Avian Foxp3 acts as a switch for IL-10 by directly binding the T3G motif: revealing a novel epigenetic modulation paradigm

BACKGROUND: Foxp3 is the master transcription factor for regulatory T (Treg) cell differentiation and function, essential for immune homeostasis and tolerance. While its role is well-characterized in mammals, its functional mechanisms in avian species remain poorly understood. METHODS: We conducted comparative genomic and functional analyses of chicken Foxp3 (chFoxp3). DNA-binding specificity was determined using motif analysis and genome-wide scanning. An H9N2 avian influenza challenge model was used to assess Foxp3 expression dynamics and CD4⁺CD25⁺ T cell frequency in vivo. The transcriptional regulation of IL-10 by chFoxp3 was investigated through promoter binding and transactivation assays, with key domains mapped via mutagenesis. RESULTS: Avian Foxp3 has evolved independently from its mammalian ortholog. Unlike human Foxp3, which binds the FKHM motif, chFoxp3 specifically recognizes a T3G microsatellite motif. Genome-wide scanning identified potential targets enriched in the FoxO signaling pathway, including IL-10. In an H9N2 infection model, Foxp3 expression and CD4⁺CD25⁺ T cell frequency exhibited biphasic dynamics. Mechanistically, chFoxp3, but not human FoxP3, directly binds the T3G motif in the IL-10 promoter to activate transcription. This transactivation function critically depends on the HNT linker within its Fork-head domain. CONCLUSIONS: Our study reveals a novel mode of transcriptional regulation by chFoxp3 via T3G motif recognition, underscoring its functional diversification during evolution. These findings provide a foundation for understanding immune regulation in birds under both physiological and pathological conditions, such as avian influenza infection.