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.

Suppression of experimental autoimmune myasthenia gravis by autologous T regulatory cells.

Adoptive transfer of regulatory T (Treg) cells have been employed effectively for suppression of several animal models for autoimmune diseases. In order to employ Treg cell therapy in patients, it is necessary to generate Treg cells from the patient's own cells (autologous) that would be able to suppress effectively the disease in vivo, upon their reintroduction to the patient. Towards this objective, we report in the present study on a protocol for a successful immune-regulation of experimental autoimmune myasthenia gravis (EAMG) by ex vivo--generated autologous Treg cells. For this protocol bone marrow (BM) cells, are first cultured in the presence of GM-CSF, giving rise to a population of CD11c(+)MHCII(+)CD45RA(+)CD8(-) DCs (BMDCs). Splenic CD4(+) T cells are then co-cultured with the differentiated BM cells and expand to 90% of Foxp3(+) Treg cells. In vitro assay exhibits a similar dose dependent manner in the suppression of T effector cells proliferation between Treg cells obtained from either healthy or sick donors. In addition, both Treg cells inhibit similarly the secretion of IFN-γ from activated splenocytes. Administration of 1 × 10(6) ex-vivo generated Treg cells, I.V, to EAMG rats, modulates the disease following a single treatment, given 3 days or 3 weeks after disease induction. Similar disease inhibition was achieved when CD4 cells were taken from either healthy or sick donors. The disease suppression was accompanied by reduced levels of total AChR specific antibodies in the serum. Moreover, due to the polyclonality of the described Treg cell, we have examined whether this treatment approach could be also employed for the treatment of other autoimmune diseases involving Treg cells. Indeed, we demonstrated that the ex-vivo generated autologous Treg cells suppress Adjuvant Arthritis (AA) in rats. This study opens the way for the application of induced autologous Treg cell therapy for myasthenia gravis, as well as for other human autoimmune diseases involving Treg cells.

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.

Experimental Autoimmune Myasthenia Gravis (EAMG): from immunochemical characterization to therapeutic approaches.

Myasthenia Gravis (MG) is an organ-specific autoimmune disease. In high percentage of patients there are autoantibodies to the nicotinic acetylcholine receptor (AChR) that attack AChR on muscle cells at the neuromuscular junction, resulting in muscle weakness. Experimental Autoimmune Myasthenia Gravis (EAMG) is an experimental model disease for MG. EAMG is induced in several animal species by immunization with acetylcholine receptor (AChR), usually isolated from the electric organ of electric fish, which is a rich source for this antigen. Our lab has been involved for several decades in research of AChR and of EAMG. The availability of an experimental autoimmune disease that mimics in many aspects the human disease, provides an excellent model system for elucidating the immunological nature and origin of MG, for studying various existing treatment modalities and for attempting the development of novel treatment approaches. In this review in honor of Michael Sela and Ruth Arnon, we report first on our early pioneering contributions to research on EAMG. These include the induction of EAMG in several animal species, early attempts for antigen-specific treatment for EAMG, elicitation and characterization of monoclonal antibodies and anti-idiotypic antibodies, measuring humoral and cellular AChR-specific immune responses in MG patient and more. In the second part of the review we discuss more recent studies from our lab towards developing and testing novel treatment approaches for myasthenia. These include antigen-dependent treatments aimed at specifically abrogating the humoral and cellular anti-AChR responses, as well as immunomodulatory approaches that could be used either alone, or in conjunction with antigen-specific treatments, or alternatively, serve as steroid-sparing agents.

Resistance of LPS-activated bone marrow derived macrophages to apoptosis mediated by dexamethasone.

Glucocorticoids (GC) display pleiotropic effects on the immune system. Macrophages are a major target for GC action. Here we show that dexamethasone (DEX), a synthetic GC, decreased viability of naïve bone marrow-derived macrophages (BMDM), involving an apoptotic mechanism. Administration of DEX together with lipopolysaccharide (LPS) protected BMDM against DEX-mediated cell death, suggesting that activated BMDM respond to DEX differently than naïve BMDM. An insight to the molecular basis of LPS actions was provided by a 7 fold increase in mRNA levels of glucocorticoid receptor beta (GRß), a GR dominant-negative splice variant which inhibits GRα's transcriptional activity. LPS did not inhibit all DEX-mediated effects on BMDM; DEX significantly reduced the percentage of BMDM expressing high levels of the cell surface markers F4/80 and CD11b and led to a decrease in macrophage inflammatory protein 1 alpha (MIP1-α) mRNA and protein levels. These two DEX-mediated effects were not prevented by LPS. Our finding that LPS did not reduce the DEX-induced elevation of glucocorticoid-induced leucine zipper (GILZ), a mediator of GCs anti-inflammatory actions, may provide an underlying mechanism. These findings enable a better understanding of clinical states, such as sepsis, in which macrophages are activated by endotoxins and treatment by GCs is considered.

