Author Affiliations: First Central Hospital Clinical College, Tianjin Medical University, Tianjin 300070, China (Yang Y, Yin ML and Zhang BY); Department of Organ Transplantation, Tianjin First Central Hospital, Tianjin 300192, China (Shen ZY, Wu B and Song HL)

Corresponding Author: Hong-Li Song, MD, PhD, Department of Organ Transplantation, Tianjin First Central Hospital and Tianjin Key Laboratory of Organ Transplantation, Tianjin 300192, China (Email: hlsong26@163.com)

 

© 2016, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(16)60085-1

Published online April 20, 2016.

 

 

Acknowledgments: We thank the Key Laboratory of Emergency and Care Medicine of Ministry of Health and Tianjin Key Laboratory of Organ Transplantation for allowing this work to progress in their laboratories.

Contributors: SZY and SHL conceived the research design. YY, WB, YML and ZBY performed the research. YY, SZY and SHL analyzed the data and wrote the paper. All authors contributed to the design and interpretatim of the study and to further drafts. SHL is the guarantor.

Funding: This study was supported by grants from the National Natural Science Foundation of China (81270528 and 81441022), Natural Science Foundation of Tianjin, China (08JCYBJC08400, 11JCZDJC27800 and 12JCZDJC25200), and the Technology Foundation of Health Bureau in Tianjin, China (2011KY11).

Ethical approval: All experimental procedures were carried out in according with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH publication 86-23, revised 1985), and all protocols were approved by the Animal Care and Research Committee of Tianjin First Central Hospital (Tianjin, China; Permit Number: E20140301-002A).

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Bone marrow mesenchymal stem cells (BMMSCs) exert immunosuppressive activities in transplantation. This study aimed to determine whether BMMSCs reduce acute rejection and improve outcomes of liver transplantation in rats.

 

METHODS: Orthotopic liver transplantation from Lewis to Brown Norway rats was performed, which was followed by the infusion of BMMSCs through the penile superficial dorsal vein. Normal saline infusion was used as a control. Animals were sacrificed at 0, 24, 72, or 168 hours after BMMSCs infusion. Liver grafts, and recipient serum and spleen tissues were obtained. Histopathology, apoptosis, serum liver enzymes, serum cytokines, and circulating regulatory T (Treg), Th1, Th2 and Th17 cells were assessed at each time point.

 

RESULTS: BMMSCs significantly attenuated acute rejection and improved the survival rate of allogeneic liver transplantation recipients. Liver enzymes and liver apoptosis were significantly alleviated. The levels of the Th1/Th2 ratio-associated cytokines such as IL-2 and IFN-γ were significantly reduced and IL-10 was significantly increased. The levels of the Th17/Tregs axis-associated cytokines such as IL-6, IL-17, IL-23, and TNF-α were significantly reduced, whereas TGF-β concentration was significantly increased. Moreover, flow cytometry analysis showed that the infusion of BMMSCs significantly increased Th2 and Treg cells and decreased Th1 and Th17 cells.

 

CONCLUSION: BMMSCs had immunomodulatory effects, attenuated acute rejection and improved outcomes of allogeneic liver transplantation in rats by regulating the levels of cytokines associated with Th1/Th2 and Th17/Treg ratios.

 

(Hepatobiliary Pancreat Dis Int 2016;15:257-265)

 

KEY WORDS: bone marrow mesenchymal stem cells; liver transplantation; acute rejection

Liver transplantation (LT) represents the only definitive life-saving treatment for patients with liver failure.^[1] However, post-transplant immunosuppressants put patients at risk for both acute and long-term side effects, such as nephrotoxicity, neurotoxicity, infection and post-transplant neoplasia.^[2-4] Thus, novel immunosuppressive strategies for LT are urgently needed. Immunomodulatory cell therapy is a recently described innovative approach to complement standard pharmacotherapy in solid organ transplantation. The prospect of mesenchymal stem cells (MSCs) with immunosuppressive features has renewed expectations for prolonging liver allograft survival and reducing the side effects of post-transplant immunosuppressive therapy.^{[5, 6]} MSCs are multipotent non-hematopoietic progenitor cells of a stromal origin that have been isolated from various sources, such as bone marrow,^[7] cord blood,^[8] and adipose tissue.^[9] MSCs have regenerative, anti-inflammatory, and immunomodulatory properties; therefore, they have been widely used in experimental studies of organ transplant immunosuppression.

