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Increase of peripheral Th17 lymphocytes during acute cellular rejection in liver transplant recipients
Original Article / Transplantation
Increase of peripheral Th17 lymphocytes during acute cellular rejection in liver transplant recipients Hua Fan, Li-Xin Li, Dong-Dong Han, Jian-Tao Kou, Ping Li and Qiang He Beijing, China
BACKGROUND: Although many human inflammatory and autoimmune diseases were previously considered to be mediated by T helper type 1 (Th1) cells, the recently described Th17 cells play dominant roles in several of these diseases. We and others speculated that allograft rejection after organ transplantation may also involve Th17 cells. Episodes of acute rejection occur in 30% of liver transplants. This study aimed to determine the frequency of circulating Th17 cells in patients who had received liver transplants for benign end-stage liver disease and to identify any association between acute rejection episodes and levels of Th17 cells in the peripheral blood.
CONCLUSION: Circulating Th17 cells may be useful as a surrogate marker for predicting acute rejection in liver transplant recipients.
METHODS: A prospective study compared Th17 cells from 76 consecutive benign end-stage liver disease patients who had undergone orthotopic liver transplantation from 2007 to 2011 with those from 20 age-matched healthy individuals. Peripheral blood samples were collected at different time points within one year after transplant. Blood samples and liver biopsies were also collected at the diagnosis of acute rejection. Percentages of circulating CD4+IL-17+ cells were measured by flow cytometry. The transplant patients were classified into two groups: a rejection group consisting of 17 patients who had an episode of acute rejection, and a non-rejection group comprising the remaining 59 patients with no acute rejection episodes. Percentages of circulating Th17 cells were compared between the two groups and controls.
Introduction
(Hepatobiliary Pancreat Dis Int 2012;11:606-611) KEY WORDS: liver transplantation; Th17 cells; acute cellular rejection; transplant immunology
L
iver transplantation is a viable treatment option for end-stage liver diseases. Despite the armamentarium of immunosuppressive agents, the incidence of acute rejection in patients after liver transplant is approximately 30%, and acute rejection episodes significantly decrease graft and patient survival.[1] The etiology of acute rejection is complex and involves synergistic interactions between different types of T-helper (Th) subsets and other mononuclear cells. For example, production of interferon-γ (INF-γ) by Th1 cells can initiate acute allograft rejection by augmenting antiRESULTS: The levels of circulating CD4+IL-17+ T cells in the donor cellular responses and promoting cytotoxic T-cell rejection group were higher during acute rejection than those activity. Alternatively, Th2 cells secrete interleukin-4 in the non-rejection group (2.56±0.43% versus 1.79±0.44%, (IL-4), subsequently recruit eosinophils, and promote P<0.001). The frequency of CD4+IL-17+ cells in peripheral damage of the transplanted organ.[2, 3] blood was positively correlated with the rejection activity index In recent years, the central role of effector memory (r=0.79, P=0.0002). T cells that secrete IL-17, Th17 cells has been documented in pro-inflammatory processes and the development of autoimmune diseases.[4] Th17 cells recruit neutrophils and monocytes, and their chronic presence and Author Affiliations: Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China (Fan H, Li activation augments the concentrations and effects LX, Han DD, Kou JT, Li P and He Q) of proinflammatory cytokines.[5-7] Although a few Corresponding Author: Qiang He, Professor, Department of Hepatobiliary studies[8-10] have already highlighted the possibility of a Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China (Tel: 86-10-85231504; Fax: 86-10-85231503; Email: relationship between IL-17 and acute rejection in organ transplantation, the role of Th17 cells in acute rejection [email protected]) is unknown in patients with liver transplantation. © 2012, Hepatobiliary Pancreat Dis Int. All rights reserved. doi: 10.1016/S1499-3872(12)60231-8 To elucidate the potential role of Th17 cells in acute • Hepatobiliary Pancreat Dis Int，Vol 11，No 6 • December 15，2012 • www.hbpdint.com
Th17 cells in acute rejection after liver transplant
rejection after liver transplantation, we compared the frequency of Th17 cells in peripheral blood from 4 types of participants: i) liver transplant patients during acute rejection episodes (rejectors), ii) rejectors during quiescent periods, iii) liver transplant patients who did not experience an acute rejection (non-rejectors), and iv) healthy controls.
Methods Patients A cohort of 76 liver transplant recipients (22 females and 54 males; mean age 48±13 years, range 21-69) due to benign end-stage liver disease admitted to out- and in-patient care unit of our department from 2007 to 2011, and 20 age-matched healthy controls (mean age 46±14 years) were enrolled in the study. Patients with infections, hepatitis recurrence or malignancy were excluded. The institutional review board of the hospital (2007-021) approved the study protocol, and all patients and healthy individuals provided written informed consent. Liver transplant patients provided multiple blood samples (heparinized) during the first year after transplant, and at the time of acute rejection. Patients who suffered from acute rejection were treated with methylprednisolone (Solu-Medrol, 3×1000 mg intravenous (i.v.)). A diagnosis of acute rejection was based on clinic pathological findings (jaundice, fever, irritability) and biochemical analyses (elevated levels of alanine aminotransferase, aspartate aminotransferase, and/or direct bilirubin). Evidence for acute rejection was confirmed by histological examination of liver biopsies and was scored according to the Banff classification by an experienced pathologist blinded to this study.[1] A rejection activity index (RAI) of 4 or more defined rejection. The dose of post-transplant immunosuppressant agent was initially 10 mg/kg i.v. methylprednisolone tapered to 0.3 mg/kg by 7 days for a 3-week maintenance dose. Treatment of all patients with tacrolimus (Tac) or cyclosporin A (CsA) began on postoperative day 4. Plasma trough tacrolimus concentrations were maintained between 10 and 12 ng/ mL during the first month and then reduced to 8-10 ng/ mL. Plasma trough cyclosporin A concentrations were maintained between 350 and 450 ng/mL for the first month and then tapered to 250-350 ng/mL. Seventeen patients who experienced an acute rejection within one year were defined as the rejection group. The remaining 59 patients who had no acute rejection were designated the non-rejection group. The characteristics of rejection and non-rejection groups are listed in the Table.
