MuSK EAMG: Immunological Characterization and Suppression by Induction of Oral Tolerance

Abstract

Myasthenia gravis (MG) with antibodies to the muscle-specific receptor tyrosine kinase (MuSK) is a distinct sub-group of MG, affecting 5-8% of all MG patients. MuSK, a receptor tyrosine kinase, is expressed at the neuromuscular junctions (NMJs) from the earliest stages of synaptogenesis and plays a crucial role in the development and maintenance of the NMJ. MuSK-MG patients are more severely affected and more refractory to treatments currently used for MG. Most patients require long-term immunosuppression, stressing the need for improved treatments. Ideally, preferred treatments should specifically delete the antigen-specific autoimmune response, without affecting the entire immune system. Mucosal tolerance, induced by oral or nasal administration of an auto-antigen through the mucosal system, resulting in an antigen-specific immunological systemic hyporesponsiveness, might be considered as a treatment of choice for MuSK-MG. In the present study we have characterized several immunological parameters of murine MuSK-EAMG and have employed induction of oral tolerance in mouse MuSK-EAMG, by feeding with a recombinant MuSK protein one week before disease induction. Such a treatment has been shown to attenuate MuSK-EAMG. Both induction and progression of disease were ameliorated following oral treatment with the recombinant MuSK fragment, as indicated by lower clinical scores and lower anti-MuSK antibody titers.

Keywords: T regulatory cells; muscle-specific receptor tyrosine kinase (MuSK); myasthenia gravis; neuromuscular junction (NMJ); oral tolerance.

Figure 1

Clinical characterization and antibody titers in MuSK-EAMG, induced in FVB/N mice. FVB/N female mice were immunized twice with 20–40 μg (as indicated) of recombinant MuSK in CFA, or with CFA alone, as a control (n = 6). Mice were followed up for clinical score (A) and weight loss changes (B). Anti-MuSK antibody titer was tested 4 weeks following immunization, by ELISA (C), and correlation with disease severity was tested (D). P < 0.001 in (A,B). Analyzed by the two-way ANOVA test.

Figure 2

MuSK immunized mice exhibit specific muscle damage. FVB/N mice were sacrificed 5 weeks after disease induction and RNA was isolated from masseter muscles. The expression levels of cathepsin-l (A), IL-15 (B) and MuSK (C) were analyzed by quantitative real time RT-PCR and compared to the levels obtained in CFA-immunized control mice. β-actin was used as an internal control for normalization. All data are presented as mean ± SEM. Unpaired Student t test was employed. Representative out of two experiments (n = 6).

Figure 3

Decreased Treg cell frequencies in spleens of MuSK-EAMG mice immunized mice. Spleens from control and MuSK immunized mice were harvested at the end of the experiments and analyzed by Flow cytometry and rt-PCR analyses. Representative Flow cytometry analysis of control immunized mouse (A) and MuSK immunized mouse (B). Graphical summary of the frequency of CD4+CD25+FoxP3+ cells (C). Expression levels of FoxP3, IL-18 and TGF-β (D–F, respectively) were evaluated by RT-PCR. β-actin was used as an inner control for normalization. All data are presented as mean ± SEM. Unpaired Student t test has been employed. Representative out of two experiments (n = 12).

Figure 4

Oral treatment suppresses MuSK-MG. Mice were fed with recombinant MuSK protein or OVA protein as a control, 3 times a week, starting one week before MuSK immunization and until the end of the experiment. Mean clinical score (A) and weight changes (B) in mice fed with 120μg MuSK or OVA/mouse/dose. Total MuSK specific IgG, tested by ELISA (C). n ≥ 8 mice for each group, in 3 different experiments. P < 0.001 in (A,B). Analyzed by the two-way ANOVA test. Representative out of three experiments.

Figure 5

MuSK-fed mice display increased levels of Tregs associated genes. Spleens were harvested at the end of the experiment and the RNA was isolated. Expression levels of Foxp3, IL-18, and TGF-β (A–C, respectively) were evaluated by RT-PCR. β-actin was used as an inner control for normalization. All data are presented as mean ± SEM (n ≥ 5). Unpaired Student t test has been employed.