RESUMO
564Igi mice have knocked-in immunoglobulin (Ig) heavy (H) and light (L) chain genes that encode an autoantibody recognizing RNA. Previously, we showed that these mice produce pathogenic IgG autoantibodies when activation-induced deaminase (AID) is expressed in pre-B and immature B cells but not when it is expressed only in mature B cells. AID has two functions; it is necessary for somatic hypermutation (SHM) and class switch recombination (CSR). To determine the role of each of these functions in the generation of pathogenic autoantibodies, we generated 564Igi mice that carry a mutant AID-encoding gene, Aicda (AicdaG23S), which is capable of promoting CSR but not SHM. We found that 564Igi AicdaG23S mice secreted class-switched antibodies (Abs) at levels approximately equal to 564Igi mice. However, compared to 564Igi mice, 564Igi Aicda G23S mice had increased pathogenic IgG Abs and severe systemic lupus erythematosus-like disease, including, glomerulonephritis, and early death. We suggest that in 564Igi mice SHM by AID changes Ig receptors away from self reactivity, thereby mitigating the production of autoantibody, providing a novel mechanism of tolerance.
RESUMO
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of anti-nuclear antibodies. SLE is one of many autoimmune disorders that have a strong gender bias, with 70-90% of SLE patients being female. Several explanations have been postulated to account for the severity of autoimmune diseases in females, including hormonal, microbiota, and gene dosage differences. X-linked toll-like receptors (TLRs) have recently been implicated in disease progression in females. Our previous studies using the 564Igi mouse model of SLE on a Tlr7 and Tlr9 double knockout background showed that the presence of Tlr8 on both X chromosomes was required for the production of IgG autoantibodies, Ifn-I expression and granulopoiesis in females. Here, we show the results of our investigation into the role of Tlr8 expression in SLE pathogenesis in 564Igi females. Female mice have an increase in serum pathogenic anti-RNA IgG2a and IgG2b autoantibodies. 564Igi mice have also been shown to have an increase in neutrophils in vivo, which are major contributors to Ifn-α expression. Here, we show that neutrophils from C57BL/6 mice express Ifn-α in response to 564 immune complexes and TLR8 activation. Bone marrow-derived macrophages from 564Igi females have a significant increase in Tlr8 expression compared to male-derived cells, and RNA fluorescence in situ hybridization data suggest that Tlr8 may escape X-inactivation in female-derived macrophages. These results propose a model by which females may be more susceptible to SLE pathogenesis due to inefficient inactivation of Tlr8.
RESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of pathogenic IgG antinuclear antibodies. Pathogenic IgG autoantibody production requires B-cell activation, leading to the production of activation-induced deaminase (AID) and class switching of IgM genes to IgG. To understand how and when B cells are activated to produce these IgG autoantibodies, we studied cells from 564Igi, a mouse model of SLE. 564Igi mice develop a disease profile closely resembling that found in human SLE patients, including the presence of IgG antinucleic acid Abs. We have generated 564Igi mice that conditionally express an activation-induced cytidine deaminase transgene (Aicda(tg) ), either in all B cells or only in mature B cells. Here, we show that class-switched pathogenic IgG autoantibodies were produced only in 564Igi mice in which AID was functional in early-developing B cells, resulting in loss of tolerance. Furthermore, we show that the absence of AID in early-developing B cells also results in increased production of self-reactive IgM, indicating that AID, through somatic hypermutation, contributes to tolerance. Our results suggest that the pathophysiology of clinical SLE might also be dependent on AID expression in early-developing B cells.
