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1.
J Biol Chem ; 293(39): 15136-15151, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30104412

RESUMO

Wiskott-Aldrich syndrome protein (WASP) activates the actin-related protein 2/3 homolog (Arp2/3) complex and regulates actin polymerization in a physiological setting. Cell division cycle 42 (Cdc42) is a key activator of WASP, which binds Cdc42 through a Cdc42/Rac-interactive binding (CRIB)-containing region that defines a subset of Cdc42 effectors. Here, using site-directed mutagenesis and binding affinity determination and kinetic assays, we report the results of an investigation into the energetic contributions of individual WASP residues to both the Cdc42-WASP binding interface and the kinetics of complex formation. Our results support the previously proposed dock-and-coalesce binding mechanism, initiated by electrostatic steering driven by WASP's basic region and followed by a coalescence phase likely driven by the conserved CRIB motif. The WASP basic region, however, appears also to play a role in the final complex, as its mutation affected both on- and off-rates, suggesting a more comprehensive physiological role for this region centered on the C-terminal triad of positive residues. These results highlight the expanding roles of the basic region in WASP and other CRIB-containing effector proteins in regulating complex cellular processes and coordinating multiple input signals. The data presented improve our understanding of the Cdc42-WASP interface and also add to the body of information available for Cdc42-effector complex formation, therapeutic targeting of which has promise for Ras-driven cancers. Our findings suggest that combining high-affinity peptide-binding sequences with short electrostatic steering sequences could increase the efficacy of peptidomimetic candidates designed to interfere with Cdc42 signaling in cancer.


Assuntos
Neoplasias/genética , Proteína da Síndrome de Wiskott-Aldrich/química , Síndrome de Wiskott-Aldrich/genética , Proteína cdc42 de Ligação ao GTP/química , Actinas/química , Actinas/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Cinética , Neoplasias/química , Neoplasias/patologia , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico/genética , Transdução de Sinais , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas ras/química , Proteínas ras/genética
2.
Cell Stem Cell ; 23(1): 132-146.e9, 2018 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-29979988

RESUMO

Genes that regulate hematopoietic stem cell (HSC) self-renewal, proliferation, and differentiation are tightly controlled by regulatory regions. However, mapping such regions relies on surface markers and immunophenotypic definition of HSCs. Here, we use γ-retroviral integration sites (γRV ISs) from a gene therapy trial for 10 patients with Wiskott-Aldrich syndrome to mark active enhancers and promoters in functionally defined long-term repopulating HSCs. Integration site clusters showed the highest ATAC-seq signals at HSC-specific peaks and strongly correlated with hematopoietic risk variants. Tagged genes were significantly enriched for HSC gene sets. We were able to map over 3,000 HSC regulatory regions in late-contributing HSCs, and we used these data to identify miR-10a and miR-335 as two miRNAs regulating early hematopoiesis. In this study, we show that viral insertion sites can be used as molecular tags to assess chromatin conformation on functionally defined cell populations, thereby providing a genome-wide resource for regulatory regions in human repopulating long-term HSCs.


Assuntos
Cromatina/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Diferenciação Celular , Proliferação de Células , Terapia Genética , Células HEK293 , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patologia , Síndrome de Wiskott-Aldrich/terapia
3.
Biol Blood Marrow Transplant ; 24(7): 1432-1440, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29550630

