RESUMO
OBJECTIVE: Myasthenia gravis (MG) is a neuromuscular disease mediated by antibodies against the acetylcholine receptor (AChR). The thymus plays a primary role in AChR-MG and is characterized by a type I interferon (IFN) signature linked to IFN-ß. We investigated if AChR-MG was characterized by an IFN-I signature in the blood, and further investigated the chronic thymic IFN-I signature. METHODS: Serum levels of IFN-ß and IFN-α subtypes, and mRNA expression for IFN-I subtypes and IFN-stimulated genes in peripheral mononuclear blood cells (PBMCs) were analyzed. The contribution of endogenous nucleic acids in thymic expression of IFN-I subtypes was investigated in human thymic epithelial cell cultures and the mouse thymus. By immunohistochemistry, thymic CD68+ and CD163+ macrophages were analyzed in AChR-MG. To investigate the impact of a decrease in thymic macrophages, mice were treated with an anti-CSF1R antibody. RESULTS: No IFN-I signature was observed in the periphery emphasizing that the IFN-I signature is restricted to the MG thymus. Molecules mimicking endogenous dsDNA signalization (Poly(dA:dT) and 2'3'-cGAMP), or dexamethasone-induced necrotic thymocytes increased IFN-ß and α-AChR expression by thymic epithelial cells, and in the mouse thymus. A significant decrease in thymic macrophages was demonstrated in AChR-MG. In mice, a decrease in thymic macrophages led to an increase of necrotic thymocytes associated with IFN-ß and α-AChR expression. INTERPRETATION: These results suggest that the decrease of thymic macrophages in AChR-MG impairs the elimination of apoptotic thymocytes favoring the release of endogenous nucleic acids from necrotic thymocytes. In this inflammatory context, thymic epithelial cells may overexpress IFN-ß, which specifically induces α-AChR, resulting in self-sensitization and thymic changes leading to AChR-MG. ANN NEUROL 2023;93:643-654.
Assuntos
Miastenia Gravis , Ácidos Nucleicos , Humanos , Camundongos , Animais , Timo/metabolismo , Receptores Colinérgicos , Macrófagos/metabolismoRESUMO
Acetylcholine receptor (AChR) myasthenia gravis (MG) is a chronic autoimmune disease characterized by muscle weakness. The AChR+ autoantibodies are produced by B-cells located in thymic ectopic germinal centers (eGC). No therapeutic approach is curative. The inflammatory IL-23/Th17 pathway is activated in the thymus as well as in the blood and the muscle, contributing to the MG pathogenic events. We aimed to study a potential new therapeutic approach that targets IL-23p19 (IL-23) in the two complementary preclinical MG models: the classical experimental MG mouse model (EAMG) based on active immunization and the humanized mouse model featuring human MG thymuses engrafted in NSG mice (NSG-MG). In both preclinical models, the anti-IL-23 treatment ameliorated MG clinical symptoms. In the EAMG, the treatment reduced IL-17 related inflammation, anti-AChR IgG2b antibody production, activated transduction pathway involved in muscle regeneration and ameliorated the signal transduction at the neuromuscular junction. In the NSG-MG model, the treatment reduced pathogenic Th17 cell population and expression of genes involved in eGC stabilization and B-cell development in human MG thymus biopsies. Altogether, these data suggest that a therapy targeting IL-23p19 may promote significant clinical ameliorations in AChR+ MG disease due to concomitant beneficial effects on the thymus and skeletal muscle defects.
Assuntos
Interleucina-23 , Miastenia Gravis Autoimune Experimental , Camundongos , Humanos , Animais , Subunidade p19 da Interleucina-23 , Receptores Colinérgicos , Junção Neuromuscular/patologia , AutoanticorposRESUMO
Predisposition to autoimmunity and inflammatory disorders is observed in patients with fragile X-associated syndromes. These patients have increased numbers of CGG triplets in the 5' UTR region of FMR1 (Fragile X Mental Retardation 1) gene, that affects its expression. FMR1 is decreased in the thymus of myasthenia gravis (MG) patients, a prototypical autoimmune disease. We thus analyzed the number of CGG triplets in FMR1 in MG, and explored the regulatory mechanisms affecting thymic FMR1 expression. We measured the number of CGGs using thymic DNA from MG and controls, but no abnormalities in CGGs were found in MG that could explain thymic decrease of FMR1. We next analyzed by RT-PCR the expression of FMR1 and its transcription factors in thymic samples, and in thymic epithelial cell cultures in response to inflammatory stimuli. In control thymuses, FMR1 expression was higher in males than females, and correlated with CTCF (CCCTC-binding factor) expression. In MG thymuses, decreased expression of FMR1 was correlated with both CTCF and MAX (Myc-associated factor X) expression. Changes in FMR1 expression were supported by western blot analyses for FMRP. In addition, we demonstrated that FMR1, CTCF and MAX expression in thymic epithelial cells was also sensitive to inflammatory signals. Our results suggest that FMR1 could play a central role in the thymus and autoimmunity. First, in relation with the higher susceptibility of females to autoimmune diseases. Second, due to the modulation of its expression by inflammatory signals that are known to be altered in MG thymuses.