Assuntos
Anticorpos Antinucleares/imunologia , Linfócitos B/imunologia , Citidina Desaminase/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Animais , Formação de Anticorpos/imunologia , Autoanticorpos/biossíntese , Autoanticorpos/imunologia , Autoantígenos/imunologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Tolerância Imunológica/imunologia , Switching de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease with a high incidence in females and a complex phenotype. Using 564Igi mice, a model of SLE with knock-in genes encoding an autoreactive anti-RNA Ab, we investigated how expression of Toll-like receptors (TLRs) in B cells and neutrophils affects pathogenesis. We established that TLR signaling through MyD88 is necessary for disease. Autoantibody was produced in mice with single deletions of Tlr7, Tlr8, or Tlr9 or combined deletions of Tlr7 and Tlr9. Autoantibody was not produced in the combined absence of Tlr7 and Tlr8, indicating that TLR8 contributes to the break in tolerance. Furthermore, TLR8 was sufficient for the loss of B-cell tolerance, the production of class-switched autoantibody, heightened granulopoiesis, and increased production of type I IFN by neutrophils as well as glomerulonephritis and death. We show that dosage of X-linked Tlr8 plays a major role in the high incidence of disease in females. In addition, we show that the negative regulation of disease by TLR9 is exerted primarily on granulopoiesis and type I IFN production by neutrophils. Collectively, we suggest that individual TLRs play unique roles in the pathogenesis of systemic lupus erythematosus, suggesting new targets for treatment.
Assuntos
Dosagem de Genes/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Caracteres Sexuais , Receptor 8 Toll-Like/imunologia , Cromossomo X/imunologia , Animais , Autoanticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Feminino , Dosagem de Genes/genética , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/patologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mielopoese/genética , Mielopoese/imunologia , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/genética , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/imunologia , Cromossomo X/genéticaRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the presence of antinucleic acid autoantibodies, high levels of circulating type I interferon (IFN-I), and an IFN-I-dependent elevated expression of activating FcγR. Increases in neutrophils and monocytes are often observed in clinical SLE, but how these contribute to autoantibody and IFN-I production is poorly understood. Here, we analyzed SLE pathogenesis in 564Igi mice, an SLE-model strain carrying gene-targeted heavy and light chain antibody genes encoding an anti-RNA autoantibody in a C57BL/6 background. Similar to human SLE patients, 564Igi mice produce anti-RNA autoantibodies and expanded neutrophil and monocyte populations. These myeloid cells produced IFN-I and exhibit increased FcγRIV expression induced via an IFN-I autocrine loop. A direct effect of IFN-I on 56 Igi BM B cells and neutrophils was supported by their upregulation of "IFN-I signature genes". In addition, 564Igi developing B cells showed upregulated TLR7 resulting in IgG2a/2b class switch recombination and autoantibody production. Our results indicate that the production of anti-RNA autoantibody is sufficient to induce an increase of BM, blood, and spleen IFN-I-producing neutrophils, and suggest a mechanism by which autoantibody and IFN-I contribute to SLE by activating B lymphocytes, neutrophils, and monocyte effector cells in vivo.
Assuntos
Linfócitos B/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Animais , Autoanticorpos/metabolismo , Comunicação Autócrina , Processos de Crescimento Celular/genética , Células Cultivadas , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Genes de Imunoglobulinas/genética , Humanos , Imunoglobulina G/metabolismo , Interferon Tipo I/genética , Interferon Tipo I/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA/imunologia , Receptores de IgG/metabolismo , Receptor 7 Toll-Like/metabolismoRESUMO
Toll-like receptor (TLR), a ligand for single-stranded RNA, has been implicated in the development of pathogenic anti-RNA autoantibodies both in systemic lupus erythematous (SLE) patients and in murine models of lupus. It is still unclear, however, where and how TLR7-mediated interactions affect the development of autoreactive B cells. We found that overexpression of TLR7 in transgenic mice (TLR7.1Tg) leads to marked alterations of transitional (T1) B cells, associated with their expansion and proliferation within the splenic red pulp (RP). This phenotype was intrinsic to the T1 subset of B cells and occurred independently of type 1 IFN signals. Overexpression of RNase in TLR7.1Tg mice significantly limited the expansion and proliferation of T1 cells, indicating that endogenous RNA complexes are driving their activation. TLR7.1Tg T1 cells were hyper-responsive to anti-IgM and TLR7 ligand stimulation in vitro and produced high concentrations of class-switched IgG2b and IgG2c, including anti-RNA antibodies. Our results demonstrate that initial TLR7 stimulation of B cells occurs at the T1 stage of differentiation in the splenic RP and suggest that dysregulation of TLR7 expression in T1 cells can result in production of autoantibodies.
