Prevention of acute liver allograft rejection by IL-10-engineered mesenchymal stem cells

Authors


Summary

Hepatic allograft rejection remains a challenging problem, with acute rejection episode as the major barrier for long-term survival in liver transplant recipients. To explore a strategy to prevent allograft rejection, we hypothesized that mesenchymal stem cells (MSCs) genetically engineered with interleukin-10 (IL-10) could produce beneficial effects on orthotopic liver transplantation (OLT) in the experimental rat model. Syngeneic MSCs transduced with IL-10 were delivered via the right jugular vein 30 min post-orthotopic transplantation in the rat model. To evaluate liver morphology and measure cytokine concentration, the blood and liver samples from each animal group were collected at different time-points (3, 5 and 7 days) post-transplantation. The mean survival time of the rats treated with MSCs–IL-10 was shown to be much longer than those treated with saline. According to Banff scheme grading, the saline group scores increased significantly compared with those in the MSCs–IL-10 group. Retinoid acid receptor-related orphan receptor gamma t (RORγt) expression was more increased in the saline group compared to those in the MSCs–IL-10 group in a time-dependent manner; forkhead box protein 3 (FoxP3) expression also decreased significantly in the saline group compared with those in the MSCs–IL-10 group in a time-dependent manner. The expression of cytokines [IL-17, IL-23, IL-6, interferon (IFN)-γ and tumour necrosis factor (TNF)-α] in the saline groups increased significantly compared with the time-point-matched MSCs–IL-10 group, whereas cytokine expression of (IL-10, TGF-β1) was deceased markedly compared to that in the MSCs–IL-10 group. These results suggest a potential role for IL-10-engineered MSC therapy to overcome clinical liver transplantation rejection.

Introduction

Currently, orthotropic liver transplantation is the only effective therapy for end-stage liver diseases, including acute liver failure, cirrhosis and liver cancer, but the recipients have to take immunosuppressive agents which prevent rejection by inhibiting the expression of interleukin (IL)-2 and the proliferation of T lymphocytes. Despite great improvements in these drugs, severe side effects inevitably occur in those patients who have undergone liver transplantation [1]. Thus, new therapeutic approaches are needed to accomplish successful liver transplantation.

Marrow-derived stem or progenitor cells are applied currently for the treatment of a number of diseases with limited or no treatment options [2, 3]. Recent studies have demonstrated that bone marrow-derived mesenchymal stem cells (MSCs) can engraft in the injured organ and even differentiate into liver cells in vivo [4, 5]. MSCs were also observed to have profound immunomodulatory effects [6] and to be protected from rejection, suggesting their therapeutic potential on liver allotransplantation [7-9].

IL-10 was described originally as a cytokine synthesis inhibitory factor, which could down-regulate a variety of immune responses. Several lines of evidence have demonstrated that genetic delivery of IL-10 to allografts leads to improved graft acceptance in animal heart [10] or liver transplantation [11] models. Given that IL-10 is a cytokine synthesis inhibitory factor, we hypothesized that genetic delivery of IL-10 may confer measurable and perceptible beneficial effects in liver transplantation treatment.

In addition, the combination of cell and gene therapy has proved to be effective in the treatment of experimental pulmonary disease [12, 13]. Thus, dual strategy not only allows direct targeting to the diseased liver for clinical intervention, but also provides a site-specific source to deliver therapeutic molecules of interest by the retained cells. Therefore, we attempted to evaluate the effects of MSCs alone and in combination with IL-10 on liver rejection in the rat model of orthotopic liver transplantation.

The objective of this study was to investigate the effects of MSCs engineered with IL-10 on survival, biological phenomena and mechanistic actions in liver transplants.

Materials and methods

Cell culture

MSCs of Dark Agouti (DA) origin were kindly provided by Dr X. F. Tang (Sichuan University, Chengdu, China) [14]. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with heat-inactivated 10% fetal bovine serum (FBS), 50 μg/ml gentamycin, 2 mM L-glutamine, 100 μM non-essential amino acids, 10 mM HEPES and 55 μM 2-mercaptoethanol. The cells were grown at 37°C in 5% CO2. The MSCs used in all in-vivo experiments were maintained in passages 8–11.

