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1.
Cell ; 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32059783

RESUMO

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

2.
J Exp Med ; 217(2)2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-31841125

RESUMO

Antibody-mediated autoimmune diseases are a major health burden. However, our understanding of how self-reactive B cells escape self-tolerance checkpoints to secrete pathogenic autoantibodies remains incomplete. Here, we demonstrate that patients with monogenic immune dysregulation caused by gain-of-function mutations in PIK3CD, encoding the p110δ catalytic subunit of phosphoinositide 3-kinase (PI3K), have highly penetrant secretion of autoreactive IgM antibodies. In mice with the corresponding heterozygous Pik3cd activating mutation, self-reactive B cells exhibit a cell-autonomous subversion of their response to self-antigen: instead of becoming tolerized and repressed from secreting autoantibody, Pik3cd gain-of-function B cells are activated by self-antigen to form plasmablasts that secrete high titers of germline-encoded IgM autoantibody and hypermutating germinal center B cells. However, within the germinal center, peripheral tolerance was still enforced, and there was selection against B cells with high affinity for self-antigen. These data show that the strength of PI3K signaling is a key regulator of pregerminal center B cell self-tolerance and thus represents a druggable pathway to treat antibody-mediated autoimmunity.

3.
Immunol Rev ; 292(1): 61-75, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31556969

RESUMO

The adaptive immune system is tasked with producing antibodies that recognize a wide scope of potential pathogens, including those never before encountered, and concurrently avoiding formation of antibodies binding host tissues. The diverse repertoire of antibodies produced by V(D)J recombination inevitably includes autoantibodies that bind to self-antigens, estimated to be as much as 70% of nascent antibodies on immature B cells. Early theoretical models of tolerance hypothesized that such self-reactive clones could not possibly be allowed to survive and mature. However from the first direct view of the fate of nascent B cells carrying a self-binding antibody it was clear that many "forbidden clones" circulate to secondary lymphoid tissues, where they adopt an IgMlow IgD+ cell surface phenotype and are prevented from secreting autoantibodies by a series of tolerance checkpoints referred to as "clonal anergy." Since anergic B cells can be reactivated to secrete pathogenic autoantibodies in certain settings, the advantage of controlling self-reactive antibodies by clonal anergy has until recently remained enigmatic. Here we review this topic and recent advances showing that anergic B cells are recruited into the germinal center to mutate away from self-reactivity, undergoing "clonal redemption" into cells making antibodies with exquisite specificity for foreign immunogens.

4.
Cancer Cell ; 35(2): 297-314.e8, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30753827

RESUMO

Promoter CpG islands are typically unmethylated in normal cells, but in cancer a proportion are subject to hypermethylation. Using methylome sequencing we identified CpG islands that display partial methylation encroachment across the 5' or 3' CpG island borders. CpG island methylation encroachment is widespread in prostate and breast cancer and commonly associates with gene suppression. We show that the pattern of H3K4me1 at CpG island borders in normal cells predicts the different modes of cancer CpG island hypermethylation. Notably, genetic manipulation of Kmt2d results in concordant alterations in H3K4me1 levels and CpG island border DNA methylation encroachment. Our findings suggest a role for H3K4me1 in the demarcation of CpG island methylation borders in normal cells, which become eroded in cancer.


Assuntos
Ilhas de CpG , Metilação de DNA , DNA de Neoplasias/metabolismo , Histonas/metabolismo , Neoplasias/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Humanos , Masculino , Metilação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Regiões Promotoras Genéticas
5.
Methods Mol Biol ; 1827: 287-309, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30196503

RESUMO

Here we describe methods for screening human blood to isolate peripheral blood mononuclear cells (PBMCs) capable of binding fluorescently labeled antigen, as well as methods for the amplification and sequencing of B cell receptor (BCR) heavy and light chain genes. Detailed protocols are provided for transient mammalian expression in a hexahistidine-tagged Fab format, purification by immobilized metal affinity chromatography (IMAC), and affinity determination by BioLayer interferometry (BLI).


