Transplantation Of Mesenchymal Stem Cells Could Affect The Function Of CD4~+CD25~+Foxp3~+Regulatory T Cells In Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a chronic, disabling disease of the central nervoussystem (CNS), It is characterized by chronic neuroinflammation with lymphocyteinfiltration into the CNS, myelin loss, liosis, various degrees of axonal andoligodendrocyte pathology and progressive neurological dysfunction. EAE is anautoimmune disease model of MS that has been used to study the mechanism of MSpathogenesis and to test the efficacy of potential therapeutic agents for the treatmentof MS. Lots of study in immunopathogenesis confirmed that autoantigen of myelinprotein in patients were recognized by CD4+T cell and induced immune response.The immune cells were abnormally activated and transmigrated into central nervoussystem then interacted with the cells expression of self-myelin protein antigens inCNS, and the final led to demyelination of CNS via a series of inflammatory factor.The demyelination of CNS was concerned with the overavtivity of effector T cells,infiltration of inflammatory cells in the CNS, increased production ofproinflammatory cytokines and inflammatory mediums. Cytokines such as interferon(IFN)-γ、tumor necrosis factor (TNF)-α、IL-2、IL-17produced by Th1and Th17cellscould agrevate the illness, even IFN-γ could induce demyelination and neuronsdamage. Th2cell and CD4+CD25+regulatary T cells (Treg) could inhibit the autoimmune reaction and limiting disease progression via production of cytokinessuch as IL-10、TGF-β1、IL-4. It has been studied that both the number and immunesupression function of Tregs were reduced in EAE mice, when input functional Tregsinto the mice, the pathogenetic condition were improved.Mesenchymal stem cell, a kind of somatic stem cell, widely existed in manyissues such as bone marrow and liver. MSCs are multipotent, self-renewing cells, andhave potentiality of multidirection differentiation. It can differentiate into varioustissues of mesodermal origin such as osteocytes, chondrocytes, adipocytes. The cellscould be easily obtained, have great expansive potential in vitro, and have weakantigenicity, so as regeneration source cells, MSCs have a good clinical applicationprospect. MSCs were also found to possess significant immunoregulatory activities.MSCs could supress the proliferations of T cell, B cell and NK cell, induceproduction of Tregs, and also could inhibit the maturation and function of antigenpresenting cell (APC). Various studies showed that MSCs have a potentialapplication prospect in the treatment of autoimmune diseases, but the mechanismsmediating such effects are still not clear. So that’s the purpose of this study.Materials and Methods1. AnimalsFemale C57BL/6J mice,6to8weeks old, and males BALB/c mice,6to8weeks old, were purchased from experimental animal institute, Chinese academy ofmedical science. Mice were housed in department of microbiology and immunology,Zhengzhou University, China. Mice were maintained in a pathogen-free environment.All work was performed in accordance with the guidelines for animal use and care.2. AntigenMouse MOG35-55peptide (MEVGWYRSPFSRVVHLYRNGK) was synthesized by Shanghai Sangon Biologocal Engineering Technology Co.,Ltd and purityconfirmed by HPLC.3. Induction of EAE and assessment of nervous functionEAE was induced in C57BL/6J mice by immunization with an emulsioncontaining200μg of purified MOG peptide in PBS and an equal volume of completeFreund’ adjuvant (CFA, Sigma) containing800μg of nonviable desiccatedMycobacterium tuberculosis H37RA (Difco). A final volume of0.2ml emulsion wasinjected subcutaneously at4sites over the flanks at day of induction (day0) and atday7. In addition,300ng of Pertusis toxin (Sigma) in0.2ml phosphate buffered saline(PBS) was injected intraperitoneally at day of induction and at day2. After EAEinduction, mice were scored daily for EAE clinical signs, according to the followingscore (16):0, no signs;1, partial loss of tail tonicity;2, tail paralysis and hindlimbweakness;3, hindlimb paralysis;4, hindlimb paralysis and forelimb weakness orparalysis;5, death due to EAE.4. Isolation and culture of mice MSCsBone marrow cells