Molecular characterization of an interleukin-4/13B homolog in grass carp (Ctenopharyngodon idella) and its role in fish against Aeromonas hydrophila infection

• We characterized an IL-4/13B homolog (CiIL-4/13B) from grass carp.
• CiIL-4/13B mRNA expression responded to Aeromonas hydrophila infection.
• rCiIL-4/13B increased fish survival and deceased bacterial load during infection.
• Grass carp MO/MФ treated with rCiIL-4/13B underwent alternative activation.

Mammalian interleukin 4 (IL-4) and interleukin 13 (IL-13) molecules are anti-inflammatory cytokines mediating the alternative activation of macrophages. However, the role of fish IL-4/13 homologs in monocytes/macrophages (MO/MФ) polarization remains unclear. In this study, we have functionally identified an IL-4/13B homolog in grass carp (Ctenopharyngodon idella), which is termed as CiIL-4/13B. Multiple alignment showed that CiIL-4/13B shared the typical characteristics and structure with other known fish IL-4/13. Phylogenetic analysis showed that CiIL-4/13B is evolutionarily closely related to zebrafish (Danio rerio) and common carp (Cyprinus carpio) IL-4/13B. CiIL-4/13B mRNA was constitutively expressed in tissues and peripheral blood lymphocytes (PBLs) examined, with its highest expression seen in PBLs. Following Aeromonas hydrophila infection, CiIL-4/13B mRNA expression was upregulated. Recombinant CiIL-4/13B (rCiIL-4/13B) was overexpressed in Escherichia coli and purified for a functional study. Using prepared anti-rCiIL-4/13B antiserum, Western blot analysis showed that native CiIL-4/13B in grass carp plasma is N-glycosylated. Intraperitoneal injection of bioactive rCiIL-4/13B significantly increased the survival rate of grass carp against A. hydrophila, and decreased the tissue bacterial load, with a higher dose having better effects. Bioactive rCiIL-4/13B treatment decreased nitrite production and mRNA expression of proinflammatory cytokines (IL-1β and TNF-α), while it increased arginase activity and mRNA expression of anti-inflammatory cytokines (TGF-β and IL-10). The phagocytosis by grass carp MO/MФ had no significant changes by the 8 h treatment of bioactive rCiIL-4/13B compared to that of the negative control, while it was significantly inhibited by the 24 h treatment of bioactive rCiIL-4/13B. The inhibitory effect of rCiIL-4/13B on MO/MФ phagocytosis may be a consequence of MO/MФ proliferation. In summary, our results suggest that CiIL-4/13B plays a protective effect in grass carp against A. hydrophila by inducing alternatively activated MO/MФ.