Mechanism of indoleamine 2, 3‐dioxygenase inhibiting cardiac allograft rejection in mice

Abstract Indoleamine 2, 3‐dioxygenase (IDO)‐mediated regulation of tryptophan metabolism plays an important role in immune tolerance in transplantation, but it has not been elucidated which mechanism specifically induces the occurrence of immune tolerance. Our study revealed that IDO exerts immunosuppressive effects through two pathways in mouse heart transplantation, ‘tryptophan depletion’ and ‘tryptophan metabolite accumulation’. The synergism between IDO+DC and TC (tryptophan catabolic products) has stronger inhibitory effects on T lymphocyte proliferation and mouse heart transplant rejection than the two intervention factors alone, and significantly prolong the survival time of donor‐derived transplanted skin. This work demonstrates that the combination of IDO+DC and TC can induce immune tolerance to a greater extent, and reduce the rejection of transplanted organs.


| INTRODUC TI ON
Organ transplantation is an effective means of clinical treatment of end-stage organ failure. In addition to the maturity of surgical techniques, the clinical application of immunosuppressive agents such as FK506, CsA and FTY720 has greatly improved the survival rate of organ transplantation. However, these inhibitors do not significantly improve the long-term survival of the graft, and many problems such as organ transplant rejection and its secondary damage have not been completely solved. 1 T cell-mediated immune responses are key factors leading to the rejection of organ transplant. Dendritic cells (DCs) presenting antigens to T cells can lead to two distinct effects, immunogenic or tolerogenic, but the mechanism of tolerogenic effect remains unclear. 2,3 Activated dendritic cells can express a large amount of indoleamine 2, 3-dioxygenase (IDO). IDO is the only rate-limiting enzyme outside the liver that catalyzes the catabolism of tryptophan (TRP) along the kynurenine (KYN) pathway and plays an important regulatory role in transplantation immune tolerance, tumour immune escape, maternal-foetal immune tolerance and autoimmune diseases. 4,5 Tryptophan is an essential amino acid for T cell activation and proliferation. Previous studies have shown that high expression of IDO can effectively reduce intracellular tryptophan concentration and down-regulate T cell immune response, while IDO-specific inhibitors can significantly induce acute transplant rejection at both the cellular and animal level. 6,7 Therefore, traditional theory suggests that IDO induces immune tolerance mainly through the tryptophan depletion.
Recent studies have found that the accumulation of tryptophan catabolic products (TC) also plays an important role in transplantation immune tolerance. The main metabolite of IDO decomposition of tryptophan is KYN. Under the action of various enzymes, KYN decomposes into 3-hydroxyanthranilic acid (3-HAA), 3-hydroxykynurenine (3-HK), anthranilic acid, et cetera. 8 A study by Brandacher G showed that the severity of postoperative rejection in kidney transplant patients was increased proportionally with the ratio of kynurenine and tryptophan (kyn/trp). 9 Animal experiments have also shown that tryptophan metabolites such as KYN, 3-HAA and 3-HK can significantly inhibit T cell proliferation and prolong the survival time of skin grafts. 10 Obviously, there are still many controversies about the mechanism by which IDO induces immune tolerance.
In this study, the effects of IDO + DC and TC combination on T cell proliferation and apoptosis, cardiac allograft rejection and graft survival were observed at the cellular and animal levels, respectively, then compared with the simple administration of IDO + DC or TC.

| Indoleamine 2, 3-dioxygenase activity detection (HPLC)
Indoleamine 2, 3-dioxygenase activity was detected by high-performance liquid chromatography (HPLC) to quantify the concentration of TRP and KYN in culture medium. C57BL/6 DCs were preincubated with Ad-IDO or Ad-Null (Adenoviral vectors without IDO gene). After 48 hours of culture, the different supernatant fluids were collected and mixed with 6% perchloric acid (PCA) at 4:1 ratio, respectively, to precipitate proteins. The acidified samples were centrifuged at 12 000 g at 4°C for 10 minutes, and the supernatant fluids were dried under nitrogen flow. The residues were dissolved in 100 μL of 1% acetonitrile in 0.1% formic acid and transferred to an autosampler vial insert; then, 20 μL of supernatant fluid was injected directly in the high-performance liquid chromatography system (Alliance separations module 2695; Waters Corp). The chromatographic separation was obtained with an Accucore PFP column (150 mm × 21 mm, 2.6 μm particle size; Thermo-Scientific) at a flow rate of 0.2 mL/min. Elution started with 99% of mobile phase A (0.1% formic acid in water) and 1% mobile phase B (100% acetonitrile) for 2 minutes, followed by an 18-minute linear gradient to 50% of phase A, a 1-minute linear gradient to 30% of phase A and a 1-minute linear gradient to 99% of phase A, which was maintained for 12 minutes to equilibrate the column. TRP and KYN were detected by UV absorption at 280 nm and 360 nm, respectively (L-4250 UV VIS Detector, Merck Hitachi).

