TNF-Receptor-Associated Factor 3 in Litopenaeus vannamei Restricts White Spot Syndrome Virus Infection Through the IRF-Vago Antiviral Pathway.

Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are vital signaling adaptor proteins for the innate immune response and are involved in many important pathways, such as the NF-κB- and interferon regulatory factor (IRF)-activated signaling pathways. In this study, the TRAF3 ortholog from the shrimp Litopenaeus vannamei (LvTRAF3) was cloned and characterized. LvTRAF3 has a transcript of 3,865 bp, with an open reading frame (ORF) of 1,002 bp and encodes a polypeptide of 333 amino acids, including a conserved TRAF-C domain. The expression of LvTRAF3 in the intestine and hemocyte was up-regulated in response to poly (I:C) challenge and white spot syndrome virus (WSSV) infection. RNAi knockdown of LvTRAF3 in vivo significantly increased WSSV gene transcription, viral loads, and mortality in WSSV-infected shrimp. Next, we found that LvTRAF3 was not able to induce the activation of the NF-κB pathway, which was crucial for synthesis of antimicrobial peptides (AMPs), which mediate antiviral immunity. Specifically, in dual-luciferase reporter assays, LvTRAF3 could not activate several types of promoters with NF-κB binding sites, including those from WSSV genes (wsv069, wsv056, and wsv403), Drosophila AMPs or shrimp AMPs. Accordingly, the mRNA levels of shrimp AMPs did not significantly change when TRAF3 was knocked down during WSSV infection. Instead, we found that LvTRAF3 signaled through the IRF-Vago antiviral cascade. LvTRAF3 functioned upstream of LvIRF to regulate the expression of LvVago4 and LvVago5 during WSSV infection in vivo. Taken together, these data provide experimental evidence of the participation of LvTRAF3 in the host defense to WSSV through the activation of the IRF-Vago pathway but not the NF-κB pathway.

MicroRNA MiR-27a-5p Alleviates the Cerulein-Induced Cell Apoptosis and Inflammatory Injury of AR42J Cells by Targeting Traf3 in Acute Pancreatitis.

Acute pancreatitis (AP), a sudden inflammatory process of pancreas, is painful and may contribute to death. The aberrant expression of miR-27a-5p has been reported in many types of cancers and diseases including AP. Thus, it is urgent to manifest the functions and mechanism of miR-27a-5p in AP. The levels of miR-27a-5p, tumor necrosis factor (TNF) receptor-associated factor 3 (Traf3) in serum of AP patient, or cerulein-treated AR42J cells were detected by qRT-PCR. Functionally, the apoptotic rate, the protein levels of Bcl-2 and Bax, the caspase-3 activity, and the levels of IL-1ß, IL-6, and TNF-α in cerulein-treated AR42J cells were measured by flow cytometry, Western blot, caspase-3 activity assay, and qRT-PCR and ELISA assay, respectively. In addition, the putative target of miR-27a-5p was predicted by TargetScan online database, and the dual luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to verify this interaction. Cerulein-treated mouse AP model was established to explore the role of miR-27a-5p in AP in vivo. The level of miR-27a-5p was notably downregulated in AP patients and cerulein-treated AR42J cells. The functional experiments indicated that miR-27a-5p mimics attenuated the promotion effects on cell apoptosis and the inflammatory response in AR42J cells caused by cerulein. The interaction between miR-27a-5p and Traf3 was predicted by TargetScan online database and validated by dual luciferase reporter assay and RIP assay. Following qRT-PCR results exhibited that Traf3 was apparently enhanced in cerulein-treated AR42J cells. The further functional experiments disclosed that Traf3 overexpression relieved the inhibitory effects on cell apoptosis and the inflammatory response induced by miR-27a-5p mimics in cerulein-treated AR42J cells. Moreover, miR-27a-5p alleviated cerulein-induced injury in vivo. In this study, we established the cerulein-treated AR42J cells as AP model in vitro. We validated that miR-27a-5p was significantly downregulated, and Traf3 was strikingly upregulated in AP patient and/or cerulein-treated AR42J cells. The further mechanistical and functional experiments unraveled that miR-27a-5p regulated Traf3 to relieve the cerulein-induced cell apoptosis and inflammatory injury of AR42J cells. Therefore, this novel regulatory network may provide therapeutic target for AP patients.

MicroRNA-214 functions as an oncogene in human osteosarcoma by targeting TRAF3.

