Yang Zhi-Jing, Li Chang-Hong, Chen Jie, Zhang Hao, Li Ming-Yun, Chen Jiong
Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, China.
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.
Fish Shellfish Immunol. 2016 Oct;57:136-147. doi: 10.1016/j.fsi.2016.08.022. Epub 2016 Aug 18.
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.
哺乳动物白细胞介素4（IL-4）和白细胞介素13（IL-13）分子是介导巨噬细胞替代性活化的抗炎细胞因子。然而，鱼类IL-4/13同源物在单核细胞/巨噬细胞（MO/MФ）极化中的作用仍不清楚。在本研究中，我们在功能上鉴定了草鱼（Ctenopharyngodon idella）中的一种IL-4/13B同源物，命名为CiIL-4/13B。多序列比对显示CiIL-4/13B与其他已知鱼类IL-4/13具有典型的特征和结构。系统发育分析表明，CiIL-4/13B在进化上与斑马鱼（Danio rerio）和鲤鱼（Cyprinus carpio）的IL-4/13B密切相关。CiIL-4/13B mRNA在检测的组织和外周血淋巴细胞（PBLs）中组成性表达，在PBLs中表达最高。嗜水气单胞菌感染后，CiIL-4/13B mRNA表达上调。重组CiIL-4/13B（rCiIL-4/13B）在大肠杆菌中过表达并纯化用于功能研究。使用制备的抗rCiIL-4/13B抗血清，蛋白质印迹分析表明草鱼血浆中的天然CiIL-4/13B是N-糖基化的。腹腔注射生物活性rCiIL-4/13B显著提高了草鱼对嗜水气单胞菌的存活率，并降低了组织细菌载量，剂量越高效果越好。生物活性rCiIL-4/13B处理降低了亚硝酸盐的产生和促炎细胞因子（IL-1β和TNF-α）的mRNA表达，同时增加了精氨酸酶活性和抗炎细胞因子（TGF-β和IL-10）的mRNA表达。与阴性对照相比，生物活性rCiIL-4/13B处理8小时后草鱼MO/MФ的吞噬作用没有显著变化，而生物活性rCiIL-4/13B处理24小时后显著抑制了吞噬作用。rCiIL-4/13B对MO/MФ吞噬作用的抑制作用可能是MO/MФ增殖的结果。总之，我们的结果表明CiIL-4/13B通过诱导替代性活化的MO/MФ对草鱼抵抗嗜水气单胞菌起到保护作用。
