Potential Therapeutic Targets Of Thymus And Humoral Immunity In Experimental Autoimmune Myasthenia Gravis

BackgroundMyasthenia gravis (MG) is an antibody-mediated,organ-specific autoimmune disease,characterized by anti-acetylcholine receptor (AChR) antibodies binding to acetylcholine receptors or to functionally related molecules in the postsynaptic membrane at the neuromuscular junction (NMJ),which lead to muscle weakness.Abnormal Tfh cells and GCs are involved in pathogenesis of MG.Follicular helper T (Tfh)cells,defined as CD4+CXCR5+ICOS+cells,maintain the germinal center (GC) and govern selection for GC B cells with increased affinity for antigen,as a result participating in the production of antibody.The chemokine receptor CXCR5 promotes the migration of Tfh cells into GC.In MG patients,thymic hyperplasia was associated with ectopic GCs,which contributes to intrathymic production of anti-AChR antibodies.Serving as a bridge between innate and adaptive immunity in the innate immune system,Toll-like receptors 9 (TLR9) detects the unmethylated CpG dinucleotides known as CpG motifs present in viral and prokaryotic genomes,whereas these dinucleotides are generally methylated in host DNA.Recognition of infection by TLR9 shapes adaptive immunity not only by directing dendritic cells (DCs) to activate naive T cells but promoting germinal centers (GCs) formation as well as B-cell activation and terminal differentiation into antibody-secreting plasma cells.The Oligodeoxynucleotide (ODN) H154 is a specific inhibitor of TLR9 signaling.It mimics the immunosuppressive activity of mammalian DNA and interferes with downstream signaling rather than by inhibiting the binding or uptake of CpG DNA.Recognition of infection by TLR9 shapes adaptive immunity not only by directing dendritic cells (DCs) to activate naive T cells but by promoting germinal centre (GC) formation as well as B-cell activation and terminal differentiation into antibody-secreting plasma cells.Recent studies have demonstrated that TLR9 activation substantially facilitated the GCs response and led to a greater proportion of high-affinity antibodies.As for MG,TLR9 expression was significantly higher in MG patients than in the healthy controls,and the mRNA expression of TLR9 is significantly higher in MG patients and positively related to the clinical severity of MG,implying that TLR9 may be involved in the pathogenesis of MGThe thymus plays a major role in the pathogenesis of MG with anti-acetylcholine receptor(AChR) antibodies.Several reports have illustrated the decreased amount and function of thymic regulatory T cells (Treg) in MG patients.nTreg cells generated in the thymus can migrate into the peripheral and suppress autoimmune responses.They play an important role in the maintenance of immune tolerance and homeostasis.Multiple factors can influence the development of thymic Treg cells.Differentiation of Treg cells is dependent on thymic stromal cells,which can be subdivided into thymic epithelial cells (TEC) and thymic dendritic cells(tDCs).Developing thymocytes are positively selected by cortical TEC (cTEC) and subsequently negatively selected by medullary TEC (mTEC) or tDCs.Treg cells are selected at the double positive (DP) stage through interaction with the MHC class ?-expressing cells within the thymic medulla,where they begin to express Foxp3 and differentiate into Foxp3+Treg cells.Exosomes,which are extracellular vesicles ranging from 30-150 nm in diameter,have been implicated in many aspects of autoimmune disease,as well as immune regulation involved in functions of T cells,B cells and DCs and so on.Previously,we have reported that statins had the ability to induce tolerogenic DCs that displayed the potential to regulate both cellular and humoral immunity to relieve clinical symptoms in experimental autoimmune myasthenia gravis(EAMG).We also used ultra-centrifugation to obtain statin-DC derived exosomes (statin-Dex)or DMSO-DC derived exosomes (DMSO-Dex).We demonstrated the ability of statin-Dex to ameliorate the clinical symptoms of EAMG rats.These therapeutic effects were associated with up-regulated levels of Foxp3+Treg cells in both the thymus and peripheral lymphoid organBased on the above evidence,the purpose is to find out new targets for MG.On one hand,we speculate that enhanced humoral immune response induced by abnormal activation of the TLR9 signaling pathway plays an important role in the pathogenesis of MG.However,few studies focused on the relationship of TLR9 and MG.On the other hand,the role of statin-Dex in inducing EAMG immune tolerance has been confirmed,but the mechanism is still unclear As the most important central immune organ,the thymus plays an important role in the pathogenesis of MG.We explored the immune regulation of TLR9 signaling pathway in MG and the regulatory mechanism of statin-Dex on thymic Treg in the study.Objective1.In this study,we