Myasthenia Gravis Thymus Research Articles

MiR-146a in myasthenia gravis thymus: from uncontrolled innate immunity to B cell-mediated autoimmunity

The thymus is the main site of autoimmunity development in myasthenia gravis (MG) associated with anti-acetylcholine receptor (AChR) autoantibodies, a prototypic autoimmune disease affecting neuromuscular junction. Most MG patients with early-onset disease presents follicular hyperplastic changes of the thymus, that are critically implicated in the initiation and perpetuation of the autoimmune response against the AChR. Uncontrolled activation of Toll-like receptor (TLR)-mediated innate immune responses, chronic inflammation and ectopic germinal center (GC) formation are key pathological features of hyperplastic thymus in MG, indicating that a close link between innate immunity and B cell-mediated autoimmunity underlies the intra-thymic pathogenesis of MG.
 MiR-146a, an “immune-miR” acting as key modulator of both innate and adaptive immunity, is a potent inhibitor of TLR signaling pathways, able to prevent and avoid an overstimulation of the inflammatory response by targeting the NF-κB signaling transducers IRAK1 and TRAF6. At the same time, miR-146a modulates the expression of c-REL, ICOS and ICOSL, which are crucial regulators of B cell function and GC response. Dysregulation of miR-146a expression is a common molecular event in several autoimmune disorders. Our recent findings revealed defective expression of miR-146a, associated with over-expression of its TLR- and B cell-related target genes, in follicular hyperplastic MG thymuses, indicating that loss of regulatory functions of this miRNA may significantly contribute to the immunopathological steps leading to MG. Of note, corticosteroids were found to increase the miR-146a expression levels, thus suggesting the miRNA capacity to mediate the effects of these drugs in inducing immunosuppression and autoimmunity control. 
 In this review, we discuss the role of miR-146a as a molecular bridge between innate and adaptive immunity, and summarize the current knowledge on the miRNA contribution to the pathogenesis of MG. Based on our and literature data, we highlight the miR-146a potential as biomarker for therapeutic monitoring, as well as target of future advanced RNA-based therapies to modulate the immune system and counteract the autoimmune response in MG.

Muscle and Muscle-like Autoantigen Expression in Myasthenia Gravis Thymus: Possible Molecular Hint for Autosensitization

The thymus is widely recognized as an immunological niche where autoimmunity against the acetylcholine receptor (AChR) develops in myasthenia gravis (MG) patients, who mostly present thymic hyperplasia and thymoma. Thymoma-associated MG is frequently characterized by autoantibodies to the muscular ryanodine receptor 1 (RYR1) and titin (TTN), along with anti-AChR antibodies. By real-time PCR, we analyzed muscle-CHRNA1, RYR1, and TTN-and muscle-like-NEFM, RYR3 and HSP60-autoantigen gene expression in MG thymuses with hyperplasia and thymoma, normal thymuses and non-MG thymomas, to check for molecular changes potentially leading to an altered antigen presentation and autoreactivity. We found that CHRNA1 (AChR-α subunit) and AIRE (autoimmune regulator) genes were expressed at lower levels in hyperplastic and thymoma MG compared to the control thymuses, and that the RYR1 and TTN levels were decreased in MG versus the non-MG thymomas. Genes encoding autoantigens that share epitopes with AChR-α (NEFM and HSP60), RYR1 (neuronal RYR3), and TTN (NEFM) were up-regulated in thymomas versus hyperplastic and control thymuses, with distinct molecular patterns across the thymoma histotypes that could be relevant for autoimmunity development. Our findings support the idea that altered muscle autoantigen expression, related with hyperplastic and neoplastic changes, may favor autosensitization in the MG thymus, and that molecular mimicry involving tumor-related muscle-like proteins may be a mechanism that makes thymoma prone to developing MG.

Central Role of Macrophages and Nucleic Acid Release in Myasthenia Gravis Thymus.

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-β. We investigated if AChR-MG was characterized by an IFN-I signature in the blood, and further investigated the chronic thymic IFN-I signature. Serum levels of IFN-β 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. 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-β 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-β and α-AChR expression. 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-β, which specifically induces α-AChR, resulting in self-sensitization and thymic changes leading to AChR-MG. ANN NEUROL 2023;93:643-654.