Table. Characteristics of patients in the rejection and non-rejection groups Parameters
Rejection group (n=17) 47.2±11.3 12/5 25.2±9.0
Age (yr) Male/Female MELD Primary liver diseases HBV 12 HCV 1 HBV+HCV 2 Primary sclerosing cholangitis 1 Autoimmune liver diseases 1 Immunosuppressive 13/4 protocol (Tac/CsA) Time point of acute rejection 2.5±3.8 episodes post-transplant (mon) NS: not significant.
Non-rejection group (n=59) 50.2±10.7 42/17 27.1±10.5 47 5 4 2 1 48/11
P value NS NS NS NS NS NS NS NS NS
Flow cytometric analysis The expression levels of CD4 and IL-17A on lymphocytes were measured by flow cytometry. Peridin chlorophyll protein (PerCP) and allophycocyanin (APC)-labeled antibodies were used: CD4 (mouse IgG1, PerCP) and CCR6 (mouse IgG1, FITC) were from Becton Dickinson, and IL-17A (mouse IgG1, APC) was from eBioscience (San Diego, CA). Appropriate isotype controls (Becton Dickinson, USA) were used. Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood by density gradient centrifugation over Ficoll-Paque plus (GE Healthcare, Uppsala Sweden). PBMCs were collected at interphase and washed twice in phosphate-buffered saline. The cells were resuspended in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL, Grand Island, NY). The cells were cultured in the absence or presence of phorbol myristate acetate (100 ng/mL) and ionomycin (1 μg/mL) (both from Alexis Biochemicals, San Diego, CA) for 5 hours. Cytokine secretion was inhibited by brefeldin A (Sigma, St. Louis, MO). Then surface staining was performed with anti-CD4 and antiCCR6. The cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD PharMingen, USA). Finally, the samples were intracellularly stained with anti-IL-17A or an appropriate isotype control. CD4 +IL-17+ T cells were sorted by a fluorescence-activated cell sorter (FACS; CaliburTM, Becton Dickinson, USA) and the FACS data were analyzed with Flow JoTM 7.3.5 software (Treestar Software Inc., USA). Statistical analysis Statistical analysis and graphic studies were done
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with Windows SPSS Version 11.5 and GraphPad PrismTM 5.0. The Mann-Whitney U test (unpaired analyses) or the Wilcoxon's signed-rank test (paired analyses) was used to assess differences between the two groups or longitudinal analysis was used in each group, respectively. Continuous variables (e.g. clinical characteristics) were compared using the Mann-Whitney U test, and discrete variables were compared using the Chi-square test or Fisher's exact test. Correlation analysis was made between different parameters by using Spearman's rank order correlation coefficient. Two-tailed P values<0.05 were considered statistically significant.
T-cell population over time (P>0.05, Fig. 2B). An example of one liver transplant patient with acute rejection and stable liver function is shown in Fig. 3. The percentage of CD4+IL-17+ T cells increased during acute rejection in comparison to that during stable liver function (Fig. 3). Next, a possible association between the frequency of CD4+IL-17+ T cells in circulation and the RAI of the liver allograft biopsy was investigated. In the rejection group, the percentage of CD4+IL-17+ T cells was positively correlated with the RAI (r=0.79, P=0.0002, Fig. 4). In addition, CD4+IL-17+ T cells were further characterized regarding CCR6 expression in the same 10 rejectors, 23 non-rejectors and 13 healthy controls. CCR6 was used to define Th17 cells. CD4+IL-17+ T cells from patients with acute rejection and from non-rejectors Results mainly expressed CCR6; only a minor percentage was + + The percentage of CD4 IL-17 T cells was significantly deficient for CCR6 (Fig. 5).[11] No significant differences higher in patients with acute rejection than both those from patients with non-rejection and healthy controls were detected between rejectors, non-rejectors, and controls (percentage of CCR6-positive cells in (CD4+IL-17+ T cells in patients with acute rejection healthy + + CD4 IL-17 T cells: rejectors versus non-rejectors, 97.64± versus those with non-rejection: 2.56±0.43% vs 1.79± 0.44%, P<0.0001; with acute rejection versus healthy controls: 2.56±0.43% vs 1.57±0.43%, P<0.0001; with non-rejection versus healthy controls: 1.79±0.44% vs 1.57±0.43%, P=0.05; Fig. 1). To assess intra-individual variation of the CD4+IL-17+ T-cell population over time, longitudinal analysis was performed. The rejection group provided 17 paired peripheral blood samples from recipients with rejection and stable liver function. In addition, 20 liver transplant patients in the non-rejection group were analyzed. In the rejection group, the CD4 +IL-17+ T-cell population was higher during rejection than stable liver function (2.56±0.43% vs 1.50±0.25%, P<0.0001, Fig. 2A). Patients in the non-rejection group maintained a stable CD4+IL-17+
Fig. 1. Relative percentages of CD4+IL-17A+ T cells in the nonrejection group, the rejection group during acute rejection, and healthy control group. *: P<0.0001.
Fig. 2. Longitudinal analysis of IL-17A+ expression within the CD4+ T-cell population over time. A: Percentage of IL-17A+ cells over time in the rejection group. Expression was assessed during acute rejection and stable liver function. *: P<0.0001. B: Percentage of IL-17A+ cells within the CD4+ T-cell population from 20 patients in the non-rejection group. Expression was assessed at two different time points in stable liver function.
