Bone marrow mesenchymal stem cells (BMMSCs) exert immunosuppressive activity in transplantation, and heme oxygenase-1 (HO-1) enhances their immunomodulatory effects. The aim of this study was to determine whether HO-1-transduced BMMSCs (HO-1/MSCs) improve rat liver transplantation (LTx) outcomes. Orthotopic LTx rejection models were treated with HO-1/MSCs, BMMSCs, HO-1, or normal saline, respectively. Our results showed a significant improvement in survival rates in the HO-1/BMMSCs group compared to the control groups. At all time points, liver function marker levels in the HO-1/MSCs group were significantly lower than in the other three groups; on POD 1, 7, and 14, the degree of rejection and apoptotic cells was significantly less in the HO-1/MSCs group than in the other three groups. Interleukin- (IL-) 10 and transforming growth factor-
Liver transplantation (LTx) is currently the only effective treatment for end-stage liver diseases, such as acute or chronic liver failure. However, the shortage of donor organs and issues of rejection and adverse reactions from immunosuppressants have hindered the use of Ltx. Immune rejection and ischemia-reperfusion injury after transplantation are two main reasons for loss of the graft [
Bone marrow mesenchymal stem cells (BMMSCs) are currently investigated in studies focused on transplantation immunity [
Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme to biliverdin and subsequently to bilirubin [
Specific pathogen-free (SPF) experimental animals were obtained from the Vital River Company (Beijing, China). Inbred adult male Lewis rats (220–250 g, 8–10 weeks old) were LTx donors, and inbred adult male Brown Norway (BN) rats (220–250 g, 8–10 weeks old) were recipients. BMMSCs were extracted from inbred adult male BN rats (100–120 g, 4–5 weeks old). Before testing, all rats were housed individually for 3 days in standard animal facilities on a 12 h light/dark cycle and provided with commercially available chow and tap water
Using the method described by Pittenger et al. [
Adenovirus/HO-1 (HO-1) obtained from Shanghai Genechem Limited Company (Shanghai, China) was diluted to 10 pfu/cell with complete culture solution and used to replace the original medium of the BMMSC cultures. After 6 to 8 h, the HO-1 culture solution was exchanged with complete culture solution for continued cultivation of the BMMSCs. After 48 h, the infection efficiency was observed under a fluorescence microscope. Molecular biological features of HO-1/MSCs were assessed by flow cytometry.
An orthotopic LTx rejection model was performed with Lewis donor rats and BN recipient rats in a sterile field under general anesthesia using 5% chloral hydrate (10 mL/kg). Food was withheld from both donor and recipient animals for 12 h prior to surgery, and differences between their weights were not greater than 10 g. The two-cuff technique for LTx was used by a single operator [
Recipient survival rates and clinical manifestations were observed in five animals per group. Humane endpoints were used for moribund animals after surgery, especially in the survival study. All animals meeting the humane endpoint criteria were euthanized by intraperitoneal injection of excess chloral hydrate when there was no unexpected death.
Recipient serum was obtained from peripheral blood. Levels of alanine transaminase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) were measured using an automatic biochemical analyzer (Olympus Au640, Tokyo, Japan).
After fixation in 10% formalin, hepatic tissues were embedded in paraffin and cut into 5
Terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end-labeling (TUNEL) staining was performed on paraffin-embedded tissue sections using the In Situ Cell Death Detection Kit (Roche Biochemicals, Mannheim, Germany), as instructed by the manufacturer. Tdt was not used in the negative controls, and deoxyribonuclease was used for the positive controls. Apoptotic nuclei would appear brown, while the staining of the cytoplasm would generally be light or absent. The slides were reviewed in a blinded fashion, and positive cells were counted in 10 randomly chosen fields under a light microscope (200x).
Serum was obtained from peripheral blood of recipients. Using ELISA, serum concentrations of cytokines related to inflammatory responses, T helper (Th)1/Th2, and Th17/Tregs were assayed at the same time points after LTx. Interleukin- (IL-) 10, TGF-
Lymphocytes were isolated from recipient spleens. Aliquots of 1 × 107 cells were resuspended in 0.1 mL PBS and labeled with antibodies specific for CD4, CD25, and Foxp3 (eBioscience, San Diego, CA, USA) or their isotype-matched control antibodies for analysis by flow cytometry (BD FACSAria III). The anti-CD4 antibody was conjugated to fluorescein isothiocyanate (FITC), and the isotype-matched antibody was mouse immunoglobulin G (IgG)2a K-FITC. The anti-CD25 antibody was conjugated to phycoerythrin (PE), and the isotype-matched antibody was mouse IgG1 K-PE. The anti-Foxp3 antibody was conjugated to PerCP-Cyanine5.5, and the isotype-matched antibody was rat IgG2a K-PerCP-Cyanine5.5.
