The Role Of Treg/Th1/Th17/Th22Cells In The Development Of Acute Rejection In Mice Following Peripheral Nerve Xenotransplantation

BACKGROUND:Peripheral nerve injuries are common chirurgic diseases characterized by the loss of motor and sensory skills. These injuries often result in long nerve segment loss; as a result, nerve ends cannot be directly sutured because of increased tension. Although previous studies in murine models showed that autografts and allografts are primary standards for nerve injures, these grafts have limited sources. In recent day, the research about xenograft nerve is increasingly concerned because of the continual improvement in transplantation immunology.The significant barriers of xenotransplant include the innate immunity (cellular and humoral immunity) and adaptive response rejection. T cells have major functions in the initiation and perpetuation of nerve graft rejection. A recent study in rodents has revealed Thl and Th17cells may have important functions in organ transplant rejection. Thl and Th17cells are the two classical lineages of the effector CD4+T cells, and these cells can express their key cytokines IFN-y and IL-17, respectively. As a new IL-22-producing T cell subset, Th22cells express the key cytokine IL-22, which can activate signal transduction and transcription3(STAT3). Present studies also demonstrated that Th22cells are implicated in skin transplant rejection. However, the function of Th22cells in the pathogenesis of nerve xenotransplantation is poorly understood. We suggest that Th22cells may partially contribute to nerve xenograft rejection, and their expressions are likely time dependent. Regulatory T cells (Tregs) are important regulators of immune responses and have major functions in maintenacing the immune steady and regulating the occurrence and development of autoimmune diseases. Tregs can suppress Thl and Th17cell-mediated responses by inhibiting transforming growth factor-β (TGF-P). Foxp3(Forked head/wings shape helix transcription factors), which play key role in controlling the development and. function of Treg cells, specifically expressed in Treg cells. Many studies have shown that TGF-β can suppress Thl and Th2cell differentiation, as well as promote Th9, Th17, and Foxp3+regulatory T cell differentiation.In contrast to TGF-β1and IL-6, TGF-β1and IL-2induce Treg cells and inhibit Th17cell differentiation. Another study has shown that IL-6/STAT3and IL-2/STAT5are necessary to induce Th17and Treg cell differentiations. IL-2can also induce Treg cells to differentiate into Thl-like cells. Thereby reveal the developmental plasticity between Treg and Thl7/Thl cells.However, the importance of Treg cells in the pathogenesis of nerve transplantation and the relationship between Treg and Th22cells remain poorly understood. In this study, a xenotransplantation model was used to investigate the biological importance of Treg and Th22cells. This model was also used to determine the potential relationship between Treg and Th22cells during peripheral nerve xenotransplant rejection.Part1the changes of immune response in actual early rejection after nerve xenotransplantationOBJECT:1. To examine the degree of mononuclear cells, Foxp3+, IL-17+, IL-22+, and IFN-γ+cells infiltration surrounding the grafts at all observation time points. 2. To examine the level of mRNA expression levels of Foxp3, RORyt, AHR, and T-bet surrounding the grafts at all observation time points.3. To analyze the percentage of Tregs, Thl,Th17, and Th22cells in the spleen cells after the treatment at all observation time points.4. To detect the level of IL-17、IL-22、IFN-γ in the serum after the treatment at all observation time points.5. Correlation analysis was performed to characterize the function of Treg cells in Thl/Thl7/Th22cells as well as the concentrations of plasma IFN-y, IL-17, and IL-22.METHODS:One hundread healthy C57mice (male,6-8weeks) were randomized into two groups:control group and experimental group. Adult male C57BL/6mice were used as the recipient for nerve xenotransplantation, and Sprague-Dawley rats were used as the donor. These nerve xenotransplanted-mice were used as the experimental groups, and those that received autograft transplant were chosen as the control group. The motor function of mice that received peripheral nerve transplantation firstly evaluated using a CatWalk system at1,3,7,14, and28d of transplantation. Then whole blood were collected using the ways of ophthalmectomy, and serum was also obtained from all of the subjects by centrifugation and stored at-80℃, cytokine levels in the serum supernatant were measured by ELISA using commercially available IFN-γ, IL-17, and IL-22ELISA kits. Splenocytes were harvested by grinding and homogenized in PBS at1×106/ml. The spleens were collected from the mice at each time point, and flow cytometry, with fluorescent conjugated monoclonal antibody present, was performed to analyze the percentages of Tregs, Thl,Th17, and Th22cells in the nerve xenografted-mice and the control