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Galectin-9 in Combination With EX-527 Prolongs the Survival of Cardiac Allografts in Mice After Cardiac Transplantation
Galectin-9 in Combination With EX-527 Prolongs the Survival of Cardiac Allografts in Mice After Cardiac Transplantation Y.-f. Taoa, F. Linb, X.-y. Yanc, X.-g. Gaod, F. Tengd, Z.-r. Fud, and Z.-x. Wanga,* a Department of General Surgery and Liver Transplant Center, Huashan Hospital, Fudan University, Shanghai, China; bDepartment of General Surgery, Taizhou First People’s Hospital, Taizhou, Zhejiang, China; cPeking University Clinical Research Institute, Health Science Center, Peking University, Beijing, China; and dDivision of Liver Transplantation, Organ Transplant Center, Changzheng Hospital, Second Military Medical University, Shanghai, China
ABSTRACT Galectin-9 (Gal-9), a member of the galectin family, has a variety of biologic activities. However, its role in allografts is not fully clarified yet. The relationship between interleukin-17 (IL-17) and Gal-9 and the role of Gal-9 in TH17-cell differentiation also remain unclear. We built a murine cardiac transplantation model, which we treated with Gal-9 and/or EX-527, a specific Sirtuin-1 inhibitor. Afterwards, flow-cytometric analysis and reverse-transcription polymerase chain reaction were used to detect the number of TH17 cells and the expression of key factors involved in the differentiation of TH17 cells; in addition, the survival times of cardiac transplanted mice in different groups were recorded. The levels of circulating TH17 cells were found to increase in the peripheral blood of mice that exhibited acute rejection (AR) after heart transplantation, which was determined to be correlated with the rejection grade. Gal-9 or EX-527 can inhibit the activation and differentiation of TH17 cells and effectively suppress TH17-cellemediated AR. These data provide new evidence for the potential regulatory effects of Gal-9 in alloimmune responses in a murine model of heart transplantation, and suggest the combined use of galectin-9 and EX-527 may be a powerful method to induce tolerance of fully mismatched murine cardiac allografts.
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EART transplantation is the treatment of choice for end-stage heart diseases, with the goal of improving survival and life quality of patients [1e5]. The incidence of acute rejection (AR) in patients after heart transplantation is w30%e40%, and AR episodes significantly decrease graft and patient survivals [6e8]. AR results from complicated synergistic interactions between different types of T-helper subsets and other mononuclear cells that are thought to be involved in the process [9e12]. For example, it has been proposed that interferon-g (INF-g) produced by TH1 cells can initiate acute allograft rejection via IFN-geinduced activation of macrophage function and the induction of MHC class II antigen expression to favor T-cell allosensitization [6]. TH2 cells secrete interleukin (IL) 4, IL-5, IL-10, and IL-13, provide help to B-cell function, and play important roles in IgG switching, IgE production, and eosinophilic inflammation [6]. Alternatively, TH2 cells secrete IL-4 to subsequently recruit eosinophils and promote damage of the transplanted organ [6,13].
In recent years, the central role of TH17 cells, which secrete IL-17, has been documented in proinflammatory processes and the occurrence and development of various heart diseases. As a newly discovered subset of CD4þ T cells, TH17 cells are distant from other T-helper cells not only in gene expression and regulation, but also regarding their biologic function, owing to the production of IL-17 and IL-17F, which are generally thought to be proinflammatory and to play an important role in host defense against The 1st 2 authors contributed equally to this work. Funding: National Natural Science Foundation of China (81001327), International Science and Technology Cooperation Foundation of Shanghai (11410708700), and Natural Science Foundation of Shanghai (11ZR1448100). *Address correspondence to Zheng-xin Wang, MD, Professor, Department of General Surgery and Liver Transplant Center, Huashan Hospital, Fudan University, Shanghai 200040, China. E-mail: [email protected]
ª 2015 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/15 http://dx.doi.org/10.1016/j.transproceed.2015.04.091
Transplantation Proceedings, 47, 2003e2009 (2015)
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Fig 1. The level of TH17 cells is elevated but the galectin-9 (Gal-9) level is decreased in murine cardiac allografts. (A) The percentage of CD4þIL-17þ T cells in circulation of cardiac-transplanted mice with (left) normal heart function and (right) acute rejection (AR) were determined by means of flow cytometry. (B) In the cardiac-transplanted mice with AR, the correlation of CD4þIL-17þ T cells and rejection activity index was analyzed by means of Spearman rank correlation assay, with the r and P values indicated. (C) Gal-9 expression in peripheral blood mononuclear cells of cardiac-transplanted mice with normal heart function and AR were determined by means of quantitative reverse-transcription polymerase chain reaction. Data are shown as mean SD (n ¼ 10) or as 1 representative experiment. Similar results were obtained in 3 independent experiments. Abbreviation: IL, interleukin.
