European Review for Medical and Pharmacological Sciences 2019; 23: 8741-8750 Netrin-1 prolongs skin graft survival by inducing the transformation of mesenchymal stem cells from pro-rejection to immune-tolerant phenotype L.- P. L IU1, W. CHEN2, Y. FAN2, B.-Y. QI2, Z. WANG2, B.-Y. SHI2 1Medical School of Chinese PLA, Beijing, China 2The Eighth Medical Center of PLA General Hospital, Beijing, China Lupeng Liu and Wen Chen contributed equally to this work Abstract. – OBJECTIVE: Mesenchymal stem MSC function. The immunohistochemistry re- cells (MSCs) induce allograft immune tolerance, sults showed that, compared with the rejection but low efficacy severely limits their wide appli- group, the T cell number in the skin graft signifi- cation. In this work, Netrin-1 was used to main- cantly decreased in the Netrin-1 group. tain MSC function in an IR environment to study CONCLUSIONS: MSC can be divided into im- its role in the immune tolerance induction of the mune-tolerant and pro-rejection types in or- allograft. gan transplantation and Netrin-1 can induce the MATERIALS AND METHODS: The experi- transformation of MSC from the pro-rejection to ments were divided into three groups: the con- immune-tolerant type and markedly prolong the trol group, the IR group and the Netrin-1 group skin graft survival time. (Netrin-1 was added to MSC medium and then cultured for 48 h). After digestion, MSCs were Key Words: mixed with TLR4 and TLR3 antibodies (BD), in- Mesenchymal stem cell, Transplantation, Netrin-1, cubated for 20 min, and washed with Phos- Immune tolerance. phate-Buffered Saline (PBS) three times. The mean fluorescence intensity (MFI) of TLR4 and TLR3 was detected by flow cytometry. Isolat- ed lymphocytes were divided into four groups: the control group (no treatment), the MSC group Introduction (lymphocytes were co-cultured with MSCs in the control group), the rejection group (lym- Organ transplantation is the most effective phocytes were co-cultured with MSCs in the IR treatment for end-stage organ diseases, and rejec- group), and the Netrin-1 group (MSCs in the IR tion is the primary independent risk factor that group) was stimulated by Netrin-1 for 48h. RESULTS: Our study found that compared affects the long-term survival of transplanted with control mice, toll-like receptor (TLR3) ex- organs. Immunosuppressive agents significantly pression in bone marrow MSCs decreased as reduce the incidence of immune rejection (IR), the expression of TLR4 increased, the secretion but it may be accompanied by many complica- of transforming growth factor-β (TGF-β) and in- tions, such as infection and tumorigenesis1. In terleukin-10 (IL-10) was reduced, while the se- recent years, scientists have been seeking a new cretion of IL-6 significantly increased in im- way to induce immune tolerance; regulatory cell mune rejection (IR) mice. MSCs in IR mice pro- moted T-cell proliferation and reduced the ra- transplantation is considered one of the most 2,3 tio of Treg cells. Netrin-1 inhibited the pro-re- promising alternatives . jection effect of these MSCs, further inhibited A mesenchymal stem cell (MSC) is a kind of T-cell proliferation and facilitated an increase in adult stem cell that can induce allograft immune the ratio of Treg cells. The animal experiment re- tolerance by inhibiting T-cell proliferation and sults showed that MSC transplantation in the re- optimizing the local immune microenvironment4. jection group would shorten the mean survival time of the skin graft and induce the infiltration MSCs increased the 4-year survival rate of a of lymphocytes. Netrin-1 prolonged the mean transplanted kidney by secreting immunosup- survival time of the skin graft by enhancing pressive molecules5. Peng et al6 found that do- Corresponding Author: Bingyi Shi, Ph.D; e-mail: [email protected] 8741 L.-P. Liu, W. Chen, Y. Fan, B.-Y. Qi, Z. Wang, B.-Y. Shi nor-derived MSCs markedly reduced the use of gle’s Medium/F12 (DMEM/F12) medium (Gibco, immunosuppressive drugs and effectively main- Grand Island, NY, USA) plus 10% fetal bovine tained the long-term survival of a transplanted serum (FBS; Gibco, Grand Island, NY, USA). kidney. However, the role of MSCs inducing Forty-eight hours later, the unattached cells were the immune tolerance in vivo is not significant, removed and the remaining cells continued to be and large amplification in vitro is needed before cultured. Fluorescein isothiocyanate (FITC)-con- treatment. jugated rat anti-mouse stem cell antigen (Sca)-1, Nerve molecules play a crucial role in revas- CD11b, phycoerythrin (PE)-conjugated rat an- cularization, immunoregulation and tissue re- ti-mouse CD29, CD44, CD34 and CD45 were generation. Netrin-1 is a secretory axon guidance used for flow cytometry analysis to identify the molecule that plays an important role in the mouse MSCs. reconstruction and repairing of neural pathways7. It was found that Netrin-1 inhibited the migration Detection of MSC