RESUMO

Our initial experience with hematopoietic stem cell transplantation (HSCT) from a matched unrelated donor (MUD; n = 12) or a haploidentical related donor (n = 6) with T cell receptor (TCR)αß+/CD19+ graft depletion in patients with Wiskott-Aldrich syndrome (WAS) (n = 18) showed a dramatic decrease in the incidence of graft-versus-host disease (GVHD) and transplantation-related mortality, with an increased overall survival (OS) of 88.9%. Unfortunately, the treatment was associated with mixed myeloid donor chimerism and secondary graft dysfunction (severe thrombocytopenia, n = 2; graft rejection, n = 5). To improve the outcome, we hypothesized that the addition of G-CSF and plerixafor to the conditioning chemotherapy would result in more complete donor stem cell engraftment. This trial was registered at www.clinicaltrials.gov (NCT03019809). A study group of patients with WAS (n = 16) underwent TCRαß+/CD19+-depleted HSCT (MUD, n = 6; haploidentical, n = 10). The conditioning regimen was treosulfan-fludarabine-rabbit antithymocyte globulin-melphalan (or thiophosphamide in 1 patient) with G-CSF (10 µg/kg/day for 5 days starting on day -8) and plerixafor (240 µg/kg/day for 3 days starting on day -6). The clinical outcomes in this study were compared to those in a historical dataset (n = 18). No patients had grade III/IV acute GVHD in either the study or the historical control group. Importantly, in the patients with WAS, there was no statistical significance in OS between those who underwent HSCT from haploidentical donors and those who underwent HSCT from MUDs (93.8% versus 88.5%; P = .612). All patients in the study group had full donor chimerism in whole blood and in the CD3+ compartments. The OS was 93.8%, and there were no cases of graft dysfunction. This study demonstrates the efficacy of adding G-CSF/plerixafor to the conditioning regimen before HSCT with TCRαß+/C D19+ graft depletion in patients with WAS.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Antígenos CD19/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Compostos Heterocíclicos/uso terapêutico , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Condicionamento Pré-Transplante/métodos , Síndrome de Wiskott-Aldrich/terapia , Fármacos Anti-HIV/farmacologia , Criança , Pré-Escolar , Feminino , Compostos Heterocíclicos/farmacologia , Humanos , Lactente , Recém-Nascido , Masculino , Resultado do Tratamento , Síndrome de Wiskott-Aldrich/patologia
4.
Cogn Behav Neurol ; 31(1): 13-17, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29561314

RESUMO

We report the neuropsychological profile of a 6-year-old girl with Wiskott-Aldrich syndrome, a rare X-linked immunodeficiency disorder associated with thrombocytopenia, eczema, recurrent infections, and malignancy. Wiskott-Aldrich syndrome occurs almost exclusively in males and is extremely rare in females, with no known research focused on cognitive and academic functioning in this population. Our patient was referred due to concerns about her memory and academic functioning. She had a history of progressive thrombocytopenia and hematopoietic stem cell transplantation at age 15 months. Standardized measures of intellectual ability, language, visual-spatial and visual-motor skills, attention, memory, and academic achievement were administered. The results showed average to above-average performance in multiple areas of cognitive and academic functioning, with weaknesses in phonological awareness and rapid naming. The advent of hematopoietic stem cell transplantation has led to considerable improvement in the long-term prognosis of children with Wiskott-Aldrich syndrome. Although the impact of this syndrome and related conditions on neurocognitive development is presently unknown, this case highlights both the importance of considering base rates for commonly occurring conditions and the significant role neuropsychology can play in identifying cognitive strengths and weaknesses in the context of the developing brain.


Assuntos
Testes Neuropsicológicos , Síndrome de Wiskott-Aldrich/diagnóstico , Criança , Feminino , Humanos , Síndrome de Wiskott-Aldrich/patologia
6.
J Allergy Clin Immunol ; 142(1): 219-234, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29248492

