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 Bortone et al. miR-146a Dysregulation in Myasthenia Gravis 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 TLRmediated 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.

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The complement system plays a key role in myasthenia gravis (MG). Anti-complement drugs are emerging as effective therapies to treat anti-acetylcholine receptor (AChR) antibody-positive MG patients, though their usage is still limited by the high costs. Here, we searched for plasma complement proteins as indicators of complement activation status in AChR-MG patients, and potential biomarkers for tailoring anti-complement therapy in MG. Plasma was collected from AChR-MG and MuSK-MG patients, and healthy controls. Multiplex immunoassays and ELISA were used to quantify a panel of complement components (C1Q, C2, C3, C4, C5, Factor B, Factor H, MBL, and properdin) and activation products (C4b, C3b, C5a, and C5b-9), of classical, alternative and lectin pathways. C2 and C5 levels were significantly reduced, and C3, C3b, and C5a increased, in plasma of AChR-MG, but not MuSK-MG, patients compared to controls. This protein profile was indicative of complement activation. We obtained sensitivity and specificity performance results suggesting plasma C2, C3, C3b, and C5 as biomarkers for AChR-MG. Our findings reveal a plasma complement “C2, C3, C5, C3b, and C5a” profile associated with AChR-MG to be further investigated as a biomarker of complement activation status in AChR-MG patients, opening new perspectives for tailoring of anti-complement therapies to improve the disease treatment.

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.

Epstein-Barr virus (EBV), a common human herpes virus latently infecting most of the world’s population with periodic reactivations, is the main environmental factor suspected to trigger and/or sustain autoimmunity by its ability to disrupt B-cell tolerance checkpoints. Myasthenia gravis (MG) is a prototypic autoimmune disorder, mostly caused by autoantibodies to acetylcholine receptor (AChR) of the neuromuscular junction, which cause muscle weakness and fatigability. Most patients display hyperplastic thymus, characterized by ectopic germinal center formation, chronic inflammation, exacerbated Toll-like receptor activation, and abnormal B-cell activation. After an overview on MG clinical features and intra-thymic pathogenesis, in the present chapter, we describe our main findings on EBV presence in MG thymuses, including hyperplastic and thymoma thymuses, in relationship with innate immunity activation and data from other autoimmune conditions. Our overall data strongly indicate a critical contribution of EBV to innate immune dysregulation and sustained B-cell-mediated autoimmune response in the pathological thymus of MG patients.