Interleukin-1? Enhances The Immunosuppressive Function Of Mesenchymal Stem Cells And Their Secreted Exosomes

Mesenchymal stem cells(MSCs)are multipotent stem cells distributed widely in almost all tissues.In addition to their differentiation functions,MSCs possess immunomodulatory properties and immune-privilege potential,and can be isolated and expanded easily in vitro.Because of these characteristics,MSCs are attractive candidates for treating many immune and inflammatory disorders.However,recent researches suggest that the immunomodulatory capacities of rest MSCs are benign,and even cannot suppress immune reactions unless they are first activated by inflammatory cytokines,such as IFN-y,TNF-a or IL-1?,et al.So finding strategies like the modification of MSCs with cytokines or drugs to improve their therapeutic efficacy is an evolving field of investigation.In our previous study,we have demonstrated that IL-1? pre-treatment could enhance the therapeutic efficacy of MSCs on DSS-induced colitis partially by promoting MSCs migrate to the inflammatory site.However,the underlying mechanism is still fragmentary and incomplete.And whether such pre-treatment could also benefit the therapeutic efficacy of MSCs on other inflammatory diseases is still unknown.Accumulating evidence has proved that there is only a transient recruitment of MSCs to the lungs and livers,and only low numbers of MSCs will actually be incorporated into injured tissues;moreover,several animal models of organ injury highlight the efficacy of conditioned media from MSCs cultures.These observations indicate that the beneficial therapeutic effects of MSCs may mainly depend on their paracrine activities.Besides soluble factors,such as well-known prostaglandin E2(PGE2),TNF-a stimulated gene/protein 6(TSG-6)and indoleamine 2,3-dioxygenase(IDO),exosomes were described as a new and an attractive mechanism of MSC functions.Exosomes are membrane vesicles with diameters of 30-150 nm that originate in the late endosomal.Numerous studies have suggested that exosomes can transfer lipids,proteins,mRNAs and microRNAs(miRNAs)to mediate local and systemic cell-to-cell communications.Recently,miRNAs have been reported as important components of exosomes.Growing evidence has suggested that MSCs-derived exosomes have capacities of tissue repair or immunomodulatory effects through transfer of important miRNAs.So we wondered whether exosomes could contribute to the improved functions of IL-1?-primed MSCs.If so,the critical biological components of exosomes were needed to be clarified.In this study,we further investigated the influence of IL-1? pre-treatment on the immunosuppresive effects of MSCs;evaluated the therapeutic efficacy of ?MSCs on sepsis;studied the immunosuppresive properties of ?MSCs-derived exosomes;analyed the critical miRNAs in the components of exosomes;and finally elucidated the possiable mechanisms involved in ?MSCs-mediated immunosuppression..We believe that our study will provide a solid basis and a new strategy for the development of MSCs-based cell therapy for inflammatory diseases.1.Interleukin-1? pretreatment enhances the immunosuppresive effects of MSCs both in vitro and in vivo:Through dose-course analysis we found that 10 ng/ml IL-1? could effectively elevated expressions of MSC functional genes,including COX-2,IDO,IL-8 and TSG-6.Mean while,we determined that this dose of IL-1? won't alter the minimal criterias of MSCs,including the capacity to adhere to plastic;the surface markers(positive expression of CD29,CD44,CD73,CD90 and CD105,and negative expression of CD11b,CD14,CD19,CD31 and HLA-DR);the capacity to differentiate into osteoblasts and adipocytes in vitro.Furthermore,IL-1? treatment did not influence cell viability,cell cycle or induce cell apoptosis.Based on these,we then evaluated the immunomodulatory effects of IL-1? pre-treated MSCs(?MSCs).Results of mixed lymphocyte reaction(MLR)demonstrated that ?MSCs or their conditioned media elevated the inhibition rate on mouse T-cell proliferation,as well as on T-cell activation markers IFN-y and CD69,compared with untreated MSCs.In addition,PMSCs,co-cultured with bone marrow-directly or in a transwell system,more significantly decreased the LPS-induced derived macrophages(BMDMs)upregulation of M1 markers TNF-a and iNOS,while increased M2 markers IL-10 and Arg-1,from both mRNA levels and protein levels.These data indicated that IL-1?pre-treatment could enhance the immunomodulatory capacities of MSCs in vitro.Notably,the predominant mechanism by which MSCs participate in T-cell inhibition and M2-like macrophage polarization seemed to be related to their paracrine activity.Given the augmented immunosuppresive properties of PMSCs in vitro,we examined the therapeutic effects of PMSCs in vivo using a cecal ligation and