Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, Aberdeen AB24 2TZ, UK.
Dev Comp Immunol. 2013 Oct;41(2):295-305. doi: 10.1016/j.dci.2013.05.013. Epub 2013 Jun 4.
Mammalian Toll-like receptor (TLR) 7 and 8 are responsible for recognizing viral single-stranded RNA (ssRNA) and are activated by anti-viral imidazoquinoline compounds, leading to a series of defensive mechanisms being launched to protect the host against viruses. In this study, we identified two TLR7 (with one probably a pseudogene) and three TLR8 genes, namely TLR8a2, TLR8b1 and TLR8b2 from Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs. Bioinformatics analysis showed that salmon TLR7 and TLR8a2 are closely related to the corresponding trout orthologs, however, salmon TLR8b1 and TLR8b2 share the highest amino acid sequence similarity to zebrafish TLR8b and formed a subfamily of the piscine TLR8 molecules in phylogenetic tree analysis. A conserved gene synteny was found with the salmon TLR7/8a members as seen in other vertebrate loci. Deduced domain organisation of salmon TLR7 and TLR8 molecules showed similar structural features, with equal numbers of leucine-rich repeats (LRRs) and insertion motifs. Individual TLR molecules were expressed in a similar pattern between parr and post-smolts, with a high expression level in immune tissues. Promoter analysis predicted several transcription factor binding sites in the TLR8a1/2 and TLR8b1 5' flanking regions, namely C/EBP, AP-1, STAT, NFκB, and IRF family, suggesting cytokine regulation of the genes. Hence, three recombinant cytokines, type I IFN, IFNγ and IL-1β were used to study the regulation of the salmon TLR gene expression levels in primary head kidney cells and the Salmon Head Kidney-1 (SHK-1) cell line. Salmon TLR7 and TLR8a1 gene expression was more sensitive to type I IFN and IFNγ treatment in primary head kidney cells and SHK-1 cells respectively, with no significant up-regulation of TLR8a2 and TLR8b2 by any of the treatments. On the other hand, salmon TLR8a1 and TLR8b1 were most sensitive to IL-1β treatment in SHK-1 cells and primary head kidney cells, respectively. TLR8b2 was undetectable in SHK-1 cells under these same conditions.
哺乳动物 Toll 样受体 (TLR) 7 和 8 负责识别病毒单链 RNA (ssRNA)，并被抗病毒咪唑并喹啉化合物激活，从而启动一系列防御机制，保护宿主免受病毒侵害。在这项研究中，我们从大西洋鲑 (Salmo salar) 全基因组鸟枪法 (WGS) 拼接体中鉴定出两个 TLR7（其中一个可能是假基因）和三个 TLR8 基因，即 TLR8a2、TLR8b1 和 TLR8b2。生物信息学分析表明，鲑鱼 TLR7 和 TLR8a2 与相应的鳟鱼同源物密切相关，然而，鲑鱼 TLR8b1 和 TLR8b2 与斑马鱼 TLR8b 的氨基酸序列相似性最高，并在系统进化树分析中形成了鱼类 TLR8 分子的一个亚家族。在与其他脊椎动物基因座的鲑鱼 TLR7/8a 成员中发现了保守的基因同线性。鲑鱼 TLR7 和 TLR8 分子的推导结构域组织显示出相似的结构特征，富含亮氨酸重复 (LRR) 和插入基序的数量相等。在幼鱼和成鱼阶段，个体 TLR 分子的表达模式相似，在免疫组织中表达水平较高。启动子分析预测了 TLR8a1/2 和 TLR8b1 5'侧翼区域的几个转录因子结合位点，即 C/EBP、AP-1、STAT、NFκB 和 IRF 家族，表明这些基因受到细胞因子的调控。因此，使用三种重组细胞因子，即 I 型干扰素、IFNγ 和 IL-1β，研究它们对鲑鱼 TLR 基因表达水平的调节作用，在原代头肾细胞和鲑鱼头肾细胞-1 (SHK-1) 细胞系中。在原代头肾细胞和 SHK-1 细胞中，鲑鱼 TLR7 和 TLR8a1 基因的表达对 I 型干扰素和 IFNγ 处理更为敏感，而任何一种处理都没有显著上调 TLR8a2 和 TLR8b2 的表达。另一方面，在 SHK-1 细胞和原代头肾细胞中，鲑鱼 TLR8a1 和 TLR8b1 对 IL-1β 处理最为敏感，而在相同条件下，TLR8b2 在 SHK-1 细胞中无法检测到。