Blocking interleukin-23 ameliorates neuromuscular and thymic defects in myasthenia gravis

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.

Altered expression of fragile X mental retardation-1 (FMR1) in the thymus in autoimmune myasthenia gravis

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.

Intrathymic Plasmablasts Are Affected in Patients With Myasthenia Gravis With Active Disease.

Background and ObjectivesTo investigate intrathymic B lymphopoiesis in patients with myasthenia gravis (MG) and explore thymus pathology associated with clinical impact.MethodsThymic lymphocytes from 15 young patients without MG, 22 adult patients without MG, 14 patients with MG without thymoma, and 11 patients with MG with thymoma were subjected to flow cytometry analysis of T follicular helper (Tfh), naive B, memory B, plasmablasts, CD19+B220high thymic B cells, B-cell activating factor receptor, and C-X-C chemokine receptor 5 (CXCR5). Peripheral blood mononuclear cells of 16 healthy subjects and 21 untreated patients with MG were also analyzed. Immunologic values were compared, and correlations between relevant values and clinical parameters were evaluated.ResultsThe frequencies of circulating and intrathymic plasmablasts were significantly higher in patients with MG than controls. On the other hand, the frequency of CD19+B220high thymic B cells was not increased in MG thymus. We observed a significant increase in CXCR5 expression on plasmablasts in MG thymus and an increased frequency of intrathymic plasmablasts that was correlated with preoperative disease activity. The frequency of intrathymic Tfh cells was significantly lower in patients who received immunosuppressive (IS) therapy than those without IS therapy. However, there was no significant difference in the frequency of intrathymic plasmablasts irrespective of IS therapy.DiscussionOur findings confirmed a correlation between increased frequency of intrathymic plasmablasts and disease activity before thymectomy. We postulate that activated intrathymic plasmablasts endow pathogenic capacity in MG.

Thymus-derived B cell clones persist in the circulation after thymectomy in myasthenia gravis

Myasthenia gravis (MG) is a neuromuscular, autoimmune disease caused by autoantibodies that target postsynaptic proteins, primarily the acetylcholine receptor (AChR) and inhibit signaling at the neuromuscular junction. The majority of patients under 50 y with AChR autoantibody MG have thymic lymphofollicular hyperplasia. The MG thymus is a reservoir of plasma cells that secrete disease-causing AChR autoantibodies and although thymectomy improves clinical scores, many patients fail to achieve complete stable remission without additional immunosuppressive treatments. We speculate that thymus-associated B cells and plasma cells persist in the circulation after thymectomy and that their persistence could explain incomplete responses to resection. We studied patients enrolled in a randomized clinical trial and used complementary modalities of B cell repertoire sequencing to characterize the thymus B cell repertoire and identify B cell clones that resided in the thymus and circulation before and 12 mo after thymectomy. Thymus-associated B cell clones were detected in the circulation by both mRNA-based and genomic DNA-based sequencing. These antigen-experienced B cells persisted in the circulation after thymectomy. Many circulating thymus-associated B cell clones were inferred to have originated and initially matured in the thymus before emigration from the thymus to the circulation. The persistence of thymus-associated B cells correlated with less favorable changes in clinical symptom measures, steroid dose required to manage symptoms, and marginal changes in AChR autoantibody titer. This investigation indicates that the diminished clinical response to thymectomy is related to persistent circulating thymus-associated B cell clones.

NK and Th17 cells in the thymus of myasthenia gravis patients

Objective: As a classical autoimmune disorder, anti-acetylcholine receptor (AChR) antibody positive myasthenia gravis (MG) has an unconventional pathophysiology that involves thymus, the central organ for immune tolerance induction. Both natural killer (NK) cells and type 17 helper T (Th17) cells possess capacity to influence autoimmune inflammation. This study aims to determine the presence of Th17 and NK cells in the thymus from MG patients. Methods: Thymectomy materials of MG patients and non-MG controls were assessed by CD56, CD16, CD2, CD3, NKG2D, NKp46 and IL-23R flow cytometry and IL-23R, IL-21R, and ROR-γ immunohistochemistry. Results: Even though NK cell infiltration was limited, the majority of these cells displayed activation markers NKG2D and NKp46. Expectedly, the amount of CD2+ lymphocytic cells were higher than CD3+ thymocytes in which a considerable percentage was carrying the receptor for IL-23 (IL-23R). In addition to IL-23R, IL-21R, and ROR-γ were also detected in MG thymus as a marker related to Th17 cells. These Th17-related markers were reduced in thymoma compared to that of detected in thymic hyperplasia or the MG thymus with normal histopathology. Conclusion: Both NK cells and Th17 cells are found in the MG thymus indicating a possible cross-regulation between these cell types that may influence the course of autoimmune reactions.