of C57BL/6J and BALB/c mice were flushed from micefemur and tibias with PBS. Red blood cells were lysed and the remaining bonemarrow cells plated in MSC expansion media and were incubated at a concentrationof106/ml at37℃in a5%CO2atmosphere. After72hours, non-adherent cells wereremoved. When the plate was80%~90%confluent, adherent cells were trypsinizedand split. Adherent MSCs were selected and expanded for10to15days. Cells werethen trypsinized and used for tail vein injections into host mice (106cells injected permouse).5. Transplantation of MSCsForty C57BL/6J mice were divided equally into4groups: A, B, C and D. Themice in group A, B and C were used for induction of EAE. Twenty and twenty-two days post EAE induction, MSCs (106MSCs in a volume of200μl) or the samevolume of PBS were injected into each mouse tail vein. The differences: MSCs fromBALB/c mice were injected into mice in group A, MSCs from C57BL/6J mice wereinjected into mice in group B and PBS were injected into mice in group C as the EAEgroup. No treatments of the mice in group D were acted as normal control.6. Flow cytometric analysisCells were isolated from thymus, spleens, mesenteric and popliteal lymphnodes of mice40days after MSCs transplantation. Single-cell suspensions wereprepared by mechanical disruption and RBC were removed from the samples withlysis buffer. The cells were washed3times in PBS using centrifugation resuspendedin PBS containing1%BSA (Sigma-Aldrich) and0.1%sodium azide. For surfacestaining of CD4and CD25, cells were incubated with fluorochrome-conjugatedantibodies (FITC-conjugated anti mouse CD4mAb0.125μg/106cells and APC-conjugated anti mouse CD25mAb0.06μg/106cells, eBioscience) to the indicated cellsurface markers at the recommended dilution of isotype control antibodies for30minutes on ice. For intracellular staining of Foxp3, cells were fixed andpermeabilized with Foxp3staining buffer (eBioscience). Permeabilized cells stainedwith PE-conjugated anti-mouse Foxp3mAbs (0.5μg/106cells; eBioscience). Stainedcells were analyzed subsequently using a FACSCalibur (BD).7. Detected the expression of Foxp3, TGF-TGF-β1and IL-10mRNA by Realtime (RT)-PCRTotal RNA was isolated respectively from107cell pellets of thymus, spleensand lymph nodes using Trizol reagent (Invitrogen) at20,40and60days after MSCstransplantation. The first strand cDNA was subsequently synthesized using RT Kit(Takara) according to the manufacture’s instructions. Expression of Foxp3, TGF-β1and IL-10mRNA were determined by RT-PCR using SYBR green I real time PCR Kit (Takara).8. Statistical analysisThe results were expressed as means±SD. Data were analyzed by analysis ofvariance (ANOVA) using SPSS13.0. A value of p<0.05was considered as asignificant difference.Results1. Characterization of MSCs after incubation and proliferation in vitroThe morphology of MSCs isolated from primary cells after24hour’sincubation were small and round, after3-4days, part of the cells attached to theculture plates and displayed a spindle-shape and a strong refraction. By days7-10,more cells attached and gradually grew to form scattered or aeratus and the size of thecells had increased. Passage of the cells were cultured continually, the number andgrowth velocity of the cells increased gradually. Cells from3to4passages acted astransplanting MSCs.2. Transplantation of MSCs could improve the clinical score of the EAE miceMice were scored daily for EAE clinical signs. The results showed that in EAEgroup, at14days after EAE induction, the animals began to show clinical signs:developed quickly from tail paralysis (score1) to hindlimb aralysis (score2) and evenhindlimb paralysis (score3), and also part of the animals showed forelimb weakness(score4) in5days. In the allogenic transplantation group and autogentictransplantation group, the results showed that the clinical score increased from14to20days after MOG immunization and decreased after MSCs transplantation (20daysafter immunization). The clinical score decreased lowest and maintained this level20days after MSCs injection. The scores had significantly decreased at20days afterMSCs injection in allogenic transplantation group and autogentic transplantation group as compared with