The transcriptome is the complete repertoire of expressed RNA transcripts in a cell. Its characterization is essential in deciphering the functional complexity of the genome and in obtaining a better understanding of cellular activities in organisms, including growth, development, disease, and immune defence. The definition of the transcriptome has long been a challenging task. Traditionally, global gene expression analysis has relied mostly on several approaches, including RNA hybridisation on high-density arrays, whole-genome tiling arrays, expressed sequence tag (EST), serial analysis of gene expression (SAGE), and SAGE-derived technologies, which include massively parallel signature sequencing (MPSS) and polony multiplex analysis of gene expression. However, these approaches have several inherent limitations. For example, the array-based approaches allow detection of specific sequences only and capture the transcriptome while ignoring splice-junction information or alternative splicing events. The EST approach provides only partial sequences of individual cDNA clones, is sensitive to cloning biases, and is associated with high costs and difficulties in data analysis. SAGE and MPSS are also costly and cannot be used for splicing events . Thus, the newly developed Solexa/Illumina RNA-seq and DGE high-throughput deep sequencing approaches have dramatically changed how functional complexity of the transcriptome can be studied. These approaches overcome many of the inherent limitations of traditional systems, making the detection of alternative splicing events and low-abundance transcripts possible. They have been applied recently to several species, such as yeast, Arabidopsis, Chlamydomonas, Zebrafish, Drosophila, Caenorhabditis, and human, for different purposes [9, 10, 18–29].
In this study, the transcriptome profile analysis of bacteria-challenged L. japonicus was conducted through these two approaches in an attempt to gain deep insights into the immunogenetics of a marine species. As expected, a large set of transcriptional sequences with complete or differing lengths of encoding regions was generated. KEGG analysis showed that more than 52% of transcripts are enrichment factors involved in approximately 219 known metabolic or signalling pathways, including cellular growth, differentiation, apoptosis, migration, endocrine, and immune system processes. Further, more than 8% of transcripts represent novel fish-specific genes that have never been described previously. Detailed analysis of immune-relevant genes and pathways showed that more than 2,673 transcripts are homologous to known immune-relevant genes, whereas approximately 2,082 transcripts can be enriched in various immune-relevant metabolic or signalling pathways. Challenging the fish with V. harveyi resulted in large alterations of the host transcriptome profile, including significant up- or down-regulation of 1,224 transcripts, among which 41 sequences might be novel immune-relevant genes in fish. In addition, several other biological processes that have not been linked to host immunity before, such as the metabolism of carbohydrates, amino acids, and lipids; activation of ATPase, NADH dehydrogenase, NAD kinase, and tyrosine protein kinase; and up-regulation of nuclear receptors, replication initiators, and ribosomal proteins, were found to be dramatically involved in host immune response. These significantly regulated transcripts might represent strong infection-responsive genes in L. japonicus, and reflect a number of immune activities during fish defence against bacterial challenge. The transcriptome profiling data sets obtained in this study provide strong basis for future genetic research in marine fish and support further in-depth genome annotation in vertebrates. Future molecular and functional characterisation of infection-responsive genes could lead to global identification of immune-relevant genes and infection markers in marine fish.
At present, transcriptome analysis in fish relies mainly on the EST approach [45, 46]. Although there have been an increasing number of ESTs sequenced in a large number of libraries in various fish species, including rainbow trout (Oncorhynchus mykiss), Atlantic salmon (Salmo salar), medaka (Oryzias latipes), and zebrafish (Danio rerio), the immune-relevant transcriptional profiling data sets obtained from fish are still insufficient. Recently, DGE- and microarray-based transcriptome profiling studies performed in zebrafish revealed that zebrafish and its developing embryo are useful in vivo models for the identification of host determinants of responses to bacterial infection [9, 10]. However, transcriptional information on immune responses to infection in a non-model marine fish remains elusive. Therefore, the large set of immune-relevant genes and their role in responses to bacterial challenge in L. japonicus presented in this study may largely improve knowledge on fish immunogenetics in other analytical systems. The present study also demonstrates the advantages of new deep sequencing approaches for gene discovery, thus providing new leads for functional studies of candidate genes involved in host-bacteria interactions. The RNA-Seq and DGE analyses conducted in this study were found to complement each other well. RNA-Seq was very effective in unravelling transcriptome complexity, and can detect a large set of genes, including numerous low-expressing genes or novel genes. DEG data can be merged with RNA-Seq data sets, indicating an affordable method for comparative gene expression study. Thus, RNA-Seq was initially performed in this study to provide strong reference transcriptome database for subsequent DGE analysis.
Emerging hallmark components and the cells necessary for innate and adaptive immunity in higher vertebrates have been identified in fish [47, 48]. This was the basis for the widely accepted notion that innate and adaptive immunity was established in teleosts about 470 million years ago. However, the exact molecular and cellular basis of immune systems in teleosts remains poorly understood. The precise regulatory mechanisms underlying the innate and adaptive immunity of teleosts remain vague due to the limited immune-relevant genetic information available in fish. The present work on the definition of high-throughput transcriptome data set of the immune system of L. japonicus may contribute greatly to better understanding of the molecular and cellular activities involved in fish immunity. Results unexpectedly showed that the fish immune system is more complex than previously thought. On one hand, the substantial amount of immune-relevant genes involved in metabolic and signalling pathways and the induction of genes encoding cell surface receptors, signalling intermediates, transcription factors, and inflammatory mediators show a clear conservation of mechanisms detected in other vertebrate models, including humans. On the other hand, a large set of novel immune response genes and infection markers that have never been linked previously to immune responses in other vertebrate systems was identified in L. japonicus, indicating the existence of numerous fish-specific immune activities during early vertebrate evolution.
