Álvarez Claudio A, Ramírez-Cepeda Felipe, Santana Paula, Torres Elisa, Cortés Jimena, Guzmán Fanny, Schmitt Paulina, Mercado Luis
Grupo de Marcadores Inmunológicos, Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile.
Grupo de Marcadores Inmunológicos, Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile.
Mol Immunol. 2017 Jul;87:102-113. doi: 10.1016/j.molimm.2017.03.010. Epub 2017 Apr 19.
Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are efficient soluble intracellular sensors that activate defense mechanisms against pathogens. In teleost fish, the involvement of NLRs in the immune response is not well understood. However, recent work has evidenced the expression of different NLRs in response to some pathogen associated molecular patterns (PAMPs). In the present work, the cDNA sequence encoding three new NOD-like receptors were identified in Oncorhynchus mykiss, namely OmNLRC3, OmNLRC5 and OmNLRX1. Results showed that their sequences coded for proteins of 1135, 836 and 1010 amino acids, respectively. The deduced protein sequences of all receptors showed characteristic domains of this receptor family, such as leucine rich repeats and NACHT domain. Phylogenetic analysis revealed a high degree of identity with other NOD-like receptors and they are clustered into different families. Transcript expression analysis indicated that OmNLRs are constitutively expressed in liver, spleen, intestine, gill, skin and brain. OmNLR expression was upregulated in kidney and gills from rainbow trout in response to LPS. In order to give new insights into the function of these new NLR members, an in vitro model of immune stimulation was established using the rainbow trout cell line RTgill-W1. Expression analysis revealed that RTgill-W1 overexpressed proinflammatory cytokines in response to LPS and poly I:C alongside with a differential overexpression of OmNLRC3, OmNLRC5 and OmNLRX1. The expression of OmNLRC5 was further verified at the protein level by immunofluorescence. Finally, the effect of the overexpressed cytokines on the OmNLR expression by RTgill-W1 cells was assessed, suggesting a regulatory mechanism on OmNLRC3 expression. Overall, results suggest that O. mykiss NOD-like receptors could play a key role in the defense mechanisms of teleost through PAMP recognition. Future studies will focus on gills which could be related with a key sensor mucosal system in one of the most environmentally fish exposed tissues.
核苷酸结合寡聚化结构域（NOD）样受体（NLRs）是高效的可溶性细胞内传感器，可激活针对病原体的防御机制。在硬骨鱼中，NLRs在免疫反应中的作用尚不清楚。然而，最近的研究表明，不同的NLRs会对一些病原体相关分子模式（PAMPs）产生反应并表达。在本研究中，在虹鳟鱼中鉴定出编码三种新的NOD样受体的cDNA序列，即OmNLRC3、OmNLRC5和OmNLRX1。结果表明，它们的序列分别编码1135、836和1010个氨基酸的蛋白质。所有受体推导的蛋白质序列均显示出该受体家族的特征结构域，如富含亮氨酸的重复序列和NACHT结构域。系统发育分析表明，它们与其他NOD样受体具有高度同源性，并聚类为不同的家族。转录表达分析表明，OmNLRs在肝脏、脾脏、肠道、鳃、皮肤和大脑中组成性表达。虹鳟鱼的肾脏和鳃中，OmNLR的表达在受到LPS刺激后上调。为了深入了解这些新的NLR成员的功能，利用虹鳟鱼细胞系RTgill-W1建立了免疫刺激的体外模型。表达分析表明，RTgill-W1在受到LPS和聚肌胞苷酸（poly I:C）刺激后，促炎细胞因子表达上调，同时OmNLRC3、OmNLRC5和OmNLRX1的表达也有差异。通过免疫荧光在蛋白质水平进一步验证了OmNLRC5的表达。最后，评估了过表达的细胞因子对RTgill-W1细胞中OmNLR表达的影响，提示存在对OmNLRC3表达的调节机制。总体而言，结果表明虹鳟鱼的NOD样受体可能通过识别PAMP在硬骨鱼的防御机制中发挥关键作用。未来的研究将聚焦于鳃，因为鳃可能与鱼类最易暴露于环境的组织之一中的关键传感器黏膜系统有关。
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