infection through the recruitment of neutrophils and macrophages to infected tissues [14]. Although a few studies have highlighted the possibility of a relationship between IL-17 and AR in organ transplantation [15], the role of TH17 cells in AR of patients after cardiac transplantation remains unclear. Galectin-9 (Gal-9) is a b-galactosideebinding protein consisting of 2 carbohydrate recognition domains connected by a linker peptide. The galectin family members are thought to be critical for the regulation of immune cell homeostasis and inflammation. Earlier studies have demonstrated that Gal-9 participates in a variety of biologic processes, such as cell adhesion, activation, chemoattraction, and apoptosis in different types of cells, including T cells, B cells, macrophages, and mast cells [16e22]. In recent years, studies of Gal-9 have revealed that it regulates the immune response by reducing the amount of TH17 cells and TH1 cells that secrete proinflammatory IL-17 and by increasing the levels of
antiinflammatory Foxp3þ regulatory T cells (Treg), which are critical in the pathogenesis of various autoimmune diseases, both in vitro and in vivo [22e26]. A recent study also has demonstrated that the Tim-3/Gal-9 pathway plays an important role in the termination of the TH17 immune response [27,28]. These findings indicate that Gal-9 may function as an important immune modulator under physiologic and pathologic conditions. In our clinical studies of cardiac transplantation, we observed that episodes of AR occur in w30%e40% of cardiac transplant patients. In addition, the levels of serum IL-17 are higher in heart transplant patients, whereas the levels of Gal-9 are markedly decreased. Based on these clinical data, we speculated that TH17 cells may be involved in AR after organ transplantation and that Gal-9 may play an important role in the regulation of TH17 cells in allogeneic heart transplant patients. However, the natural form of Gal-9 is easily inactivated by proteases owing to the labile
GALECTIN-9 IN COMBINATION WITH WITH EX-527
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Heart Transplantation and Gal-9 Treatment
Fig 2. Administration of Gal-9 alone or combine with EX-527 prolongs the survival of murine cardiac allografts. Shown are Kaplan-Meier survival curves of cardiac-transplanted mice with or without Gal-9 administration as indicated. Data were analyzed by means of log-rank test, with the P value indicated. Abbreviations: Gal-9, galectin-9; Ctrl, control; DMSO, dimethylsulfoxide; HT, heart transplantation.
nature of the linker peptide. Therefore, we elucidated the therapeutic potential of the stable form of Gal-9 and compared it with EX-527, a specific Sirtuin-1 (Sirt1) inhibitor for which it has been reported in a murine model of cardiac transplantation that deletion and inhibition of Sirt1 can inhibit T-cell function and prolong allograft survival in vivo [29]. Through the analysis of the production of TH17 cells, we evaluated the inhibitory effects of Gal-9 compared with and together with EX-527 on AR and elucidated possible mechanisms of its action. MATERIALS AND METHODS Animals and Ethics Statement BALB/c and C57BL/6J mice were purchased from the Joint Ventures Sipper BK Experimental Animal Company (Shanghai, China). All experiments with the use of animals were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of the Second Military Medical University, Shanghai, China.
Preparation of Stable-Form Human Galectin-9 Stable-form human Gal-9 was prepared as described previously [30]. Briefly, Gal-9 was cloned into the pET expression system (Novagen, Madison, Wisconsin) and transfected into E. coli BL21 (DE3) cells. The cells were grown at 30 C in 2YT broth containing 100 mg/mL ampicillin, induced at optical density 600 nm with the use of 500 mmol/L isopropylthio-beta-D-galactoside (IPTG) for 3 hours, harvested by means of centrifugation at 10,000g, lysed with a cell disrupter, and centrifuged at 20,000g for 30 minutes. The final purification of pET-hGal-9 was achieved by means of chromatography with the use of a lactose-agarose column (Seika-gaku Kogyo, Tokyo, Japan), dialysis against phosphate-buffered saline solution (PBS), and the elimination of endotoxin with the use of Cellufine ETclean-L (Chisso, Tokyo, Japan). The recombinant protein was >95% pure, as confirmed with the use of sodium dodecyl sulfatee polyacrylamide gel electrophoresis. The protein concentration was determined by means of the Bradford assay with the use of bovine serum albumin as the standard. The purified recombinant Gal-9 proteins were used in the subsequent studies.
Vascularized heart grafts were transplanted with the use of a microsurgical technique as previously described [31]. Cardiac grafts obtained from BALB/c or C57BL/6 mice were transplanted into C57BL/6 mice. Rejection was defined as complete cessation of cardiac contractility as determined through direct visualization. Recombinant stable-form Gal-9 (5 mg/kg/d) was intraperitoneally administered on postoperative days (PODs) 0e3, 5, 7, 9, 12, and 14, and solvent PBS was used as control. The study end point was defined as the complete cessation of the cardiac beat. The survival rates of the cardiac grafts were monitored by means of palpation by 2 independent observers without knowledge of the treatment protocols and were confirmed by means of histologic analysis.
Flow-Cytometric Analysis A single-cell suspension of the spleen was prepared with the use of a manual tissue homogenizer. The cells were washed with RPMI 1640 medium (Sigma, St Louis, Missouri) containing 2 mmol/L HEPES and 1% bovine serum albumin and passed through a 70-mm nylon cell strainer. The expression levels of CD4 and IL-17A on the lymphocytes were measured by means of flow cytometry. The following phycoerythrin (PE)e and fluorescein isothiocyanate (FITC)elabeled antibodies were used: IL-23 receptor (IL-23R; mouse IgG1, FITC) and CCR6 (mouse IgG1, PE), which were purchased from Becton Dickinson, and IL-17A (mouse IgG1, PE), which was obtained from eBioscience (San Diego, California). Appropriate PE- and FITC-labeled isotype control subjects (Becton Dickinson) were used. Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood by means of density-gradient centrifugation over Ficoll-Paque plus (GE Healthcare, Uppsala, Sweden). The PBMCs were collected at interphase and washed twice in PBS. The cells were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL, Grand Island, New York). The cells were cultured in the absence or presence of phorbol myristate acetate (100 ng/mL) and ionomycin (1 mg/mL; both from Alexis Biochemicals, San Diego, California) for 5 hours. Cytokine secretion was inhibited by brefeldin A (Sigma). Surface staining was then performed with anti-CD4 and anti-CCR6. The cells were fixed and permeabilized with the use of a Cytofix/Cytoperm kit (BD Pharmingen), and the samples were then intracellularly stained with antieIL-17A or an appropriate isotype control. The CD4þIL-17þ T cells were sorted with the use of a fluorescence-activated cell sorter (FACS; Calibur; Becton Dickinson), and the FACS data were analyzed with the use of the Flowjo software (Treestar Software).
