Department of Animal Biotechnology, Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Vepery High Road, Chennai 600 007, Tamil Nadu, India.
Fish Shellfish Immunol. 2012 Nov;33(5):1174-82. doi: 10.1016/j.fsi.2012.09.007. Epub 2012 Sep 24.
Sharks are a species of delight for immunologists from the evolutionary perspective since it is considered as the first species to have evolved the adaptive immune responses in addition to the innate immune system. One of the components of the highly conserved innate immune system is the toll-like receptors (TLR) which has a conserved overall protein structure throughout deuterostome evolution. There is no report that demonstrates the expression of these receptors in sharks. In this study we successfully amplified a 270 bp amplicon using a degenerate primer design strategy that corresponded to the Toll/IL-1 receptor (TIR) domain of TLR2 (GenBank ID: JF792813). BLAST analysis revealed a maximum nucleotide identity of 87% and 76% with the TLR2 of higher mammals and teleost fishes respectively. Domain prediction revealed a TIR structure between 1 and 87 amino acids that had a maximum identity of 58% and 76% with TLR2 - TIR protein of teleost fishes and higher mammals respectively. Phylogenetic analysis revealed a closer clustering of the shark TIR sequence with those from human, cattle, goat, sheep and chicken than with other fish species. Basal expression levels of the TLR2-TIR mRNA were found to be significantly higher in kidneys followed by fins, spleen and intestinal spiral valve (ISV). In tissues such as spleen and kidney the expression of the TLR2-TIR mRNA could be localized to lymphoid and macrophages like cells and tubular epithelial cells respectively. In-vivo exposure of sharks to peptidoglycan (TLR 2 ligand) resulted in 9 folds higher expression of TLR2-TIR mRNA in gills followed by 5 folds in the fins. However, when inoculated with a TLR ligand pool, the expression levels significantly increased to 12 fold in skin followed by epigonal, kidneys and ISV. These findings not only support the presence of the TLRs in sharks but also their induction upon exposure to specific ligands. Further studies are needed to identify their numbers, their ligand specificity and downstream cytokine responses.
从进化的角度来看，鲨鱼是免疫学家的研究对象，因为它被认为是除先天免疫系统之外，第一个进化出适应性免疫反应的物种。高度保守的先天免疫系统的一个组成部分是 Toll 样受体 (TLR)，它在后口动物进化过程中具有保守的整体蛋白结构。目前还没有报道表明这些受体在鲨鱼中表达。在这项研究中，我们使用简并引物设计策略成功扩增了一段 270bp 的扩增子，该策略对应于 TLR2 的 Toll/IL-1 受体 (TIR) 结构域（GenBank ID: JF792813）。BLAST 分析显示，与高等哺乳动物和硬骨鱼类的 TLR2 相比，最大核苷酸同一性分别为 87%和 76%。结构域预测显示，TIR 结构在 1 到 87 个氨基酸之间，与硬骨鱼类和高等哺乳动物的 TLR2-TIR 蛋白的最大同一性分别为 58%和 76%。系统发育分析显示，鲨鱼 TIR 序列与人类、牛、山羊、绵羊和鸡的聚类更为密切，而与其他鱼类的聚类则不密切。TLR2-TIR mRNA 的基础表达水平在肾脏中显著高于鳍、脾脏和肠螺旋瓣 (ISV)。在脾脏和肾脏等组织中，TLR2-TIR mRNA 的表达可分别定位于淋巴样细胞和巨噬样细胞以及管状上皮细胞。体内暴露于肽聚糖 (TLR2 配体) 可导致鲨鱼鳃中 TLR2-TIR mRNA 的表达增加 9 倍，鳍中增加 5 倍。然而，当接种 TLR 配体池时，皮肤中的表达水平显著增加到 12 倍，其次是肾上腺、肾脏和 ISV。这些发现不仅支持鲨鱼中 TLR 的存在，还支持它们在暴露于特定配体时的诱导。需要进一步研究以确定它们的数量、配体特异性和下游细胞因子反应。
