The exploration of the immune mechanism of interferon-γ from Nibea albiflora and the functional characteristics of its receptor 2.

Interferon-gamma (IFN-γ) serves as an inflammatory cytokine essential for modulating innate and cell-mediated immune responses by associating with a receptor complex composed of IFNGR1 and IFNGR2. In this research, the entire cDNA of IFNGR2 from Nibea albiflora was cloned and functionally analyzed (referred to as NaIFNGR2), with the complete cDNA sequence measuring 924 bp and encoding 307 amino acids. The phylogenetic analysis and multiple sequence alignment revealed a significant similarity of NaIFNGR2 with homologous sequences found in other bony fish, especially within the FNⅢ domain and the transmembrane region. Real-time PCR analysis revealed that NaIFNGR2 was consistently expressed across all examined tissues, including the head-kidney, spleen, liver, kidney, gill, muscle, and blood, with the highest levels found in the gills. Following stimulation with Polyinosinic-polycytidylic acid (Poly (I:C)), Vibrio alginolyticus, or Vibrio parahaemolyticus, the mRNA levels of NaIFNGR2 showed an increase in a time-dependent manner. Subcellular localization studies indicated that NaIFNGR2 resided on the cell membrane and NaIFN-γ, once synthesized within the cell, was transported to the membrane to interact with NaIFNGR2. Additionally, NaIFNGR1 and NaIFNGR2 were completely co-localized on the cell membrane, which was consistent with the findings that NaIFNGR1 and NaIFNGR2 could form a heterodimeric complex. Being treated with the NaIFN-γ recombinant protein, both alone and in combination with LPS, various concentrations of NaIFN-γ were non-toxic to the growth of RAW 264.7 macrophages and significantly promoted their proliferation. The expression levels of IL-1β, IL-6, and TNF-α proteins were markedly upregulated in a concentration- and time-dependent manner following 24 and 48 h of combined stimulation. Furthermore, the secretion of nitric oxide (NO) was significantly increased after 36 and 48 h of stimulation, highlighting the regulatory effect of NaIFN-γ on macrophages and its influence on the inflammatory response. Collectively, the findings indicated that the NaIFN-γ ligand-receptor system was present in N. albiflora and played a crucial role in the immune response to pathogenic bacterial infections, enhancing our comprehension of the function of IFN-γ within the immunomodulatory processes of teleosts.