Introduction
Innate immunity is evolutionarily conserved and acts as the first line of host defence against pathogen infections. Detection of microbial pathogen is an essential step in the initiation of innate immune responses and is mediated by pattern recognition receptors (PRRs; Janeway & Medzhitov 2002; Takeuchi & Akira 2010).
PRRs are microbial sensors of the innate immune system that can recognize relatively invariant molecular patterns widely shared by most types of microbial pathogens, and these relatively invariant molecular patterns are referred to as pathogen-associated molecular patterns (PAMPs).
In the past decades, several families of PRRs have been characterized and extensively studied, including Toll-like receptors (TLRs), retinoic acid-inducible gene-I-like receptors (RLRs) and Nod-like receptors (NLRs).
On activation, these PRRs initiate host innate immune responses and a series of inflammatory responses to defend against infectious pathogens, which subsequently trigger the adaptive immune system. Any dysregulation of these receptors or their downstream signalling process can potentially result in uncontrolled inflammation, autoimmune diseases or pathogen dissemination (Cook et al. 2004).
It is therefore important that these processes are tightly regulated by detailed mechanisms. There is growing evidence to indicate that many molecules have been identified as positive or negative regulators of signalling for these innate immune receptors (Jefferies et al. 2003; Wang et al. 2009; Han et al. 2010; Ramos & Gale 2011; Bonardi et al. 2012). MicroRNAs (miRNAs) have recently emerged as important regulators of gene expression essential for many biological processes, including organ development, cell differentiation and tumour progression (Bartel & Chen 2004; Calin & Croce 2006).
miRNAs are a class of non-coding RNAs of approximately 22 nucleotides in length and are highly conserved in eukaryotes. The biogenesis of miRNAs involves two processing steps by two RNase III enzymes, Drosha and Dicer. miRNAs are initially transcribed as primary transcripts (pri-miRNA) by RNA polymerase II (RNA pol II) and cropped into a short hairpin precursor miRNA transcript (pre-miRNA) in the nucleus by the RNase III Drosha (Lee et al. 2004). Pre-miRNAs are actively transported into the cytoplasm where they are cleaved by Dicer to form mature miRNAs.
The mature miRNA duplex is then incorporated into the RNA-induced silencing complex in the cytoplasm, which identifies target mRNA by base pair complementarity resulting in mRNA degradation and/or translational suppression (Filipowicz et al. 2008; Winter et al. 2009).
So far, as many as 60% of protein-coding genes are predicted to be regulated by miRNAs at the post-transcriptional and translational levels, and each miRNA can potentially target thousands of different mRNAs (Zhang & Li 2013). In the past decade, accumulating evidence has reported that miRNAs participate in mammalian immune cell differentiation, the outcome of immune responses and the development of immunological diseases (Baltimore et al. 2008; Xiao & Rajewsky 2009). Therefore, it is not surprising that miRNAs are involved in TLR, RLR and NLR signalling in the innate immune system. Toll-like receptors, known as important players, participate in controlling multiple aspects of the innate immune response.
Following recognition of TLR ligands, TLRs elicit innate immunity by activating multiple intracellular signalling cascades. In mammals, a dozen sets of miRNAs have been show to regulate TLR signalling pathways at different layers, including regulation of TLR expression, TLR-associated signalling proteins and regulatory molecules, and TLR-induced transcription factors and functional cytokines (He et al. 2014a). For example, the let-7 miRNA family, including let-7e and let-7i, has been reported to modulate TLR4 (Li & Shi 2013); MyD88 has been identified as a miR-155 target (Tang et al. 2010); miR-9 has been shown to target NF-jB1 in human monocytes and neutrophils (Bazzoni et al. 2009). The RLR sensors can recognize the genetic material of RNA viruses and induce inflammatory cytokines and type I interferon. In mammals, increasing evidence suggests that some miRNAs, such as miR-146a, miR-466l and miR-29a, have key roles in the regulation RIG-I signalling pathway and the expression of type I interferon (Hou et al. 2009; Li et al. 2012; Papadopoulou et al. 2012). With regard to NLR signalling pathway, the linkage of NOD1 or NOD2 to related ligands activates MAPK and NF-jB signalling cascades, resulting in the production of pro-inflammatory cytokines and chemokines. Other NLRs are involved in the assembly and activation of inflammasomes, which can control caspase-1 activation and subsequent processing of IL-1 and IL-18 (Franchi et al. 2012). Recently, miR-223 has been identified to target NLRP3 gene and thus inhibit IL-1b production from the inflammasome (Bauernfeind et al. 2012). Other miRNAs involved in the regulation of NLR pathways need further investigation. Fish may be regarded to be an excellent biological model in immunology studies as it is a representative population of lower vertebrates serving as an important link to early vertebrate evolution. In teleost fish, the innate immune response is a fundamental defence mechanism and is of vital importance for the disease resistance (Magnadottir 2006). Due to the significance of innate immunity in teleost fish, the related modulators and regulation mechanisms are particularly important. From the first report of miRNAs in zebrafish (Lagos-Quintana et al. 2001; Lim et al. 2003), the role of miRNAs as fine-tuning regulators of different biological processes has been clarified in teleost fish. Some miRNAs have been proposed as key switches for activating or inhibiting the innate immune response. However, these miRNAs have only been identified in few teleost species, indicating that the characterization of miRNAs in fish requires much work. Based on the strong conservation of miRNA among all vertebrates, studies of teleost miRNA can not only benefit fish, but also provide a much-needed insight into the mammal gene regulatory networks through orthologous gene functional studies. This review focuses on summarizing and comparing the recent findings regarding miRNAs associated with the innate immune receptor signalling in teleost fish. We review the current understanding of the mechanisms of miRNAs in regulating TLR, RLR and NLR signalling pathways at different levels. We also discuss current limitations to research of miRNA regulation in teleost fish.