Assuntos
Proteína do X Frágil da Deficiência Intelectual/biossíntese , Miastenia Gravis/metabolismo , Timo/metabolismo , Adolescente , Adulto , Autoimunidade/genética , Fator de Ligação a CCCTC/biossíntese , Fator de Ligação a CCCTC/genética , Células Cultivadas , DNA/química , DNA/genética , Células Epiteliais/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Caracteres Sexuais , Adulto JovemRESUMO
BACKGROUND: Myasthenia gravis (MG) is a rare autoimmune disease mainly mediated by autoantibodies against the acetylcholine receptor (AChR) at the neuromuscular junction. The thymus is the effector organ, and its removal alleviates the symptoms of the disease. In the early-onset form of MG, the thymus displays functional and morphological abnormalities such as B cell infiltration leading to follicular hyperplasia, and the production of AChR antibodies. Type-I interferon (IFN-I), especially IFN-ß, is the orchestrator of thymic changes observed in MG. As Dicer and miR-29 subtypes play a role in modulating the IFN-I signalization in mouse thymus, we investigated their expression in MG thymus. METHODS: The expression of DICER and miR-29 subtypes were thoroughly investigated by RT-PCR in human control and MG thymuses, and in thymic epithelial cells (TECs). Using miR-29a/b-1-deficient mice, with lower miR-29a/b-1 expression, we investigated their susceptibility to experimental autoimmune MG (EAMG) as compared to wild-type mice. RESULTS: DICER mRNA and all miR-29 subtypes were down-regulated in the thymus of MG patients and DICER expression was correlated with the lower expression of miR-29a-3p. A decreased expression of miR-29 subtypes was similarly observed in MG TECs; a decrease also induced in TECs upon IFN-ß treatment. We demonstrated that miR-29a/b-1-deficient mice were more susceptible to EAMG without higher levels of anti-AChR IgG subtypes. In the thymus, if no B cell infiltration was observed, an increased expression of Ifn-ß associated with Baff expression and the differentiation of Th17 cells associated with increased expression of Il-6, Il-17a and Il-21 and decreased Tgf-ß1 mRNA were demonstrated in miR-29a/b-1-deficient EAMG mice. CONCLUSIONS: It is not clear if the decreased expression of miR-29 subtypes in human MG is a consequence or a causative factor of thymic inflammation. However, our results from the EAMG mouse model indicated that a reduction in miR-29a/b1 may contribute to the pathophysiological process involved in MG by favoring the increased expression of IFN-ß and the emergence of pro-inflammatory Th17 cells.
Assuntos
MicroRNAs/biossíntese , Miastenia Gravis Autoimune Experimental/metabolismo , Miastenia Gravis/metabolismo , Adolescente , Adulto , Animais , Autoanticorpos/imunologia , Autoanticorpos/metabolismo , Células Cultivadas , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , Miastenia Gravis/genética , Miastenia Gravis/imunologia , Miastenia Gravis Autoimune Experimental/genética , Miastenia Gravis Autoimune Experimental/imunologia , Receptores Colinérgicos/imunologia , Receptores Colinérgicos/metabolismo , Timo/imunologia , Timo/metabolismo , Adulto JovemRESUMO
Thymomas are associated with a very high risk of developing Myasthenia Gravis (MG). Our objectives were to identify histological and biological parameters to allow early diagnosis of thymoma patients susceptible to developing MG. We conducted a detailed retrospective analysis from a patient database, searching for differences between patients with thymoma-associated MG (MGT, nâ¯=â¯409) and thymoma without MG (TOMA, nâ¯=â¯111) in comparison with nonthymomatous MG patients (MG, nâ¯=â¯1246). We also performed multiplex and single molecule arrays to measure the serum levels of cytokines in these groups of patients and controls (nâ¯=â¯14-22). We identified a set of parameters associated with MG development in thymoma patients: 1) detection of anti-acetylcholine receptor (AChR) antibodies, 2) development of B1 or B2 thymoma subtypes, 3) presence of ectopic thymic germinal centers (GCs), 4) local invasiveness of thymoma, and 5) being a woman under 50 years old. Among these parameters, 58.8% of MGT patients displayed GCs with a positive correlation between the number of GCs and anti-AChR titers. By immunohistochemistry, we found thymic GCs in the adjacent tissues of thymomas encircled by high endothelial venules (HEVs) that could favor peripheral cell recruitment. We also clearly associated MG symptoms with higher IFN-γ, IL-1ß and sCD40L serum levels, specifically in MGT patients compared to TOMA patients. Altogether, these analyses allowed the clear identification of histological, in particular the presence of GCs, and biological parameters that would facilitate the evaluation of the probability of the MG outcome postoperatively in thymoma patients.
