Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 63
Filtrar
Más filtros

Banco de datos
Tipo del documento
Intervalo de año de publicación
1.
Eur J Immunol ; 54(5): e2350450, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38356202

RESUMEN

The Wiskott-Aldrich syndrome protein (WASp) regulates actin cytoskeletal dynamics and function of hematopoietic cells. Mutations in the WAS gene lead to two different syndromes; Wiskott-Aldrich syndrome (WAS) caused by loss-of-function mutations, and X-linked neutropenia (XLN) caused by gain-of-function mutations. We previously showed that WASp-deficient mice have a decreased number of regulatory T (Treg) cells in the thymus and the periphery. We here evaluated the impact of WASp mutations on Treg cells in the thymus of WAS and XLN mouse models. Using in vitro Treg differentiation assays, WAS CD4 single-positive thymocytes have decreased differentiation to Treg cells, despite normal early signaling upon IL-2 and TGF-ß stimulation. They failed to proliferate and express CD25 at high levels, leading to poor survival and a lower number of Foxp3+ Treg cells. Conversely, XLN CD4 single-positive thymocytes efficiently differentiate into Foxp3+ Treg cells following a high proliferative response to IL-2 and TGF-ß, associated with high CD25 expression when compared with WT cells. Altogether, these results show that specific mutations of WASp affect Treg cell development differently, demonstrating a critical role of WASp activity in supporting Treg cell development and expansion.


Asunto(s)
Diferenciación Celular , Proliferación Celular , Linfocitos T Reguladores , Timo , Proteína del Síndrome de Wiskott-Aldrich , Animales , Linfocitos T Reguladores/inmunología , Diferenciación Celular/inmunología , Proteína del Síndrome de Wiskott-Aldrich/genética , Proteína del Síndrome de Wiskott-Aldrich/metabolismo , Ratones , Timo/inmunología , Timo/citología , Factores de Transcripción Forkhead/metabolismo , Factores de Transcripción Forkhead/genética , Interleucina-2/metabolismo , Interleucina-2/inmunología , Mutación , Factor de Crecimiento Transformador beta/metabolismo , Síndrome de Wiskott-Aldrich/inmunología , Síndrome de Wiskott-Aldrich/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-2/genética , Ratones Noqueados , Ratones Endogámicos C57BL
2.
Br J Cancer ; 128(6): 982-991, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36631633

RESUMEN

BACKGROUND: Dendritic cell (DC) vaccines for cancer therapy offer the possibility to let the patient's own immune system kill cancer cells. However, DC vaccines have shown less efficacy than expected due to failure to induce cancer cell killing and by activating T regulatory cells. METHODS: We tested if inhibition of signalling via WASp and Arp2/3 using the small molecule CK666 would enhance DC-mediated killing of tumour cells in vitro and in vivo. RESULTS: Using CK666 during the ex vivo phase of antigen processing of ovalbumin (OVA), murine and human DCs showed decreased phagosomal acidification, indicating activation of the cross-presentation pathway. When compared to untreated DCs, DCs treated with CK666 during uptake and processing of OVA-induced increased proliferation of OVA-specific CD8+ OT-I T cells in vitro and in vivo. Using the aggressive B16-mOVA melanoma tumour model, we show that mice injected with CK666-treated DCs and OVA-specific CD8+ OT-I T cells showed higher rejection of B16 melanoma cells when compared to mice receiving non-treated DCs. This resulted in the prolonged survival of tumour-bearing mice receiving CK666-treated DCs. Moreover, combining CK666-treated DCs with the checkpoint inhibitor anti-PD1 further prolonged survival. CONCLUSION: Our data suggest that the small molecule inhibitor CK666 is a good candidate to enhance DC cross-presentation for cancer therapy.


Asunto(s)
Reactividad Cruzada , Vacunas , Ratones , Animales , Humanos , Linfocitos T CD8-positivos , Células Dendríticas , Presentación de Antígeno , Ovalbúmina/metabolismo , Vacunas/metabolismo , Ratones Endogámicos C57BL
3.
Pediatr Allergy Immunol ; 34(4): e13951, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-37102395

