Transcription of NOD1 and NOD2 and their interaction with CARD9 and RIPK2 in IFN signaling in a perciform fish, the Chinese perch, Siniperca chuatsi

NOD1 and NOD2 as two representative members of nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family play important roles in antimicrobial immunity. However, transcription mechanism of nod1 and nod2 and their signal circle are less understood in teleost fish. In this study, with the cloning of card9 and ripk2 in Chinese perch, the interaction between NOD1, NOD2, and CARD9 and RIPK2 were revealed through coimmunoprecipitation and immunofluorescence assays. The overexpression of NOD1, NOD2, RIPK2 and CARD9 induced significantly the promoter activity of NF-κB, IFNh and IFNc. Furthermore, it was found that nod1 and nod2 were induced by poly(I:C), type I IFNs, RLR and even NOD1/NOD2 themselves through the ISRE site of their proximal promoters. It is thus indicated that nod1 and nod2 can be classified also as ISGs due to the presence of ISRE in their proximal promoter, and their expression can be mechanistically controlled through PRR pathway as well as through IFN signaling in antiviral immune response.


Introduction
Pathogenic infections can activate the host immune defense system, which is initiated by the recognition of pathogenic components, called pathogen-associated molecular patterns (PAMPs), by some families of pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs) and intracellular DNA sensors (1).The activated PRRs recruit adaptors and trigger downstream signaling pathways, which promote the expression of immune-related cytokines, such as interferons (IFNs) and interleukin (IL)-1b (1).IFNs are class II cytokines and play important roles in antiviral immunity (2).To date, vertebrate IFNs are identified into four types (I, II, III and IV), which use different receptors to activate Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling for the induction of IFN-stimulated gene (ISGs) through IFN-stimulated response elements (ISREs) (3)(4)(5)(6).
RLRs are the critical virus-RNA recognition receptors in cytoplasm.Almost all vertebrate RLR family has three members, including RIG-I (also known as DEAD box polypeptide 58, DDX58), melanoma differentiation-associated gene 5 (MDA5, also called IFN induced with helicase C domain 1, IFIH1) and laboratory of genetics and physiology 2 (LGP2, or DExH box polypeptide 58, DHX58), although the functional rig-I gene may have been lost in some taxa of vertebrates, such as chicken (Gallus gallus), Chinese tree shrew (Tupaia belangeri chinensis) and teleost fish in the Acanthomorphata (7).Activated RIG-I and MDA5 recruit mitochondrial antiviral signaling protein (MAVS), leading to the phosphorylation of critical kinases, such as TANK binding kinase 1 (TBK1) and some transcription factors, including IFN regulatory factor 3 (IRF3) and IRF7, which bind to the positive regulatory domains (PRDs) or ISRE in promoter region to upregulate the expression of IFNs and ISGs and inhibit virus proliferation (1).
NOD1 and NOD2 are the two representative members of NLR family and possess functional recognition to the bacterial g-Dglutamyl-meso-diaminopimelic acid (iE-DAP) and muramyl dipeptide (MDP), respectively (8).By contrast, NOD1 and NOD2 have similar domain organization with caspase activation recruitment domain (CARD) at N-terminal region, followed by a NAIP, CIITA, HET-E and TP1 (NACHT) domain and a leucinerich repeat (LRR) domain at C-terminus, although NOD2 has another CARD than NOD1 (9).The CARD of NODs is a key structure to interact with downstream adaptor, receptor-interacting serine-threonine kinase 2 (RIPK2, also known as RIP2) to activate NF-kB signaling and promote inflammatory response (9,10).Interestingly, the CARD9, a critical adaptor protein, is associated with NOD2 to trigger p38 MAPK and JNK signaling, but not NF-kB (11).In fact, CARD9 is involved in CLR-mediated activation of NF-kB pathway (11).Moreover, it is known that NOD1/NOD2 and RIPK2 complex can induce type I IFN through TRAF3-TBK1-IRF3/7 cascade (9).
Chinese perch (Siniperca chuatsi), belonging to the order Perciformes, has high economic value in Chinese freshwater aquaculture, and understanding of its immune system has attracted attention of aquaculture scientists in order to develop effective preventive and control measures against diseases (21)(22)(23).In this study, the adaptors of NODs, CARD9 and RIPK2, were cloned in Chinese perch, and the interactive relationship between CARD9/RIPK2 and NOD1/NOD2 was verified through coimmunoprecipitation and cellular colocalization, respectively.Moreover, poly(I:C) induced expression of nod1 and nod2 was examined, and the promoter sequence of nod1 and nod2 genes was bioinformatically analyzed with the possible identification of ISRE.Then, luciferase reporter assay was performed to examine if IFNs in Chinese perch could induce the promoter activity of nod1 and nod2, and vice versa if NOD1, NOD2, RIPK2 and CARD9 could induce the promoter activity of IFNs and NF-kB.These results indicated that NOD1 and NOD2 should be involved in antiviral immune response in an IFN-dependent manner at least in lower vertebrates.

