RESUMO
BACKGROUND: Cells and tissues in an inflammatory state are usually hypoxic. The hypoxic environment can affect the differentiation of immune cells and produce Hypoxia-inducible Factor-1α (HIF-1α). Inflammation is also a major contributor to the development and deterioration of Myasthenia Gravis (MG). There are limited studies on the immunopathological mechanism and targeted therapy associated with MG exacerbated with inflammation. This research aimed to explore whether BAY 87-2243 (HIF-1α inhibitor) ameliorates the symptoms of the Experimental Autoimmune Myasthenia Gravis (EAMG) inflammation model and study its regulatory mechanism on cellular immunity and humoral immunity. METHODS: We first establish the EAMG inflammation model using Lipopolysaccharide (LPS), BAY 87-2243 was applied to the EAMG inflammation model and its therapeutic effects were evaluated in vivo and in vitro experiments. RESULTS: The proportion of Treg cells was increased whereas Th1, Th17, and Th1/17 cells were decreased in BAY 87-2243-treated EAMG inflammation model. BAY 87-2243 ameliorated the acetylcholine receptors (AChRs) loss and the complement deposited at the neuromuscular junction of the EAMG inflammation model, declined the levels of IFN-γ, IL-17, and IL-6 in serum, and further attenuated responses in the germinal center and reduced the antibody levels by inhibiting the IL-6-dependent STAT3 axis. CONCLUSION: BAY 87-2243 restored the balance of CD4+T cell subsets and reduced the production of the pro-inflammatory cytokines, thus acting as both an immune imbalance regulator and anti-inflammatory. The current study suggests that HIF-1α might be a potential target for the treatment of MG exacerbated with inflammation.
Assuntos
Imunidade Humoral , Miastenia Gravis Autoimune Experimental , Animais , Interleucina-6/farmacologia , Subpopulações de Linfócitos T , Células Th1 , Miastenia Gravis Autoimune Experimental/tratamento farmacológico , Miastenia Gravis Autoimune Experimental/patologia , Inflamação/tratamento farmacológicoRESUMO
The Qiangji Jianli Decoction (QJJLD) is an effective Chinese medicine formula for treating Myasthenia gravis (MG) in the clinic. QJJLD has been proven to regulate mitochondrial fusion and fission of skeletal muscle in myasthenia gravis. In this study, we investigated whether QJJLD plays a therapeutic role in regulating mitochondrial biogenesis in MG and explored the underlying mechanism. Rats were experimentally induced to establish autoimmune myasthenia gravis (EAMG) by subcutaneous immunization with R97-116 peptides. The treatment groups were administered three different dosages of QJJLD respectively. After the intervention of QJJLD, the pathological changes of gastrocnemius muscle in MG rats were significantly improved; SOD, GSH-Px, Na+-K+ ATPase and Ca2+-Mg2+ ATPase activities were increased; and MDA content was decreased in the gastrocnemius muscle. Moreover, AMPK, p38MAPK, PGC-1α, NRF-1, Tfam and COX IV mRNA and protein expression levels were also reversed by QJJLD. These results implied that QJJLD may provide a potential therapeutic strategy through promoting mitochondrial biogenesis to alleviate MG via activating the AMPK/PGC-1α signaling pathway.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Medicamentos de Ervas Chinesas/farmacologia , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Miastenia Gravis Autoimune Experimental/tratamento farmacológico , Biogênese de Organelas , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Animais , Feminino , Regulação da Expressão Gênica , Mitocôndrias Musculares/enzimologia , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/ultraestrutura , Músculo Esquelético/enzimologia , Músculo Esquelético/ultraestrutura , Miastenia Gravis Autoimune Experimental/enzimologia , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/patologia , Fragmentos de Peptídeos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Ratos Endogâmicos Lew , Receptores Colinérgicos , Transdução de SinaisRESUMO