RESUMEN

Immunoactinopathies caused by mutations in actin-related proteins are a growing group of inborn errors of immunity (IEI). Immunoactinopathies are caused by a dysregulated actin cytoskeleton and affect hematopoietic cells especially because of their unique capacity to survey the body for invading pathogens and altered self, such as cancer cells. These cell motility and cell-to-cell interaction properties depend on the dynamic nature of the actin cytoskeleton. Wiskott-Aldrich syndrome (WAS) is the archetypical immunoactinopathy and the first described. WAS is caused by loss-of-function and gain-of-function mutations in the actin regulator WASp, uniquely expressed in hematopoietic cells. Mutations in WAS cause a profound disturbance of actin cytoskeleton regulation of hematopoietic cells. Studies during the last 10 years have shed light on the specific effects on different hematopoietic cells, revealing that they are not affected equally by mutations in the WAS gene. Moreover, the mechanistic understanding of how WASp controls nuclear and cytoplasmatic activities may help to find therapeutic alternatives according to the site of the mutation and clinical phenotypes. In this review, we summarize recent findings that have added to the complexity and increased our understanding of WAS-related diseases and immunoactinopathies.


Asunto(s)
Actinas , Síndrome de Wiskott-Aldrich , Humanos , Actinas/genética , Actinas/metabolismo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Mutación , Fenotipo
4.
J Allergy Clin Immunol ; 149(3): 1069-1084, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34384840

RESUMEN

BACKGROUND: B-cell affinity maturation in germinal center relies on regulated actin dynamics for cell migration and cell-to-cell communication. Activating mutations in the cytoskeletal regulator Wiskott-Aldrich syndrome protein (WASp) cause X-linked neutropenia (XLN) with reduced serum level of IgA. OBJECTIVE: We investigated the role of B cells in XLN pathogenesis. METHODS: We examined B cells from 6 XLN patients, 2 of whom had novel R268W and S271F mutations in WASp. By using immunized XLN mouse models that carry the corresponding patient mutations, WASp L272P or WASp I296T, we examined the B-cell response. RESULTS: XLN patients had normal naive B cells and plasmablasts, but reduced IgA+ B cells and memory B cells, and poor B-cell proliferation. On immunization, XLN mice had a 2-fold reduction in germinal center B cells in spleen, but with increased generation of plasmablasts and plasma cells. In vitro, XLN B cells showed reduced immunoglobulin class switching and aberrant cell division as well as increased production of immunoglobulin-switched plasma cells. CONCLUSIONS: Overactive WASp predisposes B cells for premature differentiation into plasma cells at the expense of cell proliferation and immunoglobulin class switching.


Asunto(s)
Linfocitos B , Neutropenia , Proteína del Síndrome de Wiskott-Aldrich , Animales , Linfocitos B/citología , División Celular , Enfermedades Genéticas Ligadas al Cromosoma X , Humanos , Inmunoglobulina A , Ratones , Neutropenia/genética , Células Plasmáticas/patología , Proteína del Síndrome de Wiskott-Aldrich/metabolismo
5.
Br J Cancer ; 127(11): 2060-2071, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36138076

RESUMEN

BACKGROUND: p53 mutants contribute to the chronic inflammatory tumour microenvironment (TME). In this study, we address the mechanism of how p53 mutants lead to chronic inflammation in tumours and how to transform it to restore cancer immune surveillance. METHODS: Our analysis of RNA-seq data from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) project revealed that mutant p53 (mtp53) cancers correlated with chronic inflammation. We used cell-based assays and a mouse model to discover a novel gain of function of mtp53 and the effect of the mtp53 reactivating compound APR-246 on the anti-tumour immune response. RESULTS: We found that tumour samples from patients with breast carcinoma carrying mtp53 showed elevated Interferon (IFN) signalling, Tumour Inflammation Signature (TIS) score and infiltration of CD8+ T cells compared to wild type p53 (wtp53) tumours. We showed that the expression of IFN and immune checkpoints were elevated in tumour cells in a mtp53-dependent manner, suggesting a novel gain of function. Restoration of wt function to mtp53 by APR-246 induced the expression of endogenous retroviruses, IFN signalling and repressed immune checkpoints. Moreover, APR-246 promoted CD4+ T cells infiltration and IFN signalling and prevented CD8+ T cells exhaustion within the TME in vivo. CONCLUSIONS: Breast carcinomas with mtp53 displayed enhanced inflammation. APR-246 boosted the interferon response or represses immune checkpoints in p53 mutant tumour cells, and restores cancer immune surveillance in vivo.


