Retinol-binding protein 4 (RBP4) is known as a highly conserved adipokine for immune activation. Aeromonas hydrophila (A. hydrophila) is the most common zoonotic pathogen in aquaculture, which causes serious economic losses to aquaculture, especially to bighead carp (Hypophthalmichthys nobilis, H. nobilis) and silver carp (Hypophthalmichthys molitrix, H. molitrix). Recent studies along with our previous findings have shown that synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN) can play a good role in aquatic animals against infection. In order to clarify the relationship between CpG ODN and RBP4 under A. hydrophila infection, firstly, full-length RBP4 cDNAs from H. nobilis and H. molitrix were cloned. And characteristics of RBP4, including sequence and structure, tissue distribution and genetic evolution were analyzed. In addition, mRNA expression levels of RBP4, cytokine, toll-like receptors (TLRs), morbidity and survival rates of H. nobilis and H. molitrix were observed post CpG ODN immunization or following challenge. The results indicated that hn/hm_RBP4 (RBP4 genes obtained from H. nobilis and H. molitrix) had the highest homology with Megalobrama amblycephala. Distribution data showed that the expression level of hn_RBP4 mRNA was higher than that of hm_RBP4. After CpG ODN immunization followed by A.hydrophila challenge, significantly higher survival was observed in both carps, together with up-regulated RBP4 expression. Meanwhile, hn/hm_IL-1β level was relatively flat (and decreased), hn/hm_IFN-γ, hn/hm_TLR4 and hn/hm_TLR9 levels increased significantly, but hn/hm_STRA6 showed no significant change, compared with control. Moreover, CpG ODN immunization could induce stronger immune protective responses (higher IFN-γ/gentle IL-1β level and lower morbidity/higher survival rate) against A. hydrophila in H. nobilis, along with higher RBP4 level, when compared with that in H. molitrix. These results demonstrated that RBP4 was well involved in the immune protection of CpG ODN. Based on the results, we speculated that in the case of A. hydrophila infection, TLR9 signaling pathway was activated by CpG ODN. Subsequently, CpG ODN up-regulated RBP4, and RBP4 activated TLR4 signaling pathway. Then TLR4 and TLR9 synergistically improved the anti-infection responses. Our findings have good significance for improving resistance to pathogen infection in freshwater fish.

视黄醇结合蛋白4（RBP4）被称为是高度保守的用于免疫激活的脂肪因子。嗜水气单胞菌（A. hydrophila）是水产养殖中最常见的人畜共患病原体，给水产养殖造成严重的经济损失，尤其是big鱼（Hypophthalmichthys nobilis，H. nobilis）和silver鱼（Hypophthalmichthys molitrix，H. molitrix）。最近的研究以及我们以前的发现表明，含有CpG模体（CpG ODN）的合成寡脱氧核苷酸可以在水生动物抗感染中发挥良好作用。为了澄清下的CpG ODN和RBP4之间的关系嗜水感染，首先，全长的cDNA RBP4从H.扇贝和克隆了H. molitrix。并分析了RBP4的特征，包括序列和结构，组织分布和遗传进化。此外，在CpG ODN免疫后或攻击后，观察到了H. nobilis和H. nobilis的RBP4，细胞因子，toll​​样受体（TLRs）的mRNA表达水平，发病率和存活率。结果表明，HN / hm_RBP4（获自RBP4基因H.扇贝和H.鲢）具有最高的同源性团头鲂。分布数据表明，hn_RBP4 mRNA的表达水平高于hm_RBP4。在对CpG ODN进行免疫后，再进行嗜水杆菌挑战中，在两个鲤鱼中观察到明显更高的存活率，以及上调的RBP4表达。同时，hn /hm_IL-1β水平相对平稳（下降），hn /hm_IFN-γ，hn / hm_TLR4和hn / hm_TLR9水平明显升高，但hn / hm_STRA6与对照组相比无明显变化。此外，CpG寡核苷酸的免疫能诱导针对更强的免疫保护性应答（更高的IFN-γ/温和IL-1β水平和较低的发病率/较高的存活率）嗜水气在H.扇贝当与在比较，具有较高的RBP4水平，沿H. molitrix。这些结果证明RBP4很好地参与了CpG ODN的免疫保护。根据结果​​，我们推测在嗜水气单胞菌的情况下感染后，CpG ODN激活了TLR9信号通路。随后，CpG ODN上调了RBP4，并且RBP4激活了TLR4信号通路。然后TLR4和TLR9协同改善抗感染反应。我们的发现对于提高淡水鱼对病原体感染的抵抗力具有重要意义。