Role of miRNAs in Normal and Myasthenia Gravis Thymus.

The thymus, a primary lymphoid organ, provides a complex environment essential for the generation of the T-cell repertoire. Thymic alterations occur during life either in the context of thymic involution upon aging or the pathophysiological context of Myasthenia Gravis (MG). These changes involve complicated regulatory networks, in which microRNAs (miRNAs) are key players. Here, we analyzed the role of miRNAs in thymocyte maturation and differentiation sustained by thymic epithelial cells. We compared data from the literature regarding the role of mouse thymic miRNAs and original data obtained from a human thymic miRnome study. We identified a set of highly expressed miRNAs defined as ThymiRs and investigated miRNA expression in infants as compared to adults to determine those associated with human thymic involution. Thymic changes are also frequently observed in MG, an autoimmune disease which results in the production of anti-acetylcholine receptor (AChR) antibodies that lead to muscle weaknesses. Alterations such as thymoma in late-onset MG patients and hyperplasia with ectopic germinal centers (GCs) in early-onset (EOMG) patients are found. Thymic miRNA expression has been studied in AChR-MG patients both in thymoma-associated MG (TAMG) and EOMG, and their function through their mRNA targets investigated. Most of the dysregulated thymic miRNAs in EOMG are associated with GC development, such as miR-7, miR-24, miR-139, miR-143, miR-145, miR-146, miR-150, miR-452, miR-548 or thymic inflammation, such as miR-125b, miR-146, or miR-29. Understanding these pathways may provide therapeutic targets or biomarkers of disease manifestations.

What's in the Neuromuscular Junction Literature?

Four retrospective studies from the United States, Europe, and Asia address outcomes in juvenile myasthenia gravis. Common features include earlier onset with ocular myasthenia gravis (MG) and generally good outcomes overall. Patients who were seronegative, had equivocal acetylcholine receptor (AChR) antibodies, or had only clustered AChR antibodies had better outcomes. An article highlighting the utility of median nerve slow-repetitive stimulation is reviewed and another showing the high sensitivity of repetitive nerve stimulation in myasthenic crisis is covered. Two articles address the thymus and thymectomy in non-AChR antibody-positive autoimmune MG. Longer term data on eculizumab as well as studies of cyclophosphamide, rituximab, and tacrolimus are summarized. Other topics include the possible role of statins in MG and central nervous system autoimmune comorbidities.

MiR-146a in Myasthenia Gravis Thymus Bridges Innate Immunity With Autoimmunity and Is Linked to Therapeutic Effects of Corticosteroids.

Toll-like receptor (TLR)-mediated innate immune responses are critically involved in the pathogenesis of myasthenia gravis (MG), an autoimmune disorder affecting neuromuscular junction mainly mediated by antiacetylcholine receptor antibodies. Considerable evidence indicate that uncontrolled TLR activation and chronic inflammation significantly contribute to hyperplastic changes and germinal center (GC) formation in the MG thymus, ultimately leading to autoantibody production and autoimmunity. miR-146a is a key modulator of innate immunity, whose dysregulation has been associated with autoimmune diseases. It acts as inhibitor of TLR pathways, mainly by targeting the nuclear factor kappa B (NF-κB) signaling transducers, interleukin 1 receptor associated kinase 1 (IRAK1) and tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6); miR-146a is also able to target c-REL, inducible T-cell costimulator (ICOS), and Fas cell surface death receptor (FAS), known to regulate B-cell function and GC response. Herein, we investigated the miR-146a contribution to the intrathymic MG pathogenesis. By real-time PCR, we found that miR-146a expression was significantly downregulated in hyperplastic MG compared to control thymuses; contrariwise, IRAK1, TRAF6, c-REL, and ICOS messenger RNA (mRNA) levels were upregulated and negatively correlated with miR-146a levels. Microdissection experiments revealed that miR-146a deficiency in hyperplastic MG thymuses was not due to GCs, but restricted to the GC-surrounding medulla, characterized by IRAK1 overexpression. We also showed higher c-REL and ICOS mRNA levels, and lower FAS mRNA levels, in GCs than in the remaining medulla, according to the contribution of these molecules in GC formation. By double immunofluorescence, an increased proportion of IRAK1-expressing dendritic cells and macrophages was found in hyperplastic MG compared to control thymuses, along with GC immunoreactivity for c-REL. Interestingly, in corticosteroid-treated MG patients intrathymic miR-146a and mRNA target levels were comparable to those of controls, suggesting that immunosuppressive therapy may restore the microRNA (miRNA) levels. Indeed, an effect of prednisone on miR-146a expression was demonstrated in vitro on peripheral blood cells. Serum miR-146a levels were lower in MG patients compared to controls, indicating dysregulation of the circulating miRNA. Our overall findings strongly suggest that defective miR-146a expression could contribute to persistent TLR activation, lack of inflammation resolution, and hyperplastic changes in MG thymuses, thus linking TLR-mediated innate immunity to B-cell-mediated autoimmunity. Furthermore, they unraveled a new mechanism of action of corticosteroids in inducing control of autoimmunity in MG via miR-146a.