BACKGROUND Myasthenia gravis (MG) is a progressive autoimmune disorder caused by the production of antibodies directed against acetylcholine receptors (AChRs), resulting in muscle weakness and fatigue. This study aimed to explore the effect and mechanism of grilled nux vomica (GNV) in experimental autoimmune myasthenia gravis (EAMG) rats. MATERIAL AND METHODS Rat 97-116 peptides were used to mediate disease in the EAMG model in SPF female Lewis rats. The treatment groups received grilled nux vomica (75 mg/kg, 150 mg/kg, and 225 mg/kg). The autoantibody and inflammatory cytokines levels were measured by enzyme-linked immunosorbent assay (ELISA). RNA profiling was performed on high-dose and model group rats. Profiling results and TLR-4/NF-kappaB signaling were validated by q-PCR and Western blot analysis. RESULTS The results showed that GNV could attenuate the symptoms of EAMG rats. There was a decreased level of AChR-ab, IFN-γ, TNF-alpha, IL-2, IL-4, and IL-17 levels, and an increased level of TGF-ß1. In total, 235 differentially expressed genes (DEGs), consisting of 175 upregulated DEGs and 60 downregulated DEGs, were identified. Functional annotation demonstrated that DEGs were largely associated with leukocyte cell-cell adhesion, NF-kappa B signaling pathway, muscle contraction, and cardiac muscle contraction pathway. Rac2, Itgb2, Lcp2, Myl3, and Tnni1 were considered as hub genes with a higher degree value in the protein-protein interaction (PPI) network. The q-PCR and Western blot results of hub genes were consistent with RNA profiles. GNV treatment also significantly reduced the TLR-4 and NF-kappaB p65 protein expression in EAMG rats. CONCLUSIONS These results indicate that grilled nux vomica ameliorates EAMG by depressing the TLR-4/NF-kappaB signaling pathway, and hub genes may serve as potential targets for MG treatment.
Assuntos
Medicamentos de Ervas Chinesas/administração & dosagem , Miastenia Gravis Autoimune Experimental/tratamento farmacológico , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Strychnos nux-vomica/química , Animais , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Músculo Esquelético/imunologia , Músculo Esquelético/patologia , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/patologia , NF-kappa B/metabolismo , RNA-Seq , Ratos , Ratos Endogâmicos Lew , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Organismos Livres de Patógenos Específicos , Receptor 4 Toll-Like/metabolismoRESUMO
The guanine nucleotide exchange factor Vav1 is essential for transducing T cell receptor (TCR) signals and plays an important role in T cell development and activation. Previous genetic studies identified a natural variant of Vav1 characterized by the substitution of an arginine (R) residue by a tryptophane (W) at position 63 (Vav1R63W). This variant impacts Vav1 adaptor functions and controls susceptibility to T cell-mediated neuroinflammation. To assess the implication of this Vav1 variant on the susceptibility to antibody-mediated diseases, we used the animal model of myasthenia gravis, experimental autoimmune myasthenia gravis (EAMG). To this end, we generated a knock-in (KI) mouse model bearing a R to W substitution in the Vav1 gene (Vav1R63W) and immunized it with either torpedo acetylcholine receptor (tAChR) or the α146-162 immunodominant peptide. We observed that the Vav1R63W conferred increased susceptibility to EAMG, revealed by a higher AChR loss together with an increased production of effector cytokines (IFN-γ, IL-17A, GM-CSF) by antigen-specific CD4+ T cells, as well as an increased frequency of antigen-specific CD4+ T cells. This correlated with the emergence of a dominant antigen-specific T cell clone in KI mice that was not present in wild-type mice, suggesting an impact on thymic selection and/or a different clonal selection threshold following antigen encounter. Our results highlight the key role of Vav1 in the pathophysiology of EAMG and this was associated with an impact on the TCR repertoire of AChR reactive T lymphocytes.
