Impaired cerebral microvascular endothelial cells integrity due to elevated dopamine in myasthenic model.

Myasthenia gravis is an autoimmune disease characterized by pathogenic antibodies that target structures of the neuromuscular junction. However, some patients also experience autonomic dysfunction, anxiety, depression, and other neurological symptoms, suggesting the complex nature of the neurological manifestations. With the aim of explaining the symptoms related to the central nervous system, we utilized a rat model to investigate the impact of dopamine signaling in the central nervous and peripheral circulation. We adopted several screening methods, including western blot, quantitative PCR, mass spectrum technique, immunohistochemistry, immunofluorescence staining, and flow cytometry. In this study, we observed increased and activated dopamine signaling in both the central nervous system and peripheral circulation of myasthenia gravis rats. Furthermore, changes in the expression of two key molecules, Claudin5 and CD31, in endothelial cells of the blood-brain barrier were also examined in these rats. We also confirmed that dopamine incubation reduced the expression of ZO1, Claudin5, and CD31 in endothelial cells by inhibiting the Wnt/ß-catenin signaling pathway. Overall, this study provides novel evidence suggesting that pathologically elevated dopamine in both the central nervous and peripheral circulation of myasthenia gravis rats impair brain-blood barrier integrity by inhibiting junction protein expression in brain microvascular endothelial cells through the Wnt/ß-catenin pathway.

An angel or a devil? Current view on the role of CD8+ T cells in the pathogenesis of myasthenia gravis.

BACKGROUND: Myasthenia gravis (MG) and the experimental autoimmune MG (EAMG) animal model are characterized by T-cell-induced and B-cell-dominated autoimmune diseases that affect the neuromuscular junction. Several subtypes of CD4+ T cells, including T helper (Th) 17 cells, follicular Th cells, and regulatory T cells (Tregs), contribute to the pathogenesis of MG. However, increasing evidence suggests that CD8+ T cells also play a critical role in the pathogenesis and treatment of MG. MAIN BODY: Herein, we review the literature on CD8+ T cells in MG, focusing on their potential effector and regulatory roles, as well as on relevant evidence (peripheral, in situ, cerebrospinal fluid, and under different treatments), T-cell receptor usage, cytokine and chemokine expression, cell marker expression, and Treg, Tc17, CD3+CD8+CD20+ T, and CXCR5+ CD8+ T cells. CONCLUSIONS: Further studies on CD8+ T cells in MG are necessary to determine, among others, the real pattern of the Vß gene usage of autoantigen-specific CD8+ cells in patients with MG, real images of the physiology and function of autoantigen-specific CD8+ cells from MG/EAMG, and the subset of autoantigen-specific CD8+ cells (Tc1, Tc17, and IL-17+IFN-γ+CD8+ T cells). There are many reports of CD20-expressing T (or CD20 + T) and CXCR5+ CD8 T cells on autoimmune diseases, especially on multiple sclerosis and rheumatoid arthritis. Unfortunately, up to now, there has been no report on these T cells on MG, which might be a good direction for future studies.

BMSCs-Derived Extracellular VesiclemiR-29a-3p Improved the Stability of Rat Myasthenia Gravis by Regulating Treg/Th17 Cells.

INTRODUCTION: Myasthenia gravis (MG) is an autoimmune disorder. Microvesicle-derived miRNAs have been implicated in autoimmune diseases. However, the role of microvesicle-derived miR-29a-3p in MG remains poorly understood. This study aimed to investigate the therapeutic effect and mechanism of miR-29a-3p derived from stem cell microvesicles (MVs) on experimental autoimmune myasthenia gravis (EAMG) rats. METHODS: EAMG was induced in rats by injection of the subunit of the rat nicotinic anti-acetylcholine receptor (AChR) R97-116 peptide.Besides the control group, EAMG rats were randomly allocated into the EAMG model group, MV group, MV-NC-agomir group, and MV- miR-29a-3p-agomir group. RESULTS: Our results found that BMSCs-MV promoted miR-29a-3p expression in gastrocnemius of EAMG rats. Bone marrow mesenchymal stem cells (BMSCs) derived microvesicle miR-29a-3p improved the hanging ability and swimming time of EMGA rats and weakened the degree of muscle fiber atrophy. Furthermore, microvesicles from miR-29a-3p overexpressing BMSCs reduced the content of AchR-Ab in the serum of EAMG rats. BMSC-derived microvesicle miR-29a-3p further suppressed the expression of IFN-γ and enhanced the IL-4 and IL-10 in the serum of EAMG rats by restoring the Th17/Treg cells balance. DISCUSSION: BMSCs-derived microvesicle miR-29a-3p improved the stability of rat myasthenia gravis by regulating Treg/Th17 cells. It may be an effective treatment for MG.

