Characterization of a new il-4/13 homologue in grass carp (Ctenopharyngodon idella) and its cooperation with M-CSF to promote macrophage proliferation.

Abstract

In this study, a new il-4/13 cDNA was isolated from grass carp (Ctenopharyngodon idella) using homologous cloning. The phylogenetic tree and sequence alignment of the deduced amino acid (aa) sequence showed that it was closer to grass carp il-4/13b (gcil-4/13b) than other homologues and therefore named gcil-4/13b-like (gcil-4/13bl). It has 399-nt coding sequence (CDS) which is less than gcil-4/13b (408\u202fnt). In addition, the cloned gcil-4/13bl gene is approximately 1,600 bp in length and has a conserved genetic structure consisting of four exons and three introns. Compared to gcil-4/13b gene, it has a variety of nucleotides variation across the CDS and contains a longer intron 3, suggesting that it is a new gcil-4/13 gene. The gcil-4/13bl transcripts were ubiquitously expressed in almost all selected tissues, and there was almost only gcil-4/13bl detected in brain and head kidney (HK). Recombinant grass carp (rgc) Il-4/13bl was prepared by using Escherichia coli (E. coli) Rosetta-gami 2 (DE3). The functional study demonstrated that rgcIl-4/13bl significantly upregulated arginase-2 gene expression and arginase activity, whilst downregulated nitric oxide (NO) production as well as the transcript levels of inducible nitric oxide synthesase (inos) and ifn-γ in freshly isolated grass carp HK monocytes/macrophages (M0/Mϕ). These data suggested that the newly cloned il-4/13bl had the conserved functions to activate M2-type but antagonize M1-type macrophages. Furthermore, rgcIl-4/13bl was able to drive the proliferation of M0/Mϕ which were pre-treated by rgcM-csf, indicating the involvement of gcIl-4/13bl in the proliferation of macrophages. Here we not only identified a new il-4/13-encoding gene in grass carp, but also for the first time revealed a novel function of fish Il-4/13 combined with M-csf engaging in M0/Mϕ proliferation.