Two interleukin-1β types reinforce innate immune response modulation in Japanese medaka, Oryzias latipes

Interleukin-1β is a pro-inflammatory cytokine with pluripotent roles in mediating immune response. In fish, IL-1β plays a major role in inflammatory responses to pathogenic infections, though its immunological functions through direct stimulation have not been studied in detail. In this study, two IL-1β genes (il1b1 and il1b2) from the Japanese medaka (Oryzias latipes) were identified and characterized. The broad immunostimulatory effects of recombinant IL-1β (OlrIL-1β) from Japanese medaka were explored using embryonic fibroblast-like cell line (OLHdrR-e3) and primary cultured kidney cells. Recombinant proteins were produced via prokaryotic expression based on predicted mature isoforms IL-1β1_Asp⁹⁵ (type II) and IL-1β2_Asp¹²¹ (type I) to investigate their distinct functional characteristics. Then, their immune-related gene expression, phagocytosis, and reactive oxygen species (ROS) production ability were evaluated in vitro and ex vivo. rIL-1β triggers the expression of not only pro-inflammatory cytokines such as il1b, tnfa, il6, il8, mcp1b, and csf1b, but also the antimicrobial peptide, def1b. Additionally, they upregulate the expression of cell specific markers, including macrophage- and granulocyte-specific genes mpeg1.1, mpx2, and epx, and nitric oxide synthase (NO) gene nos1. Moreover, medaka OlrIL-1β proteins exhibited robust phagocytosis and superoxide anion production ability than those in unstimulated control. In all evaluations, the OlrIL-1β proteins corresponding to the predicted cleavage sites exhibited strong immune activation, with type I OlIL-1β2_Asp¹²¹ demonstrating more potent response than type II. To our knowledge, this is the study to reveal the immunomodulatory functions of Japanese medaka IL-1β, highlighting its potential in immunotherapy for disease prevention and control in aquaculture.