Decreased expression of miR-29 family associated with autoimmune myasthenia gravis.

BACKGROUND: Myasthenia gravis (MG) is a rare autoimmune disease mainly mediated by autoantibodies against the acetylcholine receptor (AChR) at the neuromuscular junction. The thymus is the effector organ, and its removal alleviates the symptoms of the disease. In the early-onset form of MG, the thymus displays functional and morphological abnormalities such as B cell infiltration leading to follicular hyperplasia, and the production of AChR antibodies. Type-I interferon (IFN-I), especially IFN-ß, is the orchestrator of thymic changes observed in MG. As Dicer and miR-29 subtypes play a role in modulating the IFN-I signalization in mouse thymus, we investigated their expression in MG thymus. METHODS: The expression of DICER and miR-29 subtypes were thoroughly investigated by RT-PCR in human control and MG thymuses, and in thymic epithelial cells (TECs). Using miR-29a/b-1-deficient mice, with lower miR-29a/b-1 expression, we investigated their susceptibility to experimental autoimmune MG (EAMG) as compared to wild-type mice. RESULTS: DICER mRNA and all miR-29 subtypes were down-regulated in the thymus of MG patients and DICER expression was correlated with the lower expression of miR-29a-3p. A decreased expression of miR-29 subtypes was similarly observed in MG TECs; a decrease also induced in TECs upon IFN-ß treatment. We demonstrated that miR-29a/b-1-deficient mice were more susceptible to EAMG without higher levels of anti-AChR IgG subtypes. In the thymus, if no B cell infiltration was observed, an increased expression of Ifn-ß associated with Baff expression and the differentiation of Th17 cells associated with increased expression of Il-6, Il-17a and Il-21 and decreased Tgf-ß1 mRNA were demonstrated in miR-29a/b-1-deficient EAMG mice. CONCLUSIONS: It is not clear if the decreased expression of miR-29 subtypes in human MG is a consequence or a causative factor of thymic inflammation. However, our results from the EAMG mouse model indicated that a reduction in miR-29a/b1 may contribute to the pathophysiological process involved in MG by favoring the increased expression of IFN-ß and the emergence of pro-inflammatory Th17 cells.

Exosomes derived from statin-modified bone marrow dendritic cells increase thymus-derived natural regulatory T cells in experimental autoimmune myasthenia gravis.

BACKGROUND: The thymus plays an essential role in the pathogenesis of myasthenia gravis (MG). In patients with MG, natural regulatory T cells (nTreg), a subpopulation of T cells that maintain tolerance to self-antigens, are severely impaired in the thymuses. In our previous study, upregulated nTreg cells were observed in the thymuses of rats in experimental autoimmune myasthenia gravis after treatment with exosomes derived from statin-modified dendritic cells (statin-Dex). METHODS: We evaluated the effects of exosomes on surface co-stimulation markers and Aire expression of different kinds of thymic stromal cells, including cTEC, mTEC, and tDCs, in EAMG rats. The isolated exosomes were examined by western blot and DLS. Immunofluorescence was used to track the exosomes in the thymus. Flow cytometry and western blot were used to analyze the expression of co-stimulatory molecules and Aire in vivo and in vitro. RESULTS: We confirmed the effects of statin-Dex in inducing Foxp3+ nTreg cells and found that both statin-Dex and DMSO-Dex could upregulate CD40 but only statin-Dex increased Aire expression in thymic stromal cells in vivo. Furthermore, we found that the role of statin-Dex and DMSO-Dex in the induction of Foxp3+ nTreg cells was dependent on epithelial cells in vitro. CONCLUSIONS: We demonstrated that statin-Dex increased expression of Aire in the thymus, which may further promote the Foxp3 expression in the thymus. These findings may provide a new strategy for the treatment of myasthenia gravis.

