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

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.

Novel CXCL13 transgenic mouse: inflammation drives pathogenic effect of CXCL13 in experimental myasthenia gravis.

Abnormal overexpression of CXCL13 is observed in many inflamed tissues and in particular in autoimmune diseases. Myasthenia gravis (MG) is a neuromuscular disease mainly mediated by anti-acetylcholine receptor autoantibodies. Thymic hyperplasia characterized by ectopic germinal centers (GCs) is a common feature in MG and is correlated with high levels of anti-AChR antibodies. We previously showed that the B-cell chemoattractant, CXCL13 is overexpressed by thymic epithelial cells in MG patients. We hypothesized that abnormal CXCL13 expression by the thymic epithelium triggered B-cell recruitment in MG. We therefore created a novel transgenic (Tg) mouse with a keratin 5 driven CXCL13 expression. The thymus of Tg mice overexpressed CXCL13 but did not trigger B-cell recruitment. However, in inflammatory conditions, induced by Poly(I:C), B cells strongly migrated to the thymus. Tg mice were also more susceptible to experimental autoimmune MG (EAMG) with stronger clinical signs, higher titers of anti-AChR antibodies, increased thymic B cells, and the development of germinal center-like structures. Consequently, this mouse model finally mimics the thymic pathology observed in human MG. Our data also demonstrated that inflammation is mandatory to reveal CXCL13 ability to recruit B cells and to induce tertiary lymphoid organ development.

IL-6 and Akt are involved in muscular pathogenesis in myasthenia gravis.

INTRODUCTION: Anti-acetylcholine receptor (AChR) autoantibodies target muscles in spontaneous human myasthenia gravis (MG) and its induced experimental autoimmune model MG (EAMG). The aim of this study was to identify novel functional mechanisms occurring in the muscle pathology of myasthenia. RESULTS: A transcriptome analysis performed on muscle tissue from MG patients (compared with healthy controls) and from EAMG rats (compared with control rats) revealed a deregulation of genes associated with the Interleukin-6 (IL-6) and Insulin-Like Growth Factor 1 (IGF-1) pathways in both humans and rats. The expression of IL-6 and its receptor IL-6R transcripts was found to be altered in muscles of EAMG rats and mice compared with control animals. In muscle biopsies from MG patients, IL-6 protein level was higher than in control muscles. Using cultures of human muscle cells, we evaluated the effects of anti-AChR antibodies on IL-6 production and on the phosphorylation of Protein Kinase B (PKB/Akt). Most MG sera and some monoclonal anti-AChR antibodies induced a significant increase in IL-6 production by human muscle cells. Furthermore, Akt phosphorylation in response to insulin was decreased in the presence of monoclonal anti-AChR antibodies. CONCLUSIONS: Anti-AChR antibodies alter IL-6 production by muscle cells, suggesting a putative novel functional mechanism of action for the anti-AChR antibodies. IL-6 is a myokine with known effects on signaling pathways such as Akt/mTOR (mammalian Target of Rapamycin). Since Akt plays a key role in multiple cellular processes, the reduced phosphorylation of Akt by the anti-AChR antibodies may have a significant impact on the muscle fatigability observed in MG patients.

Impairment of regulatory T cells in myasthenia gravis: studies in an experimental model.

Myasthenia gravis (MG) is an antibody mediated, T cell dependent autoimmune disease characterized by muscle fatigability in which autoantibodies directed to the acetylcholine receptor (AChR) impair neuromuscular transmission. The identification of CD4⁺CD25⁺Foxp3⁺Treg cells as important regulators of tolerance opened a major area of investigation raising the possibility that a dysfunction in the Treg compartment is involved in the etiology and pathogenesis of autoimmune diseases, including MG. In this paper we summarize shortly Treg abnormalities that were reported in MG patients and report on our studies of Treg in experimental autoimmune MG (EAMG). Hopefully these studies would pave the way towards the development of novel Treg-based treatment modalities that will restore self-tolerance in MG and other autoimmune diseases. In our previous studies in EAMG we have shown that Treg cells transferred from healthy rat donors to myasthenic rats suppress EAMG. However, Treg cells from sick animals do not have the same in vivo suppressive activity as those from healthy donors. The objective of the present study was to further characterize quantitative and qualitative alterations in Treg cells of rats with EAMG. We found that the frequency of CD4⁺CD25⁺Foxp3⁺Treg cells within the spleen and PBL was decreased in EAMG rats as compared to naïve and CFA-immunized healthy controls. Treg cells from myasthenic rats were less effective than Treg cells from controls in suppressing the proliferation of CD4⁺T effector cells in response to ConA and of B cells in response to LPS. Moreover, CD4⁺CD25⁺ cells from EAMG rats exhibited an elevated extent of apoptosis and expressed upregulated levels of FAS and of Th17-associated cytokines. Since EAMG is an induced disease, these quantitative and qualitative alterations in Treg cells do not reflect predisposing impairments and seem to be associated with the specific autoimmune response resulting from AChR immunization.

