Liver transplantation is a widely accepted method of lifesaving procedure for patients with chronic end-stage liver diseases as well as with acute liver failures.1-4 However, the acute rejection remains to be an important comorbidity after liver transplantation. The rejection responses to allografts are initiated by a specific T-cell recognition of antigens from the donor organ.5,6 Accordingly, induction of donor-specific transplant tolerance has been suggested as a possible solution to avoid immune rejection. However, the requirements for controlling aggressive response and generating immune tolerance in allograft models are still not fully understood.
CD4+ T helper (Th) cells have been implicated in playing important roles in allograft rejection by secreting various cytokines and providing help for other effector cells.7 CD4+ Th 17 (Th17) and CD4+ Foxp3+ regulatory T (Treg) cells, 2 newly characterized T-cell subsets that are thought to have opposite effects on acute rejection as documented in clinical as well as experimental autoimmunity and transplant immunity.8-12 There was an overall increase in serum IL-17 after liver transplantation, which described a possible correlation between Th17 induction and acute liver rejection.13 Studies also have shown a link between enhanced Th17-secreted cytokine IL-17 and acute renal allograft rejection.14,15 In contrast, Treg cells and their compromised functional properties were decreased during acute rejection in liver transplant patients.16 Therefore, the imbalance of Th17/Treg cells could be a beneficial approach to understand a liver allograft’s fate. However, the exact impacts of Th17/Treg cells on liver acute rejection are not fully understood.
In this study, we explored the changes of the Th17/Treg cells and related cytokines to demonstrate Th17/Treg functional imbalance in rat liver transplantation. And for the first time, we found that inhibition of IL-17 reduced inflammatory infiltrates and alleviated acute rejection in a rat model of liver transplantation.
MATERIALS AND METHODS
Animals and Experimental Design
Inbred male Wistar rats and SD rats (220–250 g) were purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). Orthotopic liver transplantation was performed under ether anesthesia using Kamada’s method with a few modifications. Recipients were randomly divided into 3 groups (12 rats each group). The Wistar-to-SD combination as an acute rejection model could exhibit different severity of acute rejection, the SD-to-SD combination as isografts group, and the control group of normal male SD rats had a sham operation.
Liver Histological Examination
Liver tissue was fixed in 10% neutral buffered formalin and embedded paraffin. The 4-μm sections were stained with hematoxylin-eosin (H&E) for histological examination. The histological classification of hepatic H&E staining was graded according to the Banff scheme.17 The rejection activity index (RAI) was calculated from the 3 individual scores (venous endothelial inflammation, bile duct damage, and portal inflammation) by a single-blinded pathologist.
Liver paraffin-embedded slides were deparaffinized and rehydrated, followed by microwave antigen retrieval in 10 mmol/L sodium citrate buffer (pH 6.0). The sections were blocked with 5% (v/v) bovine serum albumin for 30 minutes, and then incubated with IL-17A monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight. After washing 3 times with PBS, the slides were incubated with horseradish peroxidase polymer-linked secondary antibody for 50 minutes at room temperature. The sections were then counterstained with hematoxylin and visualized with diaminobenzadine.
For Treg cells labeling, sections were post fixed in 4% buffered formaldehyde for 5 minutes and permeabilized using 0.5% Triton X-100 for 10 minutes. And then sections were incubated with anti-rat CD4-Alexa Fluor 488 and anti-rat FoxP3-Phycoerythrin(PE). The slides were mounted in 4′, 6-diamidino-2-phenylindole (DAPI)-containing mounting medium (Invitrogen) and visualized with a Zeiss microscope (Carl Zeiss Imaging, Thronwood, NY). Numbers CD4+FoxP3+ cells were counted, using Image J (Image Processing and Analysis in Java, National Institutes of Health).
Th17 and Treg Staining by Flow Cytometry
Recipients were sacrificed on day 7 postgrafting for flow cytometric analyses. Single cell suspensions of lymphocytes have been prepared as described.18 The following antibodies were used for flow cytometry: anti–CD3-allophycocyanin (APC), anti–CD4-fluorescein isothiocyanate (FITC), anti–Foxp3-PE, and anti–IL-17-PE (all from eBioscience, San Diego, CA). For Th17 staining, 2 × 106 were cultured in complete RPMI 1640 medium and stimulated for 6 hours with 25 ng/mL PMA (Sigma, St. Louis, MO) and 1 μg/mL ionomycin (Sigma) in the presence of 0.66 μL/mL Golgistop (BD PharMingen, San Diego, CA) at 37 °C in 5% CO2. Then, cells were stained for surface molecules (anti-CD3-APC and anti-CD4-FITC), fixed, and permeabilized with Cytofix/Cytoperm buffer and intracellularly stained with anti-IL-17-PE. For analysis of intracellular Foxp3, 2 × 106 cells were surface-stained with anti-CD4-FITC, then were fixed and made permeable with fixation-permeabilization buffers (eBioscience) and then stained with PE-conjugated anti-Foxp3 antibodies. Results were analyzed using FlowJo software (Tree star Inc, Ashland, OR).