• Hepatobiliary Pancreat Dis Int，Vol 11，No 6 • December 15，2012 • www.hbpdint.com
Th17 cells in acute rejection after liver transplant
Fig. 3. Representative dot-plots of multichromatic flow cytometry analysis gated on the CD4+ T-cell population. Example from one liver transplant patient with acute rejection and one with stable liver function. The percentage of CD4 +IL-17+ T cells is given in the upper right quadrant. The number of IL-17A+ T cells was increased in the liver allograft recipients who were experiencing acute rejection. Appropriate isotype controls were used.
Fig. 5. Representative dot-plots of multichromatic flow cytometry analysis gated on the CD4+IL-17+ T-cell population. Example from one liver transplant patient with acute rejection and one with stable liver function. The quadrant gates were adjusted according to the given isotype controls. Most of the CD4 +IL-17+ T cells displayed CCR6 (97.12% and 97.94%).
Fig. 4. Correlation between percentage of CD4 +IL-17+ T cells and Banff rejection activity index of liver allograft biopsy in the patients who experienced acute rejection. Each symbol represents a single individual (r=0.79, P=0.0002).
1.04% vs 98.02±1.07%, P>0.05; rejectors versus healthy controls, 97.64±1.04% vs 97.14±1.31%, P>0.05; nonrejectors versus healthy controls, 98.02±1.07% vs 97.14± 1.31%, P>0.05). Furthermore, we looked for an association between the CD4+IL-17+ T cells in circulating peripheral blood and the blood concentration of Tac or CsA within one year after liver transplant and found no correlation (data
not shown). These results were found in both rejection and non-rejection recipients. Since only four patients in the rejection group were treated with CsA, the statistical power was insufficient to expect a significant difference between subgroups.
Discussion In our study, IL-17 production by polyclonallystimulated PBMCs and activated CD4+IL-17+ T cells was markedly elevated in liver transplant recipients during the acute rejection period compared with that in those without acute rejection and healthy controls. Furthermore, circulating CD4+IL-17+ T cells were positively correlated with the grade of acute rejection.
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This discrepancy was not due to differences in clinical characteristics because they were comparable between the two groups. This finding suggested that the increase in circulating Th17 cells precedes acute rejection in liver transplant recipients and may become a surrogate marker for acute rejection with further testing. The Th1 cell is thought to be responsible for allograft rejection, but many studies investigating the role of Th17 cells challenge this dogma.[4, 12] When Th1- or Th2mediated acute rejection is neutralized in a mouse heart transplant model, acute rejection caused by heightened neutrophil responses still occurs.[13] More recently, IL-17 deficiency in mice delays recruitment of Th1-mediated cells into cardiac allografts, and may reduce the early infiltration of macrophages and neutrophils. [14] But a study demonstrated higher IL-17 concentrations during acute rejection in human liver transplant recipients, [15] but further studies are needed to demonstrate causality. Furthermore, the roles of the different IL-17 producing cell types in the rejection milieu need clarification. Studies on Th17 cells rather than the cytokine IL-17 need to be performed to decipher the role of these cells in clinical acute rejection. The contribution of Th17 cells in clinical acute rejection has not yet been widely established, but animal studies showed that they induce tissue damage by stimulating inflammatory cells to release proinflammatory cytokines and accelerate the development of severe allograft vasculopathy. [16] In our study, Th17 cell levels after nonspecific stimulation were significantly higher in patients who experienced an acute rejection episode than in non-rejectors and healthy controls. Our results are consistent with the significantly higher levels of Th17 cells in patients with acute rejection after cardiac transplantation.[17] Vanaudenaerde et al[18] reported that the bronchoalveolar lavage fluid of patients rejecting lung transplants exhibit higher IL-17 protein and mRNA levels than transplant patients without rejection. The severity of rejection correlated with the numbers of lymphocytes and neutrophils present in the bronchoalveolar lavage. These findings are consistent with our results that the frequency of circulating Th17 cells was significantly positively correlated with the RAI. Despite our finding that peripheral Th17 cells were upregulated in patients with acute rejection and in patients with nonspecific stimulation, other specific antigens may promote expansion of the IL-17 axis in acute rejection. Further studies are needed to elucidate the processes. Although Th17 cells increased among T cells in circulating peripheral blood, the mechanisms are not fully understood. Our results showed that there are two
potential hypotheses to explain these findings. First, circulating Th17 cells may be up-regulated in liver transplant recipients who experience acute rejection in an IL-6 and IL-23 laden milieu. Previous research showed that IL-6 and IL-23, which are key factors in the differentiation of Th17 cells, are significantly increased in human liver transplant recipients during acute rejection.[15, 19] Moreover, an inflammatory environment can augment the potential conversion of Tregs into Th17 cells,[20, 21] thereby increasing their local concentration and that of IL-17 cytokine, which stimulates a positive feedback loop for proinflammatory pathways. The other hypothesis is that circulating Th17 cells may be recruited from other compartments, such as the secondary lymphoid organs. Chen and colleagues [22] reported that drained lymph nodes after allocorneal rejection in mice had a large number of Th17 cells. In our study, there was insufficient evidence to support the presence of Th17 cells in the local allograft tissue. Prospective studies are needed to further test these hypotheses. The effects of calcineurin inhibitors on differentiation, activation, and effector activity of the Th17 lineage during transplantation are limited and appear to be contradictory.[23, 24] A recent report shows that calcineurin inhibitors (CNIs) can lead to a weaker ability to dampen Th17 cells versus mycophenolic acid in renal transplant patients.[25] However, the impact of CNIs remains uncertain although we found no significant correlation between circulating Th17 cells and the dose or blood concentration of CNIs. Further investigation of a larger population is needed. Our data showed an increase of circulating Th17 cells at the time of rejection and the recovery of peripheral Th17 cell levels when graft rejection resolved. Accordingly, circulating Th17 cells may be used as a surrogate marker for predicting acute rejection during the post-transplant period and assessing the immune status of liver transplant recipients. In summary, in this study the levels of circulating Th17 cells increased in the peripheral blood of patients with acute rejection after liver transplantation and were associated with rejection grade. We consider that circulating Th17 cells in the peripheral blood may serve as a monitoring tool to predict acute rejection in liver transplant recipients. However, the role of Th17 cells in initiating and promoting acute rejection and their multiple interactions with additional cell types has not yet been fully defined. Contributors: HQ proposed the study. FH and HQ performed research and wrote the first draft. FH and LLX collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. HQ is the guarantor.