mice. Total RNA was extracted from nerve grafts by using the acid guanidinium thiocyanate-phenol-chloroform method. And the mRNA expression of Foxp3, RORyt, AHR and T-bet was detected by RT-PCR.At same time, the degree of Foxp3+, IL-17+, IL-22+, and IFN-γ+cells infiltration surrounding the grafts at all observation time points were observed and immunohistochemistry. And the tissues were collected, sectioned, and visualized by routine HE staining to examine the general cellular changes around the grafts during rejection. Finally, correlation analysis was performed to characterize the function of Treg cells in Thl/Thl7/Th22cells as well as the concentrations of plasma IFN-y, IL-17and IL-22. Data from all of the experiments were expressed as median (range). The difference between the median of the groups was determined by the Wilcoxon rank sum test. Test results were reported as two-tailed p values, where p<0.05was considered statistically significant. Spearman correlation test was conducted to analyze correlation depending on data distribution.RESULTS:1. Histological analysis results revealed noticeable increment of mononuclear cell infiltration degrees surrounding nerve xenografts during the28-day observation period in all of the grafted animals compared with those in the control mice. However, no significant changes in these levels were observed in the control group during the same period.By immunohistochemistry staining, our data showed that all of the protein expressions were time dependent. IFN-γ, IL-17, IL-22, and Foxp3-positive cells were found in the infiltrating mononuclear cells surrounding the grafts, and the numbers of these cells showed statistical increment compared with those in the control mice. And the numbers of these cells showed a gradually increasing trend in the xenotransplantation model in this study except for Foxp3+cells compared with the control groups. The Foxp3+cell level in the xenotransplantation model increased and peaked at7d of xenotransplantation compared with the mice that did not receive xenotransplant. However, no significant difference in the indexes was noted in the control group during the28-day period of autotransplantation.2. The expressions of mRNA encoding Foxp3, AHR, RORyt, and T-bet were probed in the surrounding nerve graft by gel electrophoresis. The result of the antisense DNA fragment coincided with the known gene. In addition, the mRNA expression levels of AHR, RORyt, and T-bet in mice subjected to xenotransplantation remarkably increased time dependently, whereas no marked difference was noted in the control group during the28-day observation period. However, the mRNA encoding Foxp3was expressed in different ways. The mRNA expression of Foxp3increased and peaked at7d of transplantation. This level was significantly higher than that in the control group at each time point, whereas no marked difference was noted in the control group during the28-day observation period.3. The spleens were collected from the mice at each time point, and flow cytometry was performed to analyze the percentages of Tregs, Thl, Th17, and Th22cells in the nerve xenografted-mice and the control mice. We found that Th1(CD3+CD8-IFN7+) cells and Th17(CD3+CD8-IL-17+)cells in the experimental group significantly increased at1,3,7, and14d of xenotransplantation; IL-22-producing Th22cells evidently increased at7and14day of xenotransplantation compared with those in the control group.4. ELISA results revealed that the changes in IFN-y, IL-17, and IL-22during rejection exhibited statistically higher serum levels in the xenograft recipients than in the control group at3,7, and14d of xenotransplantation.5. Thus, correlation analysis was performed to characterize the function of Treg cells in Th1/Th17/Th22cells. The results showed that the low percentage of Treg cells was significantly and negatively correlated with the percentage of Th1/Th17cells at7d of xenotransplantation (P=0.028, P=0.005). In addition, the percentage of Treg cells showed a significantly negative correlation (P=0.004) with the peripheral Th22cell levels at7d of xenotransplantation. Moreover, the low percentage of Treg cells was negatively correlated with the concentrations of plasma IFN-y, IL-17, and IL-22at7d of xenotransplantation (P=0.003,P=0.029, P=0.047).Furthermore, the low percentage of Treg cells was negatively correlated with the concentrations of Thl, Thl7, and Th22cells in the spleen and the plasma IFN-y, IL-17, and IL-22levels at14d of xenotransplantation.CONCLUSION:1. These results indicated that the Treg cells involved in xenotransplant rejection2. This result suggested that Thl, Th17and Th22cells contribute to the rejection process.3. The data insighted that the percentage of CD4+CD25+Foxp3+Treg was negatively correlated with the percentages of Thl/Th17/Th22cells and levels of IL-17, IL-22, and IFN-y. Part2the effect on immune response in actual eearly rejection after nerve xenotransplantation with neutralizing antibodyOBJECT:1. To examine the degree of mononuclear cell