EAE group（p<0.01）.3. Transplantation of MSCs could affect the levels of CD4+CD25+Foxp3+T cellsin spleen, lymph nodes and thymus in EAE mice.Thymus, spleens, mesenteric and popliteal lymph nodes were collectedrespectively from of mice at20days after MSCs transplantation. Single-cellsuspensions were prepared and stained with fluorochrome-conjugated antibodies foranalysis of CD4+CD25+Foxp3+T cells. Our results showed that a significantly lowerfrequency of splenic CD4+CD25+Foxp3+T cells was observed in the EAE group ascompared with the normal control (p=0.032), allogenic transplantation group (p<0.01)and autogentic transplantation group (p=0.018). The frequency ofCD4+CD25+Foxp3+T cells from lymph nodes in EAE group was significantly lowerthan that of normal control, allogenic transplantation group and autogentictransplantation group (all p<0.01). The frequency of thymic CD4+CD25+Foxp3+Tcells in EAE group was also significantly lower than that of in normal control,allogenic transplantation group and autogentic transplantation group (p=0.025, p<0.01, p=0.036).4. Alteration of Foxp3, TGF-β1and IL-10mRNA by MSCs transplantation inEAE mice.Total RNA was isolated respectively from107cell pellets of thymus, spleens at20,40and60days after MSCs transplantation. Expression of Foxp3, TGF-β1andIL-10were detected by RT-PCR. The results showed that a stable expression ofFoxp3, TGF-β1and IL-10mRNA were displayed in the spleen, lymph nodes andthymus of normal control. Low expression of Foxp3mRNA were showed in thespleen, lymph nodes and thymus of EAE group at20days after MSCs transplantation,then decreased gradually and became lowest when60days after transplantation.Significantly difference were showed at60days after transplantation in the spleen, lymph nodes and thymus of EAE group compared with normal control (All p<0.01).Low expressions of Foxp3mRNA were showed at20days after MSCstransplantation in the spleen, lymph nodes and thymus of the allogenic transplantationgroup, then increased gradually to the normal level and had significant difference inthe three organs compared with EAE group at60days after transplantation (Allp<0.01). Similar results were displayed in the autogentic transplantation group (Allp<0.01vs EAE group in the spleen, lymph nodes and thymus at60days after MSCstransplantation). Lower expression of TGF-β1mRNA were displayed in the spleen,lymph nodes and thymus in the EAE group, allogenic transplantation group andautogentic transplantation group at20days after MSCs transplantation. In the EAEgroup, the level of TGF-β1mRNA decreased gradually and reached lowest at60daysafter MSCs transplantation (p<0.01vs normal control). The levels of TGF-β1mRNAin the allogenic transplantation group and autogentic transplantation group increasedgradually to the normal level and had significant difference compared with EAEgroup at60days after transplantation (All p<0.01vs EAE group in the three organs).We found that similar altering tendencies of IL-10was showed in the4groups in thespleen, lymph nodes and thymus and significant differences were displayed in theallogenic transplantation group and autogentic transplantation group as comparedwith EAE group (All p<0.01vs EAE group in the three organs).5. The inhibitory effect of CD4+CD25+Treg on proliferation of CD4+CD25-Teff.Both allogenic and autogenetic MSCs transplantation group could increasedthe inhibitory activity of Treg cells on proliferation of Teff cells. Compare with EAEgroup, there are significant difference, P<0.01.ConclusionsIn this study, we induce successfully Experimental Autoimmune Encephalomyelitis (EAE) animal model by injection of MOG peptide.Transplantation of MSCs (either allogenic or autogenic)20and22days after MOGimmunization on EAE mice can led to a decreased clinical score, an upregulation ofCD4+CD25+Foxp3+T cell, Foxp3, TGF-β1and IL-10mRNA in spleen, lymph nodesand thymus as compared with EAE mice. These results suggest that transplantation ofMSCs can prevent the development of EAE and may be an available method intherapy of MS. MCSs transplantation can affect the proliferation and function ofCD4+T cells in EAE and CD4+CD25+Foxp3+T cell, Foxp3, TGF-β1and IL-10maybe involved in this process.