Regulatory T cell-based immunotherapies in experimental autoimmune myasthenia gravis.

Establishment of tolerance in myasthenia gravis (MG) involves regulatory T (T(reg)) cells. Experimental autoimmune MG (EAMG) in rats is a suitable model for assessing the contribution of T(reg) cells to the immunopathology of the disease and for testing novel T(reg) cell-based treatment modalities. We have studied two immunotherapeutic approaches for targeting of T(reg) cells in myasthenia. By one approach we demonstrated that treatment of sick rats by ex vivo-generated exogenous T(reg) cells derived from healthy donors suppressed EAMG. By a different approach, we aimed at affecting the endogenous T(reg)/Th17 cell balance by targeting IL-6, which has a key role in controlling the equilibrium between pathogenic Th17 and suppressive T(reg) cells. We found that treatment of myasthenic rats by neutralizing anti-IL-6 antibodies shifted this equilibrium in favor of T(reg) cells and led to suppression of EAMG. Our results show that T(reg) cells could serve as potential targets in treating MG patients.

Experimental myasthenia gravis in Aire-deficient mice: a link between Aire and regulatory T cells.

Aire (autoimmune regulator) has a key role in the establishment of tolerance to autoantigens. Aire(-/-) mice present decreased thymic expression of AChR, significantly lower frequencies of regulatory T (T(reg)) cells, and higher expression of Th17 markers, compared to controls. We therefore predicted that Aire(-/-) mice would be more susceptible to induction of experimental autoimmune myasthenia gravis (EAMG). However, when EAMG was induced in young mice, Aire(-/-) mice presented a milder disease that wild-type (WT) controls. In contrast, when EAMG was induced in older mice, Aire(-/-) mice were more severely affected than WT mice. The relative resistance to EAMG in young Aire(-/-) mice correlated with increased numbers of T(reg) cells in their spleens compared to young controls. A similar age-related susceptibility was also observed when EAE was induced in Aire(-/-) mice, suggesting an age-related link among Aire, disease susceptibility, and peripheral T(reg) cells that may be a general feature of autoimmunity.

Blocking of IL-6 suppresses experimental autoimmune myasthenia gravis.

Suppressive regulatory T cells (Treg) and pathogenic T helper 17 (Th17) cells are two lymphocyte subsets with opposing activities in autoimmune diseases. The proinflammatory cytokine IL-6 is a potent factor in switching immune responses in vivo from the induction of Treg to pathogenic Th17 cells. We studied the Treg and Th17 cell compartments in experimental autoimmune myasthenia gravis (EAMG) and healthy control rats in order to assess whether the equilibrium between Treg and Th17 cells is perturbed in the disease. We found that Th17 cell-related genes are upregulated and Treg-related genes are downregulated in EAMG. The shift in favor of Th17 cells in EAMG could be reversed by antibodies to IL-6. Administration of anti-IL-6 antibodies to myasthenic rats suppressed EAMG when treatment started at the acute or at the chronic phase of disease. Suppression of EAMG by anti-IL-6 antibodies was accompanied by a decrease in the overall rat anti-AChR antibody titer and by a reduced number of B cells as compared with control treatment. Administration of anti-IL-6 antibodies led to down-regulation of several Th17 related genes including IL-17, IL-17R, IL-23R and IL-21 but did not affect the number of Treg cells in the lymph nodes. These data identify IL-6 as an important target for modulation of autoimmune responses.