For instance, the TLR family is the most important class of pattern recognition receptors that play crucial roles in mediating immune responses to pathogenic microorganisms [8, 49, 50]. Triggering of TLRs by ligands leads to the recruitment of adaptor proteins, resulting in the activation of a range of transcription factors, such as NF-κB, activator protein 1 (AP-1), and IFN regulatory factors (IRFs), through distinct signalling pathways. This eventually leads to the downstream activation of proinflammatory cytokines and receptors, such as IFN-α/β, TNF-α, IL-2, IL-6, IL-8, IL10, CD40, CD86, and MIP1α. To date, 13 TLRs (TLR1-13), at least five adaptor proteins (MyD88, Mal/TIRAP, TIR domain-containing adaptor protein, TRIF/TICAM1, TRAM/TICAM2, and SARM), and numerous downstream effectors have been described in mammals and humans. In the present study, a series of TLRs and corresponding adaptor proteins and downstream effectors were identified in L. japonicus. The identified TLRs include the majority seen in mammals and humans (TLR1-13), and four TLRs (TLR14, TLR18, TLR21, and TLR23) seen in fish species. Adaptor proteins and downstream effectors identified include the majority known in mammals and humans, including MYD88, BTK, TOLLIP, FADD, HMGB1, HRAS, HSPD1, CASP8, MAPK8IP3, PELI1, RIPK2, SARM1, TICAM2, TIRAP, EIF2AK2, IRAK1, IRAK2, MAP3K7, MAP3K7IP1, NR2C2, PPARA, PRKRA, TRAF6, UBE2N, and UBE2V1. These adaptor proteins and downstream effectors have been found to be well enriched in various known TLR signalling pathways. Downstream transcriptional factors and pro-inflammatory cytokines mediated by these pathways, including NF-κB, JNK/p38, NF/IL6, IRF, IFN-α/β, TNF-α, IL-2, IL-6, IL-8, and IL-10, was also be identified successfully. These suggest that TLR mechanisms are conserved from fish to mammals throughout vertebrate evolution. A putative draft of TLR signalling pathways in L. japonicus based on knowledge of TLR signalling in mammalian species was constructed (Figure 4). However, TLR signalling pathways in fish might be more intricate compared with those in mammalian species because of the novel TLRs (TLR14, TLR18, TLR21, and TLR23). An in-depth study of novel TLRs will improve understanding of fish-specific innate immunity in early vertebrates and even the complete evolutionary history of TLR-based innate immunity. DGE analysis revealed that TLR-1, -3, -13, -18, -21 and their signalling intermediates (Rac1, AKT, CASP8, IRAK1, TRAM, IRAK1, IKK alpha/beta, IRF7, and STAT1) were up- or down-regulated dramatically at different levels in the pathway upon bacterial challenge (P ≤ 0.01). This provides evidence that both conserved (TLR-1, -3, -13) and fish-specific (TLR-18, -21) TLR-based immunity participates in fish defence against bacterial challenge.
The innate immune system is generally believed to represent the evolutionarily ancient aspect of vertebrate immunity. As a representative of lower vertebrates, fish is suggested to possess stronger innate immune responses. However, fish adaptive immunity might be more primitive because of limited immunoglobulins and hallmark components necessary for adaptive immunity identified in this species . In recent years, several hallmarks for T and B cells (TCR, BCR, CD3, CD4, and CD8), antigen presenting and processing molecules (MHCI, MHCII, and DC-SIGN/CD209), co-stimulatory factors (CD80/86, CD83, CD154, and CD40), and immunoglobulins (IgM, IgD, and IgZ/T) have been identified in teleost fish, thus providing preliminary evidence that the adaptive immune system might also be well-established in fish. However, the precise molecular and cellular bases and mechanisms underlying teleost adaptive immunity are still uncharacterised and require further immunogenetic studies. The present study successfully identified a large number of adaptive immune-relevant components homologous to those in higher vertebrates, providing abundant data sets for insights into the characterisation and origin of adaptive immunity in early vertebrates. Data sets imply that adaptive immunity in teleost fish seems to be much more complicated than previously believed. The basic components and signalling pathways necessary for adaptive immunity exist in fish, and a majority showed clear conservation between fish and mammals. For instance, T cell receptor (TCR) signalling pathways regulate T cell activation, one of the most important processes in adaptive immunity . Majority of the four types of TCRs (TCR-α/β, -γδ) and numerous signalling transducers (Zap70, Lyn, LCK, SHP1, CD3, ITAM, LAT, Fyb, SLP-76, CBL, NCK, LAT, GRB2, CARMA1, NFAT, AP1, MALT1, and GRB2) discovered in humans and mammals can be identified in L. japonicus. DGE analysis showed that a number of TCR signalling pathway members, including TCR beta chain, Zap70, LCK, SHP1, CARMA1, Vav, NFAT, GRB2, MALT1, NCK, and Raf1, are induced significantly after bacterial challenge (P ≤ 0.01). These pathway members largely contribute to the proliferation and activation of T cells in mammals, thus suggesting that TCR signalling mechanisms underlying the T cell activation might be conserved between teleost fish and mammals. A putative draft of TCR signalling pathways based on knowledge of pathways known in mammals was constructed (Figure 5). Future studies on these pathways are expected to not only enrich current knowledge on fish immunology but also contribute to better understanding of the evolutionary history of adaptive immunity.