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia, which are caused by WAS mutations affecting Wiskott-Aldrich syndrome protein (WASp) expression or activity, manifest in immunodeficiency, autoimmunity, genomic instability, and lymphoid and other cancers. WASp supports filamentous actin formation in the cytoplasm and gene transcription in the nucleus. Although the genetic basis for XLT/WAS has been clarified, the relationships between mutant forms of WASp and the diverse features of these disorders remain ill-defined. OBJECTIVE: We sought to define how dysfunctional gene transcription is causally linked to the degree of TH cell deficiency and genomic instability in the XLT/WAS clinical spectrum. METHODS: In human TH1- or TH2-skewing cell culture systems, cotranscriptional R-loops (RNA/DNA duplex and displaced single-stranded DNA) and DNA double-strand breaks (DSBs) were monitored in multiple samples from patients with XLT and WAS and in normal T cells depleted of WASp. RESULTS: WASp deficiency provokes increased R-loops and R-loop-mediated DSBs in TH1 cells relative to TH2 cells. Mechanistically, chromatin occupancy of serine 2-unphosphorylated RNA polymerase II is increased, and that of topoisomerase 1, an R-loop preventing factor, is decreased at R-loop-enriched regions of IFNG and TBX21 (TH1 genes) in TH1 cells. These aberrations accompany increased unspliced (intron-retained) and decreased spliced mRNA of IFNG and TBX21 but not IL13 (TH2 gene). Significantly, increased cellular load of R-loops and DSBs, which are normalized on RNaseH1-mediated suppression of ectopic R-loops, inversely correlates with disease severity scores. CONCLUSION: Transcriptional R-loop imbalance is a novel molecular defect causative in TH1 immunodeficiency and genomic instability in patients with WAS. The study proposes that cellular R-loop load could be used as a potential biomarker for monitoring symptom severity and prognostic outcome in the XLT-WAS clinical spectrum and could be targeted therapeutically.


Assuntos
Instabilidade Genômica/genética , Células Th1/patologia , Síndrome de Wiskott-Aldrich/genética , Células Cultivadas , Dano ao DNA/genética , Humanos , Transcrição Genética , Síndrome de Wiskott-Aldrich/patologia
7.
J Leukoc Biol ; 103(3): 577-590, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28851742

RESUMO

Microthrombocytopenia is the clinical hallmark of WAS, a rare X-linked immunodeficiency that is characterized by eczema, autoimmunity, and cancer susceptibility. This disease is caused by mutations in the WAS gene, which is expressed in hematopoietic cells and regulates actin cytoskeleton remodeling thereby modulating various cellular functions, including motility, immunologic synapse assembly, and signaling. Despite extensive studies that have provided great insight into the relevance of this molecule to innate and cellular immunity, the exact mechanisms of microthrombocytopenia in WAS are still unknown. This review focuses on the recent progress made in dissecting the pathogenesis of platelet defects in patients with WAS and their murine counterparts. In parallel, we will provide an overview of the state-of-the art platelets as immune modulators at the interface between hemostasis and the immune system, which suggests that these cells may have a direct role in the pathogenesis of immune dysregulation in WAS.


Assuntos
Autoimunidade , Plaquetas/imunologia , Síndrome de Wiskott-Aldrich/imunologia , Animais , Plaquetas/patologia , Humanos , Transdução de Sinais , Síndrome de Wiskott-Aldrich/patologia
8.
Pediatr Hematol Oncol ; 34(5): 286-291, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29200320

RESUMO

BACKGROUND: The Wiskott-Aldrich syndrome (WAS) is X-linked recessive disorder associated with microplatelet thrombocytopenia, eczema, infections, and an increased risk of autoimmunity and lymphoid neoplasia. The originally described features of WAS include susceptibility to infections, microthrombocytopenia, and eczema. AIM: In this case report, we present our experience about two cases diagnosed with a new mutation. METHODS: We report phenotypical and laboratory description of two cases with WAS. RESULTS: We, for the first time, detected a new hemizygote mutation of WAS gene (NM_000377.2 p.M393lfs*102 (c.1178dupT)) in two patients. The first case was an 11-month-old boy presenting with complaints of recurrent soft tissue infection, ear infection, anemia, and thrombocytopenia with a low platelet volume. The second case was a 2-month-old boy presenting with thrombocytopenia and a low platelet volume. Both cases were the first-degree relatives: they were cousins and their mothers were sisters. CONCLUSION: Herein, we report two cases of WAS and a new gene mutation which would disrupt the WAS protein function within the Polyproline (PPP) domain. This report adds to the growing number of mutations which cause complex clinical manifestations associated with WAS.