Assuntos
Variação Genética , Miastenia Gravis Autoimune Experimental/etiologia , Miastenia Gravis Autoimune Experimental/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Suscetibilidade a Doenças , Camundongos , Miastenia Gravis Autoimune Experimental/patologia , Fenótipo , Receptores Nicotínicos/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos TRESUMO
Myasthenia gravis (MG) is an autoimmune neurological disease that is characterized by the expression of anti-acetylcholine receptor (AChR) antibodies. The immune response at AChRs of neuromuscular junction is disrupted in patients with MG, which manifests as skeletal muscle fatigue and is aggravated following periods of activity and alleviated following rest. Although a novel immune suppressant FTY720 drug, which exhibits strong immune suppression efficacy and minor adverse effects, is available, its role and mechanism in MG have not been elucidated. The aim of this study was to investigate the role of FTY720 in MG. A total of 60 healthy female Lewis rats were randomly assigned into 4 groups: Control group, Model group of experimental autoimmune myasthenia gravis (EAMG), 0.5 mg/kg FTY720-treatment EAMG group and 1.0 mg/kg FTY720treatment EAMG group. Body weight and symptoms were examined; Lennon score was used to evaluate improvement of clinical symptoms. Reverse transcriptionquantitative polymerase chain reaction and ELISA were used to test the mRNA and protein expression levels, respectively, of the helper T (Th)1 and Th2 cell cytokines, including interleukin (IL)2, interferon (IFN)γ, IL4 and IL6 in thymus tissue and serum. FTY720 treatment improved rat MG symptoms, increased body weight and decreased Lennon score. FTY720 treatments also reduced tissue and serum levels of IL2, IFNγ and IL6, but not IL4 expression levels. FTY720 suppressed the inflammatory response and improved EAMG symptoms by inhibiting the secretion of inflammatory factors.
Assuntos
Cloridrato de Fingolimode/uso terapêutico , Imunossupressores/uso terapêutico , Miastenia Gravis Autoimune Experimental/tratamento farmacológico , Células Th1/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Animais , Feminino , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/patologia , Ratos , Ratos Endogâmicos Lew , Células Th1/imunologia , Células Th1/patologia , Células Th2/imunologia , Células Th2/patologiaRESUMO
Because of the failure of many promising therapeutics identified in preclinical evaluation, funding sources have established guidelines for increased rigor in animal evaluations. The myasthenia gravis (MG) community of scientists has developed guidelines for preclinical assessment for potential MG treatments. Here, we provide a focused summary of these recommendations and the role of complement in disease development in experimental models of MG.
Assuntos
Autoanticorpos/imunologia , Proteínas Inativadoras do Complemento/farmacologia , Proteínas do Sistema Complemento/imunologia , Miastenia Gravis Autoimune Experimental/tratamento farmacológico , Miastenia Gravis Autoimune Experimental/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Receptores Colinérgicos/imunologia , Animais , Proteínas do Sistema Complemento/genética , Modelos Animais de Doenças , Imunização Passiva/métodos , Imunoglobulina G/imunologia , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Miastenia Gravis Autoimune Experimental/patologia , Ratos , Ratos Endogâmicos LewRESUMO
Recent studies have demonstrated the important role of toll-like receptor 9 (TLR9) signalling in autoimmune diseases, but its role in myasthenia gravis (MG) has not been fully established. We show herein that blocking TLR9 signalling via the suppressive oligodeoxynucleotide (ODN) H154 alleviated the symptoms of experimental autoimmune myasthenia gravis (EAMG). With the downregulation of dendritic cells (DCs), TLR9 interruption reduced follicular helper T cells (Tfh) and germinal centre (GC) B cells, leading to decreased antibody production. In addition, TLR9+ B cells as well as total B cells in the spleen were inhibited by H154. These findings highlight the critical role of TLR9 in EAMG and suggest that the inhibition of the TLR9 pathway might be a potential pharmacological strategy for the treatment of myasthenia gravis.
