Experimental Study About The Effect Of IL-17 On The Immunosuppression Of Bone Marrow Mesenchymal Stem Cells

BackgroundAt present,skin transplantation is still one of the key measures to treat large area burn and severe trauma.For patients with extensive deep burn,whether the wound can be closed as soon as possible is related to their prognosis.Closing the wound as early as possible can reduce the exudation of the wound and the loss of body fluids and nutrients,reduce the risk of infection,shorten the treatment time,relieve the pain and economic pressure of patients,improve the survival rate of patients and their quality of life.Autologous skin transplantation is the best way to close the wound of skin defect.However,extensive burns or trauma often result in serious lack of skin source,which can not meet the needs of extensive skin defect caused by burns.This is also the most important factor leading to disability or death of severe burn patients.Although xenogeneic or allogeneic skin can temporarily solve the problem of short-term wound coverage,skin is one of the most antigenic tissues,and the body has a strong immune rejection to the graft,resulting in a shorter survival time after skin transplantation.The rejection of allografts or xenografts is negative in terms of therapeutic effect.Immunosuppressive agents can significantly prolong the survival time of organ transplantation rejection.However,immunosuppressants have significant side effects.Long-term use of immunosuppressants can lead to organ damage,reduce the body’s anti-infection and anti-cancer ability,and lead to acute infection and even cancer,so the long-term use of immunosuppressants is limited.In addition,patients with extensive burns are often accompanied by systemic infection,in which case immunosuppressive agents are also carefully applied.Therefore,how to prolong the survival time of xenogenic or allogenic skin without using immunosuppressive agents is a thorny problem at present.In this case,induction of immune tolerance is particularly important.It means that the immune system does not react to tissues or substances that can induce immune response.Transplant tolerance is a stable function of allografts without immunosuppressive agents,but a complete immune response to third parties.For large area burn patients,if the wound is covered with xenogeneic or allogeneic skin without immunological rejection to achieve immune tolerance,it can not only heal the wound as soon as possible,alleviate its economic pressure,but also reduce the deformity caused by scar contracture and improve the quality of life of patients.Therefore,how to induce this immune tolerance is an urgent problem in the research of organ transplantation.Mesenchymal stem cells(MSCs)are a group of mesodermal-derived adult stem cells with self-renewal and multi-directional differentiation potential,and have broad application prospects in tissue regeneration and repair medicine.MSCs can be obtained from a variety of adult tissues,such as fat,bone marrow,synovium,pulp,placenta,amniotic fluid,cord blood,fetal lung,fetal liver and even menstrual blood.The method of separation is simple,rapid and convenient in vitro,and does not involve ethical issues that make MSCs the most promising seed cells for cell transplantation therapy.In addition,MSCs have been widely confirmed to have immunomodulation effects.Although the mechanism is still unclear,due to its clear characteristic of induction of immune tolerance,MSCs are used in the study of induction of immune tolerance after allogeneic transplantation.The focus is on its potential to treat autoimmune diseases and organ transplants.In 2002,Bartholomew found that simple infusion of allogeneic or even third-party MSCs can prolong the survival time of baboon allogeneic skin grafts,providing new options for resolving xenogeneic or allogeneic skin graft rejection.Later,Lee SM et al.showed that conditioned medium or ADSCs significantly prolonged the survival time of allograft skin compared with the control group,and pointed out that ADSCs and cytokines secreted by ADSCs have the capacity of inducing immune tolerance.A large number of studies and experiments on pretreated MSCs to improve their ability in immunosuppression and induction of