Transcriptional Expression Profiles of Olive Flounder (Paralichthys olivaceus) Toll-like Receptor 3 (TLR3) and TLR22 in Response to Extracellular Poly I:C Stimuli

Ⅰ. Introduction

Toll-like receptors (TLRs) are a type of pattern recognition receptor and a family of transmembrane sensor proteins that recognize pathogen-associated molecular patterns (PAMPs) and play crucial roles in both innate and adaptive immune systems as sentinel cells such as macrophages and dendritic cells as well as T and B lymphocytes (Schnare et al., 2001; Takeda et al., 2003; Iwasaki and Medzhitov, 2004). TLRs can detect intracellular and extracellular pathogenic factors, including the bacterial cell wall components and viral nucleotides, TLRs may play roles via. Several TLRs (from TLR1 to TLR13) have been identified both vertebrata and invertebrates.

The TLRs in host cells bind specific factors of pathogens, including single-stranded and double-stranded (ds) RNA in viruses, lipoproteins, lipopeptides, and proteins such as flagellin in bacteria or the unmethylated CpG oligodeoxynucleotide DNA in both.

In mammals, TLR3 recognizes dsRNA generated as a replication intermediate or by-product of many viruses (Alexopoulou et al., 2001; Wang et al., 2004; Schulz et al., 2005), one of the major PAMP, which trigger innate immune responses in vertebrates (Akira et al., 2006). It recruits the TIR domain-containing adaptor inducing interferon-β (TRIF) protein via interaction with its TIR domains (Yamamoto et al., 2002) and activates the TRIF dependent signaling pathway (Jacobs and Langland 1996; Sen and Sarkar 2005; Haller et al., 2006). TRIF then indirectly activates several transcription factors, such as interferon regulatory factor 3 (IRF3), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and activator protein 1 (AP-1), which modulate the transcription of interferon β (IFN-β) and inflammatory cytokines (Sharma et al., 2003; Yamamoto et al., 2003; Meylan et al., 2004; Matsuo et al., 2008).

Similar dsRNA recognizing mechanisms mediated via TLR3 have also been reported in fish (Matsuo et al., 2008). The secreted IFN regulates the expression of numerous genes encoding antiviral proteins, including dsRNA-dependent eukaryotic initiation factor kinase as protein kinase R (PKR) and Mx proteins (de Veer et al., 2001). In other words, signaling through TLR3 results in type I IFN responses in mammals (Hoebe et al., 2003). Mammalian TLR3 homologs have been reported in several fish species, such as fugu (Takifugu rubripes; Oshiumi et al., 2003), zebrafish (Danio rerio; Meijer et al., 2004; Jault et al., 2004), channel catfish (Ictalurus punctatus; Bilodeau and Waldbieser 2005), rainbow trout (Oncorhynchus mykiss; Rodriguez et al., 2005), and grass carp (Ctenopharyngodon idella; Su et al., 2009).

In addition, studies have identified an additional TLR, TLR22 in several fish species including goldfish (Stafford et al., 2003), zebrafish (Jault et al., 2004), olive flounder (Paralichthys olivaceus; Hirono et al., 2004), rainbow trout (Rebl et al., 2007), and fugu (Matsuo et al., 2008). Moreover, TLR22 has been shown to be mainly expressed in olive flounder stimulated with polyinosinic:polycytidylic (poly I:C), a dsRNA analog used in in vivo experiments to model viral infections (Chakrapani et al., 2018; Qiu et al., 2019), indicating its possible involvement in immune response in olive flounder. However, unlike TLR3, the precise role of TLR22 and the underlying mechanism in regulating the expression of immune-related genes in olive flounder has not been explored fully.

To gain a better insight into the response of immune-related genes to extracellular viral nucleotides, in the present study, we aimed to analyze the molecular patterns and changes in olive flounder TLR3 (JfTLR3) and JfTLR22 expression at the transcript level. For this purpose, the expression profiles of JfTLR3 and JfTLR22 in vivo using healthy olive flounders and in vitro using the HINAE cells stimulated with poly I:C were investigated.

Furthermore, we also investigated the functional roles of TLR3 and TLR22 in the induction of type I interferon response in HINAE cells stimulated with poly I:C through construction and transfection of short-hairpin RNA (shRNA)-producing vectors targeting the TLR22 and TLR3.