CXCR5-negative natural killer cells ameliorate experimental autoimmune myasthenia gravis by suppressing follicular helper T cells.

BACKGROUND: Recent studies have demonstrated that natural killer (NK) cells can modulate other immune components and are involved in the development or progression of several autoimmune diseases. However, the roles and mechanisms of NK cells in regulating experimental autoimmune myasthenia gravis (EAMG) remained to be illustrated. METHODS: To address the function of NK cells in experimental autoimmune myasthenia gravis in vivo, EAMG rats were adoptively transferred with splenic NK cells. The serum antibodies, and splenic follicular helper T (Tfh) cells and germinal center B cells were determined by ELISA and flow cytometry. The roles of NK cells in regulating Tfh cells were further verified in vitro by co-culturing splenocytes or isolated T cells with NK cells. Moreover, the phenotype, localization, and function differences between different NK cell subtypes were determined by flow cytometry, immunofluorescence, and ex vivo co-culturation. RESULTS: In this study, we found that adoptive transfer of NK cells ameliorated EAMG symptoms by suppressing Tfh cells and germinal center B cells. Ex vivo studies indicated NK cells inhibited CD4+ T cells and Tfh cells by inducing the apoptosis of T cells. More importantly, NK cells could be divided into CXCR5- and CXCR5+ NK subtypes according to the expression of CXCR5 molecular. Compared with CXCR5- NK cells, which were mainly localized outside B cell zone, CXCR5+ NK were concentrated in the B cell zone and exhibited higher expression levels of IL-17 and ICOS, and lower expression level of CD27. Ex vivo studies indicated it was CXCR5- NK cells not CXCR5+ NK cells that suppressed CD4+ T cells and Tfh cells. Further analysis revealed that, compared with CXCR5- NK cells, CXCR5+ NK cells enhanced the ICOS expression of Tfh cells. CONCLUSIONS: These findings highlight the different roles of CXCR5- NK cells and CXCR5+ NK cells. It was CXCR5- NK cells but not CXCR5+ NK cells that suppressed Tfh cells and inhibited the autoimmune response in EAMG models.

Exogenous IL-9 Ameliorates Experimental Autoimmune Myasthenia Gravis Symptoms in Rats.

Interleukin-9 (IL-9) is a multifunctional cytokine involved in protective immunity or immunopathology depending on the microenvironment and specific disease settings. Our early study determined that IL-9 and Th9 cells participate in and promote the progression of experimental autoimmune myasthenia gravis (EAMG). The data from this study showed that exogenous recombinant rat IL-9 (rrIL-9) acted as an IL-9 receptor antagonist, reduced the incidence of EAMG in rats, alleviated the severity of the disease, and reduced the anti-acetylcholine receptor (AChR) IgG antibody levels by altering the Th-subset distribution. These data suggest that administration of rrIL-9 may provide a novel therapeutic strategy against MG or related autoimmune diseases. Abbreviations: 2-Mercaptoethanol (2-ME); antibodies (Abs); ?-bungarotoxin (?-BTX); acetylcholine receptor (AChR); airway hyper-reactivity (AHR); allophycocyanin-conjugated (APC); antigen presenting cells (APCs); complete Freund's adjuvant (CFA); Cyanine dye 3 (Cy3); dendritic cells (DCs); experimental autoimmune encephalomyelitis (EAE); experimental autoimmune myasthenia gravis (EAMG); flow cytometry (FACS); fetal bovine serum (FBS); fetal calf serum (FCS); Fluorescein isothiocyanate (FITC); gamma chain (?c); intraperitoneally (i.p.); Incomplete Freund's adjuvant (IFA); interferon (IFN); immunoglobulin (Ig); Interleukin (IL); Janus kinase (JAK); myasthenia gravis (MG); Mononuclear cells (MNC); neuromuscular junctions (NMJ); optical density (OD); ovalbumin (OVA); phosphate-buffered saline (PBS); phycoerythrin (PE); Peridinin chlorophyll protein complex (Percp); Rat AChR ? subunit (R-AChR97-116); Recombinant Rat (rr); room temperature (RT); signal transducer and activator of transcription (STAT); T helper cells (Th).

