Inducible MicroRNA-3570 Feedback Inhibits the RIG-I-Dependent Innate Immune Response to Rhabdovirus in Teleost Fish by Targeting MAVS/IPS-1.

Effectively recognizing invading viruses and subsequently inducing innate antiviral immunity are essential for host antiviral defense. Although these processes are closely regulated by the host to maintain immune balance, viruses have evolved the ability to downregulate or upregulate these processes for their survival. MicroRNAs (miRNAs) are a family of small noncoding RNAs that play vital roles in modulating host immune response. Accumulating evidence demonstrates that host miRNAs as mediators are involved in regulating viral replication and host antiviral immunity in mammals. However, the underlying regulatory mechanisms in fish species are still poorly understood. Here, we found that rhabdovirus infection significantly upregulated host miR-3570 expression in miiuy croaker macrophages. Induced miR-3570 negatively modulated RNA virus-triggered type I interferon (IFN) and antiviral gene production, thus facilitating viral replication. Furthermore, miR-3570 was found to target and posttranscriptionally downregulate mitochondrial antiviral signaling protein (MAVS), which functions as a platform for innate antiviral signal transduction. Moreover, we demonstrated that miR-3570 suppressed the expression of MAVS, thereby inhibiting MAVS-mediated NF-κB and IRF3 signaling. The collective results demonstrated a novel regulation mechanism of MAVS-mediated immunity during RNA viral infection by miRNA.IMPORTANCE RNA viral infection could upregulate host miR-3570 expression in miiuy croaker macrophages. Induced miR-3570 negatively modulates RNA virus-triggered type I IFN and antiviral gene production, thus facilitating viral replication. Remarkably, miR-3570 could target and inhibit MAVS expression, which thus modulates MAVS-mediated NF-κB and IRF3 signaling. The collective results of this study suggest a novel regulation mechanism of MAVS-mediated immunity during RNA viral infection by miR-3570. Thus, a novel mechanism for virus evasion in fish is proposed.

Identification of Caspase-6 and Caspase-7 from miiuy croaker and evolution analysis in fish.

Apoptosis is a basic biological phenomenon of cells, which is an important component in the evolution of organisms, the stabilization of the internal environment and the development of multiple systems. In addition, the caspase protein family plays an important role in these pathways of apoptosis. Among them, apoptotic executors can directly act on specific substrates to complete the apoptotic response. In this study, we identified the Caspase-6 and Caspase-7 genes of miiuy croaker, and then analyzed the evolution of the whole Caspase family, furthermore described the evolutionary selection sites of the caspase-6 and caspase-7 genes in fish. The results showed that Caspase-6 gene appeared earlier than Caspase-7 in species evolution and gene duplication in teleost fish. Moreover, we also found that caspase-6 gene had no potential positive selection sites in the evolution of fish. Unlike the caspase-6 gene, the caspase-7 gene did not appear to be missed or replicated during the evolution of the species, while, it to be found two potential positive selection sites.

miRNA-8159 is involved in TLR signaling pathway regulation after pathogen infection by direct targeting TLR13 in miiuy croaker.

Toll-like receptors (TLRs) play a crucial role in the recognition of immune reactions against invading pathogens. The molecular regulation mechanisms of TLR expression in aquatic organisms remain unclear. MicroRNAs (miRNAs) are small non-coding RNAs that are critical adjustors of immune signaling pathway at the post-transcriptional level and play critical roles in intricate networks of host-pathogen interactions and innate immunity. The critical role of TLRs in host defense for discerning certain kinds of pathogen associated molecular patternsand striking a cascade immune response in fish have been demonstrated. Miiuy croaker TLR13 significantly increased after infection with Vibrio anguillarum, which suggests that mmiTLR13 plays an important role in innate immunity. In this study, the role of miR-8159 was explored in regulating TLR13, which is involved in inflammatory responses in miiuy croakers. Bioinformatics was used to predict miR-8159, which has a direct negative regulatory effect on TLR13 in miiuy croaker. Afterward, the dual luciferase reporter assay containing miRNA mimics or inhibitors and pre-miR-8159 showed that miR-8159 was the direct negative regulator of TLR13 in miiuy croaker. Moreover, miR-8159 downregulated the expression of TLR13 in the transcription level. The expression of miR-8159 could be upregulated by V. anguillarum challenged miiuy croaker and LPS exposure macrophages. Thus, miR-8159 could be induced by V. anguillarum and may function as a negative regulator of TLR13 in the immune response of miiuy croakers.