Assuntos
Antígenos/sangue , Linfócitos B/imunologia , Cromatografia de Afinidade/métodos , Epitopos , Análise de Sequência de Proteína/métodos , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Interferometria , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/metabolismo
6.
Science ; 360(6385): 223-226, 2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29650674

RESUMO

Antibodies have the specificity to differentiate foreign antigens that mimic self antigens, but it remains unclear how such specificity is acquired. In a mouse model, we generated B cells displaying an antibody that cross-reacts with two related protein antigens expressed on self versus foreign cells. B cell anergy was imposed by self antigen but reversed upon challenge with high-density foreign antigen, leading to germinal center recruitment and antibody gene hypermutation. Single-cell analysis detected rapid selection for mutations that decrease self affinity and slower selection for epistatic mutations that specifically increase foreign affinity. Crystal structures revealed that these mutations exploited subtle topological differences to achieve 5000-fold preferential binding to foreign over self epitopes. Resolution of antigenic mimicry drove the optimal affinity maturation trajectory, highlighting the value of retaining self-reactive clones as substrates for protective antibody responses.


Assuntos
Anticorpos/genética , Formação de Anticorpos/genética , Autoantígenos/imunologia , Centro Germinativo/imunologia , Mimetismo Molecular/genética , Tolerância a Antígenos Próprios , Animais , Anticorpos/química , Anticorpos/imunologia , Afinidade de Anticorpos/genética , Linfócitos B/imunologia , Anergia Clonal , Reações Cruzadas , Cristalografia por Raios X , Camundongos , Camundongos Mutantes , Mutação , Nucleoproteínas/genética , Nucleoproteínas/imunologia , Seleção Genética , Análise de Célula Única
7.
Bioinformatics ; 34(16): 2846-2847, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29659703

RESUMO

Motivation: The B-cell receptor (BCR) performs essential functions for the adaptive immune system including recognition of pathogen-derived antigens. The vast repertoire and adaptive variation of BCR sequences due to V(D)J recombination and somatic hypermutation necessitates single-cell characterization of BCR sequences. Single-cell RNA sequencing presents the opportunity for simultaneous capture of paired BCR heavy and light chains and the transcriptomic signature. Results: We developed VDJPuzzle, a novel bioinformatic tool that reconstructs productive, full-length B-cell receptor sequences of both heavy and light chains and extract somatic mutations on the VDJ region. VDJPuzzle successfully reconstructed BCRs from 100% (n=117) human and 96.5% (n=200) murine B cells. The reconstructed BCRs were successfully validated with single-cell Sanger sequencing. Availability and implementation: VDJPuzzle is available at https://bitbucket.org/kirbyvisp/vdjpuzzle2. Supplementary information: Supplementary data are available at Bioinformatics online.


Assuntos
RNA/genética , Receptores de Antígenos de Linfócitos B/genética , Animais , Biologia Computacional , Humanos , Camundongos , Análise de Sequência de RNA , Análise de Célula Única , Transcriptoma
8.
Arthritis Rheumatol ; 70(10): 1617-1625, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29697211

RESUMO

OBJECTIVE: Rheumatoid factors (RFs) are associated with systemic disease in primary Sjögren's syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. This study was undertaken to perform the first proteomic and transcriptomic analysis of secreted and membrane-bound IgM-RF in primary SS and identify unique heavy-chain peptide signatures for RF clonotype tracking. METHODS: Purified heavy chains of serum RFs from 15 patients with primary SS were subjected to de novo mass spectrometric sequencing. The circulating B cell Ig repertoire was determined by massively parallel sequencing of IGH RNA from matched peripheral blood mononuclear cells (n = 7). RF-specific heavy-chain third complementarity-determining region (CDR3) peptides were identified by searching RF heavy-chain peptide sequences against the corresponding IGH RNA sequence libraries. Heavy-chain CDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring. RESULTS: Serum RFs were clonally restricted and composed of shared sets of IgM heavy-chain variable region (Ig VH ) 1-69, 3-15, 3-7, and 3-74 subfamilies. Cryoprecipitable RFs from patients with mixed cryoglobulinemia (MC) were distinguishable from nonprecipitating RFs by a higher frequency of amino acid substitutions and identification of stereotypic heavy-chain CDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by multiple reaction monitoring years before patients presented with MC. Levels of Ig VH 4-34 IgM-RF decreased following immunosuppression and remission of MC. CONCLUSION: Cryoprecipitable RF clonotypes linked to vasculitis in primary SS have different molecular profiles than nonprecipitating RFs, suggesting different underlying mechanisms of production. The combined omics workflow presented herein provides molecular biomarkers for tracking and removal of pathogenic RF clones.