Isolation and Culture of CD4þIL-17þ T Cells The murine single-cell suspension of the spleen or draining lymph nodes was prepared with the use of a manual tissue homogenizer and passed through a 70-mm cell strainer. The lymphocytes were inoculated at a density of 1 106/mL in a 24-well plate, and PMA (50 ng/mL), ionomycin (1 mg/mL), and monensin (1 mL) were added to each well. The plate was then kept at 37 C with 5% CO2 for 5 hours, and the expression levels of CD4 and IL-17A in the lymphocytes were measured by means of flow cytometry. PBMCs were obtained as already described. The cells were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum. The cells were cultured in the absence or presence of transforming growth factor (TGF) b, IL-6, antieIL-4 (1 mg/mL), and antieINF-g (1 mg/mL) for 3 hours. Cell-surface staining was then performed with anti-CD4 and anti-CCR6. The cells were fixed and permeabilized with the use of a Cytofix/Cytoperm kit (BD Pharmingen), and the samples were intracellularly stained with
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GALECTIN-9 IN COMBINATION WITH WITH EX-527 antieIL-17A or an appropriate isotype control. The CD4þIL-17þ T cells were sorted with the use of FACS, and the FACS data were analyzed as described above.
Quantitative Reverse-transcription Polymerase Chain Reaction The mRNA levels were evaluated with the use of Sybr Green Iebased real-time reverse-transcription polymerase chain reaction (RT-PCR), which was performed with the use of an ABI Prism 7000 sequence detector (Applied Biosystems, Foster City, California). The total RNA from the pelleted cells was isolated with the use of the RNeasy Plus mini kit (Qiagen). The 1st-strand cDNA synthesis was performed by means of random priming with a high-capacity cDNA synthesis kit (Applied Biosystems) in the presence of Superase RNase inhibitor (Ambion). The cDNA was then used as the template in the PCR reactions. The reactions were performed with the use of the Fast Universal PCR Mastermix (Applied Biosystems) according to the manufacturer’s instruction and thermocycled in a 20-mL reaction volume in 96-well plates. The Ct values were calculated according to automatically determined threshold values with the use of the SDS software (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were used as an internal standard for calibration. The primer sequences used in the RT-PCR analysis were as follows: GAPDH: 50 -AGG TCG GTG TGA ACG GAT TTG-30 (forward), 50 TGT AGA CCA TGT AGT TGA GGT CA-30 (reverse); CCR6: 50 AAT GAA CGA TCC TGC CAG A-30 (forward), 50 -GGA GAA GCC TGA GGA CTT GT-30 (reverse); IL-23R: 50 -TCA TCC CAG AAC ACA AGC CT-30 (forward), 50 -ATT GCT GAG ATG GCT TCC CT30 (reverse); IL-17A: 50 -ATC AGG ACG CGC AAA CAT G-30 (forward), 50 -TGA TCG CTG CTG CCT TCA C-30 (reverse); IL-23; 50 -GGG AGA TGA AGA GAC TAC AAA TGA TGT-30 (forward), 50 -ATC CTT TGC AAG CAG AAC TGA CT-30 (reverse); and Gal-9: 50 -GTT TGC AAC ACG AAG CAG AA-30 (forward), 50 -GGC AGG ACG AAA GTT CTG AG-30 (reverse).
Statistical Analysis Data are presented as mean SD. Statistical comparisons between groups were analyzed by means of Student t test, and 2-tailed P < .05 was considered to indicate statistical significance. For graft survival, Kaplan-Meier graphs were constructed and analyzed by means of log-rank test. The comparison among groups was performed by means of 1-way analysis of variance. The correlation analysis was performed with the use of Spearman rank correlation coefficient assay. Data were analyzed with the use of Graphpad Prism 5.0 software.
RESULTS The Level of TH17 Cells Is Elevated Whereas the Gal-9 Level Is Decreased in Murine Cardiac Allografts
To investigate the roles of TH17 cells and Gal-9 in AR of cardiac transplantation, we detected the percentage of TH17
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cells and Gal-9 expression in circulation of murine cardiac transplantation model. As shown in Fig 1A, the percentage of CD4þIL-17þ T cells significantly increased in AR recipients with abnormal heart function compared with mice with normal heart function. Furthermore, the percentage of CD4þIL-17þ T cells in circulation was positively correlated with the rejection activity index in the heart allograft biopsy (Fig 1B), indicating that the increase of TH17 cells contributed to AR progression. Additionally, Gal-9 mRNA expression was significantly decreased in AR of heart allograft recipients (Fig 1C). These data suggest that the decrease of Gal-9 and the increase of TH17 may contribute to AR progression.
Administration of Gal-9 Alone and in Combination With EX-527 Prolongs the Survival of Murine Cardiac Allografts
To explore the impact of recombinant Gal-9 on allogeneic cardiac transplantation, a murine cardiac transplant model was used in this study. Four groups of mice models were used, treated with or without Gal-9, EX-527, and their combination after transplantation. As shown in Fig 2, administration of recombinant Gal-9 or EX-527 alone or in combination prolonged the allograft median survival time, and the mice treated with Gal-9 and EX-527 together survived longer than the other groups.
Gal-9 Alone and in Combination With EX-527 Inhibits TH17 Cell Differentiation in Vitro
The administration of Gal-9 and EX-527 in our study was demonstrated to prolong the survival time of cardiactransplanted mice, so we further evaluated the effect of Gal-9 on TH17 cells and cardiac AR in a murine model to investigate the mechanisms underlying the Gal-9emediated inhibition of AR. CD4þIL-17þ T cells from the spleens of recipient mice on POD 7 were isolated and cultured with different treatments. As shown in Fig 3A, Gal-9 and EX-527 administration significantly suppressed IL-17, C-C chemokine receptor (CCR) 6 and IL-23R expression. We also tested the mRNA levels of CCR6, IL-23R, IL-17, and IL-23 in the CD4þIL-17þ T cells with the use of real-time PCR, and the results confirmed that TH17 cell differentiation was suppressed with Gal-9 and EX-527 treatment (Fig 3B). Moreover, the suppression effect of the group treated with Gal-9 and EX-527 together was strongest. Therefore, it can be concluded that Gal-9 and EX-527 administration alone or together inhibits TH17 cell differentiation in vitro.