Muscle satellite cells are functionally impaired in myasthenia gravis: consequences on muscle regeneration.

Myasthenia gravis (MG) is a neuromuscular disease caused in most cases by anti-acetyl-choline receptor (AChR) autoantibodies that impair neuromuscular signal transmission and affect skeletal muscle homeostasis. Myogenesis is carried out by muscle stem cells called satellite cells (SCs). However, myogenesis in MG had never been explored. The aim of this study was to characterise the functional properties of myasthenic SCs as well as their abilities in muscle regeneration. SCs were isolated from muscle biopsies of MG patients and age-matched controls. We first showed that the number of Pax7+ SCs was increased in muscle sections from MG and its experimental autoimmune myasthenia gravis (EAMG) mouse model. Myoblasts isolated from MG muscles proliferate and differentiate more actively than myoblasts from control muscles. MyoD and MyoG were expressed at a higher level in MG myoblasts as well as in MG muscle biopsies compared to controls. We found that treatment of control myoblasts with MG sera or monoclonal anti-AChR antibodies increased the differentiation and MyoG mRNA expression compared to control sera. To investigate the functional ability of SCs from MG muscle to regenerate, we induced muscle regeneration using acute cardiotoxin injury in the EAMG mouse model. We observed a delay in maturation evidenced by a decrease in fibre size and MyoG mRNA expression as well as an increase in fibre number and embryonic myosin heavy-chain mRNA expression. These findings demonstrate for the first time the altered function of SCs from MG compared to control muscles. These alterations could be due to the anti-AChR antibodies via the modulation of myogenic markers resulting in muscle regeneration impairment. In conclusion, the autoimmune attack in MG appears to have unsuspected pathogenic effects on SCs and muscle regeneration, with potential consequences on myogenic signalling pathways, and subsequently on clinical outcome, especially in the case of muscle stress.

ROCK inhibitor abolishes the antibody response in experimental autoimmune myasthenia gravis.

The Rho/Rho kinase (ROCK) pathway serves as molecular switches in many biological processes including the immune response. ROCK inhibitors lead to amelioration of some autoimmune diseases. The present study was designed to define whether a selective ROCK inhibitor, fasudil, was effective in experimental autoimmune myasthenia gravis (EAMG) and investigate the underlying mechanisms. Here we found fasudil effectively attenuated the development of ongoing EAMG. Fasudil abolished the antibody production and function by decreasing follicular helper T cells and CD19(+) B cells, especially germinal center B cells. Moreover, fasudil reduced the expression of CD80 on lymph node mononuclear cells. These findings suggest the inhibition of ROCK might be a potential therapeutic strategy for antibody-mediated autoimmune diseases.

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 ß/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.

Statin-modified dendritic cells regulate humoral immunity in experimental autoimmune myasthenia gravis.

We previously demonstrated that atorvastatin induced immature dendritic cells (DCs) derived from spleen in vitro. Administration of these tolerogenic DCs led to amelioration of experimental autoimmune myasthenia gravis (EAMG). The protective effect was mainly mediated by inhibited cellular immune response, including up-regulated regulatory T cells and shifted Th1/Th17 to Th2 cytokines. The present study employed atorvastatin-modified bone marrow-derived DCs (AT-BMDCs) to explore the effect of tolerogenic DCs on humoral immune response of EAMG and further elucidate the underlying mechanisms. Our data showed that AT-BMDCs reduced the quantity and the relative affinity of pathogenic antibodies, suppressed germinal center response, decreased follicular helper T cells and IL-21, and increased regulatory B cells. These results suggest that AT-BMDCs ameliorate EAMG by regulating humoral immune response, thus providing new insights into therapeutic approaches of myasthenia gravis and other autoimmune diseases.