Transduction of MSCs with lentivirus delivered IL-10

The full-length coding sequence of IL-10 [1306 base pairs (bp), Accession no.: NM_010548·2] was cloned into a lentivirus-based plasmid construct (pHR'CS-IL-10). A vesicular stomatitis virus glycoprotein pseudotyped lentiviral vector was generated by transient transfection of three plasmids (pCMV▵8·2, pcmv.VSVG.GFP and pHR.CS-IL-10) into human embryonic kidney 293T cells with Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA). The viral vector was examined by Southern blot analysis on genomic DNA isolated from infected U2OS cells.

The lentivirus hosting the pHR'CS- IL-10 was introduced into the MSCs at a multiplicity of infection of 5 in DMEM for 24 h with 8 μg/ml Polybrene (Sigma Aldrich, St Louis, MO, USA). Infected MSCs were washed twice after 24 h, and the culture supernatant containing secreted IL-10 was examined for 7 days. IL-10 expression was validated further by enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA).

Animal experiments

Inbred male DA (RT1n) and Lewis (LEW) (RT1l) rats, weighing 250–300 g, were used as donors and recipients, respectively. All the experimental rats were maintained in specific pathogen-free conditions according to the guidelines of the Institute of Laboratory Animal Resources of Xuzhou Medical College. All animal experiments were approved by the Institutional Animal Care and Use Committee. Orthotopic rat liver transplantation was performed under ether anaesthesia according to the technique described by Kamada [15]. The recipients were divided randomly into four groups with 14 rats in each group, as follows.

  • Group A (saline): the recipients were injected with saline via the right jugular vein 30 min post-orthotopic liver transplantation.
  • Group B (MSCs): the recipients were injected with MSCs (2·5 × 105 cells in 100 μl total volume) via the right jugular vein 30 min post-orthotopic liver transplantation.
  • Group C (lentivirus vector transduced MSCs): the recipients were injected with MSCs infected with lentivirus (2·5 × 105 cells in 100 μl total volume) via the right jugular vein 30 min after orthotopic liver transplantation.
  • Group D (IL-10 engineered MSCs): MSCs infected with lentiviral vector-mediated expression of IL-10 (MSCs–IL-10) (2·5 × 105 cells in 100 μl total volume) were injected slowly via the right jugular vein 30 min post-orthotopic liver transplantation.

After injection, the cannula was withdrawn, the vein was connected and the incision was sutured using silk suture lines. To evaluate liver morphology and determine the cytokine concentration, blood and liver samples from seven rats in each group were collected at different time-points (days 3, 5 and 7 post-transplantation), and the remaining seven rats were maintained for the assessment of survival.

Isolation of hepatic T cell populations

Liver cells were dissociated from the tissue by mashing through a 100-μm mesh steel sieve. Liver leucocytes were isolated by centrifugation with washed cell suspension on 1·05 g/ml Percoll (Pharmacia Biotech, Uppsala, Sweden) for 15 min at 840 g. The resultant cell fraction containing leucocytes was washed and resuspended in red blood cell (RBC) lysis buffer [155 mM NH4Cl, 10 mM KHCO3, 0·1 mM Na2 ethylenediamine tetraacetic acid (EDTA)], as described previously [16]. The cells were then stained with fluorescein isothiocyanate-conjugated anti-CD3 antibody (CD3-FITC; Serotec, Kidlington, UK). T cell populations were collected by fluorescence-activated cell sorting using the FACS Vantage cell sorter (Becton Dickinson, North Ryde, NSW, Australia) to remove the dead cells labelled by propidium iodide (1 μg/ml).

Banff classification of liver allograft pathology

For haematoxylin and eosin (H&E) staining and histological analysis, the liver tissues were fixed with 10% neutral-buffered formalin and embedded into paraffin wax. Sections of 5-μm thickness were affixed to slides, deparaffinized, and stained with H&E to evaluate the morphological liver changes. The histological H&E staining images were graded according to the Banff scheme [17].

