IFN modulates the innate immune response via Toll-like receptors in green-spotted pufferfish (Tetraodon nigroviridis)

As an important endogenous cytokine in mammals, interferon-gamma (IFN gamma) primes and significantly enhances the recognition and response of TLRs to pathogen-associated molecular patterns (PAMPs) in leukocytes. Subsequently, a series of phenomena in the TLR signalling pathway are activated, including increased expression of TLR mRNA, activation of the downstream signalling pathway mediated by myeloid differentiation primary response protein 88 (MyD88)-tumour necrosis factor receptor-associated factor 6 (TRAF6), and nuclear translocation of the nuclear transcription factor NF-kappa B. There is only one IFN gamma isoform in mammals, while many fishes possess two IFN gamma genes, IFN gamma 1 (also called IFN gamma-rel) and IFN gamma 2. The aim of this work was to explore how these two IFN gamma isoforms regulate the TLR signalling pathway in green-spotted pufferfish (Tetraodon nigroviridis). Real-time quantitative PCR (RT-qPCR) was performed to detect the expression of TLR1, TLR2, TLR3, TLR5, TLR7, TLR8, TLR9, MyD88, and TFAF6 in T. nigroviridis after stimulation with recombinant IFN gamma 1 (rIFN gamma 1) or rIFN gamma 2. Western blot analysis showed that the expression of MyD88, TFAF6, and NF-kappa B gradually increased between 0.5 and 8 h after stimulation with rTnIFN gamma 1. However, following treatment with IFN gamma 2, the expression of NF-kappa B at the protein level initially increased rapidly, only to decline swiftly thereafter. Meanwhile, the expression of MyD88 and TFAF6 exhibited a fluctuating pattern of rapid initial decline, subsequent rapid increase, and subsequent rapid decline. Electrophoretic mobility shift assay (EMSA) analysis showed that there was a significant and rapid shift of NF-kappa B within 5 min soon after rIFN gamma 2 stimulation, while rIFN gamma 1 had no significant effect. In summary, we revealed that IFN gamma 1 and IFN gamma 2 modulate the innate immune response via TLRs in T. nigroviridis and their regulatory mechanisms are different and multileveled. IFN gamma 1 and IFN gamma 2 could be used as immunomodulators to fine-tune the immune response of T. nigroviridis in different ways.