| Mouse spleen CD4 + T cell sorting (MACS)
The spleen was taken after the mice were killed, and the precipitated cells were collected after grinding, filtration and centrifugation. The red blood cells were completely lysed by adding red blood cell lysate (BD), and the cell pellet was rinsed twice with PBS after centrifugation.
The cells were resuspended in pre-cooled binding buffer, and then the CD4 immunomagnetic beads (Miltenyi) were added and incubated for 15 minutes at 4°C. After the incubation, the cells were rinsed and resuspended in binding buffer. The cell suspension was added to the MS column (Miltenyi) until it was drained, and the column was washed three times with the washing buffer. The rinse solution was collected when rinsing the MS column with the elution buffer.

| T lymphocyte proliferation assay (MTS)
One-way mixed lymphocyte reactions (MLR) were performed in duplicate on 96-well tissue culture plates (Asahi Techno Glass) by using responder CD4 + T cells and stimulator DCs. In addition, BALB/c mice DCs were harvested as negative control, and mix-cultivated with homologous CD4 + T cells. Various cells were harvested as previously described. Allogeneic CD4 + T cells (1 × 10 6 /mL) were plated with different groups at a ratio of 10:1 (DCs at a concentration of 1 × 10 5 /mL). After incubation at 37°C in a humidified incubator with 5% CO 2 for 48 hours, cells were exposed to 20 μL of

| T cell apoptosis assay (Annexin-FITC/PI)
Detection of early apoptotic CD4 + T cells was performed using the annexin V and propidium iodide (PI) detection kit (BD Biosciences).
CD4 + T cells were harvested after mixed lymphocyte reactions with different DCs as described above. Briefly, 10 6 isolated T cells were

| Haematoxylin staining and Immunohistochemistry
Heart graft samples were collected at 7 days after transplantation and fixed in 10% buffered formaldehyde, embedded in paraffin and sectioned at 5 μm for haematoxylin (HE) staining. The ensuing morphological examination was performed using an Olympus Microscope (X51). Criteria for graft rejection included the presence of lymphocyte infiltration and interstitial oedema. Briefly, IHC was used on 5 μm sections (Leica) taken from ice-cold 4% (w/v) paraformaldehyde-fixed paraffin-embedded tissue. Sections were heated at 60°C for 1 hour, then deparaffinized in three times of xylene and rehydrated with graded concentrations of ethanol. For immunostaining (BOSTER), deparaffinized and rehydrated sections were heated in citrate buffer at 121°C for 30 minutes and incubated with 0.3% hydrogen peroxide in methanol for 20 minutes, respectively. After non-specific reactions had been blocked with 5% BSA (Solarbio), the sections were incubated with primary antibody at 4°C overnight and then with biotinylated secondary antibody at 37°C for 1 hour. The sections were counterstained with haematoxylin for detection.

| RNA Isolation and RT-PCR Analyses
The total RNA was isolated using RNA extraction kit (TIANGEN) and reverse transcribed into cDNA using reverse transcription kit (ABI). Quantitative real-time PCR analysis was performed using real-time PCR kit (ABI). The relative mRNA expression levels of IDO, IL-2, IFN-γ, IL-10, IL-4 and TNF-αwere normalized with the β-actin in the same sample. The thermal cycler parameters for the amplification of these genes were as follows: 1 cycle at 95°C for 10 minutes followed by 40 cycles at 95°C for 15 seconds, 60°C for 15 seconds and 72°C for 30 seconds. Gene expression was evaluated by the 2 −ΔΔCt method. The sequences of RT-PCR primers are the following (5′-3′, Table 1).

| Western blot
Total protein was extracted from donor heart tissues (Qiagen DNeasy kit; Qiagen) according to the manufacturer's instructions. Proteins (20 μg of total protein) were subsequently separated by SDSPAGE, and then transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk in Tris-buffered saline with Tween-20 (TBST) at room temperature for 2 hours. Then, the proteins were incubated overnight with rabbit antimouse IDO (Abcam; 1:200) at 4°C. The membranes were then incubated with HRP-conjugated goat anti-rabbit second antibody (Abbiotec; 1:1000) for 1 hour at room temperature. After washing the membrane thrice with TBST, the proteins were visualized using an ECL detection system (G: ). GAPDH (Abbiotec; 1:1000) was used as the internal control. The density of each band was determined using the corresponding GAPDH value. Bands were scanned and quantitated by densitometry using Quantity one imaging software.

| Construction of a mouse allogeneic heart transplantation model
Briefly, a midline abdominal incision was performed on the donor.

| Establishment of mouse skin transplantation model
Mice were fixed in prone position after anaesthesia. 1 cm 2 of the skin on the back of the donor was taken off after disinfection. The same size piece of skin was cut from the same part of the receptor.
Insert the donor skin in the direction of the reverse hair and make it consistent.

