鲀形目鱼类 NLRP3 炎性小体基因的分子特征与功能研究。

Molecular characterization and functional study of the NLRP3 inflammasome genes in Tetraodon nigroviridis.

机构信息

State Key Laboratory of Biocontrol and School of Life Sciences, Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Guangdong Provincial Key Laboratory for Aquatic Economic Animals and Guangdong Provincial Engineering Technology Research Center for Healthy Breeding of Important Economic Fish, Sun Yat-Sen University, Guangzhou, 510275, PR China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266373, PR China; College of Ocean, Hainan University, Haikou, 570228, PR China.

出版信息

Fish Shellfish Immunol. 2022 Dec;131:570-581. doi: 10.1016/j.fsi.2022.10.017. Epub 2022 Oct 17.

DOI:10.1016/j.fsi.2022.10.017

PMID:36257557

Abstract

Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is an important inflammasome in mammals, which is of great significance to eliminate pathogens. However, the research of the NLRP3 inflammasome in teleost is limited. Tetraodon nigroviridis has the characteristics of small genome and easy feeding, which can be used as a model for the study of fish immune function. In present study, three NLRP3 inflammasome component genes (NLRP3, ASC and caspase-1) in T. nigroviridis has been cloned. Real-time fluorescence quantitative PCR showed that TnNLRP3 (T. nigroviridis NLRP3), TnASC (T. nigroviridis ASC) and Tncaspase-1 (T. nigroviridis caspase-1) mRNA in various tissues from health T. nigroviridis were highly expressed in immune-related tissues, such as spleen, gill, head kidney and intestine. After Vibrio parahemolyticus infection, the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA in spleen, gill, head kidney reached a peak at 24 h, and the expression levels of these genes in intestine were the highest at 48 h. After the transfection of TnASC-pAcGFP-N1 monomer GFP plasmid into cos-7 cells, ASC specks, the activation marker of NLRP3 inflammasome, were observed. Bimolecular fluorescence complementarity and fluorescence colocation experiment showed that TnASC and Tncaspase-1 of TnNLRP3 inflammasome were co-located near the cell nucleus, and potentially interacted with each other. NLRP3 inflammasome inducer nigericin and agonist ATP could significantly induce the expression of TnNLRP3, TnASC and Tncaspase-1 mRNA, and activation of NLRP3 inflammasome could promote the generation of mature TnIL-1β (T. nigroviridis IL-1β). These results uncover that T. nigroviridis NLRP3 inflammasome could participate in the antibacterial immune response and the generation of mature TnIL-1β after activation.

摘要

核苷酸结合寡聚化结构域样受体蛋白 3（NLRP3）炎症小体是哺乳动物中重要的炎症小体，对于消除病原体具有重要意义。然而，硬骨鱼类 NLRP3 炎症小体的研究有限。圆斑星鲽基因组小，易于饲养，可以作为鱼类免疫功能研究的模型。本研究克隆了圆斑星鲽中三个 NLRP3 炎症小体组成基因（NLRP3、ASC 和 caspase-1）。实时荧光定量 PCR 显示健康圆斑星鲽各组织中 TnNLRP3（圆斑星鲽 NLRP3）、TnASC（圆斑星鲽 ASC）和 Tncaspase-1（圆斑星鲽 caspase-1）mRNA 在免疫相关组织中高表达，如脾脏、鳃、头肾和肠道。副溶血弧菌感染后，脾脏、鳃、头肾中 TnNLRP3、TnASC 和 Tncaspase-1 mRNA 的表达在 24 h 达到峰值，而这些基因在肠道中的表达在 48 h 时最高。转染 TnASC-pAcGFP-N1 单体 GFP 质粒到 cos-7 细胞后，观察到 ASC 斑点，即 NLRP3 炎症小体的激活标志物。双分子荧光互补和荧光共定位实验表明，圆斑星鲽 NLRP3 炎症小体中的 TnASC 和 Tncaspase-1 位于细胞核附近，可能相互作用。NLRP3 炎症小体诱导剂 Nigericin 和激动剂 ATP 可显著诱导 TnNLRP3、TnASC 和 Tncaspase-1 mRNA 的表达，并激活 NLRP3 炎症小体可促进成熟的 TnIL-1β（圆斑星鲽 IL-1β）的产生。这些结果表明，圆斑星鲽 NLRP3 炎症小体在激活后可参与抗菌免疫反应和成熟 TnIL-1β 的产生。