Asunto(s)
Neoplasias , Proteína p53 Supresora de Tumor , Ratones , Animales , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Mutación con Ganancia de Función , Neoplasias/genética , Interferones/genética , Interferones/metabolismo , Inflamación/genética , Microambiente Tumoral/genética
6.
Haematologica ; 105(5): 1339-1350, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-31582539

RESUMEN

Megakaryoblastic leukemia 1 (MKL1) is a coactivator of serum response factor and together they regulate transcription of actin cytoskeleton genes. MKL1 is associated with hematologic malignancies and immunodeficiency, but its role in B cells is unexplored. Here we examined B cells from monozygotic triplets with an intronic deletion in MKL1, two of whom had been previously treated for Hodgkin lymphoma (HL). To investigate MKL1 and B-cell responses in the pathogenesis of HL, we generated Epstein-Barr virus-transformed lymphoblastoid cell lines from the triplets and two controls. While cells from the patients with treated HL had a phenotype close to that of the healthy controls, cells from the undiagnosed triplet had increased MKL1 mRNA, increased MKL1 protein, and elevated expression of MKL1-dependent genes. This profile was associated with elevated actin content, increased cell spreading, decreased expression of CD11a integrin molecules, and delayed aggregation. Moreover, cells from the undiagnosed triplet proliferated faster, displayed a higher proportion of cells with hyperploidy, and formed large tumors in vivo This phenotype was reversible by inhibiting MKL1 activity. Interestingly, cells from the triplet treated for HL in 1985 contained two subpopulations: one with high expression of CD11a that behaved like control cells and the other with low expression of CD11a that formed large tumors in vivo similar to cells from the undiagnosed triplet. This implies that pre-malignant cells had re-emerged a long time after treatment. Together, these data suggest that dysregulated MKL1 activity participates in B-cell transformation and the pathogenesis of HL.


Asunto(s)
Infecciones por Virus de Epstein-Barr , Enfermedad de Hodgkin , Linfocitos B , Células Cultivadas , Herpesvirus Humano 4 , Enfermedad de Hodgkin/genética , Humanos
7.
Cell Commun Signal ; 18(1): 56, 2020 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-32252758

RESUMEN

BACKGROUND: AKT2 is one of the key molecules that involves in the insulin-induced signaling and the development of cancer. In B cells, the function of AKT2 is unclear. METHODS: In this study, we used AKT2 knockout mice model to study the role of AKT2 in BCR signaling and B cell differentiation. RESULTS: AKT2 promotes the early activation of B cells by enhancing the BCR signaling and actin remodeling. B cells from AKT2 KO mice exhibited defective spreading and BCR clustering upon stimulation in vitro. Disruption of Btk-mediated signaling caused the impaired differentiation of germinal center B cells, and the serum levels of both sepecific IgM and IgG were decreased in the immunized AKT2 KO mice. In addition, the actin remodeling was affected due to the decreased level of the activation of WASP, the actin polymerization regulator, in AKT2 KO mice as well. As a crucial regulator of both BCR signaling and actin remodeling during early activation of B cells, the phosphorylation of CD19 was decreased in the AKT2 absent B cells, while the transcription level was normal. CONCLUSIONS: AKT2 involves in the humoral responses, and promotes the BCR signaling and actin remodeling to enhance the activation of B cells via regulating CD19 phosphorylation. Video Abstract.


Asunto(s)
Actinas/metabolismo , Antígenos CD19/inmunología , Linfocitos B , Proteínas Proto-Oncogénicas c-akt/fisiología , Receptores de Antígenos de Linfocitos B/inmunología , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Diferenciación Celular , Inmunidad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal
8.
PLoS Biol ; 15(8): e2001750, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28821013

RESUMEN

As the central hub of the metabolism machinery, the mammalian target of rapamycin complex 2 (mTORC2) has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. Rictor is involved in B cell development, especially the peripheral development. However, the role of Rictor on B cell receptor (BCR) signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell-specfic Rictor knockout (KO) mice to investigate how Rictor regulates BCR signaling. We found that the key positive and negative BCR signaling molecules, phosphorylated Brutons tyrosine kinase (pBtk) and phosphorylated SH2-containing inositol phosphatase (pSHIP), are reduced and enhanced, respectively, in Rictor KO B cells. This suggests that Rictor positively regulates the early events of BCR signaling. We found that the cellular filamentous actin (F-actin) is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration is associated with a decreased humoral immune response in Rictor KO mice. The inhibition of actin polymerization with latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, in which Rictor regulates BCR signaling via actin reorganization.