Silencing miR-146a influences B cells and ameliorates experimental autoimmune myasthenia gravis.

MicroRNAs have been shown to be important regulators of immune homeostasis as patients with aberrant microRNA expression appeared to be more susceptible to autoimmune diseases. We recently found that miR-146a was up-regulated in activated B cells in response to rat acetylcholine receptor (AChR) α-subunit 97-116 peptide, and this up-regulation was significantly attenuated by AntagomiR-146a. Our data also demonstrated that silencing miR-146a with its inhibitor AntagomiR-146a effectively ameliorated clinical myasthenic symptoms in mice with ongoing experimental autoimmune myasthenia gravis. Furthermore, multiple defects were observed after miR-146a was knocked down in B cells, including decreased anti-R97-116 antibody production and class switching, reduced numbers of plasma cells, memory B cells and B-1 cells, and weakened activation of B cells. Previously, miR-146a has been identified as a nuclear factor-κB-dependent gene and predicted to base pair with the tumour necrosis factor receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1) genes to regulate the immune response. However, our study proved that miR-146a inhibition had no effect on the expression of TRAF6 and IRAK1 in B cells. This result suggests that the function of miR-146a in B cells does not involve these two target molecules. We conclude that silencing miR-146a exerts its therapeutic effects by influencing the B-cell functions that contribute to the autoimmune pathogenesis of myasthenia gravis.

Neutralization of interleukin-9 ameliorates symptoms of experimental autoimmune myasthenia gravis in rats by decreasing effector T cells and altering humoral responses.

Interleukin-9 (IL-9) was initially thought to be a type 2 T helper (Th2)-associated cytokine involved in the regulation of autoimmune responses by affecting multiple cell types. However, it was recently shown that IL-9-producing CD4+ T cells represent a discrete subset of Th cells, designated Th9 cells. Although Th9 cells have been shown to be important in many diseases, their roles in myasthenia gravis (MG) are unclear. The aim of this study was to determine whether IL-9 and Th9 cells promote the progression of experimental autoimmune myasthenia gravis (EAMG). The results showed that the percentage of Th9 cells changed during the progression of EAMG, accompanied by an up-regulation of IL-9. Blocking IL-9 activity with antibodies against IL-9 inhibited EAMG-associated pathology in rats and reduced serum anti-acetylcholine receptor IgG levels. Neutralization of IL-9 altered the Th subset distribution in EAMG, reducing the number of Th1 cells and increasing the number of regulatory T cells. Administration of an anti-IL-9 antibody may represent an effective therapeutic strategy for MG-associated pathologies or other T-cell- or B-cell-mediated autoimmune diseases.

Preferential production of IgG1, IL-4 and IL-10 in MuSK-immunized mice.

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness associated with acetylcholine receptor (AChR), muscle-specific receptor kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4)-antibodies. MuSK-antibodies are predominantly of the non-complement fixing IgG4 isotype. The MuSK associated experimental autoimmune myasthenia gravis (EAMG) model was established in mice to investigate immunoglobulin (Ig) and cytokine responses related with MuSK immunity. C57BL/6 (B6) mice immunized with 30µg of recombinant human MuSK in incomplete or complete Freund's adjuvant (CFA) showed significant EAMG susceptibility (>80% incidence). Although mice immunized with 10µg of MuSK had lower EAMG incidence (14.3%), serum MuSK-antibody levels were comparable to mice immunized with 30µg MuSK. While MuSK immunization stimulated production of all antibody isotypes, non-complement fixing IgG1 was the dominant anti-MuSK Ig isotype in both sera and neuromuscular junctions. Moreover, MuSK immunized IgG1 knockout mice showed very low serum MuSK-antibody levels. Sera and MuSK-stimulated lymph node cell supernatants of MuSK immunized mice showed significantly higher levels of IL-4 and IL-10 (but not IFN-γ and IL-12), than those of CFA immunized mice. Our results suggest that through activation of Th2-type cells, anti-MuSK immunity promotes production of IL-4, which in turn activates anti-MuSK IgG1, the mouse analog of human IgG4. These findings might provide clues for the pathogenesis of other IgG4-related diseases as well as development of disease specific treatment methods (e.g. specific IgG4 inhibitors) for MuSK-related MG.

