González-Stegmaier Roxana, Romero Alex, Estepa Amparo, Montero Jana, Mulero Victoriano, Mercado Luis
Grupo de Marcadores Inmunológicos, Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile; Laboratorio de Biotecnología y Patología Acuática, Instituto de Patología Animal, Universidad Austral de Chile, Valdivia, Chile; Centro FONDAP, Interdisciplinary Center for Aquaculture Research (INCAR), Chile.
Laboratorio de Biotecnología y Patología Acuática, Instituto de Patología Animal, Universidad Austral de Chile, Valdivia, Chile; Centro FONDAP, Interdisciplinary Center for Aquaculture Research (INCAR), Chile.
Fish Shellfish Immunol. 2015 Jan;42(1):144-52. doi: 10.1016/j.fsi.2014.10.034. Epub 2014 Nov 7.
Flagellin is the principal component of flagellum in Gram negative and positive bacteria, and it is also the ligand that activates the Toll-like receptor 5 (TLR5) in mammals and fish. In higher vertebrates, flagellin induces the activation of the membrane-bound TLR5 (TLR5M), which promotes the expression of proinflammatory cytokines and chemokines and the co-stimulatory molecules present in antigen-presenting cells needed for the activation of T cells. In the present study, we report the production of two recombinant proteins of Vibrio anguillarum: i) a full length flagellin B (FlaB) (rFla) and ii) the amino-terminus of the D1 domain (rND1) of the same protein, the region mainly responsible for binding to TLR5 and for the immunostimulatory activity of flagellin. The effects of these recombinant proteins were assessed in vitro using head kidney macrophages of gilthead seabream (Sparus aurata L., Perciformes, Sparidae) and rainbow trout (Oncorhynchus mykiss W., Salmoniformes, Salmonidae). In both species, 3 h of stimulation with rFla and rND1 induced expression of the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and of the chemokine IL-8. In gilthead seabream macrophages stimulated with rFla and rND1, a 900- and 6-fold increase were observed for IL-1β transcription, while a 900- and 3-fold increase were recorded for IL-8 transcription, respectively, as compared to non-stimulated macrophages. In rainbow trout, rFla increased expression of IL-8 40-fold in macrophages, whereas rND1 increased expression of the chemokine 3-fold, as compared to non-stimulated cells. The results obtained for rFla and rND1 demonstrate their modulatory capabilities in vitro, suggesting that rFla and rND1 could be evaluated as immunostimulatory candidates for use in farmed fish. However, further in vivo studies are needed to confirm and expand on the present results.
鞭毛蛋白是革兰氏阴性菌和阳性菌鞭毛的主要成分，也是激活哺乳动物和鱼类Toll样受体5（TLR5）的配体。在高等脊椎动物中，鞭毛蛋白诱导膜结合型TLR5（TLR5M）的激活，促进促炎细胞因子、趋化因子以及T细胞激活所需的抗原呈递细胞中存在的共刺激分子的表达。在本研究中，我们报道了两种鳗弧菌重组蛋白的产生：i）全长鞭毛蛋白B（FlaB）（rFla）和ii）同一蛋白D1结构域的氨基末端（rND1），该区域主要负责与TLR5结合以及鞭毛蛋白的免疫刺激活性。使用金头鲷（Sparus aurata L.，鲈形目，鲷科）和虹鳟（Oncorhynchus mykiss W.，鲑形目，鲑科）的头肾巨噬细胞在体外评估了这些重组蛋白的作用。在这两个物种中，用rFla和rND1刺激3小时诱导了促炎细胞因子白细胞介素-1β（IL-1β）和肿瘤坏死因子-α（TNF-α）以及趋化因子IL-8的表达。在用rFla和rND1刺激的金头鲷巨噬细胞中，与未刺激的巨噬细胞相比，IL-1β转录分别增加了900倍和6倍，而IL-8转录分别增加了900倍和3倍。在虹鳟中，与未刺激的细胞相比，rFla使巨噬细胞中IL-8的表达增加了40倍，而rND1使趋化因子的表达增加了3倍。rFla和rND1的结果证明了它们在体外的调节能力，表明rFla和rND1可作为养殖鱼类免疫刺激候选物进行评估。然而，需要进一步的体内研究来证实和扩展目前的结果。