Involvement of phosphodiesterases in autoimmune diseases.

We have previously shown that several phosphodiesterase (PDE) subtypes are up-regulated in muscles and lymph node cells (LNC) of rats with experimental autoimmune myasthenia gravis (EAMG). In the present study we investigated PDE expression during the course of EAMG and experimental allergic encephalomyelitis (EAE) and found that the up-regulated expression of selected PDE subtypes in both experimental models is correlated with disease severity. In EAMG, PDE expression is correlated also with muscle damage. A similar up-regulation of PDE was also observed in the respective human diseases, MG and multiple sclerosis (MS). Our findings suggest that change in PDE expression levels is a general phenomenon in autoimmune diseases and may also be used as a marker for disease severity.

A disease-specific fraction isolated from IVIG is essential for the immunosuppressive effect of IVIG in experimental autoimmune myasthenia gravis.

Intravenous immunoglobulin (IVIG) treatment is beneficially used in autoimmune disorders including myasthenia gravis (MG) although its mode of action and active components are still not fully identified. In an attempt to isolate from IVIG a disease-specific suppressive fraction, IVIG was passed on columns of IgG from rats with experimental autoimmune MG (EAMG) or from MG patients. These chromatographies resulted in depletion of the suppressive activity of IVIG on rat EAMG whereas the minute amounts of IgG fractions eluted from the EAMG- or MG-specific columns retained the immunosuppressive activity of IVIG. These results demonstrate that a minor disease-specific immunoglobulin fraction present in IVIG is essential for its suppressive activity.

DNA microarray in search of new drug targets for myasthenia gravis.

DNA microarray technology was used to identify new potential drug targets for myasthenia gravis (MG), to delineate genes involved in the pathogenesis of the disease and to possibly target their protein products for immunotherapy. In this study we compared the gene expression in lymph node cells (LNC) and muscles of rats with experimental autoimmune MG (EAMG) to those of control, healthy rats. Of the genes that were found to be deregulated in EAMG, we chose to elaborate on two gene systems: (a) The chemokine IFN-gamma-inducible protein 10 (IP-10, CXCL10), and its receptor (CXCR3) and (b) phosphodiesterases.

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.

Vaccines against myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease mediated by antibodies to nicotinic acetylcholine receptor (AChR) interfering with the neuromuscular transmission. Experimental autoimmune MG serves as an excellent animal model to study possible therapeutic modalities for MG. This review will focus on the different ways to turn off the autoimmune response to AChR, which results in suppression of myasthenia. This paper will describe the use of fragments or peptides derived from the AChR, antigen-presenting cells and anti-T cell receptor antibodies, and will discuss the underlying mechanisms of action. Finally, the authors propose new promising therapeutic prospects, including treatment based on the modulation of regulatory T cells, which have recently been found to be functionally defective in MG patients.

Overexpression of IFN-induced protein 10 and its receptor CXCR3 in myasthenia gravis.

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are autoimmune disorders in which the acetylcholine receptor (AChR) is the major autoantigen. Microarray technology was used to identify new potential drug targets for treatment of myasthenia that would reduce the need for the currently used nonspecific immunosuppression. The chemokine IFN-gamma-inducible protein 10 (IP-10; CXCL10), a CXC chemokine, and its receptor, CXCR3, were found to be overexpressed in lymph node cells of EAMG rats. Quantitative real-time PCR confirmed these findings and revealed up-regulated mRNA levels of another chemoattractant that activates CXCR3, monokine induced by IFN-gamma (Mig; CXCL9). TNF-alpha and IL-1beta, which act synergistically with IFN-gamma to induce IP-10, were also up-regulated. These up-regulations were observed in immune response effector cells, namely, lymph node cells, and in the target organ of the autoimmune attack, the muscle of myasthenic rats, and were significantly reduced after suppression of EAMG by mucosal tolerance induction with an AChR fragment. The relevance of IP-10/CXCR3 signaling in myasthenia was validated by similar observations in MG patients. A significant increase in IP-10 and CXCR3 mRNA levels in both thymus and muscle was observed in myasthenic patients compared with age-matched controls. CXCR3 expression in PBMC of MG patients was markedly increased in CD4(+), but not in CD8(+), T cells or in CD19(+) B cells. Our results demonstrate a positive association of IP-10/CXCR3 signaling with the pathogenesis of EAMG in rats as well as in human MG patients.