 

Studies have analyzed the effects of MSCs on transplanted kidney,^{[10, 11]} small bowel,^[12] islet cell,^{[13, 14]} and liver tissues.^[15,16] Many of these studies indicated that MSCs benefit the survival of the transplanted organs, or even induce immune tolerance.^{[17, 18]} However, how MSCs modulate the immune system in organ transplantation is not yet fully understood. It is generally accepted that organ-graft rejection is mediated by T cell responses, in which various subsets of CD4⁺ T helper (Th) cells (including Th1, Th2, Th17 and Treg cells) and related cytokines play pivotal roles.^[19,20] Our study aimed to determine whether bone marrow mesenchymal stem cells (BMMSCs) could reduce acute rejection and improve LT outcomes, and to examine whether this was cytokines related. We also evaluated Th1/Th2 and Th17/Treg cell ratios in liver recipients in rats.

 

 

Inbred adult male Lewis rats (RT-1^l) and male Brown Norway rats (RT-1ⁿ) weighing 200-220 g were used as donors and recipients, respectively. Immature Brown Norway rats weighing 80-100 g were used as a source of BMMSCs. The rats were purchased from Vital River Company (Beijing, China) and were humanely treated. Rat chow and tap water were provided ad libitum until fasting 12 hours before surgery. All experimental procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health (NIH publication 86-23, revised 1985), and all protocols were approved by the Animal Care and Research Committee of Tianjin First Central Hospital (Tianjin, China; Permit Number: E20140301-002A). All operations were performed under chloral hydrate anesthesia, and every effort was made to minimize animal suffering.

 

BMMSC isolation, culture and characterization were carried out using techniques previously described by our group.^[12] Briefly, BMMSCs were isolated from the femur and tibia of male Brown Norway rats (80-100 g) and were cultured in a cell culture incubator. The culture medium was changed to remove nonadherent cells and adherent cells were consistently cultured until 80% confluence. Passage 3-5 BMMSCs were trypsinized and used for all experiments when they reached 80% confluence. BMMSCs were identified by flow cytometry and the antibodies used included anti-CD29, CD34, CD45, CD90, RT1A and RT1B (Biolegend, San Diego, CA, USA). BMMSCs were also confirmed according to the plastic-adherent feature and morphology.

 

Fifty Lewis rats and paired Brown Norway rats were randomly divided into two groups. Orthotopic LT was performed from Lewis to Brown Norway rats following the two-cuff technique described by Kamada.^{[21, 22]} Briefly, transplantation was performed between a donor and recipient by supra-hepatic inferior vena cava (IVC) anastomosis with 8-0 suture, and portal vein and infra-hepatic IVC anastomosis with cuffs. The warm ischemia time was almost zero, the mean anhepatic phase time was 20.40±2.07 minutes and the mean cold ischemia time was 30.60±3.36 minutes. Recipient rats were treated with 1×10⁷ BMMSCs in the experimental group through the penile superficial dorsal vein immediately after surgery, or normal saline in controls. A total of five animals per group were euthanized at 0 (naïve), 24, 72 and 168 hours after LT.

 

Liver tissues were fixed in 10% formalin, embedded in paraffin, sliced into 5-µm sections and stained with hematoxylin and eosin (HE). The slides were blindly examined by a pathologist under a light microscope. Samples were graded and scored according to the acute liver allograft rejection index and the rejection activity index (RAI), respectively, using the Banff Schema,^[23] which is based on the density and area of inflammatory cell infiltration in the portal vein, bile duct, and venous endothelial regions.

 

TdT-mediated dUTP-X nick end labeling (TUNEL) staining was performed on paraffin-embedded tissue sections using an in situ Cell Death Detection Kit, as described by the manufacturer (Promega, Madison, WI, USA). As a negative control, terminal deoxynucleotidyl transferase was omitted, whereas positive controls were generated by treatment with deoxyribonuclease. All slides were blindly reviewed, and the number of positive cells was quantified in 10 randomly selected fields under a light microscope (original magnification ×200).

 

Liver injury was evaluated by measuring the concentrations of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL).