Assuntos
Centro Germinativo/patologia , Miastenia Gravis/etiologia , Timoma/complicações , Neoplasias do Timo/complicações , Adulto , Autoanticorpos/metabolismo , Ligante de CD40/metabolismo , Feminino , Centro Germinativo/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-1beta/metabolismo , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/metabolismo , Receptores Colinérgicos/metabolismo , Estudos Retrospectivos , Fatores de Risco , Timoma/metabolismo , Neoplasias do Timo/metabolismoRESUMO
IL-23/Th17 pathway has been identified to sustain inflammatory condition in several autoimmune diseases and therefore being targeted in various therapeutic and effective approaches. Patients affected with autoimmune myasthenia gravis exhibit a disease effector tissue, the thymus, that harbors ectopic germinal centers that sustain production of auto-antibodies, targeting proteins located in the neuromuscular junction, cause of the organ-specific chronic autoimmune disease. The present study aims to investigate the IL-23/Th17â¯cell pathway in the thymic inflammatory and pathogenic events. We found that thymuses of MG patients displayed overexpression of Interleukin-17, signature cytokine of activated Th17â¯cells. This activation was sustained by a higher secretion of Interleukin-23 by TEC, in addition to the increased expression of cytokines involved in Th17â¯cell development. The overexpression of Interleukin-23 was due to a dysregulation of interferon type I pathway. Besides, Interleukin-17 secreted, and Th17â¯cells were localized around thymic ectopic germinal centers. These cells expressed podoplanin, a protein involved in B-cell maturation and antibody secretion. Finally, production of Interleukin-23 was also promoted by Interleukin-17 secreted itself by Th17â¯cells, highlighting a chronic loop of inflammation sustained by thymic cell interaction. Activation of the IL-23/Th17 pathway in the thymus of autoimmune myasthenia gravis patients creates an unstoppable loop of inflammation that may participate in ectopic germinal center maintenance. To alleviate the physio-pathological events in myasthenia gravis patients, this pathway may be considered as a new therapeutic target.
Assuntos
Inflamação/imunologia , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Miastenia Gravis/imunologia , Células Th17/imunologia , Timo/metabolismo , Adolescente , Adulto , Células Cultivadas , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Transdução de Sinais , Timo/patologia , Adulto JovemRESUMO
OBJECTIVE: To identify novel genetic and epigenetic factors associated with Myasthenia gravis (MG) using an identical twins experimental study design. METHODS: The transcriptome and methylome of peripheral monocytes were compared between monozygotic (MZ) twins discordant and concordant for MG, as well as with MG singletons and healthy controls, all females. Sets of differentially expressed genes and differentially methylated CpGs were validated using RT-PCR for expression and target bisulfite sequencing for methylation on additional samples. RESULTS: >100 differentially expressed genes and â¼1800 differentially methylated CpGs were detected in peripheral monocytes between MG patients and controls. Several transcripts associated with immune homeostasis and inflammation resolution were reduced in MG patients. Only a relatively few genes differed between the discordant healthy and MG co-twins, and both their expression and methylation profiles demonstrated very high similarity. INTERPRETATION: This is the first study to characterize the DNA methylation profile in MG, and the expression profile of immune cells in MZ twins with MG. Results suggest that numerous small changes in gene expression or methylation might together contribute to disease. Impaired monocyte function in MG and decreased expression of genes associated with inflammation resolution could contribute to the chronicity of the disease. Findings may serve as potential new predictive biomarkers for disease and disease activity, as well as potential future targets for therapy development. The high similarity between the healthy and the MG discordant twins, suggests that a molecular signature might precede a clinical phenotype, and that genetic predisposition may have a stronger contribution to disease than previously assumed.