Enzyme-Linked Immunosorbent Assay
Serum levels of Th17-related cytokines (IL-17), Treg-related cytokines (IL-10, transforming growth factor [TGF]-β1) and IL-6 were assayed by enzyme-linked immunosorbent assay (ELISA), following the manufacturer’s instructions (all ELISA kits from R&D System, Minneapolis, MN). All samples were measured in duplicate.
Serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (T-Bili) were determined using commercially available biochemical kits (LABTEST, Minas Gerais, Brazil).
Neutralizing Anti-Rat IL-17A Antibody Administration
The neutralizing rabbit anti-rat IL-17A polyclonal antibody and its control immunoglobulin (Ig)G monoclonal antibody were purchased from Shenandoah Biotechnology. The IL-17A-neutralizing antibody (100 μg per rat) was administered intraperitoneally to each recipient from the day of transplantation until day 3 after transplantation.19 Rat receiving same amount of rabbit IgG were used as controls.
All analyses were carried out with the SPSS 15.0 software. Data were shown as mean ± SD deviation. Comparisons among the 3 groups were performed by 1-way analysis of variance, and Student-Newman-Keuls (SNK) test was used for comparison between two groups. Allograft survival was used to generate Kaplan-Meier survival curves, and comparison between groups was performed by log-rank analysis. Significant differences were as follows: *P less than 0.05; **P less than 0.01.
Allograft Rejection Correlates With the Number of Th17 and Treg Cells
Without any immunosuppression, the features of Wistar-to-SD allograft group became weakness, fatigue, and anorectic as well as passed dark, coke-colored urine; the livers of allograft group were found slightly enlarged and mottled brown, which are quite similar to those of clinical rejection. In contrast, the SD-to-SD isograft group was almost the same as the normal group.
By flow cytometry analysis, the percentages of Th17 and Treg of CD4+ T cells in splenic lymphocytes of liver graft were detected 7 days postgrafting. As shown in Figure 1A–C, the percentages of Th17 and Treg cells in SD-to-SD isograft groups were similar with those in control groups (P = 0.780; P = 0.283). However, Th17 cells in allograft groups were significantly increased when compared with isograft groups (P = 0.000), whereas Treg cells were markedly lower than those of isograft groups (P = 0.000). These results suggest that Th17/Treg cells participate in decorating, probably enhancing allograft rejection after graft.
Changes of Th17- and Treg-Related Cytokines
Th17/Treg-related cytokines in serum were detected by ELISA. As shown in Figure 2A–C, serum IL-17, IL-10, and TGF-β1 in isograft group were similar with those in the control groups (P = 0.185; P = 0.227; P = 0.085). The level of IL-17 in allograft groups was remarkably higher compared to isograft groups (P = 0.000), whereas IL-10 and TGF-β1 levels were significantly lower than those of isograft groups (P = 0.000; P = 0.000). Given the critical role of IL-6 in altering the balance between Treg and Th17 cells,20 we also compared IL-6 levels after transplantation. Result showed that IL-6 level was significantly higher in allograft groups than in controls (P = 0.000) (Figure 2D). Together, these results suggest that Th17/Treg–related cytokines contributes to allograft response after graft.
Therapy With Anti-17A Neutralizing Antibody Extended Survivals of Liver Allografts
To further examine the effects of IL-17 on allograft rejection, neutralization antibody of IL-17 was injected into allograft groups. Immunohistochemical staining results demonstrated that abundant IL-17–positive cells infiltrated the liver allografts, while few IL-17–positive cells were observed in the isograft group; however, neutralization of IL-17A significantly reduced IL-17–positive cells in the liver allografts (Figure 3A).
Survival analysis showed that rats in allograft group rejected acutely (mean survival time [MST], 8.0 ± 1.2 days) as compared with the isograft group (MST, 50.0 ± 3.8 days; χ2 = 82.38, P = 0.000) (Figure 3B), However, neutralization of IL-17A significantly prolong survival compared to the group that received isotype IgG (MST, 32.0 ± 4.8 days vs 11.0 ± 3.4 days; χ2 = 11.18, P = 0.000) (Figure 3B). In addition, liver H&E staining showed severe acute rejection in the allograft group, which was characterized by inflammatory cell infiltration and hepatocyte necrosis, whereas no histological rejection occurred in the isograft group (Figure 3C). The RAI scores in the allograft group were also significantly higher than those in the isograft group (P = 0.000) (Table 1). It is worth noting that neutralization of IL-17A can significantly mitigate acute liver injury with less infiltration of inflammatory cells (Figure 3C) and decreased RAI scores (P = 0.000) (Table 1). Similarly, the levels of AST, ALT, and T-Bili were significantly decreased after neutralization of IL-17A (P = 0.000; P = 0.000; P = 0.000) (Figure 3D).