Functional characterization and subcellular localization of miiuy croaker cytosolic MITA involved in activation NF-κB pathway.

In the innate immune responses in host protection, pattern recognition receptors are involve in a variety of sensing mechanisms to recognize and counter pathogen invasion. Recently, a resident endoplasmic reticulum adaptor, stimulator of interferon genes (STING) protein, also called MPYS, ERIS and MITA, has been indicated to play a critical role in innate immune responses. In this study, bioinformatics and functions of MITA from miiuy croaker (mmiMITA) were characterized. MmiMITA was ubiquitously expressed in the detected tissues of miiuy croaker and the highest expression in liver. Moreover, the expressions were dramatically upregulated in liver, spleen and kidney after stimulation with poly(I:C). Meanwhile, the expressions analysis of mmiMITA at the transcriptome database further prove that upon different stimuli, mmiMITA is most sensitive to the stimulation of poly(I:C) in vivo. Furthermore, the immunofluorescence of mmiMITA shows in the cytoplasm of Hela cells. Overexpression of mmiMITA can activate NF-κB reporter gene, it implying that mmiMITA might act as an important role in immune responses by activating NF-κB to induce the production of pro-inflammatory cytokines. The research of mmiMITA will enrich the information of teleost fish MITA and the functional experiments also will be helpful for researching about fish immune systems in the future.

Identification and functional characterization of miiuy croaker IRF3 as an inducible protein involved regulation of IFN response.

IFN regulatory factor (IRF) 3 as an important member of IRF family, is required for the host antiviral response. In mammals, IRF3 is known to be a critical player in regulating the transcription of IFN and IFN-stimulated genes (ISGs). However, only a few studies investigated the characteristics of IRF3 genes in fish. In this study, IRF3 from miiuy croaker was identified and characterized in bioinformatics and functions. Miiuy croaker IRF3 had conserved DBD, IAD and SRD domains with other vertebrates IRF3 genes, also miiuy croaker IRF3 had relatively conserved gene synteny and gene structures with other fish IRF3 genes. Evolutionary analysis showed IRF3 genes in mammals underwent positive selection, while IRF3 in fish underwent purifying selection. Expression analysis showed miiuy croaker IRF3 was expressed in all tested tissues and up-regulated expressed in infected liver and kidney; and up-regulated expression of miiuy croaker IRF3 was observed in head kidney macrophages which stimulated with poly(I:C) indicating that miiuy croaker IRF3 participated in the immune response to defense against poly(I:C) infection. Furthermore, luciferase reporter assay showed that overexpression of miiuy croaker IRF3 can activate the production of ISRE and IFNα, suggesting that miiuy croaker IRF3 acted as transcription activators in immune responses and maybe activate IFN signaling pathway. Immunofluorescence assay showed miiuy craoker IRF3 was localized in the cytoplasm in Hela cells. Overall, we systematically and comprehensively analyzed the bioinformatics and functions of miiuy croaker IRF3, which provided further insights into the transcriptional regulation of IRF3 gene in fish and valuable information for the study of evolution of IRF3 genes.

MicroRNA negatively regulates NF-κB-mediated immune responses by targeting NOD1 in the teleost fish Miichthys miiuy.

Inflammation is a self-protection mechanism that can be triggered when innate immune cells detect infection. Eradication of pathogen infection requires appropriate immune and inflammatory responses, but excessive inflammatory responses can cause uncontrolled inflammation, autoimmune diseases, or pathogen dissemination. Mounting evidence has shown that microRNAs (miRNAs) in mammals act as important and versatile regulators of innate immunity and inflammation. However, miRNA-mediated regulation networks are largely unknown in inflammatory responses in lower vertebrates. Here miR-144 and miR-217 are identified as negative regulators in teleost inflammatory responses. We find that Vibrio harveyi and lipopolysaccharide (LPS) treatment significantly upregulate the expression of fish miR-144 and miR-217. Upregulated miR-144 and miR-217 suppress LPS-induced inflammatory cytokine expression by targeting nucleotide-binding oligomerization domain-containing protein 1 (NOD1), thereby avoiding excessive inflammatory responses. In addition, miR-144 and miR-217 regulate inflammatory responses through NOD1-induced nuclear factor kappa (NF-kB) signaling pathways. These findings demonstrate that miR-144 and miR-217 play regulatory roles in inflammatory responses by modulating the NOD1-induced NF-κB signaling pathway.