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Th17 cells in acute rejection after liver transplant
Funding: None. Ethical approval: This study was approved by the institutional review board of the hospital (2007-021). Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
References 1 Banff schema for grading liver allograft rejection: an international consensus document. Hepatology 1997;25:658-663. 2 Piccotti JR, Chan SY, VanBuskirk AM, Eichwald EJ, Bishop DK. Are Th2 helper T lymphocytes beneficial, deleterious, or irrelevant in promoting allograft survival? Transplantation 1997;63:619-624. 3 Karczewski J, Karczewski M, Glyda M, Wiktorowicz K. Role of TH1/TH2 cytokines in kidney allograft rejection. Transplant Proc 2008;40:3390-3392. 4 Zhang F, Meng G, Strober W. Interactions among the transcription factors Runx1, RORgammat and Foxp3 regulate the differentiation of interleukin 17-producing T cells. Nat Immunol 2008;9:1297-1306. 5 Awasthi A, Kuchroo VK. Th17 cells: from precursors to players in inflammation and infection. Int Immunol 2009;21: 489-498. 6 Figueroa-Vega N, Alfonso-Pérez M, Benedicto I, SánchezMadrid F, González-Amaro R, Marazuela M. Increased circulating pro-inflammatory cytokines and Th17 lymphocytes in Hashimoto's thyroiditis. J Clin Endocrinol Metab 2010; 95:953-962. 7 Mesquita Jr D, Cruvinel WM, Camara NO, Kállas EG, Andrade LE. Autoimmune diseases in the TH17 era. Braz J Med Biol Res 2009;42:476-486. 8 Shilling RA, Wilkes DS. Role of Th17 cells and IL-17 in lung transplant rejection. Semin Immunopathol 2011;33:129-134. 9 Loverre A, Tataranni T, Castellano G, Divella C, Battaglia M, Ditonno P, et al. IL-17 expression by tubular epithelial cells in renal transplant recipients with acute antibody-mediated rejection. Am J Transplant 2011;11:1248-1259. 10 Min SI, Ha J, Park CG, Won JK, Park YJ, Min SK, et al. Sequential evolution of IL-17 responses in the early period of allograft rejection. Exp Mol Med 2009;41:707-716. 11 Singh SP, Zhang HH, Foley JF, Hedrick MN, Farber JM. Human T cells that are able to produce IL-17 express the chemokine receptor CCR6. J Immunol 2008;180:214-221. 12 Chadha R, Heidt S, Jones ND, Wood KJ. Th17: contributors to allograft rejection and a barrier to the induction of transplantation tolerance? Transplantation 2011;91:939-945. 13 Benghiat FS, Charbonnier LM, Vokaer B, De Wilde V,
Le Moine A. Interleukin 17-producing T helper cells in alloimmunity. Transplant Rev (Orlando) 2009;23:11-18. 14 Gorbacheva V, Fan R, Li X, Valujskikh A. Interleukin-17 promotes early allograft inflammation. Am J Pathol 2010;177: 1265-1273. 15 Fábrega E, López-Hoyos M, San Segundo D, Casafont F, PonsRomero F. Changes in the serum levels of interleukin-17/ interleukin-23 during acute rejection in liver transplantation. Liver Transpl 2009;15:629-633. 16 Yuan X, Paez-Cortez J, Schmitt-Knosalla I, D'Addio F, Mfarrej B, Donnarumma M, et al. A novel role of CD4 Th17 cells in mediating cardiac allograft rejection and vasculopathy. J Exp Med 2008;205:3133-3144. 17 Wang S, Li J, Xie A, Wang G, Xia N, Ye P, et al. Dynamic changes in Th1, Th17, and FoxP3+ T cells in patients with acute cellular rejection after cardiac transplantation. Clin Transplant 2011;25:E177-186. 18 Vanaudenaerde BM, Dupont LJ, Wuyts WA, Verbeken EK, Meyts I, Bullens DM, et al. The role of interleukin-17 during acute rejection after lung transplantation. Eur Respir J 2006; 27:779-787. 19 Platz KP, Mueller AR, Rossaint R, Steinmüller T, Lemmens HP, Lobeck H, et al. Cytokine pattern during rejection and infection after liver transplantation--improvements in postoperative monitoring? Transplantation 1996;62:1441-1450. 20 Li J, Lai X, Liao W, He Y, Liu Y, Gong J. The dynamic changes of Th17/Treg cytokines in rat liver transplant rejection and tolerance. Int Immunopharmacol 2011;11:962-967. 21 Afzali B, Lombardi G, Lechler RI, Lord GM. The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease. Clin Exp Immunol 2007;148:32-46. 22 Chen H, Wang W, Xie H, Xu X, Wu J, Jiang Z, et al. A pathogenic role of IL- 17 at the early stage of corneal allograft rejection. Transpl Immunol 2009;21:155-161. 23 Liu Z, Yuan X, Luo Y, He Y, Jiang Y, Chen ZK, et al. Evaluating the effects of immunosuppressants on human immunity using cytokine profiles of whole blood. Cytokine 2009;45:141-147. 24 Abadja F, Videcoq C, Alamartine E, Berthoux F, Mariat C. Differential effect of cyclosporine and mycophenolic acid on the human regulatory T cells and TH-17 cells balance. Transplant Proc 2009;41:3367-3370. 25 Hester J, Mills N, Shankar S, Carvalho-Gaspar M, Friend P, Wood KJ. Th17 cells in alemtuzumab-treated patients: the effect of long-term maintenance immunosuppressive therapy. Transplantation 2011;91:744-750. Received November 5, 2011 Accepted after revision March 30, 2012
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Increase of peripheral Th17 lymphocytes during acute cellular rejection in liver transplant recipients Hua Fan, Li-Xin Li, Dong-Dong Han, Jian-Tao Kou, Ping Li and Qiang He Beijing, China
BACKGROUND: Although many human inflammatory and autoimmune diseases were previously considered to be mediated by T helper type 1 (Th1) cells, the recently described Th17 cells play dominant roles in several of these diseases. We and others speculated that allograft rejection after organ transplantation may also involve Th17 cells. Episodes of acute rejection occur in 30% of liver transplants. This study aimed to determine the frequency of circulating Th17 cells in patients who had received liver transplants for benign end-stage liver disease and to identify any association between acute rejection episodes and levels of Th17 cells in the peripheral blood.
