All the experiments were performed based on the guidelines of our institute and the Regulation for the Administration of Affairs Concerning Experimental Animals (approved by the State Council of China). Clean grade BALB/c mice weighing 20 to 35 g (aged 30 to 40 days) were purchased from HFK Bioscience (Beijing, China) and raised for 2 days for acclimatization. Before the CLP operation, the mice were starved for 12 h. A total of 10 mice was randomly selected for the operation and anesthetized with xylazine (10 mg per kg body weight). CLP was performed as previously described (16). At day 7 post-operation, the death rate was approximately 50%. Treg cells (CD4⁺ and CD25⁺) were isolated from the peritoneal lavage and splenocytes of the 5 living mice using a flow cytometer and the Mouse Regulatory T cell Staining kit (eBioscience, San Diego, CA, USA), and maintained in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 200 µg/ml streptomycin and 0.25 µg/ml amphotericin (Invitrogen). The cells were cultured in a humidified atmosphere with 5% CO₂ at 37°C, and the medium was changed every 2 days.

At 48 h post-transfection, the cells were treated with ATP (substrate of CD39) or AMP (substrate of CD73; Sigma-Aldrich, Shanghai, China) at a concentration of 1 mM for 1 h. The supernatant was then collected for the detection of the ATP, AMP and adenosine concentrations using the HPLC SpectraSYSTEM (Thermo Fisher Scientific, Waltham, MA, USA). Tests were conducted in the octadecyl silane-C18 column at room temperature. The injection volume of each test was 20 µl. Potassium phosphate buffer (50 mM, pH 6.5) was used as the mobile phase, with a flow velocity of 1 ml/min. The ultraviolet radiation wavelength for detection was 254 nm.

Peritoneal lavage and splenocytes were collected from the mice based on the methods of a previous study (17). Briefly, the peritoneal lavage and the spleen tissue of the 5 living mice were collected after the mice were anesthetized and sacrificed. For the collection of the peritoneal lavage, 5 ml phosphate-buffered saline was injected intraperitoneally and samples were obtained after 30 min of gently pressing the abdomen. The cells were then isolated and cultured according to the abovementioned method. Treg cells were isolated using the CD4⁺CD25⁺ Regulatory T Cell Isolation kit (Miltenyi Biotec, Teterow, Germany). αCD4 (RM4-5) fluorochrome-labeled antibody (BD Biosciences, Heidelberg, Germany), CD25 (PC61.5) antibody and Armenian and Syrian hamster IgG (BioLegend, San Diego, CA, USA) were used to label the Treg cells. The purity of the Treg cells was detected using a flow cytometer (BD FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA).

The Treg cells were treated with CGS21680 (100 nM, Biochempartner, Shanghai, China), or adenosine (10 µM, MCE, Shanghai, China) at 37°C. Transcription factors in the promoter sequences of CD39 and CD73 were predicted using UCSC genome browser (http://genome.ucsc.edu) and VISTA Enhancer Brower (http://genome.lbl.gov/vista/index.shtml). The E2F-1- and CREB-specific siRNA and negative control siRNA were purchased from Cell Signaling Technology (CST, Boston, MA, USA). The Treg cells were plated on 60-mm dishes and transfected with the corresponding siRNA treated with Lipofectamine^® RNAiMAX Transfection Reagent (Invitrogen) after 24 h of incubation at the concentration of 1 pmol per 0.3 µl reagent.

The cells were transfected with Cignal E2F Reporter (luc) kit or CREB-luc (Qiagen, Shanghai, China) using Lipofectamine 2000 transfection reagent (Invitrogen). The transfected cells were lysed and the luciferase activities were analyzed using the Dual-Luciferase^® reporter assay system (Promega, Madison, WI, USA) and measured as relative light units using a luminometer (Turner Designs, Sunnyvale, CA, USA).

The protein samples were extracted from the cells using lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P 40 (NP-40), 0.1% sodium dodecyl sulfate (SDS), and 0.5% sodium deoxycholate. The proteins of the same amounts were then separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes. The blots were blocked with 5% skim milk at 4°C overnight and incubated with the primary antibodies [GAPDH (sc-365062), CD39 (sc-33558), CD73 (sc-14684), E2F-1 (sc-251), signal transducer and activator of transcription (STAT)5 (sc-836), p-STAT5 (sc-12893), CREB (sc-377154) or p-CREB-specific (sc-7978), Santa Cruz Biotechnology, Dallas, TX, USA] at room temperature for 2 h, followed by incubation with peroxidase-conjugated secondary antibody (anti-β-actin antibody, Sigma-Aldrich) at room temperature for 1 h. GAPDH was used as the internal reference. Positive bands were detected using enhanced chemiluminescence reagents and analyzed using the Kodak Digital Imaging System (Rochester, NY, USA) in triplicate.

All experiments were repeated 3 times and the results are presented as the means ± standard deviation (SD). Statistical analyses were performed using Statistical Product and Service Solutions (SPSS) 19.0 software and one-way analysis of variance (ANOVA). Differences were considered statistically significant at P<0.05.

Western blot analysis was used to examine the effects of adenosine and CGS21680 on the expression of CD39 and CD73. At 24 h post-treatment with adenosine or CGS21680, both the CD39 and CD73 expression levels were upregulated compared to the control group (Fig. 1A and B), indicating that CD39 and CD73 are upregulated by adenosine and CGS21680. We then performed further experiments to determine the direct regulatory factors of CD39 and CD63.

