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As an important endogenous cytokine in mammals, interferon-γ (IFNγ) 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-κB. There is only one IFNγ isoform in mammals, while many fishes possess two IFNγ genes, IFNγ1 (also called IFNγ-rel) and IFNγ2. The aim of this work was to explore how these two IFNγ 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 IFNγ1 (rIFNγ1) or rIFNγ2. Western blot analysis showed that the expression of MyD88, TFAF6, and NF-κB gradually increased between 0.5 and 8 h after stimulation with rTnIFNγ1. However, following treatment with IFNγ2, the expression of NF-κB 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-κB within 5 min soon after rIFNγ2 stimulation, while rIFNγ1 had no significant effect. In summary, we revealed that IFNγ1 and IFNγ2 modulate the innate immune response via TLRs in T. nigroviridis and their regulatory mechanisms are different and multileveled. IFNγ1 and IFNγ2 could be used as immunomodulators to fine-tune the immune response of T. nigroviridis in different ways. [ABSTRACT FROM AUTHOR]