Molecular cloning and expression analysis of Foxp3 from Nile tilapia

Foxp3 is a crucial transcription factor for the development and function of CD4+CD25+ regulatory T cells (Tr) which play a key role in preventing autoimmune diseases and maintaining maternal-fetal tolerance in mammals. However, the knowledge of Foxp3 and its regulation in teleosts is limited. In the present study, the Foxp3 cDNA was cloned from Nile tilapia and characterized. The full length cDNA of Nile tilapia (nt)Foxp3 contained a 5′UTR of 104bp, a 3′UTR of 239bp and an open reading frame of 1134bp. The putative ntFoxp3 contained a C2H2 zinc finger domain, a winged-helix/fork head domain and a leucine zipper-like domain with a consensus of V-x(6)-L-x(6)-L-x(6)-L, which are typical motifs of a Foxp3. The highest mRNA expression of ntFoxp3 was observed in the spleen, with moderate expression in the head kidney, intestine, kidney, liver and brain. Stimulation of peripheral blood mononuclear cells with PHA and LPS led to a significant increase of ntFoxp3 mRNA expression at 6 and 24h, respectively. In contrast, stimulation with PMA had no effect during the sampling period. After injecting 1×107cells of live Aeromonas hydrophila into adult female Nile tilapias, the mRNA expression of ntFoxp3 significantly increased in the gill and intestine at 6h, while unchanged in the spleen. In all the tissues examined, a clearly up-regulation of ntFoxp3 expression was detected at 24h after the second challenge. These results suggest that ntFoxp3 might be involved into lymphocyte activation and immune responses as reported in mammals. Meanwhile, the correlation between the expression profile of ntFoxp3 and serum estrogen (17-beta-estradiol, E2) concentration was investigated by real-time PCR and enzyme immunoassay. The results showed that E2 could regulate the expression of ntFoxp3 in the intestine and kidney but not in the spleen. This work will help future investigations into Tr cells and extend our understanding of interactions between sex hormones and immune related molecules in teleosts.