Assuntos
Cadeias Pesadas de Imunoglobulinas/sangue , Leucócitos Mononucleares/fisiologia , Fator Reumatoide/sangue , Síndrome de Sjogren/sangue , Adulto , Linfócitos B/metabolismo , Compostos de Boro/metabolismo , Rastreamento de Células , Feminino , Perfilação da Expressão Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Imunoglobulina M/imunologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteômica , Fator Reumatoide/imunologia , Síndrome de Sjogren/imunologia
9.
J Autoimmun ; 79: 99-104, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28118945

RESUMO

The detection of cardiac conduction defects in an 18-24 week old foetus in the absence of structural abnormalities predicts with near certainty the presence of autoantibodies against 60kD and 52kD SSA/Ro in the mother regardless of her health status. Previous studies have emphasized these autoantibodies as key mediators of tissue injury. The aim of this study was to focus on the anti-Ro52 response to determine whether these autoantibodies originate from progenitors that are inherently self-reactive or from B-cells that acquire self-reactivity during an immune response. We traced the evolution of two anti-Ro52 autoantibodies isolated from circulating IgG1-switched B-cells from an asymptomatic mother of a child with third degree congenital heart block. The autoantibodies were expressed as their immune form and as pre-immune ancestors by reverting somatic mutations to germline sequence. The reactivity of pre-immune and immune antibodies for Ro52, Ro60, La and DNA was measured. Both anti-Ro52 autoantibodies exhibited a low frequency of somatic mutations (3-4%) and utilised the same heavy and light chain genes but represented distinct clones based on differing complementarity determining region sequences. Pre- and post-immune antibodies showed specific binding to Ro52 with no measurable reactivity for other autoantigens. Ro52 binding was higher for immune antibodies compared to pre-immune counterparts demonstrating that autoreactivity was enhanced by affinity maturation. These data indicate that Ro52 reactivity is an intrinsic property of the germline antibody repertoire in a mother with a pathogenic antibody defined by cardiac injury in her offspring, and implies defects in both central and peripheral tolerance mechanisms.


Assuntos
Autoanticorpos/imunologia , Autoimunidade , Exposição Materna , Mães , Células Precursoras de Linfócitos B/imunologia , Ribonucleoproteínas/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Autoanticorpos/sangue , Autoanticorpos/química , Autoanticorpos/genética , Doenças Autoimunes/sangue , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Feminino , Humanos , Lactente , Lúpus Eritematoso Sistêmico/congênito , Células Precursoras de Linfócitos B/metabolismo , Hipermutação Somática de Imunoglobulina/genética
10.
Nat Commun ; 7: 13381, 2016 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-27830696

RESUMO

Self-tolerance by clonal anergy of B cells is marked by an increase in IgD and decrease in IgM antigen receptor surface expression, yet the function of IgD on anergic cells is obscure. Here we define the RNA landscape of the in vivo anergy response, comprising 220 induced sequences including a core set of 97. Failure to co-express IgD with IgM decreases overall expression of receptors for self-antigen, but paradoxically increases the core anergy response, exemplified by increased Sdc1 encoding the cell surface marker syndecan-1. IgD expressed on its own is nevertheless competent to induce calcium signalling and the core anergy mRNA response. Syndecan-1 induction correlates with reduction of surface IgM and is exaggerated without surface IgD in many transitional and mature B cells. These results show that IgD attenuates the response to self-antigen in anergic cells and promotes their accumulation. In this way, IgD minimizes tolerance-induced holes in the pre-immune antibody repertoire.


