Netrin-1 prolongs skin graft survival by inducing the transformation of mesenchymal stem cells from pro-rejection to immune-tolerant phenotype.

Mesenchymal stem cells (MSCs) induce allograft immune tolerance, but low efficacy severely limits their wide application. In this work, Netrin-1 was used to maintain MSC function in an IR environment to study its role in the immune tolerance induction of the allograft.The experiments were divided into three groups: the control group, the IR group and the Netrin-1 group (Netrin-1 was added to MSC medium and then cultured for 48 h). After digestion, MSCs were mixed with TLR4 and TLR3 antibodies (BD), incubated for 20 min, and washed with Phosphate-Buffered Saline (PBS) three times. The mean fluorescence intensity (MFI) of TLR4 and TLR3 was detected by flow cytometry. Isolated lymphocytes were divided into four groups: the control group (no treatment), the MSC group (lymphocytes were co-cultured with MSCs in the control group), the rejection group (lymphocytes were co-cultured with MSCs in the IR group), and the Netrin-1 group (MSCs in the IR group) was stimulated by Netrin-1 for 48h.Our study found that compared with control mice, toll-like receptor (TLR3) expression in bone marrow MSCs decreased as the expression of TLR4 increased, the secretion of transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) was reduced, while the secretion of IL-6 significantly increased in immune rejection (IR) mice. MSCs in IR mice promoted T-cell proliferation and reduced the ratio of Treg cells. Netrin-1 inhibited the pro-rejection effect of these MSCs, further inhibited T-cell proliferation and facilitated an increase in the ratio of Treg cells. The animal experiment results showed that MSC transplantation in the rejection group would shorten the mean survival time of the skin graft and induce the infiltration of lymphocytes. Netrin-1 prolonged the mean survival time of the skin graft by enhancing MSC function. The immunohistochemistry results showed that, compared with the rejection group, the T cell number in the skin graft significantly decreased in the Netrin-1 group.MSC can be divided into immune-tolerant and pro-rejection types in organ transplantation and Netrin-1 can induce the transformation of MSC from the pro-rejection to immune-tolerant type and markedly prolong the skin graft survival time.