Quantitation of mRNA by real-time quantitative PCR

The mRNA expression of T helper type 17 (Th17)-related transcription factor [retinoid-acid receptor-related orphan receptor gamma t (RORγt)] and regulatory T cell (Treg)-related transcription factor [forkhead box protein 3 (FoxP3)] were detected by SYBR Green polymerase chain reaction (PCR). To analysis the expression of FoxP3 and RORγt, CD4+/CD25+ T cells and Th17 cells were further sorted from the spleens using FACS, as described above.

Total RNA was prepared by Trizol reagent (Invitrogen), according to the manufacturer's instructions. RNA concentration and quality were assessed using a spectrophotometer based on the ratio of the absorbance at 260 and 280 nm (A260/280). RNA was reverse-transcribed at 37°C for 60 min followed by 95°C for 5 min to inactivate the reverse transcriptase. Real-time quantitative PCR was performed using the LightCycler 2·0 (Roche Applied Science, Penzburg, Germany) in 20 μl volume containing 50 nmol/l of primers,10 ng of cDNA, nucleotides, Taq DNA polymerase and SYBR Green I. The PCR reaction condition was: predenaturation phase at 94°C for 5 min, followed by denaturation at 94°C for 45 s; annealing at 58°C for 60 s; and extension at 72°C for 60 s in a total of 40 cycles.

The changes in fluorescence of SYBR Green I dye in each cycle were monitored by the software from PE Applied Biosystems (Framingham, MA, USA). Gene expression was normalized using beta_actin. Sequence detection primers for the cytokines were as follows: RORγt: 5′-CATTGACCGAACCAGCCGCAACCGA-3′ (sense), 5′-CGGGTGGTATAAGGTTATGGAACCG-3′ (anti-sense); and FoxP3: 5′-AGCCTGCCTCTGACAAGAACCCAAT-3′ (sense), 5′-AAGACGACGGTGACCCCAGAAGAGG-3′ (anti-sense).

Effect on survival through intravenous blockade of IL-17 signalling by anti-IL17 antibody

To assess the potential therapeutic effect mediated by IL-17, we used anti-IL-17 antibody to block IL-17 signalling by IL-17 neutralization. The recipients who underwent liver transplantation were subsequently divided randomly into three groups of 14 rats for each: intravenous injection with anti-IL-17 antibody (50 mg/rat), isotype control antibody (50 mg/rat) or saline 30 min after orthotopic liver transplantation. The anti-IL-17 antibody and isotype control antibody were purchased from eBiosciences (San Jose, CA, USA). Censored survival analysis was conducted for 60 days post-transplantation.

Cytokine expression in T cell populations from recipients’ liver

The protein levels of Th17/Treg-related cytokines [IL-17, IL-23, IL-6, IL-10, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, transforming growth factor (TGF-β1)] in T cell populations were measured by Western blotting. In brief, the protein lysate was mixed with loading buffer, boiled for 5 min and loaded onto sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) with equal amounts of protein, and run at 200 V for 60 min followed by transferring to nitrocellulose membranes (Amersham Biosciences, Little Chalfont, UK) at 100 V for 30 min at room temperature and incubated with primary antibodies. Primary antibodies included anti-IL-17, anti-IL-23, anti-IL-6, anti-IL-10, anti-IFN-γ, anti-TNF-α and anti-TGF-β1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies [anti-goat/-rabbit/-mouse immunoglobulin G (IgG)-horseradish peroxidase (HRP)] were purchased from Zhongshan Company (Beijing, China). The protein expression levels were detected by chemiluminescence (ECL system; Amersham Biosciences) and quantified using Quantity one software (Bio-Rad, Berkeley, CA, USA); the expression level of interested genes was normalized using β-actin.