Assuntos
Linfócitos B/imunologia , Anergia Clonal/imunologia , Imunoglobulina D/imunologia , Imunoglobulina M/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Anergia Clonal/genética , Perfilação da Expressão Gênica/métodos , Imunoglobulina D/genética , Imunoglobulina M/genética , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Camundongos Endogâmicos C57BL , Mutação , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Tolerância a Antígenos Próprios/genética , Tolerância a Antígenos Próprios/imunologia , Sindecana-1/genética , Sindecana-1/imunologia , Sindecana-1/metabolismo
11.
Clin Immunol ; 173: 57-63, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27609500

RESUMO

We have used high-resolution mass spectrometry to sequence precipitating anti-Ro60 proteomes from sera of patients with primary Sjögren's syndrome and compare immunoglobulin variable-region (IgV) peptide signatures in Ro/La autoantibody subsets. Anti-Ro60 were purified by elution from native Ro60-coated ELISA plates and subjected to combined de novo amino acid sequencing and database matching. Monospecific anti-Ro60 Igs comprised dominant public and minor private sets of IgG1 kappa and lambda restricted heavy and light chains. Specific IgV amino acid substitutions stratified anti-Ro60 from anti-Ro60/La responses, providing a molecular fingerprint of Ro60/La determinant spreading and suggesting that different forms of Ro60 antigen drive these responses. Sequencing of linked anti-Ro52 proteomes from individual patients and comparison with their anti-Ro60 partners revealed sharing of a dominant IGHV3-23/IGKV3-20 paired clonotype but with divergent IgV mutational signatures. In summary, anti-Ro60 IgV peptide mapping provides insights into Ro/La autoantibody diversification and reveals serum-based molecular markers of humoral Ro60 autoimmunity.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Região Variável de Imunoglobulina/imunologia , RNA Citoplasmático Pequeno/imunologia , Ribonucleoproteínas/imunologia , Síndrome de Sjogren/imunologia , Autoanticorpos/sangue , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Mapeamento de Peptídeos , Proteoma , Síndrome de Sjogren/sangue
12.
J Exp Med ; 213(7): 1255-65, 2016 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-27298445

RESUMO

Clonal anergy is an enigmatic self-tolerance mechanism because no apparent purpose is served by retaining functionally silenced B cells bearing autoantibodies. Human autoantibodies with IGHV4-34*01 heavy chains bind to poly-N-acetyllactosamine carbohydrates (I/i antigen) on erythrocytes and B lymphocytes, cause cold agglutinin disease, and are carried by 5% of naive B cells that are anergic. We analyzed the specificity of three IGHV4-34*01 IgG antibodies isolated from healthy donors immunized against foreign rhesus D alloantigen or vaccinia virus. Each IgG was expressed and analyzed either in a hypermutated immune state or after reverting each antibody to its unmutated preimmune ancestor. In each case, the preimmune ancestor IgG bound intensely to normal human B cells bearing I/i antigen. Self-reactivity was removed by a single somatic mutation that paradoxically decreased binding to the foreign immunogen, whereas other mutations conferred increased foreign binding. These data demonstrate the existence of a mechanism for mutation away from self-reactivity in humans. Because 2.5% of switched memory B cells use IGHV4-34*01 and >43% of these have mutations that remove I/i binding, clonal redemption of anergic cells appears efficient during physiological human antibody responses.


Assuntos
Anticorpos Antivirais/imunologia , Autoanticorpos/imunologia , Linfócitos B/imunologia , Imunização , Imunoglobulina G/imunologia , Sistema do Grupo Sanguíneo Rh-Hr/administração & dosagem , Hipermutação Somática de Imunoglobulina/imunologia , Vírus Vaccinia/imunologia , Anergia Clonal/imunologia , Feminino , Humanos , Memória Imunológica/efeitos dos fármacos , Memória Imunológica/imunologia , Masculino , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Hipermutação Somática de Imunoglobulina/efeitos dos fármacos
13.
J Autoimmun ; 73: 30-41, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27289167

