Molecular characterization and differential immunomodulatory functions of TGF-β2 and TGF-β3 following Vibrio challenge in Takifugu obscurus.

Transforming growth factor-β (TGF-β) genes play critical roles in immune regulation, but their functions in teleost fish, particularly TGF-β2 and TGF-β3, remain poorly understood. In this study, two TGF-β genes, namely ToTGF-β2 and ToTGF-β3 were identified in pufferfish, Takifugu obscurus. Bioinformatics analysis revealed that both genes possess conserved TGFb_propeptide and TGFB domains, with ToTGF-β2 additionally containing a low-complexity region. Tissue distribution analysis by RT-qPCR indicated that ToTGF-β2 showed the highest expression in the intestine, whereas ToTGF-β3 was most abundant in the head kidney. Following challenge with Vibrio parahaemolyticus or Vibrio harveyi, both genes were significantly upregulated in head kidney and spleen in a time-dependent manner, suggesting active participation in antibacterial immune responses. To further investigate their functional roles, in vivo RNA interference was performed. Knockdown of ToTGF-β2 significantly reduced the expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and impaired bacterial clearance, whereas knockdown of ToTGF-β3 markedly increased cytokine levels and enhanced bacterial elimination. These results demonstrate that ToTGF-β2 acts as a positive regulator of inflammation and host defense, while ToTGF-β3 functions as a negative regulator. This study provides novel insights into the functional diversification of TGF-β genes in fish antibacterial immunity.