Molecular characterization and antibacterial function of the interleukin-8 in humpback grouper (Cromileptes altivelis)

Interleukin-8 (IL8) is a key factor in the promotion and regulation of vertebrate inflammatory and immune responses. In this study, a novel interleukin-8 ( Ca IL8) isolated from the humpback grouper ( Cromileptes altivelis ) was successfully cloned and characterized. The gene comprises a 375 bp open reading frame, encoding a protein of 124 amino acids with an estimated molecular weight of 11.2 kDa and contains four conserved cysteine residues common in other known IL8s. Phylogenetic analysis revealed that Ca IL8 is most closely related to IL8 from Epinephelus lanceolatus , and that both group within a fish-specific IL8 clade. Expression analysis demonstrated that CaIL8 is ubiquitously expressed in all the tested tissues from healthy humpback groupers, with high expression levels in the blood, intestine, brain, and liver. Upon infection with Vibrio harveyi , CaIL8 expression was upregulated in various tissues, and the recombinant Ca IL8 (r Ca IL8) exhibited significant binding to and antibacterial activity against the tested bacterial strains. Furthermore, r Ca IL8 enhanced the chemotaxis and proliferation of the head kidney leukocytes and increased the phagocytic activity of macrophages. In vivo experiments indicated that overexpression of Ca IL8 enhances the fish's ability to inhibit V. harveyi replication, while knocking it down reduces antibacterial capacity. This suggests that in teleosts, Ca IL8 acts as a proinflammatory cytokine, activating immune cells against bacterial infections.