EFFECTS OF INDOLEAMINE 2, 3-DIOXYGENASE GENE MODIFIED BONE MAEEOW MESENCHYMAL STEM CELLS IN RAT COMPOSITE TISSUE ALLOGRAFT REJECTION_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HANFu , WANGYaojun , WANGYunchuan , JIAYanhui , YANGLonglong , JIAWenbin , FANGXiaobing , BAIXiaozhi ,  HUDahai

Department of Burns and Cutaneous Surgery, Xijing Hospital, the Fourth Military Medical University, Xi'an Shaanxi, 710032, P. R. China;

Corresponding author：

HUDahai, Email: Hudhai@fmmu.edu.cn

Keywords：

Bone marrow mesenchymal stem cells; Indoleamine 2,3-dioxygenase; T lymphocytes; Composite tissue allograft; Immunological rejection; Rat

DOI：

10.7507/1002-1892.20140166

Video：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

Objective To evaluate the effects and mechanism of indoleamine 2, 3-dioxygenase (IDO) modified rat bone marrow mesenchymal stem cells (BMSCs) in composite tissue allograft rejection. Methods BMSCs isolated from Brown Norway (BN) rats (aged, 4-6 weeks) were infected by IDO[green fluorescent protein (GFP)]-lentivirus. The high expression target gene and biological activity cell line (IDO-BMSCs) were screened. IDO mRNA and protein expressions were detected by RT-PCR and Western blot. The biological activity of IDO in supernatant was detected by measuring the amount of kynurenine generation. In mixed lymphocyte reaction system, different numbers of IDO-BMSCs mixed with responding cells (peripheral blood mononuclear cell isolated from 4-6-week-old LEWIS rats, as recipient) and stimulating cells (peripheral blood mononuclear cell isolated from BN rats, as donor), with the cells ratios of 1:5:5, 1:10:10, 1:50:50, and 1:100:100 (as experimental groups 1, 2, 3, and 4, respectively). Each reaction system was blocked by 1 mmol/L 1-methyl-tryptophan (1-MT) (IDO specific inhibitor). IDO-BMSCs mixed with responding cells (1:5) as the negative control group, responding cells mixed with stimulating cells (1:1) as positive control group; and IDO-BMSCs were cultured in RPMI 1640 medium alone as blank control group. MTT assay was used to detect the T lymphocytes proliferation at 5 days. Furthermore, GFP-BMSCs (group A), IDO-BMSCs (group B), and normal saline (group C) were infused via the tail vein of allogeneic limb transplantation rats, and graft survival time and rejection were observed in each group. Results The IDO expression of BMSCs after genetic modification was higher than that before genetic modification. IDO-BMSCs could significantly improved kynurenine concentration in culture medium supernatant when compared with GFP-BMSCs (P<0.05). Before adding 1-MT, with the ratio of IDO-BMSCs to responding cells decreased, T lymphocytes proliferation rate increased in experimental groups 1, 2, and 3, showing significant differences between groups (P<0.05); there was no significant difference between experimental group 4 and the positive control group (P>0.05). After adding 1-MT, T lymphocytes proliferation rate was significantly higher than that before adding 1-MT in the other experimental groups (P<0.05) except experimental group 4 (P>0.05). In vivo, IDO-BMSCs could promote colonization in allograft, inhibit transplantation rejection, and prolong survival time of composite tissue allograft; the survival time of composite tissue allograft was (11.5±0.6) days in group A, (14.5±0.8) days in group B, and (9.0±0.3) days in group C, and it was significantly longer in group B than in groups A and C, and in group A than in group C (P<0.05). Conclusion IDO-BMSCs can promote the survival of allogeneic composite tissue grafts in rats, and its mechanism may involve in inhibition of T lymphocytes proliferation and promotion their own colonization in allograft.