Assuntos
Hemizigoto , Mutação , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Humanos , Lactente , Masculino , Domínios Proteicos , Síndrome de Wiskott-Aldrich/patologia
9.
Sci Rep ; 7(1): 11978, 2017 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-28931895

RESUMO

Wiskott-Aldrich syndrome protein (WASp) is a key regulator of the actin cytoskeleton. Defective T - cell function is a major cause for immune deficiency in Wiskott-Aldrich syndrome (WAS) patients. T cells originate in the bone marrow and develop in the thymus, and then migrate to peripheral tissues. TCR excision circles (TRECs) present in thymic output cells stably, which is used as a molecular marker for thymic output. We found that CD8+ T naïve cells of classic WAS patients were significantly reduced, and TRECs in patients with classic WAS and X-linked thrombocytopenia (XLT) dramatically decreased compared with that of HCs. TRECs were also reduced in WAS (KO) mice. These suggest that defective thymic output partially accounts for T cell lymphopenia in WAS patients. However, the correlation between the defect of thymic output and actin organization still remains elusive. We found that the subcellular location and the levels of of F-actin were altered in T cells from both WAS and XLT patients compared to that of HCs with or without stimulation. Our study shows that WASp plays a critical role in thymic output, which highly correlates with the subcellular location and level of F-actin in T cells.


Assuntos
Actinas/metabolismo , Linfopenia/fisiopatologia , Linfócitos T/patologia , Timo/patologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/patologia , Animais , Humanos , Lactente , Recém-Nascido , Camundongos
10.
Nat Commun ; 8: 14816, 2017 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-28368018

RESUMO

Human actin-related protein 2/3 complex (Arp2/3), required for actin filament branching, has two ARPC1 component isoforms, with ARPC1B prominently expressed in blood cells. Here we show in a child with microthrombocytopenia, eosinophilia and inflammatory disease, a homozygous frameshift mutation in ARPC1B (p.Val91Trpfs*30). Platelet lysates reveal no ARPC1B protein and greatly reduced Arp2/3 complex. Missense ARPC1B mutations are identified in an unrelated patient with similar symptoms and ARPC1B deficiency. ARPC1B-deficient platelets are microthrombocytes similar to those seen in Wiskott-Aldrich syndrome that show aberrant spreading consistent with loss of Arp2/3 function. Knockout of ARPC1B in megakaryocytic cells results in decreased proplatelet formation, and as observed in platelets from patients, increased ARPC1A expression. Thus loss of ARPC1B produces a unique set of platelet abnormalities, and is associated with haematopoietic/immune symptoms affecting cell lineages where this isoform predominates. In agreement with recent experimental studies, our findings suggest that ARPC1 isoforms are not functionally interchangeable.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/deficiência , Transtornos Plaquetários/metabolismo , Plaquetas/metabolismo , Inflamação/patologia , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Plaquetas/efeitos dos fármacos , Plaquetas/patologia , Plaquetas/ultraestrutura , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Forma Celular , Suscetibilidade a Doenças , Fibrinogênio/farmacologia , Técnicas de Inativação de Genes , Humanos , Megacariócitos/efeitos dos fármacos , Megacariócitos/metabolismo , Megacariócitos/patologia , Mutação/genética , Vasculite/patologia , Síndrome de Wiskott-Aldrich/patologia
11.
Pediatr Blood Cancer ; 64(1): 106-109, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27566838

RESUMO

Patients with Wiskott-Aldrich syndrome (WAS) are predisposed to malignancy and autoimmunity in addition to infections. We report a male child with WAS, who had presented with recurrent pneumonia, eczema, thrombocytopenia, autoimmune hemolytic anemia, and vasculitic skin lesions. Genetic analysis revealed a classical genotype WAS 155C>T; R41X. At 2 years of follow-up, he developed persistent headache and progressive hepatomegaly. Brain imaging showed a mass in the right frontal region, which on histopathology was shown to be high-grade non-Hodgkin lymphoma. Magnetic resonance cholangiopancreatography showed features of sclerosing cholangitis. This report extends the clinical spectrum and highlights unusual manifestations of sclerosing cholangitis and intracranial lymphoma in a patient with WAS.