Results are expressed as the mean ± standard deviation (SD) and compared by one-way analysis of variance. Kaplan-Meier analysis was used to compare survival rates of recipients, and log-rank testing was used to determine significant differences between groups. All statistical analyses were performed using SPSS statistical software, version 17.0 (SPSS GmbH, Munich, Germany), with
Cells isolated from rat bone marrow were confirmed as BMMSCs based on their spindle-shaped morphology, adherence to plastic, and phenotypic characterization by flow cytometry (Figure
Morphological (100x) and flow cytometric analysis of BMMSCs. (a) Morphology of third-passage BMMSCs. (b) Morphology of BMMSCs after transduction with HO-1 in a bright field. BMMSCs exhibited spindle-shaped morphology and were arranged in whorls when transfected with HO-1. (c) Morphology of BMMSCs after transduction with HO-1 in a fluorescent field. Over 85% of BMMSCs after transduction with HO-1 emitted green fluorescence. (d) The proportion of CD29-positive and CD34-negative cells was 97.5%. (e) The proportion of CD90-positive and CD45-negative cells was 93.3%. (f) The proportion of RT1A-positive and RT1B-negative cells was 96.7%.
At 48 h after HO-1 transduction, BMMSCs were observed by fluorescence microscopy, which showed that the infection efficiency was about 85% (Figures
From POD 3 to 5, recipients in the HO-1 and normal saline groups had poor appetite, decreased activity, untidy and lusterless hair, jaundice, and lethargy. Most of them died within one month. However, most of the recipients in groups treated with HO-1/MSCs and BMMSCs survived for more than two months and were more active and responded more quickly to stimulation. The median recipient survival times were 77, 61, 25, and 21 days in the HO-1/MSCs, BMMSCs, HO-1, and normal saline groups, respectively. The survival time of the HO-1/MSCs group was significantly different from that of the other groups (
Kaplan-Meier survival curve of recipients. (A) Normal saline group; (B) HO-1 group; (C) BMMSCs group; (D) HO-1/MSCs group. (a) Median recipient survival times were 77, 61, 25, and 21 days in the HO-1/MSCs group, BMMSCs group, HO-1, and normal saline groups, respectively (
Within 7 days of LTx, levels of ALT and AST in all groups decreased at first, then increased, and finally decreased again. The level of TBIL in the normal saline and HO-1 groups increased steadily between POD 5 and 7, increased sharply on POD 10, and then decreased significantly but remained at a relatively high level. During that time, TBIL in the HO-1/MSCs and BMMSCs groups increased steadily to the maximum on POD 28, except for a slight decrease on POD 7. At all time points liver function marker levels in the HO-1/MSCs group were lower than in the other three groups (
ACR was classified by the grade of inflammatory infiltrate in the portal space, around biliary ducts and in vessel walls [
Histological sections of the rat liver and grading of ACR after LTx. (A) Normal saline group; (B) HO-1 group; (C) BMMSCs group; (D) HO-1/MSCs group. (H&E staining, 100x). (a) In the normal saline and HO-1 groups, ACR grading on day 7 was moderate to severe, with abundant mixed lymphocytes in the portal area, interlobular bile duct inflammation and damage, inflammatory cell infiltration in the vein area, and necrosis of liver cells. In the BMMSCs group, ACR grading was mild until day 7, was aggravated sharply after day 7, and was moderate to severe on day 14. ACR was attenuated in the HO-1/MSCs group and decreased at all time points, compared to control groups (
ACR is associated with increased apoptosis in the graft. Using TUNEL staining, we evaluated apoptotic cells in the hepatic tissue after LTx (Figure
Apoptotic cells in liver grafts. (A) Normal saline group; (B) HO-1 group; (C) BMMSCs group; (D) HO-1/MSCs group. (a) Histological sections from rats of four groups at each time point were subjected to the TUNEL assay (200x). (b) Histogram showing the percentage of positive apoptotic signals in each group on days 1, 7, and 14. Apoptotic cell numbers in the HO-1/MSCs group were significantly lower compared with control groups on POD 1, 7, and 14, respectively (
ELISAs were performed to assay the serum concentrations of cytokines related to inflammatory responses and differentiation of T cells, such as Th1/Th2 and Th17/Tregs (Figure
IL-10, TGF-
Using flow cytometry, CD4+CD25+Foxp3+Tregs were assayed at various time points after LTx (Figure
Proportion of Tregs in recipient splenocytes. (A) Normal saline group; (B) HO-1 group; (C) BMMSCs group; (D) HO-1/MSCs group. Percentages of CD4+CD25+Foxp3+ cells in recipient spleens were measured by flow cytometry. (a) Scatter plots on POD 1, 7, and 14 showing that the percentage of Tregs was higher in HO-1/MSC-treated rats than in the control groups. (b) Histogram showing percentages of Tregs on POD 3, 5, 7, 10, 14, and 28. The number of Tregs in the HO-1/MSCs group first increased and then decreased but peaked on POD 14 and were significantly higher than those of other groups on POD 3, 5, 7, 10, 14, and 28 (