 

Recipient serum was obtained from the peripheral blood at each time point and stored at -80 �� for measurement. Concentrations of interleukin (IL)-10, transforming growth factor (TGF)-β, IL-2, IL-6, IL-17, IL-23, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ were measured using enzyme-linked immunosorbent assay (ELISA) kits as described by the manufacturer (R&D System, Minneapolis, MN, USA). The optical density was measured under 450 nm. The intra-assay coefficient of variation for each ELISA was ≤6.

 

Lymphocytes were isolated from recipient spleens and mashed into single-cell suspensions. Aliquots (1×10⁶ cells) were resuspended in 0.1 mL PBS and incubated with anti-CD4-FITC, anti-CD25-PE, and anti-Foxp3-PE-Cy5 antibodies (eBioscience, San Diego, CA, USA). Isotype-matched control antibodies served as staining controls. The CD4⁺CD25⁺Foxp3⁺ Treg cells were analyzed using flow cytometry (FACSCalibur; Becton-Dickenson, San Jose, CA, USA).

 

Lymphocytes were prepared in the same way as described for Treg cells detection, immunostained with various combinations of the following fluorescence-conjugated antibodies: CD4-FITC, IFN-γ-APC, IL-4-PE and IL-17-PE (eBioscience). Before intracellular cytokine staining, cells were stimulated in culture medium containing phorbol myristate acetate (25 ng/mL; Sigma-Aldrich), ionomycin (250 ng/mL; Sigma-Aldrich), or monensin (GolgiStop, 1 µL/mL; BD PharMingen) in an incubator with 5% CO₂ at 37 �� for 4 hours. Intracellular staining was performed using an intracellular staining kit (eBioscience) according to the manufacturer’s protocol. Flow cytometry was performed in order to detect IFN-γ⁺CD4⁺ Th1 cells, IL-4⁺CD4⁺ Th2 cells and IL-17⁺CD4⁺ Th17 cells.

 

All statistical analyses were performed using SPSS Statistical Software version 17.0 (SPSS, Chicago, IL, USA). Data were expressed as mean±standard deviation and compared by one-way analysis of variance. The survival rates of recipients were compared between the two groups (n=5 in each group) by the Kaplan-Meier method, and the log-rank test was used to identify significant differences between the groups. Differences were considered statistically significant when P<0.05.

 

 

Cells isolated from rat bone marrow were purified by adherence to plastic culture flasks and were confirmed as BMMSCs based on spindle-shaped fibroblastic morphologies with a swirl pattern after expansion. Phenotypic analysis by flow cytometry showed that BMMSCs expressed high levels of CD29, CD90, and RT1A, but little or no CD34, CD45, and RT1B (Fig. 1).

 

Recipients in the allogeneic control group exhibited emaciation, skin yellowing, and ascites within 7 days. In the BMMSCs-treated group, these symptoms attenuated in one recipient or were delayed until days 15, 17, 21 and 24 in the other four recipients. The median recipient survival was 19 vs 56 days, in the control and BMMSCs groups. Recipient survival rates were significantly improved with BMMSCs therapy (Fig. 2A). The levels of serum ALT, AST, and TBIL were significantly lower in the BMMSCs-treated group than in the control group, especially at 168 hours postoperatively (Fig. 2B).

 

Allogeneically transplanted animals treated with normal saline showed gradual increases in acute rejection, with severe acute rejection observed at day 7. By pathological examination, we noted a series of changes, including the infiltration of mixed types of inflammatory cells in the portal area, subendothelial lymphocytic infiltration of the interlobular and central veins, and interlobular bile duct inflammation and destruction. Acute rejection was significantly attenuated in the BMMSCs-treated group with alleviated inflammatory cell infiltration in the portal area (Fig. 3A). RAI was significantly decreased at 72 and 168 hours postoperatively compared with the control group (Fig. 3B).

 

We identified apoptotic cells in liver allografts by TUNEL staining. The Claybank-positive cells were counted in a high power field. Numerous apoptotic cells were apparent at 168 hours in the control group, while significantly fewer TUNEL-positive cells were observed in the BMMSCs-treated recipients (P<0.05 at 72 and 168 hours, Fig. 4).

 

Th1/Th2-related cytokines, IL-2 and IFN-γ were increased rapidly and peaked at 168 hours after transplantation in the control group, whereas IL-10 exhibited a downward trend. In contrast, serum concentrations of IL-2 and IFN-γ were significantly lower in BMMSCs-treated group than in the control group, and IL-10 rose sharply in the BMMSCs-treated group (Fig. 5).

