Junxia Cui

MicroRNA-3570 Modulates the NF-κB Pathway in Teleost Fish by Targeting MyD88

The inflammatory response, a protective process to clear detrimental stimuli, constitutes the defense against infectious pathogens. However, excessive inflammation disrupts immune homeostasis, which may induce autoimmune and inflammatory diseases. In this study, we report that microRNA (miR)-3570 plays a negative role in the bacteria-induced inflammatory response of miiuy croaker. Upregulation of miR-3570 by Vibrio anguillarum and LPS inhibits LPS-induced inflammatory cytokine production, thus avoiding an excessive inflammation response. Evidence showed that miR-3570 targets MyD88 and posttranscriptionally downregulates its expression. Overexpression of miR-3570 in macrophages suppresses the expression of MyD88, as well as its downstream signaling of IL-1R–associated kinases 1 and 4 and TNFR-associated factor 6. These results suggest that miR-3570 plays a regulatory in the bacteria-induced inflammatory response through the MyD88-mediated NF-κB signaling pathway by targeting MyD88.

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

ABSTRACT 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‐&kgr;B 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. HighlightsTLR1 could response to LPS stimulation in miiuy croaker.miR‐8159‐5p and miR‐217‐5p negatively co‐regulated TLR1.Expression profiles of two miRNAs responding to LPS was conducted in vitro and in vivo.

Inducible microRNA-214 contributes to the suppression of NF-κB-mediated inflammatory response via targetingmyd88gene in fish

Upon recognition of bacterial pathogens by pattern recognition receptors, cells are activated to produce pro-inflammatory cytokines and type I IFN by multiple signaling pathways. Every step of the process must be precisely regulated to prevent dysregulation. MicroRNAs (miRNAs) have been shown to be important regulators with profound effects on inflammatory response. Nevertheless, the miRNA-mediated regulatory mechanism remains unclear in fish species. Here, we addressed the role of miiuy croaker miR-214 in the bacteria triggered inflammatory response. miR-214 could significantly be up-regulated by Vibro harveyi and LPS stimulation. Up-regulating miR-214 subsequently inhibits the production of inflammatory cytokines by targeting myd88 to avoid excessive inflammation. Moreover, the negative regulatory mechanism of miR-214 has been demonstrated to be via the myd88-mediated NF-κB pathway. This is the first to focus on miR-214 acting as the negative regulator involved in the bacteria-triggered inflammatory response and thus may provide knowledge on the host-cell regulator responses to microbial infection.

IRF9 as a negative regulator involved in TRIF-mediated NF-κB pathway in a teleost fish, Miichthys miiuy

&NA; Proinflammatory cytokines and type I IFNs were produced by TLR signaling and these responses are crucial for host defensive responses against pathogens. In order to avoid harmful and inappropriate inflammatory responses, there are multiple mechanisms to negatively regulate TLR signaling. In this paper, we have firstly studied IRF9 functions as a negative regulator involved in TRIF‐mediated NF‐&kgr;B pathway. Moreover, we found inhibitory effect of IRF9 primary depends on DBD domain. Interestingly, we also demonstrated that else mutants of IRF9, except for IRF9‐&Dgr;DBD, have different inhibitory effects upon TRIF‐mediated NF‐&kgr;B pathway. This study provides a novel evidence about the negatively regulation of innate immune signaling pathway in teleost fish. In addition, this finding provides new insights into the regulatory mechanism in mammals.

Genome-guided transcriptome analysis of miiuy croaker provides insights into pattern recognition receptors and cytokines in response to Vibrio anguillarum

&NA; Miiuy croaker (Miichthys miiuy), as an economically important marine fish is affected by numerous bacterial diseases. Infection by bacterial pathogen Vibrio anguillarum causes high mortality and great economic loss in aquaculture. To understand the immune response of the miiuy croaker to V. anguillarum infection, we analyzed the transcriptomic profile of V. anguillarum‐challenged M. miiuy, and two cDNA libraries, namely, the normal library (Ctrl) and V. anguillarum challenged library (Van) were constructed. Combined with the whole genome‐guided assembly, total 47,971 unigenes were acquired. Moreover, 2482 significantly differentially expressed unigenes were identified based on the expression patterns. Immune‐related differentially expressed genes (DEGs) that were significantly up‐ or down‐regulated after V. anguillarum injection were identified via enrichment analysis using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases. To further understand the expression profiles that underlie the response to V. anguillarum infection, we investigated the DEGs that are related to pattern‐recognition receptors and cytokines. The expression patterns of relevant DEGs were validated by qRT‐PCR. Additionally, the expression profiles of DEGs that are related to pattern‐recognition receptors and cytokines were further measured in miiuy croaker macrophages. This study is the first to identify and characterize the transcriptome of miiuy croaker in response to V. anguillarum infection. The results expand the general understanding of the immune defense mechanisms of miiuy croaker and provide helpful information for counteracting V. anguillarum infection. Highlights2482 DEGs were identified within 1313 up‐regulated unigenes.Immune‐related DEGs were identified and classified.DEGs related to PPRs and cytokines were analyzed in detail.

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

Abstract 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‐&kgr;B 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. HighlightsNOD1 can recognized iE‐DAP in teleost fish.NOD1 can activate the immune response to defense G− bacteria infection in teleost fish.NOD1 activation of NF‐&kgr;B signaling pathway by recruit downstream adaptor protein RIP2.

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

&NA; 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. HighlightsmiR‐21 could response to poly(I:C) stimulation in teleost fish.miR‐21 negatively regulate the TLR signaling pathway in teleost fish.miR‐21 could inhibit the expression of cytokines and antiviral genes.

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

ABSTRACT 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.

The inducible microRNA-203 in fish represses the inflammatory responses to Gram-negative bacteria by targeting IL-1 receptor-associated kinase 4

Innate immune responses are the first defense against pathogenic invaders. Activation and termination of these immune responses are regulated by several mechanisms. MicroRNAs (miRNAs), a group of small non-coding RNAs, have been implicated in the regulation of a spectrum of both physiological and pathological conditions, including immune responses. Although the immune regulatory miRNA networks in higher vertebrates have been well described, regulation of these responses in fish species is poorly understood. In the present study, we investigated the role of the miRNA miR-203 involved in inflammatory responses in miiuy croaker (Miichthys miiuy). We found that the Gram-negative bacterium Vibrio anguillarum and lipopolysaccharide significantly up-regulated host miR-203 expression. The increased miR-203 expression suppressed the production of inflammatory cytokines and thereby prevented mounting of a full immune response. Mechanistically, we identified and validated IL-1 receptor-associated kinase 4 (IRAK4) as a target of miR-203. We observed that miR-203 post-transcriptionally controls IRAK4 expression and thereby inhibits the activation of nuclear factor κB (NF-κB) signaling. In summary, our findings reveal that miR-203 in fish is a critical suppressor of innate immune responses to bacterial infection by suppressing a feedback to IRAK4–NF-κB–mediated signaling.

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

Abstract 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. HighlightsTLR13 was direct target gene of miR‐8159 in miiuy croaker.pri‐miR‐8159‐5p showed remarkable inhibitory effects than mimics.The regulation of miR‐8159 to TLR13 presented dose and time dependent.