Assuntos
Imunidade Humoral/efeitos dos fármacos , Miastenia Gravis Autoimune Experimental/tratamento farmacológico , Miastenia Gravis Autoimune Experimental/imunologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , Receptor Toll-Like 9/antagonistas & inibidores , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Feminino , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/patologia , Imunidade Humoral/genética , Miastenia Gravis Autoimune Experimental/genética , Miastenia Gravis Autoimune Experimental/patologia , Ratos , Ratos Endogâmicos Lew , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Receptor Toll-Like 9/genéticaRESUMO
INTRODUCTION: Agrin is essential for the formation and maintenance of neuromuscular junctions (NMJs). NT-1654 is a C-terminal fragment of mouse neural agrin. In this study, we determined the effects of NT-1654 on the severity of experimental autoimmune myasthenia gravis (EAMG). METHODS: EAMG was induced in female Lewis rats by immunization with the Torpedo acetylcholine receptor (tAChR) and complete Freund's adjuvant (CFA). NT-1654 was dissolved in phosphate-buffered saline (PBS) and injected daily subcutaneously into tAChR immunized rats during the first 10 days after immunization, and then every other day for the following 20 days. RESULTS: We showed that NT-1654 attenuated clinical severity, effectively promoted the clustering of AChRs at NMJs, and alleviated the impairment of NMJ transmission and the reduction of muscle-specific kinase (MuSK) in EAMG rats. DISCUSSION: We demonstrated that NT-1654 attenuated clinical severity, effectively promoted the clustering of AChRs at NMJs, and alleviated the impairment of NMJ transmission and the reduction of muscle-specific kinase (MuSK) in EAMG rats. Muscle Nerve 57: 814-820, 2018.
Assuntos
Agrina/uso terapêutico , Imunização/efeitos adversos , Miastenia Gravis Autoimune Experimental/tratamento farmacológico , Miastenia Gravis Autoimune Experimental/patologia , Fragmentos de Peptídeos/uso terapêutico , Potenciais de Ação/fisiologia , Agrina/biossíntese , Agrina/química , Animais , Autoanticorpos/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eletromiografia , Feminino , Adjuvante de Freund/toxicidade , Regulação da Expressão Gênica/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/etiologia , Atrofia Muscular/terapia , Proteínas do Tecido Nervoso/metabolismo , Neurofibromina 1/metabolismo , Junção Neuromuscular/patologia , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/química , Ratos , Ratos Endogâmicos Lew , Receptores Colinérgicos/imunologia , Receptores Colinérgicos/metabolismoRESUMO
The transcription factor NFAT1 plays a pivotal role in the homeostasis of T lymphocytes. However, its functional importance in non-CD4+ T cells, especially in systemic immune disorders, is largely unknown. In this study, we report that NFAT1 regulates dendritic cell (DC) tolerance and suppresses systemic autoimmunity using the experimental autoimmune myasthenia gravis (EAMG) as a model. Myasthenia gravis and EAMG are T cell-dependent, Ab-mediated autoimmune disorders in which the acetylcholine receptor is the major autoantigen. NFAT1-knockout mice showed higher susceptibility to EAMG development with enhanced Th1/Th17 cell responses. NFAT1 deficiency led to a phenotypic alteration of DCs that show hyperactivation of NF-κB-mediated signaling pathways and enhanced binding of NF-κB (p50) to the promoters of IL-6 and IL-12. As a result, NFAT1-knockout DCs produced much higher levels of proinflammatory cytokines such as IL-1ß, IL-6, IL-12, and TNF-α, which preferentially induce Th1/Th17 cell differentiation. Our data suggest that NFAT1 may limit the hyperactivation of the NF-κB-mediated proinflammatory response in DCs and suppress autoimmunity by serving as a key regulator of DC tolerance.