Immunosuppression of rat myasthenia gravis by oral administration of a syngeneic acetylcholine receptor fragment.

A syngeneic rat recombinant fragment of the extracellular domain of the acetylcholine receptor (AChR) alpha-subunit (Ralpha1-205), administered orally, suppresses ongoing experimental autoimmune myasthenia gravis (EAMG) in rats. The underlying mechanism is a shift from Th1 to Th2 regulation as evidenced by downregulated mRNA expression levels of IFN-gamma and TNF-alpha, upregulated IL-10, changes in anti-AChR IgG isotypes and diminished Th1 signaling via CD28/CTLA-4:B7. Unlike the xenogeneic fragment, the syngeneic Ralpha1-205 does not induce elevation in TGF-beta and elicitation of autoregulatory cells. The ability to suppress EAMG by a non-immunogenic syngeneic fragment of AChR is encouraging and may in the future be applied for the treatment of myasthenia gravis in humans.

The binding site of acetylcholine receptor: from synthetic peptides to solution and crystal structure.

Our group has been employing short synthetic peptides, encompassing sequences from the acetylcholine receptor (AChR) alpha-subunit for the analysis of the binding site of the AChR. A 13-mer peptide mimotope, with similar structural motifs to the AChR binding region, was selected by alpha-bungarotoxin (alpha-BTX) from a phage-display peptide library. The solution structure of a complex between this library-lead peptide and alpha-BTX was solved by NMR spectroscopy. On the basis of this NMR study and on structure-function analysis of the AChR binding site, and in order to obtain peptides with higher affinity to alpha-BTX, additional peptides resulting from systematic residue replacement in the lead peptide were designed and characterized. Of these, four peptides, designated high-affinity peptides (HAPs), homologous to the binding region of the AChR, inhibited the binding of alpha-BTX to the AChR with an IC(50) of 2 nM. The solution and crystal structures of complexes of alpha-BTX with HAP were solved, demonstrating that the HAP fits snugly to alpha-BTX and adopts a beta-hairpin conformation. The X-ray structures of the bound HAP and the homologous loop of the acetylcholine binding protein (AChBP) are remarkably similar. Their superposition results in a model indicating that alpha-BTX wraps around the receptor binding-site loop and, in addition, binds tightly at the interface of two of the receptor subunits, where it inserts a finger into the ligand-binding site. Our proposed model explains the strong antagonistic activity of alpha-BTX and accommodates much of the biochemical data on the mode of interaction of alpha-BTX with the AChR.

Suppression of myasthenia gravis by antigen-specific mucosal tolerance and modulation of cytokines and costimulatory factors.

We have shown that mucosal administration of recombinant fragments corresponding to the human acetylcholine receptor (AChR) alpha subunit suppresses chronic ongoing experimental autoimmune myasthenia gravis (EAMG) in rats. Treated animals exhibit a Th1 to Th2/Th3 shift in their cytokine profile and downregulation of costimulatory factors. However, application of a xenogeneic recombinant fragment may have limitations when considered as a possible approach for the treatment of MG in humans. We therefore tested the potential of a syngeneic fragment and of long synthetic peptides to suppress EAMG. We found that a syngeneic fragment corresponding to the extracellular region of the rat AChR alpha subunit was as effective as the formerly described human xenogeneic fragment in suppressing ongoing EAMG. This is encouraging in view of the potential use of mucosally administered recombinant AChR fragments for the treatment of MG in humans. However, in severely affected individuals, this antigen-specific approach may need to be supported by direct modulation of cytokines and costimulatory factors known to be involved in the pathogenesis of EAMG. To test the potential of this approach, myasthenic rats were injected by antibodies either to the proinflammatory cytokine IL-18 or to the costimulatory factor CD40L. These treatments act via different mechanisms, but both lead to the alleviation of clinical symptoms even when given at the chronic phase of EAMG. We suggest that antagonists to key cytokines and/or costimulatory factors be used to augment antigen-specific treatments of myasthenia such as mucosal administration of AChR recombinant fragments.