infiltration surrounding the tissues of xenografts at all observation time points after using IFN-y, IL-17, and IL-22neutralizing antibodys.2. To examine the degree of Foxp3+, IL-17+, IL-22+, and IFN-y+cells infiltration surrounding the tissues of xenografts at all observation time points after using IFN-y, IL-17, and IL-22neutralizing antibodys.3. To examine the level of mRNA expression levels of Foxp3, RORyt, AHR, and T-bet surrounding the tissues of xenografts at all observation time points after using IFN-y, IL-17, and IL-22neutralizing antibodys.4. To analyze the percentage of Tregs, Thl, Th17, and Th22cells in the spleen cells after the treatment at all observation time points.5. To evaluate the efficacy on the motor function of nerve xenografted-mice after they were subjected or not subjected to treatment with IFN-y, IL-17, and IL-22neutralizing antibodys.METHODS150healthy C57mice (male,6-8weeks) were randomized into three groups: IFN-y Abs group, IL-17Abs group and IL-22Abs group, and each of these groups were experimental group. Adult male C57BL/6mice were used as the recipient for nerve xenotransplantation, and Sprague-Dawley rats were used as the donor. These nerve xenotransplanted-mice were used as the experimental groups, and those that received autograft transplant were chosen as the control group. Not, all of the animals were pre-treated with IFN-y, IL-17, and IL-22before the experiment was conducted. The motor function of mice that received peripheral nerve transplantation firstly evaluated using a CatWalk system at1,3,7,14, and28d of transplantation. Splenocytes were harvested by grinding and homogenized in PBS at1×106/ml. The spleens were collected from the mice at each time point, and flow cytometry, with fluorescent conjugated monoclonal antibody present, was performed to analyze the percentages of Tregs, Thl, Th17, and Th22cells in the nerve xenografted-mice and the control mice. Total RNA was extracted from nerve grafts by using the acid guanidinium thiocyanate-phenol-chloroform method. And the mRNA expression of Foxp3, RORyt, AHR and T-bet was detected by RT-PCR.At same time, the degree of Foxp3+, IL-17+, IL-22+, and IFN-γ+cells infiltration surrounding the grafts at all observation time points were observed and immunohistochemistry. And the tissues were collected, sectioned, and visualized by routine HE staining to examine the general cellular changes around the grafts during rejection. Data from all of the experiments were expressed as median (range). The difference between the median of the groups was determined by the Wilcoxon rank sum test. Test results were reported as two-tailed p values, where p<0.05was considered statistically significant.Results:1. CatWalk-assisted gait analysis Nerve xenografted-and IFN-γ, IL-17, and IL-22neutralizing antibodys-treated mice showed better neurological function recovery, compared with those in the recipients without antibodies.2. The levels of mononuclearcells infiltration were detected by H&E staining. We found that the number of mononuclear cells significantly decreased at3,7, and14d of treatment with IFN-y and IL-17neutralizing antibodies as well as at7and14d of treatment with IL-22neutralizing antibodies.3. By immunohistochemistry staining, our data showed that IFN-y+, IL-17+, and IL-22+cells were evidently decreased in xenografted mice since7d of treatment with IFN-y, IL-17, IL-22neutralizing antibodies. Foxp3+cells level in the recipients with IFN-y, IL-17, and IL-22neutralizing antibodies by abdominal injection clearly increased. Moreover, the Treg cell level peaked on the7d and then progressively decreased. In addition, the amount of Foxp3+cells infiltration surrounding the grafts in the recipients with IL-17neutralizing antibodies is significantly higher than EFN-y and IL-22neutralizing antibodies.4. By RT-PCR, we found that the mRNA expressions of AHR, RORyt, and T-bet decreased in the recipients of intraperitoneally injected IFN-y, IL-17, and IL-22neutralizing antibodys at all observation time points except for1and3day after xenotransplantation; by contrast, Foxp3expression increased.The mRNA expression of Foxp3increased and peaked at7d of transplantation. 5. By FCM, we found that the levels of Thl, Th17, or Th22cells at different time points of xenotransplantation were statistically decreased in the xenograft recipients with IFN-y, IL-17, or IL-22neutralizing antibodies compared with those in the recipients without antibodies. Moreover, the percentage of Treg cells was significantly higher in the xenograft recipients with IFN-y, IL-17, and IL-22neutralizing antibodies compared with that of the recipients without antibodies at different time points of xenotransplantation.CONCLUSION:1.IFN-γ, IL-17, and IL-22neutralizing antibodys showed better neurological function recovery.2.IFN-γ, IL-17, and IL-22neutralizing antibodys inhibit the lymphocyte infiltration.3. Evident improvement in the rejection of peripheral never xenograft after IFN-y, IL-17, and IL-22neutralizing antibodys were injected.