Assuntos
Anemia Hemolítica Autoimune/patologia , Neoplasias Encefálicas/patologia , Colangite Esclerosante/patologia , Linfoma não Hodgkin/patologia , Trombocitopenia/patologia , Síndrome de Wiskott-Aldrich/complicações , Adolescente , Anemia Hemolítica Autoimune/etiologia , Neoplasias Encefálicas/etiologia , Colangite Esclerosante/etiologia , Humanos , Linfoma não Hodgkin/etiologia , Masculino , Mutação/genética , Prognóstico , Trombocitopenia/etiologia , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética
12.
Stem Cell Reports ; 7(2): 139-48, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27396937

RESUMO

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease caused by mutations in the gene encoding the WAS protein (WASp). Here, induced pluripotent stem cells (iPSCs) were derived from a WAS patient (WAS-iPSC) and the endogenous chromosomal WAS locus was targeted with a wtWAS-2A-eGFP transgene using zinc finger nucleases (ZFNs) to generate corrected WAS-iPSC (cWAS-iPSC). WASp and GFP were first expressed in the earliest CD34(+)CD43(+)CD45(-) hematopoietic precursor cells and later in all hematopoietic lineages examined. Whereas differentiation to non-lymphoid lineages was readily obtained from WAS-iPSCs, in vitro T lymphopoiesis from WAS-iPSC was deficient with few CD4(+)CD8(+) double-positive and mature CD3(+) T cells obtained. T cell differentiation was restored for cWAS-iPSCs. Similarly, defects in natural killer cell differentiation and function were restored on targeted correction of the WAS locus. These results demonstrate that the defects exhibited by WAS-iPSC-derived lymphoid cells were fully corrected and suggests the potential therapeutic use of gene-corrected WAS-iPSCs.


Assuntos
Terapia Genética , Células-Tronco Pluripotentes Induzidas/patologia , Linfopoese , Síndrome de Wiskott-Aldrich/patologia , Síndrome de Wiskott-Aldrich/terapia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Humanos , Células Matadoras Naturais/metabolismo , Linfócitos T/imunologia , Proteína da Síndrome de Wiskott-Aldrich/genética
13.
Blood ; 127(25): 3180-91, 2016 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-27170596

RESUMO

Wiskott-Aldrich syndrome protein (WASp) is a hematopoietic-specific regulator of actin nucleation. Wiskott-Aldrich syndrome (WAS) patients show immunodeficiencies, most of which have been attributed to defective T-cell functions. T follicular helper (Tfh) cells are the major CD4(+) T-cell subset with specialized B-cell helper capabilities. Aberrant Tfh cells activities are involved in immunopathologies such as autoimmunity, immunodeficiencies, and lymphomas. We found that in WAS patients, the number of circulating Tfh cells was significantly reduced due to reduced proliferation and increased apoptosis, and Tfh cells were Th2 and Th17 polarized. The expression of inducible costimulator (ICOS) in circulating Tfh cells was higher in WAS patients than in controls. BCL6 expression was decreased in total CD4(+) T and Tfh cells of WAS patients. Mirroring the results in patients, the frequency of Tfh cells in WAS knockout (KO) mice was decreased, as was the frequency of BCL6(+) Tfh cells, but the frequency of ICOS(+) Tfh cells was increased. Using WAS chimera mice, we found that the number of ICOS(+) Tfh cells was decreased in WAS chimera mice, indicating that the increase in ICOS(+) Tfh cells in WAS KO mice was cell extrinsic. The data from in vivo CD4(+) naive T-cell adoptive transfer mice as well as in vitro coculture of naive B and Tfh cells showed that the defective function of WASp-deficient Tfh cells was T-cell intrinsic. Consistent findings in both WAS patients and WAS KO mice suggested an essential role for WASp in the development and memory response of Tfh cells and that WASp deficiency causes a deficient differentiation defect in Tfh cells by downregulating the transcription level of BCL6.