Asunto(s)
Actinas/metabolismo , Linfocitos B/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Agammaglobulinemia Tirosina Quinasa , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes , Membrana Celular/metabolismo , Inmunidad Humoral , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Polimerizacion , Proteína Asociada al mTOR Insensible a la Rapamicina , Tiazolidinas
9.
Circ Res ; 122(10): 1385-1394, 2018 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-29618596

RESUMEN

RATIONALE: Regulatory T (Treg) cells suppress immune responses and have been shown to attenuate atherosclerosis. The Treg cell lineage-specification factor FOXP3 (forkhead box P3) is essential for Treg cells' ability to uphold immunologic tolerance. In humans, FOXP3 exists in several different isoforms, however, their specific role is poorly understood. OBJECTIVE: To define the regulation and functions of the 2 major FOXP3 isoforms, FOXP3fl and FOXP3Δ2, as well as to establish whether their expression is associated with the ischemic atherosclerotic disease. METHODS AND RESULTS: Human primary T cells were transduced with lentiviruses encoding distinct FOXP3 isoforms. The phenotype and function of these cells were analyzed by flow cytometry, in vitro suppression assays and RNA-sequencing. We also assessed the effect of activation on Treg cells isolated from healthy volunteers. Treg cell activation resulted in increased FOXP3 expression that predominantly was made up of FOXP3Δ2. FOXP3Δ2 induced specific transcription of GARP (glycoprotein A repetitions predominant), which functions by tethering the immunosuppressive cytokine TGF (transforming growth factor)-ß to the cell membrane of activated Treg cells. Real-time polymerase chain reaction was used to determine the impact of alternative splicing of FOXP3 in relation with atherosclerotic plaque stability in a cohort of >150 patients that underwent carotid endarterectomy. Plaque instability was associated with a lower FOXP3Δ2 transcript usage, when comparing plaques from patients without symptoms and patients with the occurrence of recent (<1 month) vascular symptoms including minor stroke, transient ischemic attack, or amaurosis fugax. No difference was detected in total levels of FOXP3 mRNA between these 2 groups. CONCLUSIONS: These results suggest that activated Treg cells suppress the atherosclerotic disease process and that FOXP3Δ2 controls a transcriptional program that acts protectively in human atherosclerotic plaques.


Asunto(s)
Empalme Alternativo , Factores de Transcripción Forkhead/genética , Placa Aterosclerótica/metabolismo , Linfocitos T Reguladores/metabolismo , Amaurosis Fugax/metabolismo , Amaurosis Fugax/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Células Cultivadas , Factores de Transcripción Forkhead/fisiología , Regulación de la Expresión Génica , Vectores Genéticos/farmacología , Humanos , Células Jurkat , Placa Aterosclerótica/inmunología , Placa Aterosclerótica/patología , Isoformas de Proteínas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/metabolismo , Linfocitos T Reguladores/patología , Transcripción Genética
10.
J Autoimmun ; 98: 86-94, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30616979

RESUMEN

CTLA-4 is required for CD4+Foxp3+ regulatory T (Treg) cell function, but its mode of action remains incompletely defined. Herein we generated Ctla-4ex2fl/flFoxp3-Cre mice with Treg cells exclusively expressing a naturally occurring, ligand-independent isoform of CTLA-4 (liCTLA-4) that cannot interact with the costimulatory molecules CD80 and CD86. The mice did not exhibit any signs of effector T cell activation early in life, however, at 6 months of age they exhibited excessive T cell activation and inflammation in lungs. In contrast, mice with Treg cells completely lacking CTLA-4 developed lymphoproliferative disease characterized by multi-organ inflammation early in life. In vitro, Treg cells exclusively expressing liCTLA-4 inhibited CD80 and CD86 expression on dendritic cells (DC). Conversely, Treg cells required the extra-cellular part of CTLA-4 to up-regulate expression of the co-inhibitory molecule PD-L2 on DCs. Transcriptomic analysis of suppressed DCs revealed that Treg cells induced a specific immunosuppressive program in DCs.


Asunto(s)
Antígeno CTLA-4/metabolismo , Células Dendríticas/inmunología , Trastornos Linfoproliferativos/inmunología , Neumonía/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos CD4/metabolismo , Antígeno CTLA-4/genética , Diferenciación Celular , Células Cultivadas , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Activación de Linfocitos , Trastornos Linfoproliferativos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neumonía/genética , Proteína 2 Ligando de Muerte Celular Programada 1/genética , Proteína 2 Ligando de Muerte Celular Programada 1/metabolismo , Isoformas de Proteínas/genética
11.
Blood ; 127(2): 216-20, 2016 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-26468226

RESUMEN

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.