Serum cytokine levels in liver allograft recipients

Serum levels of cytokines (IL-17, IL-23, IL-6, IL-10, IFN-γ, TNF-α, TGF-β1) were examined by enzyme-linked immunosorbent assay (ELISA), following the manufacturer's instructions (R&D Systems).The minimum detectable concentrations were 1·6 pg/ml for IL-17, 7·8 pg/ml for IL-23, 0·7 pg/ml for IL-6, 5 pg/ml for IL-10, 6·7 pg/ml for IFN-γ, 11·8 pg/ml for TNF-α and 4·61 pg/ml for TGF-β1. Intra- and interassay coefficients of variation for all ELISAs were <5 and <10%, respectively. All samples were measured in triplicate.

Retention of transplanted mesenchymal stem cells (MSCs)

The rat liver tissues were digested into single cells using dispase II enzyme (3·6 U/ml; Roche), according to previously published protocol [18]. The number of total cells recovered was determined by haemocytometer. Isolated cells were analysed by flow cytometer (FC500; Beckman Coulter, Brea, CA, USA), with a minimum collection of 30 000 events per sample. Cell nuclei were counterstained with nuclear dye TO-PRO-3 (Invitrogen), and confocal microscopic images were obtained with a Leica laser scanning confocal microscope.

Fluorescent levels of carboxyfluorescein succinimidyl ester (CFSE)-labelled cells could represent apoptotic cell debris. To avoid this bias, annexin V-FITC was used to examine live/dead discrimination of ex-vivo MSCs. The annexin V-FITC assay was performed according to the manufacturer's instructions (Invitrogen). Briefly, MSCs were collected and then washed with cold phosphate-buffered saline (PBS), precipitated by centrifugation, resuspended in 100 μl of binding buffer containing 2·5 μl FITC-conjugated annexin V and 1 μl 100 μg/ml propidium iodide (PI) and incubated for 15 min at room temperature in the dark. A total of at least 10 000 events was collected and analysed by flow cytometry (Becton Dickinson, San Jose, VA, USA).

Statistics

Experimental data were shown as mean ± standard deviation and evaluated using one-way analysis of variance (anova) for significant differences; differences of P < 0·05 were considered statistically significant. Graft survival was calculated according to the Kaplan–Meier method. Statistical analysis was performed using the spss version 12·0 software package.

Results

IL-10 expression in MSCs transduced with IL-10

At 24 h after transduced MSCs with IL-10, IL-10 protein (280·0 ± 23·3 pg/ml) was detected from cell culture supernatant (from 5 × 105 cells), and sustained expression of IL-10 was observed for more than 7 days. In contrast, far fewer IL-10 proteins were detected in either non-transduced or null-transduced MSCs with empty vector (Fig. 1).

Figure 1.

The expression of supernatant cytokine [interleukin (IL-10)] increased significantly in the mesenchymal stem cells (MSCs)–IL-10 group compared with the day-matched saline or MSCs groups. *P < 0·05 versus day-matched saline or MSCs groups.

Survival rate for transplant recipients

To evaluate the effect of a combination of MSCs and IL-10 on recipient survival, we injected the IL-10-engineered MSCs into the liver allograft through the jugular vein. In agreement with the report from both our group and others [19], the liver allograft rejection in LEW recipients of DA livers occurs within 14 days post-transplant in control groups, as shown in the historical data.

The mean survival time of rats treated with MSCs–IL-10 (112 ± 17·3 days) was prolonged significantly compared with that of rats treated with saline (8 ± 3·9 days) (P < 0·0001), although mild portal infiltration and proliferation of bile ducts were observed in the MSCs–IL-10 treatment group, while the survival time of rats in the MSCs (67 ± 8·5 days) group showed no difference from that in the MSCs–lentiviral group (76 ± 7·7 days), indicating that the administration of MSCs–IL-10 had a beneficial effect on liver allograft survival (Fig. 2) and the survival advantage is not due to lentival vectors. In addition, no obvious aberrant liver architecture was recognized by historical staining in the rats with longer-term survival (Fig. 3).

Figure 2.

Lewis (LEW) recipients of Dark Agouti (DA) livers died as a result of the rejection of their liver allografts within 14 days of transplantation (n = 7). LEW recipients of DA livers treated with mesenchymal stem cells (MSCs)–interleukin (IL)-10 had significantly (P < 0·001, log-rank test) prolonged survival (n = 7).