Fig 3. Gal-9 alone or combined with EX-527 inhibits TH17-cell differentiation in vitro. (A) CD4þIL-17þ T cells were isolated from the spleens of the recipient mice on day 7 after transplantation and cultured with Gal-9 alone or combined with EX-527. CCR6þ, IL-23Rþ, and IL-17þ cells in the CD4þIL-17þ T-cell population were analyzed with the use of flow cytometry. (B) Expressions of CCR6, IL-23R, IL-17, and IL-23 were determined with the use of quantitative reverse-transcription polymerase chain reaction. Data are shown as mean SD (n ¼ 10) or 1 representative experiment. Similar results were obtained in 3 independent experiments. Abbreviations: CCR6, C-C chemokine receptor 6; IL-17, interleukin-17; IL-23, interleukin-23; IL-23R, interleukin-23 receptor; other abbreviations as in Fig 2.
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Fig 4. Gal-9 inhibits the number of TH17 cells in vivo alone or together with EX-527. Percentage of CD4þIL-17þ T cells in circulation of the cardiac-recipient mice on day 7 after transplantation was analyzed with the use of flow cytometry. Data are shown as mean SD (n ¼ 10) or 1 representative experiment. Similar results were obtained in 3 independent experiments. Abbreviations as in Figs 2 and 3. NS, no statistically significant difference of pairwise comparisons; *P < .05.
Gal-9 Decreased the Number of TH17 Cells in Vivo Alone or Together With EX-527
To examine the effect of Gal-9 or the combination of EX-527 and Gal-9 administration on TH17 cell number in the murine cardiac transplantation model, we detected the percentage of CD4þIL-17þ T cells in circulation of the recipient mice on POD 7. As shown in Fig 4, Gal-9 and EX-527 administration or the combination of EX-527 and Gal-9 administration in vivo significantly inhibited TH17 cell population in the heart transplant recipient mice.
DISCUSSION
In this study, we observed that activated CD4þIL17þ T cells were significantly elevated in cardiac transplant mice during the period of AR, and the levels of CD4þIL17þ T cells were positively correlated with AR grade. These data suggest that an increased level of circulating TH17 cells may be a novel marker for AR. Earlier studies demonstrated that TH1 cells are responsible for AR, and some recent studies have suggested that TH17 cells may also be involved in the process [32e35]. The contribution of TH17 cells to clinical AR has not yet been well documented, but animal studies have shown that TH17 cells induce tissue damage by stimulating inflammatory cells to release proinflammatory cytokines and accelerate the development of allograft vasculopathy [33]. Based on previous and the present research, we speculate that TH17 cells are involved in the proinflammatory process of AR. Moreover, a significantly decreased level of Gal-9 was expressed in cardiac transplant mice during the period of AR in the present study. And the administration of Gal-9
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and EX-527 in our study was demonstrated to prolong the survive time of cardiac-transplanted mice (Fig 2). Therefore we further evaluated the effect of Gal-9 on TH17 cells and cardiac AR in this murine model. As reported in earlier reports [36,37], TH17 cells were activated during AR of cardiac transplantation. However, the underlying mechanisms responsible for the increased proportion of TH17 cells within the T-cell population are still not well understood. Earlier studies discovered that IL-17, which is primarily produced by TH17 cells, takes part in variety of inflammatory reactions and disease development, including rejection in organ transplantation [15]. The expression of IL-23 and its receptor (IL-23R) was reported to increase the secretion of IL-17 by TH17 cells [38] as well as to induce the expression of CCR6, which is the surface receptor of TH17 cells, thus promoting the formation and development of inflammatory autoimmune disease [39]. In the present study, the key factors involved in the differentiation of TH17 cells, including CCR6, IL-17, IL-23, and IL-23R, were significantly decreased in heart transplant recipient mice treated with Gal-9 and EX-527. Additionally, the systemic administration of a stable form of human Gal-9 clearly ameliorates AR in the TH17-mediated cardiac transplantation model. Based on these data, we hypothesize that the inflammatory environment promotes the conversion of Tregs into TH17 cells, which results in an increase in the local level of IL-17 cytokines and thereby stimulates proinflammatory pathways. Another possibility is that the TH17 cells may be recruited from other organs. And further studies are needed to confirm these hypotheses. The present study also showed the similar inhibition effect of administration of Gal-9 and EX-527 in suppressing activation and number of TH17 cells and prolongation of the survival time of cardiac-transplanted mice. Sirtuins are NADþ-dependent class III histone/protein deacetylase enzymes that are highly conserved across eukaryotic species [31]. Sirt1 is a key factor in the caloric restriction response and life span control, and it is a therapeutic target in various cancers [30,32]. The combined use of Gal-9 and EX-527 had a better inhibition effect on the proliferation of TH17 cells and better effect on the survival time of cardiac-transplanted mice, providing a more effective treatment of alloimmune responses, and more research is needed to figure out the underlying mechanism. In summary, the levels of circulating TH17 cells were found to increase in the peripheral blood of mice that exhibited AR after heart transplantation, and the levels were determined to be correlated with the rejection grade. The circulating TH17 cells in the peripheral blood may be used as a marker for the prediction of AR in heart transplant recipients. Gal-9 or EX527 can inhibit the activation and differentiation of TH17 cell and effectively suppress TH17 cellemediated AR. These data provide new evidence for the potential regulatory effects of Gal-9 in alloimmune responses in a murine model of heart transplantation and suggest that the combined use of Gal-9 and EX-527 may be a powerful method to induce tolerance of fully mismatched murine cardiac allografts.