Assuntos
Autoanticorpos/biossíntese , Subpopulações de Linfócitos B/imunologia , Glicoproteínas de Membrana/metabolismo , Células Precursoras de Linfócitos B/imunologia , Receptor 7 Toll-Like/metabolismo , Animais , Apoptose , Subpopulações de Linfócitos B/citologia , Diferenciação Celular , Proliferação de Células , Citidina Desaminase/genética , Humanos , Switching de Imunoglobulina , Interferon Tipo I/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células Precursoras de Linfócitos B/citologia , RNA/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Baço/citologia , Baço/imunologia , Proteínas com Domínio T/genética , Receptor 7 Toll-Like/genética , Regulação para CimaRESUMO
Activation-induced cytidine deaminase (AID) is required for immunoglobulin (Ig) gene class switch recombination (CSR), somatic hypermutation (SHM), and somatic hyperconversion. In general, high AID expression is found in mature B cells that are responding to antigens. However, AID expression and SHM have also been detected in developing B cells from transgenic mice that have a limited Ig repertoire. Here we demonstrate that AID expression, ongoing CSR, and active SHM occur in developing B cells from wild-type mice. Further, our results suggest that somatic variants arising from developing B cells in the bone marrow further diversify in the spleen of unimmunized mice. AID expression in developing B cells is T cell independent but involves engagement of B cell receptors and Toll-like receptors. Early AID expression can increase the preimmune repertoire of developing B cells, may provide an innate population of IgG- and IgA-expressing cells, and could be involved in receptor editing of self-reactive immature B cells.
Assuntos
Linfócitos B/imunologia , Switching de Imunoglobulina/genética , Receptores de Antígenos de Linfócitos B/fisiologia , Recombinação Genética/imunologia , Hipermutação Somática de Imunoglobulina/genética , Receptores Toll-Like/fisiologia , Animais , Linfócitos B/enzimologia , Citidina Desaminase/biossíntese , Citidina Desaminase/genética , Citidina Desaminase/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos NusRESUMO
Systemic lupus erythematosus (SLE) is characterized by the production of autoantibodies that are frequently directed against nucleic acid-associated antigens. To better understand how B cells reactive with such antigens are regulated, we generated a model system in which heavy and light chain genes encoding 564 immunoglobulin have been targeted to the heavy and light chain loci of the nonautoimmune C57BL/6 mouse strain. This antibody recognizes RNA, single-stranded DNA, and nucleosomes. We show that B cells expressing this immunoglobulin were activated, producing class-switched autoantibody in vivo despite the apparently normal induction of anergy. This autoantibody production was largely dependent on Toll-like receptor 7 (TLR7). We further show that production of these autoantibodies was sufficient to cause kidney pathology in these mice. These results demonstrate that the particular threat of nucleic acid-containing autoantigens lies in their ability to bind both antigen receptor and TLR7.
Assuntos
Autoanticorpos/biossíntese , Linfócitos B/imunologia , Tolerância Imunológica , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Glicoproteínas de Membrana/fisiologia , Receptor 7 Toll-Like/fisiologia , Animais , Autoanticorpos/fisiologia , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Tolerância Imunológica/genética , Lúpus Eritematoso Sistêmico/patologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptor 7 Toll-Like/deficiência , Receptor 7 Toll-Like/genéticaRESUMO
Somatic hypermutation contributes to the generation of antibody diversity and is strongly associated with the maturation of antigen-specific immune responses. We asked whether somatic hypermutation also plays a role in the generation of the murine immunoglobulin repertoire during B cell development. To facilitate identification of somatic mutations, we examined mouse systems in which only antibodies expressing lambda1, lambda2, and lambdax light chains can be generated. Somatic mutations were found in cells, which, by surface markers, RAG expression, and rapid turnover, had the phenotype of immature B cells. In addition, expression of AID was detected in these cells. The mutations were limited to V regions and were localized in known hotspots. Mutation frequency was not diminished in the absence of T cells. Our results support the idea that somatic hypermutation can occur in murine immature B cells and may represent a mechanism for enlarging the V gene repertoire.