Asunto(s)
Autoinmunidad/genética , Linfocitos B/inmunología , Proteína Neuronal del Síndrome de Wiskott-Aldrich/fisiología , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/inmunología , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Eliminación de Gen , Ratones , Ratones Noqueados , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/inmunología , Síndrome de Wiskott-Aldrich/patología , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genética
12.
FASEB J ; 31(2): 491-504, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27825104

RESUMEN

Dendritic cells (DCs) involved in proinflammatory immune responses derive mainly from peripheral monocytes, and the cells subsequently mature and migrate into the inflammatory micromilieu. Here we report that suppressing of 15-lipoxygenase-1 led to a substantial reduction in DC spreading and podosome formation in vitro. The surface expression of CD83 was significantly lower in both sh-15-lipoxygenase-1 (15-LOX-1)-transduced cells and DCs cultivated in the presence of a novel specific 15-LOX-1 inhibitor. The T-cell response against tetanus-pulsed DCs was only affected to a minor extent on inhibition of 15-LOX-1. In contrast, endocytosis and migration ability of DCs were significantly suppressed on 15-LOX-1 inhibition. The expression of 15-LOX-1 in DCs was also demonstrated in affected human skin in atopic and contact dermatitis, showing that the enzyme is indeed expressed in inflammatory diseases in vivo. This study demonstrated that inhibiting 15-LOX-1 led to an impaired podosome formation in DCs, and consequently suppressed antigen uptake and migration capacity. These results indicated that 15-LOX-1 is a potential target for inhibiting the trafficking of DCs to lymphoid organs and inflamed tissues and decreasing the inflammatory response attenuating symptoms of certain immunologic and inflammatory disorders such as dermatitis.-Han, H., Liang, X., Ekberg, M., Kritikou, J. S., Brunnström, Å., Pelcman, B., Matl, M., Miao, X., Andersson, M., Yuan, X., Schain, F., Parvin, S., Melin, E., Sjöberg, J., Xu, D., Westerberg, L. S., Björkholm, M., Claesson, H.-E. Human 15-lipoxygenase-1 is a regulator of dendritic-cell spreading and podosome formation.


Asunto(s)
Araquidonato 15-Lipooxigenasa/metabolismo , Citocinas/metabolismo , Células Dendríticas/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Podosomas/fisiología , Araquidonato 15-Lipooxigenasa/genética , Movimiento Celular/fisiología , Citocinas/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Células de Langerhans/metabolismo , Monocitos , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/genética , Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismo
13.
Ann Rheum Dis ; 76(10): 1755-1763, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28760805

RESUMEN

OBJECTIVES: Vaccination of patients with rheumatic disease has been reported to result in lower antibody titres than in healthy individuals. However, studies primarily include patients on immunosuppressive therapy. Here, we investigated the immune response of treatment-naïve patients diagnosed with primary Sjögren's syndrome (pSS) to an H1N1 influenza vaccine. METHODS: Patients with Sjögren's syndrome without immunomodulatory treatment and age-matched and gender-matched healthy controls were immunised with an H1N1 influenza vaccine and monitored for serological and cellular immune responses. Clinical symptoms were monitored with a standardised form. IgG class switch and plasma cell differentiation were induced in vitro in purified naïve B cells of untreated and hydroxychloroquine-treated patients and healthy controls. Gene expression was assessed by NanoString technology. RESULTS: Surprisingly, treatment-naïve patients with Sjögren's syndrome developed higher H1N1 IgG titres of greater avidity than healthy controls on vaccination. Notably, off-target B cells were also triggered resulting in increased anti-EBV and autoantibody titres. Endosomal toll-like receptor activation of naïve B cells in vitro revealed a greater propensity of patient-derived cells to differentiate into plasmablasts and higher production of class switched IgG. The amplified plasma cell differentiation and class switch could be induced in cells from healthy donors by preincubation with type 1 interferon, but was abolished in hydroxychloroquine-treated patients and after in vitro exposure of naïve B cells to chloroquine. CONCLUSIONS: This comprehensive analysis of the immune response in autoimmune patients to exogenous stimulation identifies a mechanistic basis for the B cell hyperactivity in Sjögren's syndrome, and suggests that caution is warranted when considering vaccination in non-treated autoimmune patients.


