Alveolar echinococcosis (AE) is a zoonosis caused by Echinococcus multilocularis (E. multilocularis) and is a rare but life-threatening disease in humans.1,2 The disease is acquired through ingestion of parasite eggs shed by the definitive host (mainly foxes and dogs). Following oral uptake, a released oncosphere penetrates the intestinal tissue, enters blood and lymphatic vessels, and ends up primarily in the liver. Humans acquire AE by accidentally ingesting viable parasite eggs. AE is mostly characterized by an infiltrative, tumour-like growth of the E. multilocularis metacestode affecting the liver of intermediate hosts such as small mammals or humans, causing organ dysfunction, and occasionally leading to death.3,4 Liver transplantation may be the only effective treatment for the end-stage hepatic AE.5
Generally, a transplant recipient must receive additional immunosuppressive therapy throughout their life time. However, the immunosuppressive agents that are commonly used in the clinic are non-specific. Despite treatments that have a certain efficacy, an increased incidence of infection and tumors and varying degrees of toxicity to the transplanted organs could be the reasons for loss of graft function during immunosuppressive therapy. These drawbacks severely limit the clinical efficacy of this approach to long term tolerance of a graft. Consequently, attention has been paid to develop a new approach in the transplant field that would avoid using a large dose of immunosuppressive drugs and would prolong the survival of the grafts.
Parasites can coexist with the host for a long time, and we were inspired by the phenomenon of chronic parasitic infections to investigate the immune tolerance to infection. In particular, we are interested in immune modulatory mechanisms that the parasite can stimulate in the host body6 that may be applicable for improving graft survival. A recent report showed five patients with residual/recurrent AE lesions who survived for more than 15 years after liver transplantation,7 suggesting that E. multilocularis may induce a state of tolerance that increases the graft survival time. The objectives of this study were to determine the mechanism of prolongation of graft survival in a rat heart allografting model with E. multilocularis infection, to assess influence of infection on lymphocyte subsets, eosinophils, the levels of cytokines, and regulatory T cells, and to evaluate the mechanisms of E. multilocularis induced tolerance for potential clinical use.
Animals and materials
E. multilocularis infected gerbils were provided by the University of Salford, UK. Lewis (LEW) and Brown Norway (BN) inbred male rats (weighing 180 g to 250 g) were purchased from the Beijing Vital River Experimental Animal Center, China. Animal care and experiments were performed in accordance with the Principles of Laboratory Animal Care and Guide for Care and Use of Laboratory Animals. All experiments and sampling procedures were approved by the Experimental Animal Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University, China (No. A-20101020011). Rat lymphocyte separation medium was obtained from Sigma (Germany), and CD4-PE, CD25-fluorensein isothiocyanate (FITC) from eBioscience (San Diego, USA).
Establishment of the animal models
Rat models of AE
One gerbil with E. multilocularis infection and a bulging abdomen was sacrificed after ether anesthesia, and E. multilocularis infected tissue was removed under sterile conditions. After removal of the necrotic lesions, the infected tissue was weighted, cut into pieces, pestled, sieved, and homogenized, then rinsed three times with saline to remove blood cells. A 20% E. multilocularis suspension was prepared from the E. multilocularis infected tissue with sterile saline containing penicillin. The suspension was determined microscopically and it was immediately used for infection of the rats. We injected the suspension intraperitoneally into the right lower abdomen of LEW rats at 1 ml/ 50–80 g body weight. The control group received an intraperitoneal injection of saline. The rat model of AE was considered successful if E. multilocularis lesions, confirmed by pathological examination, were found in the livers of the rats three months after inoculation.
Rat models of allograft heart transplantation
BN rats served as donors (12 donors) and LEW rats were recipients (12 recipients). All animals were inbred males and weighed 180 to 250 g. Donor heart grafts were transplanted into the recipient abdominal aorta and inferior vena cava by the methods described by Ono and Lindsey.8 In the experimental group, the heart grafts were transplanted from BN rats to E. multilocularis infected LEW rats. In the control group, the heart grafts were transplanted from BN rats to healthy LEW rats. Heart graft function was monitored twice daily (morning and evening) by palpation through the abdominal wall to assess graft survival, and when the hearts ceased to beat it was considered that the hearts were rejected.9 The recipient was sacrificed when rejection was determined, and final graft survival time was a record as one day earlier. The heart grafts and peripheral blood samples were collected. The heart grafts were fixed in 10% formalin or quick frozen and serial sections prepared for histopathological examination. Blood was taken from the inferior vena cava for cytokine testing and determination of CD4+CD25+ regulatory T cells by flow cytometry.
