Effect of miR-322-5p on inhibiting Th17 differentiation by targeting Akt3 in experimental autoimmune encephalomyelitis interfered by interferon-β / 上海交通大学学报（医学版）

Objective:To investigate the effect of miR-322-5p which targets Akt3 on Th17 differentiation in experimental autoimmune encephalomyelitis (EAE) interfered by interferon-β (IFN-β). Methods:The effect of IFN-β on EAE mice which were randomly divided into IFN-β group and PBS group was examine. The percents of Th17 in the two groups were compared by fluorescence activated cell sorting. The miRNA array was made to find different miRNAs between those two groups. MiR-322-5p was screened for further research. The target gene of miR-322-5p was predicted using softwares and the common predicted target gene Akt3 was got. The expression of Akt3 was detected after IFN-β intervention and miR-322-5p overexpression. The target relationship between Akt3 and miR-322-5p was verified by luciferase reporter assay. At last, the effect of Akt3 on Th17 differentiation was explored in vitro. Results:Compared to PBS group, the percent of Th17 was significantly downregulated, the expression of miR-322-5p was significantly upregulated and Akt3 was significantly downregulated in IFN-β group. The expression of Akt3 was obviously decreased after overexpressing miR-322-5p. Luciferase reporter assay showed that Akt3 was directly targeted by miR-322-5p. The percent of Th17 differentiation was greatly promoted by Akt3 in vitro. Conclusion:IFN-β significantly ameliorates the severity of EAE by affecting miR-322-5p which can inhibit Th17 differentiation by targeting Akt3.

Identification of new type I interferon-stimulated genes and investigation of their involvement in IFN-β activation

Virus infection induces the production of type I interferons (IFNs). IFNs bind to their heterodimeric receptors to initiate downstream cascade of signaling, leading to the up-regulation of interferon-stimulated genes (ISGs). ISGs play very important roles in innate immunity through a variety of mechanisms. Although hundreds of ISGs have been identified, it is commonly recognized that more ISGs await to be discovered. The aim of this study was to identify new ISGs and to probe their roles in regulating virus-induced type I IFN production. We used consensus interferon (Con-IFN), an artificial alpha IFN that was shown to be more potent than naturally existing type I IFN, to treat three human immune cell lines, CEM, U937 and Daudi cells. Microarray analysis was employed to identify those genes whose expressions were up-regulated. Six hundred and seventeen genes were up-regulated more than 3-fold. Out of these 617 genes, 138 were not previously reported as ISGs and thus were further pursued. Validation of these 138 genes using quantitative reverse transcription PCR (qRT-PCR) confirmed 91 genes. We screened 89 genes for those involved in Sendai virus (SeV)-induced IFN-β promoter activation, and PIM1 was identified as one whose expression inhibited SeV-mediated IFN-β activation. We provide evidence indicating that PIM1 specifically inhibits RIG-I- and MDA5-mediated IFN-β signaling. Our results expand the ISG library and identify PIM1 as an ISG that participates in the regulation of virus-induced type I interferon production.

Hydroquinone suppresses IFN-β expression by targeting AKT/IRF3 pathway

Previous studies have demonstrated the role of hydroquinone (HQ), a hydroxylated benzene metabolite, in modulating various immune responses; however, its role in macrophage-mediated inflammatory responses is not fully understood. In this study, the role of HQ in inflammatory responses and the underlying molecular mechanism were explored in macrophages. HQ down-regulated the expression of interferon (IFN)-β mRNA in LPS-stimulated RAW264.7 cells without any cytotoxicity and suppressed interferon regulatory factor (IRF)-3-mediated luciferase activity induced by TIR-domain-containing adapter-inducing interferon-β (TRIF) and TANK-binding kinase 1 (TBK1). A mechanism study revealed that HQ inhibited IRF-3 phosphorylation induced by lipopolysaccharide (LPS), TRIF, and AKT by suppressing phosphorylation of AKT, an upstream kinase of the IRF-3 signaling pathway. IRF-3 phosphorylation is highly induced by wild-type AKT and poorly induced by an AKT mutant, AKT C310A, which is mutated at an inhibitory target site of HQ. We also showed that HQ inhibited IRF-3 phosphorylation by targeting all three AKT isoforms (AKT1, AKT2, and AKT3) in RAW264.7 cells and suppressed IRF-3-mediated luciferase activities induced by AKT in HEK293 cells. Taken together, these results strongly suggest that HQ inhibits the production of a type I IFN, IFN-β, by targeting AKTs in the IRF-3 signaling pathway during macrophage-mediated inflammation.

Mechanism of RING finger protein 34 involved in innate immunity:a preliminary study / 军事医学

Objective To study the mechanism of RING finger protein 34 ( RNF34 ) involved in innate immunity . Methods Recombinant PCR was used and transient expression of the plasmid was achieved in HEK 293T cells.The cells were stimulated with Sendai virus ( SeV) or N-RIG-Ⅰfor the indicated time while luciferase activity was observed using the dual-luciferase reporter assay kit .Results We constructed the plasmid pcDNA 3-Flag-RNF34 and its three mutations .The study found that when stimulated by SeV , RNF34 could inhibit the activity of NF-κB and IFN-βmore significantly than RNF34-ΔFYVE, RNF34-ΔCID and RNF34-ΔRING.We also found that RNF 34 and its three mutants had similar inhibitory effect when the activation of NF-κB and IFN-βwas stimulated by the N-RIG-Ⅰ.Conclusion RNF34 negatively regulates innate immunity by acting on the RIG-Ⅰ-MAVS signaling pathway .