Citation： HANFu, WANGYaojun, WANGYunchuan, JIAYanhui, YANGLonglong, JIAWenbin, FANGXiaobing, BAIXiaozhi, HUDahai. EFFECTS OF INDOLEAMINE 2, 3-DIOXYGENASE GENE MODIFIED BONE MAEEOW MESENCHYMAL STEM CELLS IN RAT COMPOSITE TISSUE ALLOGRAFT REJECTION. Chinese Journal of Reparative and Reconstructive Surgery, 2014, 28(6): 745-751. doi: 10.7507/1002-1892.20140166 Copy

1.	De Miguel MP, Fuentes-Julian S, Blazquez-Martinez A, et al. Immunosuppressive properties of mesenchymal stem cells:advances and applications. Curr Mol Med, 2012, 12(5):574-591.
2.	Gebler A, Zabel O, Seliger B. The immunomodulatory capacity of mesenchymal stem cells. Trends Mol Med, 2012, 18(2):128-134.
3.	Curran TA, Jalili RB, Farrokhi A, et al. IDO expressing fibroblasts promote the expansion of antigen specific regulatory T cells. Immunobiology, 2014, 219(1):17-24.
4.	Rezakhanlou AM, Habibi D, Lai A, et al. Highly efficient stable expression of indoleamine 2, 3 dioxygenase gene in primary fibroblasts. Biol Proced Online, 2010, 12(1):9028.
5.	Däubener W, Wanagat N, Pilz K, et al. A new, simple, bioassay for human IFN-gamma. J Immunol Methods, 1994, 168(1):39-47.
6.	Meisel R, Zibert A, Laryea M, et al. Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2, 3-dioxygenase-mediated tryptophan degradation. Blood, 2004, 103(12):4619-4621.
7.	Yuan Y, Lu X, Tao CL, et al. Forced expression of indoleamine-2, 3-dioxygenase in human umbilical cord-derived mesenchymal stem cells abolishes their anti-apoptotic effect on leukemia cell lines in vitro. In Vitro Cell Dev Biol Anim, 2013, 49(10):752-758.
8.	Wang Y, Zheng Z, Wang Y, et al. Role of donor-specific regulatory T cells in long-term acceptance of rat hind limb allograft. PLoS One, 2012, 7(8):e43825.
9.	Buttemeyer R, Jones NF, Min Z, et al. Rejection of the component tissues of limb allografts in rats immunosuppressed with FK-506 and cyclosporine. Plast Reconstr Surg, 1996, 97(1):139-151.
10.	Lee WP, Pan YC, Kesmarky S, et al. Experimental orthotopic transplantation of vascularized skeletal allografts:functional assessment and long-term survival. Plast Reconstr Surg, 1995, 95(2):336-353.
11.	Cendales LC, Kirk AD, Moresi JM, et al. Composite tissue allotransplantation:classification of clinical acute skin rejection. Transplantation, 2006, 81(3):418-422.
12.	Gander B, Brown CS, Vasilic D, et al. Composite tissue allotransplantation of the hand and face:a new frontier in transplant and reconstructive surgery. Transpl Int, 2006, 19(11):868-880.
13.	Swearingen B, Ravindra K, Xu H, et al. Science of composite tissue allotransplantation. Transplantation, 2008, 86(5):627-635.
14.	Lee WP, Yaremchuk MJ, Pan YC, et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg, 1991, 87(3):401-411.
15.	Wang Y, Liu J, Xu C, et al. Bone marrow transplantation combined with mesenchymal stem cells induces immune tolerance without cytotoxic conditioning. J Surg Res, 2011, 171(1):e123-131.
16.	Griffin MD, Ryan AE, Alagesan S, et al. Anti-donor immune responses elicited by allogeneic mesenchymal stem cells:what have we learned so far? Immunol Cell Biol, 2013, 91(1):40-51.
17.	Fallarino F, Grohmann U, Puccetti P. Indoleamine 2, 3-dioxygenase:from catalyst to signaling function. Eur J Immunol, 2012, 42(8):1932-1937.
18.	Pallotta MT, Orabona C, Volpi C, et al. Indoleamine 2, 3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells. Nat Immunol, 2011, 12(9):870-878.
19.	François M, Romieu-Mourez R, Li M, et al. Human MSC suppression correlates with cytokine induction of indoleamine 2, 3-dioxygenase and bystander M₂ macrophage differentiation. Mol Ther, 2012, 20(1):187-195.

