Experimental Autoimmune Myasthenia Gravis Pathogenesis Research Articles

Osteopontin exacerbates the progression of experimental autoimmune myasthenia gravis by affecting the differentiation of T cell subsets

Osteopontin (OPN) is a multifunctional extracellular matrix phosphoprotein that has a specific and complicated structure, and contributes to numerous physiological and pathological activities. The mechanism of OPN in many diseases has been confirmed; however, the role of OPN in myasthenia gravis (MG) remains unclear. In this study, we recombined rat OPN protein in vitro, and assessed how OPN affects the development of autoimmunity using an experimental autoimmune myasthenia gravis (EAMG) rat model. The results showed that the concentration of OPN in serum was up-regulated. Both mRNA and protein levels in splenocytes increased in the EAMG model. OPN treatment in vitro strongly promoted the differentiation of Th1 cells, and inhibited the differentiation of Treg cells. Intraperitoneal injection of OPN revealed the early incidence of EAMG, and more serious disease. This effect was accompanied by an increased percentage of Th1 cells. In conclusion, OPN likely exacerbates the pathogenesis of EAMG by promoting the differentiation of Th1 cells and inhibiting the differentiation of Treg cells.

IL-17-producing CD4(+) T cells contribute to the loss of B-cell tolerance in experimental autoimmune myasthenia gravis.

The role of Th17 cells in the pathogenesis of autoantibody-mediated diseases is unclear. Here, we assessed the contribution of Th17 cells to the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), which is induced by repetitive immunizations with Torpedo californica acetylcholine receptor (tAChR). We show that a significant fraction of tAChR-specific CD4(+) T cells is producing IL-17. IL-17(ko) mice developed fewer or no EAMG symptoms, although the frequencies of tAChR-specific CD4(+) T cells secreting IL-2, IFN-γ, or IL-21, and the percentage of FoxP3(+) Treg cells were similar to WT mice. Even though the total anti-tAChR antibody levels were equal, the complement fixating IgG2b subtype was reduced in IL-17(ko) as compared to WT mice. Most importantly, pathogenic anti-murine AChR antibodies were significantly lower in IL-17(ko) mice. Furthermore, we confirmed the role of Th17 cells in EAMG pathogenesis by the reconstitution of TCR β/δ(ko) mice with WT or IL-17(ko) CD4(+) T cells. In conclusion, we show that the level of IgG2b and the loss of B-cell tolerance, which results in pathogenic anti-murine AChR-specific antibodies, are dependent on IL-17 production by CD4(+) T cells. Thus, we describe here for the first time how Th17 cells are involved in the induction of classical antibody-mediated autoimmunity.

IgG1 deficiency exacerbates experimental autoimmune myasthenia gravis in BALB/c mice.

Myasthenia gravis is an autoimmune disease characterized by muscle weakness due to neuromuscular junction (NMJ) damage by anti-acetylcholine receptor (AChR) auto-antibodies and complement. In experimental autoimmune myasthenia gravis (EAMG), which is induced by immunization with Torpedo AChR in CFA, anti-AChR IgG2b and IgG1 are the predominant isotypes in the circulation. Complement activation by isotypes such as IgG2b plays a crucial role in EAMG pathogenesis; this suggested the possibility that IgG1, which does not activate complement through the classical pathway, may suppress EAMG. In this study, we show that AChR-immunized BALB/c mice genetically deficient for IgG1 produce higher levels of complement-activating isotypes of anti-AChR, especially IgG3 and IgG2a, and develop increased IgG3/IgG2a deposits at the NMJ, as compared to wild type (WT) BALB/c mice. Consistent with this, AChR-immunized IgG1(-/-) BALB/c mice lose muscle strength and muscle AChR to a greater extent than AChR-immunized WT mice. These observations demonstrate that IgG1 deficiency leads to increased severity of EAMG associated with an increase in complement activating IgG isotypes. Further studies are needed to dissect the specific role or mechanism of IgG1 in limiting EAMG and that of EAMG exacerbating role of complement activating IgG3 and IgG2a in IgG1 deficiency.

