-/-) IL-21(-/-) mice. This likely results from the presence of IgG autoantibodies against a limited set of non-DNA Ags. These studies identify a specific role for IL-21 in the class switching of anti-DNA B cells and demonstrate that neither IL-21 nor anti-DNA IgG is required for kidney damage in lyn(-/-) mice.
Assuntos
Anticorpos Antinucleares/imunologia , DNA/imunologia , Imunoglobulina G/imunologia , Interleucinas/imunologia , Rim/imunologia , Quinases da Família src/genética , Tirosina Quinase da Agamaglobulinemia , Animais , Autoanticorpos/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , DNA/genética , Glomerulonefrite/genética , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Interleucina-6/deficiência , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-6/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Rim/metabolismo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Baço/imunologia , Baço/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Quinases da Família src/imunologia , Quinases da Família src/metabolismoRESUMO
Mice deficient in Lyn, a tyrosine kinase that limits B cell activation, develop a lupus-like autoimmune disease characterized by the accumulation of splenic plasma cells and the production of autoantibodies. Lyn-/- mice have reduced numbers of marginal zone (MZ) B cells, a B cell subset that is enriched in autoreactivity and prone to plasma cell differentiation. We hypothesized that this is due to unchecked terminal differentiation of this potentially pathogenic B cell subpopulation. However, impairing MZ B cell development in Lyn-/- mice did not reduce plasma cell accumulation or autoantibodies, and preventing plasma cell differentiation did not restore MZ B cell numbers. Instead, Lyn-/- mice accumulated B-1a cells when plasma cell differentiation was impaired. Similar to MZ B cells, B-1a cells tend to be polyreactive or weakly autoreactive and are primed for terminal differentiation. Our results implicate B-1a cells, but not MZ B cells, as contributors to the autoreactive plasma cell pool in Lyn-/- mice.
Assuntos
Subpopulações de Linfócitos B , Plasmócitos , Animais , Camundongos , Linfócitos B , Baço , AutoanticorposRESUMO
The levels of transcription factor Ets1 are high in resting B and T cells, but are downregulated by signaling through antigen receptors and Toll-like receptors (TLRs). Loss of Ets1 in mice leads to excessive immune cell activation and development of an autoimmune syndrome and reduced Ets1 expression has been observed in human PBMCs in the context of autoimmune diseases. In B cells, Ets1 serves to prevent premature activation and differentiation to antibody-secreting cells. Given these important roles for Ets1 in the immune response, stringent control of Ets1 gene expression levels is required for homeostasis. However, the genetic regulatory elements that control expression of the Ets1 gene remain relatively unknown. Here we identify a topologically-associating domain (TAD) in the chromatin of B cells that includes the mouse Ets1 gene locus and describe an interaction hub that extends over 100 kb upstream and into the gene body. Additionally, we compile epigenetic datasets to find several putative regulatory elements within the interaction hub by identifying regions of high DNA accessibility and enrichment of active enhancer histone marks. Using reporter constructs, we determine that DNA sequences within this interaction hub are sufficient to direct reporter gene expression in lymphoid tissues of transgenic mice. Further analysis indicates that the reporter construct drives faithful expression of the reporter gene in mouse B cells, but variegated expression in T cells, suggesting the existence of T cell regulatory elements outside this region. To investigate how the downregulation of Ets1 transcription is associated with alterations in the epigenetic landscape of stimulated B cells, we performed ATAC-seq in resting and BCR-stimulated primary B cells and identified four regions within and upstream of the Ets1 locus that undergo changes in chromatin accessibility that correlate to Ets1 gene expression. Interestingly, functional analysis of several putative Ets1 regulatory elements using luciferase constructs suggested a high level of functional redundancy. Taken together our studies reveal a complex network of regulatory elements and transcription factors that coordinate the B cell-specific expression of Ets1.
RESUMO
Ets1 is a key transcription factor in B cells that is required to prevent premature differentiation into Ab-secreting cells. Previously, we showed that BCR and TLR signaling downregulate Ets1 levels and that the kinases PI3K, Btk, IKK, and JNK are required for this process. PI3K is important in activating Btk by generating the membrane lipid phosphatidylinositol (3,4,5)-trisphosphate, to which Btk binds via its PH domain. Btk in turn is important in activating the IKK kinase pathway, which it does by activating phospholipase Cγ2âprotein kinase Cß signaling. In this study, we have further investigated the pathways regulating Ets1 in mouse B cells. Although IKK is well known for its role in activating the canonical NF-κB pathway, IKK-mediated downregulation of Ets1 does not require either RelA or c-Rel. We also examined the potential roles of two other IKK targets that are not part of the NF-κB signaling pathway, Foxo3a and mTORC2, in regulating Ets1. We find that loss of Foxo3a or inhibition of mTORC2 does not block BCR-induced Ets1 downregulation. Therefore, these two pathways are not key IKK targets, implicating other as yet undefined IKK targets to play a role in this process.
