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

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 (NOD2) and RIG-I (RIG-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 NOD2 and RIG-I. Results showed that NOD2 and RIG-I shared conserved structural hallmarks with their mammalian counterparts, and activated RIG-I signaling can induce NOD2 production. Surprisingly, NOD2-initiated signaling can also induce RIG-I expression, indicating that a mutual regulatory mechanism may exist between them. Studies conducted using HEK293T cells and zebrafish embryos showed that RIG-I could negatively regulate NOD2-activated NF-κB signaling, and NOD2 could inhibit RIG-I-induced IFN signaling. Moreover, knocking down RIG-I expression by morpholino could enhance NOD2-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.