GALECTIN-9 IN COMBINATION WITH WITH EX-527
ACKNOWLEDGMENTS The authors thank Prof Quan-xing Wang and Mrs Fang Liu for the preparation of stable-form human Gal-9 and Dr Shao-hua Song for expert technical assistance in operation of the animal experiments.
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ABSTRACT Galectin-9 (Gal-9), a member of the galectin family, has a variety of biologic activities. However, its role in allografts is not fully clarified yet. The relationship between interleukin-17 (IL-17) and Gal-9 and the role of Gal-9 in TH17-cell differentiation also remain unclear. We built a murine cardiac transplantation model, which we treated with Gal-9 and/or EX-527, a specific Sirtuin-1 inhibitor. Afterwards, flow-cytometric analysis and reverse-transcription polymerase chain reaction were used to detect the number of TH17 cells and the expression of key factors involved in the differentiation of TH17 cells; in addition, the survival times of cardiac transplanted mice in different groups were recorded. The levels of circulating TH17 cells were found to increase in the peripheral blood of mice that exhibited acute rejection (AR) after heart transplantation, which was determined to be correlated with the rejection grade. Gal-9 or EX-527 can inhibit the activation and differentiation of TH17 cells and effectively suppress TH17-cellemediated AR. These data provide new evidence for the potential regulatory effects of Gal-9 in alloimmune responses in a murine model of heart transplantation, and suggest the combined use of galectin-9 and EX-527 may be a powerful method to induce tolerance of fully mismatched murine cardiac allografts.
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EART transplantation is the treatment of choice for end-stage heart diseases, with the goal of improving survival and life quality of patients [1e5]. The incidence of acute rejection (AR) in patients after heart transplantation is w30%e40%, and AR episodes significantly decrease graft and patient survivals [6e8]. AR results from complicated synergistic interactions between different types of T-helper subsets and other mononuclear cells that are thought to be involved in the process [9e12]. For example, it has been proposed that interferon-g (INF-g) produced by TH1 cells can initiate acute allograft rejection via IFN-geinduced activation of macrophage function and the induction of MHC class II antigen expression to favor T-cell allosensitization [6]. TH2 cells secrete interleukin (IL) 4, IL-5, IL-10, and IL-13, provide help to B-cell function, and play important roles in IgG switching, IgE production, and eosinophilic inflammation [6]. Alternatively, TH2 cells secrete IL-4 to subsequently recruit eosinophils and promote damage of the transplanted organ [6,13].
In recent years, the central role of TH17 cells, which secrete IL-17, has been documented in proinflammatory processes and the occurrence and development of various heart diseases. As a newly discovered subset of CD4þ T cells, TH17 cells are distant from other T-helper cells not only in gene expression and regulation, but also regarding their biologic function, owing to the production of IL-17 and IL-17F, which are generally thought to be proinflammatory and to play an important role in host defense against The 1st 2 authors contributed equally to this work. Funding: National Natural Science Foundation of China (81001327), International Science and Technology Cooperation Foundation of Shanghai (11410708700), and Natural Science Foundation of Shanghai (11ZR1448100). *Address correspondence to Zheng-xin Wang, MD, Professor, Department of General Surgery and Liver Transplant Center, Huashan Hospital, Fudan University, Shanghai 200040, China. E-mail: [email protected]
ª 2015 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/15 http://dx.doi.org/10.1016/j.transproceed.2015.04.091
Transplantation Proceedings, 47, 2003e2009 (2015)
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Fig 1. The level of TH17 cells is elevated but the galectin-9 (Gal-9) level is decreased in murine cardiac allografts. (A) The percentage of CD4þIL-17þ T cells in circulation of cardiac-transplanted mice with (left) normal heart function and (right) acute rejection (AR) were determined by means of flow cytometry. (B) In the cardiac-transplanted mice with AR, the correlation of CD4þIL-17þ T cells and rejection activity index was analyzed by means of Spearman rank correlation assay, with the r and P values indicated. (C) Gal-9 expression in peripheral blood mononuclear cells of cardiac-transplanted mice with normal heart function and AR were determined by means of quantitative reverse-transcription polymerase chain reaction. Data are shown as mean SD (n ¼ 10) or as 1 representative experiment. Similar results were obtained in 3 independent experiments. Abbreviation: IL, interleukin.
infection through the recruitment of neutrophils and macrophages to infected tissues [14]. Although a few studies have highlighted the possibility of a relationship between IL-17 and AR in organ transplantation [15], the role of TH17 cells in AR of patients after cardiac transplantation remains unclear. Galectin-9 (Gal-9) is a b-galactosideebinding protein consisting of 2 carbohydrate recognition domains connected by a linker peptide. The galectin family members are thought to be critical for the regulation of immune cell homeostasis and inflammation. Earlier studies have demonstrated that Gal-9 participates in a variety of biologic processes, such as cell adhesion, activation, chemoattraction, and apoptosis in different types of cells, including T cells, B cells, macrophages, and mast cells [16e22]. In recent years, studies of Gal-9 have revealed that it regulates the immune response by reducing the amount of TH17 cells and TH1 cells that secrete proinflammatory IL-17 and by increasing the levels of
antiinflammatory Foxp3þ regulatory T cells (Treg), which are critical in the pathogenesis of various autoimmune diseases, both in vitro and in vivo [22e26]. A recent study also has demonstrated that the Tim-3/Gal-9 pathway plays an important role in the termination of the TH17 immune response [27,28]. These findings indicate that Gal-9 may function as an important immune modulator under physiologic and pathologic conditions. In our clinical studies of cardiac transplantation, we observed that episodes of AR occur in w30%e40% of cardiac transplant patients. In addition, the levels of serum IL-17 are higher in heart transplant patients, whereas the levels of Gal-9 are markedly decreased. Based on these clinical data, we speculated that TH17 cells may be involved in AR after organ transplantation and that Gal-9 may play an important role in the regulation of TH17 cells in allogeneic heart transplant patients. However, the natural form of Gal-9 is easily inactivated by proteases owing to the labile
GALECTIN-9 IN COMBINATION WITH WITH EX-527
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Heart Transplantation and Gal-9 Treatment
Fig 2. Administration of Gal-9 alone or combine with EX-527 prolongs the survival of murine cardiac allografts. Shown are Kaplan-Meier survival curves of cardiac-transplanted mice with or without Gal-9 administration as indicated. Data were analyzed by means of log-rank test, with the P value indicated. Abbreviations: Gal-9, galectin-9; Ctrl, control; DMSO, dimethylsulfoxide; HT, heart transplantation.
