肥大细胞对重组口蹄疫病毒VP1-VP4应答的蛋白质表达谱检测

Protein expression profile of mast cells in response to recombinant VP1-VP4 of foot-and-mouth disease virus

摘要:

为揭示肥大细胞抗口蹄疫病毒VP1-VP4蛋白的天然免疫作用，以重组口蹄疫病毒VP1-VP4蛋白刺激小鼠腹腔肥大细胞 (Peritoneal mast cells, PMCs)，用高通量ELISA芯片检测PMCs的蛋白质表达谱。结果显示，VP1-VP4蛋白刺激的PMCs (VP1-VP4组) 表达CCL19、L-selectin、CCL17和TNF-α的水平极显著低于对照组 (PMCs) (P<0.001)，而VP1-VP4蛋白刺激经甘露糖受体 (Mannose receptor, MR) 抑制剂预处理的PMCs (MR组) 表达CCL19、IL-15、IL-9、G-CSF和Galectin-1的水平则极显著高于对照组 (P<0.01)，IL-10表达水平也有显著升高 (P<0.05)。MR组与VP1-VP4组相比，PMCs表达IL-10、IL-17、CCL20、IL-15、IL-9、L-selectin、CCL17、TNF-α和CCL19的水平极显著升高 (P<0.01)，CCL21和G-CSF的表达也显著高于VP1-VP4组 (P<0.05)。生物信息学差异表达分析结果显示，与对照组相比，VP1-VP4组PMCs表达的L-selectin和CCL17为下调性差异表达蛋白 (Log2(ratio)≤–1)。MR组与VP1-VP4相比，PMCs表达的CCL20、CCL19、L-selectin和IL-15为上调性差异表达蛋白 (Log2(ratio)≥1)。这表明，PMCs可自发分泌CCL19、L-selectin、CCL17和TNF-α，而VP1-VP4则对PMCs的天然免疫功能具有抑制作用。由于阻断MR后PMCs的蛋白质表达水平显著升高，所以VP1-VP4对小鼠PMCs的免疫抑制作用可能是由MR介导的。

Abstract:

To reveal the innate immunity of mast cells against recombinant VP1-VP4 protein of foot-and-mouth disease virus (FMDV), mouse peritoneal mast cells (PMCs) were pulsed with recombinant VP1-VP4 protein. The supernatants harvested from PMCs cultures were applied to the high throughput ELISA array. Our results show that the expression levels of CCL19, L-selectin, CCL17, and TNF alpha released from PMCs pulsed with recombinant VP1-VP4 were significantly down-regulated compared with PMCs alone (P<0.001). Surprisingly, in comparison with PMCs alone, the expression levels of CCL19, IL-15, IL-9, G-CSF, and Galectin-1 in PMCs with the mannose receptor (MR) inhibitor were significantly up-regulated (P<0.01), and the expression level of IL-10 was also remarkably up-regulated (P<0.05). Importantly, the protein expression levels in PMCs treated with MR inhibitor were higher than PMCs pulsed with VP1-VP4, including IL-10, IL-17, CCL20, IL-15, IL-9, L-selectin, CCL17, TNF alpha, and CCL19 (P<0.01) as well as CCL21, and G-CSF (P<0.05). Differential expression analysis in bioinformatics shows that both L-selectin and CCL17 were recognized as differentially expressed protein molecules (Log2(ratio)≤–1) when compared with PMCs alone. Furthermore, the up-regulation of the expression levels of CCL20, CCL19, L-selectin, and IL-15 in PMCs treated with MR inhibitor was defined as differential expression (Log2(ratio)≥1). These data indicate that PMCs are capable of secreting CCL19, L-selectin, CCL17, and TNF alpha spontaneously and the recombinant VP1-VP4 has an inhibitive potential to PMCs during their performance of innate immune response. Given the protein expression levels from PMCs pre-treated with MR inhibitor were significantly increased, it can be deduced that immunosuppression of FMDV is presumably initiated by the VP1 recognition of MR on mast cells.