used the EAMG rat model with or without interference of TLR9 signaling,to explore the mechanisms of TLR9 in EAMG immunopathology and to seek therapeutic approaches of human MG.2.The aim of this study was to explore how statin-Dex up-regulates Treg cells in the thymus in EAMG,which may provide a new strategy in the treatment of myasthenia gravis.MethodsPart ?1.Induction of EAMG rats and evaluation of clinical scores.Female Lewis rats were immunized by injection at the base of the tail with a 200 ?l emulsified R-AChR97-116 peptide followed by a boost on day 30.Before immunization (day 0) and thereafter every other day,body weights and clinical scores were recorded and assessed as follows:0,normal strength and no abnormalities;1,mildly decreased activity and weak grip or cry,more evident at the end of exercise;2,clinical signs present before exercise (tremor,head down,hunched posture,weak grip);3,severe clinical signs present before exercise,no grip,moribund;4,dead.2.ODN treatmentFollowing immunization,at days 5,12,19,26,30,37 and 44,each group of rats received intraperitoneal injection of 100 ?g GC-control containing ODN (5'-TCC-ATG-AGC-TTC-CTG-ATGCT-3'),or equal dose of suppressive H154-thioate-ODN (5'-CCT-CAA-GCT-TGA-GGGG-3').[1]3.Preparation of the lymph node and spleen mononuclear cellsRats were sacrificed on day 46 p.i.and their inguinal lymph nodes and spleens were removed under aseptic conditions.Single-cell suspensions of mononuclear cells (MNCs) from individual rats were prepared and re-suspended in complete medium for the following experiments.For spleen cells,erythrocytes were osmotically lysed and the remaining cells were resuspended in complete medium.4.In vitro treatmentsTriplicate aliquots of MNC suspensions were placed into 24-well round bottom plates,and stimulated with R-AChR97-116 in the presence of control or suppressive ODNs.After incubated for 48 h,cells were collected and prepared for FACS analysis.5.Flow cytometric analysis and cell sortingFor intracellular TLR9 analysis,MNCs were fixed with 2% paraformaldehyde for 20 min at 4? and permeabilized with intracellular permeabilization wash buffer.Then these permeabilized cells were incubated with anti-TLR9 antibody.FACS analysis was performed on BD FACScan.For isolation of B cells,spleen MNCs were prepared as previously described.After exclusion of T cell contamination,MNCs were purified through magnetic isolation to obtain B cells.The purity of B cells was>90%,as determined by flow cytometry6.ImmunofluorescenceLymph nodes and spleens were frozen in liquid nitrogen and embedded in OCT Compound at-80?.Sections (8 ?m) were incubated with PNA,IgM,and TLR9,respectively.All images were captured with fluorescence microscopy7.ELISASerum was obtained on day 46 p.i.and anti-R-AChR97-116 antibody levels were assessed by a standard ELISA technique.Meanwhile,serums at different time points (days 16,26,36,and 46 p.i.) were collected for detection of anti-R-AChR97-116 peptide IgG antibody8.Statistical analysisStatistical analysis of the data was performed using SPSS 22.0 software.A two-tailed unpaired Student's t test with a 95% confidence interval or a nonparametric two-tailed Mann-Whitney test was used to calculate P-values.Error bars represent standard deviation (SD)Part II1.EAMG induction and exosome administrationEAMG models were induced by a subcutaneous immunization at the base of the tail (two sites) with AChR 97-116 peptide emulsified in Complete Freud Adjuvant containing Mycobacterium Tuberculosis.Female Lewis rats were divided into three groups.One group of rats were used as PBS control;the other two groups received exosomes derived from either DMSO or statin-treated BMDCs.The exosomes were administered on day 5,10,and 15 p.i..The rats were sacrificed on day 20 p.i.2.The culture of bone marrow dendritic cell and isolation of exosomesTo isolate and culture BMDCs,tibias and femurs were removed from 6-8 week old Lewis rats.The whole bone marrow was flushed out and filtered through a 70 ?m Cell Strainer.After lysis of erythrocytes,cells were re-suspended in medium containing recombinant rat (rr) GM-CSF and rrIL-4.After a 72 h incubation,non-adherent cells were gently removed and adherent cells were further cultured.On day 7,non-adherent and loosely adherent cells were harvested and incubated with atorvastatin dissolved in dimethylsulfoxide (DMSO) or DMSO (only in medium).After 48 h cultivation,the supernatant was collected for exosomes isolation.To isolate and identify exosomes,the supernatant was centrifuged at 2,000 g for 10 min and 10,000 g for 30 min to remove whole cells and debris.The resultant supernatant fluid was then centrifuged at 100,000 g for 70 min.Exosome pellets were rinsed with PBS and re-centrifuged at 100,000 g for 70 min.Finally,exosomes were suspended in sterile PBS and quantified by the K5600 MicroSpectroPhotoMeter.Exosomes were stored at -20?.3.Identification