Ⅱ. Materials and methods

2. Expression analysis of genes after poly I:C stimulation in olive flounder

Three fish, each from the poly I:C and PBS groups, were sampled at 12 and 24 h post-treatment and kidney tissues were collected and immediately frozen in liquid nitrogen until further use. Total RNA was extracted from the kidney tissues using RNAiso Plus reagent according to the manufacturer’s instruction (Takara, USA). Total RNA (0.5 μg) was then reverse-transcribed to cDNA using HyperScriptTM kit (GeneAll Biotechnology, Seoul, Korea). Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) was performed to determine the relative mRNA expression levels of TLR3, TLR22, Interferon-stimulated gene 15 (ISG15), Mx, and 18S rRNA (internal control) genes of olive flounder using the gene-specific primers shown in <Table 1>. Total reaction volume of 20 μL for PCR comprised 2×Prime Taq Premix (Genet Bio, Daejeon, Korea) and 1 μL of 10 times diluted cDNA template. The thermal cycling conditions for the semi-quantitative RT-PCR were 1 cycle of 4 min at 95°C (initial denaturation), followed by 17 cycles (for 18S rRNA), 26 cycles (for ISG15 and MX), or 32 cycles (for TLR3 and TLR22) of 95°C for 30 s, 60°C for 30 s, 72°C for 30 s with a final extension of 72°C for 7 min. PCR products were electrophoresed on 1% agarose gels and visualized with ethidium bromide (Et-Br) staining.

<Table 1>

Oligonucleotide primer pairs used in this study

4. Construction of shRNA-producing vector targeting the JfTLR22 and JfTLR3

Using Ambion’s “siRNA Target Finder and Design Tool,” siRNA target sequences for JfTLR22 and JfTLR3 genes were designed <Table 2>. The fugu U6 promoter-driven shRNA expression vector (Zenke and Kim, 2008) was used to construct plasmids producing shRNA targeting the JfTLR22 and JfTLR3 genes using the shRNA system used to silence target gene expression via RNA interference (RNAi). The amplification conditions were 5 cycle at 95°C for 3 min (initial denaturation), 5 cycles at 95°C for 30 s, 48°C for 30 s, 72°C for 30 s, followed by 30 cycles at 95°C for 30 s, 65°C for 30 s, 72°C for 30 s.

<Table 2 >

Target sequences used for the production of pFuguU6shRNA vectors

Each shRNA-producing cassette was prepared following three-step PCR reactions. Using cloned fugu U6 promoter as the template, the first-step PCR was performed with two primers, U6F-SphI and each of shRNA R1, containing the last 16 bps of fugu U6 promoter, shRNA sense and loop. Next, each amplified product from the first PCR was purified and used as the template for second-step PCR with U6F-SphI and each of shRNA R2 containing 10 bps of shRNA sense, loop and 4 bps shRNA antisense. One-tenth of the second PCR product was then used as the template for the third-step PCR with U6F-SphI and each of the shRNA R3-SacII containing 5 bps of shRNA sense, loop, shRNA antisense and termination signal. Intermediately after the U6 promoter sequence, one guanine residue, which is predicted to be a transcription start point of shRNA, was included in this construct. Finally, each PCR product was gel-purified and cloned into pGEM T-easy vector (Promega), resulting in vector pFuguU6shRNA vectors (pshTLR3·577, pshTLR3·939, pshTLR3·939cont, pshTLR22·903, pshTLR22·1726, and pshTLR22·1726cont). The pshTLR22·1726cont or pshTLR3·939cont vector differed from pshTLR22·1726 or pshTLR3·939 for two nucleotides, each. To select shRNA clones that effectively suppressed JfTLR22 expression, a dual vector harboring each shRNA-producing cassette and JfTLR22 gene expressing cassette was constructed. The JfTLR22 or JfTLR3 gene ORFs containing HindIII and BamHI sites were cloned into pGEM T-easy vector and then inserted into the pcDNA 3.1(+) vector (Invitrogen). Then, the cassette containing the CMV promoter, JfTLR22 ORF and bovine growth hormone polyadenylation signal was digested using BglII and SalI restriction enzymes and inserted into each of the above-mentioned shRNA expression vectors, resulting in pshTLR3·577-fTLR3, pshTLR3·939-fTLR3, pshTLR3·939cont-fTLR3, pshTLR22·903-fTLR22, pshfTLR22·1726-fTLR22, and pshTLR22·1726cont-fTLR22, and control vector is p-fTLR3 or fTLR22. A neomycin-resistant cassette, including the SV40 early promoter and the SV40 polyadenylation signal in the pcDNA 3.1 vector, was inserted into the ApaI and AatII sites of each shRNA expression vector and the resulting vectors were named pNeoshTLR3·939, pNeoshTLR22·1726, pNeoshfTLR22·1726cont, and control vector was named pNeo. The integrity of the DNA sequences of the vectors was confirmed by sequencing using an automatic sequencer (Applied Biosystems).