Assuntos
Células Dendríticas/imunologia , Ativação Linfocitária , Miastenia Gravis Autoimune Experimental/imunologia , Fatores de Transcrição NFATC/imunologia , Transdução de Sinais/imunologia , Animais , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/patologia , Tolerância Imunológica/genética , Camundongos , Camundongos Transgênicos , Miastenia Gravis Autoimune Experimental/genética , Miastenia Gravis Autoimune Experimental/patologia , NF-kappa B/genética , NF-kappa B/imunologia , Fatores de Transcrição NFATC/genética , Transdução de Sinais/genética , Células Th1/imunologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/patologiaRESUMO
Experimental autoimmune myasthenia gravis (EAMG), an animal model of myasthenia gravis (MG), can be induced in C57BL/6 (B6, H-2 b) mice by 2-3 injections with Torpedo californica AChR (tAChR) in complete Freund's adjuvant. Some EAMG mice exhibit weight loss with muscle weakness. The loss in body weight, which is closely associated with bone structure, is particularly evident in EAMG mice with severe muscle weakness. However, the relationship between muscle weakness and bone loss in EAMG has not been studied before. Recent investigations on bone have shed light on association of bone health and immunological states. It is possible that muscle weakness in EAMG developed by anti-tAChR immune responses might accompany bone loss. We determined whether reduced muscle strength associates with decreased bone mineral density (BMD) in EAMG mice. EAMG was induced by two injections at 4-week interval of tAChR and adjuvants in two different age groups. The first tAChR injection was either at age 8 weeks or at 15 weeks. We measured BMD at three skeletal sites, including femur, tibia, and lumbar vertebrae, using dual energy X-ray absorptiometry. Among these bone areas, femur of EAMG mice in both age groups showed a significant decrease in BMD compared to control adjuvant-injected and to non-immunized mice. Reduction in BMD in induced EAMG at a later-age appears to parallel the severity of the disease. The results indicate that anti-tAChR autoimmune response alone can reduce bone density in EAMG mice. BMD reduction was also observed in adjuvant-injected mice in comparison to normal un-injected mice, suggesting that BMD decrease can occur even when muscle activity is normal. Decreased BMD observed in both tAChR-injected and adjuvant-injected mice groups were discussed in relation to innate immunity and bone-related immunology involving activated T cells and tumour necrosis factor-related cytokines that trigger osteoclastogenesis and bone loss.
Assuntos
Densidade Óssea/imunologia , Reabsorção Óssea/patologia , Debilidade Muscular/patologia , Miastenia Gravis Autoimune Experimental/patologia , Absorciometria de Fóton , Fatores Etários , Animais , Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/diagnóstico por imagem , Reabsorção Óssea/imunologia , Fêmur/diagnóstico por imagem , Fêmur/imunologia , Fêmur/patologia , Proteínas de Peixes/administração & dosagem , Adjuvante de Freund/administração & dosagem , Vértebras Lombares/diagnóstico por imagem , Vértebras Lombares/imunologia , Vértebras Lombares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Debilidade Muscular/induzido quimicamente , Debilidade Muscular/diagnóstico por imagem , Debilidade Muscular/imunologia , Miastenia Gravis Autoimune Experimental/induzido quimicamente , Miastenia Gravis Autoimune Experimental/diagnóstico por imagem , Miastenia Gravis Autoimune Experimental/metabolismo , Receptores Colinérgicos/administração & dosagem , Índice de Gravidade de Doença , Tíbia/diagnóstico por imagem , Tíbia/imunologia , Tíbia/patologia , Fatores de Tempo , Torpedo/metabolismoRESUMO
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.
Assuntos
Músculo Esquelético/fisiopatologia , Miastenia Gravis Autoimune Experimental/fisiopatologia , Miastenia Gravis/fisiopatologia , Células Satélites de Músculo Esquelético/fisiologia , Adulto , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Tamanho Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Músculo Esquelético/patologia , Miastenia Gravis/patologia , Miastenia Gravis Autoimune Experimental/patologia , Miogenina/metabolismo , RNA Mensageiro/metabolismo , Receptores Colinérgicos/imunologia , Regeneração/imunologia , Células Satélites de Músculo Esquelético/patologia , Soro/imunologia , Adulto JovemRESUMO
BACKGROUND: Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ), characterized with muscle weakness. While MG develops due to acetylcholine receptor (AChR) antibodies in most patients, antibodies to muscle-specific receptor tyrosine kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4) may also be identified. Experimental autoimmune myasthenia gravis (EAMG) has been previously induced by both LRP4 immunization and passive transfer of LRP4 antibodies. OBJECTIVE: Our aim was to confirm previous results and to test the pathogenic effects of LRP4 immunization in a commonly used mouse strain C57BL/6 (B6) using a recombinantly expressed human LRP4 protein. METHODS: B6 mice were immunized with human LRP4 in CFA, Torpedo Californica AChR in CFA or only CFA. Clinical and pathogenic aspects of EAMG were compared among groups. RESULTS: LRP4- and AChR-immunized mice showed comparable EAMG clinical severity. LRP4-immunized mice displayed serum antibodies to LRP4 and NMJ IgG and complement factor C3 deposits. IgG2 was the dominant anti-LRP4 isotype. Cultured lymph node cells of LRP4- and AChR-immunized mice gave identical pro-inflammatory cytokine (IL-6, IFN-γ and IL-17) responses to LRP4 and AChR stimulation, respectively. CONCLUSION: Our results confirm the EAMG-inducing action of LRP4 immunization and identify B6 as a LRP4-EAMG-susceptible mouse strain. Demonstration of complement fixing anti-LRP4 antibodies in sera and complement/IgG deposits at the NMJ of LRP4-immunized mice indicates complement activation as a putative pathogenic mechanism. We have thus developed a practical LRP4-induced EAMG model using a non-conformational protein and a widely available mouse strain for future investigation of LRP4-related MG.
