Humoral Immunoregulation In Experimental Autoimmune Myasthenia Gravis

Background:Myasthenia gravis is a classical organ-specific autoimmune disease, which is antibody-mediated, T cell-dependent and complement-involved. It impairs the postsynaptic nicotinic acetylcholine receptor (AchR) at the neuromuscular junction (NMJ), leading to neuromuscular transmission failure. Current immunosuppressive agents for MG treatment, such as corticosteroids and cytotoxic reagents, are more pan-immunosuppressive than specific cell targeted and desire a long-term therapy, thus causing numerous side effects, which drives us to search new strategies and approaches.Dendritic cells (DCs) are the most efficient antigen-presenting cells, and they can induce immunity as well as tolerance, which at least partially depends on their maturation state. Statins are widely used in clinical practice for the treatment of atherosclerosis. Accumulating evidence has shown the antiatherosclerosis effect was more dependent on its anti-inflammatory and immune-modulatory effect. Previously, we found that atorvastatin could induce immature spleen-derived DCs in vitro and atorvastatin-modified spleen-derived DCs successfully ameliorated EAMG by inhibiting lymphocyte proliferation, up-regulating Treg cells and shifting Th1/Th17 to Th2 cytokines. However, the underlying mechanisms by which statin-modified dendritic cells decrease antibody production, especially the effects on humoral immunity were still unknown.Recently, a distinct T cell subset, named follicular helper T (Tfh) cells and characterized by expression of transcription factor B-cell lymphoma 6 (Bcl6), surface molecules including CXC chemokine receptor 5 (CXCR5), inducible T-cell costimulator (ICOS) and programmed death protein-1 (PD-1), and cytokine IL-21, is indicated to supply direct assistance for antibody production of B cells and play an important role in autoimmune diseases. Moreover, the discovery of regulatory B cells (Breg), a novel negatively modulatory subset, has challenged the conventional roles of B cells as antigen-presenting cells and antibody-secreting cells. The present study also focused on the effect of tolerogenic DCs on Tfh and Breg in EAMG.Objective:The aim of our present study was to explore the effect of tolerogenic DCs on humoral immune response of EAMG and further elucidate the underlying mechanisms.Methods:1. EAMG induction and assessment EAMG was induced by immunization with R97-116 peptide in Lewis rats on day 0 and boosted on day 11 post immunization (p.i.). Rats were observed for clinical sighs every other day in a blinded fashion, and disease severity was evaluated as Baggi described. Results were expressed as the mean score of each group at each time point.2. Bone marrow-derived DC induction, modification and characterization Femurs and tibias from ongoing EAMG rats were aseptically removed and bone marrow cells were flushed out. Erythrocytes were osmotically lysed and the remained cells were cultured for 72 h incubation. Non-adherent cells were gently removed and adherent cells were further cultured. On day 8, atorvastatin or dimethysulfoxide (DMSO) was added. After 48 h incubation, non-adherent cells were harvested and collected for phenotype analysis by flow cytometry (FACS).3. EAMG treatmentEAMG rats were randomly divided into 3 groups (n= 5 in each group).Rats in AT-BMDC group and 0-BMDC group were injected with corresponding BMDCs intraperitoneally (i.p.) on days 5 and 16 p.i.. The control rats in EAMG group received the same volume of RPMI 1640 medium.4. Detection of levels and affinity of anti-R97-116 IgG by ELISASerum was collected on day 43 p.i. to detect anti-R97-116 IgG antibody by ELISA. The relative affinity of anti-R97-116 antibodies was determined by ELISA using thiocyanate elution.5. Detection of germinal centers (GCs) by immunofluorescenceSpleens were collected and cryopreserved in Tissue-Tek OCT Compound at-80℃. Cryostat sections were incubated with peanut agglutinin (PNA) and examined under an immunofluorescence microscopy to detect B cells in GCs.6. Detection of Tfh cells, IL-21+ cells and Breg by FACSSingle-cell suspensions of MNC were prepared from inguinal lymph nodes of individual rat on day 43 p.i. for FACS analysis. Tfh cells were defined by staining of anti-CD4, anti-CXCR5 and anti-ICOS antibodies. B-Foxp3 cells were defined by staining of anti-CD19 and anti-Foxp3 antibodies. B10 cells were defined by staining of anti-CD 19 and anti-IL-10 antibodies. IL-21+ cells were detected by IL-21 polyclonal antibody. Then cells were analyzed by a FACScan.7. Statistical analysisThe SPSS 17.0 computer program was used for all calculations and statistical evaluations. Differences among different groups were tested by one-factor analysis of variance (ANOVA) or nonparametric Kruskal-Wallis test. A level of p< 0.05 was considered significant.Results:1. Atorvastatin induces immature BMDCs in vitroIn the presence of atorvastatin, CD80, CD86 and MHCII expressed on DCs were inhibited compared with those on DCs cultured without atorvastatin. The results indicated that atorvastatin induced immature BMDCs in vitro.2. AT-BMDC treatment suppresses the development of ongoing EAMGRats in AT-BMDC group exhibited lower scores compared with EAMG group and differences were statistically significant on day 13,24,34,36,38 and 42 p.i.. When compared with 0-BMDC group, AT-BMDC group showed lower clinical scores, however, the difference was not significant. Moreover, there was no significant difference between 0-BMDC group and EAMG group.3. AT-BMDC treatment inhibits the quantity and affinity of anti-R97-116 IgG in EAMG ratsRats in AT-BMDC group produced less anti-R97-116 IgG compared with rats in EAMG and 0-BMDC group. Moreover, the relative affinity was determined by ELISA using thiocyanate elution and showed similar results. There was no difference between EAMG group and 0-BMDC group.4. AT-BMDC treatment inhibits GC response in EAMG ratsPNA+GC-B cells were significantly decreased in AT-BMDC group compared with EAMG group and 0-BMDC group, which implied that the formation of GCs was abolished by AT-BMDC treatment.5. AT-BMDC treatment decreases Tfh cells and IL-21+ cells in EAMG ratsThe percentage of Tfh cells among CD4+cells in AT-BMDC group was lower than that in EAMG group and 0-BMDC group. In addition, there were less IL-21+ cells in AT-BMDC group compared with EAMG group and 0-BMDC group.6. AT-BMDC treatment increases Breg in EAMG ratsThe results showed that AT-BMDCs decreased CD19+ B cells compared with 0-BMDC group. However, when compared with EAMG group, there was a slight decrease without significant difference. Interestingly, the percentages of B-Foxp3 cells were increased in AT-BMDC group and 0-BMDC group. B10 cells were also slightly up-regulated in AT-BMDC group compared with EAMG group and 0-BMDC group, however, there was no significant difference.Conclusions:1. Atorvastatin successfully induced immature BMDCs in vitro, which induced immune tolerance in EAMG rats.2. AT-BMDCs suppressed the quantity and the relative affinity of anti-R97-116 IgG, inhibited germinal center response, down-regulated Tfh cells and IL-21, and up-regulated Breg to regulate humoral immune response in EAMG rats.Background and objectives:Rho-associated coiled-coil containing kinase (ROCK) is an important downstream substrate of Rho GTPase. ROCK phosphorylates various target proteins, and serves as molecular switches in fundamental cellular processes. Accumulating evidence indicates that the Rho/ROCK pathway is also involved in immunocytes, and plays an important role in the development of T cells and B cells. The activity of Rho/ROCK pathway is elevated in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients. Experiments indicate that ROCK inhibitors ameliorate autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE) and SLE.Myasthenia gravis (MG) is a classic antibody-mediated organ-specific autoimmune disease, characterized by muscle weakness and fatigue. The role of the Rho/ROCK pathway in MG is still unclear nowadays. Researches show that statins exhibit anti-inflammatory and immunomodulatory effect, which partly depends on the inhibition of the Rho/ROCK pathway. However, the muscle toxicity of statins limit the application in MG. Fasudil is the only specific ROCK inhibitor which is approved for clinical treatment of cerebrovascular spasm after subarachnoid hemorrhage. In the present study, we choose fasudil to treat experimental autoimmune myasthenia gravis (EAMG), aiming to explore the role of the Rho/ROCK pathway in MG and the effect of ROCK inhibitor on the humoral immunity of EAMG, and try to elucidate the underlying mechanisms.Methods:1. EAMG induction and assessmentEAMG was induced by immunization with R97-116 peptide in Lewis rats on day 0 and boosted on day 11 post immunization (p.i.). Rats were observed for clinical sighs every other day in a blinded fashion, and disease severity was evaluated as Baggi described. Results were expressed as the mean score of each group at each time point.2. EAMG treatmentEAMG rats were randomly divided into 3 groups (n=5 in each group). Fasudil at doses of 10 or 30 mg/kg was administered daily from day 6 post immunization onward. The control rats in EAMG group received the same volume of saline solution.3. Detection of CD80, CD86 and MHCII by FACSSingle-cell suspensions of mononuclear cells (MNC) were prepared from inguinal lymph nodes of individual rat on day 43 p.i. for FACS analysis. The expressions of CD80, CD86 and MHCII were determined by FACS.4. Detection of intracellular cytokines by FACSSingle-cell suspensions of mononuclear cells (MNC) were incubated with cell stimulation cocktail (plus protein transport inhibitors) for 4 h, then collected and labeled as CD4+ IFN-γ+(Th1),CD4+ IL-4+(Th2) and CD4+ IL-17+(Th17). Intracellular cytokines IL-1β, IL-6 and TNF-α were also determined by FACS.5. Detection of levels and affinity of anti-R97-116 IgG by ELIS ASerum was collected on day 43 p.i. to detect anti-R97-116 IgG antibody by ELISA. The relative affinity of anti-R97-116 antibodies was determined by ELISA using thiocyanate elution.6. Detection of follicular helper T cells (Tfh) and regulatory B cells (Breg) by FACSSingle-cell suspensions of MNC were prepared from inguinal lymph nodes of individual rat on day 43 p.i. for FACS analysis. Tfh cells were defined by staining of anti-CD4, anti-CXCR5 and anti-ICOS antibodies. B-Foxp3 cells were defined by staining of anti-CD 19 and anti-Foxp3 antibodies. B10 cells were defined by staining of anti-CD 19 and anti-IL-10 antibodies. Then cells were analyzed by a FACScan.7. Detection of germinal centers (GCs) by immunofluorescenceSpleens were collected and cryopreserved in Tissue-Tek OCT Compound at -80℃. Cryostat sections were incubated with peanut agglutinin (PNA) and examined under an immunofluorescence microscopy to detect B cells in GCs.8. Statistical analysisThe SPSS 17.0 computer program was used for all calculations and statistical evaluations. Differences among different groups were tested by one-factor analysis of variance (ANOVA) or nonparametric Kruskal-Wallis test. A level ofp<0.05 was considered significant.Results:1. Fasudil suppresses the development of ongoing EAMGFasudil showed an inhibitory effect on the development of EAMG compared with the vehicle control. Rats in the high-dose group exhibited significantly lower clinical scores compared with rats in the control group on day 24 p.i. and from day 28 to day 43 p.i.. Severity of rats in the low-dose group was lower than severity of rats in the control group, however, there was statistically significant difference only on day 35 p.i.. The clinical severity of rats in the high-dose group and the-low dose group did not differ significantly.2. Fasudil decreases the expression of CD80 on MNC in EAMG ratsFasudil treatment at high dose inhibited the expression of CD80 on MNC when compared with the vehicle control. There were no significant differences in the expression of CD86 and MHCⅡ among three groups.3. Effects of fasudil on Thl/Th2/Th17 cytokines and pro-inflammatory cytokinesThere were no significant differences in the percentages of CD4+ IFN-γ+ cells, CD4+ IL-4+ cells and CD4+ IL-17+ cells in EAMG rats with or without fasudil treatment. Moreover, the production of TNF-α, IL-1β and IL-6 in lymph node MNC was not altered by fasudil treatment in EAMG rats.4. Fasudil reduces Tfh cells in EAMG ratsThe percentage of Tfh cells among CD4+T cells in the high-dose group was down-regulated significantly compared with that in the control group and the low-dose group.5. Fasudil decreases CD19+ B cells and abolished GC responseCD19+ B cells were decreased in the high-dose group compared with the control group and the low-dose group. PNA+ GC-B cells were significantly decreased in the the high-dose group compared with EAMG group and the low-dose group.6. Fasudil impairs antibody production and function in EAMG ratsThe production of anti-R97-116 IgG in EAMG rats was inhibited by fasudil at high dose, while rats in the low-dose group produced lower levels of IgG than those in the control group but without significant difference. The affinity of anti-R97-116 IgG was inhibited after fasudil treatment at low and high dose.Conclusions:Fasudil, a specific ROCK inhibitor, was effective in amelioration of EAMG. Fasudil treatment decreased Tfh cells and CD19+ B cells, especially GC B cells. The antibody response in EAMG was inhibited by fasudil as both the production and the function of anti-R97-116 IgG were impaired. Moreover, the expression of CD80 in lymph node MNC was inhibited by fasudil. Taken together, inhibition of the Rho/ROCK pathway led to suppressed antibody response and resulted in amelioration of ongoing EAMG.