of exosomesFor transmission electron microscopy analysis,carbon-coated copper grids were placed in the exosome suspensions fixed with 2% paraformaldehyde overnight.The grids were then negatively stained by 2% phosphotungstic acid for 5 min,and air-dried for 1 min at room temperature.The exosome samples were visualized by a Tecnai 20U-TWIN operated at 80 kV.The exosome size was measured by dynamic light scattering (DLS).Briefly,exosomes(1 ?g) were re-suspended in 1mL-filtered PBS at pH 7.4.The sizes of the particles were analyzed by DLS Nano sizer.4.Tracking analysis in the thymusPurified exosomes were labeled with membrane-targeted red fluorescent dye PKH26.For uptake studies,labeled exosomes were injected to EAMG rats.Following 3 days of intravenous injection,thymuses removed from rats in different groups were visualized with a fluorescence microscope to determine the distribution of exosomes.5.Preparation of thymic stromal cellsThymuses were dissected from freshly killed rats and trimmed of fat and connective tissue.Tissues were gently agitated on 70-?m filter with a vitreous stirrer.Thymocytes were collected under the filter.The thymic stromal cells were isolated through enzyme digestion.Both thymocytes and thymic stromal cells were counted and prepared for antibody labeling.6.In vitro Treg cells induction experimentsTo determine the functions of exosomes on the differentiation of thymic Treg cells,thymocytes were cultured with exosomes and TECs,exosomes and BMDCs,or exosomes only.After digestion described above,thymic CD45- stromal cells were labeled and sorted by FACS.Thymocytes and BMDCs or CD45- stroma cells were suspended in the medium with exosomes.After 2 days,the cells were collected and the percentages of CD4+Foxp3+ T cells were determined by FACS.7.FACSFor cell surface marker analysis,thymic TEC and DCs were stained using conventional methods.tDCs were defined as OX62 positive cells,cTEC as CD45-APA+,and mTEC as CD45-UEAl+.For Treg cells,thymocytes were stained with CD4,CD8 and CD25 before being fixed and permeabilized with Perm/Fix solution.Finally,antibody specific for Foxp3 was added for 30 min in the dark.8.ImmunofluorescenceThymus tissues were cryo-sectioned and processed for the immunofluorescence (IF) assay.Samples were incubated with antibody Aire.The fluorescence microscopy examination was performed in a blinded manner.9.Western blottingA western blot was performed to determine thymic Aire from thymus tissues.Anti-Aire and anti-GADPH were used as primary antibodies;horseradish peroxidase-coupled goat anti-mouse or goat anti-rabbit as secondary antibodies.Bands were quantified with Image J software.10.Statistical analysisStatistical tests were performed using GraphPad Prism 7.0.Differences between controls and experimental groups were determined with one-way ANOVA and followed by Bonferroni test as a post-hoc test.P <0.05 was considered statistically significant.ResultsPart ?1.Inhibition of TLR9 signalling suppressed the development of EAMGThe average clinical scores of H154-treated rats were lower than control ODN-treated rats from days 34 to 46 post immunization (p.i.),even though disease in both groups occurred at the same time (days 34 p.i.).At the end of the experiment,the mean clinical score was 0.7±0.27 in the H154 group,and it was 1.58± 0.20 in the control group.The differences between the two groups were statistically significant on days 36,38,42,44 and 46 p.i..2.Blocking the TLR9 signaling impaired antibodies production in EAMGRats in the H154 group had lower levels of not only IgG?*P<0.05),but IgG2a and IgG2b(*p<0.05 and p=0.62,respectively).Moreover,the serum antibody levels of IgG maintained an increasing trend along with the disease progression,especially the outburst after boost immunization,ascertaining the EAMG animal model could mimic the clinical illness.3.Suppressive ODN reduced both Tfh and germinal center B cells in spleenThe percentages of Tfh cells among spleen CD4+T cells were significantly suppressed by H154.In addition,we observed lower B cell frequency in spleen GCs with H154 treatment than in the control group.We further confirmed the downregulation of B cell clusters in GC in the H154-treated group with immunofluorescence.However,we did not find significant differences in percentages of plasma cells between these two groups.These results suggest that blocking TLR9 signalling could efficiently inhibit humoral immunity in the spleen.4.Suppressive ODN inhibited Tfh in lymph nodes of EAMG ratsIn lymph nodes,only levels of Tfh decreased in the H154 group,with no significant difference in other items in humoral immunity.5.Suppressive ODN decreased the percentages of both B cells and DCs among spleen cells.Both percentages of B ceils and DCs were reduced by TLR9 interruption,which might account for the downregulation of Tfh in the H154 group.6.Suppressive ODN downregulated spleen