6. Transfection of HINAE cells with shRNA expression vectors and selection

HINAE cells were cultured on 6-well plates (2×10⁶ cells/well) at 20°C containing L-15 medium supplemented with 20 mM HEPES and 10% FBS. At 80% confluence, the cells were transfected with pNeo, pNeoshTLR3·939, pNeoshTLR22·1726, and pNeoshTLR22·1726cont vectors using FuGENE 6 (Roche) according to the manufacturer’s instructions. To select HINAE cells harboring shRNA expressing vectors, 400 μg/mL G-418 (geneticin) antibiotic was added to the culture medium of cells transfected with each pFuguU6shRNA-neomycin plasmid. The surviving cells were then trypsinized using TrypLE Express (Gibco) and plated in new T25 and were grown in L-15 medium containing 10% FBS, antibiotics (penicillin 100 U/mL and streptomycin 100 μg/mL) and 400 μg/mL of G-418. The selected cells were maintained for six months.

[Fig. 1]

Transfection of HINAE cells with shRNAproducing vectors, selection and expression analysis of genes after simulated poly I:C.

Ⅲ. Results

1. Expression of TLR3, TLR22, ISG15, and Mx genes in poly I:C injected olive flounder

Expression analysis of JfTLR3, JfTLR22, JfISG15, and JfMx genes using semi-quantitative RT-PCR revealed no or very weak expression of these genes in the PBS group throughout the experimental period. However, poly I:C stimulation upregulated the expression of JfTLR22 and JfTLR3 genes at 12 and 24 h, wherein JfTLR22 showed a higher expression than JfTLR3. In addition, the expression of ISG15 and Mx genes was highly elevated by poly I:C stimulation [Fig. 2].

[Fig. 2]

Effect of poly I:C injection on the expression of TLR3, TLR22, ISG15, and Mx genes in olive flounder kidney. At 12 and 24 h post-injection with poly I:C or PBS, the expression of each gene in the kidney was analyzed by semi-quantitative RT-PCR.

2. Expression of TLR3, TLR22, IRF3, IRF7 and ISG15 genes in poly I:C stimulated HINAE cells

Semi-quantitative RT-PCR results of the JfTLR3, JfTLR22, JfIRF3, JfIRF7 and JfISG15 genes in HINAE cells stimulated with poly I:C are shown in Fig. 2. The results revealed that the expression of JfTLR22, IRF3, IRF7, and ISG15 genes was upregulated at 6 and 12 h post-stimulation, and that of the TLR3 gene was approximately increased 5 times compared with the TLR22 gene at 24 and 48 h post-stimulation [Fig. 3].

[Fig. 3]

Effect of poly I:C on the expression of TLR3, TLR22, IRF3, IRF7, and ISG15 genes in HINAE cells. At 6, 12, 24, 48 h post-stimulation, expression of the above genes and 18s rRNA gene (control) was analyzed by RT-PCR. ‘- sign’ indicates PBS-stimulation and ‘+ sign’ indicates poly I:C stimulation.

3. Selection of an effective shRNA clone targeting JfTLR3 or JfTLR22 in HINAE cell

The expression analysis of the JfTLR22 or JfTLR3 genes in HINAE cells transfected with p-fTLR3 or fTLR22, pshTLR3·577-fTLR3, pshTLR3·939-fTLR3, pshTLR3·939cont-fTLR3, pshTLR22·903-fTLR22, pshTLR22·1726-fTLR22, and pshTLR22·1726cont-fTLR22 revealed that both JfTLR3 or JfTLR22 genes were downregulated in HINAE cell transfected with pshTLR3·939-fTLR3 or pshTLR22 ·1726-fTLR22. However, cells transfected with pshTLR3·577-fTLR3, pshTLR3·939cont-fTLR3, pshTLR22·939-fTLR22, and pshTLR22·1726cont-fTLR22 vectors showed no reduction in expression of the JfTLR22 gene [Fig. 4].

[Fig. 4]

Interference effects of JfTLR3 and JfTLR22 genes in each transfected HINAE cells. HINAE cells transfected with p-fTLR3 (lane 1), pshTLR3·577-fTLR3 (lane 2), pshTLR3·939-fTLR3 (lane 3), pshTLR3·939cont-fTLR3 (lane 4), p-fTLR22 (lane 5), pshTLR22·903-fTLR22 (lane 6), pshfTLR22 ·1726-fTLR22 (lane 7) and pshTLR22·1726cont-fTLR22 (lane 8) vectors. Expression of (A) 18S rRNA, (B) JfTLR3, and (C) JfTLR22 genes analyzed by semi-quantitative RT-PCR.