Assuntos
Ativação do Complemento/efeitos dos fármacos , Imunização/métodos , Imunoglobulina G/biossíntese , Proteínas Relacionadas a Receptor de LDL/administração & dosagem , Miastenia Gravis Autoimune Experimental/imunologia , Receptores Colinérgicos/administração & dosagem , Animais , Complemento C3/metabolismo , Adjuvante de Freund/administração & dosagem , Humanos , Isotipos de Imunoglobulinas/biossíntese , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-17/biossíntese , Interleucina-17/imunologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Proteínas Relacionadas a Receptor de LDL/imunologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miastenia Gravis Autoimune Experimental/induzido quimicamente , Miastenia Gravis Autoimune Experimental/patologia , Cultura Primária de Células , Receptores Colinérgicos/imunologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Índice de Gravidade de Doença , Torpedo/metabolismoRESUMO
Properly sustained impulse transmission at the neuromuscular junction (NMJ) is crucial for successful muscle contraction. To guarantee this, NMJs not only possess a considerable safety factor in transmission but also have the ability to adjust the presynaptic acetylcholine release level to cope with any changes in the postsynaptic neurotransmitter sensitivity. This review will provide overview on the discovery and characterization of this synaptic homeostatic mechanism, especially in the condition of the neuromuscular disorder myasthenia gravis (MG) where the postsynaptic transmitter sensitivity at the NMJ becomes severely reduced due to autoimmune attack of acetylcholine receptors. Because homeostatic signalling and adaptation is presumably maximally active in this condition, NMJs from MG animal models are important study objects. Although candidate post- and presynaptic factors as well as the retrograde signals have been proposed, the homeostatic mechanism at the MG NMJ is still incompletely understood. Further identification and functional characterization of key factors is important because these may form new therapeutic targets in MG.
Assuntos
Miastenia Gravis/fisiopatologia , Junção Neuromuscular/fisiopatologia , Acetilcolina/metabolismo , Animais , Homeostase , Humanos , Contração Muscular , Miastenia Gravis/patologia , Miastenia Gravis Autoimune Experimental/patologia , Miastenia Gravis Autoimune Experimental/fisiopatologia , Junção Neuromuscular/ultraestrutura , Transdução de Sinais , Transmissão SinápticaRESUMO
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that involves the selective loss of the upper and lower motor neurons (MNs). Neuroinflammation has been implicated in the pathogenesis of the sporadic form of the disease. We earlier developed immune-mediated animal models of ALS and demonstrated humoral and cellular immune reactions in the nervous system and in the sera of patients and animals. The accumulation of immunoglobulin G (IgG), an elevated intracellular level of calcium, ultrastructural alterations in the MNs, and activation of the microglia were noted in the spinal cord of ALS patients. Similar alterations developed in mice inoculated intraperitoneally with IgG from ALS patients or from an immune-mediated goat model. METHODS: We have now examined whether the intraperitoneal injection of mice with IgG from sporadic ALS patients or from immunized goats with the homogenate of the anterior horn of the bovine spinal cord is associated with changes in the pro-inflammatory (TNF-α and IL-6) and anti-inflammatory (IL-10) cytokines in the spinal cord and serum of the mice. The levels of cytokines were measured by ELISA. RESULTS: Intraperitoneally administered IgG from the ALS patients induced subclinical signs of MN disease, while the injection of IgG from immunized goats resulted in a severe respiratory dysfunction and limb paralysis 24 h after the injections. Significantly increased levels of TNF-α and IL-10 were detected in the spinal cord of the mice injected with the human ALS IgG. The level of IL-6 increased primarily in the serum. The IgG from the immunized goats induced highly significant increases in the levels of all three cytokines in the serum and the spinal cord of mice. CONCLUSIONS: Our earlier experiments had proved that when ALS IgG or IgG from immune-mediated animal models was inoculated into mice, it was taken up in the MNs and had the ability to initiate damage in them. The pathological process was paralleled by microglia recruitment and activation in the spinal cord. The present experiment revealed that these forms of IgG cause significant increases in certain cytokine levels locally in the spinal cord and in the serum of the inoculated mice. These results suggest that IgG directed to the MNs may be an initial element in the damage to the MNs both in human ALS and in its immune-mediated animal models.