Tissue plasminogen activator involvement in experimental autoimmune myasthenia gravis: Aggravation and therapeutic potential

Tissue plasminogen activator (tPA), a component of the PA/plasmin system, is elevated in inflammatory areas and plays a role in inflammatory neurological disorders. In the present study we explored the involvement of tPA and the potential immunomodulatory activity of tPA in experimental autoimmune myasthenia gravis (EAMG). Mice deficient in tPA (tPA−/−) present with a markedly more severe disease than wild type EAMG mice. In an attempt to treat EAMG with an 18aa peptide derived from the PA system inhibitor (PAI-1), designed to tether out the endogenous inhibitor, a significant suppression of disease severity was demonstrated. The more severe disease in tPA−/− mice was accompanied by a higher level of anti-AChR antibodies and increased expression of B-cell markers. In view of the essential role of B-cell activating factor (BAFF) in B-cell maturation, the expression of BAFF family components was tested. An increase in BAFF and BAFF receptor was observed in EAMG tPA−/− mice, whereas BCMA expression was reduced, consistent with the increased level of pathogenic antibodies and the more severe disease. Given the importance of T regulatory cells (Tregs) in EAMG, they were evaluated and their number was reduced in tPA−/− mice, in which EAMG was aggravated, whereas following PAI-1dp treatment, Tregs were replenished and the disease was ameliorated. The results show the involvement of tPA in EAMG, implying a protective role for tPA in EAMG pathogenesis. The amelioration of EAMG by PAI-1dp treatment suggests that the PA system may be considered a potential site for therapeutic intervention in neuroimmune diseases.

Identification of Novel MicroRNA Signatures Linked to Experimental Autoimmune Myasthenia Gravis Pathogenesis: Down-Regulated miR-145 Promotes Pathogenetic Th17 Cell Response

Emerging evidence demonstrates that miRNAs, a new family of key mRNA regulatory molecules, have crucial roles in controlling and modulating immunity. Their contribution to myasthenia gravis (MG), a T cell-dependent, antibody-mediated nervous system autoimmune disease, has not been thoroughly investigated. In the present study, using a highly sensitive microarray-based approach, we identified 11 miRNAs with differential expression between Peripheral Blood Mononuclear Cells (PBMC) from experimental autoimmune MG (EAMG) rats and control rats. miR-145 is one of the most significantly down-regulated miRNAs in PBMC from EAMG rats. Down-regulation of miR-145 expression was confirmed in PBMC and CD4+CD25- T cells (T effector cells) from both EAMG rats and MG patients by real-time PCR. Bioinformatics target prediction identified two crucial immune-related molecules-CD28 and NFATc1, as putative targets of miR-145. Furthermore, miR-145 inhibited CD28 and NFATc1 expression by directly targeting their 3'-UTRs, which was abolished by mutation of the miR-145 and CD28/NFATc1 binding sites. In vitro up-regulation of miR-145 in CD4+ T cells can significantly reduce CD28 protein levels accompanied by decreased proliferative response. In a dendritic cell (DC)-T cell coculture system, overexpression of miR-145 in AChR-specific CD4+ T cells suppresses NFATc1 expression and T Helper 17 cells level. Finally, we observed that administration of lentiviral-miR-145 decreased the severity of ongoing, established EAMG with decreased IL-17 production, and also decreased serum anti-AChR IgG levels. Our studies provide an important new insight into the pathogenesis of EAMG and MG, which may open a new perspective for the development of effective gene therapy for EAMG/MG.