immune tolerance are being carried out in full blast.Studies have shown that MSCs combined with gene transfection,drug or cytokine treatment may increase their ability to induce immune tolerance.For example,Bian L’s experimental results showed that hepatocyte growth factor-transfected MSCs can significantly prolong the survival time of rat allograft skin.Zhang Y et al.demonstrated that Stro-1(+)MSCs can significantly prolong the survival time of rat skin grafts compared with untreated MSCs.Han X et al found that IL-17 significantly enhanced the immunosuppressive effect of MSCs in a mouse model of hepatitis induced by ConA.In this study,bone marrow mesenchymal stem cells(BMSCs)pretreated with IL-17 were injected into allogeneic skin graft model mice to explore the feasibility of IL-17 pretreated BMSCs enhancing the immune tolerance induced by BMSCs and prolonging the survival time of allogeneic skin.ObjectiveTo verify and confirm BMSCs have the effect of prolonging the survival time of transplanted skin.It was also explored whether BMSCs pretreated with IL-17 could enhance the induction of immune tolerance by BMSCs and prolong the skin survival time of allogeneic skin grafts.This experiment explored the preliminary mechanism of interleukin-17 enhancing the induction of immune tolerance by BMSCs.Methods1.Isolation,culture and identification of BMSCsThree healthy Balb/c mice were sacrificed by cervical dislocation.The femur,tibia and humerus were obtained sterilely and washed twice with PBS.Then the marrow cavity was exposed by cutting the ends of the backbone.Then the bone marrow was washed carefully by 1ml syringe with pre-cooled PBS 3-5 times and then the cell suspension was collected.The cell suspension was centrifuged at 1000 rpm for 5 minutes,and then the supernatant was discarded.The cells were cultured in DMEM low glucose medium supplemented with 10%fetal bovine serum(FBS)and 1:100 penicillin and streptomycin in a humid atmosphere with 5%CO2 at 37℃.BMSCs were identified by their multi-directional differentiation and surface marker antigen detection.In vitro differentiation:Osteogenic,chondrogenic and adipogenic differentiation were performed using BALB/c Mouse BMSCs Osteogenic,Chondrogenic and Adipogenic Differentiation Basal Medium,separately following the instructions.Cells were stained respectively for Alizarin Red S,Alcian blue and Oil Red O to confirm cell differentiation potential.Surface marker antigen detection:The BALB/C mouse BMSCs were digested,then the supernatant was discarded after centrifugation.The cells were resuspended.The surface marker antigen was detected with anti-mouse monoclonal antibody Anti-CD31-FITC,Anti-CD117-FITC,Anti-CD29-FITC and Anti-CD44-FITC with flow cytometry.2.IL-17 pretreatment of BMSCs2×105 BMSCs grown in logarithmic phase of the 4th-8th generation were inoculated in 25 cm2 flask.After 24 hours,the medium was changed and IL-17 was added to the medium(final concentration was 50ng/ml).The morphology and growth trend of BMSCs after treatment were observed every day.After 5 days,the medium was removed,and BMSCs-17 was collected by trypsin for the following experiment.3.Labeling and tracing of BMSCs with CM-DilBMSCs and BMSCs-17 were labeled by 5μg/mL CM-Dil.After labeling,BMSCs were injected into C57BL/6J from tail vein.To track the cells,the frozen section analysis of the grafts was performed at day 7.The BMSCs were observed under a fluorescence microscope.4.Establishment of the allograft skin graft modelsThe mice were anesthetized using 4%Chloral hydrate and disinfected with betadine.Then the 1.5×1.5cm2 dorsal full-thickness skin graft were acquired from the donor BALB/c while the full-thickness dorsal dermal wounds were created of recepient C57BL/6J.Then the skin transplant surgery was performed.The mice were fed in separate cages.5.Cell infusion and experimental groupingBMSCs of BALB/C mice were injected into C57BL/6J mice from tail vein one day before allograft skin transplantation.C57BL/6J mice were randomly divided into 3 groups:group A was injected with PBS into the tail vein,and allogeneic skin transplantation;group B was injected with BMSCs into the tail vein,and allogeneic skin transplantation;group C was injected with BMSCs-17 into the tail vein,and