1. De Miguel MP, Fuentes-Julian S, Blazquez-Martinez A, et al. Immunosuppressive properties of mesenchymal stem cells:advances and applications. Curr Mol Med, 2012, 12(5):574-591.
2. Gebler A, Zabel O, Seliger B. The immunomodulatory capacity of mesenchymal stem cells. Trends Mol Med, 2012, 18(2):128-134.
3. Curran TA, Jalili RB, Farrokhi A, et al. IDO expressing fibroblasts promote the expansion of antigen specific regulatory T cells. Immunobiology, 2014, 219(1):17-24.
4. Rezakhanlou AM, Habibi D, Lai A, et al. Highly efficient stable expression of indoleamine 2, 3 dioxygenase gene in primary fibroblasts. Biol Proced Online, 2010, 12(1):9028.
5. Däubener W, Wanagat N, Pilz K, et al. A new, simple, bioassay for human IFN-gamma. J Immunol Methods, 1994, 168(1):39-47.
6. Meisel R, Zibert A, Laryea M, et al. Human bone marrow stromal cells inhibit allogeneic T-cell responses by indoleamine 2, 3-dioxygenase-mediated tryptophan degradation. Blood, 2004, 103(12):4619-4621.
7. Yuan Y, Lu X, Tao CL, et al. Forced expression of indoleamine-2, 3-dioxygenase in human umbilical cord-derived mesenchymal stem cells abolishes their anti-apoptotic effect on leukemia cell lines in vitro. In Vitro Cell Dev Biol Anim, 2013, 49(10):752-758.
8. Wang Y, Zheng Z, Wang Y, et al. Role of donor-specific regulatory T cells in long-term acceptance of rat hind limb allograft. PLoS One, 2012, 7(8):e43825.
9. Buttemeyer R, Jones NF, Min Z, et al. Rejection of the component tissues of limb allografts in rats immunosuppressed with FK-506 and cyclosporine. Plast Reconstr Surg, 1996, 97(1):139-151.
10. Lee WP, Pan YC, Kesmarky S, et al. Experimental orthotopic transplantation of vascularized skeletal allografts:functional assessment and long-term survival. Plast Reconstr Surg, 1995, 95(2):336-353.
11. Cendales LC, Kirk AD, Moresi JM, et al. Composite tissue allotransplantation:classification of clinical acute skin rejection. Transplantation, 2006, 81(3):418-422.
12. Gander B, Brown CS, Vasilic D, et al. Composite tissue allotransplantation of the hand and face:a new frontier in transplant and reconstructive surgery. Transpl Int, 2006, 19(11):868-880.
13. Swearingen B, Ravindra K, Xu H, et al. Science of composite tissue allotransplantation. Transplantation, 2008, 86(5):627-635.
14. Lee WP, Yaremchuk MJ, Pan YC, et al. Relative antigenicity of components of a vascularized limb allograft. Plast Reconstr Surg, 1991, 87(3):401-411.
15. Wang Y, Liu J, Xu C, et al. Bone marrow transplantation combined with mesenchymal stem cells induces immune tolerance without cytotoxic conditioning. J Surg Res, 2011, 171(1):e123-131.
16. Griffin MD, Ryan AE, Alagesan S, et al. Anti-donor immune responses elicited by allogeneic mesenchymal stem cells:what have we learned so far? Immunol Cell Biol, 2013, 91(1):40-51.
17. Fallarino F, Grohmann U, Puccetti P. Indoleamine 2, 3-dioxygenase:from catalyst to signaling function. Eur J Immunol, 2012, 42(8):1932-1937.
18. Pallotta MT, Orabona C, Volpi C, et al. Indoleamine 2, 3-dioxygenase is a signaling protein in long-term tolerance by dendritic cells. Nat Immunol, 2011, 12(9):870-878.
19. François M, Romieu-Mourez R, Li M, et al. Human MSC suppression correlates with cytokine induction of indoleamine 2, 3-dioxygenase and bystander M₂ macrophage differentiation. Mol Ther, 2012, 20(1):187-195.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECTS OF INDOLEAMINE 2, 3-DIOXYGENASE GENE MODIFIED BONE MAEEOW MESENCHYMAL STEM CELLS IN RAT COMPOSITE TISSUE ALLOGRAFT REJECTION

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content