CONCLUSION: Circulating Th17 cells may be useful as a surrogate marker for predicting acute rejection in liver transplant recipients.
METHODS: A prospective study compared Th17 cells from 76 consecutive benign end-stage liver disease patients who had undergone orthotopic liver transplantation from 2007 to 2011 with those from 20 age-matched healthy individuals. Peripheral blood samples were collected at different time points within one year after transplant. Blood samples and liver biopsies were also collected at the diagnosis of acute rejection. Percentages of circulating CD4+IL-17+ cells were measured by flow cytometry. The transplant patients were classified into two groups: a rejection group consisting of 17 patients who had an episode of acute rejection, and a non-rejection group comprising the remaining 59 patients with no acute rejection episodes. Percentages of circulating Th17 cells were compared between the two groups and controls.
Introduction
(Hepatobiliary Pancreat Dis Int 2012;11:606-611) KEY WORDS: liver transplantation; Th17 cells; acute cellular rejection; transplant immunology
L
iver transplantation is a viable treatment option for end-stage liver diseases. Despite the armamentarium of immunosuppressive agents, the incidence of acute rejection in patients after liver transplant is approximately 30%, and acute rejection episodes significantly decrease graft and patient survival.[1] The etiology of acute rejection is complex and involves synergistic interactions between different types of T-helper (Th) subsets and other mononuclear cells. For example, production of interferon-γ (INF-γ) by Th1 cells can initiate acute allograft rejection by augmenting antiRESULTS: The levels of circulating CD4+IL-17+ T cells in the donor cellular responses and promoting cytotoxic T-cell rejection group were higher during acute rejection than those activity. Alternatively, Th2 cells secrete interleukin-4 in the non-rejection group (2.56±0.43% versus 1.79±0.44%, (IL-4), subsequently recruit eosinophils, and promote P<0.001). The frequency of CD4+IL-17+ cells in peripheral damage of the transplanted organ.[2, 3] blood was positively correlated with the rejection activity index In recent years, the central role of effector memory (r=0.79, P=0.0002). T cells that secrete IL-17, Th17 cells has been documented in pro-inflammatory processes and the development of autoimmune diseases.[4] Th17 cells recruit neutrophils and monocytes, and their chronic presence and Author Affiliations: Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China (Fan H, Li activation augments the concentrations and effects LX, Han DD, Kou JT, Li P and He Q) of proinflammatory cytokines.[5-7] Although a few Corresponding Author: Qiang He, Professor, Department of Hepatobiliary studies[8-10] have already highlighted the possibility of a Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China (Tel: 86-10-85231504; Fax: 86-10-85231503; Email: relationship between IL-17 and acute rejection in organ transplantation, the role of Th17 cells in acute rejection [email protected]) is unknown in patients with liver transplantation. © 2012, Hepatobiliary Pancreat Dis Int. All rights reserved. doi: 10.1016/S1499-3872(12)60231-8 To elucidate the potential role of Th17 cells in acute • Hepatobiliary Pancreat Dis Int，Vol 11，No 6 • December 15，2012 • www.hbpdint.com
Th17 cells in acute rejection after liver transplant
rejection after liver transplantation, we compared the frequency of Th17 cells in peripheral blood from 4 types of participants: i) liver transplant patients during acute rejection episodes (rejectors), ii) rejectors during quiescent periods, iii) liver transplant patients who did not experience an acute rejection (non-rejectors), and iv) healthy controls.
Methods Patients A cohort of 76 liver transplant recipients (22 females and 54 males; mean age 48±13 years, range 21-69) due to benign end-stage liver disease admitted to out- and in-patient care unit of our department from 2007 to 2011, and 20 age-matched healthy controls (mean age 46±14 years) were enrolled in the study. Patients with infections, hepatitis recurrence or malignancy were excluded. The institutional review board of the hospital (2007-021) approved the study protocol, and all patients and healthy individuals provided written informed consent. Liver transplant patients provided multiple blood samples (heparinized) during the first year after transplant, and at the time of acute rejection. Patients who suffered from acute rejection were treated with methylprednisolone (Solu-Medrol, 3×1000 mg intravenous (i.v.)). A diagnosis of acute rejection was based on clinic pathological findings (jaundice, fever, irritability) and biochemical analyses (elevated levels of alanine aminotransferase, aspartate aminotransferase, and/or direct bilirubin). Evidence for acute rejection was confirmed by histological examination of liver biopsies and was scored according to the Banff classification by an experienced pathologist blinded to this study.[1] A rejection activity index (RAI) of 4 or more defined rejection. The dose of post-transplant immunosuppressant agent was initially 10 mg/kg i.v. methylprednisolone tapered to 0.3 mg/kg by 7 days for a 3-week maintenance dose. Treatment of all patients with tacrolimus (Tac) or cyclosporin A (CsA) began on postoperative day 4. Plasma trough tacrolimus concentrations were maintained between 10 and 12 ng/ mL during the first month and then reduced to 8-10 ng/ mL. Plasma trough cyclosporin A concentrations were maintained between 350 and 450 ng/mL for the first month and then tapered to 250-350 ng/mL. Seventeen patients who experienced an acute rejection within one year were defined as the rejection group. The remaining 59 patients who had no acute rejection were designated the non-rejection group. The characteristics of rejection and non-rejection groups are listed in the Table.