Assuntos
Linfócitos B , Ativação Linfocitária , NF-kappa B , Proteína Proto-Oncogênica c-ets-1 , Animais , Camundongos , Linfócitos B/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina , Fosfatidilinositol 3-Quinases , Proteína Proto-Oncogênica c-ets-1/genética , Quinase I-kappa B/metabolismoRESUMO
Accumulation of plasma cells and autoantibodies against nuclear antigens characterize both human and murine lupus. Understanding how these processes are controlled may reveal novel therapeutic targets for this disease. Mice deficient in Lyn, a negative regulator of B and myeloid cell activity, develop lupus-like autoimmune disease. Here, we show that lyn(-) (/) (-) mice exhibit increased splenic plasmablasts and plasma cells and produce IgM against a wide range of self-antigens. Both events require Btk, a target of Lyn-dependent inhibitory pathways. A Btk-dependent increase in the expression of the plasma cell survival factor IL-6 by lyn(-) (/) (-) splenic myeloid cells was also observed. Surprisingly, IL-6 was not required for plasma cell accumulation or polyclonal IgM autoreactivity in lyn(-/-) mice. IL-6 was, however, necessary for the production of IgG autoantibodies, which we show are focused towards a limited set of nucleic acid-containing and glomerular autoantigens in lyn(-) (/) (-) mice. A similar uncoupling of plasma cell accumulation from IgG autoantibodies was seen in lyn(+/-) mice. Plasma cell accumulation and polyclonal IgM autoreactivity are therefore controlled separately from, and are insufficient for, the production of IgG against lupus-associated autoantigens. Regulators of either of these two checkpoints may be attractive therapeutic targets for lupus.
Assuntos
Anticorpos Antinucleares/biossíntese , Lúpus Eritematoso Sistêmico/imunologia , Plasmócitos/metabolismo , Baço/patologia , Quinases da Família src/metabolismo , Tirosina Quinase da Agamaglobulinemia , Animais , Anticorpos Antinucleares/sangue , Anticorpos Antinucleares/genética , Autoantígenos/imunologia , Células Cultivadas , Modelos Animais de Doenças , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Interleucina-6/biossíntese , Interleucina-6/genética , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise em Microsséries , Plasmócitos/imunologia , Plasmócitos/patologia , Proteínas Tirosina Quinases/metabolismo , Quinases da Família src/genética , Quinases da Família src/imunologiaRESUMO
Strict control of B lymphocyte development is required for the ability to mount humoral immune responses to diverse foreign antigens while remaining self-tolerant. In the bone marrow, B lineage cells transit through several developmental stages in which they assemble a functional B cell receptor in a stepwise manner. The immunoglobulin heavy chain gene is rearranged at the pro-B stage. At the large pre-B stage, cells with a functional heavy chain expand in response to signals from IL-7 and the pre-BCR. Cells then cease proliferation at the small pre-B stage and rearrange the immunoglobulin light chain gene. The fully formed BCR is subsequently expressed on the surface of immature B cells and autoreactive cells are culled by central tolerance mechanisms. Once in the periphery, transitional B cells develop into mature B cell subsets such as marginal zone and follicular B cells. These developmental processes are controlled by transcription factor networks, central to which are IRF4 and IRF8. These were thought to act redundantly during B cell development in the bone marrow, with their functions diverging in the periphery where IRF4 limits the number of marginal zone B cells and is required for germinal center responses and plasma cell differentiation. Because of IRF4's unique role in mature B cells, we hypothesized that it may also have functions earlier in B cell development that cannot be compensated for by IRF8. Indeed, we find that IRF4 has a unique role in upregulating the pre-B cell marker CD25, limiting IL-7 responsiveness, and promoting migration to CXCR4 such that IRF4-deficient mice have a partial block at the pre-B cell stage. We also find that IRF4 acts in early transitional B cells to restrict marginal zone B cell development, as deletion of IRF4 in mature B cells with CD21-cre impairs plasma cell differentiation but has no effect on marginal zone B cell numbers. These studies highlight IRF4 as the dominant IRF family member in early B lymphopoiesis.