Age dependent course of EAE in Aire-/- mice.

This study explores the consequences of deficiency in the autoimmune regulator (Aire) on the susceptibility to experimental autoimmune encephalomyelitis (EAE). Increased susceptibility to EAE was found in Aire knockout (KO) compared to wild type (WT) in 6month old mice. In contrast, 2month old Aire KO mice were less susceptible to EAE than WT mice, and this age-related resistance correlated with elevated proportions of T regulatory (Treg) cells in their spleen and brain. Combined with our previous findings in experimental autoimmune myasthenia gravis, we suggest an age-related association between Aire and Treg cells in the susceptibility to autoimmunity.

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.

The susceptibility of Aire(-/-) mice to experimental myasthenia gravis involves alterations in regulatory T cells.

The autoimmune regulator (Aire) is involved in the prevention of autoimmunity by promoting thymic expression of tissue restricted antigens which leads to elimination of self-reactive T cells. We found that Aire knockout (KO) mice as well as mouse strains that are susceptible to experimental autoimmune myasthenia gravis (EAMG) have lower thymic expression of acetylcholine receptor (AChR- the main autoantigen in MG), compared to wild type (WT) mice and EAMG-resistant mouse strains, respectively. We demonstrated that Aire KO mice have a significant and reproducible lower frequency of CD4+Foxp3+ cells and a higher expression of Th17 markers in their thymus, compared to wild type (WT) mice. These findings led us to expect that Aire KO mice would display increased susceptibility to EAMG. Surprisingly, when EAMG was induced in young (2 month-old) mice, EAMG was milder in Aire KO than in WT mice for several weeks until the age of about 5 months. However, when EAMG was induced in relatively aged (6 month-old) mice, Aire KO mice presented higher disease severity than WT controls. This age-related change in susceptibility to EAMG correlated with an elevated proportion of Treg cells in the spleens of young but not old KO, compared to WT mice, suggesting a role for peripheral Treg cells in the course of disease. Our observations point to a possible link between Aire and Treg cells and suggest an involvement for both in the pathogenesis of myasthenia.

Insertion of an NPVY sequence into the cytosolic domain of the erythropoietin receptor selectively affects erythropoietin-mediated signalling and function.

EPO (erythropoietin), the major hormone regulating erythropoiesis, functions via activation of its cell-surface receptor (EPO-R) present on erythroid progenitor cells. One of the most striking properties of EPO-R is its low expression on the cell surface, as opposed to its high intracellular levels. The low cell-surface expression of EPO-R may thus limit the efficacy of EPO that is routinely used to treat primary and secondary anaemia. In a recent study [Nahari, Barzilay, Hirschberg and Neumann (2008) Biochem. J. 410, 409-416] we have shown that insertion of an NPVY sequence into the intracellular domain of EPO-R increases its cell-surface expression. In the present study we demonstrate that this NPVY EPO-R insert has a selective effect on EPO-mediated downstream signalling in Ba/F3 cells expressing this receptor (NPVY-EPO-R). This is monitored by increased phosphorylation of the NPVY-EPO-R (on Tyr479), Akt, JAK2 (Janus kinase 2) and ERK1/2 (extracellular-signal-regulated kinase 1/2), but not STAT5 (signal transducer and activator of transcription 5), as compared with cells expressing wild-type EPO-R. This enhanced signalling is reflected in augmented proliferation at low EPO levels (0.05 units/ml) and protection against etoposide-induced apoptosis. Increased cell-surface levels of NPVY-EPO-R are most probably not sufficient to mediate these effects as the A234E-EPO-R mutant that is expressed at high cell-surface levels does not confer an augmented response to EPO. Taken together, we demonstrate that insertion of an NPVY sequence into the cytosolic domain of the EPO-R confers not only improved maturation, but also selectively affects EPO-mediated signalling resulting in an improved responsiveness to EPO reflected in cell proliferation and protection against apoptosis.

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.