Rab5a promotes expression of pro-inflammatory cytokines and type Ⅰ IFN in CpG induced macrophages / 中国免疫学杂志

Objective:Using the macrophage cell lines RAW264.7 stably expressing Rab5a and its dominant negative mutant Rab5aN133I as models to analyze the effect and the mechanism of Rab 5a,Rab5aN133I on CpG-induced production of pro-inflammatory cytokines and type Ⅰ IFN.Methods: The eukaryotic expression vectors of Rab5a and Rab5aN133I were transfected into RAW264.7 cells by liposome,and screened with G418.The G418-resistant colonies were obtained and amplified.The transformed cell lines were i-dentified by RT-PCR,Real time-PCR and Western blot.The production of cytokines were measured after transformed cell lines of Rab5a and Rab5aN133I was stimulation with CpG for 8 h.Results: Rab5a expression in transfected cells was significantly higher than the control cell group (P<0.05).Overexpression of Rab5a significantly promoted the production of TNF -α,IL1-β(P<0.01) and IFN-β( P<0.05) in CpG stimulated RAW264.7.The production of cytokines was restored in Rab 5aN133I transfected cell line.Conclusion:Rab 5a promotes CpG-induced pro-inflammatory cytokines and typeⅠIFN in macrophages,it may be act as a positive regulator in TLR9 signaling pathway.

Helicobacter pylori induces NOD1/NF-κB activation and IFN-βand IP-10 production in gastrics of mice / 中国免疫学杂志

Objective:To construct the Helicobacter pylori infected C57BL/6 mice model to observe the activation of NOD1/NF-κB signaling pathways in the gastric tissues,and study its roles in inflammatory response during Hp infection.Methods:6-8 week-old C57BL/6 mice were randomly divided into two groups,the Hp infection group and the control group,and mice were given by gavage every 48 h for five times with Hp or PBS,respectively.All the animals were sacrificed at different time point and the gastric tissue were stained with hematoxylin-eosin( HE);The mRNA expression of NOD1 and RIP2 in gastric tissues were examined by RT-PCR;Levels of IFN-βand IP-10 in mice serum were assessed by ELISA;Nuclear translocation of p65 in gastric tissue was detected by Western blot.Results:Hp infection elicits an inflammatory cell response,glands in gastric tissue were reduced or atrophic,as compared with that in the control group.The levels of IP-10 and IFN-βincreased in the model group, and peaked at 16 weeks after Hp infection.Hp infection increased the mRNA expression of NOD1 and the p65 content in nuclear between 24-120 h(P<0.05),and the highest level at 48 h,subsequently the expression levels were began to decrease.The mRNA expression level of RIP2 was up-regulated after Hp was administrated, peaked at 48 h and declined after 72 h.However, the expression levels would rise again at 120 h.Conclusion: Hp infection can activate the NOD1/NF-κB signaling pathways and induce the production of IFN-βand IP-10 in gastrics of mice.

Post-Marketing Surveillance of Natural IFN-β Preparation for Chronic Hepatitis C / 薬剤疫学

Objective : Post-marketing surveillance was conducted for the purpose of demonstrating the relationship between the therapeutic effect of natural IFN-β preparation on chronic active hepatitis C and HCV subtype or viral load as well as various predictors of its efficacy.<BR>Design : Cohort studies.<BR>Methods : Questionnaires were sent to all medical institutions at which IFN-β ('IFNβMochida') was administered to patients with chronic active hepatitis C once daily for at least 8 weeks and its therapeutic effect was judged based on the rate of virological sustained response (VSR) and the rate of biochemical (ALT) sustained response (BSR).<BR>Results : Questionnaires for 2, 076 patients were collected from 244 medical institutions all over the country. Of these questionnaires, those for 1, 503 patients, 930 men (61.9%) and 573 women (38.1%), collected from 229 institutions could be evaluated regarding the therapeutic effect of IFNβ Mochida. The patients' mean age was 50.2 years. The average VSR were 31% for all of the patients (1, 503 patients), 61% for those with a low viral load (HCV-RNA level before IFN treatment ; <106 copies/ml) and 14% for those with a high viral load (≥106 copies/ml) ; with the subtypes 1 b, 2 a and 2 b accounting for 18, 55 and 29% respectively. BSR were 45, 69 and 32%, respectively ; with the subtypes 1 b, 2 a and 2 b accounting for 32, 66 and 56%. As for the therapeutic effect in patients with the same level of viral load but different viral subtype, at each level of viral load VSR was the highest in subtype 2 a, followed by 2 b and 1 b, showing a significant difference between 2 a and 1 b or 2 b, depending on the level of viral load. BSR of 2 a and 2 b were similarly high, showing a significant difference between 2 a or 2 b and 1 b, depending on the level of viral load. In patients with subtype 1 a or 1 b, patients who were administered IFN-β≥339 MU obtained a higher VSR than those who were administered IFN-β ≤336MU. Adverse drug reactions were observed in 89% of the total 2, 076 patients, however, these symptoms disappeared immediately after the completion of the treatment. Univariate and multivariate logistic regression analyses conducted to detect the predictors on the therapeutic effect (VSR) of IFN-β revealed that the subtype, viral load and age were significant factors for all the patients and that the viral load and NS5A mutation were significant factors for the patients with subtype 1 b. However, the NS5A mutant type viral load was significantly less than that of the other types, showing no difference in the therapeutic effect in the comparison at the same level of viral load.<BR>Conclusion : It was confirmed that the therapeutic effect of the natural IFN-β preparation on chronic active hepatitis C varied widely depending on the viral load and viral subtype. This information will play an important role in the development of therapy for chronic hepatitis C in the future.