Table. Characteristics of patients in the rejection and non-rejection groups Parameters
Rejection group (n=17) 47.2±11.3 12/5 25.2±9.0
Age (yr) Male/Female MELD Primary liver diseases HBV 12 HCV 1 HBV+HCV 2 Primary sclerosing cholangitis 1 Autoimmune liver diseases 1 Immunosuppressive 13/4 protocol (Tac/CsA) Time point of acute rejection 2.5±3.8 episodes post-transplant (mon) NS: not significant.
Non-rejection group (n=59) 50.2±10.7 42/17 27.1±10.5 47 5 4 2 1 48/11
P value NS NS NS NS NS NS NS NS NS
Flow cytometric analysis The expression levels of CD4 and IL-17A on lymphocytes were measured by flow cytometry. Peridin chlorophyll protein (PerCP) and allophycocyanin (APC)-labeled antibodies were used: CD4 (mouse IgG1, PerCP) and CCR6 (mouse IgG1, FITC) were from Becton Dickinson, and IL-17A (mouse IgG1, APC) was from eBioscience (San Diego, CA). Appropriate isotype controls (Becton Dickinson, USA) were used. Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood by density gradient centrifugation over Ficoll-Paque plus (GE Healthcare, Uppsala Sweden). PBMCs were collected at interphase and washed twice in phosphate-buffered saline. The cells were resuspended in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL, Grand Island, NY). The cells were cultured in the absence or presence of phorbol myristate acetate (100 ng/mL) and ionomycin (1 μg/mL) (both from Alexis Biochemicals, San Diego, CA) for 5 hours. Cytokine secretion was inhibited by brefeldin A (Sigma, St. Louis, MO). Then surface staining was performed with anti-CD4 and antiCCR6. The cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD PharMingen, USA). Finally, the samples were intracellularly stained with anti-IL-17A or an appropriate isotype control. CD4 +IL-17+ T cells were sorted by a fluorescence-activated cell sorter (FACS; CaliburTM, Becton Dickinson, USA) and the FACS data were analyzed with Flow JoTM 7.3.5 software (Treestar Software Inc., USA). Statistical analysis Statistical analysis and graphic studies were done
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with Windows SPSS Version 11.5 and GraphPad PrismTM 5.0. The Mann-Whitney U test (unpaired analyses) or the Wilcoxon's signed-rank test (paired analyses) was used to assess differences between the two groups or longitudinal analysis was used in each group, respectively. Continuous variables (e.g. clinical characteristics) were compared using the Mann-Whitney U test, and discrete variables were compared using the Chi-square test or Fisher's exact test. Correlation analysis was made between different parameters by using Spearman's rank order correlation coefficient. Two-tailed P values<0.05 were considered statistically significant.
T-cell population over time (P>0.05, Fig. 2B). An example of one liver transplant patient with acute rejection and stable liver function is shown in Fig. 3. The percentage of CD4+IL-17+ T cells increased during acute rejection in comparison to that during stable liver function (Fig. 3). Next, a possible association between the frequency of CD4+IL-17+ T cells in circulation and the RAI of the liver allograft biopsy was investigated. In the rejection group, the percentage of CD4+IL-17+ T cells was positively correlated with the RAI (r=0.79, P=0.0002, Fig. 4). In addition, CD4+IL-17+ T cells were further characterized regarding CCR6 expression in the same 10 rejectors, 23 non-rejectors and 13 healthy controls. CCR6 was used to define Th17 cells. CD4+IL-17+ T cells from patients with acute rejection and from non-rejectors Results mainly expressed CCR6; only a minor percentage was + + The percentage of CD4 IL-17 T cells was significantly deficient for CCR6 (Fig. 5).[11] No significant differences higher in patients with acute rejection than both those from patients with non-rejection and healthy controls were detected between rejectors, non-rejectors, and controls (percentage of CCR6-positive cells in (CD4+IL-17+ T cells in patients with acute rejection healthy + + CD4 IL-17 T cells: rejectors versus non-rejectors, 97.64± versus those with non-rejection: 2.56±0.43% vs 1.79± 0.44%, P<0.0001; with acute rejection versus healthy controls: 2.56±0.43% vs 1.57±0.43%, P<0.0001; with non-rejection versus healthy controls: 1.79±0.44% vs 1.57±0.43%, P=0.05; Fig. 1). To assess intra-individual variation of the CD4+IL-17+ T-cell population over time, longitudinal analysis was performed. The rejection group provided 17 paired peripheral blood samples from recipients with rejection and stable liver function. In addition, 20 liver transplant patients in the non-rejection group were analyzed. In the rejection group, the CD4 +IL-17+ T-cell population was higher during rejection than stable liver function (2.56±0.43% vs 1.50±0.25%, P<0.0001, Fig. 2A). Patients in the non-rejection group maintained a stable CD4+IL-17+
Fig. 1. Relative percentages of CD4+IL-17A+ T cells in the nonrejection group, the rejection group during acute rejection, and healthy control group. *: P<0.0001.
Fig. 2. Longitudinal analysis of IL-17A+ expression within the CD4+ T-cell population over time. A: Percentage of IL-17A+ cells over time in the rejection group. Expression was assessed during acute rejection and stable liver function. *: P<0.0001. B: Percentage of IL-17A+ cells within the CD4+ T-cell population from 20 patients in the non-rejection group. Expression was assessed at two different time points in stable liver function.
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Th17 cells in acute rejection after liver transplant
Fig. 3. Representative dot-plots of multichromatic flow cytometry analysis gated on the CD4+ T-cell population. Example from one liver transplant patient with acute rejection and one with stable liver function. The percentage of CD4 +IL-17+ T cells is given in the upper right quadrant. The number of IL-17A+ T cells was increased in the liver allograft recipients who were experiencing acute rejection. Appropriate isotype controls were used.