Mid-left ventricle heart tissue samples were sent for histological determination/gradation of the rejection. Tissue samples were excised, fixed in formalin, and embedded in paraffin. Tissue sections were cut and stained with hematoxylin and eosin. Cardiac histology was based on standardization of Billingham et al.10
Strep avidin-biotin-peroxidase complex (SABC) immunohistochemistry kit was purchased from Boster Biological Technology Co., Ltd., Wuhan, China. Formanlin-fixed and paraffin embedded sections were deparaffinized and rehydrated with distilled water. Endogenous peroxidase was blocked using blocking buffer (3% H2O2). The slides were washed with distilled water three times. Normal serologic blocking fluid was added and incubated for 20 minutes at room temperature. The primary rabbit IgG antibody was then added and incubated overnight in a wetting case at 4°C. The slides were washed with phosphate-buffered saline (PBS) three times, for three minutes each time, and incubated with biotinylated goat anti-rabbit IgG secondary antibody for 30 minutes in a wetting case at 37°C. Then the slides were incubated with SABC for 30 minutes in a wetting case at 37°C. After washing four times with PBS for five minutes we added 3,2-diaminobenzidine (DAB) following the standard immunohistochemical techniques, then washed with distilled water after coloration for five minutes at room temperature. The slides were then counterstained with hematoxylin for 10 seconds, dehydrated with absolute ethyl alcohol for two minutes and cleared with acetone for two minutes before mounting. For each slide, ten integral and non-overlapping visual fields were chosen at random to determine the number of positive cells.
Flow cytometric analysis
The peripheral blood was collected from recipients when the heart grafts were considered rejected. After centrifugation on density gradient material, the cells of the interface layer were harvested and suspended at 2×106/ml in PBS. One milliliter of cells were incubated at 4°C for 30 minutes with a saturating concentration of PE-conjugated anti-rat CD4 antibody (Pharmingen, USA) in combination with FITC-conjugated anti-rat CD25 antibody (Pharmingen) diluted in PBS containing 1% fetal calf serum. After washing, the cells were suspended in 1 ml of PBS, followed by analysis with flow cytometry (FACSscan, Becton Dickinson, USA). The dead cells were excluded from the analysis using propidium iodide fluorescence.
Concentration of interferon (IFN)-γ and interleukin (IL)-4 in serum
The enzyme-linked immunosorbent assay (ELISA) was employed to detect the concentration of the cytokines IFN-γ and IL-4 in serum of rats according to the method of Šoltýs and Quinn.11 Results were expressed as picogram per milliliter using murine recombinant IFN-γ and IL-4 (BD Biosciences, USA) as standard.
Statistical analyses were performed using Statistical Product and Service Solutions (SPSS) 17.0 (SPSS Inc., Chicago, IL, USA). Measurement data are presented as mean ± standard deviation (SD). Statistical evaluations for the differences between groups were assessed using the Student's t-test and one-way analysis of variance (ANOVA). A statistical level of significance was defined as P <0.05. P <0.001 was considered a highly significant statistical difference.
Growth ofE. multilocularis metacestode
E. multilocularis metacestode tissue was extracted from gerbil and a 20% E. multilocularis suspension was prepared and injected intraperitoneally into the right lower abdomen of LEW rats. It was shown that physical growth of E. multilocularis infected LEW rats was slower than healthy LEW rats and the body weight was lower in E. multilocularis septic rats than in normal rats of the same age. The infection rate was 96% in LEW rats three months after inoculation (Table 1).
In the E. multilocularis infected samples, pathology revealed that a high number of metacestodes was seen, often surrounded by host connective tissue, with a clear discernible laminated and germinal layer (Figure 1). The laminated layer was very gracile with incomplete structure mostly. The increase in the number of vesicles was due to exogenous budding of small daughter vesicles from larger parental vesicles. We could find many portoscolex in vesicles (Figure 1).
AE rat models and heart allograft survival
E. multilocularis infection was found in the recipient LEW rats previously inoculated, and there was a 100% success for allograft heart transplantation. All rats were included in the final analysis. The average heart allograft survival time in the experimental group was (16.17±3.19) days. In contrast, the average allograft survival time was (7.92±1.93) days in the control group. The survival time in the experimental group was significantly longer compared with the control group (P <0.05).
Sections of heart grafts were stained with H&E and observed with light microscopy. H&E staining results are shown in Figure 2. The myocardial lesion in the experimental group was slight and the inflammatory reaction was less compared with the control group. The average rejection grade of the grafts in the control group was 3.79±0.40 (range from 2.00 to 4.00), and the average rejection grade was 3.38±0.43 (range 2.00 to 4.00) in the experimental group (P >0.05).
Histology and immunohistochemistry studies
Histological examination of the rejected allografts revealed features of acute cellular rejection but there was no statistically significant difference in the number in graft-infiltrating CD4+ T cells between the experimental group heart grafts and the control group grafts (P >0.05). But decreased numbers of graft infiltrating CD8+ T cells in the rejected grafts in the experimental group were observed. We also quantified the total number of graft infiltrating eosinophile granulocytes (CD15+), and as expected, found the numbers of infiltrating eosinophile granulocytes were increased in the experimental group (Figure 3A and 3B) compared to the control group (P <0.05).