Asunto(s)
Anticuerpos Antivirales/sangre , Linfocitos B , Citocinas/sangre , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Síndrome de Sjögren/inmunología , Antígenos CD19/análisis , Antirreumáticos/farmacología , Autoanticuerpos/biosíntesis , Autoantígenos/inmunología , Linfocitos B/química , Linfocitos B/fisiología , Estudios de Casos y Controles , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Femenino , Expresión Génica , Antígenos HLA-DR/análisis , Herpesvirus Humano 4/inmunología , Humanos , Hidroxicloroquina/farmacología , Inmunoglobulina D/análisis , Inmunoglobulina G/sangre , Interferón-alfa/metabolismo , Interferón-alfa/farmacología , Interleucina-10/farmacología , Activación de Linfocitos , Recuento de Linfocitos , Ribonucleoproteínas/inmunología , Transducción de Señal/genética , Síndrome de Sjögren/genética , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 9/metabolismo , Transcriptoma , Vacunación , Antígeno SS-B
14.
J Immunol ; 194(10): 4750-8, 2015 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-25870239

RESUMEN

The Rho GTPase Cdc42 coordinates regulation of the actin and the microtubule cytoskeleton by binding and activating the Wiskott-Aldrich syndrome protein. We sought to define the role of intrinsic expression of Cdc42 by mature B cells in their activation and function. Mice with inducible deletion of Cdc42 in mature B cells formed smaller germinal centers and had a reduced Ab response, mostly of low affinity to T cell-dependent Ag, compared with wild-type (WT) controls. Spreading formation of long protrusions that contain F-actin, microtubules, and Cdc42-interacting protein 4, and assumption of a dendritic cell morphology in response to anti-CD40 plus IL-4 were impaired in Cdc42-deficient B cells compared with WT B cells. Cdc42-deficient B cells had an intact migratory response to chemokine in vitro, but their homing to the B cell follicles in the spleen in vivo was significantly impaired. Cdc42-deficient B cells induced a skewed cytokine response in CD4(+) T cells, compared with WT B cells. Our results demonstrate a critical role for Cdc42 in the motility of mature B cells, their cognate interaction with T cells, and their differentiation into Ab-producing cells.


Asunto(s)
Linfocitos B/inmunología , Activación de Linfocitos/inmunología , Proteína de Unión al GTP cdc42/inmunología , Animales , Western Blotting , Diferenciación Celular/inmunología , Movimiento Celular/inmunología , Citometría de Flujo , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes
15.
PLoS Biol ; 11(11): e1001704, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24223520

RESUMEN

Negative regulation of receptor signaling is essential for controlling cell activation and differentiation. In B-lymphocytes, the down-regulation of B-cell antigen receptor (BCR) signaling is critical for suppressing the activation of self-reactive B cells; however, the mechanism underlying the negative regulation of signaling remains elusive. Using genetically manipulated mouse models and total internal reflection fluorescence microscopy, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is coexpressed with WASP in all immune cells, is a critical negative regulator of B-cell signaling. B-cell-specific N-WASP gene deletion causes enhanced and prolonged BCR signaling and elevated levels of autoantibodies in the mouse serum. The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization. Upon BCR activation, WASP is activated first, followed by N-WASP in mouse and human primary B cells. The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation. Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.


Asunto(s)
Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Proteína Neuronal del Síndrome de Wiskott-Aldrich/fisiología , Actinas/metabolismo , Animales , Anticuerpos Antinucleares/sangre , Antígenos/inmunología , Autoanticuerpos/sangre , Linfocitos B/inmunología , Células Cultivadas , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Transporte de Proteínas , Síndrome de Wiskott-Aldrich/inmunología , Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismo
16.
J Immunol ; 193(9): 4732-8, 2014 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-25252954

RESUMEN

We describe a spontaneously derived mouse line that completely failed to induce Ig class switching in vitro and in vivo. The mice inherited abolished IgG serum titers in a recessive manner caused by a spontaneous G → A transition mutation in codon 112 of the aicda gene, leading to an arginine to histidine replacement (AID(R112H)). Ig class switching was completely reconstituted by expressing wild-type AID. Mice homozygous for AID(R112H) had peripheral B cell hyperplasia and large germinal centers in the absence of Ag challenge. Immunization with SRBCs elicited an Ag-specific IgG1 response in wild-type mice, whereas AID(R112H) mice failed to produce IgG1 and had reduced somatic hypermutation. The phenotype recapitulates the human hyper-IgM (HIGM) syndrome that is caused by point mutations in the orthologous gene in humans, and the AID(R112H) mutation is frequently found in HIGM patients. The AID(R112H) mouse model for HIGM provides a powerful and more precise tool than conventional knockout strategies.