| Statistical analysis
Data were compared and reported as mean ± standard deviation.
Statistical comparison between various groups was performed by one-way analysis of variance with Tukey's post hoc adjustment for multiple comparisons, as appropriate, using the SPSS software (SPSS, Inc). Allograft survival among experimental groups was compared using log-rank (Mantel-Cox) testing. Differences were considered statistically significant when P value was <.05. suggesting that the transfected DC cells have higher IDO activity ( Figure 1A). The decrease of tryptophan concentration in the IDO + DC group was not significant, probably because the initial concentration of tryptophan in the medium was much higher than the number of IDO + DC cells.

| Combined application of IDO + DC and TC has a stronger inhibitory effect on T lymphocytes than the two intervention factors alone
In vitro experiments showed that the T cell stimulation index of  Figure 1B). This result was also veri-  Figure 2B). This suggested that the combination of IDO + DC and TC might induce the occurrence of immune tolerance in vivo, but it still needed to be further explored.

| Combined application of IDO + DC and TC has a stronger inhibitory effect on cardiac allograft rejection in mice than the two intervention factors alone
To further study the effect of IDO on animal organ transplant re- group, and the increase was more obvious in the IDO + DC+TC group ( Figure 3A,B). Immunohistochemical staining showed that IDO protein was mainly expressed in the cytoplasm of the cardiomyocytes and the vessel wall. The positive rate and staining intensity of IDO in the CsA group, the TC group, the IDO + DC group and the IDO + DC+TC group were significantly higher than other groups, and it was most significant in the IDO + DC+TC group ( Figure 3C). It was suggested that the IDO overexpressing mouse heterotopic heart transplantation model was successfully constructed. Kaplan-Meier survival analysis showed that the survival time of the transplanted hearts in the TC group and the IDO + DC group was significantly longer than that in the PBS group and the DC group ( Figure 3D).
Moreover, the median survival time of the transplanted hearts in the IDO + DC+TC group was significantly longer than that in the TC group and the IDO + DC group (24 days vs 11 and 17.5 days, respectively, P < .01, Figure 3E).
In addition, the phenotype and recovery status of transplanted hearts in the CsA group, the TC group, the IDO + DC group and the IDO + DC+TC group were much better than control groups ( Figure 4A).
Histopathological examination showed multifocal inflammatory cell infiltration and severe myocardial damage in the PBS group and the untransfected DC group manifested as moderate-to-severe acute rejection. While the cardiomyocytes in the IDO + DC group had slight focal lesions and a small amount of inflammatory cell infiltration, which showed mild rejection. In contrast, the degree of myocardial damage was even weaker in the IDO + DC+TC group, and allogeneic rejection was more significantly inhibited ( Figure 4B and Table 2). This result showed that the apoptosis rate of receptor T cells was significantly higher than that of the control group after IDO + DC and TC intervention, and the degree of apoptosis was more obvious in the IDO + DC+TC group (P < .01, Figure 5A). All of the above data

| Combined application of IDO + DC and TC can significantly prolong the survival time of donorderived transplanted skin
To further clarify whether IDO + DC+TC has specific immunosuppression against donor antigens, we re-established the model Total RNA and protein were extracted from heart tissues and then subjected to RT-PCR and Western blot. The ratio of IDO/GAPDH in the IDO + DC+TC group was significantly higher compared with other groups, which indicated that combined application of IDO + DC and TC make IDO highly expressed in donor hearts than the two intervention factors alone after transplantation. C, Immunohistochemical staining of IDO in transplanted hearts (×400). The expression of IDO protein in the IDO + DC+TC group was significantly higher compared with TC and IDO + DC groups, respectively. Furthermore, in the donor hearts, IDO was mainly expressed in heart muscle cells and also weakly stained in blood vessels. D, Kaplan-Meier analysis of transplanted heart survival in each group. E, Quantification of median survival in (D). Data are shown as mean ± SD for six independent experiments; *P < .05, **P < .01 tissue hierarchy and vascular structure. Fourteen days after surgery, the transplanted skin showed mild scarring and hair loss, and only a small amount of inflammatory cells infiltrated in the subcutaneous tissue ( Figure 6A and 6). However, the C3H/Hederived transplanted skin showed rejection only 1 week after surgery, with dispersed tissue and vascular, and the subcutaneous tissue was accompanied by a large amount of inflammatory cell infiltration. The transplanted skin was completely scarred and shedding 10 days after surgery ( Figure 6A and 6). The above results indicated that IDO + DC and TC interventions have differential immune defenses for different sources of stimulation. It was suggested that the combination of IDO + DC and TC could induce the occurrence of immune tolerance to a certain extent, but its long-term effect and specific mechanism still needed to be further explored.

ACK N OWLED G EM ENTS
The authors thank Yujie Qiu, Hui Liang and Na Zhao for technical assistance, and Marrion for language modification. This research work was supported by the National Natural Science Foundation (No. 81070377) of China.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the finding of this study are available from the corresponding author upon reasonable request.