Two functional Il-35 molecules negatively regulate Ifn-γ-induced macrophage immune responses in large yellow croaker (Larimichthys crocea).

Il-35 is an immunosuppressive cytokine composed of Ebi3 and Il-12p35, which plays a critical role in regulating the functions of mammalian macrophages, yet its functions in macrophages of teleost fish remain unclear. In this study, two functional Il-35 molecules were identified in large yellow croaker (Larimichthys crocea), which are formed by Ebi3 (encoded by ebi3) paired with Il-12p35a (encoded by il12a1) or Il-12p35b (encoded by il12a2), respectively, designated as Il-35a and Il-35b. Their regulatory effects on macrophage functions were characterized. Through gene cloning, sequence alignment, phylogenetic analysis and synteny analysis, the il12a1 and il12a2 genes of large yellow croaker were identified, and the proteins encoded by both genes were confirmed to share similar structures. qRT-PCR analysis revealed that ebi3, il12a1 and il12a2 are constitutively expressed in multiple tissues, and their expression can be upregulated upon lipopolysaccharide (LPS) stimulation. Co-immunoprecipitation assays demonstrated that Ebi3 can bind to Il-12p35a and Il-12p35b separately to generate two distinct Il-35 molecules. Both recombinant Il-35a and Il-35b proteins markedly induce the expression of their own subunit genes, with Il-35b exhibiting a stronger inductive effect. Functional assays indicated that Il-35a and Il-35b significantly suppress Ifn-γ-triggered phagocytic activity and intracellular bactericidal capacity of macrophages, alongside reduced production of ROS and NO, and Il-35b exerts an overall stronger inhibitory effect than Il-35a. In conclusion, this study is the first to identify and functionally characterize two distinct Il-35 molecules in large yellow croaker, demonstrating their conserved yet functionally divergent roles in suppressing Ifn-γ-mediated macrophage immune responses. These findings provide new insights into the evolution and functional diversification of Il-35 in teleost, and establish Il-35 as a key negative regulator of fish innate immunity.