Introduction: With the widespread development of organ donation after cardiac death (DCD), the quality of organs has attracted more and more attention. The preservation time of DCD organs is significantly longer than that of living related donation, and the organic ischemia-reperfusion injury (IRI) is more significant. IRI is one of the main factors of chronic rejection in DCD-related kidney transplantation. The chronic rejection is an important cause of long-term failure of renal transplantation and the treatment is very difficult. During DCD kidney transplantation, an emergency response was initiated and inflammatory factors were highly expressed in vivo, especially the increase of IFN-γ. Umbilical cord mesenchymal stem cells (UC-MSCs) can rapidly inhibit the inflammatory response and secrete high concentrations of prostaglandin PGE2, which can mediate the hypermethylation of MDSCs to regulate the differentiation of M2 macrophages and their subpopulations. By injecting pre-sensitized UC-MSCs with IFN-γ, the method can inhibit or delay chronic rejection and induce specific immune tolerance while protecting IRI of transplanted kidney.

Methods: MDSC primary cells were prepared from fresh umbilical cord tissue, and the cells were passaged to the third passage (P3). The cell concentration was adjusted to 1×105cells/mL. UC-MSCs were pretreated with 100 ng/mL IFN-γ for 48 hours, and then transfused into DCD kidney transplanted C57BL/6 recipient mice, and three control groups were set up for observation. The peripheral blood, transplanted kidney and local tissues of the above-mentioned rats were isolated, and the expression abundance of PGE2 was detected by RT-PCR at the 3rd,7th,14th and 28th days after operation. The proportion, distribution, methylation and phenotypic changes of M2 macrophage cells and MDSC were also identified by single-cell sequencing technology. Pathological specimens were used to analyze T lymphocyte, neutrophil, and macrophage infiltration. Biochemical samples were used to detect the expression of inflammatory factors, oxidative stress (SOD, MPO, MDA), and the expression of apoptosis-related proteins (caspase-3, caspase-9, Bcl-2, Bax) in kidney cells were detected by western-blotting.

Results: The rat model of DCD kidney transplantation was successfully established. The hypermethylation status of MDSCs in the IFN-γ-UC-MSCs kidney transplantation group was higher than that in the PBS and UC-MSCs groups(P<0.05). The abundance of PGE2 expression in the transplanted kidney and local tissue in the IFN-γ-UC-MSCs group was higher than that in the other two groups. The ratio of M2/M1 macrophages was significantly higher than that in the other two groups (P<0.05).M2 macrophages were mainly distributed in the microenvironment around the transplanted kidney, and the phenotypes were dominated by M2b and M2c subtypes. From the 3rd day to the 7th day of transplantation, the expression levels of IL-12, IL-2 and IFN-γ cytokines in the serum of each group were increased. The increased levels of the IFN-γ-UC-MSCs group and the UC-MSCs group was significantly lower than the PBS group (P<0.05). On the 14th day of transplantation, the serum level of IL-10 in the IFN-γ-UC-MSCs group was significantly higher than other two groups (P<0.05). On the 28th day, CD4+CD25+ Tregs and CD4+CD25+Foxp3+ Tregs the spleen accounted for the highest proportion of CD4+ T cells, which was significantly different from other groups (P<0.05). Foxp3 mRNA expression in IFN-γ-UC-MSCs group was significantly higher than that in UC-MSCs and PBS groups (P<0.05). The ratio of M2/M1 macrophages in IFN-γ -UC-Mscs group was significantly higher than that in other groups (P<0.05). The HE staining of the transplanted kidney showed that the inflammatory response of the transplant was significantly lower than that of the PBS and UC-MSCs groups.

Conclusions: By injecting UC-MSCs pre-sensitized with IFN-γ, the DCD kidney transplanted rats can secrete a large amount of PGE2, which mediate the hypermethylation of MDSCs to induce an increase in the M2 macrophages. The increase of M2 macrophages forms a local immunosuppressive microenvironment, inhibit the occurrence of chronic rejection and induce long-term specific immune tolerance.