Study On The Effects And Mechanisms Of IFN-? Primed HUCMSCs Transplantation On Experimental Autoimmune Encephalomyelitis In A Mouse Model

BackgroundMultiple sclerosis(MS)is an inflammatory and demyelinating disorder in the central nervous system,which is induced by the repeated activation of the autoimmune system.MS is one of the most common progressive neurological disorders in young people.Experimental autoimmune encephalomyelitis(EAE)is an animal model of MS that is mainly used for pathological studies and screening drugs for MS.Until recently,the clinical treatments of MS can only delay the disease progression with various disease-modifying drugs(DMD).Therefore,it is necessary to find other therapeutic methods for MS.Recent studies have shown that mesenchymal stem cells(MSCs)could exert immunomodulatory and anti-inflammatory effects in various tissues.MSCs from umbilical cord tissue and bone marrow could reduce CNS leukocyte infiltration and lower clinical scores in the EAE mouse.In addition,the umbilical cord mesenchymal stem cells(UCMSCs)has been used in clinical trials of treatment for patients with MS,the results showed that UCMSCs patients symptoms improved after transplantation.After the transplantation,the MS patients did not have new clinical symptoms,the scores of expanded disability status scale(EDSS)decreased,and MRI showed a reduction in the number of lesions.Therefore,MSCs transplantation may be a potential method for EAE/MS treatmentThe regulatory mechanism mediated by MSCs is complexStudies have shown that MSCs from human tissues could exert immune regulatory effects through the indoleamine 2,3-dioxygenase(IDO1),and the IDO1 could be detected after exposure to interferon-?(IFN-?).Therefore,we speculated that the expression of IDO1 in MSCs was closely related to IFN-? exposure.What would happen to the expression of IDO1 in IFN-? primed human UCMSCs?Does IFN-?-hUCMSCs have therapeutic effects on EAE mice?According to the research background,hUCMSCs were primed by IFN-? at different concentrations and time in order to detect the expression of IDO1 firstly.Then,in order to observe whether IFN-?-hUCMSCs transplantation could exert much more treatment effects on MS than hUCMSCs,the EAE model was established and therapeutic effects of IFN-?-hUCMSCs and hUCMSCs transplantation were compared.We observed the clinical symptoms and behavioral scores of EAE mice,changes in electromyography and concentration of inflammatory factors.Then,we investigated the therapeutic effects of IFN-?-hUCMSCs transplantation on EAE mice and possible immunological mechanism.Objective1.To observe the primed effects of IFN-? on hUCMSCs in vitro.The hUCMSCs were isolated,cultured,and identified differentiation potential.Then,hUCMSCs were primed by IFN-y at different concentrations and time to observe the expression of IDO1,and investigated whether the IDO1 expression was related to the concentration and primed time of IFN-?.2.To investigate the potential therapeutic effects of IFN-?-hUCMSCs transplantation on EAE mice.IFN-?-hUCMSCs with appropriate treatment concentration and primed time were transplanted to EAE mice via tail vein.The body weight,clinical symptoms,EMG,and inflammatory factors of mice were observed.3.To investigate the immunological mechanism of IFN-?-hUCMSCs transplantation on EAE mice.Methods1.Isolation,culture and identification of hUCMSCs.Human umbilical cord specimens were obtained from healthy young females under sterile condition,who received cesarean operation at the obstetrical department.Then,hUCMSCs were isolated and amplified.The ability of hUCMSCs to differentiate into osteoblasts and adipose cells was detected.The surface markers of hUCMSCs,including CD11b,CD19,CD34,CD45,CD73,CD90,CD105 and HLA-DR,were detected by flow cytometry2.To observe the primed effect of IFN-? on hUCMSCs.hUCMSCs were induced by IFN-? at different concentrations(20ng/ml,50ng/ml)and different time(24h,48h,72h).The expression of IDO1 was observed.RT-PCR and Western blot were used to detect the expression of IDO1 in the IFN-?-hUCMSCs and compared the differences among groups.Then we chose the appropriate treatment condition to induce hUCMSCs and prepared for the transplantation.3.To observe the therapeutic effects of IFN-?-hUCMSC on EAE mice.Female C57BL/6J mice aged 6-8 weeks were randomly divided into 4 groups after adaptive feeding.Then the EAE mouse model was established by immunological induction of myelin oligodendrocyte glycoprotein(MOG)35-55.Mice in the blank control group were not induced by MOG35-55.The hUCMSCs and IFN-?-hUCMSCs were transplanted via tail vein in the treatment groups,and mice in the control groups were given equal volume PBS instead.The body weight,clinical symptoms and behavioral scores of the mice were observed daily.The nerve demyelination was detected by EMG.4,In order to detect the inflammatory reaction in EAE mice after transplantation with IFN-?