 

Th17/Treg-associated cytokine, IL-6, IL-17, IL-23 and TNF-α were progressively increased, whereas, TGF-β was decreased in the control group. BMMSCs significantly inhibited the secretion of IL-6, IL-17, IL-23, and TNF-α, but enhanced the production of TGF-β (Fig. 5).

 

The expression of IL-4⁺CD4⁺ Th2 cells was higher in the BMMSCs-treated group than that in the control group. In contrast, spleen cells from the BMMSCs-treated group showed decreased expression of IFN-γ⁺CD4⁺ Th1 cells and IL-17⁺CD4⁺ Th17 cells (Fig. 6).

 

The frequencies of CD4⁺CD25⁺Foxp3⁺ Treg cells in the spleens were significantly increased in the BMMSCs-treated group compared with the control group (Fig. 7). The frequencies of Treg cells were significantly higher at 72 and 168 hours (P<0.05).

 

 

LT is currently the only effective therapy for end-stage liver diseases. Although LT outcomes have markedly improved since the introduction of immunosuppressants to prevent acute rejection, the consequent severe toxic and side effects of the procedure cannot be ignored. Therefore, safer and more effective methods are needed to better prevent and control rejection. Current MSC-based approaches have the potential to induce tolerance to transplanted organs. Our present study was designed to investigate the role of immunoregulatory BMMSCs in LT and potential associations with Th cell differentiation.

 

Orthotopic LT were performed in a combination of major histocompatibility complex (MHC)-disparate rat strains (Lewis to Brown Norway). Pathological analysis showed gradually aggravated acute rejection in liver allograft tissues. Increased amounts of liver enzymes, TBIL, and apoptosis indicated that hepatocyte damage had occurred in liver allografts. BMMSC therapy significantly reduced acute rejection and RAI, markedly improved liver function and apoptosis, and ultimately improved the survival rate of recipients.

 

Generally, allograft rejection in organ transplantation is thought to be mediated by T cell responses directed against donor organ-derived antigens. Various subsets of CD4⁺ Th cells, including Th1, Th2, Th17, and Treg cells, can exert different effects on immunopathologies. The ratios of Th1/Th2 and Th17/Treg cells play important roles in regulating T cell responses in transplantation.^{[19, 20]}

 

The Th1 cell subset mainly secretes IL-2, IFN-γ and TNF-α, whereas Th2 cells mainly produce IL-4, IL-10, and IL-13. The Th1/Th2 paradigm and the cytokines related to these cells have been used to explain transplantation-related phenomena in immunity.^[24-27] Increased serum concentrations of IL-2 have been strongly correlated with the severity of rejection.^[28] Indeed, elevated serum IFN-γ levels have been shown to precede rejection in both liver and kidney transplant patients and to participate in graft rejection,^{[29, 30]} whereas elevated IL-10 levels have been noted to be preferentially associated with non-rejection and the tolerance of solid allografts.^{[24, 31]} Our findings showed that Th1 cell levels were lower, but Th2 cell levels were higher in the BMMSCs-treated group than those in the control group. Similarly, the concentrations of the Th1-associated cytokines IL-2 and IFN-γ were significantly decreased, whereas the concentration of the Th2-associated cytokine IL-10 was markedly increased after BMMSCs treatment.

 

Th17 cells are characterized by the production of IL-17, IL-21, and IL-22,^{[32, 33]} and their differentiation requires TGF-β and IL-6.^[34] Stabilization of the Th17 cell phenotype is driven by IL-23, along with TNF-α and IL-1β.^[35] In contrast, Treg cells that express Foxp3 have anti-inflammatory functions and can promote tolerance to “self” by contact-mediated suppression or secretion of the anti-inflammatory cytokines IL-10 and TGF-β. Transplant rejection is likely to be mediated by Th17 cells and the associated cytokines IL-6,^[36] IL-17,^{[37, 38]} and IL-23,^[38] whereas it can potentially be prevented by Treg cells.^{[39, 40]} It has been suggested that the Th17/Treg ratio could be a sensitive and specific biomarker of acute graft-versus-host disease (GVHD).^[41] The factors and cytokines that affect the Th17/Treg balance have been implicated in both solid organ and hematopoietic stem cell transplantation.^[42-44] Our study aimed to measure serum Th17/Treg-associated cytokine concentrations and spleen Th17/Treg cell frequencies in liver transplant recipients. Our findings showed down-regulation of Th17 cells and Th17-associated cytokines (IL-6 and IL-17) in the BMMSCs-treated group but, in contrast, up-regulation of Th17 cells and Th17-associated cytokines in the control group. Consequently, Th17 cell levels and Th17-associated cytokine concentrations were much lower in the BMMSCs-treated group than in the control group. In addition, the increased Treg cell frequency was greater in the BMMSCs-treated group than in the control group, particularly at 168 hours.

