Mutual Regulation of NOD2 and RIG-I in Zebrafish Provides Insights into the Coordination between Innate Antibacterial and Antiviral Signaling Pathways

Abstract

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and retinoic acid-inducible gene I (RIG-I) are two important cytosolic pattern recognition receptors (PRRs) in the recognition of pathogen-associated molecular patterns (PAMPs), initiating innate antibacterial and antiviral signaling pathways. However, the relationship between these PRRs, especially in teleost fish models, is rarely reported. In this article, we describe the mutual regulation of zebrafish NOD2 (DrNOD2) and RIG-I (DrRIG-I) in innate immune responses. Luciferase assays were conducted to determine the activation of NF-κB and interferon signaling. Morpholino-mediated knockdown and mRNA-mediated rescue were performed to further confirm the regulatory roles between DrNOD2 and DrRIG-I. Results showed that DrNOD2 and DrRIG-I shared conserved structural hallmarks with their mammalian counterparts, and activated DrRIG-I signaling can induce DrNOD2 production. Surprisingly, DrNOD2-initiated signaling can also induce DrRIG-I expression, indicating that a mutual regulatory mechanism may exist between them. Studies conducted using HEK293T cells and zebrafish embryos showed that DrRIG-I could negatively regulate DrNOD2-activated NF-κB signaling, and DrNOD2 could inhibit DrRIG-I-induced IFN signaling. Moreover, knocking down DrRIG-I expression by morpholino could enhance DrNOD2-initiated NF-κB activation, and vice versa, which could be rescued by their corresponding mRNAs. Results revealed a mutual feedback regulatory mechanism underlying NOD2 and RIG-I signaling pathways in teleosts. This mechanism reflects the coordination between cytosolic antibacterial and antiviral PRRs in the complex network of innate immunity.

Multiple Sclerosis patients carry an increased burden of exceedingly rare genetic variants in the inflammasome regulatory genes

“…RIPK2 is essential for the function of NOD2, with whom it forms a protein complex. While commonly associated with response to bacterial infection, NOD2 is increasingly recognized in response to both RNA and DNA viruses 50–52 . By interacting with both the DDX58 and MAVS on the one hand 53 and with CARD18, PYCARD and CASP1 on the other 54 , the NOD2/RIPK2 complex is centrally positioned to serve as the integrator of both pathways.…”

Nucleotide‐binding and oligomerization domain (NOD)‐like receptors in teleost fish: Current knowledge and future perspectives

“…Interestingly, RIG‐I negatively regulates NOD2‐induced NF‐κB signalling in mammals (Morosky, Zhu, Mukherjee, Sarkar, & Coyne, ). It has reported that knocking down RIG‐I expression by morpholino could enhance NOD2‐initiated NF‐κB activation, and vice versa (Nie, Xu, Xiang, Shao, & Chen, ). Therefore, the effects of RIG‐I on NOD2‐mediated NF‐κB activation were similar both in mammals and fish species.…”

香鱼NOD2在活化NF-κB和MAPK信号通路及抗细菌先天免疫中的功能研究

“…Studies in mammals and zebrafish have shown that the LRR motif maintains NOD2 in an autoinhibition state, such that it cannot initiate signaling under physiological state without the stimulation of its ligand MDP (Girardin et al, 2003; Nie et al, 2017). After recognition of MDP by LRR, NOD2 undergoes self-oligomerization and recruits the downstream adaptor receptor-interacting serine/threonine-protein kinase 2 (RIPK2) through CARD-CARD interaction (Park et al, 2007).…”

“…Compared with other teleost species, studies on NOD2 in zebrafish ( Danio rerio ) are the most comprehensive. For example, the structural model of the NACHT domain and ATP binding orientations, antibacterial and protective functions, induced NF-κB signaling pathways, and mutual regulation between NOD2 and RIG-I signaling in zebrafish have been well studied (Maharana et al, 2015; Nie et al, 2017; Oehlers et al, 2011; Zou et al, 2016). Other than zebrafish, preliminary studies on NOD2 in other teleosts, including grass carp ( Ctenopharyngodon idella ), Nile tilapia ( Oreochromis niloticus ), Indian major carp ( Catla catla ), rohu ( Labeo rohita ), orange-spotted grouper ( Epinephelus coioides ), miiuy croaker ( Miichthys miiuy ), and rainbow trout ( Oncorhynchus mykiss ) have been carried out (Basu et al, 2016; Chang et al, 2011; Chen et al, 2010; Gao et al, 2018; Hou et al, 2012; Li et al, 2015; Maharana et al, 2013).…”