Causes and Consequences of miR-150-5p Dysregulation in Myasthenia Gravis.

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.

The pathology of the thymus in myasthenia gravis

Myasthenia gravis (MG) is an autoantibody mediated autoimmune disease characterized by skeletal muscle fatigability and weakness through different types of immune attacks against various proteins of the postsynaptic membrane or in the neuromuscular cleft of the neuromuscular junction. In a subset of patients with autoantibodies against the muscle type acetylcholine receptors (AChRs) there is strong evidence that the thymus is involved in the pathogenesis of MG, while its role is questionable or unknown in MG due to autoantibodies against muscle specific kinase (MuSK), lipoprotein receptor like protein 4 and agrin. Here we describe the thymic alterations that have repeatedly been encountered in MG, i.e., thymic follicular hyperplasia (TFH) (thymitis), ‘thymic atrophy’, thymoma and thymolipoma. The potential link of non-thymic tumors [e.g., follicular dendritic cell (FDC) sarcoma] with MG will be discussed.

MicroRNA and mRNA expression associated with ectopic germinal centers in thymus of myasthenia gravis.

BackgroundA characteristic pathology of early onset myasthenia gravis is thymic hyperplasia with ectopic germinal centers (GC). However, the mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. Dysregulation of small, non-coding microRNAs (miRNAs) and their target genes has been identified in the pathology of several autoimmune diseases. We assessed the miRNA and mRNA profiles of the MG thymus and have investigated their role in GC formation and maintenance.MethodsMG thymus samples were assessed by histology and grouped based upon the appearance of GC; GC positive and GC negative. A systems biology approach was used to study the differences between the groups. Our study included miRNA and mRNA profiling, quantitative real-time PCR validation, miRNA target identification, pathway analysis, miRNA-mRNA reciprocal expression pairing and interaction.ResultsThirty-eight mature miRNAs and forty-six annotated mRNA transcripts were differentially expressed between the two groups (>1.5 fold change, ANOVA p<0.05). The miRNAs were found to be involved in immune response pathways and identified in other autoimmune diseases. The cellular and molecular functions of the mRNAs showed involvement in cell death and cell survival, cellular proliferation, cytokine signaling and extra-cellular matrix reorganization. Eleven miRNA and mRNA pairs were reciprocally regulated. The Regulator of G protein Signalling 13 (RGS13), known to be involved in GC regulation, was identified in specimens with GC and was paired with downregulation of miR-452-5p and miR-139-5p. MiRNA target sites were validated by dual luciferase assay. Transfection of miRNA mimics led to down regulation of RGS13 expression in Raji cells.ConclusionOur study indicates a distinct miRNA and mRNA expression pattern in ectopic GC in MG thymus. These miRNAs and mRNAs are involved in regulatory pathways common to inflammation and immune response, cell cycle regulation and anti-apoptotic pathways suggesting their involvement in support of GC formation in the thymus. We demonstrate for the first time that miR-139-5p and miR-452-5p negatively regulate RGS13 expression.

Regulatory B cells in myasthenia gravis are differentially affected by therapies.