microRNA-375 modulates the NF-κB pathway in miiuy croaker by targeting DUSP1 gene.

microRNAs (miRNAs) are highly conserved, small non-coding endogenous molecule, and can participate in a variety of biological processes in organisms such as development, growth and immune response. Dual-Specificity Phosphatases (DUSPs) are enzymes that can remove phosphate groups from phosphatases. Research found that DUSP1 is an important molecule in the process of MAPK regulation. However, as a significant regulatory factor, the study of DUSP1 was very few in fish. Consequently, in this study, the regulatory role of miRNAs on DUSP1 has been verified through dual-luciferase reporter assay and western blotting analysis. Furethermore, we found that miR-375 mimics and pre-miR-375 plasmid can negatively regulate the target gene DUSP1 in miiuy croaker through combining with 3'untranslated region of DUSP1 gene. These experiment results directly indicate the negative regulatory function of miR-375 to DUSP1. Moreover, miR-375 can negatively regulate NF-κB signaling pathway via target to DUSP1. This study can increase our knowledge and help us to understand complexity of genomic and complex gene expression regulatory networks in teleost fish.

Recognition of Lipopolysaccharide and Activation of NF-κB by Cytosolic Sensor NOD1 in Teleost Fish.

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria. This molecule can induce strong immune response and various biological effects. In mammals, TLR4 can recognize LPS and induce inflammatory response. However, the innate receptor in fish for recognizing LPS remains ambiguous. LPS can invade the cytoplasm via outer membrane vesicles produced by Gram-negative bacteria and could be detected by intracellular receptor caspase-11 in mammals, so, there may also exist the intracellular receptors that can recognize LPS in fish. NOD1 is a member of NOD-like receptors family and can recognize the iE-DAP in the cytoplasm in mammals. In fish, NOD1 can also respond to infection of Gram-negative bacteria and may play an important role in the identification of bacterial components. In this study, to study whether NOD1 is a recognition receptor for LPS, we detected the expression of NOD1 and several cytokines at transcript levels to determine whether LPS can induce inflammatory response in teleost fish and NOD1 can respond to LPS. Then, we perform the binding analysis between NOD1 and ultrapure LPS by using Streptavidin pulldown assay and enzyme-linked immunosorbent assay to prove that NOD1 can be combined with LPS, and using dual luciferase reporter gene assay to verify the signal pathways activated by NOD1. Next, through cell viability analysis, we proved that LPS-induced cytotoxicity can be mediated by NOD1 in fish. The results showed that NOD1 can identify LPS and activate the NF-κB signal pathway by recruiting RIPK2 and then promoting the expression of inflammatory cytokines to induce the resistance of organism against bacterial infection.

MicroRNA-148 as a negative regulator of the common TLR adaptor mediates inflammatory response in teleost fish.

MicroRNAs are small endogenous noncoding RNAs implicating in the regulation of diverse biological processes, including proliferation, differentiation, cancer, apoptosis, and viral infections. MicroRNAs regulate gene expression by either mRNA degradation or inhibition of protein translation. Although microRNAs have emerged as important controller involved in regulation of inflammatory response, the microRNA-mediated regulatory mechanism remains less clear in teleost. Here, we report that miR-148 targets MyD88 and down-regulates its expression by inhibition protein translation rather than degradation mRNA in miiuy croaker. Additionally, we found that miR-148 was significantly upregulated in miiuy croaker after treated with Vibro harveyi, as well as LPS. Overexpression of miR-148 inhibited LPS-induced inflammatory cytokines production, such as IL-6 and IL-1ß, which then avoid excessive inflammation response. miR-148 has also been identified to suppress NF-κB pathway through targeting and repressing MyD88 expression. Taken together, our findings indicate that miR-148 participates in bacteria-induced inflammatory response and act as a negative regulator for MyD88-mediated NF-κB signaling, which may clarify the mechanism of microRNAs for avoiding excessive inflammation in teleost fish.

