Molecular cloning of Japanese eel Anguilla japonica TNF-α and characterization of its expression in response to LPS, poly I:C and Aeromonas hydrophila infection

As a potent pleiotropic cytokine, tumor necrosis factor-alpha (TNF-alpha) plays an important role in innate immune responses. The cDNA sequence and genomic structure of the TNF-alpha gene (Aj TNF-alpha) in the Japanese eel (Anguilla japonica) were identified and characterized. The full-length AjTNF-alpha cDNA was 1 546 bp, including a 5'-untranslated region (UTR) of 13 bp, a 3'-UTR of 879 bp and an open reading frame of 654 bp encoding a protein of 218 amino acids. The full-length genomic sequence of AjTNF-alpha was 2 392 bp and included four exons and three introns. The putative AjTNF-alpha protein contained TNF family signature motifs, including a protease cleavage site, a transmembrane domain and two conserved cysteine residues. Quantitative real-time reverse transcription PCR analysis revealed AjTNF-alpha expression in a wide range of tissues, with predominant expression in blood and liver. Lower levels of expression were seen in spleen, gills, kidney, intestine, heart, and skin, with very low levels in muscle. The modulation of AjTNF-alpha expression after injection of eels with lipopolysaccharide (LPS), the viral mimic, poly I:C, or Aeromonas hydrophila was assessed in blood, liver, and kidney. In blood, TNF-alpha mRNA levels increased rapidly and then rapidly decreased after stimulation with LPS, poly I:C or A. hydrophila. However, the response to LPS and A. hydrophila peaked at 6 h while for poly I:C the peak was at 12 h. In liver, after injection with A. hydrophila, an up- and down-regulation of AjTNF-alpha expression occurred twice, peaking at 6 h and 24 h, respectively. No remarkable increase of AjTNF-alpha expression appeared in liver until 72 h after LPS or poly I:C treatment. In kidney, AjTNF-alpha expression increased significantly only at 72 h post-stimulation with LPS or A. hydrophila. Our results suggest that AjTNF-alpha plays an important role in fish in the defense against viral and bacterial infection.