Fig. 5. Representative dot-plots of multichromatic flow cytometry analysis gated on the CD4+IL-17+ T-cell population. Example from one liver transplant patient with acute rejection and one with stable liver function. The quadrant gates were adjusted according to the given isotype controls. Most of the CD4 +IL-17+ T cells displayed CCR6 (97.12% and 97.94%).
Fig. 4. Correlation between percentage of CD4 +IL-17+ T cells and Banff rejection activity index of liver allograft biopsy in the patients who experienced acute rejection. Each symbol represents a single individual (r=0.79, P=0.0002).
1.04% vs 98.02±1.07%, P>0.05; rejectors versus healthy controls, 97.64±1.04% vs 97.14±1.31%, P>0.05; nonrejectors versus healthy controls, 98.02±1.07% vs 97.14± 1.31%, P>0.05). Furthermore, we looked for an association between the CD4+IL-17+ T cells in circulating peripheral blood and the blood concentration of Tac or CsA within one year after liver transplant and found no correlation (data
not shown). These results were found in both rejection and non-rejection recipients. Since only four patients in the rejection group were treated with CsA, the statistical power was insufficient to expect a significant difference between subgroups.
Discussion In our study, IL-17 production by polyclonallystimulated PBMCs and activated CD4+IL-17+ T cells was markedly elevated in liver transplant recipients during the acute rejection period compared with that in those without acute rejection and healthy controls. Furthermore, circulating CD4+IL-17+ T cells were positively correlated with the grade of acute rejection.
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This discrepancy was not due to differences in clinical characteristics because they were comparable between the two groups. This finding suggested that the increase in circulating Th17 cells precedes acute rejection in liver transplant recipients and may become a surrogate marker for acute rejection with further testing. The Th1 cell is thought to be responsible for allograft rejection, but many studies investigating the role of Th17 cells challenge this dogma.[4, 12] When Th1- or Th2mediated acute rejection is neutralized in a mouse heart transplant model, acute rejection caused by heightened neutrophil responses still occurs.[13] More recently, IL-17 deficiency in mice delays recruitment of Th1-mediated cells into cardiac allografts, and may reduce the early infiltration of macrophages and neutrophils. [14] But a study demonstrated higher IL-17 concentrations during acute rejection in human liver transplant recipients, [15] but further studies are needed to demonstrate causality. Furthermore, the roles of the different IL-17 producing cell types in the rejection milieu need clarification. Studies on Th17 cells rather than the cytokine IL-17 need to be performed to decipher the role of these cells in clinical acute rejection. The contribution of Th17 cells in clinical acute rejection has not yet been widely established, but animal studies showed that they induce tissue damage by stimulating inflammatory cells to release proinflammatory cytokines and accelerate the development of severe allograft vasculopathy. [16] In our study, Th17 cell levels after nonspecific stimulation were significantly higher in patients who experienced an acute rejection episode than in non-rejectors and healthy controls. Our results are consistent with the significantly higher levels of Th17 cells in patients with acute rejection after cardiac transplantation.[17] Vanaudenaerde et al[18] reported that the bronchoalveolar lavage fluid of patients rejecting lung transplants exhibit higher IL-17 protein and mRNA levels than transplant patients without rejection. The severity of rejection correlated with the numbers of lymphocytes and neutrophils present in the bronchoalveolar lavage. These findings are consistent with our results that the frequency of circulating Th17 cells was significantly positively correlated with the RAI. Despite our finding that peripheral Th17 cells were upregulated in patients with acute rejection and in patients with nonspecific stimulation, other specific antigens may promote expansion of the IL-17 axis in acute rejection. Further studies are needed to elucidate the processes. Although Th17 cells increased among T cells in circulating peripheral blood, the mechanisms are not fully understood. Our results showed that there are two
potential hypotheses to explain these findings. First, circulating Th17 cells may be up-regulated in liver transplant recipients who experience acute rejection in an IL-6 and IL-23 laden milieu. Previous research showed that IL-6 and IL-23, which are key factors in the differentiation of Th17 cells, are significantly increased in human liver transplant recipients during acute rejection.[15, 19] Moreover, an inflammatory environment can augment the potential conversion of Tregs into Th17 cells,[20, 21] thereby increasing their local concentration and that of IL-17 cytokine, which stimulates a positive feedback loop for proinflammatory pathways. The other hypothesis is that circulating Th17 cells may be recruited from other compartments, such as the secondary lymphoid organs. Chen and colleagues [22] reported that drained lymph nodes after allocorneal rejection in mice had a large number of Th17 cells. In our study, there was insufficient evidence to support the presence of Th17 cells in the local allograft tissue. Prospective studies are needed to further test these hypotheses. The effects of calcineurin inhibitors on differentiation, activation, and effector activity of the Th17 lineage during transplantation are limited and appear to be contradictory.[23, 24] A recent report shows that calcineurin inhibitors (CNIs) can lead to a weaker ability to dampen Th17 cells versus mycophenolic acid in renal transplant patients.[25] However, the impact of CNIs remains uncertain although we found no significant correlation between circulating Th17 cells and the dose or blood concentration of CNIs. Further investigation of a larger population is needed. Our data showed an increase of circulating Th17 cells at the time of rejection and the recovery of peripheral Th17 cell levels when graft rejection resolved. Accordingly, circulating Th17 cells may be used as a surrogate marker for predicting acute rejection during the post-transplant period and assessing the immune status of liver transplant recipients. In summary, in this study the levels of circulating Th17 cells increased in the peripheral blood of patients with acute rejection after liver transplantation and were associated with rejection grade. We consider that circulating Th17 cells in the peripheral blood may serve as a monitoring tool to predict acute rejection in liver transplant recipients. However, the role of Th17 cells in initiating and promoting acute rejection and their multiple interactions with additional cell types has not yet been fully defined. Contributors: HQ proposed the study. FH and HQ performed research and wrote the first draft. FH and LLX collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. HQ is the guarantor.