Novel animal models of acetylcholine receptor antibody–related myasthenia gravis

Experimental autoimmune myasthenia gravis (EAMG) in mice has been used to unravel the pathogenic mechanisms and to be used as a preclinical model of myasthenia gravis (MG). Induction of predominantly ocular EAMG in HLA-DQ8 transgenic mice immunized with acetylcholine receptor (AChR) subunits demonstrated the importance of nonconformationally expressed AChR subunits in extraocular muscle involvement. Wild-type (WT) and CD4(+) T cell knockout (KO) C57BL/6 mice developed EAMG upon immunization with AChR in incomplete Freund's adjuvant plus lipopolysaccharide. AChR-specific IgG2(+) B cell frequencies, estimated by Alexa-conjugated AChR, and AChR-reactive IgG2b levels significantly correlated with the clinical grades of EAMG in WT mice. CD4(+) T cell-deficient EAMG mice exhibited AChR antibodies mainly of the IgG2b isotype, emphasizing T helper-independent B cell activation pathways in EAMG induction. These novel EAMG models have suggested that diverse immunopathological mechanisms might contribute to EAMG or MG pathogenesis.

BM stromal cells ameliorate experimental autoimmune myasthenia gravis by altering the balance of Th cells through the secretion of IDO

In addition to their capacity to differentiate, BM stromal cells (BMSC) have immunosuppressive qualities that make them strong candidates for use in cell therapy against human autoimmune diseases. We studied the immunoregulatory activities of BMSC on experimental autoimmune myasthenia gravis (EAMG) in vitro and in vivo. Intravenous administration of syngenic BMSC to EAMG-model rats on the day of their second immunization was effective in ameliorating the pathological features of the disease. In vitro, the proliferative ability of T cells or B cells from EAMG rats was inhibited when they were cocultured with BMSC at proper ratios. This inhibitory effect was at least partially dependent on the secretion of IDO. We also determined that the development of EAMG is accompanied by an imbalance among the Th1, Th2, Th17, and Treg cell subsets, and that this can be corrected by the administration of BMSC, which leads to an increase of Th2 (IL-4) and Treg (Foxp3) cells, and a reduction of Th1 (IFN-gamma) and Th17 (IL-17) cells, through an IDO-dependent mechanism. These results provide further insights into the pathogenesis of MG, EAMG, and other immune-mediated diseases, and support a potential role for BMSC in their treatment.

C5a is not involved in experimental autoimmune myasthenia gravis pathogenesis

C5 deficient mice are highly resistant to experimental autoimmune myasthenia gravis (EAMG) despite intact immune response to acethylcholine receptor (AChR), validating the pivotal role played by membrane attack complex (MAC, C5b-9) in neuromuscular junction destruction. To distinguish the significance of C5a from that of C5b in EAMG pathogenesis, C5a receptor (C5aR) knockout (KO) and wild-type (WT) mice were immunized with AChR to induce pathogenic anti-AChR antibodies. In contrast with C5 deficient mice, C5aR KO mice were equally susceptible to EAMG as WT mice and exhibited comparable antibody and lymphocyte proliferation response to AChR implicating that C5a is not involved in EAMG development.

Complement regulator CD59 deficiency fails to augment susceptibility to actively induced experimental autoimmune myasthenia gravis

Complement deficient mice are resistant to experimental autoimmune myasthenia gravis (EAMG), suggesting a pivotal role for the membrane attack complex (MAC) in EAMG pathogenesis. To test the significance of MAC regulation in EAMG pathogenesis, CD59 KO and wild type mice were immunized with acetylcholine receptor (AChR). Interestingly, deletion of CD59, the regulator of MAC assembly, failed to augment EAMG susceptibility. The CD59 KO mice had reduced serum anti-AChR IgG1, IgG2b and complement levels. Their lymph node cell IL-2 production and lymphocyte proliferation response to AChR were reduced. The data challenge the current paradigm that CD59 is solely involved in MAC regulation and suggest a role for this molecule in antigen-driven T cell and B cell activation.