Assuntos
Centro Germinativo/patologia , Linfócitos T Auxiliares-Indutores/patologia , Linfócitos T Auxiliares-Indutores/fisiologia , Síndrome de Wiskott-Aldrich/imunologia , Animais , Linfócitos B , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/fisiologia , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Centro Germinativo/imunologia , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Interleucinas/metabolismo , Linfonodos/imunologia , Linfonodos/patologia , Camundongos , Camundongos Knockout , Fator 1 de Ligação ao Domínio I Regulador Positivo , Receptores CXCR5/metabolismo , Proteínas Repressoras/metabolismo , Síndrome de Wiskott-Aldrich/sangue , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética
14.
Biol Cell ; 108(8): 205-18, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27061938

RESUMO

The trans-Golgi network (TGN) is a major sorting, packing and delivering station of newly synthesised proteins and lipids to their final destination. These cargo molecules follow the secretory pathway, which is a vital part of cellular trafficking machinery in all eukaryotic cells. This secretory pathway is well conserved in all eukaryotes from low-level eukaryotes, such as yeast, to higher level eukaryotes like mammals. The molecular mechanisms of protein sorting by adaptor proteins, membrane elongation and transport to the final destinations by motor proteins and the cytoskeleton, and membrane pinching-off by scission proteins must be choreographically managed for efficient cargo delivery, and the understanding of these detailed processes is not yet completed. Functionally, defects in these mechanisms are associated with the pathology of prominent diseases such as acute myeloid leukaemia, Charcot-Marie-Tooth disease, I-cell disease and Wiskott-Aldrich syndrome. The present review points out the recent advances in our knowledge of the molecular mechanisms involved in the transportation of the cargo from the TGN towards the endosome.


Assuntos
Doença de Charcot-Marie-Tooth/metabolismo , Endossomos/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Rede trans-Golgi/metabolismo , Animais , Transporte Biológico Ativo , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Endossomos/genética , Endossomos/patologia , Humanos , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patologia , Rede trans-Golgi/genética , Rede trans-Golgi/patologia
17.
Blood ; 127(2): 216-20, 2016 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-26468226

RESUMO

Mutations of the Wiskott-Aldrich syndrome gene (WAS) are responsible for Wiskott-Aldrich syndrome (WAS), a disease characterized by thrombocytopenia, eczema, immunodeficiency, and autoimmunity. Mice with conditional deficiency of Was in B lymphocytes (B/WcKO) have revealed a critical role for WAS protein (WASP) expression in B lymphocytes in the maintenance of immune homeostasis. Neural WASP (N-WASP) is a broadly expressed homolog of WASP, and regulates B-cell signaling by modulating B-cell receptor (BCR) clustering and internalization. We have generated a double conditional mouse lacking both WASP and N-WASP selectively in B lymphocytes (B/DcKO). Compared with B/WcKO mice, B/DcKO mice showed defective B-lymphocyte proliferation and impaired antibody responses to T-cell-dependent antigens, associated with decreased autoantibody production and lack of autoimmune kidney disease. These results demonstrate that N-WASP expression in B lymphocytes is required for the development of autoimmunity of WAS and may represent a novel therapeutic target in WAS.


Assuntos
Autoimunidade/genética , Linfócitos B/imunologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/fisiologia , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/imunologia , Animais , Linfócitos B/metabolismo , Linfócitos B/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Deleção de Genes , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/imunologia , Síndrome de Wiskott-Aldrich/patologia , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética
18.
PLoS One ; 10(10): e0139729, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26448644

RESUMO

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema, and high incidence of malignancy and autoimmunity. The cellular mechanisms underlying autoimmune complications in WAS have been extensively studied; however, they remain incompletely defined. We investigated the characteristics of IL-10-producing CD19+CD1dhighCD5+ B cells (CD1dhighCD5+ Breg) obtained from Was gene knockout (WKO) mice and found that their numbers were significantly lower in these mice compared to wild type (WT) controls. Moreover, we found a significant age-dependent reduction of the percentage of IL-10-expressing cells in WKO CD1dhighCD5+ Breg cells as compared to age-matched WT control mice. CD1dhighCD5+ Breg cells from older WKO mice did not suppress the in vitro production of inflammatory cytokines from activated CD4+ T cells. Interestingly, CD1dhighCD5+ Breg cells from older WKO mice displayed a basal activated phenotype which may prevent normal cellular responses, among which is the expression of IL-10. These defects may contribute to the susceptibility to autoimmunity with age in patients with WAS.