Fish, cell lines and virus
Chinese perch were obtained from a fish farm in Hubei Province, China, and maintained in aerated tanks at 28 °C with recirculating fresh water for two weeks prior to the experiments.All animal experiments were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals and were approved by the Institute of Hydrobiology, Chinese Academy of Sciences.

RNA extraction, cDNA synthesis and gene cloning
DNase I reagent (Thermo Scientific) was used at 37°C to remove genomic DNA remnants in total RNA samples, which were extracted from fish tissues or cells using TRIzol ® reagent (Ambion) following the manufacturer's protocol.Next, the RevertAid ™ First Strand cDNA Synthesis Kit (Thermo Scientific) was used to synthesize first strand cDNA according to the manufacturer's instruction.Full-length open reading frames (ORFs) of ripk2, card9, nod1 and nod2 were generated through PCR amplification by using Phanta Max Master Mix high-fidelity DNA polymerase (Vazyme) with gene-specific primers (referring to Supplementary Table 1) which were designed based on the predicted sequences (XM_044218208.1 and XM_044195794.1 from NCBI database) and the reported sequences (KY974318 and KY974317) (25).All PCR products were confirmed by sequencing.

Immunofluorescence assay
MFF-1 cells were seeded onto coverslips in 24-well plates and were co-transfected with 0.25 µg of each plasmid in same combinations as described above.24 hours after transfection, the supernatants were removed, and the cells were washed with PBS and fixed by using 4% paraformaldehyde at 28°C for 40 min.Next, the cells were washed four times with PBS and incubated with 0.2% Triton X-100 to permeate the cell membrane at 28°C for 12 min.After being washed with PBS for four times, the cells were blocked by 5% BSA at 37°C for 1 hour and incubated with the tag-antibodies described above and the fluorescein-labelled secondary antibodies, including Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor ™ 594 (Thermo Fisher Scientific) or Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor ™ 488 (Thermo Fisher Scientific).Subsequently, the coverslips with labelled cells were sticked on the microscope slides by using ProLong ™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific).The cells were observed under a Leica confocal microscope (40 × magnification, oil immersion lens).

Gene expression analyses
To investigate the tissue distribution of nod1, nod2, card9 and ripk2, three healthy Chinese perch with a body length of about 10 cm were sacrificed to collect different organs/tissues, including fin, head-kidney, truck-kidney, spleen, skin, liver, gill, eye, and intestine, for qRT-PCR detection.To investigate whether the four genes (nod1, nod2, card9 and ripk2) could be induced by poly(I:C), IFN-g, IFNh and IFNc, MFF-1 cells (5.0 × 10 5 ) were seeded in 12well plates and were separately transfected with 50 mg (final concentration of 41.67 mg/mL) poly(I:C) by using 1 mL Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) or directly incubated with 100 mL prepared culture medium supernatant containing the recombinant protein of IFNg/IFNh/IFNc, as described above.The samples were collected at 3, 6, 12 and 24 hours post-stimulation, respectively.For IHSV infection, MFF-1 cells (5.0 × 10 5 ) were seeded in 12-well plates and were infected with IHSV (MOI = 0.01) or without virus (control group), and the samples were collected at 12, 24, 36 and 48 hours post-infection, respectively.