OBJECTIVE: Osteosarcoma is a malignant bone tumor with high incidence. The prognosis of osteosarcoma is very poor when it is diagnosed with metastasis. Numerous studies have demonstrated that aberrant expressions of microRNAs are involved in cancer initiation and development. However, the potential role of miR-214 in osteosarcoma remains largely unrevealed. The current study investigated the relationship between the miR-214 and TNF receptor-associated factor 3 (TRAF3) in osteosarcoma tissues and cell lines. We also aimed to evaluate the potential roles of miR-214 on the occurrence and metastasis in osteosarcoma and verify its effect on the regulation of TRAF3. PATIENTS AND METHODS: The miR-214 expression and TRAF3 expression in osteosarcoma tissue samples and cell line were measured using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Followed by transfection assays, transwell assay was conducted to detect the migration and invasion abilities of osteosarcoma cells. Subsequently, Western blotting and luciferase reporter assay were performed in osteosarcoma cells to confirm the target of miR-214. RESULTS: The results showed that miR-214 expression levels were significantly increased not only in osteosarcoma tissues but also in osteosarcoma cell lines as compared with adjacent normal tissues and matched cell lines, respectively. On the contrary, the TRAF3 expression levels in osteosarcoma tissues and cell lines were frequently decreased compared to the control group. Moreover, TRAF3 was identified as a direct target of miR-214 and the inverse relationship between them was also observed in osteosarcoma tissues. Additionally, we found that miR-214 restoration could significantly promote osteosarcoma cell invasion and migration via targeting TRAF3. CONCLUSIONS: MicroRNA-214 functioned as an oncogene in osteosarcoma via targeting TRAF3, which may provide new insights into osteosarcoma prevention and treatment.

Acanthoic acid suppresses lipin1/2 via TLR4 and IRAK4 signalling pathways in EtOH- and lipopolysaccharide-induced hepatic lipogenesis.

OBJECTIVES: In alcoholic liver disease, alcohol and lipopolysaccharide (LPS) are major stimulation factors of hepatic lipogenesis. Our objective was to determine the protective mechanism of acanthoic acid (AA) in EtOH- and LPS-induced hepatic lipogenesis. METHODS: HSC-T6 cells were treated with ethanol (200 mm) plus LPS (1 µg/ml) for 1 h, followed by AA (10 or 20 µm) for another 6 h. C57BL/6 mice were pretreated with of AA (20 and 40 mg/kg) or equal volume of saline and then exposed to three doses of ethanol (5 g/kg body weight) within 24 h. The mice were sacrificed at 6 h after the last ethanol dosing. KEY FINDINGS: Acanthoic acid significantly decreased the expressions of α-SMA, collagen-I, SREBP-1, and lipin1/2 induced, also decreased fat droplets caused by EtOH/LPS. AA treatment decreased the protein expressions of TLR4, CD14, IRAK4, TRAF3, p-TAK1 and NF-κB increased by EtOH/LPS on HSC cells. Results in vivo were consistent with results in vitro. CONCLUSIONS: Our data demonstrated that AA might modulate hepatic fibrosis and lipid deposition in HSC-T6 cell stimulated with ethanol combined with LPS by decreasing lipin1/2 via TLR4 and IRAK4 signalling pathways, and AA might be considered as a potential therapeutic candidate for alcoholic liver disease.

Activation-induced tumor necrosis factor receptor-associated factor 3 (Traf3) alternative splicing controls the noncanonical nuclear factor κB pathway and chemokine expression in human T cells.

The noncanonical nuclear factor κB (ncNFκB) pathway regulates the expression of chemokines required for secondary lymphoid organ formation and thus plays a pivotal role in adaptive immunity. Whereas ncNFκB signaling has been well described in stromal cells and B cells, its role and regulation in T cells remain largely unexplored. ncNFκB activity critically depends on the upstream NFκB-inducing kinase (NIK). NIK expression is negatively regulated by the full-length isoform of TNF receptor-associated factor 3 (Traf3) as formation of a NIK-Traf3-Traf2 complex targets NIK for degradation. Here we show that T cell-specific and activation-dependent alternative splicing generates a Traf3 isoform lacking exon 8 (Traf3DE8) that, in contrast to the full-length protein, activates ncNFκB signaling. Traf3DE8 disrupts the NIK-Traf3-Traf2 complex and allows accumulation of NIK to initiate ncNFκB signaling in activated T cells. ncNFκB activity results in expression of several chemokines, among them B cell chemoattractant (CxCL13), both in a model T cell line and in primary human CD4(+) T cells. Because CxCL13 plays an important role in B cell migration and activation, our data suggest an involvement and provide a mechanistic basis for Traf3 alternative splicing and ncNFκB activation in contributing to T cell-dependent adaptive immunity.

HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia.

BACKGROUND: HIV-1 Tat protein is known to be associated with neuroinflammation, a condition that develops in almost half of patients infected with HIV-1. HIV-1 Tat can alter glial neuroprotective functions, leading to neurotoxicity within the CNS. HIV-1 Tat is known to be secreted from productively infected cells and can affect neighboring uninfected cells by modulating cellular gene expression in a bystander fashion. METHODS: We were interested to study whether exogenous exposure to HIV-1 Tat-C protein perturbs the microRNA (miRNA) expression profile of human microglial cells, leading to altered protein expression. We used protein expression and purification, miRNA overexpression, miRNA knockdown, transfection, site-directed mutagenesis, real-time PCR, luciferase assay and western blotting techniques to perform our study. RESULTS: HIV-1 Tat-C treatment of human microglial cells resulted in a dose-dependent increase in miR-32 expression. We found that tumor necrosis factor-receptor-associated factor 3 TRAF3) is a direct target for miR-32, and overexpression of miR-32 in CHME3 cells decreased TRAF3 both at the mRNA and the protein level. Recovery of TRAF3 protein expression after transfection of anti-miR-32 and the results of the luciferase reporter assay provided direct evidence of TRAF3 regulation by miR-32. We found that the regulation of interferon regulatory factor 3 (IRF3) and IRF7 is controlled by cellular levels of TRAF3 protein in microglial cells, as after overexpression of miR-32 and application of anti-miR-32, expression levels of IRF3 and IRF7 were inversely regulated by expression levels of TRAF3. Thus, our results suggest a novel miRNA mediated mechanism for regulation of TRAF3 in human microglial cells exposed to HIV-1 Tat C protein. These results may help to elucidate the detrimental neuroinflammatory consequences of HIV-1 Tat C protein in bystander fashion. CONCLUSION: HIV-1 Tat protein can modulate TRAF3 expression through miRNA mediated pathway and can change the downstream expression of IRF3 and IRF7. This study demonstrates a novel mechanism of HIV-1 Tat C protein-mediated perturbation of miRNA, resulting in dysregulation of cellular TRAF3.

Hepatitis C virus reveals a novel early control in acute immune response.

Recognition of viral RNA structures by the intracytosolic RNA helicase RIG-I triggers induction of innate immunity. Efficient induction requires RIG-I ubiquitination by the E3 ligase TRIM25, its interaction with the mitochondria-bound MAVS protein, recruitment of TRAF3, IRF3- and NF-κB-kinases and transcription of Interferon (IFN). In addition, IRF3 alone induces some of the Interferon-Stimulated Genes (ISGs), referred to as early ISGs. Infection of hepatocytes with Hepatitis C virus (HCV) results in poor production of IFN despite recognition of the viral RNA by RIG-I but can lead to induction of early ISGs. HCV was shown to inhibit IFN production by cleaving MAVS through its NS3/4A protease and by controlling cellular translation through activation of PKR, an eIF2α-kinase containing dsRNA-binding domains (DRBD). Here, we have identified a third mode of control of IFN induction by HCV. Using HCVcc and the Huh7.25.CD81 cells, we found that HCV controls RIG-I ubiquitination through the di-ubiquitine-like protein ISG15, one of the early ISGs. A transcriptome analysis performed on Huh7.25.CD81 cells silenced or not for PKR and infected with JFH1 revealed that HCV infection leads to induction of 49 PKR-dependent genes, including ISG15 and several early ISGs. Silencing experiments revealed that this novel PKR-dependent pathway involves MAVS, TRAF3 and IRF3 but not RIG-I, and that it does not induce IFN. Use of PKR inhibitors showed that this pathway requires the DRBD but not the kinase activity of PKR. We then demonstrated that PKR interacts with HCV RNA and MAVS prior to RIG-I. In conclusion, HCV recruits PKR early in infection as a sensor to trigger induction of several IRF3-dependent genes. Among those, ISG15 acts to negatively control the RIG-I/MAVS pathway, at the level of RIG-I ubiquitination.These data give novel insights in the machinery involved in the early events of innate immune response.