Figure 3.

(a) Histological grading of the liver allografts using the Banff score revealed no significant difference in the rejective activity index (RAI) score between mesenchymal stem cells (MSCs) and MSCs–lentiviral-treated recipients. Administration of MSCs–interleukin (IL)-10 was associated with a lower score (*P < 0·05 versus saline, MSC or MSC–lentiviral groups). (b) Control grafts showed parenchymal necrosis, severe endothelitis and destruction of bile ducts. Recipients treated with MSCs–IL-10 had virtually normal histology. Recipients treated with MSCs or MSCs–lentiviral was characterized by a prominent portal infiltrate with varying degrees of endothelitis and bile duct inflammation.

Morphological changes in liver

As shown in Fig. 3, prominent acute rejection accompanied by progressive development occurred in the rat livers of the saline group, whereas barely visible acute rejection was observed in hepatic tissue from the MSCs–IL-10 group. Dramatic morphological changes were found in hepatic tissue from the saline group compared with those from the MSCs, MSCs–lentiviral and MSCs–IL-10 groups at day 3 post-transplantation. Severe degeneration and vacuolation occurred in hepatocytes, with hepatic sinusoids gravely expanded and filled with many erythrocytes. Portal areas were infiltrated by a few inflammatory cells; the sinusoidal endothelial cells were markedly swollen. The rejection grading scores increased significantly in the saline group compared with those in the MSCs, MSCs–lentiviral and MSCs–IL-10 groups, which is consistent with the corresponding morphological changes in each group (P < 0·05) (Fig. 3).

Expression of RORγt and FoxP3

RORγt is a master transcription factor regulating Th17 differentiation, as is FoxP3, the transcription factor for Treg, so we evaluated the expression of RORγt and FoxP3 in the above four groups. As shown in Fig. 4, RORγt expression levels increased time-dependently and were much higher in the saline groups than those in the MSCs–IL-10 group at the time-points (3, 5 and 7 days) post-transplantation (P < 0·05). In contrast, FoxP3 expression levels decreased time-dependently and were markedly lower in the saline group compared with the MSCs–IL-10 group at the same time-points (P < 0·05) (Fig. 4).

Figure 4.

Expression of retinoid-acid receptor-related orphan receptor gamma t (RORγt) and forkhead box protein 3 (FoxP3) between four groups. (a) The ratios of FoxP3/β-actin mRNA were compared in four groups at days 3, 5 and 7 post-transplantation. *P < 0·05 versus the day-matched saline group. (b) The ratios of RORγt/β-actin mRNA were compared in two groups at days 3, 5 and 7 post-transplantation. *P < 0·05 versus the day-matched saline group.

Superior effect of intravenous IL-17 antibody on graft survival

The recipients administered intravenously with anti-IL-17 antibodies 30 min after orthotopic liver transplantation revealed improved overall survival through blocking the IL-17 pathway.

The mean survival time of recipients treated with anti-IL-17 antibodies (43 ± 5·3 days) shows a distinct difference from that of rats treated with saline (8 ± 3·9 days) (P < 0·001) or those treated with isotype control antibody (9·5 ± 3·1 days) (P < 0·001) (Fig. 5).

Figure 5.

Lewis (LEW) recipients of Dark Agouti (DA) livers treated with saline or treated with isotype control antibody died as a result of the rejection of their liver allografts within 14 days of transplantation (n = 14). LEW recipients of DA livers that were treated with anti-interleukin (IL)-17 antibodies had significantly (P < 0·001, log-rank test) prolonged survival (n = 14).

Changes of Th17/Treg-associated cytokine expression in intragraft

The protein levels of IL-17, IL-23, IL-6, IL-10, IFN-γ, TNF-α and TGF-β1 in T cells from rat liver were measured by Western blot at the indicated time-points. As shown in Fig. 6, the expression of cytokines (IL-17, IL-23, IL-6, IFN-γ, TNF-α) in the saline group increased significantly compared with the time-matched MSCs–IL-10 group and accumulated a maximal value at days 3, 5, 7 post-transplantation (P < 0·05), whereas the expression of cytokines (IL-10, TGF-β1) was markedly lower than those of the MSCs–IL-10 group (P < 0·05) (Fig. 6).