Quantitative real-time PCR
The cDNA synthesis from total RNA was performed with Allin-One RT SuperMix Perfect for qPCR kit (Vazyme).A total of 20 mL reaction system consisted of 10 mL PowerUP ™ SYBR ™ Green Master Mix (ABI), 2 mL primer mixture, 7 mL sterile water, and 1 mL cDNA template.The qRT-PCR was performed on the QuantStudiO ™ 3 Real-Time PCR Instrument (96-Well Block, ABI) with the following protocol: initial denaturation for 2 min at 50°C, 2 min at 95°C, 50 cycles for 15 s at 95°C, 30 s at 58°C, followed by a further 15 s at 95°C, 1 min at 60°C, 1 s at 95°C.The b-actin was used as the internal reference gene to calculate the folding change of expression level of target genes using the 2 (-DDCt) method.The specific primers are listed in Supplementary Table 1.

Statistical analyses
All experiments were repeated separately at least three times.Results were analyzed statistically with Student's t-test in SPSS 16.0 software.Significant differences were indicated with * P < 0.05, and ** P < 0.01, respectively.The data were presented as mean ± standard error (SE).

Identification of ripk2 and card9 in Chinese perch
The nucleotide sequences of two adaptors, RIPK2 and CARD9, were cloned from mixed cDNA samples of Chinese perch tissues and were deposited in the GenBank database with the accession numbers, OR713117 and OR713116, respectively.The full-length ORF of ripk2 and card9 contained 1806 and 1623 base pairs, respectively, which were predicted to encode 601 and 540 amino acids.The RIPK2 and CARD9 protein sequences shared medium identity with human (46.6%/35.6%),mouse (46.4%/37.6%),chicken (44.6%/35.8%)and zebrafish (69.8%/43.2%),respectively.The multiple alignments and domain prediction revealed that vertebrate RIPK2 has a conserved serine-threonine kinase domain (KD) at N-terminal region and a conserved C-terminal CARD domain, and CARD9 has a N-terminal CARD domain and a coiledcoil (CC) domain, while no transmembrane motif (TM) was found in the two molecules (Supplementary Figures 1, 2).Phylogenetic analysis showed that RIPK2 and CARD9 were formed into a separate clade with their fish homologues, respectively (Figure 1).Moreover, in synteny analysis, vertebrate card9 was found to be linked with conserved small nuclear RNA activating complex polypeptide 4 (snapc4) from fish to human, and tetrapod ripk2 is located on a conserved genetic region which contains matrix metallopeptidase 16 (mmp16) and oxidative stress induced growth inhibitor family member 2 (osgin2) (Figure 2A).However, fish ripk2 was sandwiched with agouti signaling protein, nonagouti homolog (mouse) 2b (asip2b) and coiled-coil domain containing 39 (ccdc39) (Figure 2B).