Assuntos
Metilação de DNA , Miastenia Gravis/genética , Transcriptoma , Gêmeos Monozigóticos , Adulto , Idoso , Estudos de Casos e Controles , Ilhas de CpG , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Miastenia Gravis/metabolismo , Transdução de Sinais , Receptor Gatilho 1 Expresso em Células Mieloides/genética , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Adulto JovemRESUMO
Myasthenia gravis (MG) is an autoimmune disease in which the thymus frequently presents follicular hyperplasia and signs of inflammation and T cells display a defect in suppressive regulation. Defects in a suppressive assay can indicate either the defective function of Treg cells or the resistance of Tconv cells to suppression by Treg cells. The aim of this study was to determine which cells were responsible for this defect and to address the mechanisms involved. We first performed cross-experiment studies using purified thymic Treg cells and Tconv cells from controls (CTRL) and MG patients. We confirmed that MG Treg cells were defective in suppressing CTRL Tconv proliferation, and we demonstrated for the first time that MG Tconv cells were resistant to Treg cell suppression. The activation of MG Tconv cells triggered a lower upregulation of FoxP3 and a higher upregulation of CD4 and CD25 than CTRL cells. To investigate the factors that could explain these differences, we analyzed the transcriptomes of purified thymic Treg and Tconv cells from MG patients in comparison to CTRL cells. Many of the pathways revealed by this analysis are involved in other autoimmune diseases, and T cells from MG patients exhibit a Th1/Th17/Tfh signature. An increase in IL-17-related genes was only observed in Treg cells, while increases in IFN-γ, IL-21, and TNF-α were observed in both Treg and Tconv cells. These results were confirmed by PCR studies. In addition, the role of TNF-α in the defect in Tconv cells from MG patients was further confirmed by functional studies. Altogether, our results indicate that the immunoregulatory defects observed in MG patients are caused by both Treg cell and Tconv cell impairment and involve several pro-inflammatory cytokines, with TNF-α playing a key role in this process. The chronic inflammation present in the thymus of MG patients could provide an explanation for the escape of thymic T cells from regulation in the MG thymus.
Assuntos
Miastenia Gravis/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Células Th1/imunologia , Células Th17/imunologia , Adolescente , Adulto , Antígenos CD4/metabolismo , Células Cultivadas , Criança , Pré-Escolar , Feminino , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica , Humanos , Tolerância Imunológica , Lactente , Recém-Nascido , Mediadores da Inflamação/metabolismo , Interleucina-17/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
Myasthenia gravis (MG) is an autoimmune disease mainly mediated by anti-acetylcholine receptor (AChR) antibodies. In the late onset, a thymoma, tumor of the thymus, is quite frequent. However, the events leading to thymoma and MG are not understood. As thymoma-associated MG (MG-T) patients also display anti-interferon type I (IFN-I) neutralizing antibodies, we investigated if MG-T could be associated with an anti-viral signature. RT-PCR analyses demonstrated huge increases of IFN-I subtypes, IFN-α2, -α8, -ω and -ß, in thymoma-associated MG but not in thymomas without MG or in control thymuses. Next, we investigated if dsRNA signaling pathway involvement could be observed in MG-T, as recently observed in early-onset MG. We observed an abnormal regulation of dsRNA-sensing molecules with an increase of toll-like receptor 3 (TLR3), and a decrease of protein kinase R (PKR) and dsRNA helicases (RIG-I and MDA5) in thymoma from MG patients. We also detected a decreased expression of p53, the tumor suppressor that is known to be down-regulated by dsRNA. Altogether, these results strongly suggest that MG-T could be linked to a viral infection. As p16 (CDKN2A), a marker of HPV infections, was up-regulated in MG-T, we thus screened DNA from thymomas for human papillomavirus (HPV) by real-time PCR using HPV consensus SPF10 primers. RT-PCR results were negative for all samples tested. We confirmed the absence of HPV DNA detection by end point PCR using FAP primers to amplify a larger panel of HPV genotypes. Our data clearly demonstrate INF-I overexpression together with the activation of innate immunity pathways in thymoma-associated MG suggesting that MG might develop after a pathogen infection. We were not able to relate thymoma to HPV infections and the implication of other pathogens is discussed.
