Immune correlates of NF-κB and TNFα promoter DNA methylation in Japanese flounder (Paralichthys olivaceus) muscle and immune parameters change response to vibrio anguillarum infection

Vibrio anguillarum infection can activate NF-kappa B/TNF alpha pathway in the immune organs of fish. Fish muscle is also an important immune organ, but the research on its immune function is few. Our aim was to study regulating mechanism of NF-kappa B and TNF alpha gene expressions in the muscle of Japanese flounder (Paralichthys olivaceus) which was under Vibrio anguillarum infection (0, 24, 48, 72 and 96 h). The results showed that the expressions of NF-kappa B and TNF alpha increased significantly at 48 h, and there was a significant positive correlation between them. In situ hybridization confirmed the co-existence of NF-kappa B and TNF alpha genes in Japanese flounder muscle. Interestingly, the expression of the TNF alpha gene was regulated by the DNA methylation and its methylation level was negatively correlated with the expression. The lowest methylation level of TNF alpha occurred at 48 h under Vibrio anguillarum infection (P < 0.05). And more, when the fragment (-2122 similar to -730) was deleted on TNF alpha gene promoter, double luciferase activity was the highest, indicating that fragment (- 730-0) was the transcription factor binding region. The site (-78 similar to -69) on the fragment (- 730-0) binding NF-kappa B was mutated, and double luciferase activity decreased significantly. The results confirmed that the site (-78 similar to -69) was indeed an important binding site for NF-kappa B. In addition, the activity of TNF alpha in the serum of Japanese flounder changed with the prolongation of vibrio anguillarum infection, and the concentration of other immune factors such as ALP, ALT, AST and LDH also changed in the muscle under vibrio anguillarum infection. They all showed a trend of first increasing and then decreasing. Above studies implied that Japanese flounder responded to Vibrio anguillarum infection at the immune level with the change of its methylation status and the activation of transcription factor. By studying the mechanism of immune pathways, understanding the response to immune stress is great significant to the research of fish breeding for disease resistance.

Automated summary

Infection by Vibrio anguillarum can activate the NF-kappa B and TNF alpha pathway in the fish immune organs, leading to significant upregulation of these genes in the muscle of Japanese flounder. The expression of TNF alpha gene in the muscle is regulated by DNA methylation and its methylation level is negatively correlated with expression. The study highlighted the importance of understanding immune response mechanisms in fish breeding for disease resistance.