Delivery of an miR155 inhibitor by anti-CD20 single-chain antibody into B cells reduces the acetylcholine receptor-specific autoantibodies and ameliorates experimental autoimmune myasthenia gravis.

MicroRNA-155 (miR155) is required for antibody production after vaccination with attenuated Salmonella. miR155-deficient B cells generated reduced germinal centre responses and failed to produce high-affinity immunoglobulin (Ig)G1 antibodies. In this study, we observed up-regulation of miR155 in the peripheral blood mononuclear cells (PBMCs) of patients with myasthenia gravis (MG), and miR155 was also up-regulated in torpedo acetylcholine receptor (T-AChR)-stimulated B cells. We used an inhibitor of miR155 conjugated to anti-CD20 single-chain antibody to treat both the cultured B cells and the experimental autoimmune MG (EAMG) mice. Our results demonstrated that silencing of miR155 by its inhibitor impaired the B cell-activating factor (BAFF)-R-related signalling pathway and reduced the translocation of nuclear factor (NF)-κB into the nucleus. Additionally, AChR-specific autoantibodies were reduced, which may be related to the altered amounts of marginal zone B cells and memory B cells in the spleens of EAMG mice. Our study suggests that miR155 may be a promising target for the clinical therapy of MG.

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.

Atorvastatin-modified dendritic cells in vitro ameliorate experimental autoimmune myasthenia gravis by up-regulated Treg cells and shifted Th1/Th17 to Th2 cytokines.

Conventional therapies for autoimmune diseases produce nonspecific immune suppression, which are usually continued lifelong to maintain disease control, and associated with a variety of adverse effects. In this study, we found that spleen-derived dendritic cells (DCs) from the ongoing experimental autoimmune myasthenia gravis (EAMG) rats can be induced into tolerogenic DCs by atorvastatin in vitro. Administration of these tolerogenic DCs to EAMG rats on days 5 and 13 post immunization (p.i.) resulted in improved clinical symptoms, which were associated with increased numbers of CD4(+)CD25(+) T regulatory (Treg) cells and Foxp3 expression, decreased lymphocyte proliferation among lymph node mononuclear cells (MNC), shifted cytokine profile from Th1/Th17 to Th2 type cytokines, decreased level of anti-R97-116 peptide (region 97-116 of the rat acetylcholine receptor α subunit) IgG antibody in serum. These tolerogenic DCs can migrate to spleen, thymus, popliteal and inguinal lymph nodes after they were injected into the EAMG rats intraperitoneally. Furthermore, these tolerogenic DCs played their immunomodulatory effects in vivo mainly by decreased expression of CD86 and MHC class II on endogenous DCs. All these data provided us a new strategy to treat EAMG and even human myasthenia gravis (MG).

Recombinant Acetylcholine Receptor Immunization Induces a Robust Model of Experimental Autoimmune Myasthenia Gravis in Mice.

Myasthenia gravis (MG) is a prototypical autoimmune disease of the neuromuscular junction (NMJ). The study of the underlying pathophysiology has provided novel insights into the interplay of autoantibodies and complement-mediated tissue damage. Experimental autoimmune myasthenia gravis (EAMG) emerged as a valuable animal model, designed to gain further insight and to test novel therapeutic approaches for MG. However, the availability of native acetylcholine receptor (AChR) protein is limited favouring the use of recombinant proteins. To provide a simplified platform for the study of MG, we established a model of EAMG using a recombinant protein containing the immunogenic sequence of AChR in mice. This model recapitulates key features of EAMG, including fatigable muscle weakness, the presence of anti-AChR-antibodies, and engagement of the NMJ by complement and a reduced NMJ density. Further characterization of this model demonstrated a prominent B cell immunopathology supported by T follicular helper cells. Taken together, the herein-presented EAMG model may be a valuable tool for the study of MG pathophysiology and the pre-clinical testing of therapeutic applications.