Genetic evidence for the involvement of Fcγ receptor III in experimental autoimmune myasthenia gravis pathogenesis

Immune complexes and classical complement pathway play vital roles in experimental autoimmune myasthenia gravis (EAMG). To analyze the role of immune complex receptors in EAMG, FcγRIII knockout (KO) mice were immunized with AChR and were found out to be resistant to EAMG induction. This was associated with reduced neuromuscular junction deposits, lymph node cell (LNC) IL-6 production and serum complement levels. EAMG resistance of anti-C1q Ab-administered mice was also associated with reduced LNC IL-6 production and neuromuscular junction deposits, indicating C1q involvement in EAMG resistance. The data provide the first direct genetic evidence for Fcγ receptor involvement in EAMG pathogenesis.

Overexpression of phosphodiesterases in experimental autoimmune myasthenia gravis: suppression of disease by a phosphodiesterase inhibitor

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are T cell-dependent antibody-mediated autoimmune disorders, in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. DNA microarray analysis revealed increased levels of several phosphodiesterase (PDE) subtypes in lymph node cells (LNC) and muscles of EAMG rats compared with healthy controls. Quantitative real-time PCR analysis indicated that EAMG is characterized by an increase of PDE subtypes 1, 3, 4, and 7 in LNC and of PDE subtypes 2, 3, 4, and 7 in muscles. Pentoxifylline (PTX), a general PDE inhibitor, inhibited the progression of EAMG when treatment started at either the acute or chronic stages of disease. This suppression was associated with down-regulation of humoral and cellular AChR-specific responses, as well as down-regulation of PDE4, TNF-alpha, IL-18, IL-12, and IL-10 in LNC and of PDEs 1, 4, 7, and TNF-alpha in muscles. The expression of Foxp3, a transcription factor essential for CD4+CD25+ regulatory T cell function, was increased in splenocytes although the number of these cells remained unchanged. PTX also reduced the expression of the endopeptidase cathepsin-l, a marker of muscle damage, in EAMG muscles. This study demonstrates the involvement of PDE regulation in EAMG pathogenesis and suggests that PDE inhibitors may be considered for immunotherapy of MG.

Decreased expression of Src homology 2 domain-containing protein tyrosine phosphatase 1 reduces T cell activation threshold but not the severity of experimental autoimmune myasthenia gravis

Myasthenia gravis (MG) and its murine model experimental autoimmune myasthenia gravis (EAMG) are T cell-dependent, antibody-mediated autoimmune diseases. Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) is a cytosolic tyrosine phosphatase that is involved in regulating the T cell activation cascade from signals initiated through the TCR. To study the role of SHP-1 in EAMG pathogenesis, we immunized C57BL/6 (B6) mice heterozygous for deletion of the SHP-1 gene ( me v+/− ) and their littermate wild type B6 mice with torpedo acetylcholine receptor (TAChR). T cell proliferation and IFNγ production were significantly increased in B6. me v+/− mice after immunization with AChR compared to that of wild type littermates. However, clinical incidence and severity of the disease were not changed. There also were no significant differences in AChR-specific antibodies produced between wild type and me v+/− mice. These data suggest that deficiency in SHP-1 expression does decrease the activation threshold of autoreactive T cells in EAMG, but the increased frequency of autoreactive T cells does not aggravate EAMG in terms of clinical score, incidence, or antibody titers.

Resistance to experimental autoimmune myasthenia gravis in IL-6-deficient mice is associated with reduced germinal center formation and C3 production.

To provide direct genetic evidence for a role of IL-6 in experimental autoimmune myasthenia gravis (EAMG), IL-6 gene KO (IL-6(-/-)) mice in the C57BL/6 background were immunized with Torpedo californica acetylcholine receptor (AChR) and evaluated for EAMG. Only 25% of AChR-immunized IL-6(-/-) mice developed clinical EAMG compared to 83% of C57BL/6 (wild-type) mice. A significant reduction in the secondary anti-AChR Ab of IgG, IgG(2b), and IgG(2c), but not the primary or secondary IgM response was observed in AChR-immunized IL-6(-/-) mice, suggesting a possible defect in T cell help and class switching to anti-AChR IgG(2) isotype. The AChR-specific lymphocyte proliferative response, IFN-gamma, and IL-10 production were suppressed in AChR-immunized IL-6(-/-) mice. EAMG resistance in IL-6(-/-) mice was associated with a significant reduction in germinal center formation and decreased serum complement C3 levels. The data provide the first direct genetic evidence for a key role of IL-6 in the autoimmune response to AChR and in EAMG pathogenesis.