Assuntos
Alphapapillomavirus/imunologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Interferon Tipo I/metabolismo , Miastenia Gravis/imunologia , Infecções por Papillomavirus/imunologia , Timoma/imunologia , Neoplasias do Timo/imunologia , Adulto , Idoso , Alphapapillomavirus/genética , Anticorpos Antivirais/metabolismo , Autoanticorpos/metabolismo , Células Cultivadas , Pré-Escolar , Inibidor p16 de Quinase Dependente de Ciclina/genética , DNA Viral/análise , Feminino , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Lactente , Recém-Nascido , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/etiologia , Miastenia Gravis/genética , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/genética , Receptores Colinérgicos/imunologia , Timoma/etiologia , Timoma/genética , Neoplasias do Timo/etiologia , Neoplasias do Timo/genética , Adulto JovemRESUMO
Myasthenia gravis (MG) is an autoimmune disease characterized by muscle fatigability due to acetylcholine receptor (AChR) autoantibodies. To better characterize juvenile MG (JMG), we analyzed 85 pre- and 132 post-pubescent JMG (with a cutoff age of 13) compared to 721 adult MG patients under 40 years old using a French database. Clinical data, anti-AChR antibody titers, thymectomy, and thymic histology were analyzed. The proportion of females was higher in each subgroup. No significant difference in the anti-AChR titers was observed. Interestingly, the proportion of AChR+ MG patients was notably lower among adult MG patients aged between 30 and 40 years, at 69.7%, compared to over 82.4% in the other subgroups. Thymic histological data were examined in patients who underwent thymectomy during the year of MG onset. Notably, in pre-JMG, the percentage of thymectomized patients was significantly lower (32.9% compared to more than 42.5% in other subgroups), and the delay to thymectomy was twice as long. We found a positive correlation between anti-AChR antibodies and germinal center grade across patient categories. Additionally, only females, particularly post-JMG patients, exhibited the highest rates of lymphofollicular hyperplasia (95% of cases) and germinal center grade. These findings reveal distinct patterns in JMG patients, particularly regarding thymic follicular hyperplasia, which appears to be exacerbated in females after puberty.
Assuntos
Autoanticorpos , Miastenia Gravis , Receptores Colinérgicos , Timectomia , Timo , Humanos , Miastenia Gravis/patologia , Miastenia Gravis/epidemiologia , Feminino , Masculino , Adulto , França/epidemiologia , Timo/patologia , Timo/cirurgia , Adolescente , Autoanticorpos/imunologia , Autoanticorpos/sangue , Receptores Colinérgicos/imunologia , Adulto Jovem , Criança , Estudos de Coortes , Centro Germinativo/patologia , Centro Germinativo/imunologiaRESUMO
Myasthenia Gravis (MG) is a neurological autoimmune disease characterized by disabling muscle weaknesses due to anti-acetylcholine receptor (AChR) autoantibodies. To gain insight into immune dysregulation underlying early-onset AChR+ MG, we performed an in-depth analysis of peripheral mononuclear blood cells (PBMCs) using mass cytometry. PBMCs from 24 AChR+ MG patients without thymoma and 16 controls were stained with a panel of 37 antibodies. Using both unsupervised and supervised approaches, we observed a decrease in monocytes, for all subpopulations: classical, intermediate, and non-classical monocytes. In contrast, an increase in innate lymphoid cells 2 (ILC2s) and CD27- γδ T cells was observed. We further investigated the dysregulations affecting monocytes and γδ T cells in MG. We analyzed CD27- γδ T cells in PBMCs and thymic cells from AChR+ MG patients. We detected the increase in CD27- γδ T cells in thymic cells of MG patients suggesting that the inflammatory thymic environment might affect γδ T cell differentiation. To better understand changes that might affect monocytes, we analyzed RNA sequencing data from CD14+ PBMCs and showed a global decrease activity of monocytes in MG patients. Next, by flow cytometry, we especially confirmed the decrease affecting non-classical monocytes. In MG, as for other B-cell mediated autoimmune diseases, dysregulations are well known for adaptive immune cells, such as B and T cells. Here, using single-cell mass cytometry, we unraveled unexpected dysregulations for innate immune cells. If these cells are known to be crucial for host defense, our results demonstrated that they could also be involved in autoimmunity.
