Toll-like Receptor Preconditioning Enhances The Modulatory Function Of Adipose-derived Mesenchymal Stem Cells And The Study Of Mechanism

Objective:Donor-specific tolerance is the ultimate goal in organ transplantation.Diverse approaches,including the use of mesenchymal stem cells(MSCs),have been investigated to induce graft tolerance.Non-stimulated MSCs showed limited regulatory functions through interaction with multiple immune regulatory cells,such as the regulatory T cells(Tregs).In order to augment their functions,MSCs have been preconditioned with toll-like receptor(TLR3/4)agonist in autoimmune disease models but reached controversial results.MSCs can be divided into MSC1 and MSC2 according to the stimulation of toll-like receptor(TLR)on MSC surface.The activation of TLR4 on the surface of MSC will modify the MSCs into pro-inflammatory MSC1,while activation of TLR3 will propagate MSCs into anti-inflammatory MSC2.We aimed to investigate the effects of activation/inhibition of TLR3 and TLR4 on the immunomodulatory function of ADSCs in the context of mouse MHC fully mismatched heterotopic heart transplantation.Also,we would like to know whether TLR3 stimulator plus TLR4 blocker can induce stronger effects on ADSCs than using TLR3 stimulator alone.In addition,we try to identify the molecules exerting the major effect in these immunomodulatory procedures.In this report,we isolated ADSCs from mice,confirmed their phenotypes and multilineage differentiating capabilities.Then we proved their immunomodulatory functions in both ex vivo and in vivo models.TLR3 stimulation alone induced the highest regulatory effects in these ADSCs,even better than the combination of TLR3stimulator with TLR4 blocker.In addition,expression of FGL2,a reported effector molecule of Tregs,was significantly increased in ADSCs stimulated with TLR3 agonist.The results of these studies clarified the roles of TLR3/4 stimulations in MSC-related immunomodulation,shedding lights for the effector molecule,and provide important insights for designing MSC related therapeutics in the field of organ transplantation.Materials:1.AnimalsMale C57BL/6 mice and BALB/c mice,6–8week old,were purchased from the department of laboratory animal science of China Medical University(Shenyang,China).The animals were maintained under standard conditions.2.ADSC culture in vitroFirst,ADSCs were isolated from C57BL/6 using the criteria of International Society for Cellular Therapy(ISCT)for the isolation of MSCs from humans.ADSCs were obtained from the abdominal subcutaneous lipid tissue isolated from 6–8-week-old C57BL/6 mice through mechanical and enzymatic digestion.After tissue mincing,type I collagenase was added for 1 h at 37°C with agitation.The digested adipose tissue was centrifuged at1000 rpm for 5 min.The resulting cell pellet was cultured in DMEM supplemented with10%fetal bovine serum(FBS),antibiotic solution(100 U/mL penicillin,and 100?g/mL streptomycin),and incubated at 37°C in a humidified environment containing 5%CO2.Subsequently,the cells between passages 3 and 6 were used for further experiments.3.ADSC characterizationThe ADSCs were differentiated towards adipogenic,chondrogenic,and osteogenic lineages.ADSC phenotypes were analyzed by flow cytometry according to the Cell Surface Immunofluorescence Staining Protocol(BioLegend).The expression level of four surface markers,including CD29(beta-1 integrin),stem cells antigen-1(Sca-1),CD34,and CD45 were measured.4.Antibodies and flow cytometric analysisFITC anti-mouse CD29,FITC anti-mouse Sca-1,PE anti-mouse CD34,PE anti-mouse CD45,FITC anti-mouse CD4,and Alexa Fluor 647anti-mouse Foxp3 were purchased from BioLegend.FACS CantoII was used for flow cytometry,and data were analyzed by BD FACSDiva software and FlowJo(v X.0.7)software.5.Treatment of ADSCsADSCs were grown to sub-confluence in DMEM and incubated with agonists/antagonists for 1 h.Poly(I:C)(10?g/mL)was used as agonist for TLR3.LPS(500 ng/mL)was used as agonist for TLR4.TAK242(1?M)was used as an antagonist for TLR4.The cells were washed twice in growth medium before using for subsequent assays.6.CD4~+T cell sorting from C57BL/6 miceSingle splenic lymphocytes were prepared from C57BL/6 mice.CD4+T cells were isolated using the MojosortTM mouse CD4 T cell isolation kit(BioLegend),with>95%purity.Next,the cells were labeled with carboxyfluorescein succinimidyl ester(CFSE).Freshly purified T cells were resuspended in 0.1%BSA-PBS at a density of 2×10~6cells/mL and incubated with CFSE for 15 mins at 37°C.Subsequently,the cells were washed and resuspended in 1640 Medium for 10 mins to stabilize the CFSE staining.Then,these CFSE labeled CD4~+T cells were seeded in 96-well plate at a density of 2×10~5cells/well,as