The alpha7 nicotinic acetylcholine receptor in schizophrenia: decreased mRNA levels in peripheral blood lymphocytes.

Recent studies have suggested that the alpha7 nicotinic acetylcholine receptor (alpha7 AChR) may play a role in the pathogenesis of schizophrenia. In search for peripheral biological markers for schizophrenia we have investigated alpha7 mRNA levels in peripheral blood lymphocytes (PBLs) of schizophrenic patients and healthy controls. Peripheral blood samples were collected from medicated and non-medicated (drug naive) schizophrenic patients as well as from healthy (non-mentally ill) smokers and non-smokers. RNA was prepared from isolated lymphocytes. Polymerase chain reaction products specific for human alpha7 AChR were quantified by densitometry using Scion image-analysis (shared NIH software). We observed a significant decrease of alpha7 mRNA levels on PBLs of schizophrenic patients compared with controls. The decrease in alpha7 mRNA levels was not a result of medication management, because non-medicated schizophrenic patients displayed the same level of reduction in alpha7 mRNA as did patients receiving medication. In addition, we exclude the possibility that the observed decrease in alpha7 mRNA levels resulted from nicotine consumption in smoking, because healthy smokers exhibited the same levels of alpha7 mRNA as non-smokers. We propose that alpha7 AChR may be involved in the pathophysiology of the disease and may serve as a reliable peripheral biological marker in schizophrenia.

Design and synthesis of peptides that bind alpha-bungarotoxin with high affinity and mimic the three-dimensional structure of the binding-site of acetylcholine receptor.

Alpha-bungarotoxin (alpha-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind alpha-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the alpha-BTX/AChR system yielded 13-mer peptides that bind to alpha-BTX with high affinity and are potent inhibitors of alpha-BTX binding to AChR (IC(50) of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel beta-hairpin structure when bound to alpha-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.

Coupling of dopamine receptors to G proteins: studies with chimeric D2/D3 dopamine receptors.

D2 and D3 dopamine receptors belong to the superfamily of G protein-coupled receptors; they share a high degree of homology and are structurally similar. However, they differ from each other in their second messenger coupling properties. Previously, we have studied the differential coupling of these receptors to G proteins and found that while D2 receptor couples only to inhibitory G proteins, D3 receptor couples also to a stimulatory G protein, Gs. We aimed to investigate the molecular basis of these differences and to determine which domains in the receptor control its coupling to G proteins. For this purpose four chimeras were constructed, each composed of different segments of the original D2 and D3 receptors. We have demonstrated that chimeras with a third cytoplasmic loop of D2 receptor couple to Gi protein in a pattern characteristic of D2 receptor. On the other hand chimeras containing a third cytoplasmic loop of D3 receptor have coupling characteristics like those of D3 receptor, and they couple also to Gs protein. These findings demonstrate that the third cytoplasmic loop determines and accounts for the coupling of dopamine receptors D2 and D3 to G proteins.

betagamma Dimers mediate synergy of dopamine D2 and adenosine A2 receptor-stimulated PKA signaling and regulate ethanol consumption.

Dopamine release is activated by ethanol and addicting drugs, but molecular mechanisms linking dopaminergic signaling to neuronal responses and drinking behavior are poorly understood. We report that dopamine-D2 receptors induce PKA Calpha translocation and increase CRE-regulated gene expression. Ethanol also activates PKA signaling. Subthreshold concentrations of the D2 agonist NPA and ethanol, without effect alone, together cause synergistic PKA translocation and CRE-mediated gene transcription. D2 or adenosine A2 receptor blockade, pertussis toxin, Rp-cAMPS, or overexpression of dominant-negative peptides that sequester betagamma dimers prevent synergy. Importantly, overexpression of a betagamma inhibitor peptide in the nucleus accumbens strikingly reduces sustained alcohol consumption. We propose that synergy of D2 and A2 confers ethanol hypersensitivity and that betagamma dimers are required for voluntary drinking.