RESUMO

At birth, the human immune system already contains substantial levels of polymeric IgM, that include autoantibodies to neo-epitopes on apoptotic cells (ACs) that are proposed to play homeostatic and anti-inflammatory roles. Yet the biologic origins and developmental regulation of these naturally arising antibodies remain poorly understood. Herein, we report that levels of IgM-antibodies to malondialdehyde (MDA) protein adducts, a common type of in vivo generated oxidative stress-related neoepitope, directly correlate with the relative binding of neonatal-IgM to ACs. Levels of IgM to phosphorylcholine (PC), a natural antibody prevalent in adults, were relatively scant in cord blood, while there was significantly greater relative representation of IgM anti-MDA antibodies in newborns compared to adults. To investigate the potential interrelationships between neonatal IgM with pathogenic IgG-autoantibodies, we studied 103 newborns born to autoimmune mothers with IgG anti-Ro (i.e., 70 with neonatal lupus and 33 without neonatal lupus). In these subjects the mean levels of IgM anti-Ro60 were significantly higher than in the newborns from non-autoimmune mothers. In contrast, levels of IgM anti-MDA in IgG anti-Ro exposed neonates were significantly lower than in neonates from non-autoimmune mothers. The presence or absence of neonatal lupus did not appear to influence the total levels of IgM in the anti-Ro exposed newborns. Taken together, our studies provide evidence that the immune development of the natural IgM-repertoire may be affected, and become imprinted by, the transfer of maternal IgG into the fetus.


Assuntos
Apoptose/imunologia , Autoanticorpos/imunologia , Epitopos/imunologia , Feto/imunologia , Imunoglobulina M/imunologia , Troca Materno-Fetal/imunologia , Estresse Oxidativo/imunologia , Ribonucleoproteínas/imunologia , Adulto , Anticorpos Anti-Idiotípicos/sangue , Autoanticorpos/sangue , Autoantígenos/imunologia , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Feminino , Sangue Fetal/imunologia , Humanos , Imunoglobulina G/imunologia , Recém-Nascido , Malondialdeído/efeitos adversos , Malondialdeído/química , Malondialdeído/imunologia , Mães , Fosforilcolina/efeitos adversos , Fosforilcolina/sangue , Gravidez , Complicações na Gravidez , Ribonucleoproteínas/química
15.
J Autoimmun ; 67: 36-45, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26432597

RESUMO

Based on the consistent demonstration of fibrosis of the atrioventricular node surrounded by macrophages and multinucleated giant cells in anti-Ro antibody exposed fetuses dying with heart block, this study focuses on macrophage signaling stimulated by ssRNA associated with the Ro60 protein and the impact of antagonizing innate cell drivers such as TLR7/8. Transcriptome and epigenetic modifications which affect transcription factors, NF-κB and STAT1, were selected to evaluate the phenotype of macrophages in which TLR7/8 was ligated following treatment with either anti-Ro60/Ro60/hY3 RNA immune complexes or transfection with hY3. Based on microarray, TNF and IL6 were among the most highly upregulated genes in both stimulated conditions, each of which was significantly inhibited by preincubation with hydroxychloroquine (HCQ). In contrast, following stimulation of macrophages with either TNF-α or IFN-α, which do not signal through TLR, the resultant gene expression was refractory to HCQ. Ligation of TLR7/8 resulted in increased histone methylation as measured by increased H3K4me2, a requirement for binding of NF-κB at certain promoters, specifically the kB1 region in the TNF promoter (ChIP-qPCR), which was significantly decreased by HCQ. In summary, these results support that the HCQ-sensitive phenotype of hY3 stimulated macrophages reflects the bifurcation of TLR downstream signals involving NF-κB and STAT 1 pathways and for the former dimethylation of H3K4. Accordingly, HCQ may act more as a preventive measure in downregulating the initial production of IFN-α or TNF-α and not affect the resultant autocoid stimulation reflected in TNF-α and IFN-α responsive genes. The beneficial scope of antimalarials in the prevention of organ damage, inclusive of heart block in an anti-Ro offspring or more broadly SLE, may include in part, a mechanism targeting TLR-dependent epigenetic modification.