Asunto(s)
Citidina Desaminasa/genética , Modelos Animales de Enfermedad , Síndrome de Inmunodeficiencia con Hiper-IgM/genética , Síndrome de Inmunodeficiencia con Hiper-IgM/inmunología , Cambio de Clase de Inmunoglobulina/genética , Cambio de Clase de Inmunoglobulina/inmunología , Mutación , Hipermutación Somática de Inmunoglobulina , Animales , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Citidina Desaminasa/metabolismo , Análisis Mutacional de ADN , Femenino , Centro Germinal/inmunología , Síndrome de Inmunodeficiencia con Hiper-IgM/metabolismo , Inmunofenotipificación , Patrón de Herencia , Recuento de Linfocitos , Masculino , Ratones , Linaje , Fenotipo , Carácter Cuantitativo Heredable
17.
J Autoimmun ; 63: 23-30, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26149776

RESUMEN

The forkhead/winged-helix transcription factor FOXP3 confers suppressive ability to CD4(+)FOXP3(+) regulatory T (Treg) cells. Human Treg cells express several different isoforms of FOXP3 that differ in function. However, the regulation and functional consequences of FOXP3 isoform expression remains poorly understood. In order to study the function of the FOXP3Δ2Δ7 isoform in vivo we generated mice that exclusively expressed a Foxp3 isoform lacking exon 2 and 7. These mice exhibited multi-organ inflammation, increased cytokine production, global T cell activation, activation of antigen-presenting cells and B cell developmental defects, all features that are shared with mice completely deficient in FOXP3. Our results demonstrate that the mouse counterpart of human FOXP3Δ2Δ7 is unable to confer suppressive ability to Treg cells.


Asunto(s)
Factores de Transcripción Forkhead , Linfocitos T Reguladores/metabolismo , Animales , Exones , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Humanos , Activación de Linfocitos/genética , Ratones , Ratones Transgénicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Linfocitos T Reguladores/inmunología
18.
J Autoimmun ; 62: 81-92, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26143192

RESUMEN

Humoral immunodeficiency caused by mutations in the Wiskott-Aldrich syndrome protein (WASp) is associated with failure to respond to common pathogens and high frequency of autoimmunity. Here we addressed the question how deficiency in WASp and the homologous protein N-WASp skews the immune response towards autoreactivity. Mice devoid of WASp or both WASp and N-WASp in B cells formed germinal center to increased load of apoptotic cells as a source of autoantigens. However, the germinal centers showed abolished polarity and B cells retained longer and proliferated less in the germinal centers. While WASp-deficient mice had high titers of autoreactive IgG, B cells devoid of both WASp and N-WASp produced mainly IgM autoantibodies with broad reactivity to autoantigens. Moreover, B cells lacking both WASp and N-WASp induced somatic hypermutation at reduced frequency. Despite this, IgG1-expressing B cells devoid of WASp and N-WASp acquired a specific high affinity mutation, implying an increased BCR signaling threshold for selection in germinal centers. Our data provides evidence for that N-WASp expression alone drives WASp-deficient B cells towards autoimmunity.


Asunto(s)
Autoanticuerpos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Eliminación de Gen , Centro Germinal/inmunología , Centro Germinal/metabolismo , Inmunoglobulina M/inmunología , Proteína del Síndrome de Wiskott-Aldrich/genética , Animales , Anticuerpos Antinucleares/sangre , Anticuerpos Antinucleares/inmunología , Formación de Anticuerpos , Antígenos CD19/genética , Apoptosis/genética , Apoptosis/inmunología , Autoanticuerpos/sangre , Autoantígenos/inmunología , Linfocitos B/citología , Trasplante de Médula Ósea , Diferenciación Celular , Haptenos , Hemocianinas/inmunología , Inmunoglobulina M/sangre , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Ratones Transgénicos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Quimera por Trasplante
19.
Cell Physiol Biochem ; 34(6): 2017-26, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25562150