TLR9+B cells as well as the expression of TLR9 on spleen B cells in vivo and in vitro,and altered the phenotype of B cells in vitro.Rats in the H154 group showed decreased percentages of TLR9+B cells among spleen MNCs,as well as depressed TLR9 expression in B cells (**p<0.01).However,the data in lymph nodes showed no significant difference (data not shown).The expression of TLR9 was assessed by immunofluorescence and was found an obvious localization in the B cell zone in lymph nodes.Furthermore,consistent with results in vivo,we observed a decline in the level of TLR9 percentages as well as the mean fluorescence intensity(MFI) both in lymph nodes and spleens B cells after H154 treatment in vitro.Next,we sorted B220+B cells from spleen MNCs and treated with H154.The expression of TLR9 and surface marker MHCII was analyzed.H154 made both TLR9 and MHC ? decline on B cells,suggesting H154 could directly inhibit TLR9 expression and influence the B cell activation.Part ?1.Identification of exosomes derived from DCs culture medium.The diameters of particles in the DMSO-Dex and the statin-Dex groups were primarily distributed in the range of 70-140 nm.The morphology observed under an electron microscope was shown to be intact,with the diameter about 100 nm.To confirm the exosome-enriched fractions,TSG101 (a well-established marker for exosomes) was determined by western blot.Exosomes from both the DMSO-Dex and the statin-Dex groups expressed the TSG101 with size of 49 kDa.2.Exosomes are distributed mostly in the cortex and the junction between cortical and medulla in the thymusExosomes were mainly located at the cortex and the junction between the cortex and medulla within the thymus.There is no difference in the distribution between the DMSO-Dex group and the statin-Dex group.Furthermore,exosomes were shown to be engulfed by the keratin positive thymic epithelial cells.3.Statin-Dex increases Foxp3 expression in thymocytesThere were no statistical differences in CD4+CD8-CD25+thymocytes (thymic Treg cells)among the three groups.However,the percentage of Foxp3+Treg cells among CD4+CD8-thymocytes was significantly higher in the statin-Dex group than in the PBS-treated control group.4.MHC ? and B7 molecules remain unchanged in the control group,the DMSO-Dex group,and statin-Dex groups.There were no significant differences among the three experimental groups,suggesting that MHC ? and B7 molecules may not be involved in the regulation of Foxp3+Treg cells by exosomes.5.DC-derived exosomes up-regulate CD40 expression on thymic medullary epithelial cell and thymic DCs.Compared to the control group,both DMSO-Dex and statin-Dex increased the expression of CD40 on the mTEC and tDCs,but there was no alteration of CD40 expression on the cTEC in the thymus.6.Statin-Dex increase Aire in the thymic medulla.Immunofluorescence results demonstrated an obviously increased Aire expression in thymic medullary epithelial cells in the statin-Dex group when compared to both the control group and the DMSO-Dex group.Increased Aire expression in the thymus in the statin-Dex group was further confirmed by the western quantitative analysis compared to the control group and the DMSO-Dex group.7.DC-derived exosomes promote the development of Foxp3+Treg cells among thymocytes by TEC in vitro.Thymocytes were cultured with DMSO-Dex,statin-Dex and PBS respectively and there was no difference in the percentages of Foxp3+Treg cells among the three experimental groups,which indicates that exosomes may not directly affect Foxp3+Treg cells differentiation in the thymus.Then,BMDCs and thymocytes were co-cultured with DMSO-Dex,statin-Dex and PBS,respectively.After 48 hours of cultivation,no differences in the percentages of Foxp3+ Treg cells in the three experimental groups were observed,which suggested that exosomes could not promote Foxp3+ Treg cell expansion through regulation of DCs.CD45- cells (mainly TEC)were isolated and co-cultured with thymocytes in the presence of PBS,DMSO-Dex and statin-Dex.The percentages of both CD4+Foxp3+ Treg cells among total thymocytes and Foxp3+ Treg cells among CD4+ thymocytes were higher in the two exosome groups compared to the control.Taken together,these data provided strong evidence that upregulated Foxp3+Treg cells by exosomes in thymocytes is mainly mediated by TEC.Conclusions1.The TLR9 pathway may be critical for the pathogenesis of EAMG.Interfering with the TLR9 pathway using suppressive ODNs H154 decreased Tfh cells and GC B cells,abated anti-AChR antibody production and class switch,and terminally alleviated myasthenia manifestation.2.We confirmed the effects of statin-Dex in inducing Foxp3+Treg cells.Both statin-Dex and DMSO-Dex could up-regulate CD40 but only statin-Dex increases Aire expression in thymic stromal cells in vivo.The role of statin-Dex and DMSO-Dex in the induction of Foxp3+Treg cells was dependent on epithelial cells in vitro.