puncture(CLP)-induced sepsis model.Mice were injected with human MSCs,pMSCs,or PBS via the tail vein 4 hours after a mid-grade CLP operation.Results showed that,compared with MSCs,PMSCs significantly improved the symptoms of sepsis,including increased survival rates and bacterial clearance;reduced serum levels of TNF-a,IL-6,aspartate aminotransferase(AST)and alanine aminotransferase(ALT);recovery of liver and lung injury.Of interest,PMSCs more significantly decreased macrophage population in both lungs and livers,suppressed M1 markers and promoted M2 phenotype.All these indicate that IL-1? pretreatment enhances the immunosuppressive effects of MSCs in vitro and improves the therapeutic effects in mouse sepsis.2.Exosomes serve as vehicles of the immunosuppresive properties of ?MSCs:The above data suggest that ?MSCs may release soluble factors or vesicles to regulate T-cell activation and macrophage polarization,perhaps by secreting exosomes.To confirm this speculation,we blocked exosome secretion using GW4869,an inhibitor of neutral sphingomyelinase 2(nSMase2)which controls exosome secretion.As expect,the inhibitory effect on T-cell activation by MSCs or ?MSCs was partially impaired by GW4869;meanwhile,the promotion of macrophage polarization from M1 to M2 phenotype was also negated by GW4869 treatment.Moreover,the influences by GW4869 were more obviously on ?MSCs than on MSCs.These indicate the involvement of exosomes in the immunomodulatory effects of MSCs,especially of ?MSCs.Next,we purified exosomes from the culture supernatants of MSCs and ?MSCs respectively,to evaluate their direct effects on T-cells and BMDMs.We found that the effects of exosomes were consistent with their cellular counterparts.Compared with exosomes derived from MSCs(MSCs-exo),exosomes derived from ?MSCs(?MSCs-exo)more significantly inhibited T-cell activation,down-regulated M1 markers and up-regulated M2 markers in BMDMs,and more importantly,improved survival rate in septic mice.All these indicate that exosomes are the key mediators in PMSCs-induced immunomosuppression.3.MiR-146a is the critical molecule in the immunosuppressive effects of exosomes derived from ?MSCs:It is convinced that miRNAs are critically involved in the immunomodulation effect of MSCs.Moreover,recent studies have demonstrated that miRNAs can be selectively packaged into exosomes.So we tend to determine whether any pivotal miRNAs are contained in exosmes to contribute to ?MSCs-induced immunomodulatory effects.We first measured the level of several famous immunoregulatory miRNAs(including miR-21,miR-143,miR-146a,miR-147a and miR-149-5p)in MSCs.The level of miR-21,miR-146a and miR-149-5p were all increased by IL-1? stimulation,among which miR-146a was the most strongly up-regulated.Over-expression of miR-146a by transfecting miR-146a mimics improved MSCs-elicited down-regulation of TNF-a and up-regulation of IL-10;while inhibition of miR-146a by transfecting miR-146a inhibitors obviously impaired?MSCs-elicited down-regulation of TNF-? and up-regulation of IL-10.In vivo study,miR-146a over-expressed MSCs(MSC-mi-146a)were observed to improve the survival rate,compared with negative control transfected MSCs;while knock-down of miR-146a partially negated the protection effect of ?MSCs.These data indicate that miR-146a is indeed involved in the immunosuppresive effects of IL-1?-primed MSCs.Interstingly,we found high level of miR-146a in both MSCs and ?MSCs derived exosomes.Of note,it was more redundant in ?MSCs-exo than in MSCs-exo.When added to cultured BMDMs,exosomes were internalized by BMDMs,resulted in the increased intracellular level of miR-146a,and the down-regulation of well-known miR-146a targets IL-1 receptor-associated kinase 1(IRAK1),TNF receptor-associated factor 6(TRAF6)and interferon regulatory factor 5(IRF5)in LPS-stimulated BMDMs.To further confirm the role of miR-146a in exosomes,we subsequently isolated exosomes from the culture supernatants of miR-146a inhibitors transfected and ?MSCs(in-146a-?MSC-exo).When added to BMDMs,the down-regulation of M1 markers and the up-regulation of M2 markers induced by in-NC-?MSC-exo were partially negated by in-146a-?MSC-exo.These indicate that exosomes may transfer miR-146a to macrophages so that inhibit M1 polarization but promote M2 polarization in vitro.Finally,we evaluated the influence of miR-146a in pMSC-exo mediated protective effect in septic mice.As expected,compared with in-NC-?MSC-exo treated group,lower survival rate was observed in in-146a-?MSC-exo treated group.Taken together,IL-1?pre-treatment enhances MSCs-induced T-cell inhibition and alternative macrophage polarization.Moreover,IL-1?-primed MSCs effectively improved the therapeutic effects on sepsis.Mechanistically,IL-1?-upregulated miR-146a shuttered by exosomes plays an essential role.