Figure 6.

Protein levels of cytokine in graft. (a–g) Protein levels of intragraft expression at days 3, 5 and 7 post-transplantation. *P < 0·05 versus the day-matched saline group.

Changes of Th17/Treg-associated cytokine concentration in serum

IL-17, IL-23, IL-6, IL-10, IFN-γ, TNF-α and TGF-β1 expression in serum from the above four groups were determined at days 3, 5 and 7 post-transplantation by ELISA. As shown in Fig. 7, serum concentrations of cytokines IL-17, IL-23, IL-6, IFN-γ and TNF-α in the MSCs–IL-10 group were markedly lower compared with the concentrations in saline group at days 3, 5 and 7 post-transplantation (P < 0·05), while other cytokines IL-10 and TGF-β1 were significantly higher than those in the saline group at the same points (P < 0·05).

Figure 7.

Mesenchymal stem cells (MSCs)–interleukin (IL)-10 functional imbalance in serum. (a,b) The expression of serum cytokine [IL-10, transforming growth factor (TGF)-β1] increased significantly in the MSCs–IL-10 group compared with the day-matched saline group. *P < 0·05 versus the day-matched saline group. (c–g) The expression of serum cytokine [IL-17, IL-23, interferon (IFN)-γ, tumour necrosis factor (TNF)-α and IL-6, respectively] clearly decreased in the MSCs–IL-10 group compared with the day-matched saline group. *P < 0·05 versus the day-matched saline group.

Persistence of MSCs in rats with or without liver transplantation

Retention of MSCs in the liver after central venous injection was evaluated by confocal microscopy and flow cytometry. MSCs labelled with the green fluorescent fluorescein diacetate [CellTracker Green carboxyfluorescein diacetate, succinimidyl ester (CFDA SE)] were observed in liver sections from both naive and liver-transplanted rats killed 24 h post-transplantation (initial retention, Fig. 8a); the labelled cells were much less abundant, although they were still detectable 7 days after injection (Fig. 8c). No cell-specific green fluorescence was detectable from the animals that did not receive CFDA SE-labelled cells (unpublished data). The percentage of injected MSCs retained in the liver was then quantified by flow cytometry following dispase liver digestion. On average, 47% of injected cells were found in the liver shortly after MSCs delivery in transplanted mouse liver compared to 38% in naive rats, although the difference was not statistically significant. Regardless of liver injury, the majority of MSCs were lost from the liver after 7 days, with fewer than 8% of cells remaining (Fig. 8c).

Figure 8.

Retention of injected mesenchymal stem cell (MSCs) in mice with or without transplanted liver MSCs were labelled with the cell tracing dye carboxyfluorescein diacetate, succinimidyl ester (CFDA SE) (green) prior to injection. Nuclei were stained with TO-PRO-3 (blue). Scale bars in photomicrographs = 20 μm. White arrows indicate labelled MSCs. (a) Labelled MSCs were observed in 5 lm, paraformaldehyde (PFA)-fixed lung sections from lipopolysaccharide (LPS)-injured mice killed at 24 h (initial retention). Image obtained with a Leica laser scanning confocal microscope with a ×20 objective. (b) Interleukin (IL)-10-transfected MSCs labelled with the green fluorescent cell tracker CFDA SE were observed in liver sections from liver transplanted mice killed at 7 days. Photomicrographs were taken on the Z-axis with a ×20 objective, then images were stacked using Leica confocal software. (c) Livers from each animal were enzyme-digested into single cells before MSCs were counted by flow cytometry; n = 7 per group. (d) Viability of MSCs in the liver-transplanted/MSCs–24-h group, naive/MSCs–24-h group, liver-transplanted/MSCs–IL-10 7 days group and naive/MSCs–IL-10 7 days group were monitored by flow cytometry. LL is indicative of viability percentage.