Assuntos
Anticorpos Antinucleares/imunologia , Epigênese Genética , Marcação de Genes , Bloqueio Cardíaco/etiologia , Bloqueio Cardíaco/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Fatores de Transcrição/genética , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Linhagem Celular , Endocitose/imunologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Ligação Proteica , Ribonucleoproteínas/imunologia , Receptor 7 Toll-Like/antagonistas & inibidores , Receptor 8 Toll-Like/antagonistas & inibidores
16.
J Immunol ; 191(1): 110-6, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23698747

RESUMO

Cardiac neonatal lupus (NL) is presumed to arise from maternal autoantibody targeting an intracellular ribonucleoprotein, Ro60, which binds noncoding Y RNA and only becomes accessible to autoantibodies during apoptosis. Despite the importance of Ro60 trafficking in the development of cardiac NL, the mechanism underlying cell surface exposure is unknown. To evaluate the influence of Y RNA on the subcellular location of Ro60 during apoptosis and activation of macrophages, stable Ro60 knockout murine fibroblasts expressing wild-type or mutated FLAG-Ro60 were assessed. FLAG3-Ro60(K170A R174A) binds Y RNA, whereas FLAG3-Ro60(H187S) does not bind Y RNA; fibroblasts expressing these constructs showed equivalent intracellular expression of Ro60. In contrast, apoptotic fibroblasts containing FLAG3-Ro60(K170A R174A) were bound by anti-Ro60, whereas FLAG3-Ro60(H187S) was not surface expressed. RNA interference of mY3 RNA in wild-type fibroblasts inhibited surface translocation of Ro60 during apoptosis, whereas depletion of mY1 RNA did not affect Ro60 exposure. Furthermore, Ro60 was not exposed following overexpression of mY1 in the mY3-depleted fibroblasts. In an in vitro model of anti-Ro60-mediated injury, Y RNA was shown to be an obligate factor for TLR-dependent activation of macrophages challenged with anti-Ro60-opsonized apoptotic fibroblasts. Murine Y3 RNA is a necessary factor to support the surface translocation of Ro60, which is pivotal to the formation of immune complexes on apoptotic cells and a TLR-dependent proinflammatory cascade. Accordingly, the Y3 RNA moiety of the Ro60 ribonucleoprotein imparts a critical role in the pathogenicity of maternal anti-Ro60 autoantibodies.


Assuntos
Cardiopatias/imunologia , Cardiopatias/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , RNA não Traduzido/metabolismo , Ribonucleoproteínas/metabolismo , Adulto , Animais , Animais Recém-Nascidos , Autoanticorpos/metabolismo , Células Cultivadas , Criança , Técnicas de Cocultura , Cardiopatias/patologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Knockout , Ribonucleoproteínas/antagonistas & inibidores , Ribonucleoproteínas/imunologia , Frações Subcelulares/imunologia , Propriedades de Superfície
17.
Rheumatology (Oxford) ; 52(8): 1448-53, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23598443

RESUMO

OBJECTIVE: Cardiac neonatal lupus (cardiac-NL), initiated by surface binding of anti-Ro60 autoantibodies to apoptotic cardiocytes during development, activates the urokinase plasminogen activator/urokinase plasminogen activator receptor (uPA/uPAR) system. Subsequent accumulation of apoptotic cells and plasmin generation facilitates increased binding of anti-Ro60 by disrupting and cleaving circulating ß2-glycoprotein I (ß2GPI) thereby eliminating its protective effect. The association of soluble levels of components of the uPA/uPAR system with cardiac-NL was examined. METHODS: Levels of the uPA/uPAR system were assessed by ELISA in cord blood and immunohistological evaluation of autopsies. RESULTS: uPA, uPAR and plasminogen levels were each significantly higher in cord blood from cardiac-NL (n = 35) compared with non-cardiac-NL (n = 26) anti-Ro-exposed neonates: 3.3 ± 0.1 vs 1.9 ± 0.05 ng/ml (P < 0.0001), 6.6 ± 0.3 vs 2.1 ± 0.2 ng/ml (P < 0.0001) and 435 ± 34 vs 220 ± 19 ng/ml (P < 0.0001), respectively. In three twin pairs discordant for cardiac-NL, the twin with cardiac-NL had higher levels of uPA, uPAR and plasminogen than the unaffected twin (3.1 ± 0.1 vs 1.9 ± 0.05 ng/ml; P = 0.0086, 6.2 ± 1.4 vs 2.2 ± 0.7 ng/ml; P = 0.147 and 412 ± 61 vs 260 ± 27 ng/ml; P = 0.152, respectively). Immunohistological evaluation of three hearts from fetuses dying with cardiac-NL revealed macrophages and giant cells expressing uPA and plasminogen in the septal region. CONCLUSION: Increased soluble uPA, uPAR and plasminogen in cord blood and expression in affected tissue of fetuses with cardiac-NL supports the hypothesis that fetal cardiac injury is in part mediated by plasmin generation initiated by anti-Ro binding to the apoptotic cardiocyte.