 

In addition to the immunoregulatory effects of BMMSCs, its regenerative and anti-inflammatory properties are also responsible for the improvement of outcome of rat LT.^[16] It has been reported that BMMSCs can ameliorate hepatic ischemia/reperfusion injuries and promote liver cell regenerations.^{[45, 46]} We found the levels of liver enzymes, TBIL and apoptosis were lower or less in the BMMSCs-treated group than in the control group.

 

In summary, our data indicated that the infusion of BMMSCs reduced acute rejection, improved liver function, prevented apoptosis, and increased the survival rate of recipients, which all contributed to improved outcomes of liver transplantation in rats. We concluded that the immunoregulatory effects of BMMSCs took place via the modification of the ratios of Th1/Th2 and Th17/Treg cells, along with their associated cytokines. However, there were some limitations to our study. The tracing of BMMSCs in vivo and detecting cytokines in liver grafts may provide more persuasive evidence. Moreover, the long-term effects of BMMSCs remain unknown.

 

 

1 Lee WM, Squires RH Jr, Nyberg SL, Doo E, Hoofnagle JH. Acute liver failure: summary of a workshop. Hepatology 2008;47:1401-1415. PMID: 18318440

2 Webber A, Hirose R, Vincenti F. Novel strategies in immunosuppression: issues in perspective. Transplantation 2011;91: 1057-1064. PMID: 21412186

3 Grinyó JM, Cruzado JM. Mycophenolate mofetil and calcineurin-inhibitor reduction: recent progress. Am J Transplant 2009;9:2447-2452. PMID: 19775321

4 Vajdic CM, van Leeuwen MT. Cancer incidence and risk factors after solid organ transplantation. Int J Cancer 2009;125:1747-1754. PMID: 19444916

5 Detry O, Jouret F, Vandermeulen M, Erpicum P, Delens L, Grégoire C, et al. Mesenchymal stromal cells and organ transplantation. Rev Med Liege 2014;69:53-56. PMID: 25796799

6 Benseler V, Obermajer N, Johnson CL, Soeder Y, Dahlke MD, Popp FC. MSC-based therapies in solid organ transplantation. Hepatol Int 2014;8:179-184. PMID: 26202500

7 Comite P, Cobianchi L, Avanzini MA, Zonta S, Mantelli M, Achille V, et al. Isolation and ex vivo expansion of bone marrow-derived porcine mesenchymal stromal cells: potential for application in an experimental model of solid organ transplantation in large animals. Transplant Proc 2010;42:1341-1343. PMID: 20534296

8 Zeddou M, Briquet A, Relic B, Josse C, Malaise MG, Gothot A, et al. The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood. Cell Biol Int 2010;34:693-701. PMID: 20187873

9 de Mattos Carvalho A, Alves AL, Golim MA, Moroz A, Hussni CA, de Oliveira PG, et al. Isolation and immunophenotypic characterization of mesenchymal stem cells derived from equine species adipose tissue. Vet Immunol Immunopathol 2009;132:303-306. PMID: 19647331

10 Perico N, Casiraghi F, Gotti E, Introna M, Todeschini M, Cavinato RA, et al. Mesenchymal stromal cells and kidney transplantation: pretransplant infusion protects from graft dysfunction while fostering immunoregulation. Transpl Int 2013;26:867-878. PMID: 23738760

11 Perico N, Casiraghi F, Introna M, Gotti E, Todeschini M, Cavinato RA, et al. Autologous mesenchymal stromal cells and kidney transplantation: a pilot study of safety and clinical feasibility. Clin J Am Soc Nephrol 2011;6:412-422. PMID: 20930086