Tumor necrosis factor receptor p55 and p75 deficiency protects mice from developing experimental autoimmune myasthenia gravis

The precise pathogenic role of proinflammatory cytokines belonging to the tumor necrosis factor (TNF) family has not been investigated yet in antibody-mediated myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG). In this study we report that TNF receptor p55 −/− p75 −/− mice were resistant to the development of clinical EAMG induced by acetylcholine receptor (AChR) immunizations. The resistance was associated with reduced serum levels of IgG, IgG 1, IgG 2a, and IgG 2b anti-AChR antibody isotypes. However, IgM anti-AChR antibodies were not reduced, suggesting defective anti-AChR IgG class switching in TNF receptor p55 −/− p75 −/− mice. We thus demonstrate the genetic evidence for the role of TNF receptor p55 and p75 in EAMG pathogenesis, and their requirement for the generation of anti-AChR IgG antibodies.

Tumor necrosis factor receptor-1 is critically involved in the development of experimental autoimmune myasthenia gravis.

Tumor necrosis factor receptor-1 (TNFR1, CD120a) has been implicated in the pathogenesis of several experimental models of T cell-mediated autoimmune disorders, but its role in antibody-mediated autoimmune diseases has not been addressed. Experimental autoimmune myasthenia gravis (EAMG), an autoantibody-mediated T cell-dependent neuromuscular disorder, represents an animal model for myasthenia gravis in human. To investigate the role of TNFR1 in the pathogenesis of EAMG, TNFR1(-/-) and wild-type mice were immunized with TORPEDO: acetylcholine receptor (AChR) in complete Freund's adjuvant. TNFR1(-/-) mice failed to develop EAMG. Lymphoid cells from TNFR1(-/-) mice produced low amounts of T(h)1 (IFN-gamma, IL-2 and IL-12)-type cytokines, but elevated levels of T(h)2 (IL-4 and IL-10)-type cytokines compared with lymphoid cells of wild-type mice. Accordingly, the levels of anti-AChR IgG2 antibodies were severely reduced and the level of anti-AChR IgG1 antibodies were moderately reduced. Co-injection of recombinant mouse IL-12 with AChR in adjuvant restored T cell responses to AChR and promoted development of EAMG in TNFR1(-/-) mice. These results demonstrate that the TNF/TNFR1 system is required for the development of EAMG. The lack of a functional TNF/TNFR1 system can, at least in part, be substituted by IL-12 at the stage of initial priming with AChR and adjuvant.

Role for interferon-gamma in rat strains with different susceptibility to experimental autoimmune myasthenia gravis.

Experimental autoimmune myasthenia gravis (EAMG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular postsynaptic membrane and represents an animal model of myasthenia gravis in human. Recent studies highlighted the roles of TH1 cytokines (IFN-gamma, IL-12), rather than TH2 cytokines (IL-4), in the pathogenesis of EAMG by using homozygous (-/-) knockout mice with an EAMG-susceptible genetic background. To further evaluate a role for IFN-gamma, we injected recombinant rat IFN-gamma (rrIFN-gamma) at the time of immunization with AChR in complete Freund's adjuvant to EAMG-susceptible Lewis rats and EAMG-resistant Wistar Furth (WF) rats. RrIFN-gamma enhanced Lewis rat EAMG. The exacerbated muscular weakness was associated with higher levels of anti-AChR IgG and enhanced TNF-alpha responses. Anti-AChR IgG antibody levels were augmented to a similar extent as in Lewis rats, however, the identical immunization and IFN-gamma injection induced only mild and transient EAMG in WF rats due to the default TH3 phenotype development and inherent low TH1 responses. We conclude that IFN-gamma plays a major role in the pathogenesis of EAMG in the Lewis rat, but fails to break disease resistance in the WF rat.