Assuntos
Sangue Fetal/imunologia , Fibrinolisina/imunologia , Cardiopatias/imunologia , Lúpus Eritematoso Sistêmico/congênito , Receptores de Ativador de Plasminogênio Tipo Uroquinase/imunologia , Ribonucleoproteínas/imunologia , Anticorpos Antinucleares/imunologia , Anticorpos Antinucleares/metabolismo , Biomarcadores , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Fibrinolisina/metabolismo , Cardiopatias/mortalidade , Cardiopatias/patologia , Humanos , Imuno-Histoquímica , Recém-Nascido , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/mortalidade , Lúpus Eritematoso Sistêmico/patologia , Macrófagos/imunologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/metabolismo , Gravidez , Receptores de Ativador de Plasminogênio Tipo Uroquinase/sangue , Valores de Referência , Ribonucleoproteínas/metabolismo , Taxa de Sobrevida , Ativador de Plasminogênio Tipo Uroquinase/sangue , Ativador de Plasminogênio Tipo Uroquinase/imunologia
18.
J Biol Chem ; 288(13): 9077-83, 2013 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-23386618

RESUMO

Toll-like receptor (TLR) signaling is an important component in the inflammatory response generated in diseases characterized by autoantibody reactivity to proteins such as SSA/Ro in complex with endogenous nucleic acids. Complement receptor 3 (CR3), a genetic variant of which has been identified as a risk factor in systemic lupus erythematosus, has been shown to induce tolerogenic responses in dendritic cells and suppress TLR4 responses in a murine sepsis model. Accordingly, this study addressed the hypothesis that activation of CR3, influenced by genotype of CD11b, negatively regulates TLR7/8-dependent effector function. Allosteric activation of CD11b via pretreatment with the small molecule, leukadhedrin 1 (LA1), significantly attenuated TLR7/8-induced (hY3 RNA, R848) secretion of TNFα in THP-1 cells and human macrophages isolated from donors homozygous for the ancestral common ITGAM allele at rs1143679. This inhibition was accompanied by profound degradation of the adaptor protein MyD88, an effect not observed with direct inhibition of TLR ligation by an antagonist oligonucleotide. In contrast, the addition of LA1 after incubation with the TLR agonists did not result in MyD88 degradation and subsequent attenuation of TNFα secretion. In TLR7/8-stimulated macrophages isolated from donors heterozygous for the CD11b variant, pretreatment with LA1 did not down-regulate TNFα release. These novel findings support a negative cross-talk between CR3 and TLR pathways likely to be induced by antibodies reactive with ribonucleoproteins and point to the development of CR3-specific agonists as potential therapeutics for diseases such as neonatal lupus.