The predicted transcription factors, E2F-1 and STAT5 in the CD39 promoter, and CREB in the CD73 promoter (data not shown), were selected for this study. We conjectured that these factors may be regulated by adenosine or CGS21680, and may thus regulate CD39 and CD73. We thus performed a serious of experiments for verification. First, the expression levels of E2F-1 and STAT5 were measured by western blot analysis to verify whether they are regulated by adenosine or CGS21680 (Fig. 2A). The results revealed that adenosine upregulated the expression of p-E2F-1 and E2F-1. CGS21680 upregulated p-E2F-1; however, it did not affect E2F-1 expression. On the contrary, the expression patterns of p-STAT5 and STAT5 were not affected by either adenosine or CGS21680, indicating that STAT5 is not regulated by adenosine or CGS21680. Similarly, the expression levels of CREB and p-CREB were both upregulated by adenosine and CGS21680 (Fig. 2B). Thus, we only referred to E2F-1 and CREB in the following experiments. Dual-luciferase reporter assay indicated that E2F-1 was expressed at significantly higher levels in the Treg cells treated with adenosine or CGS21680 (P<0.01, Fig. 2C), which further confirmed tht E2F-1 is regulated by adenosine and CGS21680. Furthermore, the expression of CREB was significantly increased by adenosine and CGS21680 (P<0.01, Fig. 2D), inferring that adenosine and CGS21680 upregulate CREB. Taken together, these data indicate that the two transcription regulatory factors, E2F-1 and CREB, may be regulated by adenosine and CGS21680.

We then verified the effects of E2F-1 and CREB on CD39 and CD73 using their specific siRNA. The knockdown of E2F-1 led to the downregulation of both p-E2F-1 and E2F-1 (Fig. 3A), and the knockdown of CREB inhibited the expression of p-CREB and CREB protein as expected (Fig. 3B). We then examined the effect of E2F-1 or CREB knockdown on CD39 and CD73, and found that the inhibition of E2F-1 resulted in the downregulation of CD39 (Fig. 3C). There was no change in CD39 expression when the transfected cells were treated with adenosine (si-E2F-1 + adenosine vs. si-E2F-1), while CGS21680 compensated to some extent for the inhibition of E2F-1. These results indicate that CGS21680 and E2F-1 are both positive regulators of CD39. As for CREB, its knockdown also downregulated the expression of CD73 (Fig. 3D), indicating that CREB regulates CD73. Adenosine and CGS21680 slightly increased the expression of CD73 in the transfected cells (si-CREB + adenosine or si-CREB + CGS21680 vs. si-CREB), implying that adenosine and CGS21680 upregulated CD73 by promoting CREB. Taken together, our data indicate that adenosine and CGS21680 promote CD39 and CD73 by upregulating E2F-1 or CREB, constituting the CD39/CD73/adenosine pathway. Therefore, the regulatory functions of adenosine, CGS21680, E2F-1 and CREB are the possible reasons for the high expression of CD39 and CD73 on the Treg cell surface.

After the cells were treated with ATP, the production of ATP and AMP in the supernatant was detected and compared, as shown in Fig. 4A. The amount of ATP decreased sharply in both the CGS21680-treated and untreated cells when detected at 5 min after ATP treatment. In particular, the changes in the amount of ATP in the CGS21680-treated cells were more significant than those in the untreated cells (P<0.05), lasting until the final detection at 80 min post-ATP treatment. Furthermore, the amount of AMP, the hydrolysate of ATP, kept increasing until 40 min post-ATP treatment and the CGS21680-treated cells exhibited significant differences before this time point when compared with the untreated cells (P<0.05). Similarly, after adding AMP to the cells, AMP decreased gradually and adenosine, the hydrolysate of ATP, increased accordingly (Fig. 4B). Significant differences were observed between the CGS21680-treated and untreated cells in the changes in the amount of both AMP and adenosine (P<0.05). These results indicated that the adenosine cycle was accelerated in the presence of CGS21680.

Based on the above-mentioned results that E2F-1 may regulate CD39, and that CREB may regulate CD73, we conducted further analysis by adding ATP and AMP, the corresponding substrates of CD39 and CD73. First, the changes in the adenosine cycle were analyzed after ATP treatment in the 3 groups of cells, namely the cells with no pre-treatment, the cells transfected with si-E2F-1 and the transfected cells treated with CGS21680 (Fig. 5A). After the addition of ATP, the amount of ATP in the supernatant decreased, while the amount of AMP increased. Significant differences were observed between the untreated cells and the si-E2F-1-transfected cells (P<0.05). Slight differences were observed between the transfected cells and the transfected cells treated with CGS21680, with no significance (P>0.05). A similar detection was also performed after AMP treatment among the cells with no pre-treatment, the si-CREB-transfected cells and the transfected cells treated with CGS21680 (Fig. 5B). The trends in the changes in AMP or adenosine content were consistent among the 3 groups, with significant differences only observed in the amount of adenosine between the untreated cells and the si-CREB-transfected cells at 5, 10 and 40 min post-AMP treatment (P<0.05). Therefore, the promoting effects of CREB on the adenosine cycle were ambiguous. Overall, these results suggest that E2F-1, CREB may promote the adenosine cycle and the effects of their inhibition may be compensated by CGS21680 to a certain extent.