4. Effect of shRNAs targeting JfTLR22 and JfTLR3 on the expression of type I IFN-induced genes in HINAE cells

The poly I:C stimulation for 12 h of HINAE cells harboring pNeo, pNeoshTLR22·1726, pNeoshTLR22·1726cont, and pNeoshTLR3·939 selected using G-418 supplementation demonstrated that the expression of JfTLR22 gene was reduced in HINAE cells harboring pNeoshTLR22·1726, whereas the cells harboring pNeoshTLR22 ·1726cont showed no reduction in JfTLR22 expression compared with that of 18S rRNA [Fig. 5].

[Fig. 5]

Effects of shRNAs targeting JfTLR22 or JfTLR3 on the expression of JfTLR22, JfTLR3, ISG15, and Mx genes in HINAE cells analyzed by semi-quantitative RT-PCR at 12 h post-stimulation of HINAE cells with poly I:C. Lane 1, HINAE cells (no handling); lane 2, HINAE cells harboring pNeo; lane 3, HINAE cells harboring pNeoshTLR22·1726; lane 4, HINAE cells harboring pNeoshTLR22 ·1726cont; lane 5, HINAE cells harboring pNeoshTLR3·939.

Furthermore, despite the stimulation of poly I:C both ISG15 and Mx genes were highly down-regulated by knock-down of JfTLR22 (in cells harboring pNeoshTLR22·1726); however, the JfTLR3 knock-down (in cells harboring pNeoshTLR3·939) could only reduce the expression of these genes by 0.5 times compared with those in the control cells (in cells harboring pNeo).

Ⅳ. Conclusion and Suggestions

In mammals, including humans, TLR3 is located in the endosomal membrane or the cell surface and recognizes dsRNA (Matsumoto et al., 2014). However, in fish, an additional TLR, TLR22, is also known to respond to dsRNA. Recently, Matsuo et al. (2008) have reported that fish TLR22 might play a similar role in fish as that played by TLR3 in humans. Although the precise locations of proteins derived from these genes vary among vertebrates (Paria et al.,2018), they are mostly located in the intracellular vesicle or cell membrane surface (Li et al., 2017; Hu et al., 2015). And we were analysis gene information and performed new cloning of the olive flounder TLR22 gene, it was mostly consistent with TLR3 of NCBI accession no. AB109396.1. It seems that the genetic information of TLR22 was incorrectly labeled as TLR3 in the NCBI gene bank. In the present study, we analyzed the changes in expression of the JfTLR3 and JfTLR22 genes at transcription level after poly I:C stimulation in vivo in olive flounders and in vitro in HINAE cells. The findings demonstrated that the mRNA levels of JfTLR3 and JfTLR22 were upregulated by extrinsically administered poly I:C; however, the increase was lower for JfTLR3 at 6 and 12 h post-stimulation, which became stronger at 24 and 48 h post-stimulation. These results suggest that JfTLR22 is a more sensitive receptor and recognizes extracellular earlier than JfTLR3. Therefore, it can be inferred that JfTLR3 might be located on the inner surface of the endosomal membrane in the cytoplasm, whereas JfTLR22 might be located on the outer surface of cells.

And plasmid vector-based shRNAs widely used to induce RNAi in vertebrate cells provide a useful tool to develop continuous immortalized cell lines in which the suppression of a target gene is stably maintained by RNAi (Taxman et al., 2010). Previously, we have demonstrated successful transcription of shRNA by fugu U6 promoter in bluegill fry (BF-2) cells and efficient knock-down of a target gene by the shRNA in BF-2, grunt fin (GF), and Chinook salmon embryo (CHSE) cells (Zenke and Kim, 2008). In the present study, we established HINAE cells that stably express shRNAs by transfection with the fugu U6 promoter-driven shRNA expressing vector and selection with G-418 The present study employing RNAi demonstrated the important role of TLR22 in recognizing extracellular poly I:C and induction of type I IFN response. HINAE cells harboring the JfTLR22-targeting shRNA vector showed markedly reduced JfTLR22 expression, which resulted in a large reduction of ISG15 and Mx genes expression, suggesting that JfTLR22 plays an important role in the induction of type I IFN response to extracellular dsRNA (poly I:C) stimulation for 12 h. On the contrary, at 12h after poly I:C stimulation, inhibition of TLR3 expression in HINAE cells with the JfTLR3-targeting shRNA vector weakly reduced the expression of ISG15 and Mx genes, suggesting that TLR3 partially participate in the induction of type I IFN response in cells.

Acknowledgments

This work was supported by the the National Research Foundation of Korea(NRF) grant funded by the Korea government under Grant number NRF-2021R1I1A2045209.

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