Expression analysis of grass carp Foxp3 and its biologic effects on CXCL-8 transcription in non-lymphoid cells

Teleost Forkhead box protein P3 (Foxp3) expression was discovered not only in regulatory T cells (Tregs) but also in other cells. Compared to the extensive study on its roles in lymphoid cells, the expression pattern and biological roles of Foxp3 in non-lymphoid cells have not been elucidated in both mammals and fish species. In the present study, grass carp Foxp3 (gcFoxp3) mRNA expression was detected in different cell types, showing that it has a moderate expression level in peripheral blood leukocytes (PBLs), head kidney leukocytes (HKLs) and grass carp fibroblast-like kidney cells (CIK cells). Interestingly, gcFoxp3 mRNA and protein expression could be significantly stimulated by polyinosinic-polycytidylic acid (poly I:C) in CIK cells, indicating its participation in poly I:C-induced immune response in non-lymphoid cells. To further investigate the function of gcFoxp3, its overexpression plasmid was constructed and transfected into CIK cells. After 24 h of transfection, grass carp C-X-C chemokine ligand (CXCL) 8 (gcCXCL-8) mRNA expression was elevated, implying the modulatory role of gcFoxp3 in gcCXCL-8 mRNA expression. This notion was further supported by the features of gcCXCL-8 promoter which contained a putative Foxp3 binding site at -2196 to -2190 region. Poly I:C or overexpression of gcFoxp3 obviously stimulated gcCXCL-8 promoter activity and deletion of gcFoxp3 binding region on the promoter abolished this stimulation, revealing that Foxp3 is pivotal for transcription of CXCL-8 induced by poly I:C. In conclusion, our results collectively demonstrate expression pattern of teleost Foxp3, and illuminate novel immune function of fish Foxp3 in regulating chemokine transcription in non-lymphoid cells.