Assuntos
Miastenia Gravis , Doenças do Sistema Nervoso , Neoplasias do Timo , Humanos , Imunidade Inata , Linfócitos , Receptores Colinérgicos , AutoanticorposRESUMO
The thymus is involved in autoimmune Myasthenia gravis (MG) associated with anti-acetylcholine (AChR) antibodies. In MG, thymic regulatory T cells (Treg) are not efficiently suppressive, and conventional T cells (Tconv) are resistant to suppression. To better understand the specific role of the thymus in MG, we compared the phenotype and function of peripheral and thymic Treg and Tconv from controls and MG patients. Suppression assays with thymic or peripheral CD4 + T cells showed that the functional impairment in MG was more pronounced in the thymus than in the periphery. Phenotypic analysis of Treg showed a significant reduction of resting and effector Treg in the thymus but not in the periphery of MG patients. CD31, a marker lost with excessive immunoreactivity, was significantly reduced in thymic but not blood resting Treg. These results suggest that an altered thymic environment may explain Treg differences between MG patients and controls. Since thymic epithelial cells (TECs) play a major role in the generation of Treg, we co-cultured healthy thymic CD4 + T cells with control or MG TECs and tested their suppressive function. Co-culture with MG TECs consistently hampers regulatory activity, as compared with control TECs, suggesting that MG TECs contribute to the immune regulation defects of MG CD4 + T cells. MG TECs produced significantly higher thymic stromal lymphopoietin (TSLP) than control TECs, and a neutralizing anti-TSLP antibody partially restored the suppressive capacity of Treg derived from co-cultures with MG TECs, suggesting that TSLP contributed to the defect of thymic Treg in MG patients. Finally, a co-culture of MG CD4 + T cells with control TECs restored numbers and function of MG Treg, demonstrating that a favorable environment could correct the immune regulation defects of T cells in MG. Altogether, our data suggest that the severe defect of thymic Treg is at least partially due to MG TECs that overproduce TSLP. The Treg defects could be corrected by replacing dysfunctional TECs by healthy TECs. These findings highlight the role of the tissue environment on the immune regulation.
Assuntos
Células Sanguíneas/imunologia , Células Epiteliais/fisiologia , Miastenia Gravis/imunologia , Linfócitos T Reguladores/imunologia , Timo/imunologia , Adolescente , Adulto , Autoanticorpos/metabolismo , Células Cultivadas , Criança , Técnicas de Cocultura , Citocinas/metabolismo , Feminino , Homeostase , Humanos , Imunomodulação , Recém-Nascido , Masculino , Receptores Colinérgicos/imunologia , Adulto Jovem , Linfopoietina do Estroma do TimoRESUMO
Autoimmune Myasthenia gravis (MG) is a chronic neuromuscular disease mainly due to antibodies against the acetylcholine receptor (AChR) at the neuromuscular junction that induce invalidating muscle weaknesses. In early-onset MG, the thymus is the effector organ and is often characterized by B-cell infiltrations leading to ectopic germinal center (GC) development. The microRNA miR-150-5p has been previously characterized as a biomarker in MG due to its increase in the serum of patients and its decrease after thymectomy, correlated with an improvement of symptoms. Here, we investigated the causes and consequences of the miR-150 increase in the serum of early-onset MG patients. We observed that miR-150 expression was upregulated in MG thymuses in correlation with the presence of thymic B cells and showed by in situ hybridization experiments, that miR-150 was mainly expressed by cells of the mantle zone of GCs. However, we did not observe any correlation between the degree of thymic hyperplasia and the serum levels in MG patients. In parallel, we also investigated the expression of miR-150 in peripheral blood mononuclear cells (PBMCs) from MG patients. We observed that miR-150 was down-regulated, especially in CD4+ T cells compared to controls. These results suggest that the increased serum levels of miR-150 could result from a release from activated peripheral CD4+ T cells. Next, we demonstrated that the in vitro treatment of PBMCs with miR-150 or antimiR-150 oligonucleotides, respectively, decreased or increased the expression of one of its major target gene: the proto-oncogene MYB, a well-known actor of hematopoiesis. These results revealed that increased serum levels of miR-150 in MG patients could have a functional effect on PBMCs. We also showed that antimiR-150 caused increased cellular death of CD4+ and CD8+ T cells, along with the overexpression of pro-apoptotic genes targeted by miR-150 suggesting that miR-150 controlled the survival of these cells. Altogether, these results showed that miR-150 could play a role in MG both at the thymic level and in periphery by modulating the expression of target genes and peripheral cell survival.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , MicroRNAs/imunologia , Miastenia Gravis/imunologia , Adolescente , Adulto , Feminino , Centro Germinativo/imunologia , Centro Germinativo/patologia , Humanos , Masculino , Miastenia Gravis/patologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myb/imunologia , Receptores Nicotínicos/imunologia , Timo/imunologia , Timo/patologiaRESUMO
