NLRC3-like 1 inhibits NOD1-RIPK2 pathway via targeting RIPK2

https://doi.org/10.1016/j.dci.2020.103769Get rights and content

Highlights

  • Zebrafish NLRC3-like 1 consists of a fish-specific FISNA, a conserved NACHT and C-terminal LRRs domains.

  • NLRC3-like 1 overexpression facilitated bacterial growth and dissemination, and reduced larvae survival.

  • Zebrafish NLRC3-like 1 negatively regulated the expression of proinflammatory cytokines.

  • Zebrafish NLRC3-like 1 interacted with RIPK2, but not with NOD1.

  • Zebrafish NLRC3-like 1 inhibited NOD1-mediated NF-κB and MAPK pathways and proinflammatory cytokine production.

Abstract

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.

Introduction

The nucleotide oligomerization domain (NOD)-like receptor (NLR) family proteins are intracellular pattern recognition receptors (PRRs) that have many functions including pattern recognition, antigen presentation, autophagy, immune modulation and embryonic development (Kufer and Sansonetti, 2011; Meunier and Broz, 2017). The most commonly studied subfamily of NLRs is NOD or NLRC subfamily, which includes NOD1 (NLRC1), NOD2 (NLRC2), NLRC3 (NOD3), NLRC4 (IPAF), NLRC5 (NOD27) and NLRX1 (Meunier and Broz, 2017). Among them, NOD1 and NOD2 are the best-characterized members as positive regulatory NLRs, and have been shown to form a multiprotein complex to modulate IFN and NF-κB signaling following bacterial or viral infection (Hasegawa et al., 2008; Coutermarsh-Ott et al., 2016).

In contrast to NOD1 and NOD2, NLRC3 was reported to function by attenuating signaling cascades initiated by other families of PRRs such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs). NLRC3 consisted of an N-terminal caspase activation and recruitment domain (CARD), a central nucleotide-binding domain (NBD or NACHT) and a C-terminal leucine-rich repeat (LRR) domain (Conti et al., 2005; Schneider et al., 2012). In mammals, NLRC3 was originally identified as a suppressor of T cell activation (Conti et al., 2005). Subsequent studies revealed that NLRC3 inhibited TLR signaling through the interaction with TRAF6 to regulate NF-κB (Schneider et al., 2012), and reduced STING-dependent innate immune activation in response to cytosolic DNA, cyclic di-GMP (c-di-GMP) and DNA viruses (Zhang et al., 2014). Among well-known inflammasome components, NLRC3 interacted with ASC, caspases 1 and 5, which negatively regulated NLRP3-mediated inflammatory responses (Gültekin et al., 2015; Eren et al., 2017). Furthermore, NLRC3 negatively regulated the PI3K–mTOR axis through the interaction of NLRC3 with PI3K subunits (Karki et al., 2016, 2017).

In teleosts, multiple NLRC3-like genes were identified through transcriptome sequencing (Hu et al., 2017; Wu et al., 2018). A few studies examined the function of piscine NLRC3 or NLRC3-like genes. In Japanese flounder (Paralichthys olivaceus), JfNLRC (containing NACHT and LRRs domains) and poNLRC3 (containing NACHT, FISNA, LRRs and PRY/SPRY domains) positively regulated the expression of proinflammatory cytokines (Unajak et al., 2011; Li et al., 2016a). Zebrafish NLRC3-like gene, which contains the canonical pyrin (PYD) and NACHT domains but lacks the common LRRs, can bind the inflammasome component ASC to regulate the inflammasome and is required for microglia development (Shiau et al., 2013). In goldfish, NLRC3L consisting of FISNA, NACHT, PRY, SPRY and LRRs domains interacted with ASC and cooperated with RIPK2 to regulate NF-κB activity (Xie and Belosevic, 2018). Since the functional diversities exist for NLRC3 or NLRC3-like genes, there is a need to reveal the functions and molecular mechanisms for the expanding analogues of piscine NLRC3-like genes.

In our previous studies, transcriptomic analysis and qRT-PCR results revealed that multiple NLRC3-like genes were regulated by NOD1 and RIPK2 deficiency (Hu et al., 2017; Wu et al., 2018). The present study focused on investigating the function of a zebrafish NLRC3-like gene consisting of FISNA, NACHT and LRRs domains (named as NLRC3-like 1) in bacterial infection. Our data suggest that zebrafish NLRC3-like 1 plays a negative role in bacterial infection and inhibits NOD1-RIPK2 antibacterial pathway via targeting RIPK2.

Section snippets

Maintenance of zebrafish

Wild-type AB/TU adult zebrafish were raised and maintained at 28 °C in the system water. Zebrafish embryos were obtained by artificial insemination and reared in the petri dishes at 28 °C.

Plasmid construction and sequence analysis

Based on zebrafish sequence derived by automated computational analysis using gene prediction method (GenBank accession No: XM_003200085), the forward and reverse primers NLRC3-like 1F/NLRC3-like 1R (Table 1) were used for cloning the open reading frame (ORF) and inserted into p3XFLAG-CMV™-14 Expression Vector

Features of zebrafish NLRC3-like 1

In our previous study, transcriptomic analysis revealed that multiple NLRC3-like (namely NOD3-like) genes were found in zebrafish database (Hu et al., 2017). Among them, a EST sequence (gene26713) was consistent with the Danio rerio si:ch211-181d7.3 sequence (GenBank accession number XM_003200085), which was cloned by us. Phylogenetic analysis confirmed that this gene was the analogue of piscine NLRC3 (Fig. 1A), and named as NLRC3-like 1. The coding region of zebrafish NLRC3-like 1 consists of

Discussion

Both NLRC3 and NOD1 belong to NLRC subfamily according to their phylogenetic relationships or regulatory NLR subfamily based on their best-characterized function (Schroder and Tschopp, 2010; Coutermarsh-Ott et al., 2016). Here we identified and characterized a novel NLRC3 gene (termed as NLRC3-like 1) from the zebrafish, and investigated the expression and function of NLRC3-like 1 in response to E. piscicida infection in vivo and in vitro. The correlation between zebrafish NLRC3-like 1 and NOD1

Acknowledgements

This work was supported by National Natural Science Foundation of China (31672687 and 31873046), and Fund for State Key Laboratory of Freshwater Ecology and Biotechnology (2019FBZ04).

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