The Cytokines Secreted From Bone Marrow Mesenchymal Stem Cell Attenuate Alloimmune Responses Through The Mechanism Of RAP1/NF-κB Signaling Pathway

Background Mesenchymal stem cells have the great application prospect in the field of transplantation immunity with their property of low immunogenicity and immunosuppression. There is close relationship between the immunoregulationability of MSCs and NF-κB signaling pathway. Recent studies have identified Rapl as an essential modulator of NF-KB-mediated pathways. NF-κB is induced by ectopic expression of Rapl, whereas its activity is inhibited by Rapl depletion. Through comparing the immunoregulation between RKO-MSCs and RWT-MSCs, we intend to study the roles of NF-κB signaling pathway in MSC-mediated immune rejection response.Materials and Results First, bone marrow mesenchymal stem cells from Rapl knockout mice (RKO-MSCs) and their homologous wild type mice (RWT-MSCs) were isolated and well characterized. Then we compared the differences of immunoregulation ability between RKO-MSCs and RWT-MSCs by using in vitro mixed lymphocyte reaction (MLR).RKO-MSCs and RWT-MSCs demonstrated equivalent ability to inhibit proliferation of MLR (p>0.05). However, culture supernatants of RWT-MSCs had better effect in suppressing MLR proliferation than RKO-MSCs (p<0.01). This phenomenon could be partially explained by the differences of soluble factor between RKO-MSCs and RWT-MSCs as flow cytomix showed that RWT-MSCs secreted more pro-inflammatory cytokines and anti-inflammatory cytokines than RKO-MSCs(p<0.05).In mouse heart transplantation model, rapamycin (Rapa) in low/high dosage was combined with MSCs, grouped as following:1) lx Rapa (low Rapa);2)2x Rapa (high Rapa);3) lx Rapa+RWT-MSCs;4)2x Rapa+RWT-MSCs;5) lx Rapa+RKO-MSCs;6)2x Rapa+RKO-MSCs.2x Rapa+RWT-MSCs(group4) can obviously prolong the cardiac allograft survival than other groups (p<0.01)The worst group was RWT-MSCs combined with low dose of rapamycin (group3), corresponding the shortest allograft survival of6days, whereas the longest allograft survival up to30days was appeared in RWT-MSCs combined with high dose of Rapa (group4), with less inflammatory cells infiltration and increased expression of Foxp3, detected by HE and immunefluorescence staining(P<0.01), respectively. Correspondingly, the lowest serum protein level of pro-inflammatory factors, including IFN-y, IL-10, TNF-a and IL-6was detected in RWT-MSCs combined high dose of Rapa group (group4).(p<0.001)Aiming at emphasizing paracrine differences between RKO-MSCs and RWT-MSCs in immonoregulation, we used biological material to capsulize MSCs for heart transplantation modeling, and achieved similar tendency of survival curve.2x Rapa+Encapsulated RWT-MSCs can obviously prolong the cardiac allograft survival than other groups (p<0.05).Conclusions RWT-MSCs processed more stable and reliable immune-suppressing capability than RKO-MSCs by secreting cytokines to inhibit inflammatory.