Phenotype and recruitment of inflammatory cells through The experiments were divided into three the mitogen-activated protein kinase (MAPK) groups: the control group, the IR group and the and eukaryotic protein kinase (ERK) pathway; Netrin-1 group (Netrin-1 was added to MSC me- it also inhibited excessive local inflammatory re- dium and then cultured for 48 h). After digestion, sponse8,9. In addition, Netrin-1 maintained MSC MSCs were mixed with TLR4 and TLR3 antibod- function under ischemic conditions and promoted ies (BD), incubated for 20 min and then washed the reconstruction of nerves and blood vessels10. with PBS three times. The mean fluorescence In this work, Netrin-1 was used to maintain the intensity (MFI) of TLR4 and TLR3 was detected MSC function in an IR environment to study its by flow cytometry (FACSCalibur; BD Bioscienc- role in the immune tolerance induction of the es, Franklin Lakes, NJ, USA). allograft. After culturing for 48 h, the MSC medium in each group was collected and centrifuged at 3000 rpm for 5 min; then, the supernatant was collect- Materials and Methods ed. The transforming growth factor-β (TGF-β), interleukin-10 (IL-10) and IL-6 concentrations in Skin Transplantation the supernatant were measured by enzyme-linked All animal experiments were performed in immunosorbent assay (ELISA; Neobioscience, accordance with the regulations on animal exper- Shenzhen, China). iments of the Eighth Medical Center of the PLA General Hospital (Beijing, China) and were ap- Cell Co-Culture proved by the hospital’s Ethics Committee. After At 48 h after skin transplantation, the heart anesthesia by pentobarbital sodium, the Balb/c blood of mice was collected and then lymphocytes mice back skin was taken and transplanted to were isolated. Isolated lymphocytes were divided the backs of C57BL/6 mice. Interrupted sutures into four groups: the control group (no treatment), were carried out with the operation line in the the MSC group (lymphocytes were co-cultured connection of the skin, and the mice were reared with MSCs in the control group), the rejection under suitable conditions. The judgment of skin group (lymphocytes were co-cultured with MSCs allograft survival was defined by Pilon et al11: in the IR group), and the Netrin-1 group (MSCs Skin grafts were monitored daily by visual and in the IR group was stimulated by Netrin-1 for tactile examination. The rejection was defined as 48 h. After being washed with PBS three times, greater than 80% graft necrosis. MSCs were co-cultured with lymphocytes). After culturing for 48 h, the lymphocytes were collect- Isolation and Identification of ed. We used the mouse regulatory T cell staining Bone Marrow MSCs kits (eBioscience 88-8111-40, San Diego, CA, MSCs were isolated from the femurs of Balb/c USA) for the instructions and flow cytometry to mice. After removing the ends of the femurs, the detect the ratio of CD4+CD25+Foxp3+Treg cells. bone marrow residues were rinsed with Phos- phate-Buffered Saline (PBS; Gibco, Grand Is- Cell Proliferation Experiment land, NY, USA) and then centrifuged at 1500 rpm To study the effect of MSC on T-cell prolif- for 20 min. After washing them three times, BM- eration, an EDU proliferation experiment was MSCs were cultured in Dulbecco’s Modified Ea- carried out. Cell grouping was the same as be- 8742 Netrin-1 prolongs skin graft survival fore. MSCs were cultured with lymphocytes for Statistical Analysis 24 h, T cells were sorted by magnetic beads The t-test and variance analysis were performed and a 5-Ethynyl-2’- deoxyuridine (EdU) staining using Statistical Product and Service Solutions reaction solution (R&D Systems, Minneapolis, (SPSS) 12.0 software (SPSS Inc., Chicago, IL, MN, USA) was added and then cultured for 24 USA). All experimental data were expressed as h. The supernatant was discarded and the cells means ± standard deviation. A p-value of less were washed with PBS three times. Then, 0.5% than 0.05 was considered statistically significant. TritonX-100 was added and penetrated for 10 min; then, a staining reaction solution was added and incubated in a dark place for 30 min. After Results fully washing with 0.5% TritonX-100, it was stained with DAPI for 5 min, washed with PBS Isolation and Identification of three times, section sealed and observed under a Mouse BM-MSCs fluorescence microscope. After 48 hours of culturing, non-adherent cells were removed and adherent cells were selected. Animal Experiment The MSCs were expanded to six generations C57BL/6 mice were randomly divided into four and then digested by trypsin. Surface markers groups: the control group (prior to skin transplan- were identified by flow cytometry. We found tation, saline was injected), the MSC group (prior the BM-MSCs were CD45-CD11b -CD34-Sca-1+C- to skin transplantation, 2×106 MSCs in the control D44+CD29+ cells (Figure 1), consistent with the group were injected from the caudal vein), the previous report12.