Identification and characterization of fish-specific IL-23 isoforms and their roles in fish immunity

Abstract IL-23 is well known as a regulator in inflammation response and host defense by promoting the development of Th17 cells. In fish, three genes coding p40 (p40a, p40b and p40c) are originated from fish-specific genome duplication, thereby generating three IL-23 isoforms (IL-23a, IL-23b and IL-23c). Considering the functional diversity of fish IL-23, it is possible that the fish-specific immunoregulatory mechanism of IL-23 may have existed. In this study, we isolated and identified the subunits of IL-23 from grass carp. In response to bacterial infection in vivo, the mRNA levels of gcp19 were upregulated in headkidney, liver and gill, while those of p40a were significantly induced in the selected tissues except liver. Surprisingly, the levels of gcp40b and gcp40c did not show marked change in tissues. What’s more, LPS, PHA stimulation in vitro, the mRNA levels of these subunits also showed a significant increase in periphery blood lymphocytes. Meanwhile, the result of Co-IP showed p19 could couple with each of the three p40 to form IL-23 and be secreted into the supernatants, verifying the existence of three IL-23 isoforms in grass carp. In order to assess their functions in fish immunity, we constructed plasmids containing gcp19 CDS combined with gcp40a/b/c CDS by a linker sequence and over-expressed them in CHO cells. The positive stable transfectants were selected, and the recombinant proteins were produced and purified from the culture medium. At present, we have gained these grass carp IL-23 proteins. Their bioactivities were investigated in grass carp headkidney cells, showing same or different functions in the immune regulation of grass carp. Finally, our work has provided a basis for elucidating the roles of IL-23 isoforms in fish immunity.