Furthermore, considering the critical role of IL-17 in the acceleration of allograft rejection by suppressing regulatory T cell expansion,21 we hypothesized that neutralization of allograft recipients with IL-17A may affect Treg cells. Indeed, neutralization with IL-17A resulted in increased Treg cells but had no significant influence on Th17 cells in the spleen of allograft group (P = 0.000; P =0.684) (Figure 3E-G). We also found that inhibition of IL-17A can significantly increase Treg cells in the liver of allograft group (Figure 3H). Together, these results indicated that neutralization of IL-17A extended the survivals of liver allografts by shifting the Th17/Treg cell balance from Th17 predominance to Treg cell.
Liver transplantation is a viable treatment option for end-stage liver diseases. However, acute rejection significantly decreases graft and recipient survival.17 Although the etiology of acute rejection is complex and involves synergistic interactions between Th1 and Th2 cells,22 whether other CD4+T cell subsets, such as Th17 and Treg cells, involved in acute rejection remains not fully understood. In this study, we provide evidence that Th17/Treg cells and related cytokines participated in the allogeneic liver rejection. And for the first time, we found that neutralization of IL-17 in vivo prolonged survival and improved the liver function with increased Treg cells in a rat grafts model.
The balance of Th17/Treg cells controls inflammation and may play critical roles in the development/prevention of transplant rejection. Th17-secreted cytokine IL-17 levels were enhanced in human and experimental renal allograft rejection, which suggested critical roles of Th17 cells in promoting graft rejection.14,15 In contrast, peripheral Treg cells and their compromised functional properties were decreased during renal and liver transplant patients.23,24 To explore whether Th17/Treg balance affect the liver function in rat liver transplantation, we detected Th17/Treg cells on different levels including Th17/Treg cells, related cytokines, serum transaminase levels, and recipient survival rate postoperation. Results showed that allografts groups had a lower survival rate, accompanied by increased Th17 cells, IL-17 level, and hepatic function. These results are in agreement with the observations derived from experimental models that alloreactive Th17 cells and related IL-17 level were increased during skin or liver allograft.13,25,26 In addition, we also found reduced Treg cells and related cytokines in allografts groups, which indicated that enhanced Th17 cells favor rejection through inhibiting Treg cells. Together, these results suggested that IL-17/Treg balance play critical roles in allograft rejection.
Numerous animal experiments and clinical trials have found IL-17 levels were increased in the kidney, lung, heart, and cornea after transplantation. And recently, IL-17 has reported to be a potential target for therapeutic intervention in allograft rejection.27-31 Therefore, we monitored the survival rates in allografts rats after administration of anti–IL-17A neutralizing antibody. We found that neutralization of IL-17A can inhibit intragraft production of IL-17, reduce liver damage and inflammatory infiltrates, as well as prolong graft survival, which provide direct evidence that inhibition of IL-17 may be beneficial for allograft survival. We do not exclude that animals treated with the IL-17 antibody may be died of some inflammation-free factors, as there was rarely inflammatory infiltrate after neutralization of IL-17A when compared to control allograft group, or indicators of liver functions (AST and T-Bili) are still higher than normal rats. It was reported that IL-17, a potent proinflammatory cytokine, has been demonstrated to accelerate allograft rejection by suppressing Treg cell expansion21; we found that neutralization with IL-17A resulted in increased Treg cells both in spleen and liver in allograft rats, suggesting enhanced activity of Treg cells attributed to the improvement of allograft survival after neutralization of IL-17 in allograft rats. Similar observations have been made in a rodent model of heart transplantation, which reported that inhibition of IL-17 activity reduced inflammatory infiltrates and delayed acute allograft rejection.32 Altogether, these results indicated that neutralization of IL-17A extended the survivals of liver allografts by shifting the Th17/Treg balance from Th17 predominance to Treg cells, which have a protective effect against graft rejection.
In summary, our results show that the imbalance of Th17/Treg cells and related cytokines, such as IL-17/IL-10 and TGF-β1, plays a crucial role in immune regulation in rat liver transplantation, indicating the shift toward predominantly Th17 cytokine profile may be 1 mechanism of graft rejection. Our data suggested inhibition of IL-17A as a potential therapeutic target for the treatment of liver graft.
The authors acknowledge the assistance from Dr. Zhipeng Xu (Department of Pathogen Biology and Immunology, Nanjing Medical University, China) for his analyses of our data.
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