12 Yang Y, Song HL, Zhang W, Wu BJ, Fu NN, Zheng WP, et al. Reduction of acute rejection by bone marrow mesenchymal stem cells during rat small bowel transplantation. PLoS One 2014;9:e114528. PMID: 25500836

13 Perez-Basterrechea M, Obaya AJ, Meana A, Otero J, Esteban MM. Cooperation by fibroblasts and bone marrow-mesenchymal stem cells to improve pancreatic rat-to-mouse islet xenotransplantation. PLoS One 2013;8:e73526. PMID: 24009755

14 Wu H, Wen D, Mahato RI. Third-party mesenchymal stem cells improved human islet transplantation in a humanized diabetic mouse model. Mol Ther 2013;21:1778-1786. PMID: 23765442

15 Wang Y, Zhang A, Ye Z, Xie H, Zheng S. Bone marrow-derived mesenchymal stem cells inhibit acute rejection of rat liver allografts in association with regulatory T-cell expansion. Transplant Proc 2009;41:4352-4356. PMID: 20005397

16 Obermajer N, Popp FC, Johnson CL, Benseler V, Dahlke MH. Rationale and prospects of mesenchymal stem cell therapy for liver transplantation. Curr Opin Organ Transplant 2014;19:60-64. PMID: 24231429

17 Le Blanc K, Frassoni F, Ball L, Locatelli F, Roelofs H, Lewis I, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet 2008;371:1579-1586. PMID: 18468541

18 Reinders ME, de Fijter JW, Roelofs H, Bajema IM, de Vries DK, Schaapherder AF, et al. Autologous bone marrow-derived mesenchymal stromal cells for the treatment of allograft rejection after renal transplantation: results of a phase I study. Stem Cells Transl Med 2013;2:107-111. PMID: 23349326

19 Yi T, Chen Y, Wang L, Du G, Huang D, Zhao D, et al. Reciprocal differentiation and tissue-specific pathogenesis of Th1, Th2, and Th17 cells in graft-versus-host disease. Blood 2009;114:3101-3112. PMID: 19602708

20 Lim JY, Park MJ, Im KI, Kim N, Jeon EJ, Kim EJ, et al. Combination cell therapy using mesenchymal stem cells and regulatory T-cells provides a synergistic immunomodulatory effect associated with reciprocal regulation of TH1/TH2 and th17/treg cells in a murine acute graft-versus-host disease model. Cell Transplant 2014;23:703-714. PMID: 23452894

21 Kamada N, Calne RY. Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage. Transplantation 1979;28:47-50. PMID: 377595

22 Kamada N, Calne RY. A surgical experience with five hundred thirty liver transplants in the rat. Surgery 1983;93:64-69. PMID: 6336859

23 Banff schema for grading liver allograft rejection: an international consensus document. Hepatology 1997;25:658-663. PMID: 9049215

24 Chen Y, Chen J, Liu Z, Liang S, Luan X, Long F, et al. Relationship between TH1/TH2 cytokines and immune tolerance in liver transplantation in rats. Transplant Proc 2008;40:2691-2695. PMID: 18929837

25 Chen Y, Yan T, Shi LJ, Liu Z, Liang SY, Luan XF, et al. Knockdown of interleukin-2 by shRNA-mediated RNA interference prolongs liver allograft survival. J Surg Res 2010;159:582-587. PMID: 19589546

26 Liao W, Zeng F, Kang K, Qi Y, Yao L, Yang H, et al. Lipoxin A4 attenuates acute rejection via shifting TH1/TH2 cytokine balance in rat liver transplantation. Transplant Proc 2013;45:2451- 2454. PMID: 23742835

27 Li B, Tian L, Diao Y, Li X, Zhao L, Wang X. Exogenous IL-10 induces corneal transplantation immune tolerance by a mechanism associated with the altered Th1/Th2 cytokine ratio and the increased expression of TGF-β. Mol Med Rep 2014;9:2245- 2250. PMID: 24676597

28 Hu A, Li Q, Shi H, Tai Q, Wu L, Xiong J, et al. Donor-derived bone marrow transfusion produces mixed chimerism and promotes a Th2 shift in Th1/Th2 balance in rat heterotopic small bowel transplantation. Dig Liver Dis 2012;44:988-994. PMID: 22954489