Population of CD4+CD25+ regulatory T cells in peripheral lymphocytes
We performed FACS analysis using the peripheral lymphocytes from graft recipients in the two groups. The proportion of CD4+CD25+ regulatory T cells was 10.8% on average in the experimental group, significantly higher than that in the control group (6.1%, Figure 4).
Concentration of IFN-γ and IL-4 in serum
Figure 5 shows the concentration of IL-4 was significantly higher in the experimental group than in the control group (P <0.05). The concentration of IFN-γ was significantly lower in the experimental group than in the control group (P <0.05).
The present study showed that the survival time of grafts was significantly prolonged, demonstrating that E. multilocularis infection may be able to induce immune tolerance of grafts, and the E. multilocularis infected recipients immune status was changed, including elevated IL-4 secretion, decreased level of IFN-γ, increased CD4+CD25+ regulatory T cells, reduced CD8+ T cells infiltrating. These findings may bring an inspiration to some interest in the field of organ transplantation. To our knowledge, this is the first report on the immune responses to allograft heart transplantation in experimental rats infected with E. multilocularis.
T helper (Th) cell is playing a crucial role in host immune response in parasite infections,12 and Th1/Th2 polarized type immune response largely determines the fate of grafts in organ transplantation. In this study, we analyzed the pattern of cytokine secretion to investigate whether a Th1/Th2-polarized type response was stimulated in E. multilocularis infected rats. We wish to point out that the allograft survival prolongation was correlated with a Th1/Th2 imbalance. We demonstrated the Th1/Th2 balance by determining the serum IFN-γ and IL-4 concentration. In this context, it was interesting to note that a shift to a Th2-type pattern was observed: the secretion of IL-4 increased dramatically while that of IFN-γ decreased in the experimental group (Figure 5), in accordance with some studies of several types of parasites.13–15 In addition, it is well established that the emergence of an immune response dominated by a Th2-type profile is characteristic of many helminth infections.16–18 Differentiated Th2 excretes IL-4, participates in maintaining a lower immune response in rejection.19Fasciola hepatica was found through induction of IL-4 inhibiting the activation of macrophages and suppressing IFN-γ production by Th1 cells.20 It looks like that IL-4 is capable of converting Th1 cells to the Th2 subtype. These data indicate that the heart-graft acceptance in the presence of E. multilocularis infection was strongly associated with Th2 differentiation, which had been demonstrated to be important in tolerance induction and maintenance.21
The transplantation immune response is mediated largely by T cells and a variety of inflammatory stimuli that induce T cells to infiltrate the graft tissue.22,23 Honey et al24 proposed that CD8+ T cell mediated rejection could be suppressed by Th2 polarization of the immune response and CD8+ T cells depletion could induce skin transplantation tolerance. We examined effector T cells infiltrating the rejected allografts by immunohistochemical examination, and found that the number of CD4+ T cells was not different between the two groups (P >0.05). However, a significant decrease in the number of infiltrating CD8+ T cells was observed in the experimental group (Figure 3). We speculate that the prolongation of graft survival might be induced by CD8+ T cells suppression.
Visibly higher number of eosinophils in the blood and in the region of the lesion is a significant immunological feature in E. multilocularis infection.12 There was strong evidence that IL-5 and eosinophils reject mouse major histocompatibility complex (MHC) mismatched allogeneic heart transplants under conditions of the absence of CD8+ T cell.25 A relatively increased number of CD15+ infiltrating eosinophil granulocytes in the experimental group were found by immunohistochemical analysis in rejected myocardial tissue compared with those in the control group (Figure 3), suggesting that eosinophils were involved in the process of graft rejection.
CD4+CD25+ regulatory T cells have been proposed to be essential for T cell homeostasis as well as self-tolerance and transplantation.26,27 The number of CD4+CD25+ regulatory T cells increased significantly in mice that were infected by Schistosoma japonicum.28 We examined the population of CD4+CD25+ regulatory T cells in the peripheral blood of E. multilocularis infected recipients and found that the proportion of CD4+CD25+ regulatory T cell was markedly increased (Figure 4). Additionally, the secretion of the type Th1 cytokine IFN-γ was significantly decreased, and the type Th2 cell cytokine IL-4 level was increased in E. multilocularis septic recipient models (Figure 5). Previous reports suggested that the CD4+CD25+ regulatory T cells cause Th2 type immune response polarization in Schistosoma mansoni infection through inhibition of the Th1 type immune response; their inhibition is stronger on Th1 cells than on Th2 cells.29,30 This may explain what we found in our research. Although very preliminary, our observation would support the idea of the role of CD4+CD25+ regulatory T cells in the suppression on Th1 cells. Current experiments are investigating the mechanisms of regulatory T cells in parasite infection.
In conclusion, E. multilocularis infection could prolong the allograft survival time through the polarization of Th1/Th2-type cells and induction of CD4+CD25+ regulatory T cells. This strategy may provide a new idea for establishing transplantation tolerance.
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