responding cells in the mixed lymphocyte reaction(MLR).7.Allogeneic mixed lymphocyte and ADSC reactionsAn allogeneic MLR assay was used to determine the effect of poly(I:C),LPS or TAK242preconditioning on the immunomodulatory properties of ADSCs.Splenocytes were prepared from BALB/c mice.The cells were cultured with mitomycin C and used as donor stimulating cells at 1:1 ratio(CD4~+T cell from C57BL/6 mice:splenocytes from BALB/c mice).Then 5 groups of ADSCs,including ADSCs(as control group),ADSC-poly(I:C),ADSC-LPS,ADSC-TAK242,ADSC-poly(I:C)plus TAK242 were utilized in the MLR assay at ratios 1:0.1,1:0.2,1:1,1:2(CD4~+T cells:ADSC)in RPMI medium.After 5 days,the peak of CFSE on the flow cytometric histograms were evaluated,representing the division cycles for CD4~+T cells.8.Quantitative real-time PCR(qRT-PCR)Total RNA was extracted from each group of ADSCs.Using the method of qRT-PCR,expressions of three cytokines,including Fgl2,Cox-2,and IL-10 were analyzed.9.ELISA for cytokinesThe cell culture supernatants of ADSC following the treatments with different combination of TLR3/4 activators/blocker were collected.PGE2,FGL2,and IL-10 levels were measured,using an ELISA kit according to the manufacturer's protocol.10.Vascularized cardiac transplantation and treatment schedule of ADSCsHeterotopic transplantations of intact allogeneic BALB/c hearts into C57BL/6 recipients were performed as described previously.BALB/c hearts were transplanted into BALB/c recipients as technical control group.In tolerant control group,BALB/c hearts were transplanted into C57BL/6 recipients,and rapamycin(0.4mg/kg)was injected intraperitoneally on postoperative day(POD)0,1,2,4,6,8,10,12,14 and 16.A total of0.5×106 ADSCs were injected into the recipient via the caudal vein on POD1.A subset of the recipients was sacrificed on POD 7 to obtain spleens and hearts,and the other recipients were investigated until allografts stopped beating.11.Ex vivo analysis of CD4~+Foxp3~+Treg cell from the spleens of recipient miceSplenocytes were freshly isolated from the spleens of recipient mice.The cells were stained by FITC anti-mouse CD4 according to the Cell Surface Immunofluorescence Staining Protocol,followed by staining with Alexa Fluor 647anti-mouse Foxp3according to True-Nuclear?Transcription Factor Staining Protocol.After that,the percentage of CD4+Foxp3+Treg cells were evaluated by flow cytometry.12 Histopathological analysis and damage scoreThe grafted hearts were harvested POD7.The graft was formalin fixated and embedded in paraffin.Sections of 3 mm were taken at one-third of the distance from the base to the apex of the heart and stained with hematoxylin and eosin(HE).According to the standardized grading system for the pathologic diagnosis of rejection in cardiac biopsies of International Society for Heart and Lung Transplantation(ISHLT),acute cellular rejection could be divided into Grade 0 R(no rejection);Grade 1 R(mild:interstitial and/or perivascular infiltrate with up to 1 focus of myocyte damage);Grade 2R(moderate:two or more foci of infiltrate with associated myocyte damage);Grade 3R(severe:diffuse infiltrate with multifocal myocyte damage±edema,±hemorrhage±vasculitis).Two observers evaluated the histological slides individually,with five fields being checked in each slide.The average scores were calculated;final results were denoted as mean±standard deviation(SD).Results:1.ADSCs have the full capabilities of MSCs.2.The addition of ADSCs significantly inhibited the proliferation of CD4~+T cells in a dose-dependent manner as compared to that in the control condition.3.Preconditioning of ADSCs with poly(I:C)had a maximal inhibitory effect on CD4+T cells in MLR.4.FGL2 but not PGE2 or IL-10 is the main effector molecule in ADSCs preconditioned with TLR3 activator.5.Intravenous injection of ADSCs pretreated with poly(I:C)prolonged the survival of cardiac allograft.6.Preconditioning of ADSCs with poly(I:C)increased the proportion of CD4+Foxp3+Treg cells in the mouse spleen after heart transplantation.7.ADSCs preconditioned with TLR3 stimulator show protective effect in graft histological findings.Conclusion:1.Preconditioning of ADSCs with poly(I:C)had a maximal inhibitory effect in vitro and in vivo experiments.2.Preconditioning of ADSCs with poly(I:C)and TAK242 couldn't have a maximal inhibitory effect.3.Although the goal of graft tolerance was not reached,using one dose TLR3 activated ADSCs prolonged the graft survival from 7 days up to 12.3 days.In the future,multiple injections with different dosage through varied administrating routes could be attempted.4.Our results suggested that there might be a correlation between the IFN-?expression induced by TLR3 activation and the over expression of FGL2.