nature of the linker peptide. Therefore, we elucidated the therapeutic potential of the stable form of Gal-9 and compared it with EX-527, a specific Sirtuin-1 (Sirt1) inhibitor for which it has been reported in a murine model of cardiac transplantation that deletion and inhibition of Sirt1 can inhibit T-cell function and prolong allograft survival in vivo [29]. Through the analysis of the production of TH17 cells, we evaluated the inhibitory effects of Gal-9 compared with and together with EX-527 on AR and elucidated possible mechanisms of its action. MATERIALS AND METHODS Animals and Ethics Statement BALB/c and C57BL/6J mice were purchased from the Joint Ventures Sipper BK Experimental Animal Company (Shanghai, China). All experiments with the use of animals were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of the Second Military Medical University, Shanghai, China.
Preparation of Stable-Form Human Galectin-9 Stable-form human Gal-9 was prepared as described previously [30]. Briefly, Gal-9 was cloned into the pET expression system (Novagen, Madison, Wisconsin) and transfected into E. coli BL21 (DE3) cells. The cells were grown at 30 C in 2YT broth containing 100 mg/mL ampicillin, induced at optical density 600 nm with the use of 500 mmol/L isopropylthio-beta-D-galactoside (IPTG) for 3 hours, harvested by means of centrifugation at 10,000g, lysed with a cell disrupter, and centrifuged at 20,000g for 30 minutes. The final purification of pET-hGal-9 was achieved by means of chromatography with the use of a lactose-agarose column (Seika-gaku Kogyo, Tokyo, Japan), dialysis against phosphate-buffered saline solution (PBS), and the elimination of endotoxin with the use of Cellufine ETclean-L (Chisso, Tokyo, Japan). The recombinant protein was >95% pure, as confirmed with the use of sodium dodecyl sulfatee polyacrylamide gel electrophoresis. The protein concentration was determined by means of the Bradford assay with the use of bovine serum albumin as the standard. The purified recombinant Gal-9 proteins were used in the subsequent studies.
Vascularized heart grafts were transplanted with the use of a microsurgical technique as previously described [31]. Cardiac grafts obtained from BALB/c or C57BL/6 mice were transplanted into C57BL/6 mice. Rejection was defined as complete cessation of cardiac contractility as determined through direct visualization. Recombinant stable-form Gal-9 (5 mg/kg/d) was intraperitoneally administered on postoperative days (PODs) 0e3, 5, 7, 9, 12, and 14, and solvent PBS was used as control. The study end point was defined as the complete cessation of the cardiac beat. The survival rates of the cardiac grafts were monitored by means of palpation by 2 independent observers without knowledge of the treatment protocols and were confirmed by means of histologic analysis.
Flow-Cytometric Analysis A single-cell suspension of the spleen was prepared with the use of a manual tissue homogenizer. The cells were washed with RPMI 1640 medium (Sigma, St Louis, Missouri) containing 2 mmol/L HEPES and 1% bovine serum albumin and passed through a 70-mm nylon cell strainer. The expression levels of CD4 and IL-17A on the lymphocytes were measured by means of flow cytometry. The following phycoerythrin (PE)e and fluorescein isothiocyanate (FITC)elabeled antibodies were used: IL-23 receptor (IL-23R; mouse IgG1, FITC) and CCR6 (mouse IgG1, PE), which were purchased from Becton Dickinson, and IL-17A (mouse IgG1, PE), which was obtained from eBioscience (San Diego, California). Appropriate PE- and FITC-labeled isotype control subjects (Becton Dickinson) were used. Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood by means of density-gradient centrifugation over Ficoll-Paque plus (GE Healthcare, Uppsala, Sweden). The PBMCs were collected at interphase and washed twice in PBS. The cells were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL, Grand Island, New York). The cells were cultured in the absence or presence of phorbol myristate acetate (100 ng/mL) and ionomycin (1 mg/mL; both from Alexis Biochemicals, San Diego, California) for 5 hours. Cytokine secretion was inhibited by brefeldin A (Sigma). Surface staining was then performed with anti-CD4 and anti-CCR6. The cells were fixed and permeabilized with the use of a Cytofix/Cytoperm kit (BD Pharmingen), and the samples were then intracellularly stained with antieIL-17A or an appropriate isotype control. The CD4þIL-17þ T cells were sorted with the use of a fluorescence-activated cell sorter (FACS; Calibur; Becton Dickinson), and the FACS data were analyzed with the use of the Flowjo software (Treestar Software).