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.

The overexpression of Fra1 disorders the inflammatory cytokine secretion by mTEC of myasthenia gravis thymus.

The thymus of a myasthenia gravis (MG) patient is often accompanied by and effected with follicular hyperplasia. Inflammatory cytokines in thymus induce the formation of germinal centres (GC). MG thymic inflammatory cytokines are predominantly secreted by stromal cells. Our previous studies revealed that the expression level of the Fra1 protein, which is a Fos member of the activator protein 1 transcription factors (AP-1), was higher in the MG thymus compared with that of the normal thymus. Based on that, we demonstrated that Fra1 was mainly expressed in medulla thymic epithelial cells (mTECs) and that the rate of Fra1 positive mTECs in the MG thymus was higher than normal. In vitro, we found that the expression of CCL-5, CCL-19 and CCL-21 could be regulated by Fra1 in mTEC and that IL-1β, IL-6, IL-8 and ICAM1 were downregulated in the Fra1 overexpression group and upregulated in the Fra1 knock-down group. Meanwhile, we detected that the expression levels of suppressor of cytokine signalling 3 (SOCS3) were significantly upregulated along with the overexpression of Fra1. Hence, we considered that the overexpression of Fra1 disrupted inflammatory cytokine secretion by mTEC in the MG thymus and that STAT3 and SOCS3 were strongly involved in this process.

Analysis of microRNA expression in the thymus of Myasthenia Gravis patients opens new research avenues.

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.

An imbalance between regulatory T cells and T helper 17 cells in acetylcholine receptor-positive myasthenia gravis patients.

A chronic autoimmune disease, myasthenia gravis (MG) is characterized in 85% of patients by antibodies directed against the acetylcholine receptor (AChR) located at the neuromuscular junction. The functional and effective balance between regulatory T cells (Treg cells) and effector T cells (Teff cells) is lost in the hyperplastic thymus of MG patients with antibodies specific for the AChR (AChR+ MG patients). The objective of this review is to describe how Treg cells and inflammatory T cells participate in this imbalance and contribute to induce a chronic inflammatory state in the MG thymus. We discuss the origins and characteristics of Treg cells and their reported dysfunctions in AChR+ MG patients. We also review the inflammatory condition observed in MG thymus, including overexpression of interleukin (IL)-1β, IL-6, and IL-23, cytokines that promote the differentiation of T helper 17 (TH 17) cells and the expression of IL-17. We summarize the preclinical models used to determine the implication of expression of cytokines, such as IL-6, IL-12 (IL-23 subunit), IL-17, and interferon γ to the development of experimental autoimmune MG. Finally, we suggest that biological agents, such as humanized monoclonal antibodies that target the IL-23/TH 17 pathway, should be investigated in the context of MG, as they have proven efficiency in other autoimmune diseases.

Toll-like receptors 7 and 9 in myasthenia gravis thymus: amplifiers of autoimmunity?

Pathogen infections and dysregulated Toll-like receptor (TLR)-mediated innate immune responses are suspected to play key roles in autoimmunity. Among TLRs, TLR7 and TLR9 have been implicated in several autoimmune conditions, mainly because of their ability to promote abnormal B cell activation and survival. Recently, we provided evidence of Epstein-Barr virus (EBV) persistence and reactivation in the thymus of myasthenia gravis (MG) patients, suggesting an involvement of EBV in the intrathymic pathogenesis of the disease. Considerable data highlight the existence of pathogenic crosstalk among EBV, TLR7, and TLR9: EBV elicits TLR7/9 signaling, which in turn can enhance B cell dysfunction and autoimmunity. In this article, after a brief summary of data demonstrating TLR activation in MG thymus, we provide an overview on the contribution of TLR7 and TLR9 to autoimmune diseases and discuss our recent findings indicating a pivotal role for these two receptors, along with EBV, in driving, perpetuating, and/or amplifying intrathymic B cell dysregulation and autoimmune responses in MG. Development of therapeutic approaches targeting TLR7 and TLR9 signaling could be a novel strategy for treating the chronic inflammatory autoimmune process in myasthenia gravis.

Thymus involvement in early-onset myasthenia gravis.

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)-β is overexpressed in MG thymus and could orchestrate thymic changes associated with MG. Knowledge about how IFN-β 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.