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Th17 cells in acute rejection after liver transplant
Funding: None. Ethical approval: This study was approved by the institutional review board of the hospital (2007-021). Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.
References 1 Banff schema for grading liver allograft rejection: an international consensus document. Hepatology 1997;25:658-663. 2 Piccotti JR, Chan SY, VanBuskirk AM, Eichwald EJ, Bishop DK. Are Th2 helper T lymphocytes beneficial, deleterious, or irrelevant in promoting allograft survival? Transplantation 1997;63:619-624. 3 Karczewski J, Karczewski M, Glyda M, Wiktorowicz K. Role of TH1/TH2 cytokines in kidney allograft rejection. Transplant Proc 2008;40:3390-3392. 4 Zhang F, Meng G, Strober W. Interactions among the transcription factors Runx1, RORgammat and Foxp3 regulate the differentiation of interleukin 17-producing T cells. Nat Immunol 2008;9:1297-1306. 5 Awasthi A, Kuchroo VK. Th17 cells: from precursors to players in inflammation and infection. Int Immunol 2009;21: 489-498. 6 Figueroa-Vega N, Alfonso-Pérez M, Benedicto I, SánchezMadrid F, González-Amaro R, Marazuela M. Increased circulating pro-inflammatory cytokines and Th17 lymphocytes in Hashimoto's thyroiditis. J Clin Endocrinol Metab 2010; 95:953-962. 7 Mesquita Jr D, Cruvinel WM, Camara NO, Kállas EG, Andrade LE. Autoimmune diseases in the TH17 era. Braz J Med Biol Res 2009;42:476-486. 8 Shilling RA, Wilkes DS. Role of Th17 cells and IL-17 in lung transplant rejection. Semin Immunopathol 2011;33:129-134. 9 Loverre A, Tataranni T, Castellano G, Divella C, Battaglia M, Ditonno P, et al. IL-17 expression by tubular epithelial cells in renal transplant recipients with acute antibody-mediated rejection. Am J Transplant 2011;11:1248-1259. 10 Min SI, Ha J, Park CG, Won JK, Park YJ, Min SK, et al. Sequential evolution of IL-17 responses in the early period of allograft rejection. Exp Mol Med 2009;41:707-716. 11 Singh SP, Zhang HH, Foley JF, Hedrick MN, Farber JM. Human T cells that are able to produce IL-17 express the chemokine receptor CCR6. J Immunol 2008;180:214-221. 12 Chadha R, Heidt S, Jones ND, Wood KJ. Th17: contributors to allograft rejection and a barrier to the induction of transplantation tolerance? Transplantation 2011;91:939-945. 13 Benghiat FS, Charbonnier LM, Vokaer B, De Wilde V,
Le Moine A. Interleukin 17-producing T helper cells in alloimmunity. Transplant Rev (Orlando) 2009;23:11-18. 14 Gorbacheva V, Fan R, Li X, Valujskikh A. Interleukin-17 promotes early allograft inflammation. Am J Pathol 2010;177: 1265-1273. 15 Fábrega E, López-Hoyos M, San Segundo D, Casafont F, PonsRomero F. Changes in the serum levels of interleukin-17/ interleukin-23 during acute rejection in liver transplantation. Liver Transpl 2009;15:629-633. 16 Yuan X, Paez-Cortez J, Schmitt-Knosalla I, D'Addio F, Mfarrej B, Donnarumma M, et al. A novel role of CD4 Th17 cells in mediating cardiac allograft rejection and vasculopathy. J Exp Med 2008;205:3133-3144. 17 Wang S, Li J, Xie A, Wang G, Xia N, Ye P, et al. Dynamic changes in Th1, Th17, and FoxP3+ T cells in patients with acute cellular rejection after cardiac transplantation. Clin Transplant 2011;25:E177-186. 18 Vanaudenaerde BM, Dupont LJ, Wuyts WA, Verbeken EK, Meyts I, Bullens DM, et al. The role of interleukin-17 during acute rejection after lung transplantation. Eur Respir J 2006; 27:779-787. 19 Platz KP, Mueller AR, Rossaint R, Steinmüller T, Lemmens HP, Lobeck H, et al. Cytokine pattern during rejection and infection after liver transplantation--improvements in postoperative monitoring? Transplantation 1996;62:1441-1450. 20 Li J, Lai X, Liao W, He Y, Liu Y, Gong J. The dynamic changes of Th17/Treg cytokines in rat liver transplant rejection and tolerance. Int Immunopharmacol 2011;11:962-967. 21 Afzali B, Lombardi G, Lechler RI, Lord GM. The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease. Clin Exp Immunol 2007;148:32-46. 22 Chen H, Wang W, Xie H, Xu X, Wu J, Jiang Z, et al. A pathogenic role of IL- 17 at the early stage of corneal allograft rejection. Transpl Immunol 2009;21:155-161. 23 Liu Z, Yuan X, Luo Y, He Y, Jiang Y, Chen ZK, et al. Evaluating the effects of immunosuppressants on human immunity using cytokine profiles of whole blood. Cytokine 2009;45:141-147. 24 Abadja F, Videcoq C, Alamartine E, Berthoux F, Mariat C. Differential effect of cyclosporine and mycophenolic acid on the human regulatory T cells and TH-17 cells balance. Transplant Proc 2009;41:3367-3370. 25 Hester J, Mills N, Shankar S, Carvalho-Gaspar M, Friend P, Wood KJ. Th17 cells in alemtuzumab-treated patients: the effect of long-term maintenance immunosuppressive therapy. Transplantation 2011;91:744-750. Received November 5, 2011 Accepted after revision March 30, 2012
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