Effect Of SOCS1 On Mesenchymal Stem Cells Immunomodulation

Mesenchymal stem cells(MSCs)exist in almost all tissues and have been isolated from bone marrow,adipose tissue,umbilical cord,placenta and amniotic fluid.MSCs can differentiate into osteoblast,adipocyte and chondrocyte.Recent studies have found that MSCs have potent immunoregulatory capacity and possess therapeutic potential for various inflammation related diseases.These cells can block T cell proliferation and B cell terminal differentiation,NK cell cytotoxicity,and dendritic cell maturation.Although various molecules including indoleamine 2,3-dioxygenase(IDO),inducible nitric oxide synthase(iNOS),prostaglandin E2(PGE2),transforming growth factor-b(TGF-b),and programmed death-1 ligand(PD-L1)have been shown to be involved to the mechanisms of MSC-mediated immunosuppression,the detailed mechanisms are still not fully understood.The suppressor of cytokine signaling 1(SOCS1)controls signaling of many cytokines,including IFNr,IFNa and IL-10,with a feedback loop mechanism.The expression of SOCS1 is upregulated when a cytokine is excessive.SOCS1 then inhibits the production of the cytokine by Janus tyrosine kinase(JAK)and signal transducers and activators of transcription(STAT)signaling.Dendritic cells(DCs)are professional antigen presenting cells with key regulatory roles in the maintenance of tolerance to self-antigens and in the activation of innate and adaptive immunity.Previous studies have shown that SOCS1 is induced in DCs and silencing of SOCS1 enhances antigen presentation by DCs and antigen-specific anti-tumor immunity.Downregulating the production of SOCS1 in DCs allows increased signaling to T cells and resulting in expanded memory T cell populations.Since MSCs are responsive to proinflammatory cytokines and regulate immune cells,we hypothesize that SOCS1 may play an important role in the immunoregulatory function of MSCs.Experiment one:Effect of SOCS1 on the basic biology properties of MSCs.Some studies have found that SOCS1 can regulate the development of many immune cells.However,the effect of SOCS1 on the basic biology properties of MSCs is stil unclear.Lentivirus expressing SOCS1 shRNA and green fluorescent protein(GFP)were used to knock down SOCS1.Then we carried out these experiments as following:1)We detected the GFP expression in MSCs expressing SOCS1 shRNA(MSC/SOCSlsh)and MSCs expressing scramble shRNA(MSC/CTLsh)by fluorescence microscope and flow cytometry,and found that Green fluorescence was expressed on almost all the cells after transduction and selection by flow cytometry.2)Real-time PCR and western blot analysis showed that SOCS1 was significantly reduced in MSC/SOCSlsh compared with MSC/CTLsh.All these demonstrated the efficiency of our knockdown protocol.3)By flow cytometry analyse,we found that MSC/SOCSlsh and MSC/CTLsh comprised the similar immunophenotype.This suggests that SOCS1 has not an impact on the immunophenotype of MSCs.4)To examine the effect of SOCS1 on MSC proliferation,cell counting,CCK-8 assay and BrdU incorporation were performed.MSC/SOCSlsh did not show a significant increase in their proliferative activity compared with MSC/CTLsh.5)By osteogenic,adipogenic induction,we did not observe the significant changes in both MSC/SOCS1sh and MSC/CTLsh.This indicates that SOCS1 did not affect the differentiation of MSCs.Experiment two:effect of SOCS1 on MSCs immunomodulation on T cells and the mechanism.Our previous study showed that SOCS1 had no influence on immunophynotype,proliferation and differentiation of MSCs.However,it is unclear whether SOCS1 effects MSCs immunoregulation.So,we carried out the experimentsas following.1)SOCS1 has been described as being induced in various antigen presenting cells,We investigated whether expression of SOCS1 was up-regulated or not in MSCs,following stimulation with inflammatory cytokines.Primary cultured mouse MSCs and murine MSC cell line,C3H10T1/2,were exposed to IFNr plus TNFa(2 ng/ml each)at different time-points(12 hand 24 h),and C3H10T1/2 was stimulated with varied concentrations of IFNr/TNFa at 24h.Then SOCS1 expression was detected by real-time PCR and western blot.Our data showed that SOCS1 expression was up-regulated in primary MSCs and C3H10T1/2 after simulation with IFNr plus TNFa in the above experiments.2)T cells were stained with CFSE and stimulated with PMA and inomyosin,and then MSC/SOCS1sh and MSC/CTLsh were added at various ratios.As expected,T cell proliferation was inhibited as indicated by the slower reduction in CFSE intensity by MSC/CTLsh.However,instead of stimulating T cell proliferation,MSC/SOCS1sh exhibited stronger immunosuppressive activity.This indicated that SOCS1 silence contribute to inhibition of MSCs to T cells proliferation in vitro.3)As MSC/SOCSlsh showed stronger immunosuppressive activity in vitro,we tested their effect in vivo in a mouse model of melanoma.B16-FO melanoma cells were co-administered with MSC/SOCS1sh or MSC/CTLsh and 16 days later,the resultant tumors were weighed.Both MSCs were found to promote tumor growth,and MSC/SOCSlsh exhibited stronger effect.Then we further tested the immunosuppressive effect in the DTH response.OVA-immunized.mice were injected in the footpad with OVA alone or OVA with MSC/SOCS1sh