Among teleost NLRs, NLR-C subfamily is a large group of proteins that were teleost-specific and evolution analysis showed that NLR-Cs are most likely to evolve from NLRC3 gene (thus also called as NLRC3Ls). Presently, although there have been rich studies investigating teleost NLRC3 and NLRC3L, the data on the regulatory mechanism was limited. In this study, immune regulation of inflammatory signaling pathway mediated by common carp NLRC3L gene (CcNLRC) has been investigated. Confocal microscopy analysis showed that CcNLRC was located in cytoplasm, and in HEK293T cells, dual-luciferase reporter assay showed the regulation of NF-κB signaling by CcNLRC, in which CcNLRC could alter/decrease RIPK2-induced activation of NF-κB. These results indicated that CcNLRC may function as a negative NLR in the regulation of inflammatory response in common carp. Our data will allow to gain more insights into the molecular mechanism of teleost specific NLR (NLRC3L).

Both NLRC3 and NOD1 belong to regulatory NLR subfamily based on their best-characterized function. In mammals, NLRC3 was reported to function by attenuating signaling cascades initiated by other families of PRRs. In teleosts, multiple NLRC3-like genes were identified through transcriptome sequencing. However, the functions of many NLRC3-like genes, especially the fish-specific NLRC3-like genes, remain unclear. In the present study, we report the functional characterization of a novel category of NLRC3-like proteins (named as NLRC3-like 1) from the zebrafish, which consists of a fish-specific FISNA, a conserved NACHT and five C-terminal LRRs domains. The expression of zebrafish NLRC3-like 1 was inducible in response to Edwardsiella piscicida infection. During bacterial infection, the in vitro and in vivo studies revealed that zebrafish NLRC3-like 1 overexpression facilitated bacterial growth and dissemination, together with the decreased survival rate of zebrafish larvae infected with E. piscicida. The attenuated response by zebrafish NLRC3-like 1 in response to bacterial infection were characterized by the impaired expression of antibacterial genes, proinflammatory cytokines and Nox genes. Furthermore, zebrafish NLRC3-like 1 interacted with the adaptor protein RIPK2 of NODs signaling via the FISNA (Fish-specific NACHT associated domain) and NACHT domains. However, the interaction between zebrafish NLRC3-like 1 and RIPK2 inhibited the assembly of the NOD1-RIPK2 complex. Importantly, zebrafish NLRC3-like 1 inhibited NOD1-mediated antibacterial activity, NF-κB and MAPK pathways and proinflammatory cytokine production. All together, these results firstly demonstrate that zebrafish NLRC3-like 1 inhibits NOD1-RIPK2 antibacterial pathway via targeting the adaptor protein RIPK2.

NLRC (the nucleotide-oligomerization domain (NOD)-like receptor subfamily C) consists of teleost-specific NLRs (NOD-like receptors) and plays pivotal roles in microbial recognition and regulation of innate immune response. In this study, we cloned and characterized a NLRC3-like gene (MamNLRC3-like) from blunt snout bream (Megalobrama amblycephala) by using the quantitative real-time PCR method, and analyzed the correlation between its polymorphisms and resistance to Aeromonas hydrophila infection. The full length cDNA of MamNLRC3-like was 2863 bp, with a 5\'-UTR of 169 bp, ORF of 2301 bp and 3\'-UTR of 393 bp, encoding 766 amino acid residues. MamNLRC3-like consisted of a conserved NACHT domain, a fish-specific NACHT associated domain (FISNA) and a PYRIN effective domain. During early developmental stages, MamNLRC3-like was most highly expressed at 39.4 hpf (hours post fertilization, hatching stage) and constitutively detected in various healthy tissues. The expression of MamNLRC3-like was strongly induced by A. hydrophila infection and peaked in the head kidney, liver, intestine, and trunk kidney at 12 h post infection. Six SNPs (single nucleotide polymorphisms) from MamNLRC3-like, with 2 in introns and 4 in exons, were identified by direct sequencing, in which 6515C/T was a novel non-synonymous mutation. HRM (high resolution melting) genotyping of the 6 loci showed that locus 6515C/T SNP was associated with the resistance/susceptibility of blunt snout bream to A. hydrophila infection (P < 0.01). The CC genotype fish were more resistant than CT carriers (P < 0.01). This study suggests that the MamNLRC3-like gene might play an important role in the innate immunity of blunt snout bream and could be used as a candidate marker for genetic selection of A. hydrophila-resistant blunt snout bream.