Animal Models of Myasthenia Gravis

Myasthenia gravis (MG) is an antibody-mediated, autoimmune neuromuscular disease. Animal models of experimental autoimmune myasthenia gravis (EAMG) can be induced in vertebrates by immunization with Torpedo californica acetylcholine receptors (AChR) in complete Freund's adjuvant. The MHC class II genes influence the cellular and humoral immune response to AChR and are involved in the development of clinical EAMG in mice. A dominant epitope within the AChR alpha146-162 region activates MHC class II-restricted CD4 cells and is involved in the production of pathogenic anti-AChR antibodies by B cells. Neonatal or adult tolerance to this T-cell epitope could prevent EAMG. During an immune response to AChR in vivo, multiple TCR genes are used. The CD28-B7 and CD40L-CD40 interaction is required during the primary immune response to AChR. However, CTLA-4 blockade augmented T- and B-cell immune response to AChR and disease. Cytokines IFN-gamma and IL-12 upregulate, while IFN-alpha downregulates, EAMG pathogenesis. However, the Th2 cytokine IL-4 fails to play a significant role in the development of antibody-mediated EAMG. Systemic or mucosal tolerance to AChR or its dominant peptide(s) has prevented EAMG in an antigen-specific manner. Antigen-specific tolerance and downregulation of pathogenic cytokines could achieve effective therapy of EAMG and probably MG.

Is pathogenic humoral autoimmunity a Th1 response? Lessons from (for) myasthenia gravis

Myasthenia gravis (MG) is mediated by autoantibodies to the nicotinic acetylcholine receptor (AChR). Although AChR-specific T cells are necessary for disease development, the role of cytokines is not clear. Here, Balaji Balasa and Nora Sarvetnick discuss the T helper 1 (Th1)/Th2 paradigm and emerging evidence that interferon γ (IFN-γ) and interleukin 12 (IL-12) are important in experimental autoimmune MG pathogenesis.

The role of B-cells in experimental myasthenia gravis in mice

Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are caused by auto-antibodies against the nicotinic acetylcholine receptor (AChR) at the postsynaptic membrane. To evaluate the extent to which the humoral immune response against AChR operates in the pathogenesis of EAMG, we immunized B-cell knockout (microMT) and wild type C57BL/6 mice with AChR in complete Freund's adjuvant. The ability of AChR-primed lymph node cells to proliferate and secrete IFN-gamma in response to AChR and its dominant peptide alpha 146-162 were intact in microMT as in wild type mice. Similar levels of mRNA for IFN-gamma, IL-4 and IL-10 in AChR-reactive lymph node cells were detected in microMT and wild type mice. However, microMT mice had no detectable anti-AChR antibodies and never developed clinical EAMG. We conclude that B-cells are critically required for the genesis of clinical EAMG, but not for AChR-specific T-cell priming.

Experimental autoimmune myasthenia gravis induction in B cell-deficient mice.

Experimental autoimmune myasthenia gravis (EAMG) is an animal model for human myasthenia gravis (MG). Autoantibody-induced functional loss of nicotinic acetylcholine receptor (AChR) at the postsynaptic membrane is an important pathogenic feature of both MG and EAMG. To evaluate the extent at which the humoral immune response against AChR operates in the pathogenesis of EAMG, we immunized B cell knockout (muMT) and wild-type C57BL/6 mice with AChR and complete Freund's adjuvant. The ability of AChR-primed lymph node cells to proliferate and secrete IFN-gamma in response to AChR and its dominant peptide alpha146-162 were intact in muMT mice as in wild-type mice. Similar amounts of mRNA for IFN-gamma, IL-4 and IL-10 in AChR-reactive lymph node cells were detected in muMT and wild-type mice. However, muMT mice had no detectable anti-AChR antibodies and remained completely free from clinical EAMG. We conclude that B cells are critically required for the genesis of clinical EAMG, but not for AChR-specific T cell priming.