Assuntos
Envelhecimento , Linfócitos B Reguladores/imunologia , Síndrome de Wiskott-Aldrich/patologia , Animais , Antígenos CD19/metabolismo , Antígenos CD1d/metabolismo , Linfócitos B Reguladores/citologia , Linfócitos B Reguladores/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD5/metabolismo , Técnicas de Cocultura , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Interleucina-10/análise , Masculino , Camundongos , Camundongos Knockout , Síndrome de Wiskott-Aldrich/metabolismo
19.
J Exp Med ; 212(10): 1663-77, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26371186

RESUMO

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder frequently associated with systemic autoimmunity, including autoantibody-mediated cytopenias. WAS protein (WASp)-deficient B cells have increased B cell receptor (BCR) and Toll-like receptor (TLR) signaling, suggesting that these pathways might impact establishment of the mature, naive BCR repertoire. To directly investigate this possibility, we evaluated naive B cell specificity and composition in WASp-deficient mice and WAS subjects (n = 12). High-throughput sequencing and single-cell cloning analysis of the BCR repertoire revealed altered heavy chain usage and enrichment for low-affinity self-reactive specificities in murine marginal zone and human naive B cells. Although negative selection mechanisms including deletion, anergy, and receptor editing were relatively unperturbed, WASp-deficient transitional B cells showed enhanced proliferation in vivo mediated by antigen- and Myd88-dependent signals. Finally, using both BCR sequencing and cell surface analysis with a monoclonal antibody recognizing an intrinsically autoreactive heavy chain, we show enrichment in self-reactive cells specifically at the transitional to naive mature B cell stage in WAS subjects. Our combined data support a model wherein modest alterations in B cell-intrinsic, BCR, and TLR signals in WAS, and likely other autoimmune disorders, are sufficient to alter B cell tolerance via positive selection of self-reactive transitional B cells.


Assuntos
Linfócitos B/metabolismo , Linfócitos B/patologia , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores Toll-Like/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Adolescente , Adulto , Animais , Fator Ativador de Células B/sangue , Linfócitos B/imunologia , Estudos de Casos e Controles , Criança , Pré-Escolar , Citoproteção , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/metabolismo , Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Adulto Jovem
20.
Eur J Immunol ; 45(10): 2773-9, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26256668

RESUMO

Patients with Wiskott-Aldrich syndrome (WAS) exhibit prominent defects in splenic marginal zone (MZ), resulting in abnormal T-cell-independent antibody responses and increased bacterial infections. B-cell-intrinsic deletion of the affected gene WAS protein (WASp) markedly reduces splenic MZ B cells, without impacting the rate of MZ B-cell development, suggesting that abnormal B-cell retention within the MZ accounts for MZ defects in WAS. Since WASp regulates integrin-dependent actin cytoskeletal rearrangement, we previously hypothesized that defective B-cell integrin function promotes MZ depletion. In contrast, we now report that B-cell-intrinsic deletion of the TLR signaling adaptor MyD88 is sufficient to restore the MZ in WAS. We further identify TLR7, an endosomal single-stranded RNA (ssRNA) receptor, as the MyD88-dependent receptor responsible for WAS MZ depletion. These findings implicate spontaneous activation of MZ B cells by ssRNA-containing self-ligands (likely derived from circulating apoptotic material) as the mechanism underlying MZ depletion in WAS. Together, these data suggest a previously unappreciated role for B-cell intrinsic TLR signals in MZ homeostasis, of relevance to both pathogen responses and to the development of systemic autoimmunity.


Assuntos
Linfócitos B/imunologia , Glicoproteínas de Membrana/imunologia , Transdução de Sinais/imunologia , Baço/imunologia , Receptor 7 Toll-Like/imunologia , Síndrome de Wiskott-Aldrich/imunologia , Animais , Linfócitos B/patologia , Citoesqueleto/genética , Citoesqueleto/imunologia , Modelos Animais de Doenças , Integrinas/genética , Integrinas/imunologia , Depleção Linfocítica , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Transdução de Sinais/genética , Baço/patologia , Receptor 7 Toll-Like/genética , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patologia
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