Dihydroartemisinin ameliorates experimental autoimmune myasthenia gravis by regulating CD4+ T cells and modulating gut microbiota.

BACKGROUND: Dihydroartemisinin (DHA), a derivative and active metabolite of artemisinin, possesses various immunomodulatory properties. However, its role in myasthenia gravis (MG) has not been clearly explored. Here, we investigated the role of DHA in experimental autoimmune myasthenia gravis (EAMG) and its potential mechanisms. METHODS: The AChR97-116 peptide-induced EAMG model was established in Lewis rats and treated with DHA. Flow cytometry was used to assess the release of Th cell subsets and Treg cells, and 16S rRNA gene amplicon sequence analysis was applied to explore the relationship between the changes in the intestinal flora after DHA treatment. In addition, network pharmacology and molecular docking were utilized to explore the potential mechanism of DHA against EAMG, which was further validated in the rat model by immunohistochemical and RT-qPCR for further validation. RESULTS: In this study, we demonstrate that oral administration of DHA ameliorated clinical symptoms in rat models of EAMG, decreased the expression level of Th1 and Th17 cells, and increased the expression level of Treg cells. In addition, 16S rRNA gene amplicon sequence analysis showed that DHA restored gut microbiota dysbiosis in EAMG rats by decreasing Ruminococcus abundance and increasing the abundance of Clostridium, Bifidobacterium, and Allobaculum. Using network pharmacology, 103 potential targets of DHA related to MG were identified, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that PI3K-AKT signaling pathway was related to the treatment of DHA on EAMG. Meanwhile, molecular docking verified that DHA has good binding affinity to AKT1, CASP3, EGFR, and IGF1. Immunohistochemical staining showed that DHA treatment significantly inhibited the phosphorylated expression of AKT and PI3K in the spleen tissues of EAMG rats. In EAMG rats, RT-qPCR results also showed that DHA reduced the mRNA expression levels of PI3K and AKT1. CONCLUSIONS: DHA ameliorated EAMG by inhibiting the PI3K-AKT signaling pathway, regulating CD4+ T cells and modulating gut microbiota, providing a novel therapeutic approach for the treatment of MG.

ATRA alters humoral responses associated with amelioration of EAMG symptoms by balancing Tfh/Tfr helper cell profiles.

All-trans retinoic acid (ATRA) is a vitamin A metabolite with diverse immunomodulatory actions used therapeutically in the treatment of some autoimmune diseases. However, the effects that ATRA may have on diminishing myasthenia gravis (MG) symptoms remain undefined. This study investigated the effect of ATRA on experimental autoimmune myasthenia gravis (EAMG) in vivo and in vitro. Data presented in this study demonstrated that intraperitoneal injection of ATRA ameliorated EAMG pathology in rats associated with reduced total anti-acetylcholine receptor (AChR) serum IgG levels. We observed that EAMG development was accompanied by an increase in follicular helper T cells (Tfh, defined as CD4(+)CXCR5(+)ICOS(high)) and a decrease of follicular regulatory T cells (Tfr, defined as CD4(+)Foxp3(+)CXCR5(+)ICOS(median)) and that the Tfh:Tfr ratio was altered following ATRA administration. In addition, ATRA treatment restored the Th1/Th2/Th17/Treg balance. In vitro, ATRA inhibited AChR-specific cell proliferation and eliciting apoptosis in these cells without affecting the cell cycle. ATRA also altered the Th distribution in animals presenting with EAMG resulting in a reduction in Th1/Th17/Tfh cells and increasing the number of Th2/Treg/Tfr cell types. These results suggested that ATRA reduced EAMG severity by regulating Th cell profiles thereby providing new insights into the development of novel MG (or related) therapies.