MicroRNA-21 contributes to suppress cytokines production by targeting TLR28 in teleost fish.

Toll-like receptors (TLRs) as important pattern recognition receptors, play critical roles in identifying pathogens and activating the immune response. However, when the dysregulation was occurred in this process, it could lead to excessive immune response, so it need many regulatory factors to control this process. Recently, microRNAs (miRNAs) have been shown to act as an important regulator in TLRs signaling pathway. As a member of TLRs family, TLR28 has been newly discovered in teleost fish, and play an important role in the immune response. In this study, we found that the expression of miR-21 was up-regulated after poly(I:C) stimulation, and miR-21 could inhibit the expression of cytokines. Then we predicted the target genes of miR-21, and found that TLR28 is a direct target of miR-21, which could be significantly down-regulated by both miR-21 mimics and pre-miR-21. These results suggested that miR-21 can inhibit the expression of cytokines by negative regulation of TLR28, thereby inhibiting the generation of excessive immunity and maintaining the balance of the body. This study is the first to demonstrate that miRNA can suppresses cytokines by regulating the TLR signaling pathway in teleost fish, and also can provides some new ideas for the research of the regulation of miRNA and immune system in mammals.

Up-regulated of miR-8159-5p and miR-217-5p by LPS stimulation negatively co-regulate TLR1 in miiuy croaker.

Toll-like receptors (TLRs) are a group of pattern-recognition receptors which play vital roles in ligand recognition and activation of the innate immune response. As an important member of TLRs family, TLR1 is mainly responsible for PAMPs from bacteria and play a pivotal role in sensing microbial products. Recent studies revealed that TLR1 could perceive LPS stimulation and transfer signals to activate the NF-κB pathway, whereas ligands and signaling pathway of TLR1 are still unclear in fish. Growing evidence has shown that miRNAs (microRNAs) play as negative regulators in controlling the diverse of biophysical and biochemical processes at the post-transcriptional level. In this study, we used a combination of bioinformatics and experimental techniques to exhibit that both miR-8159-5p and miR-217-5p were the direct negative regulators of TLR1 in miiuy croaker. Furthermore, dual-luciferase reporter assays showed that combining miR-8159-5p and miR-217-5p exhibited a greater negative regulatory effect on TLR1 than only miR-8159-5p or miR-217-5p. Additionally, we also demonstrated that the expression of both the two miRNAs could be up-regulated by LPS stimulation in either LPS-stimulation spleen tissue or LPS-treated cultured macrophage, which indicating that miR-8159-5p and miR-217-5p could be induced by LPS and may be as the negative regulators of TLR1 involved in the immune response to LPS stimulation. These results would enhance our understanding of the miRNA regulation in fish TLR signaling pathways.

NOD1 is the innate immune receptor for iE-DAP and can activate NF-κB pathway in teleost fish.

The innate immune system is the first line for organisms defense against microbial infection, and NOD-like receptors (NLRs) protein family is an important member of innate immunity effector molecules. It has been proved that NLRs are located in the endochylema and can senses of microbial products. NOD1 is one of the representatives of this family, it has been proved that in mammals, NOD1 can distinguish a specific muropeptide (G-d-glutamyl-meso-diaminopimelic acid, iE-DAP) which was derived from bacterial peptidoglycans. However, the NOD-mediated intracellular recognition of microorganisms remains largely uncharacterized in teleost fishes. In this study, we use miiuy croaker (Miichthys miiuy) as a model to determine NOD1 can response to the infection of Gram-negative bacteria and it is the receptor that can recognize of iE-DAP by LRRs domain, it can activate the NF-κB signaling pathway through recruit RIP2 to induce inflammatory response in teleost fishes. Results showed that NOD1 can recognize the components of Gram-negative bacteria and activate inflammatory response to resistance of bacterial infection. Our study can improve the knowledge on immune system of fishes and provide a theoretical basis for the study of prevention and treatment of fish diseases.