29 Lin ML, Zhan Y, Nutt SL, Brady J, Wojtasiak M, Brooks AG, et al. NK cells promote peritoneal xenograft rejection through an IFN-gamma-dependent mechanism. Xenotransplantation 2006;13:536-546. PMID: 17059581

30 Zhang XG, Lü Y, Wang B, Li H, Yu L, Liu C, et al. Cytokine production during the inhibition of acute vascular rejection in a concordant hamster-to-rat cardiac xenotransplantation model. Chin Med J (Engl) 2007;120:145-149. PMID: 17335659

31 Ding Y, Chen D, Tarcsafalvi A, Su R, Qin L, Bromberg JS. Suppressor of cytokine signaling 1 inhibits IL-10-mediated immune responses. J Immunol 2003;170:1383-1391. PMID: 12538698

32 Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17 family cytokines and the expanding diversity of effector T cell lineages. Annu Rev Immunol 2007;25:821-852. PMID: 17201677

33 Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annu Rev Immunol 2009;27:485-517. PMID: 19132915

34 Bettelli E, Carrier Y, Gao W, Korn T, Strom TB, Oukka M, et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 2006;441:235-238. PMID: 16648838

35 Aggarwal S, Ghilardi N, Xie MH, de Sauvage FJ, Gurney AL. Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17. J Biol Chem 2003;278:1910-1914. PMID: 12417590

36 Booth AJ, Grabauskiene S, Wood SC, Lu G, Burrell BE, Bishop DK. IL-6 promotes cardiac graft rejection mediated by CD4+ cells. J Immunol 2011;187:5764-5771. PMID: 22025555

37 Yang JJ, Feng F, Hong L, Sun L, Li MB, Zhuang R, et al. Interleukin-17 plays a critical role in the acute rejection of intestinal transplantation. World J Gastroenterol 2013;19:682-691. PMID: 23429965

38 Fábrega E, López-Hoyos M, San Segundo D, Casafont F, Pons-Romero F. Changes in the serum levels of interleukin-17/interleukin-23 during acute rejection in liver transplantation. Liver Transpl 2009;15:629-633. PMID: 19479806

39 Abe Y, Urakami H, Ostanin D, Zibari G, Hayashida T, Kitagawa Y, et al. Induction of Foxp3-expressing regulatory T-cells by donor blood transfusion is required for tolerance to rat liver allografts. PLoS One 2009;4:e7840. PMID: 19956764

40 Li W, Kuhr CS, Zheng XX, Carper K, Thomson AW, Reyes JD, et al. New insights into mechanisms of spontaneous liver transplant tolerance: the role of Foxp3-expressing CD25+CD4+ regulatory T cells. Am J Transplant 2008;8:1639-1651. PMID: 18557727

41 Ratajczak P, Janin A, Peffault de Latour R, Leboeuf C, Desveaux A, Keyvanfar K, et al. Th17/Treg ratio in human graft-versus-host disease. Blood 2010;116:1165-1171. PMID: 20484086

42 Li J, Lai X, Liao W, He Y, Liu Y, Gong J. The dynamic changes of Th17/Treg cytokines in rat liver transplant rejection and tolerance. Int Immunopharmacol 2011;11:962-967. PMID: 21376155

43 Kim KW, Chung BH, Kim BM, Cho ML, Yang CW. The effect of mammalian target of rapamycin inhibition on T helper type 17 and regulatory T cell differentiation in vitro and in vivo in kidney transplant recipients. Immunology 2015;144:68-78. PMID: 24974886

44 Liu Y, Cai Y, Dai L, Chen G, Ma X, Wang Y, et al. The expression of Th17-associated cytokines in human acute graft-versus-host disease. Biol Blood Marrow Transplant 2013;19:1421-1429. PMID: 23792271

45 Yu J, Yin S, Zhang W, Gao F, Liu Y, Chen Z, et al. Hypoxia preconditioned bone marrow mesenchymal stem cells promote liver regeneration in a rat massive hepatectomy model. Stem Cell Res Ther 2013;4:83. PMID: 23856418

46 Pan GZ, Yang Y, Zhang J, Liu W, Wang GY, Zhang YC, et al. Bone marrow mesenchymal stem cells ameliorate hepatic ischemia/reperfusion injuries via inactivation of the MEK/ERK signaling pathway in rats. J Surg Res 2012;178:935-948. PMID: 22658855