The live/dead discrimination of ex-vivo MSCs were evaluated by annexin V-FITC (Fig. 8d) and the viability of MSC was measured by flow cytometry. The mean viability of MSCs was 96·42% in the 24 h post-liver-transplanted MSCs group and 94·11% in the 7 days post-liver-transplanted MSCs–IL-10 group. There was no significant difference in the viability of MSCs between the 24 h post-liver-transplanted MSCs group and the 7 days post-liver-transplanted MSCs–IL-10 group (P > 0·05).

Discussion

Acute rejection still remains a major cause of early graft loss and a predominant risk factor for long-term recipient and graft survival post-liver transplantation. Although many approaches, such as donor-specific antigen infusion, co-stimulatory blockade and immunosuppressant, have been used widely to prevent rejection, the rejection incidence rate remains high and the underlying mechanism is so far uncertain [20, 21].

To potentially overcome severe side effects from immunosuppressive therapy, stem cell-based approaches provide the potential for tolerance induction of organ transplantation without non-specific immunosuppression, or at least with a reduced dose for long-term immunosuppression.

Several studies show that the stem cells could restore the function of damaged tissue in preclinical models of various diseases [12]; therefore, combining gene therapy with stem cells may confer additional beneficial effects in liver transplantation. As yet, however, stem cells have only been applied successfully to solid organ transplantation, due to several limitations, including the following.

  • Cell-based gene transfer can overcome some of the limitations of direct gene therapy for liver diseases, achieving selective and durable transgene expression in the liver [22, 23]
  • Mesenchymal stem cells (MSCs) prove to be an important candidate for cell-based immunotherapy promoting tolerance of solid allografts [24, 25] by inhibiting the proliferation and function of T cells, B cells and natural killer cells [26, 27].
  • Selection of the most optimal target gene is critical for the potential efficacy of gene therapy strategy, and requires a profound understanding of the mechanistic actions of the target gene regulating the disease.
  • The mechanism of MSCs-mediated immune suppression is linked to non-specific anti-proliferative effects, which is the consequence of cyclin D2 inhibition [16]. Several lines of study have suggested that prostaglandin E2, nitric oxide, insulin-like growth factor binding protein and tolerability of antigen-presenting cells and indoleamine 2,3-dioxygenase are involved in this mechanism [16].
  • In addition, MSCs also have tremendous potential for regeneration from marginal organ transplantation, thus improving the overall clinical outcome. Although the physiological significance of immunosuppression is unclear, the underlying mechanism is associated with stromal cell function. It appears to play its role by increasing the survival and renewal of parenchymal stem cells.

In this study, we have demonstrated that intravenous administration of MSCs could partially prevent the transplant rejection and promote tolerance development in the rat model; moreover, treatment with IL-10-engineered MSCs resulted in further improvement in liver transplantation tolerance. These findings shed great light on post-liver-transplantation management.

Through introducing IL-10 engineered MSCs into the liver allograft, we observed that the mean survival time in rats treated with MSCs–IL-10 (112 ± 17·3 days) differed significantly from that in rats treated with saline (8 ± 3·9 days), despite mild portal infiltration and proliferation of bile ducts being found in the MSCs–IL-10 treatment group. In addition, the effects of IL-10-engineered MSCs compared with the saline group are reflected in the expression changes of various inflammatory cytokines in the blood and graft, suggesting that the introduction of MSCs–IL-10 into liver remarkably improved the survival of liver allograft by regulating inflammatory pathways.

The role of MSCs–IL-10 in preventing the rejection of liver transplantation may not be mediated directly by IL-10. Nevertheless, it should be noted that MSCs alone resulted in an almost complete imbalance of Th17/Treg-related proinflammatory cytokine expression, which could be altered further in the rats transduced with IL-10 engineered MSCs.

Control of the Treg/Th17 balance involved in inflammation and autoimmune diseases may play an important role in promoting or suppressing immune rejection. To study the possible disruption of the balance present in rat liver transplantation, Th17/Treg regulatory functions were assessed from several aspects, including their related cytokine secretion and key transcription factors in four experimental groups at days 3, 5 and 7 after operation.

