Advanced oxidation protein products inhibit the converting of CD4+ CD25-T cells into CD4+ CD25+ regulatory T cells

Abstract

Objective To investigate the effect and mechanism of advanced oxidation protein products (AOPP) on the converting of CD4+ CD25-T cells into CD4+ CD25+ regulatory T cells (Tregs). Methods CD4+ CD25-T cells were cultured in 96-well plates (1×106 cells/well), and were divided according to the presence of AOPP into 3 different groups: Control group (200 μg/ml serum albumin), AOPP group (200 μg/ml AOPP), Antioxidant group (pretreated with 100 μmol/L diphenyleneiodonium (DPI) 1 h before AOPP was added). Flow cytometry was used to detect the phenotype of CD4+ CD25-T cells and the DHE fluorescence intensity. The induced Tregs were collected and cultured with CD4+ CD25-T cells as the effective cells (Teff), the incorporation of [3H] thymidine was used to evaluate the suppressive function of iTregs on the Teff. Western blotting was used to observe the expression of phosphorylated signal transducer and activators of transcription 5 (p-STAT5). Results In AOPP group, (28.6±4.5)% of CD4 cells expressed CD25 and (10.7±1.7)% of CD4+ CD25+ T cells expressed Foxp3, which were significantly lower than those of Control group [(73.8±10.7)%, (64.3±9.4)%](P=0.003, 0.001). In Antioxidant group, these values were (41.3±6.4)% and (22.3±3.0)%, which were significantly more than those of AOPP group(P=0.049, 0.004), but lower than those of Control group (P=0.011, 0.002). When Tregs and Teff were incubated at a 1∶1 ratio, the Counts per minute (CPM) of Teff in AOPP group was 17 521.0±1 703.8, which was significantly more than that of Control group 9 932.3±1 142.9 (P=0.003). The CPM of Teff in Antioxidant group was 15 709.7±1 272.9, which was more than that of Control group (P=0.004) and no significant difference as compared with AOPP group (P=0.214). The DHE fluorescence intensity of AOPP group was (163.0±11.5)%, which was higher than that (51.1±4.0)% of Control group (P=0.000); while the DHE fluorescence intensity (113.4±7.8%) of the Antioxidant group was lower than that of the AOPP group (P=0.003) and was higher than that of the Control group (P=0.000). Compared with Control group, addition of AOPP significantly downregulated the expression of p-STAT5 in CD4+ CD25-T cells (P=0.006). After antioxidants was added, the expression of p-STAT5 was significantly upregulated compared with AOPP group (P=0.008) and lower than that of Control group (P=0.043). Conclusion AOPP significantly inhibit the converting of CD4+ CD25-T cells into Tregs through downregulating p-STAT5 via oxidative stress in vitro. Key words: Advanced oxidation protein products; Interleukin-15; Regulatory T cells; T cells; Phosphorylated signal transducer and activators of transcription 5