Su Bor-Chyuan, Lin Wen-Chun, Huang Han-Ning, Chen Jyh-Yih
Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, 23-10 Dahuen Road, Jiaushi, Ilan 262, Taiwan.
Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, 23-10 Dahuen Road, Jiaushi, Ilan 262, Taiwan.
Fish Shellfish Immunol. 2017 May;64:276-286. doi: 10.1016/j.fsi.2017.03.032. Epub 2017 Mar 18.
Serum amyloid A (SAA) is an acute-phase protein that plays a crucial role in the inflammatory response. In this study, we identified an SAA homolog from Epinephelus lanceolatus (ElSAA). Molecular characterization revealed that ElSAA contains a fibronectin-like motif that is typical of SAAs. Recombinant ElSAA protein (rElSAA) was produced in E. coli BL21 (DE3) cells and purified as a soluble protein. To analyze its biological activity, mouse Raw264.7 macrophage cells were treated with various concentrations of rElSAA. Expression of several inflammation-related cytokines, including tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6, and IL-10, was induced by rElSAA. This protein also triggered macrophage differentiation, as evidenced by increases in cell size and complexity. To determine whether rElSAA regulates macrophage polarization, we assessed gene expression of M1 and M2 markers. The results demonstrated that rElSAA induced the expression of both M1 and M2 markers, suggesting that it promotes the differentiation of macrophages into a mixed M1/M2 phenotype. To evaluate whether rElSAA enhances phagocytosis via an opsonization-dependent mechanism, GFP-labeled E. coli cells were pretreated with rElSAA, followed by incubation with Raw264.7 cells. Flow cytometry was used to monitor the phagocytic uptake of GFP-labeled E. coli by macrophages. Surprisingly, incubating E. coli with rElSAA did not enhance bacterial uptake by macrophages. However, preincubating Raw264.7 cells with various concentrations of rElSAA, followed by infection with E. coli (multiplicity of infection = 20 or 40), resulted in a clear enhancement of macrophage phagocytic capacity. In conclusion, we have identified SAA from E. lanceolatus and have demonstrated that rElSAA promotes inflammatory cytokine production and macrophage differentiation. In addition, rElSAA enhances phagocytosis of bacteria by macrophages via an opsonization-independent mechanism.
血清淀粉样蛋白A（SAA）是一种急性期蛋白，在炎症反应中起关键作用。在本研究中，我们从波纹唇鱼中鉴定出一种SAA同源物（ElSAA）。分子特征表明，ElSAA含有一个SAA典型的纤连蛋白样基序。重组ElSAA蛋白（rElSAA）在大肠杆菌BL21（DE3）细胞中产生，并作为可溶性蛋白进行纯化。为了分析其生物学活性，用不同浓度的rElSAA处理小鼠Raw264.7巨噬细胞。rElSAA诱导了几种炎症相关细胞因子的表达，包括肿瘤坏死因子α（TNF-α）、白细胞介素（IL）-1β、IL-6和IL-10。这种蛋白还引发了巨噬细胞分化，细胞大小和复杂性增加证明了这一点。为了确定rElSAA是否调节巨噬细胞极化，我们评估了M1和M2标志物的基因表达。结果表明，rElSAA诱导了M1和M2标志物的表达，表明它促进巨噬细胞分化为混合的M1/M2表型。为了评估rElSAA是否通过调理作用依赖性机制增强吞噬作用，用rElSAA预处理绿色荧光蛋白（GFP）标记的大肠杆菌细胞，然后与Raw264.7细胞孵育。流式细胞术用于监测巨噬细胞对GFP标记的大肠杆菌的吞噬摄取。令人惊讶的是，将大肠杆菌与rElSAA孵育并没有增强巨噬细胞对细菌的摄取。然而，用不同浓度的rElSAA预孵育Raw264.7细胞，然后用大肠杆菌感染（感染复数=20或40），导致巨噬细胞吞噬能力明显增强。总之，我们从波纹唇鱼中鉴定出了SAA，并证明rElSAA促进炎症细胞因子的产生和巨噬细胞分化。此外，rElSAA通过一种非调理作用依赖性机制增强巨噬细胞对细菌的吞噬作用。
