Wang Sutian, Deng Shoulong, Cao Yang, Zhang Rui, Wang Zhixian, Jiang Xiaojing, Wang Jiahao, Zhang Xiaosheng, Zhang Jinlong, Liu Guoshi, Lian Zhengxing
Beijing Key Laboratory for Animal Genetic Improvement, National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, Beijing, China.
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
Cell Physiol Biochem. 2018;49(2):662-677. doi: 10.1159/000493032. Epub 2018 Aug 30.
BACKGROUND/AIMS: Phagocytosis of bacteria by monocytes/macrophages can trigger the immune response and the clearance of bacteria. This innate immune response involves Toll-like receptor 4 (TLR4). However, much remains unknown about the mechanism of TLR4-regulated phagocytosis of Salmonella enterica serovar Typhimurium (S. typhimurium) within sheep monocytes/macrophages. Here, we aimed to address these knowledge gaps by infecting transgenic sheep overexpressing TLR4 with S. typhimurium and examining the phagocytic mechanisms involved.
Transgenic sheep were generated by microinjection of the constructed plasmids into fertilized eggs. Monocytes/macrophages isolated from sheep blood were stimulated with LPS and S. typhimurium. Phagocytosis-related factor expression, phagocytic ability, and adhesion were then determined. TLR4/phosphatidylinositide 3-kinase (PI3K) signaling was inhibited to investigate if this pathway is involved in changes in bacterial internalization in sheep.
We found that TLR4 overexpression effectively activated the PI3K signaling pathway and upregulated the expression of scavenger receptors. Additionally, actin polymerization and adhesive capacity were both enhanced in TLR4-overexpressing sheep monocytes/macrophages. TLR4 inhibition decreased S. typhimurium phagocytosis by reducing the actin polymerization and adhesive capacity of cells. Furthermore, inhibition of PI3K markedly impaired TLR4-dependent phagocytosis by restraining actin polymerization and scavenger receptor expression and reduced the adhesive capacity of the monocytes/macrophages.
Our findings indicate that overexpression of TLR4 enhances phagocytosis through PI3K signaling and the subsequent activation of actin polymerization and scavenger receptors in sheep monocytes/macrophages infected with S. typhimurium.
背景/目的：单核细胞/巨噬细胞对细菌的吞噬作用可触发免疫反应并清除细菌。这种先天性免疫反应涉及Toll样受体4（TLR4）。然而，关于TLR4调节绵羊单核细胞/巨噬细胞内鼠伤寒沙门氏菌吞噬作用的机制仍知之甚少。在此，我们旨在通过用鼠伤寒沙门氏菌感染过表达TLR4的转基因绵羊并研究其中涉及的吞噬机制来填补这些知识空白。
通过将构建的质粒显微注射到受精卵中来培育转基因绵羊。用脂多糖（LPS）和鼠伤寒沙门氏菌刺激从绵羊血液中分离出的单核细胞/巨噬细胞。然后测定吞噬相关因子的表达、吞噬能力和黏附情况。抑制TLR4/磷脂酰肌醇3激酶（PI3K）信号传导以研究该途径是否参与绵羊体内细菌内化的变化。
我们发现TLR4的过表达有效激活了PI3K信号通路并上调了清道夫受体的表达。此外，过表达TLR4的绵羊单核细胞/巨噬细胞中肌动蛋白聚合和黏附能力均增强。抑制TLR4可通过降低细胞的肌动蛋白聚合和黏附能力来减少鼠伤寒沙门氏菌的吞噬作用。此外，抑制PI3K可通过抑制肌动蛋白聚合和清道夫受体表达显著损害TLR4依赖性吞噬作用，并降低单核细胞/巨噬细胞的黏附能力。
我们的研究结果表明，TLR4的过表达通过PI3K信号传导以及随后在感染鼠伤寒沙门氏菌的绵羊单核细胞/巨噬细胞中激活肌动蛋白聚合和清道夫受体来增强吞噬作用。