allogeneic skin transplantation.Three other mice were injected with CM-Dil-labeled BMSCs via the tail vein in group B and C respectively.6.The indexes of postoperative observation and detectionThe indexes of postoperative observation and detection included the appearance and HE staining of the skin graft,the proportion of the regulatory T cells(CD4 CD25 Foxp3 Treg cells)in the recipient mice,and the content of the cytokines(TGF-beta,IL-10,IFN-gamma)in the peripheral blood of recipient mice.Results1.BMSCs of BALB/c mice have multi-directional differentiation potential,and can differentiate into adipocytes,osteoblasts and chondroblasts in the corresponding induction medium.2.In vitro,BMSCs can significantly inhibit T lymphocyte proliferation(stimulated by ConA)without restriction of MHC.In addition,the inhibition is reluctant with the increase of MSCs cell concentration.BMSCs pretreated with IL-17 significantly enhanced the inhibitory effect of MSCs on T lymphocyte proliferation.3.The survival time of control group was almost 11.8±0.834 days;the survival time of MSCs group was almost 15.8±0.783 days;the survival time of BMSCs-17 group was significantly prolonged to 19.2±1.012 days.The survival time of BMSCs-17 is much longer than control group(P<0.001)and BMSCs group(P<0.01).In summary,IL-17 induced MSCs could dramatically prolong the allografts survival time.7 days after skin grafting,the graft appearance of control group turned black,hard and necrotizing;the grafts of BMSCs group and BMSCs-17 group survived well from general observation.By H&E staining,the control group had a lot of inflammatory cell infiltrates,exfoliation and no angiopoiesis;the BMSCs group had a little of inflammatory cell infiltrates and angiopoiesis;the BMSCs-17 group had little inflammatory cell infiltrates and much more angiopoiesis.After induced by IL-17,much more BMSCs homed to the grafts.The number of BMSCs homing to implant was increased after IL-17 treatment.IL-17 can enhance the homing ability of BMSCs.7 days after transplantation,the Treg subpopulations percentage of control group was much lower than the other two groups(P<0.001).Injection of IL-17 induced MSCs could increase the Treg subpopulations more than injection of BMSCs(P<0.001).The anti-inflammatory and proinflammatory cytokines in the serum were examined.TGF-β and IL-10 of control group were much lower than the other two groups(P<0.001).TGF-β and IL-10 of BMSCs-17 group were significantly higher than MSCs group(P<0.001).However,IFN-y of control group was much higher than BMSCs group and BMSCs-17 group(P<0.001).IFN-y of BMSCs-17 group was much lower than BMSCs group(P<0.001).So,IL-17 could enhance the immune suppression of BMSCs.Conclusion1.BMSCs can be differentiated into adipocytes,osteoblasts and chondrocytes in specific differentiation medium,and express specific surface antigens.2.The inhibition experiment of splenic lymphocyte proliferation showed that IL-17 significantly enhanced the inhibitory effect of BMSCs on lymphocyte proliferation.3.IL-17 can alleviate immune rejection and prolong the survival time of transplanted tissues or organs by improving the immunosuppressive effect and the homing ability of BMSCs after allotransplantation.SignificanceSkin is one of the organs with the strongest immunogenicity in the whole body.If the problem of skin transplantation tolerance or prolonging the survival time of allograft skin can be solved,this method can also be applied to other organ transplantation theoretically to alleviate or even eliminate immune rejection,which has far-reaching significance for organ transplantation.Therefore,we apply the above theory of "IL-17 can enhance the immunosuppressive effect of BMSCs" to the model of allograft skin transplantation to explore whether IL-17 can enhance the immune tolerance induced by BMSCs and prolong the survival time of transplanted skin.DeficienciesThe deeper mechanism of "IL-17 enhances the immunosuppression of BMSCs" in this experiment still needs to be further explored.It is believed that this mechanism will have a better therapeutic value for the clinical treatment of autoimmune diseases and organ transplantation rejection with IL-17 and BMSCs.In the future,we will continue to study these mechanisms.