title: Molecular characterization of an interleukin-4/13B homolog in grass carp (Ctenopharyngodon idella) and its role in fish against Aeromonas hydrophila infection.
abstract: 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.
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China.
authors.forename: Zhi-Jing
authors.initials: ZJ
authors.lastname: Yang
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China.
authors.forename: Chang-Hong
authors.initials: CH
authors.lastname: Li
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China; Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University, Ningbo 315211, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Chen
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China.
authors.forename: Ming-Yun
authors.initials: MY
authors.lastname: Li
authors.affiliation: Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China; Collaborative Innovation Center for Zhejiang Marine High-efficiency and Healthy Aquaculture, Ningbo University, Ningbo 315211, China. Electronic address: jchen1975@163.com.
authors.forename: Jiong
authors.initials: J
authors.lastname: Chen
mesh_terms: D016980:Aeromonas hydrophila / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D058491:Bacterial Load; D002347:Carps; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005393:Fish Diseases / Q000235:genetics / Q000276:immunology / Q000382:microbiology; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016905:Gram-Negative Bacterial Infections / Q000235:genetics / Q000276:immunology / Q000382:microbiology / Q000662:veterinary*; D007113:Immunity, Innate; D015847:Interleukin-4 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment / Q000662:veterinary
chemical_list: D018076:DNA, Complementary; D029941:Fish Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015847:Interleukin-4
keywords: Aeromonas hydrophila infection; Bacterial load; Grass carp; Interleukin-4/13B; M2 polarization; Monocyte/macrophage; Sequence analysis; Survival rate