RESUMEN

BACKGROUND/AIMS: Diabetes mellitus (DM) is characterized by hyperglycemia, associated to a lack or inefficiency of the insulin to regulate glucose metabolism. DM is also marked by alterations in a diversity of cellular processes that need to be further unraveled. In this study, we examined the autophagy pathway in diabetic rat macrophages before and after treatment with insulin. METHODS: Bone marrow-derived macrophages (BMM), bronchoalveolar lavage (BAL) and splenic tissue of diabetic male Wistar rats (alloxan, 42 mg/kg, i.v., 10 days) and control rats (physiological saline, i.v.). Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 8 h before experiments. For characterization of the model and evaluation of the effect of insulin on the autophagic process, the following analyzes were performed: (a) concentrations of cytokines: interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-6, IL-4, IL-10, cytokine-induced neutrophil chemoattractant (CINC)-1 and CINC-2 in the BAL supernatant was measured by ELISA; (b) characterization of alveolar macrophage (AM) of the BAL as surface antigens (MHCII, pan-macrophage KiM2R, CD11b) and autophagic markers (protein microtubule-associated light chain (LC)3, autophagy protein (Atg)12 by flow cytometry and confocal microscopy (c) study of macrophages differentiated from the bone marrow by flow cytometry and confocal microscopy (d) histology of the spleen by immunohistochemistry associated with confocal microscopy. RESULTS: Interestingly, insulin exerted antagonistic effects on macrophages from different tissues. Macrophages from bronchoalveolar lavage (BAL) enhanced their LC3 autophagosome bound content after treatment with insulin whereas splenic macrophages from red pulp in diabetic rats failed to enhance their Atg 12 levels compared to control animals. Insulin treatment in diabetic rats did not change LC3 content in bone marrow derived macrophages (BMM). M1 and M2 macrophages behaved accordingly to the host they were derived from. Diabetic M1 BMM had their LC3 vesicle-bound content diminished and M2 BMM enhanced their LC3 levels and insulin treatment failed to rescue autophagy to control levels. Insulin normalizes CINC-2 level but does not modulate autophagy markers. CONCLUSION: Taking these results together, diabetic macrophages derived from different compartments show different levels of autophagy markers compared to healthy animals, therefore, they suffer distinctively in the absence of insulin.


Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Glucosa/metabolismo , Insulina/administración & dosificación , Macrófagos/efectos de los fármacos , Aloxano/toxicidad , Animales , Autofagia/efectos de los fármacos , Autofagia/genética , Lavado Broncoalveolar , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/genética , Humanos , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Macrófagos/patología , Ratas , Bazo/efectos de los fármacos , Bazo/metabolismo
20.
Blood ; 119(17): 3966-74, 2012 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-22411869

RESUMEN

The Wiskott-Aldrich syndrome protein (WASP) is a key cytoskeletal regulator of hematopoietic cells. Although WASP-knockout (WKO) mice have aberrant B-cell cytoskeletal responses, B-cell development is relatively normal. We hypothesized that N-WASP, a ubiquitously expressed homolog of WASP, may serve some redundant functions with WASP in B cells. In the present study, we generated mice lacking WASP and N-WASP in B cells (conditional double knockout [cDKO] B cells) and show that cDKO mice had decreased numbers of follicular and marginal zone B cells in the spleen. Receptor-induced activation of cDKO B cells led to normal proliferation but a marked reduction of spreading compared with wild-type and WKO B cells. Whereas WKO B cells showed decreased migration in vitro and homing in vivo compared with wild-type cells, cDKO B cells showed an even more pronounced decrease in the migratory response in vivo. After injection of 2,4,6-trinitrophenol (TNP)-Ficoll, cDKO B cells had reduced antigen uptake in the splenic marginal zone. Despite high basal serum IgM, cDKO mice mounted a reduced immune response to the T cell-independent antigen TNP-Ficoll and to the T cell-dependent antigen TNP-keyhole limpet hemocyanin. Our results reveal that the combined activity of WASP and N-WASP is required for peripheral B-cell development and function.


Asunto(s)
Linfocitos B/citología , Linfocitos B/fisiología , Proteína Neuronal del Síndrome de Wiskott-Aldrich/fisiología , Proteína del Síndrome de Wiskott-Aldrich/fisiología , Animales , Western Blotting , Movimiento Celular , Proliferación Celular , Células Cultivadas , Quimiotaxis , Ficoll/análogos & derivados , Ficoll/farmacología , Citometría de Flujo , Hematopoyesis/fisiología , Inmunización , Técnicas para Inmunoenzimas , Integrasas/metabolismo , Ratones , Ratones Noqueados , Trinitrobencenos/farmacología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA