| BackgroundGuillain-Barre syndrome(GBS)is an acute immune-mediated inflammatorydemyelinating disease of the peripheral nervous system,presenting as rapidly progressive symmetric motor dysfunction in the limbs,hyporeflexia or areflexia,often accompanied with dysfunction of autonomic and sensory nervous system.Histological examination of peripheral nerves shows demyelination,axon fracture,and inflammatory cell infiltration including activated macrophages,and T lymphocyte.The exact pathogen and pathogenesis are still not clear.Despite most patients have good outcomes after conventional treatments like plasma exchange and intravenous immunoglobulin therapy,some patients still die of severe complications of EAN.Therefore,more acceptable and efficacious therapy is needed.Immune cell activation and differentiation occurs concurrently with metabolic reprogramming to meet the energy and substrate needs in the immune responses.These changes offer a promising opportunity for selective regulation of immune subsets,and shine a light on the therapy of autoimmune diseases.Enhanced glycolysis is usually related to the differentiation and function of inflammatory cells,for example,hypoxia-inducible factor la(HIF-la)-dependent glycolystic pathway orchestrates a metabolic checkpoint in Th17 differentiation;pyruvate kinase 2(PKM2)participates in the inflammatory cytokine secretion of macrophages;mammalian target of rapamycin(mTOR)-dependent glycolysis contributes to the differentiation of T follicular helper(Tfh)cells.Although the enhanced glycolysis has been reported in systemic lupus erythematosus(SLE)model and autoimmune arthritis model,up to now,no research about the metabolic reprogramming in GBS can be found.Experimental autoimmune neuritis(EAN)is the classic model of GBS,widely used for the pathogenesis study and new therapy exploration.Considering the major pathogenic cells of EAN as Thl,Thl7,Tfh cells and activated macrophages depend on glycolysis to differentiate and exert function,we assumed that glycolysis contributed to the pathogenesis of EAN,and glycolysis inhibition would inhibit the initiation and progression of EAN by affecting the differentiation of lymphocytes and the activation of macrophages.As a glucose analogue,2-Deoxy-D-glucose(2-DG)is competitively phosphorylated to 2-DG-phosphate which cannot be further metabolized.The accumulated 2-DG-phosphate leads to the inhibition of glycolysis.As the inhibitor of glycolysis,2-DG has been widely used in the experimental studies.ObjectiveTo explore the roles of glycolysis in the pathogenesis of EAN,we intend to apply 2-DG in preventive and therapeutic patterns and evaluated clinical scores,pathological changes,cellular and humoral immune responses in vivo.Effects of glycolysis inhibition on DC and macrophages will to be explored in vitro also.Methods1.Induction of EAN and evaluation of clinical signsTo induce EAN in rats,200?l inoculum containing BPM,Freud adjuvant and Mycobacterium tuberculosis was injected into the base of tail subcutaneously.Clinical scores were graded as follows:0 = normal;1 = reduced tonus of the tail;2 = partial tail paralysis;3 = complete tail paralysis or absent righting reflex;4 = gait ataxia;5 =mild paresis of the hind limbs;6 = moderate paraparesis;7 = severe paraparesis of the hind limbs;8 = tetraparesis;9 = moribund;10 = death.2.2-DG treatmentFor preventive treatment,the rats were divided into control group and preventive group.2-DG solution was administered daily from the day of immunization to day 13 post-immunization(p.i.)when the symptoms peaked.For therapeutic treatment,the rats were divided into control group and therapeutic group.2-DG was given daily from day 10 p.i.when the first clinical sign appeared,to day 21 p.i.when all rats showed signs of recovery.Weigh and assess all the rats every day.At the end point of the experiments,serum,ingunal lymph nodes,spleens and sciatic nerves were harvested for further study.3.Histopathological assessmentSciatic nerves from both groups were harvested,fixed,dehydrated,embedded with paraffin and sliced.Hematoxylin-eosin(H&E)staining to evaluate the inflammatory infiltration.Immunohistochemistry staining with anti-CD68 antibody was performed to investigate the macrophage infiltration.Luxol fast blue(LFB)staining were applied to evaluate the demyelination.The histological scores were acquired according to a semiquantitative grading system:0 = normal;1 = demyelinated fibres less than 25%;2 = demyelinated fibres 25-50%;3 = demyelinated fibres 50-75%;4 = demyelinated fibres more than 75%.4.Maturation of dendritic cells and lymphocyte subset distributionThe bilateral lymph nodes and spleens were harvested and grinded,and after erythrocytes deletion by Osmotic lysis method,the mononuclear cells were prepared for further study.Surface staining of CD4,CD25,OX62,MHC II,CD86,CD80,CXCR5,ICOS and intracellular staining of Foxp3,IFN-? and IL-17 were carried out for flow cytometry.The maturation of dendritic cells were evaluated by the expression of MHC II,CD86 and(or)CD80 on OX62 positive cells.Treg cells were marked with CD4,CD25 and Foxp3 staining,and Tfh cells were marked with CD4,CXCR5 and ICOS staining.After staining,the cells were fixed with paraformaldehyde and detected by flow cytometry.5.Anti-BPM antibody by ELISAFlat-bottomed polystyrene 96-well plates were first coated with BPM.Then,the levels of anti-BPM antibody in the serum were examined by ELISA.6.Measurement of glucose-uptakeTo obtain activated macrophages,peritoneal macrophages were first stimulated by Lipopolysaccharides(LPS).Then,the glucose-uptake activity was measured by fluorescent d-glucose analogue with flow cytometry.the culture medium was harvested for the examination of glucose concentration and pH values.7.Maturation of dendritic cells after glycolysis inhibition in vitroTo acquire DC,bone marrow cells were induced by GM-CSF and IL-4 for 7 days.Then the floating cells were harvested and treated with LPS with or without 2-DG The expression of MHC ?,CD80 and CD86 was detected by flow cytometry.8.Phagocytosis of macrophages after glycolysis inhibition in vitroThe peritoneal macrophages or LPS-stimulated RAW264.7 cells were first treated with 2-DG.The differences in the phagocytosis activities were determined by the uptake of fluorescent dextran assessed by flow cytometry.9.NO-production assay in vitroFor NO-production assay,the isolated peritoneal macrophages and RAW264.7 cells were stimulated by LPS with or without 2-DG.All the above culture medium was harvested and examined with nitric oxide(NO)assay kit according to the instruction manual.10.TNF-a secretion in vitro by ELISAAfter LPS stimulation on the peritoneal macrophages pretreated by glycolysis inhibitor,the level of TNF-a was determined by ELISA.11.Statistical analysisStatistical analysis was performed with GraphPad Prim 6.0.Differences between two groups were tested by two-tailed Student t test and among three groups by one-factor analysis of variance(ANOVA).Data were expressed as mean ± SD,and p<0.05 was considered significant.Results1.2-DG inhibited the increased glycolysis in LPS-stimulated peritoneal macrophagesUsing fluorescence-labeled glucose analogue,2-NBDG,we found that after stimulation,glucose-uptake was significantly increased in the peritoneal macrophages.Meanwhile,the glucose concentration and the pH values of the culture medium declined significantly,which were both inhibited by 2-DG.2.2-DG inhibits the initiation of EAN and the inflammatory infiltrationCompared to the rats of control group,the rats treated with 2-DG developed symptoms later,and lost less weight.The clinical scores and weight loss differed significantly on day 10,11,12 and 13 post immunization.The infiltrated mononuclear cells in the sciatic nerves of 2-DG group were less than control group showed by H&E staining,as well as the CD68 positive macrophages showed by immunohistochemical staining.3.Preventive treatment of 2-DG modulates both cellular and humoral immune response in vivoResults showed a significant decrease in the percentages of Thl cells,Th 17 cells and Tfh cells(CD4+CXCR5+ICOS+),accompanied by a significant increase in the percentage of Treg cells,after 2-DG treatment.2-DG treatment also significantly reduced the expression of MHC ? on the spleen DC and the level of anti-BPM antibody in the serum.4.2-DG halts the progression of ongoing EAN and improves histological changes2-DG treatment nearly completely suppressed EAN progressing from day 13 to 21 p.i..The maximal clinical score was significantly reduced in 2-DG group than that in control group.2-DG-treatment also significantly attenuated the loss of body weight frorm day 13 to 21 p.i.There's no difference in the amount of infiltrated mononuclear cells between both groups,while the demyelination was reduced in 2-DG-treated rats.5.Therapeutic treatment of 2-DG modulates immune response in vivoCompared with control group,there is no significant inhibition of Thl or Thl7 cells after therapeutic treatment with 2-DG While,the percentage of Treg cells was increased and the expression of MHC class ?I on OX62+ DC was also inhibited following 2-DG treatment.6.2-DG inhibits the LPS-stimulated maturation of dendritic cells in vitro2-DG significantly decreased the LPS-induced expression of MHC ?,CD80,CD86 on DC determined by FC.7.2-DG inhibits phagocytosis and NO-production of macrophage in vitroData showed 2-DG significantly inhibited the uptake of FITC-dextran by the isolated peritoneal macrophages and the LPS-activated RAW264.7 cells.NO-production of both peritoneal macrophages and RAW264.7 cells was significantly reduced by 2-DG treatment,but reversed by glucose addition.ConclusionThe enhanced Glycolysis may be involved in the pathogenesis of EAN.Glycolysis inhibition inhibits the initiation of EAN and halts the progression of ongoing EAN.The underlying mechanisms include suppressing Thl,Thl7,Tfh cells development,promoting Treg cells differentiation,inhibiting DC maturation and decreasing the NO-production and phagocytosis of macrophages.Glycolysis or the pathway underlying may be a target in GBS therapy.BackgroundMyasthenia gravis(MG)is an antibody-mediated,T-cell-dependent and complement-involved autoimmune disease.The production of function-blocking antibody against the acetylcholine receptor(AChR)at the neuromuscular junction(NMJ)leads to loss of AChR and the neuromuscular transmission failure,thereby triggering excessive muscle weakness and fatigue.The current therapies for MG including corticosteroids,immunosuppressive drugs(azathioprine,tacrolimus,cyclophosphamide etc.)and thymusectomy are effective in most patients,but not in all.Therefore,new target for MG treatment is needed.Experimental autoimmune myasthenia gravis(EAMG)of rat can be induced by immunization with torpedo acetylcholine receptor(TAChR)or a synthetic peptide corresponding to the rat AChR?97-116(R97-116)region,which is a reliable model for investigating novel therapeutic strategies.Proteasomes,multisubunit proteases,are responsible for the degradation of many abnormal/misfolded proteins in every eukaryotic cell.In vertebrates,each catalytic subunit of the proteasome is encoded by two genes.Most cell types express the constitutive subunits(?1c,?2c,?5c),however,in dendritic cells(DCs),lymphocytes and monocyte-derived cells,the constitutive subunits are replaced by the immunosubuits(?1i,?2i,?5i)to form immunoproteasomes.Additionally,immunoproteasomes can also be induced in most cell types after exposue to inflammatory cytokines,such as interferon(IFN)-y and tumour necrosis factor(TNF)-a.Accumulating evidence reveals that immunoproteasomes contribute to the regulation of cytokine production(IL-6,IFN-?,TNF-a and IL-23)and the differentiation of Thl,Th17 and Treg cells.ONX-0914,a newly developed selective inhibitor of the immunosubit ?5i(also known as low-molecular mass polypeptide-7(LMP7)),has shown inspiring therapeutic effects in animal models of rheumatoid arthritis,systemic lupus erythematosus,multiple sclerosis,Hashimoto's thyroiditis and inflammatory bowel disease.Whether ONX-0914 brings benefits to EAMG or MG has not been reported.ObjectivesWith the administration of the selective immunoproteasome inhibitor ONX-0914 and the assess of clinical symptoms and immune responses of EAMG,we tried to explore the feasibility of immunoproteasome inhibition for MG treatment.Methods1.Immunohistochemistry on thymic sectionImmunohistochemistry was carried out on the sections of MG patient's thymus,including deparaffinizing,antigen retrieval and antibodies incubation etc..Counterstaining was performed with hematoxylin to check the expression of LMP7 in the ectopic germinal centers in the MG thymus.2.Induction and clinical assessment of EAMGFemale Lewis rats,aged six to eight weeks were selected for EAMG induction.Lewis rats were anesthetized by isoflurane and immunized subcutaneously at the base of tail with inoculum containing R97-116 peptide,Mycobacterium tuberculosis in incomplete Freund's adjuvant on day 0 followed by a boost on day 30 only with the same peptide in incomplete Freund's adjuvant.Clinical scoring of both groups were evaluated every other day,according to Baggi F described.3.ONX-0914 treatmentThe EAMG rats were divided into control group and treatment group.For ONX-0914 administration,ONX-0914 were dissolved and administered every 3 days throughout the course of the experiment,starting from Day 5 post immunization(p.i.).4.Flow cytometric analysis of spleen and lymph node MNCFor mononuclear cell(MNC)preparation,the spleen and bilateral inguinal lymph nodes of individual rat were harvested and grinded through the cell strainer.Osmotic lysis method was used to deplete the erythrocytes to get spleen MNC.Surface staining of CD4,Peanut Agglutinin(PNA),B220,OX62,MHC II,CD86,CXCR5,ICOS and intracellular staining of Foxp3,IFN-y,IL-4 and IL-17 were carried out for flow cytometry.The maturation of dendritic cells were evaluated by the expression of MHC II,CD80 and CD86 on OX62 positive cells.Treg cells were marked with CD4 and Foxp3 staining,and Tfh cells were marked with CD4,CXCR5 and ICOS staining.Germinal center B cells were marked with PNA and B220.After staining,the cells were fixed with paraformaldehyde and detected by flow cytometry.5.Detection of levels and affinity of anti-R97-116 IgG by ELISABloods were harvested to get the serums in different time points.The levels of anti-R97-115 IgG were detected by ELISA,and the relative affinity of serum anti-R97-116 antibodies was determined by ELISA using thiocyanate elution.6.Cytokine assay by ELISASpleen MNC from both EAMG or ONX-0914 group rats were cultured in the presence of AchR R97-116 sterilely.The supernatant was harvested for IFN-? and IL-17 detection by ELISA.7.Statistical analysisStatistical analysis was performed with GraphPad Prism 6.Differences between two groups were tested by two-tailed Student t test.Results were displayed as means±SD.The significance level was set at p<0.05.Results1.LMP7 was expressed in the ectopic germinal centers of MG patient 's thymusHigher expression of LMP7 in the ectopic germinal center was observed in the MG thymus compared to the tissue nearby and the normal thymus by immunohistochemistry.2.LMP7 inhibitor ONX-0914 suppressed the development of ongoing EAMGAfter ONX-0914 administration,rats in the treatment group exhibited significantly lower milder clinical symptoms on day 34,38,46 post immunization and in the later days,than control group.3.ONX-0914 treatment effected autoantibody production and antibody affinityWe found that ONX-0914 significantly inhibited the antibody production on day 25 p.i..However,after the second immunization at day 30,the level of anti-R97-116 IgG rapidly increased to be the same with EAMG group on day 35 and 50 p.i..However,the affinity of antibody against R97-116 was still significantly brought down on day 50 p.i..4.ONX-0914 decreased Tfh cells and germinal center B cellsAfter ONX-0914 administration,Tfh cells marked as CD4+CXCR5+ICOS+ cells were significantly reduced,as well as GC-B cells characterized by PNA and B220 staining,determined by FC.5.ONX-0914 decreased DC and MHC ?+ cellsONX-0914 treatment significantly decreased the percentage of OX62+DC in the spleens and the MHC ?+ cells in the lymph nodes,determined by FC.6.ONX-0914 treatment altered Th subset distributionONX-0914 significantly reduced Th17 cells and the section of IL-17 in the spleen MNC,accompanied with the mild increase in Thl cells.While,no difference of Thl and Treg cells was observed.ConclusionONX-0914 significantly reduced DC,MHC ?+ cells,inhibited the differentiation of Tfh and Th17,and declined the relative affinity of anti-R97-116 antibody,ameliorated the severity of EAMG.Therefore,immunoproteasomes may be a new target for MG treatment. |
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