Course of Biochemistry and Applied Biosciences, Graduate School of Agriculture, University of Miyazaki, 1-1 Gakuen kibanadai-nishi, Miyazaki, 889-2192, Japan.
Interdisciplinary Graduate School of Agriculture and Engineering, University of Miyazaki, 1-1 Gakuen kibanadai-nishi, Miyazaki, 889-2192, Japan.
Fish Shellfish Immunol. 2020 Oct;105:438-445. doi: 10.1016/j.fsi.2020.07.009. Epub 2020 Jul 9.
Currently, circadian regulation of immune molecules in lower vertebrates, particularly, diurnal oscillation in the immune status of a fish, is not well understood. In this study, the diurnal oscillation of toll-like receptor (Tlr) 9, which plays a role in pathogen recognition, was investigated in the Japanese medaka fish (Oryzias latipes). We confirmed the expression of tlr9 and clock genes (bmal1 and clock1) in the central and peripheral tissues of medaka. These genes were expressed in a diurnal manner in medaka acclimated to a 12-h:12-h light-dark (12:12 LD) cycle. In addition, increased tlr9 expression was detected in medaka embryo cells (OLHdrR-e3) overexpressing both bmal1 and clock1 genes; however, this result was not obtained when only one or neither of the genes was overexpressed. This suggests that the increase in expression was mediated by the Bmal1 and Clock1 proteins together. In vitro stimulation of the head kidney with CpG-oligodeoxynucleotides (CpG-ODNs) at different zeitgeber times (ZTs; ZT0 = light on, ZT12 = light off) affected the degree of tlr9 gene expression, showing high and low responsiveness to CpG-ODN stimulation at ZT6/10 and ZT18/22, respectively. Similarly, bacterial infection at different ZT points induced a difference in the expression of Tlr9 signaling pathway-related genes (tlr9 and myd88). These results suggested that fish tlr9 exhibits diurnal oscillation, which is regulated by clock proteins, and its responsiveness to immune-stimulation/pathogen infection depends on the time of the day.
目前，较低等脊椎动物（尤其是鱼类）的免疫分子的昼夜节律调节机制尚不清楚，鱼类的免疫状态是否存在昼夜节律波动也尚不明确。在这项研究中，我们研究了日本青鳉（Oryzias latipes）中参与病原体识别的 toll 样受体（Tlr）9 的昼夜节律波动。我们证实了 tlr9 和生物钟基因（bmal1 和 clock1）在青鳉中央和外周组织中的表达具有昼夜节律性，并且在适应 12:12 LD 光暗循环的青鳉中这些基因呈昼夜节律性表达。此外，在过表达 bmal1 和 clock1 基因的青鳉胚胎细胞（OLHdrR-e3）中检测到 tlr9 表达增加，但当仅过表达一个或两个基因时，均未观察到该结果。这表明表达的增加是由 Bmal1 和 Clock1 蛋白共同介导的。在不同光照时间（ZT；ZT0=光照开始，ZT12=光照结束）下用 CpG-寡脱氧核苷酸（CpG-ODN）体外刺激头肾，影响 tlr9 基因表达的程度，分别在 ZT6/10 和 ZT18/22 时对 CpG-ODN 刺激具有高和低的反应性。类似地，在不同的 ZT 点进行细菌感染诱导了 Tlr9 信号通路相关基因（tlr9 和 myd88）表达的差异。这些结果表明，鱼类 tlr9 表现出昼夜节律波动，该波动受生物钟蛋白调节，其对免疫刺激/病原体感染的反应性取决于一天中的时间。