Our study has demonstrated that the MSCs–IL-10 group exhibited a higher mean survival time and significantly increased expression of Treg cell-associated cytokines (IL-10 and TGF-β1) and transcription factor (FoxP3), accompanied by remarkably decreased expression of Th17 cell-related cytokines (IL-17, IL-6, IFN-γ, TNF-α and IL-23) and transcription factors (RORγt) when compared to those in the saline group from day 3 post-transplantation.

Many recent studies have shown that CD4+CD25+FoxP3+ Treg cells play a predominant role in the prevention of autoimmune diseases and transplant rejection [28-30]. Adoptive transfer of natural or antigen-specific induced Treg cells can reduce acute rejection and prolong recipient survival rate in animal models. In this study we found that the expression of Treg cell-related cytokines (IL-10, TGF-β1) and transcription factor (FoxP3) in MSCs–IL-10 were significantly higher compared with day-matched saline groups, indicating that Treg cells play an important role in inhibiting acute graft rejection, in agreement with a previous report [31].

Treg cells function partly through anti-inflammatory cytokine secretion (IL-10 and TGF-β1) [32], while TGF-β1 is a critical regulator in the initiation and maintenance of FoxP3 expression, a master control transcription factor for Treg induction.

The switch of the Th17/Treg axis plays an important role in regulating immune responses and autoimmune disease [33, 34]. The Th17 cell-expressed RORγt is a distinct subset, differing from Th1 and Th2 cells that play a key role in triggering autoimmunity and allergic reactions by producing IL-17 and, to a lesser extent, IL-6 [35, 36]. Therefore, increased IL-17 expression is correlated with the initiation and progression of autoimmune disease [37, 38]. However, only limited studies were conducted regarding the impact of IL-17 on transplant rejection, with delayed rejection via IL-17 antagonism. In transplant rejection, IL-17 may mediate acute, rather than chronic, transplant rejection through rapid recruitment of myeloid cells. In this study, we found that LEW recipients of DA livers treated with anti-IL-17 antibodies exhibited significantly prolonged survival compared with saline or treated with isotype control antibody, indicating that IL-17 plays an important role in inhibiting acute graft rejection.

Recent clinical studies have shown a significant increase of Th17-related cytokines (IL-17 and IL-23) in serum from the patients receiving liver transplantation [39].

We aimed to decipher the functional alterations of Th17 cells in an animal model of liver transplantation. There was a time-dependent and significant increase in Th17-related cytokines (IL-17, IL-6 and IL-23) and RORγt expression in the saline group when compared with that in the MSCs–IL-10 group from day 3 post-transplantation, suggesting the role of Th17 in promoting acute transplant rejection.

IL-6 and IL-23 act by amplifying and/or stabilizing the response of differentiated Th17 cells. Our study has also demonstrated simultaneous up-regulation of IL-6, IL-23 and IL-17 during acute liver rejection, which is consistent with a growing body of research on experimental models, and clinical observations that Th17 lymphocytes are involved in graft damage upon renal, cardiac and lung allograft rejection [40, 41].

We have also investigated whether the observed effects on liver rejection are dependent upon the retention of MSCs. The obviously higher proportion of MSC cells are retained in transplanted liver 24 h post-operation, yet the difference in cell populations between transplanted versus untransplanted liver was not significant, and in both cases the majority of MSC cells were lost from the liver after 7 days; these observations are also in agreement with previous reports [42].

Notably, we find that the high-level and long-term presence of transduced MSCs are not required for the positive effects generated by cell-based therapy in the liver transplantation model. This observation is consistent with several studies of bone marrow cell therapy [43], which demonstrated important features for the paracrine actions of transplanted cells on neovascularization and tissue healing, although the precise mechanism through which genetically engineered MSCs confer a therapeutic benefit in the treatment of liver transplantation model remains to be explored.

Acknowledgements

This study was supported by grants from the National Science Foundation of China (30801125, 30800446, 81170468). No support has been received from any company.

Disclosure

The authors have no financial conflicts of interest to disclose.

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