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

Abstract

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.

Expression and Clinical Significance of miR-146a and Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6) in Myasthenia Gravis Patient Serum

“…Zhang et al [ 5 ] further improved clinical muscle weakness symptoms in experimental autoimmune MG mice by silencing the expression of miR-146a. In addition, Bortone et al [ 7 ] found that miR-146a expression was significantly downregulated in the serum of patients with proliferative MG. All of these studies confirmed that miR-146a may be helpful in diagnosing MG and predicting progression. Previously, miR-146a has been identified as a nuclear factor-kb-dependent gene that could be involved in the regulation of interleukin 1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor- (TNF-) associated factor 6 (TRAF6) [ 8 ].…”

Role of miRNAs in Normal and Myasthenia Gravis Thymus

“…Interestingly, the expression of miR-146 and these target genes were normalized in EOMG patients under corticosteroid treatment. Altogether, these results suggest that miR-146 could modulate TLR signaling via IRAK1 and cREL and GC formation via ICOS (93). In addition, miR-146 is of great interest in MG as it is known to control the activation of IFN-I signaling pathways (94).…”

Reduced plasmablast frequency is associated with seronegative myasthenia gravis

“…An alternative possibility is that SN MG involves other non‐antibody mediated immune mechanisms, such as innate immunity, to a greater extent. Prior studies of innate immune function in other forms of human MG and experimental models support this possibility, 20‐24 and this should be pursued in the future.…”