Isolation and Culture of CD4þIL-17þ T Cells The murine single-cell suspension of the spleen or draining lymph nodes was prepared with the use of a manual tissue homogenizer and passed through a 70-mm cell strainer. The lymphocytes were inoculated at a density of 1 106/mL in a 24-well plate, and PMA (50 ng/mL), ionomycin (1 mg/mL), and monensin (1 mL) were added to each well. The plate was then kept at 37 C with 5% CO2 for 5 hours, and the expression levels of CD4 and IL-17A in the lymphocytes were measured by means of flow cytometry. PBMCs were obtained as already described. The cells were resuspended in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum. The cells were cultured in the absence or presence of transforming growth factor (TGF) b, IL-6, antieIL-4 (1 mg/mL), and antieINF-g (1 mg/mL) for 3 hours. Cell-surface staining was then performed with anti-CD4 and anti-CCR6. The cells were fixed and permeabilized with the use of a Cytofix/Cytoperm kit (BD Pharmingen), and the samples were intracellularly stained with
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GALECTIN-9 IN COMBINATION WITH WITH EX-527 antieIL-17A or an appropriate isotype control. The CD4þIL-17þ T cells were sorted with the use of FACS, and the FACS data were analyzed as described above.
Quantitative Reverse-transcription Polymerase Chain Reaction The mRNA levels were evaluated with the use of Sybr Green Iebased real-time reverse-transcription polymerase chain reaction (RT-PCR), which was performed with the use of an ABI Prism 7000 sequence detector (Applied Biosystems, Foster City, California). The total RNA from the pelleted cells was isolated with the use of the RNeasy Plus mini kit (Qiagen). The 1st-strand cDNA synthesis was performed by means of random priming with a high-capacity cDNA synthesis kit (Applied Biosystems) in the presence of Superase RNase inhibitor (Ambion). The cDNA was then used as the template in the PCR reactions. The reactions were performed with the use of the Fast Universal PCR Mastermix (Applied Biosystems) according to the manufacturer’s instruction and thermocycled in a 20-mL reaction volume in 96-well plates. The Ct values were calculated according to automatically determined threshold values with the use of the SDS software (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were used as an internal standard for calibration. The primer sequences used in the RT-PCR analysis were as follows: GAPDH: 50 -AGG TCG GTG TGA ACG GAT TTG-30 (forward), 50 TGT AGA CCA TGT AGT TGA GGT CA-30 (reverse); CCR6: 50 AAT GAA CGA TCC TGC CAG A-30 (forward), 50 -GGA GAA GCC TGA GGA CTT GT-30 (reverse); IL-23R: 50 -TCA TCC CAG AAC ACA AGC CT-30 (forward), 50 -ATT GCT GAG ATG GCT TCC CT30 (reverse); IL-17A: 50 -ATC AGG ACG CGC AAA CAT G-30 (forward), 50 -TGA TCG CTG CTG CCT TCA C-30 (reverse); IL-23; 50 -GGG AGA TGA AGA GAC TAC AAA TGA TGT-30 (forward), 50 -ATC CTT TGC AAG CAG AAC TGA CT-30 (reverse); and Gal-9: 50 -GTT TGC AAC ACG AAG CAG AA-30 (forward), 50 -GGC AGG ACG AAA GTT CTG AG-30 (reverse).
Statistical Analysis Data are presented as mean SD. Statistical comparisons between groups were analyzed by means of Student t test, and 2-tailed P < .05 was considered to indicate statistical significance. For graft survival, Kaplan-Meier graphs were constructed and analyzed by means of log-rank test. The comparison among groups was performed by means of 1-way analysis of variance. The correlation analysis was performed with the use of Spearman rank correlation coefficient assay. Data were analyzed with the use of Graphpad Prism 5.0 software.
RESULTS The Level of TH17 Cells Is Elevated Whereas the Gal-9 Level Is Decreased in Murine Cardiac Allografts
To investigate the roles of TH17 cells and Gal-9 in AR of cardiac transplantation, we detected the percentage of TH17
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cells and Gal-9 expression in circulation of murine cardiac transplantation model. As shown in Fig 1A, the percentage of CD4þIL-17þ T cells significantly increased in AR recipients with abnormal heart function compared with mice with normal heart function. Furthermore, the percentage of CD4þIL-17þ T cells in circulation was positively correlated with the rejection activity index in the heart allograft biopsy (Fig 1B), indicating that the increase of TH17 cells contributed to AR progression. Additionally, Gal-9 mRNA expression was significantly decreased in AR of heart allograft recipients (Fig 1C). These data suggest that the decrease of Gal-9 and the increase of TH17 may contribute to AR progression.
Administration of Gal-9 Alone and in Combination With EX-527 Prolongs the Survival of Murine Cardiac Allografts
To explore the impact of recombinant Gal-9 on allogeneic cardiac transplantation, a murine cardiac transplant model was used in this study. Four groups of mice models were used, treated with or without Gal-9, EX-527, and their combination after transplantation. As shown in Fig 2, administration of recombinant Gal-9 or EX-527 alone or in combination prolonged the allograft median survival time, and the mice treated with Gal-9 and EX-527 together survived longer than the other groups.
Gal-9 Alone and in Combination With EX-527 Inhibits TH17 Cell Differentiation in Vitro
The administration of Gal-9 and EX-527 in our study was demonstrated to prolong the survival time of cardiactransplanted mice, so we further evaluated the effect of Gal-9 on TH17 cells and cardiac AR in a murine model to investigate the mechanisms underlying the Gal-9emediated inhibition of AR. CD4þIL-17þ T cells from the spleens of recipient mice on POD 7 were isolated and cultured with different treatments. As shown in Fig 3A, Gal-9 and EX-527 administration significantly suppressed IL-17, C-C chemokine receptor (CCR) 6 and IL-23R expression. We also tested the mRNA levels of CCR6, IL-23R, IL-17, and IL-23 in the CD4þIL-17þ T cells with the use of real-time PCR, and the results confirmed that TH17 cell differentiation was suppressed with Gal-9 and EX-527 treatment (Fig 3B). Moreover, the suppression effect of the group treated with Gal-9 and EX-527 together was strongest. Therefore, it can be concluded that Gal-9 and EX-527 administration alone or together inhibits TH17 cell differentiation in vitro.