This review is dedicated to John Newsom-Davis, who was an exceptional colleague and friend, always exchanging ideas with respect and consideration. We shall not forget his involvement and passion in search for the truth on the role of thymectomy in the management of Myasthenia Gravis (MG). In this short review, we shall summarize what we learnt from DNA microarrays applied to MG thymus. We shall focus on three main comparisons of the thymic transcriptomes: 1) highly hyperplastic MG patients versus non-MG adults; 2) corticosteroid-treated versus untreated seropositive MG patients; and 3) seronegative versus seropositive MG patients.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Miastenia Gravis/genética , Análise de Sequência com Séries de Oligonucleotídeos , Timo/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Interferons/farmacologia , Masculino , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miastenia Gravis/tratamento farmacológico , Miastenia Gravis/patologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Extratos do Timo/genética , Extratos do Timo/metabolismo , Timo/efeitos dos fármacos , Timo/imunologiaRESUMO
The thymus is frequently hyperplastic in young female myasthenia gravis (MG) patients presenting with anti-acetylcholine receptor (AChR) antibodies. This thymic pathology is characterized by the presence of ectopic germinal centers (GCs) containing B cells involved at least partially in the production of pathogenic anti-AChR antibodies. Our recent studies have furthered our understanding of the mechanisms leading to GC formation in the hyperplastic thymus. First, we showed that CXCL13 and CCL21, chemokines involved in GC formation, are overexpressed in MG thymus. Second, we demonstrated an increase in pro-inflammatory activity in the thymus from MG patients and its partial normalization by glucocorticoids, as evidenced by gene expression profile. Third, we found that pro-inflammatory cytokines are able to upregulate the expression of AChR subunits in thymic epithelial and myoid cells. Fourth, we showed that the function of T regulatory (Treg) cells, whose role is to downregulate the immune response, is severely impaired in the thymus of MG patients; such a defect could explain the chronic immune activation observed consistently in MG thymic hyperplasia. Altogether, these new data suggest that CXCL13 and CCL21, which are produced in excess in MG thymus, attract peripheral B cells and activated T cells, which are maintained chronically activated in the inflammatory thymic environment because of the defect in suppressive activity of Treg cells. Presence of AChR in the thymus and upregulation of its expression by the pro-inflammatory environment contribute to the triggering and maintenance of the anti-AChR autoimmune response.
Assuntos
Miastenia Gravis/imunologia , Miastenia Gravis/patologia , Corticosteroides/uso terapêutico , Distribuição por Idade , Quimiocinas/imunologia , Quimiocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Miastenia Gravis/complicações , Miastenia Gravis/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Linfócitos T Reguladores/imunologia , Hiperplasia do Timo/complicações , Hiperplasia do Timo/epidemiologia , Hiperplasia do Timo/patologia , Receptor fas/metabolismoRESUMO
Autoimmune diseases (AIDs) are chronic disorders characterized by inflammatory reactions against self-antigens that can be either systemic or organ specific. AIDs can differ in their epidemiologic features and clinical presentations, yet all share a remarkable complexity. AIDs result from an interplay of genetic and epigenetic factors with environmental components that are associated with imbalances in the immune system. Many of the pathogenic mechanisms of AIDs are also implicated in myasthenia gravis (MG), an AID in which inflammation of the thymus leads to a neuromuscular disorder. Our goal here is to highlight the similarities and differences between MG and other AIDs by reviewing the common transcriptome signatures and the development of germinal centers and by discussing some unresolved questions about autoimmune mechanisms. This review will propose hypotheses to explain the origin of regulatory T (Treg ) cell defects and the causes of chronicity and specificity of AIDs.
Assuntos
Autoimunidade/genética , Linfócitos B/imunologia , Predisposição Genética para Doença/genética , Lúpus Eritematoso Sistêmico/genética , Miastenia Gravis/genética , Linfócitos T Reguladores/imunologia , Autoimunidade/imunologia , Centro Germinativo/imunologia , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária/imunologia , Miastenia Gravis/imunologia , Miastenia Gravis/patologia , Sarcoidose/genética , Sarcoidose/imunologiaRESUMO
Thymic epithelial cells are one of the main components of the thymic microenvironment required for T-cell development. In this work, we describe an efficient method free of enzymatic and Facs-sorted methods to culture human medullary thymic epithelial cells without affecting the cell phenotypic, physiologic and functional features. Human medulla thymic epithelial cells (mTECs) are obtained by culturing thymic biopsies explants. After 7 days of primo-culture, mTECs keep their ability to express key molecules involved in immune tolerance processes such as autoimmune regulator, tissue-specific antigens, chemokines, and cytokines. In addition, the cells sensor their cultured environment and consequently adjust their gene expression network. Therefore, we describe and provide a human mTEC model that may be used to test the effect of various molecules on thymic epithelial cell homeostasis and physiology. This method should allow the investigations of the specificities and the knowledge of human mTECs in normal or pathological conditions and therefore discontinue the extrapolations done on the murine models.