-hUCMSCs,concentration of IL-17A and TNF-? in serum were detected by ELISA.The concentration of IL-10 and IL-17A in the spleen macrophages supernatant was detected by ELISA too.5.In order to explore the possible immunological mechanism of IFN-?-hUCMSCs transplantation on EAE mice,RT-PCR was used to detect the mRNA expression of STAT3,ROR-yt and Foxp3 genes.Then the expressions were statistically analyzed to explore the possible immune mechanism.Results1.Isolation and culture of hUCMSCsThe hUCMSCs were isolated and cultured in the cell culture medium.At the 4th day of primary culture,spindle or triangular cells were adherent to the wall of the medium.Amplification cycle of hUCMSCs was about 3 days.The cell culture medium was replaced twice a week.When the growth density of adherent cells reached 70-80%,the cells were subcultured.hUCMSCs were removed to identify immunophenotype at the 4th passage.The purity of cultured hUCMSCs would be over 95%.Microscopic observation showed that the adherent cells exhibited fibroblast-like morphology.The results showed that the hUCMSCs grew well and were suitable for subsequent experimental studies.2.Immunophenotype identification of hUCMSCsResults from flow cytometry showed that the expression of CD73,CD90 and CD105 were all above 95%,while the expression of CDllb,CD19,CD34,CD45 and HLA-DR were all below 3%.These results suggested that the cultured cells were actually hUCMSCs,which were suitable for the following cell transplantation.3.Identification the differentiation of hUCMSCs3.1 hUCMSCs differentiate into osteoblastsThe hUCMSCs can differentiate into osteoblasts under certain condition.Under the inverted microscope,the osteoblasts differentiated from hUCMSCs were brown-red with alizarin red staining.3.2 hUCMSCs can differentiate into adipocytesThe hUCMSCs can differentiate into adipocytes under certain condition,Under the inverted microscope,it can be seen that hUCMSCs differentiate into adipocytes,which can be stained red with oil red O.The above results indicated that the hUCMSCs had good differentiation potential.4.IFN-? could up-regulate mRNA expression of IDO1 in hUCMSCs.In order to observe the effect of IFN-? on hUCMSCs,hUCMSCs were primed with different concentrations(20ng/ml,50ng/ml)and different time(24h,48h,72h)of IFN-?.The mRNA expression of IDO1 in hUCMSCs was detected by RT-PCR.Our results showed that,compared with the control group,IFN-? could significantly up-regulate the mRNA expression of IDO1 in hUCMSCs,and the expression was positively correlated with the concentration and the primed time of IFN-?.Our results showed that IFN-y could up-regulate the mRNA expression of IDO1 in hUCMSCs.5.IFN-? could increase the protein expression of IDO1 in hUCMSCs.The hUCMSCs were primed with different concentrations and different time of IFN-?.Then we observed the effects of IFN-? on the protein expression of IDO1 in hUCMSCs.Western blot was used to detect the protein expression of IDO1 in the control group and the experimental groups.Our results showed that the protein expression of IDO1 was consistent with mRNA.Compared with the control group,IFN-? could significantly increase the protein expression of IDO1 in hUCMSCs,which was positively correlated with the concentration and primed time of IFN-?.The quantitative analysis of Western blot showed that the IDO1protein expression of the IFN-? primed group(20ng/ml,48h)and the group(20ng/ml,72h)were significantly higher than the group(20ng/ml,24h).However,there was no significant difference between the expression of the IFN-? primed group(20ng/ml?48h)and the group(20ng/ml,72h),as well as between the IFN-? primed group(20ng/ml,48h)and the group(50ng/ml,48h).The results showed that IFN-? could increase the protein expression of IDO1 in hUCMSCs.Referring to the results of RT-PCR and Western blot,we found that the expression of IDO1 in hUCMSCs was significantly up-regulated which was time-dose dependent.In addition,we found that under the same concentration of IFN-?,the protein expression of IDO1 in 48h and 72h groups was significantly higher than that in the 24h group,but there was no significant difference between the 48h and 72h groups.Moreover,when the primed time was 48h,there was no statistical difference in IDO1 expression between the 20ng/ml group and the 50ng/ml group.Therefore,we selected IFN-?(20ng/ml,48h)primed hUCMSCs for subsequent animal studies to observe the therapeutic effect of IFN-?-hUCMSCs on EAE mice.6.EAE mouse modelingFemale C57BL/6J mice aged 6-8 weeks were immunized with MOG35-55.The mice generally developed diseases around the 14th day after immunization,characterized by weight loss,decreased or disappeared tail tension,and gradually developed gait clumsiness,ataxia,limb weakness and urinary incontinence.7.IFN-?-hUCMSCs transplantation could alleviate the weight loss and clinical symptoms of EAE mice.On the 14th day after immunization,hUCMSCs and IFN-?