Expression of nod1, nod2, ripk2 and card9 genes
The qRT-PCR was used to detect the expression level of nod1, nod2, ripk2 and card9 genes in Chinese perch organs/tissues, including head-kidney, trunk-kidney, spleen, liver, intestine, fin, gill, eye, and skin.As shown in Figure 3A, the four genes were constitutively expressed in all detected organs/tissues, and card9 mRNA was mainly enriched in head-kidney, spleen, and gill, while ripk2 gene was expressed with highest transcription level in spleen and gill.Interestingly, nod1 and nod2 genes showed high expression levels in head-kidney.
To investigate whether nod1, nod2, ripk2 and card9 can be induced by PAMPs and virus, the expression of the four genes was detected in poly(I:C)-treated and IHSV-infected MFF-1 cells, respectively.Compared to the control, poly(I:C) stimulation and IHSV infection all resulted in significant increase in the expression of ripk2 and card9 genes at different time points, and the induced expression of nod1 and nod2 genes was also observed (Figures 3B,  C, Supplementary Figure 3).For further investigating whether nod1, nod2, ripk2 and card9 genes could be upregulated by IFNs, the expression of the four genes was detected in MFF-1 cells incubated with recombinant type I (IFNh and IFNc) and type II (IFN-g) IFN proteins, which have been confirmed to induce ISGs, such as mx, pkr and viperin (Supplementary Figures 4A-E).It was observed that all the IFNs significantly induced the expression of nod1, nod2, ripk2 and card9 genes at 3 and 6 hours post-stimulation (Figure 4).

Identification of nod1 and nod2 as ISGs
For understanding inducible regulatory elements of nod1 and nod2 genes, 5'-flanking sequences of the proximal promoter were identified.A potential ISRE site was found in the proximal promoters of both nod1 and nod2 genes, which were cloned to construct luciferase reporter plasmids with wildtype (wt) or mutant (mut) ISRE site (Figures 5A, B).The recombinant IFN proteins (IFNh, IFNc and IFN-g) significantly activated ISRE-wt promoter plasmids of both nod1 and nod2 genes, but not the ISRE-mut plasmids (Figures 5C-H).

Discussion
In mammals, NOD1 and NOD2 mediated signaling is involved in antimicrobial immunity (9).However, the transcription and signaling complex of NOD1 and NOD2 are quite unclear in teleost fish.In the present study, with the cloning of card9 and ripk2 genes, the interaction of NOD1 and NOD2 with CARD9 and RIPK2 was revealed through colocalization and Co-IP.Interestingly, these four genes were found to be upregulated by type I and type II IFNs, i.e.IFNh and IFNc, and IFN-g in Chinese perch.An ISRE was identified in the proximal promoter of nod1 and nod2 genes, and the overexpression of NOD1, NOD2, RIPK2 and CARD9 all resulted in the increase in reporter activity of NF-kB, and IFNh and IFNc, revealing the antiviral signaling of NOD1 and NOD2 in the perciform Chinese perch (Figure 11).
In response to viral infection, IFN signaling leads to the induction of a large group of genes called ISGs, some of which are responsible for interfering with viral replication and providing protection for host cells (2).A mass of ISGs with antiviral activity have been discovered in mammals, while in fish, their number is comparatively limited (31, 32).The proximal promoter of typical ISGs may contain specific DNA elements, i.e.ISRE, which can respond to IFN signaling by binding with a family of transcription Gene synteny of card9 (A) and ripk2 (B) loci in vertebrates.The functional genes with transcription direction were indicated in arrow symbols, and the card9, ripk2 and linked genes were dyed in red, blue, and gray, respectively.In mammals, NOD1/NOD2 interacts with RIPK2, leading to the production of proinflammatory cytokines, to play roles in autophagy and anti-bacterial, -viral and -parasitic infections (9,10,35).In bony fish, the interaction between RIPK2 and The subcellular colocalization of NOD1-CARD9 (A), NOD2-RIPK2 (B), NOD2-CARD9 (C), NOD2-RIPK2 (D) and RIPK2-CARD9 (E).MFF-1 cells were co-transfected with expression plasmids including NOD1-Myc, NOD2-Myc, CARD9-HA, RIPK2-HA, RIPK2-Flag, or empty vectors, for 24 hours.The cells were fixed by using 4% paraformaldehyde and were permeated by using 0.2% Triton X-100 to perform immunofluorescence assays with the fluorescein-labelled secondary antibodies.The cells were observed under a Leica confocal microscope (Scale bars = 10 mm).