Assuntos
Doenças Autoimunes/metabolismo , Antígeno CD11b/biossíntese , Regulação da Expressão Gênica , Antígeno de Macrófago 1/metabolismo , Macrófagos/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , Sítio Alostérico , Anti-Inflamatórios/farmacologia , Adesão Celular , Regulação para Baixo , Heterozigoto , Humanos , Inflamação , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/metabolismo , Macrófagos/citologia , Modelos Genéticos , Fator 88 de Diferenciação Mieloide/metabolismo , RNA/metabolismo , Transdução de Sinais
19.
Arthritis Care Res (Hoboken) ; 64(9): 1373-81, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22511615

RESUMO

OBJECTIVE: Maternal anti-Ro autoantibodies are associated with cardiac manifestations of neonatal lupus (cardiac NL), yet only 2% of women with this reactivity have an affected child. Identification of a more specific marker would channel intense monitoring to fetuses at greater risk. This study aimed to determine whether autoantibodies against Ro 52 amino acids 200-239 (p200) confer added risk over autoantibodies to full-length Ro 52, Ro 60, or La. METHODS: Anti-Ro-exposed pregnancies resulting in cardiac NL or no cardiac manifestations were identified from the Research Registry for Neonatal Lupus and the PR Interval and Dexamethasone Evaluation study. Umbilical cord (n = 123) and maternal (n = 115) samples were evaluated by enzyme-linked immunosorbent assay. RESULTS: The frequencies of p200, Ro 52, Ro 60, and La autoantibodies were not significantly different between affected and unaffected children. However, neonatal anti-Ro 52 and Ro 60 titers were highest in cardiac NL and their unaffected siblings compared to unaffected neonates without a cardiac NL sibling. Although both maternal anti-Ro 52 and p200 autoantibodies were less than 50% specific for cardiac NL, anti-p200 was the least likely of the Ro autoantibodies to be false-positive in mothers who have never had an affected child. Titers of anti-Ro 52 and p200 did not differ during a cardiac NL or unaffected pregnancy from the same mother. CONCLUSION: Maternal reactivity to p200 does not confer an added risk to fetal conduction defects over full-length Ro 52 or Ro 60 autoantibodies. Mothers who may never be at risk for having an affected child have lower anti-Ro 60 titers and may require less stringent echocardiographic monitoring compared to women with high-titer autoantibodies.


Assuntos
Anticorpos Antinucleares/sangue , Sangue Fetal/imunologia , Bloqueio Cardíaco/etiologia , Epitopos Imunodominantes , Lúpus Eritematoso Sistêmico/congênito , Fragmentos de Peptídeos/imunologia , Ribonucleoproteínas/imunologia , Biomarcadores/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Bloqueio Cardíaco/sangue , Bloqueio Cardíaco/imunologia , Humanos , Modelos Lineares , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Gravidez , Sistema de Registros , Medição de Risco , Fatores de Risco , Estados Unidos
20.
Immunol Cell Biol ; 90(3): 304-9, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22249199

RESUMO

Ro/SSA and La/SSB comprise a linked set of autoantigens that are clinically important members of the extractable nuclear antigen family and key translational biomarkers for lupus and primary Sjögren's syndrome. Autoantibodies directed against the Ro60 and La polypeptide components of the Ro/La ribonucleoprotein complex, and the structurally unrelated Ro52 protein, mediate tissue damage in the neonatal lupus syndrome, a model of passively acquired autoimmunity in humans in which the most serious manifestation is congenital heart block (CHB). Recent studies have concentrated on two distinct pathogenic mechanisms by which maternal anti-Ro/La autoantibodies can cause CHB: by forming immune complexes with apoptotic cells in developing fetal heart; and/or by acting as functional autoantibodies that cross-react with and inhibit calcium channels. Although the precise role of the individual autoantibodies is yet to be settled, maternal anti-Ro60 and anti-Ro52 remain the most likely culprits. This article will discuss the molecular pathways that culminate in the development of CHB, including the recent discovery of ß2 glycoprotein I as a protective factor, and present a proteomic approach based on direct mass spectrometric sequencing, which may give a more representative snapshot of the idiotype repertoire of these autoantibodies than genomic-based technologies.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Bloqueio Cardíaco/congênito , Imunidade Materno-Adquirida , Ribonucleoproteínas/imunologia , Animais , Complexo Antígeno-Anticorpo/imunologia , Reações Cruzadas , Feminino , Bloqueio Cardíaco/imunologia , Humanos , Recém-Nascido , Lúpus Eritematoso Sistêmico/congênito , Lúpus Eritematoso Sistêmico/imunologia , Proteômica
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