Fig 3. Gal-9 alone or combined with EX-527 inhibits TH17-cell differentiation in vitro. (A) CD4þIL-17þ T cells were isolated from the spleens of the recipient mice on day 7 after transplantation and cultured with Gal-9 alone or combined with EX-527. CCR6þ, IL-23Rþ, and IL-17þ cells in the CD4þIL-17þ T-cell population were analyzed with the use of flow cytometry. (B) Expressions of CCR6, IL-23R, IL-17, and IL-23 were determined with the use of quantitative reverse-transcription polymerase chain reaction. Data are shown as mean SD (n ¼ 10) or 1 representative experiment. Similar results were obtained in 3 independent experiments. Abbreviations: CCR6, C-C chemokine receptor 6; IL-17, interleukin-17; IL-23, interleukin-23; IL-23R, interleukin-23 receptor; other abbreviations as in Fig 2.
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Fig 4. Gal-9 inhibits the number of TH17 cells in vivo alone or together with EX-527. Percentage of CD4þIL-17þ T cells in circulation of the cardiac-recipient mice on day 7 after transplantation was analyzed with the use of flow cytometry. Data are shown as mean SD (n ¼ 10) or 1 representative experiment. Similar results were obtained in 3 independent experiments. Abbreviations as in Figs 2 and 3. NS, no statistically significant difference of pairwise comparisons; *P < .05.
Gal-9 Decreased the Number of TH17 Cells in Vivo Alone or Together With EX-527
To examine the effect of Gal-9 or the combination of EX-527 and Gal-9 administration on TH17 cell number in the murine cardiac transplantation model, we detected the percentage of CD4þIL-17þ T cells in circulation of the recipient mice on POD 7. As shown in Fig 4, Gal-9 and EX-527 administration or the combination of EX-527 and Gal-9 administration in vivo significantly inhibited TH17 cell population in the heart transplant recipient mice.
DISCUSSION
In this study, we observed that activated CD4þIL17þ T cells were significantly elevated in cardiac transplant mice during the period of AR, and the levels of CD4þIL17þ T cells were positively correlated with AR grade. These data suggest that an increased level of circulating TH17 cells may be a novel marker for AR. Earlier studies demonstrated that TH1 cells are responsible for AR, and some recent studies have suggested that TH17 cells may also be involved in the process [32e35]. The contribution of TH17 cells to clinical AR has not yet been well documented, but animal studies have shown that TH17 cells induce tissue damage by stimulating inflammatory cells to release proinflammatory cytokines and accelerate the development of allograft vasculopathy [33]. Based on previous and the present research, we speculate that TH17 cells are involved in the proinflammatory process of AR. Moreover, a significantly decreased level of Gal-9 was expressed in cardiac transplant mice during the period of AR in the present study. And the administration of Gal-9
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and EX-527 in our study was demonstrated to prolong the survive time of cardiac-transplanted mice (Fig 2). Therefore we further evaluated the effect of Gal-9 on TH17 cells and cardiac AR in this murine model. As reported in earlier reports [36,37], TH17 cells were activated during AR of cardiac transplantation. However, the underlying mechanisms responsible for the increased proportion of TH17 cells within the T-cell population are still not well understood. Earlier studies discovered that IL-17, which is primarily produced by TH17 cells, takes part in variety of inflammatory reactions and disease development, including rejection in organ transplantation [15]. The expression of IL-23 and its receptor (IL-23R) was reported to increase the secretion of IL-17 by TH17 cells [38] as well as to induce the expression of CCR6, which is the surface receptor of TH17 cells, thus promoting the formation and development of inflammatory autoimmune disease [39]. In the present study, the key factors involved in the differentiation of TH17 cells, including CCR6, IL-17, IL-23, and IL-23R, were significantly decreased in heart transplant recipient mice treated with Gal-9 and EX-527. Additionally, the systemic administration of a stable form of human Gal-9 clearly ameliorates AR in the TH17-mediated cardiac transplantation model. Based on these data, we hypothesize that the inflammatory environment promotes the conversion of Tregs into TH17 cells, which results in an increase in the local level of IL-17 cytokines and thereby stimulates proinflammatory pathways. Another possibility is that the TH17 cells may be recruited from other organs. And further studies are needed to confirm these hypotheses. The present study also showed the similar inhibition effect of administration of Gal-9 and EX-527 in suppressing activation and number of TH17 cells and prolongation of the survival time of cardiac-transplanted mice. Sirtuins are NADþ-dependent class III histone/protein deacetylase enzymes that are highly conserved across eukaryotic species [31]. Sirt1 is a key factor in the caloric restriction response and life span control, and it is a therapeutic target in various cancers [30,32]. The combined use of Gal-9 and EX-527 had a better inhibition effect on the proliferation of TH17 cells and better effect on the survival time of cardiac-transplanted mice, providing a more effective treatment of alloimmune responses, and more research is needed to figure out the underlying mechanism. In summary, the levels of circulating TH17 cells were found to increase in the peripheral blood of mice that exhibited AR after heart transplantation, and the levels were determined to be correlated with the rejection grade. The circulating TH17 cells in the peripheral blood may be used as a marker for the prediction of AR in heart transplant recipients. Gal-9 or EX527 can inhibit the activation and differentiation of TH17 cell and effectively suppress TH17 cellemediated AR. These data provide new evidence for the potential regulatory effects of Gal-9 in alloimmune responses in a murine model of heart transplantation and suggest that the combined use of Gal-9 and EX-527 may be a powerful method to induce tolerance of fully mismatched murine cardiac allografts.
GALECTIN-9 IN COMBINATION WITH WITH EX-527
ACKNOWLEDGMENTS The authors thank Prof Quan-xing Wang and Mrs Fang Liu for the preparation of stable-form human Gal-9 and Dr Shao-hua Song for expert technical assistance in operation of the animal experiments.
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