-hUCMSCs were transplanted to EAE mice respectively in the treatment groups via tail vein.Mice of the control groups were given equal volume of PBS instead.We found that compared with the blank control group,the averaged body weight of EAE mice was declined.The transplantations of hUCMSCs and IFN-?-hUCMSCs could both elevate the body weight of mice,and the IFN-y-hUCMSCs group alleviated much more significantly.For the clinical symptoms assessment,compared with the EAE mice group,the mean behavior score was lower in the treatment groups,and the IFN-?-hUCMSCs group could significantly reduce the behavior scores.The behavior scores reduction of IFN-?-hUCMSCs group suggested that the clinical symptoms of mice were alleviated.Taken together,these results showed that,transplantation of IFN-?-hUCMSCs could significantly alleviate the body weight loss and clinical symptoms impairment in the EAE mice,much better than hUCMSCs transplantation group.8.The nerve demyelination in EAE mice was alleviated after IFN-?-hUCMSCs transplantationOn the 28th day after immunization(14th day after transplantation),we attempted to detect the demyelination of EAE mice by EMG.Electrical stimulation experiments were conducted with a single channel stimulator.Stimulation electrodes were placed in the motor cortex and silver needle electrodes were made through the skin,then reaching the muscle belly,and the latency was recorded.Because the muscle atrophy of EAE mice was obvious,the compound muscle action potential(CMAP)amplitude could not reflect the nerve injury state.We used the latency of motor evoked potentials(MEP)for detection,which could reflect the degree of demyelination in EAE mice.Our results showed that,compared with the control group,the averaged latency was significantly reduced in the treatment groups,and the IFN-?-hUCMSCs group was significantly lower than the hUCMSCs group.Our results suggested that the demyelination of EAE mice were gradually improved after IFN-?-hUCMSCs transplantation compared with the hUCMSCs transplantation group.9.IFN-?-hUCMSCs transplantation reduced the inflammatory reaction in EAE miceIn order to explore the inflammatory reaction in EAE mice,we detected inflammatory cytokines(IL-17A and TNF-?)in the peripheral blood of mice and inflammatory cytokines(IL-10 and IL-17A)in mice spleen macrophages supernatant.We found that compared with the blank control group,pro-inflammatory factors(IL-17A and TNF-?)in peripheral blood of EAE mice increased significantly,which suggested obvious inflammation of EAE mice.After transplantation of hUCMSCs and IFN-?-hUCMSCs,pro-inflammatory factors(IL-17A and TNF-?)of EAE mice decreased significantly.The pro-inflammatory factors decreased more significantly in the IFN-?-hUCMSCs transplantation group than the hUCMSCs transplantation group.In addition,we also detected the level of IL-10 and IL-17A in mice spleen macrophages supernatant.We found that compared with the blank control mice,the IL-10 concentration of EAE mice reduced,while the IL-17A concentration significantly increased.After transplantation of hUCMSCs and IFN-?-hUCMSCs,the concentration of anti-inflammatory factor IL-10 increased significantly,while the concentration of pro-inflammatory factor IL-17A decreased significantly,and the effect of IFN-?-hUCMSCs transplantation group was much more significant than the hUCMSCs transplantation group.Taken together,the results showed that,compared with hUCMSCs transplantation group,IFN-?-hUCMSCs transplantation could much more significantly increase anti-inflammatory factor and decrease pro-inflammatory factors,thus reduce the inflammatory response in EAE mice.10.IFN-?-hUCMSCs transplantation alleviated the inflammatory response through the STAT3/ROR-?t/Foxp3 signaling pathway in EAE miceWe detected the mRNA expression of STAT3,ROR-?t and Foxp3 in the lumbar medulla of EAE mice by RT-PCR.We found that compared with the blank control group,mRNA expressions of STAT3 and ROR-?t increased in the EAE mice,while the mRNA expression of Foxp3 decreased.After transplantation of hUCMSCs and IFN-?-hUCMSCs,mRNA expressions of STAT3 and ROR-?t significantly decreased,but the mRNA expression of Foxp3 was up-regulated.The IFN-?-hUCMSCs transplantation group was much more significant than the hUCMSCs group.Therefore,IFN-?-hUCMSCs transplantation might regulate Thl7/Tregs imbalance and reduce inflammatory response through the STAT3/ROR-?t/Foxp3 signaling pathway.Conclusion1.IFN-? could significantly increase the mRNA and protein expression of IDO1 in hUCMSCs which was time-dose dependent.2.IFN-?-hUCMSCs could significantly improve the clinical symptoms,reduce inflammation and alleviate the demyelination of EAE mice.3.IFN-y-hUCMSCs transplantation might regulate Th17/Tregs imbalance and reduce inflammatory response through the STAT3/ROR-?t/Foxp3 signaling pathway.SignificanceOur study primarily demonstrated that IFN-? could increase the expression of IDO1 in hUCMSCs.IFN-?-hUCMSCs transplantation could significantly improve outcomes of EAE mice,which provided an important basis for the treatment of IFN-?-hUCMSCs in MS.