A B C
NOD1/NOD2 has been reported in zebrafish, goldfish (Carassius auratus), and miiuy croaker (Miichthys (36)(37)(38).Structurally, NOD1 and NOD2 all contain a nucleotide oligomerization NACHT, a LRR domain, and one CARD/two CARDs, which is/are critical site(s) to interact with the CARD of RIPK2 (9).In mammal, CARD9 binds to NOD2 through NACHT or CARD-NACHT linker region of NOD2, but not through CARD-CARD interaction (39).Structurally, the two adaptor proteins have conserved CARD domain and possess additional CC or KD domain (9,11,40,41).It is obvious that an N-terminal CARD and a Cterminal CC domain, and an N-terminal KD and a C-terminal CARD domain are also found in CARD9 and RIPK2 in Chinese perch, respectively.The conserved domain composition, and conserved gene loci for CARD9 and RIPK2 in fish as in mammals may imply their similar functional characteristics in vertebrates.

A B C
The As mentioned above, NOD1, NOD2, CARD9 and RIPK2 Chinese perch possess conserved domains, which may provide the binding basis for the proteins.Expectedly, NOD1 and NOD2 were associated with RIPK2, and the interaction between CARD9 and NOD1/NOD2/RIPK2 was also observed through Co-IP and immunofluorescence assay, suggesting that teleost CARD9 and RIPK2 are likely to be the components of signal transduction complex of both NOD1 and NOD2, as reported in mammals.
It has been reported that mammal NOD1 and NOD2 can induce antiviral response via RIPK2-TRAF3-IRF3/IRF7-IFN pathway to suppress the replication of viruses, such as human cytomegalovirus (HCMV), respiratory syncytial virus (RSV), or influenza A virus (IAV) (29,(42)(43)(44)(45).To date, NOD1 has been demonstrated to be able to mediate RLR-associated antiviral signaling in bony fish, and this is accomplished by binding viral RNA and regulating the interaction between MDA5 and MAVS, thereby promoting antiviral signaling (20).NOD2 and RIPK2 can also activate NF-kB and type I IFN promoters in fish, inducing significant antiviral defense against SVCV infection (46).In this study, the overexpression of NOD1, NOD2, and RIPK2 significantly induced the activity of NF-kB, IFNh and IFNc promoter reporters, indicating the conserved signal pathway mediated by NOD1 and NOD2 in teleost.Interestingly, mammal CARD9 was reported to be involved in the process of RIG-I mediated NF-kB activation against  Expression regulation model of nod1 and nod2 in Chinese perch.NOD1, NOD2, CARD9 and RIPK2 show interactive relationship and contribute to the expression of type I IFNs.Furthermore, nod1 and nod2 genes contain ISRE in their promoters, and can be induced by type I IFNs, which in turn can be upregulated also by CARD9, RIPK2, and even by NOD1/NOD2 themselves.
RNA viruses (47).In this study, CARD9 was found to be interactive with all three key molecules, NOD1, NOD2 and RIPK2, implying that teleost CARD9 is involved in NOD1 and NOD2 mediated signaling, and CARD9 can also induce the promoter activity of NF-kB and type I IFNs.Furthermore, NOD1 and NOD2 as the critical PRRs were induced by MDA5, CARD9, RIPK2, IFNs, as well as NOD1/NOD2 themselves through ISRE in Chinese perch, suggesting the positive-feedback regulation pathway of NOD1/ NOD2-IFNs in teleost.
In summary, with the identification of CARD9 and RIPK2 in Chinese perch, the interactive relationship between NOD1/NOD2 and CARD9/RIPK2 was demonstrated, contributing to the expression of type I IFNs.Furthermore, nod1 and nod2 genes contain ISRE in their promoters, and can be induced by type I IFNs, which in turn can be upregulated also by MDA5, CARD9, RIPK2, and even by NOD1/NOD2 themselves.