Assuntos
Esclerose Lateral Amiotrófica/sangue , Citocinas/metabolismo , Imunoglobulina G/administração & dosagem , Medula Espinal/metabolismo , Esclerose Lateral Amiotrófica/imunologia , Análise de Variância , Animais , Cálcio/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Cabras , Humanos , Injeções Intraperitoneais , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Atividade Motora/efeitos dos fármacos , Neurônios Motores/patologia , Neurônios Motores/ultraestrutura , Miastenia Gravis Autoimune Experimental/sangue , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Sera of myasthenia gravis (MG) patients with muscle-specific receptor kinase-antibody (MuSK-Ab) predominantly display the non-complement fixing IgG4 isotype. Similarly, mouse IgG1, which is the analog of human IgG4, is the predominant isotype in mice with experimental autoimmune myasthenia gravis (EAMG) induced by MuSK immunization. The present study was performed to determine whether IgG1 anti-MuSK antibody is required for immunized mice to develop EAMG. Results demonstrated a significant correlation between clinical severity of EAMG and levels of MuSK-binding IgG1+, IgG2+ and IgG3+ peripheral blood B cells in MuSK-immunized wild-type (WT) mice. Moreover, MuSK-immunized IgG1 knockout (KO) and WT mice showed similar EAMG severity, serum MuSK-Ab levels, muscle acetylcholine receptor concentrations, neuromuscular junction immunoglobulin and complement deposit ratios. IgG1 and IgG3 were the predominant anti-MuSK isotypes in WT and IgG1 KO mice, respectively. These observations demonstrate that non-IgG1 isotypes can mediate MuSK-EAMG pathogenesis.
Assuntos
Imunoglobulina G/imunologia , Miastenia Gravis Autoimune Experimental/induzido quimicamente , Miastenia Gravis Autoimune Experimental/imunologia , Receptores Proteína Tirosina Quinases/toxicidade , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Autoanticorpos/sangue , Linfócitos B/metabolismo , Linfócitos B/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Adjuvante de Freund/toxicidade , Imunização , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miastenia Gravis Autoimune Experimental/patologia , Junção Neuromuscular/imunologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Receptores Colinérgicos , Índice de Gravidade de Doença , Estatísticas não ParamétricasRESUMO
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.