miRNAs associated with immune responses in teleost fish
The next-generation sequencing is a highly rapid and effective method to explore small-RNA (sRNA) populations, and hence, it has been widely used in animals. In mammals, most miRNAs are identified and discovered using highthroughput technologies. Similarly, an increasing number of miRNAs have been identified in different fish species using the sequencing technology. Recent studies of miRNAs in teleost fish have revealed the involvement of miRNAs in immune processes and their link to inflammatory disorders (Table 1). To date, most studies have only focused on identifying the miRNAs that are differentially expressed between infected and uninfected individuals. The underlying hypothesis of these studies is that miRNAs may participate in regulating immune system gene networks when a change occurs in their expression upon pathogen infection.
Zebrafish (Danio rerio)
The zebrafish is an important model organism for studying vertebrate innate immunity, and it provides several advantages for the study of miRNA functions. In the first weeks of zebrafish development, innate immunity is the primary line of defence against infections, when there was no functional adaptive immunity (Renshaw & Trede 2012). The initial investigations for miRNAs in zebrafish have been conducted as early as 2001, which have revealed the conservation of several miRNAs between mammals and zebrafish (Lagos-Quintana et al. 2001). Subsequently, more detailed analyses of zebrafish miRNAs have been performed. Based mainly on sequencing of small-RNA cDNA libraries, approximately 217 different miRNAs have been identified from several developmental stages (Chen et al. 2005; Fjose & Zhao 2010). To gain insights into function of miRNAs in immunity, the screening of miRNAs was conducted after challenge with pathogenic bacteria. Ordas et al. (2013) reported the zebrafish miR-21, miR-29 and miR-146 families could be obviously induced by infection of zebrafish embryos with Salmonella typhimurium. Wu et al. (2012) screened miRNAs after Vibrio harveyi infection and found 3 specific zebrafish miRNAs (miR-122, miR-192 and miR194a) showing the different expression between infected and uninfected zebrafish. Moreover, Fernandes et al. (2013) examined the effect of LPS immunostimulation on miRNA expression and found dre-miR-92a downregulated while dre-miR-99, dre-miR-125b and dre-miR-222 upregulated in larvae exposed to LPS. To further investigate the immunological role of zebrafish miRNAs under Gram-positive bacterial infection, the effect of Staphylococcus aureus on miRNA expression profiles was tested, and the data identified a total of 30 differentially expressed miRNAs during infection (Zhang et al. 2019). In addition, to understand the relationships between miRNAs and their targets during infection, researchers have used zebrafish as an infection model to characterize the miRNA and mRNA transcriptomes upon Vibrio parahaemolyticus infection (Ji et al. 2019). Collectively, these data provide basic insight into the regulation roles of miRNAs in zebrafish innate immune responses against bacterial infection.
Olive flounder (Paralichthys olivaceus)
Olive flounder has a peculiar anatomy due to the metamorphosis that changes its body form from larvae to juvenile (Kim et al. 2009; Xie et al. 2011). The species is the economically important teleost fish farmed widely in the world. miRNA in the species was first identified in 2011, which was found to be related to the asymmetric development (Xie et al. 2011). To preliminarily know the effect of pathogen infection on microRNA expression profile in olive flounder, Zhang et al. firstly reported the change of cellular miRNA expression profiles in response to megalocytivirus (DNA virus, Iridoviridae). The results showed that a total of 381 host miRNAs were identified, among which 121 miRNAs were significantly altered post-viral infection (Zhang et al. 2014a). To study the effect of Edwardsiella tarda infection on miRNA expression profile, Japanese flounder were injected intraperitoneally with E. tarda, and the data showed that a total of 164 mature miRNAs were identified, of which 17 miRNAs were differentially expressed after infection. Meanwhile, Liu et al. (2019) also conducted global profiling of Japanese flounder kidney microRNAs upon E. tarda infection and identified a total of 96 differently expression miRNAs. To gain insights into the miRNA expression profiles upon RNA virus infection, the effect of VHSV infection on the miRNA expression profile was measured at different time points post-infection. Najib et al. (2016) reported that a total of 372 mature miRNAs have been identified, among which 63 miRNAs were differentially expressed during viral infection. These miRNAs exhibited dynamic changes in their expression during viral infection, suggesting the profound effects of miRNAs on host immune defence during viral infection.