Li Shuo, Chen Xiaoli, Hao Gaixiang, Geng Xuyun, Zhan Wenbin, Sun Jinsheng
Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin 300387, China.
Tianjin Key Laboratory of Animal and Plant Resistance, College of Life Sciences, Tianjin Normal University, 393 West Binshui Road, Xiqing District, Tianjin 300387, China.
Fish Shellfish Immunol. 2016 Mar;50:79-90. doi: 10.1016/j.fsi.2016.01.029. Epub 2016 Jan 25.
Nucleotide oligomerization domain (NOD)-like receptor (NLR) family with a caspase activation and recruitment domain (CARD) containing 3 (NLRC3) protein is an important cytosolic pattern recognition receptor that negatively regulates innate immune response in mammals. Hitherto, the immunological significance of NLRC3 protein in fish remains largely uncharacterized. Here we identified and characterized a novel NLRC3 gene (named poNLRC3) implicated in regulation of fish innate immunity from Japanese flounder Paralichthys olivaceus. The poNLRC3 protein is a cytoplasmic protein with an undefined N-terminal domain, a NACHT domain, a fish-specific NACHT associated domain, six LRR motifs, and a C-terminal fish-specific PYR/SPYR (B30.2) domain but only shares less than 40% sequence identities with the known Japanese flounder NLRC proteins. poNLRC3 gene is ubiquitously expressed in all tested tissues and is dominantly expressed in the Japanese flounder head kidney macrophages (HKMs). We for the first time showed that poNLRC3 expression was significantly modulated by the stimulation of extracellular ATP, an important danger/damage-associated molecular pattern in activating innate immunity in P. olivaceus. Importantly, we revealed that poNLRC3 plays an important role in positively regulating ATP-induced IL-1beta and IL-6 gene expression, suggesting the involvement of poNLRC3 in extracellular ATP-mediated immune signaling. In addition, we showed that poNLRC3 mRNA expression was up-regulated in response to LPS and Edwardsiella tarda immune challenges. Finally, we showed that down-regulating the endogenous poNLRC3 expression with small interfering RNA significantly reduced LPS-induced proinflammatory cytokine gene expression in the Japanese flounder HKM cells. Altogether, we have identified a novel inducible fish NLR member, poNLRC3, which is involved in extracellular ATP-mediated immune signaling and may positively regulate the LPS-induced innate immune response in the Japanese flounder HKM cells.
含半胱天冬酶激活和募集结构域（CARD）的核苷酸寡聚化结构域（NOD）样受体（NLR）家族成员3（NLRC3）蛋白是一种重要的胞质模式识别受体，在哺乳动物中负向调节先天免疫应答。迄今为止，NLRC3蛋白在鱼类中的免疫学意义仍 largely未被阐明。在此，我们从牙鲆（Paralichthys olivaceus）中鉴定并表征了一个与鱼类先天免疫调节相关的新型NLRC3基因（命名为poNLRC3）。poNLRC3蛋白是一种胞质蛋白，具有一个未定义的N端结构域、一个NACHT结构域、一个鱼类特异性的NACHT相关结构域、六个LRR基序以及一个C端鱼类特异性的PYR/SPYR（B30.2）结构域，但与已知的牙鲆NLRC蛋白的序列同一性小于40%。poNLRC3基因在所有检测组织中均有广泛表达，且在牙鲆头肾巨噬细胞（HKMs）中优势表达。我们首次表明，细胞外ATP（一种在激活牙鲆先天免疫中重要的危险/损伤相关分子模式）刺激可显著调节poNLRC3的表达。重要的是，我们揭示poNLRC3在正向调节ATP诱导的IL-1β和IL-6基因表达中起重要作用，表明poNLRC3参与细胞外ATP介导的免疫信号传导。此外，我们表明poNLRC3 mRNA表达在LPS和迟缓爱德华氏菌免疫刺激下上调。最后，我们表明用小干扰RNA下调内源性poNLRC3表达可显著降低牙鲆HKM细胞中LPS诱导的促炎细胞因子基因表达。总之，我们鉴定了一个新型的可诱导鱼类NLR成员poNLRC3，其参与细胞外ATP介导的免疫信号传导，并可能正向调节牙鲆HKM细胞中LPS诱导的先天免疫应答。