Assuntos
Linfócitos B/patologia , Quimiocina CXCL13/fisiologia , Inflamação/complicações , Miastenia Gravis Autoimune Experimental/patologia , Hiperplasia do Timo/fisiopatologia , Animais , Linfócitos B/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Citometria de Fluxo , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Humanos , Técnicas Imunoenzimáticas , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miastenia Gravis Autoimune Experimental/etiologia , Miastenia Gravis Autoimune Experimental/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
IL-10-competent subset within CD1d(hi)CD5(+) B cells, also known as B10 cells, has been shown to regulate autoimmune diseases. In our previous study, adoptive transfer of CD1d(hi)CD5(+) B cells expanded in vivo by GM-CSF prevented and suppressed experimental autoimmune myasthenia gravis (EAMG). The goal of this study was to further examine the role and mechanism of IL-10 in the regulatory function of B10 cells in EAMG. We found that only IL-10 competent CD1d(hi)CD5(+) B cells sorted from WT mice, but not IL-10 deficient CD1d(hi)CD5(+) B cells exhibited regulatory function in vitro and in vivo. Adoptive transfer of IL-10 competent CD1d(hi)CD5(+) B cells led to higher frequency of Tregs and B10 cells, and low levels of proinflammatory cytokines and autoantibody production. We conclude that IL-10 production within CD1d(hi)CD5(+) B cells plays an important role in immune regulation of EAMG.
Assuntos
Antígenos CD1d/metabolismo , Subpopulações de Linfócitos B/química , Antígenos CD5/metabolismo , Interleucina-10/metabolismo , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/patologia , Transferência Adotiva/métodos , Animais , Proliferação de Células/fisiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Interleucina-10/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Miastenia Gravis Autoimune Experimental/induzido quimicamente , Miastenia Gravis Autoimune Experimental/fisiopatologia , Fragmentos de Peptídeos/imunologia , Receptores Nicotínicos/imunologiaRESUMO
It is expensive to induce experimental autoimmune myasthenia gravis (EAMG) by active immunity, and difficult to obtain natural acetylcholine receptor (AChR). We sought a new method of inducing EAMG by immunizing rats with artificially synthesized AChR. The AChR mRNA in TE671 cells was extracted and reverse transcribed. The inclusion body was purified and protein concentration was determined, and the EAMG animal model was used for induction. The serum was extracted from rat blood. The antibody titer was determined using enzyme-linked immunosorbant assay (ELISA). The concentration of decay accelerating factor (DAF) in the rat serum was determined by ELISA, and the metabolism of serum rDAF was determined by western blot. We evaluated the inhibition of rDAF by determining the 50% complement hemolysis unit in the rat serum. The extracellular domain (ECD) nucleotide sequence clone produced by polymerase chain reaction was completely consistent with that in the human gene bank; it was induced by isopropyl ß-D-1-thiogalactopyranoside to express the protein after insertion into vector pET16b. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the inclusion body protein was the exact target. The ECD protein was able to bind with mAb35 after dialysis and renaturation, which demonstrated protein activity. The soluble ECD protein was used to immunize rats and obtain the EAMG models. The inhibitory effect of the complement was unsatisfactory owing to high decay rate after rDAF injection into the EAMG models. It is easy to induce the EAMG model by obtaining the AChRTEα1 subunit ECD protein using the substitution method.
Assuntos
Antígenos CD55/uso terapêutico , Miastenia Gravis Autoimune Experimental/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Animais , Antígenos CD55/administração & dosagem , Antígenos CD55/sangue , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Injeções Intravenosas , Miastenia Gravis Autoimune Experimental/sangue , Miastenia Gravis Autoimune Experimental/patologia , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , Renaturação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Ratos Endogâmicos Lew , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
Immature dendritic cell-derived exosomes (iMDEX) display a certain degree of immunosuppressive activity in autoimmune diseases. However, the role of iMDEX in experimental autoimmune myasthenia gravis (EAMG) is still unclear. Therefore, we tested the effects of mouse bone marrow (BM)-derived iMDEX on tolerance induction in a mouse model of EAMG. In this study, we found that the CELLine culture system produced more exosomes, the morphology and phenotype of these exosomes were found to be identical when compared with traditional cell culture. And, iMDEX(1000) ameliorated the progression of EAMG by reducing AChR-reactive lymphocyte proliferation, AChR antibody levels and pro-inflammatory cytokine levels.
Assuntos
Células Dendríticas/imunologia , Exossomos/imunologia , Miastenia Gravis Autoimune Experimental/imunologia , Miastenia Gravis Autoimune Experimental/prevenção & controle , Animais , Células Cultivadas , Células Dendríticas/patologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Miastenia Gravis Autoimune Experimental/patologiaRESUMO
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.