LTx is an effective treatment for end-stage liver diseases, but rejection and ischemia-reperfusion injury can affect survival in LTx recipients and mainly account for transplantation failures. Although the use of immunosuppressive drugs can mitigate immune rejection, their long-term application can also result in high costs and side effects, such as immune deficiency and tumor susceptibility. Induction of tolerance, a long-term and specific state of anergy of the immune system, in graft recipients should be achieved while maintaining their ability to respond to other foreign antigens. Thus, inducing a lasting and stable tolerance without drugs is an ideal goal [
In the current study, an orthotopic liver LTx rejection model was established with major histocompatibility complex- (MHC-) disparate rat strains (Lewis to BN). Histopathological analyses showed that the ACR reaction gradually increased in the normal saline group, with moderate to severe ACR occurring on day 7. The trend in the HO-1 group was nearly the same as that in the normal saline group, in which the degree of ACR was slight and not obviously different, suggesting that the effect of infusing pure HO-1 was not ideal. BMMSCs could attenuate the allograft rejection until day 7. From day 10, however, the improvement was attenuated, likely due to the reduction in numbers of BMMSCs and their diminished effects
The pathogenesis of organ transplant rejection has been shown to be mainly mediated by T cell immune responses [
Tregs (CD4+CD25+Foxp3+ T cells) exert negative immune regulation and account for approximately 5–10% of peripheral CD4+T cells. By inhibiting the activation and proliferation of other immune effector cells, Tregs play an important role in the induction of immune tolerance to transplanted tissue. Studies to date have implicated the following mechanisms for Treg-mediated inhibition of other immune effector cells: inhibiting IL-2 production by close contact among cells; preventing antigen presenting cells from expressing stimulus molecules and preventing dendritic cells from maturing; destroying target cells via the granular enzyme/perforin death pathways; consuming local IL-2 to cause loss of stimulatory signals to effector T cells; and inducing production of inhibitory cytokines such as TGF-
In summary, in this study, compared with BMMSCs, HO-1/MSCs improved the pathology of the transplanted liver, reduce cell apoptosis, improved liver functions, decreased levels of Th1 and Th17 cytokines, and promoted the generation of Tregs. Therefore, we reasoned that HO-1/MSCs could improve the outcome of allogeneic LTx by attenuating the inflammatory response and ACR, with better and more prolonged effects compared to BMMSCs. However, further studies exploring mechanism are needed to fully elucidate how HO-1 helps BMMSCs to attenuate the inflammatory response and ACR. In addition studies using higher doses of HO-1 are required. We believe that these findings provide a potentially new and effective strategy for suppressing ACR and improving outcomes after LTx.
Bone marrow mesenchymal stem cells
Brown Norway
Enzyme-linked immunosorbent assay
Green fluorescent protein
Heme oxygenase-1
Liver transplantation
Acute cellular rejection
Postoperative days
Interferon-
Interleukin-2
Interleukin-6
Interleukin-10
Interleukin-17
Interleukin-23
Phosphate-buffered saline
Transforming growth factor-
Tumor necrosis factor-
T regulatory cells
Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling.
The authors declare no conflict of interests.
Bin Wu participated in performance of the research, data analysis, and writing of the paper. Hong-Li Song participated in research design, data analysis, and writing of the paper. Yang Yang, Ming-Li Yin, Bo-Ya Zhang, and Yi Cao participated in performance of the research. Chong Dong participated in research design. Zhong-Yang Shen participated in research design, data analysis, and writing of the paper. All authors have read and approved the final paper.
This work was supported by grants from the National Natural Science Foundation of China (nos. 81270528 and 81441022), the National Science Foundation of Tianjin, China (nos. 08JCYBJC08400, 11JCZDJC27800, and 12JCZDJC25200), and the Technology Foundation of Health Bureau in Tianjin, China (no. 2011KY11). The authors thank the Key Laboratory of Emergency and Care Medicine of Ministry of Health and the Tianjin Key Laboratory of Organ Transplantation for allowing this work to progress in their laboratories.