or MSC/CTLsh,and the resultant DTH response measured by footpad thickness increment.As expected,administration of MSC/CTLsh resulted in reduced inf lammation.MSC/SOCS1sh exhibited stronger immunosuppressive effect,showing as sharp reduction of footpad thickness increment.Collectively,MSCs with SOCS1 knockdown showed enhanced immunosuppressive activity in vivo.4)Next we examined the iNOS expression after SOCS1 knockdown.iNOS mRNA and iNOS protein expression were examined by real-time PCR and western blotting analysis respectively.Our results showed the significant increase of iNOS mRNA and iNOS protein expression in MSC/SOCSlsh.The supernatant from MSC/SOCS1sh and MSC/CTLsh stimulated with inflammatory cytokines was examined for their nitrate concentration.NO production was increased dramatically after SOCS1 knockdown.To further confirm the NO-dependent manner of the strong immunosuppressive function of MSC/SOCSlsh,the iNOS inhibitor NG-monomethyl-L-arginine acetate salt(L-NMMA)was added to the T cell proliferation assay.As expected,L-NMMA restored.T-cel1 proliferation in the co-culture assay.Therefore,SOCS1 might attenuate the immunosuppressive capacity of MSCs by inhibiting iNOS expression and thereby NO release.5)Recent evidence suggests that NO is generated in the inflamed joint in patients with RA.Our data also showed 2-5 fold NO in the joint fluid in patient with RA compared with those in patient with OA.We next examined whether MSCs isolated from joint fluid in patient with RA(RAMSC)contributed to the high N0.Compared with MSCs isolated from bone marrow(BMMSC)of healthy donor RAMSC secreted NO after stimulated with inflammatory cytokines.We found no detectable levels of NO in BMMSC.We further tested SOCS1 expression in RAMSC.SOCS1 mRNA level in RAMSC was dramatically lower than that in OAMSC.Experiment three:effect of SOCS1 in the immunomodulatory function of MSCs on B cells proliferation,activation and ultimate differentiation.Our previous results showed that SOCS1 silence contribute prohibition of MSCs to T cells proliferation.What impact has SOCS1 had on B cells proliferation,activation and ultimate differentiation?1)B cells were stained with CFSE and stimulated with LPS,LPS/IL4,LPS/TGF ? 1,respectively.then MSC/SOCS1sh and MSC/CTLsh were added at various ratios.As a result,both MSC/CTLsh and MSC/SOCSlsh inhibit B cell proliferation as indicated by the reduction in CFSE intensity in all stimulatory groups.Compared with MSC/CTLsh,MSC/SOCS1sh exhibited a much stronger immunosuppressive activity.2)The impact of MSC/CTLsh or MSC/SOCS1sh on B cell activation has yet to be defined.B220+B cells were incubated alone or together with MSC/CTLsh or MSC/SOCS1sh at two ratios(MSCs:B=1:10 or 1:40)in the presence of LPS,LPS/IL4,LPS/TGF ? 1,respectively.Then CD40 and CD80 expression of B cells were analyzed by flow cytometry.Our results showed that the CD40 and CD80 expression of B cells did not display obvi.ous difference between MSC/CTLsh and MSC/SOCSlsh in each sitmulation group,regardless of the ratios of MSC against B cells.at the same time,CD40 and CD80 expression rate of B cells from co-culture group were almost same with that of the alone culture group.These data suggested that SOCS1 did not affect activation of B cells.3)The effect of SOCS1 on B cell ultimate differentiation was assessed by flow cytometry.The B cells were exposed to LPS,LPS/IL4,LPS/TGF ?1,respectively.23 hours later,MSC/CTLsh or MSC/SOCSlsh was seed on the B cells at different ratios(MSCs:B=1:10 or 1:40).2 days later,B cells were collected and the B220+CD138+ B cells were detected for assessing the ultimate differentiation.Our results showed that CD138+B cells percent of alone B cells group is significant lower than that of all co-culture groups in individual sitimulaltion group.These results indicated that MSCs contributed to differentiation of B cells to plasm cells.It was noteworthy that CD138 expression in MSC/SOCSlsh group was higher than that in MSC/CTLsh group,regardless of the concentration grade of MSCs.However,CD138 expression of B cells had not difference in both MSC/SOCSlsh group and MSC/CTLsh group with LPS or LPS/TGF ?1 stimualtion.These data suggested that the effect of SOCS1 on B cells was dependent on stimulators species.Conclusion:our results showed that S0CS1 was induced in MSCs treated with inflammatory cytokines.Knockdown of SOCS1 did not bring much difference on the immunophenotype,proliferation and differentiation properties of MSCs.However,MSCs with SOCS1 knockdown exhibited enhanced immunosuppressive capacity,showing as inhibiting T and B cell proliferation in vitro,no affecting B cell activation,prohibiting or no prohibiting B cell differentiation dependent on sitmualtors species,significantly promoting tumor growth and inhibiting delayed-type hypersensitivity response in vivo.We further demonstrated that SOCS1 inhibited the immunosuppressive capacity of MSCs by reducing inducible nitric oxide synthase(iNOS)expression.Additionally,we found the significantly lower SOCS1 expression and higher nitric oxide(NO)production in MSCs isolated from synovial fluid of rheumatoid arthritis patients.Collectively,our data revealed a novel role of SOCS1 in regulating the immune modulatory activities of MSCs.