Suppression of experimental autoimmune myasthenia gravis by inhibiting the signaling between IFN-gamma inducible protein 10 (IP-10) and its receptor CXCR3.

We have previously demonstrated that the chemokine IFN-gamma inducible protein 10 (IP-10) and its receptor CXCR3, are overexpressed in myasthenia gravis (MG) and its animal model experimental autoimmune MG (EAMG). We now studied the potential of modulating rat EAMG by interference in CXCR3/IP-10 signaling. Two different approaches were used: 1) blocking IP-10 by IP-10-specific antibodies and 2) inhibiting the CXCR3 chemokine receptor by a CXCR3 antagonist. Treatment by either of these reagents led to suppression of EAMG suggesting that inhibition of CXCR3/IP-10 signaling can be considered as a potential treatment modality for MG.

Suppression of experimental myasthenia gravis by a B-cell epitope-free recombinant acetylcholine receptor.

Myasthenia gravis (MG) and experimental autoimmune MG (EAMG) are antibody-mediated autoimmune diseases in which the nicotinic acetylcholine receptor (AChR) is the major autoantigen. Previously we have revealed that oral treatment with the less native recombinant fragment of the extracellular domain of the human AChR (Halpha1-205) suppressed ongoing EAMG, whereas the more native recombinant Trx-Halpha1-210 exacerbated EAMG. In this study, we speculated on the role of B-cell epitopes in oral tolerogens for the induction of oral tolerance in EAMG. We developed a B-cell epitope-free AChR fragment (BF-AChR) by removing two major B-cell epitopes (67-76 and 129-145) from Trx-Halpha1-210. BF-AChR exhibited a poor response to EAMG sera and to AChR-specific B- and T-cells while its parent fragment, Trx-Halpha1-210, showed much higher reactivity. Oral administration of BF-AChR ameliorated the symptoms in ongoing myasthenic rats accompanied by a significant decrease in AChR-specific humoral and Th1 cellular responses. The underlying mechanism for BF-AChR-induced oral tolerance was mediated by a shift from Th1 to regulatory T-cell (IL-10(+), CD4(+) TGF-beta(+) or Foxp3(+)) responses. This shift was assessed by changes in the cytokine profile and a deviation in the anti-AChR IgG isotypes from IgG2a/IgG2b to IgG1. Our results suggest that the removal of pathogenic B-cell epitopes from AChR fragments increases tolerogenicity by reducing the activation and proliferation of autoreactive B- and T-cells. Collectively, careful consideration of the immunogenicity of a tolerogen is necessary to induce successful oral tolerance in autoimmune disorders.

Suppression of experimental autoimmune myasthenia gravis by combination therapy: pentoxifylline as a steroid-sparing agent.

Myasthenia gravis (MG) is frequently treated by corticosteroids such as methylprednisolone. However, continuous treatment with steroids often results in adverse effects. In the present study we evaluated the therapeutic potential of a combination of suboptimal doses of methylprednisolone (Solumedrol) and Pentoxifylline (PTX), a general phosphodiesterase (PDE) inhibitor, in rat experimental autoimmune MG (EAMG). This combined treatment resulted in a pronounced suppressive effect on EAMG and was by far more effective than each of the drugs administered separately at these low doses. The suppressive effect on EAMG was accompanied by decreased humoral and cellular responses to AChR as well as down-regulated mRNA expression levels of Th1 cytokines and IL-10 in lymph node cells and of PDE-4 and cathepsin-l in the muscle. This study demonstrates the potential of PTX as a steroid-sparing agent in the management of myasthenia gravis.

Immunosuppression of EAMG by IVIG is mediated by a disease-specific anti-immunoglobulin fraction.

Intravenous immunoglobulin (IVIG) administration has been beneficially used for the treatment of a variety of autoimmune diseases including myasthenia gravis (MG). We have demonstrated that IVIG administration in experimental autoimmune MG (EAMG) results in suppression of disease that is accompanied by decreased Th1 cell and B cell proliferation. Chromatography of pooled human immunoglobulins (IVIG) on immobilized IgG, isolated from rats with EAMG or from MG patients, results in a depletion of the suppressive activity of the IVIG. Moreover, reconstitution of the activity-depleted IVIG with the eluted minute IVIG fractions that had been adsorbed onto the EAMG- or MG-specific columns recovers the depleted immunosuppressive activity. This study supports the notion that the therapeutic effect of IVIG is mediated by an antigen-specific anti-immunoglobulin (anti-idiotypic) activity that is essential for its suppressive activity.