Assuntos
Proliferação de Células , Fatores Reguladores de Interferon/metabolismo , Linfopoese , Células Precursoras de Linfócitos B/metabolismo , Receptores de Complemento 3d/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL12/farmacologia , Quimiotaxia de Leucócito , Regulação da Expressão Gênica no Desenvolvimento , Fatores Reguladores de Interferon/genética , Interleucina-7/farmacologia , Linfopoese/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/imunologia , Receptores de Complemento 3d/genética , Transdução de SinaisRESUMO
While apoptosis plays a role in B-cell self-tolerance, its significance in preventing autoimmunity remains unclear. Here, we report that dysregulated B cell apoptosis leads to delayed onset autoimmune phenotype in mice. Our longitudinal studies revealed that mice with B cell-specific deletion of pro-apoptotic Bim (BBimfl/fl ) have an expanded B cell compartment with a notable increase in transitional, antibody secreting and recently described double negative (DN) B cells. They develop greater hypergammaglobulinemia than mice lacking Bim in all cells and accumulate several autoantibodies characteristic of Systemic Lupus Erythematosus (SLE) and related Sjögren's Syndrome (SS) including anti-nuclear, anti-Ro/SSA and anti-La/SSB at a level comparable to NODH2h4 autoimmune mouse model. Furthermore, lymphocytes infiltrated the tissues including submandibular glands and formed follicle-like structures populated with B cells, plasma cells and T follicular helper cells indicative of ongoing immune reaction. This autoimmunity was ameliorated upon deletion of Bruton's tyrosine kinase (Btk) gene, which encodes a key B cell signaling protein. These studies suggest that Bim-mediated apoptosis suppresses and B cell tyrosine kinase signaling promotes B cell-mediated autoimmunity.
Assuntos
Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Apoptose/fisiologia , Doenças Autoimunes/imunologia , Autoimunidade/imunologia , Linfócitos B/imunologia , Proteína 11 Semelhante a Bcl-2/fisiologia , Tirosina Quinase da Agamaglobulinemia/deficiência , Tirosina Quinase da Agamaglobulinemia/fisiologia , Animais , Especificidade de Anticorpos , Autoanticorpos/sangue , Linfócitos B/enzimologia , Linfócitos B/patologia , Proteína 11 Semelhante a Bcl-2/deficiência , Divisão Celular , Células Cultivadas , Hipergamaglobulinemia/imunologia , Tolerância Imunológica/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos/imunologia , Síndrome de Sjogren/imunologia , Linfócitos T/imunologiaRESUMO
Though B cells play key roles in lupus pathogenesis, the molecular circuitry and its dysregulation in these cells as disease evolves remain poorly understood. To address this, a comprehensive scan of multiple signaling axes using multiplexed Western blotting was undertaken in several different murine lupus strains. PI3K/AKT/mTOR (mTOR, mammalian target of rapamycin), MEK1/Erk1/2, p38, NF-kappaB, multiple Bcl-2 family members, and cell-cycle molecules were observed to be hyperexpressed in lupus B cells in an age-dependent and lupus susceptibility gene-dose-dependent manner. Therapeutic targeting of the AKT/mTOR axis using a rapamycin (sirolimus) derivative ameliorated the serological, cellular, and pathological phenotypes associated with lupus. Surprisingly, the targeting of this axis was associated with the crippling of several other signaling axes. These studies reveal that lupus pathogenesis is contingent upon the activation of an elaborate network of signaling cascades that is shared among genetically distinct mouse models and raise hope that targeting pivotal nodes in these networks may offer therapeutic benefit.
Assuntos
Linfócitos B/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Transdução de Sinais/imunologia , Fatores Etários , Animais , Linfócitos B/patologia , Modelos Animais de Doenças , Dosagem de Genes/imunologia , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/imunologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Fenótipo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Quinases/genética , Proteínas Quinases/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sirolimo/farmacologia , Sirolimo/uso terapêutico , Serina-Treonina Quinases TORRESUMO
B cell antigen receptor (BCR) cross-linking promotes proliferation and survival of mature B cells. Phosphoinositide-3-kinase-mediated down-regulation of pro-apoptotic and anti-mitogenic genes such as the Foxo family of transcription factors is an important component of this process. Previously, we demonstrated that BCR signaling decreases expression of transcripts for Foxo1, Foxo3 and Foxo4. We now show that BCR-induced down-regulation of Foxo3 and Foxo4 mRNA expression occurs via distinct mechanisms from those established for Foxo1. While Foxo1, Foxo3 and Foxo4 bind the same DNA sequence, the differential control of their expression upon B cell activation suggests that they may have unique functions in the B lineage. To begin to address this issue, we evaluated B cell development and function in Foxo3-/- mice. No effect of Foxo3 deficiency was observed with respect to the following parameters in the splenic B cell compartment: sub-population distribution, proliferation, in vitro differentiation and expression of the Foxo target genes cyclin G2 and B cell translocation gene 1. However, Foxo3-/- mice demonstrated increased basal levels of IgG2a, IgG3 and IgA. A significant reduction in pre-B cell numbers was also observed in Foxo3-/- bone marrow. Finally, recirculating B cells in the bone marrow and peripheral blood were decreased in Foxo3-/- mice, perhaps due to lower than normal expression of receptor for sphingosine-1 phosphate, which mediates egress from lymphoid organs. Thus, Foxo3 makes a unique contribution to B cell development, B cell localization and control of Ig levels.