RESUMO
We analyzed the number and functionality of regulatory B (Breg) cells in well-defined myasthenia gravis patients. We first showed a decreased number of circulating CD19+ CD24++ CD38++ Breg cells and an altered functionality of Breg cells in untreated myasthenia gravis patients. Next, we demonstrated that the proportion of circulating Breg cells was restored in myasthenia gravis patients after thymectomy, probably as Breg cells could be sequestered in the myasthenia gravis thymus. In contrast, corticosteroid treatments did not restore and decreased even more the proportion of Breg cells in myasthenia gravis patients. These results clearly demonstrated that two distinct immunomodulatory therapies affect differentially Breg cells.
RESUMO
It has long been established that the thymus plays a central role in autoimmune myasthenia gravis (MG) because of either thymoma or thymic hyperplasia of lymphoproliferative origin. In this review, we discuss thymic changes associated with thymic hyperplasia and their implications in the development of an autoimmune response against the acetylcholine receptor (AChR).The hyperplastic MG thymus displays all the characteristics of tertiary lymphoid organs (TLOs): neoangiogenic processes with high endothelial venule and lymphatic vessel development, chemokine overexpression favoring peripheral cell recruitment, and ectopic germinal center development. As thymic epithelial cells or myoid cells express AChR, a specific antigen presentation can easily occur within the thymus in the presence of recruited peripheral cells, such as B cells and T follicular helper cells. How the thymus turns into a TLO is not known, but local inflammation seems mandatory. Interferon (IFN)-ß is overexpressed in MG thymus and could orchestrate thymic changes associated with MG. Knowledge about how IFN-ß is induced in MG thymus and why its expression is sustained even long after disease onset would be of interest in the future to better understand the etiological and physiopathological mechanisms involved in autoimmune MG.
Assuntos
Miastenia Gravis/etiologia , Timo/imunologia , Adulto , Idade de Início , Quimiocinas/genética , Feminino , Centro Germinativo/imunologia , Centro Germinativo/patologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Interferon beta/imunologia , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Miastenia Gravis/imunologia , Miastenia Gravis/patologia , Neovascularização Patológica , Receptores Colinérgicos/imunologia , Linfócitos T/imunologia , Timo/irrigação sanguínea , Timo/patologia , Hiperplasia do Timo/complicações , Hiperplasia do Timo/imunologia , Hiperplasia do Timo/patologia , Receptores Toll-Like/genética , Regulação para CimaRESUMO
In early-onset Myasthenia Gravis (MG) with anti-acetylcholine receptor antibodies, thymic abnormalities associated with ectopic germinal centers are frequent. miRNAs by acting as post-transcriptional regulators are involved in autoimmunity. To investigate the implication of miRNAs in thymic changes associated with early-onset MG, we performed a miRnome study and data were analyzed with different approaches. miRNAs of interest were further investigated by RT-PCR and transfection experiments for functional tests. First, analyzing specific dysregulated miRNAs, we focused our attention on miR-7-5p and miR-125a-5p, and confirmed by RT-PCR their respective down- and up-regulation in MG thymuses. miR-7 was the most down-regulated thymic miRNA in MG and we observed an inverse correlation between its expression and CCL21 mRNA expression. We next showed that miR-7 down-regulation was due to thymic epithelial cells and by transfecting these cells with miR-7, we demonstrated that it controlled CCL21 release. As CCL21 is essential for germinal center development, we suggested that miR-7 could be involved in thymic changes associated with MG. miR-125a was up-regulated in MG thymuses and is of great interest as it is known to regulate FoxP3 expression, and to modulate the different inflammatory signaling pathways. Thanks to this thymic miRnome study, we also showed the specific dysregulation of miRNA clusters. In particular, we observed that miRNAs localized at the extremity of the X chromosome were down-regulated. This effect seemed linked to their close localization to the fragile X mental retardation 1 gene (FMR1) and the DNA methylation status. Altogether, this miRnome analysis demonstrated that specific thymic miRNAs can be associated with MG and provides novel insights into the pathogenesis of MG.