Oxidant tone regulates RANTES gene expression in airway epithelial cells infected with respiratory syncytial virus. Role in viral-induced interferon regulatory factor activation

Respiratory syncytial virus (RSV) produces intense pulmonary inflammation, in part, through its ability to induce chemokine synthesis in infected airway epithelial cells. RANTES (regulated upon activation, normal T-cells expressed and secreted) is a CC chemokine which recruits and activates monocytes, lymphocytes, and eosinophils, all cell types present in the lung inflammatory infiltrate induced by RSV infection. In this study we investigated the role of reactive oxygen species in the induction of RANTES gene expression in human type II alveolar epithelial cells (A549), following RSV infection. Our results indicate that RSV infection of airway epithelial cells rapidly induces reactive oxygen species production, prior to RANTES expression, as measured by oxidation of 2',7'-dichlorofluorescein. Pretreatment of airway epithelial cells with the antioxidant butylated hydroxyanisol (BHA), as well a panel of chemically unrelated antioxidants, blocks RSV-induced RANTES gene expression and protein secretion. This effect is mediated through the ability of BHA to inhibit RSV-induced interferon regulatory factor binding to the RANTES promoter interferon-stimulated responsive element, that is absolutely required for inducible RANTES promoter activation. BHA inhibits de novo interferon regulator factor (IRF)-1 and -7 gene expression and protein synthesis, and IRF-3 nuclear translocation. Together, these data indicates that a redox-sensitive pathway is involved in RSV-induced IRF activation, an event necessary for RANTES gene expression.     

TSA通过抑制STAT1磷酸化与核转位下调人肝癌细胞HepG2内 IDO的表达

目的:研究曲古抑菌素A (Trichostatin A, TSA)下调γ干扰素(interferon-gamma, IFN-γ)诱导的人肝癌细胞HepG2内吲哚胺2, 3-双加氧酶(indoleamine 2, 3-dioxygenase, IDO)表达的分子机制。方法: Western blot 检测TSA在IFN-γ诱导的HepG2细胞中IDO的表达、信号转导及转录激活子1(STAT1)的磷酸化和干扰素调节因子1(IRF-1)的诱导表达情况。用免疫细胞化学法检测TSA处理HepG2细胞后对IDO表达的影响。流式细胞术分析TSA处理后IFN-γ受体2表达量的变化,进一步在激光共聚焦显微镜下观察TSA对STAT1核转位的影响,利用双荧光素酶报告基因系统检测TSA对IFN-γ激活位点(γ-activated sites, GAS)、干扰素刺激应答元件(interferon stimulated response elements, ISRE)和核因子-κB (NF-κB)的激活的影响。结果:TSA以剂量依赖方式下调HepG2细胞内IFN-γ诱导的IDO表达、能明显抑制STAT1第701位酪氨酸的磷酸化和STAT1的核转位,但是上调IFN-γ受体2受体的表达。双荧光素酶报告基因分析和Western blot结果表明:TSA能显著抑制GAS和IRF-1 的激活却不能抑制NF-κB和ISRE的激活。 结论:TSA能下调IFN-γ诱导的HepG2细胞中IDO的表达,其机制可能是与其抑制STAT1的磷酸化和核转位,以及抑制STAT1与GAS的结合有关,而不是通过下调IFN-γ受体的表达来实现的。     

3-nitrotyrosine attenuates respiratory syncytial virus infection in human bronchial epithelial cell line

3-nitrotyrosine (NO2Tyr), an L-tyrosine derivative during nitrative stress, can substitute the COOH-terminal tyrosine of alpha-tubulin, posttranslationally altering microtubular functions. Because infection of the cells by respiratory syncytial virus (RSV) may require intact microtubules, we tested the hypothesis that NO2Tyr would inhibit RSV infection and intracellular signaling via nitrotyrosination of alpha-tubulin. A human bronchial epithelial cell line (BEAS-2B) was incubated with RSV with or without NO2Tyr. The release of chemokines and viral particles and activation of interferon regulatory factor-3 (IRF-3) were measured. Incubation with NO2Tyr increased nitrotyrosinated alpha-tubulin, and NO2Tyr colocalized with microtubules. RSV-infected cells released viral particles, RANTES, and IL-8 in a time- and dose-dependent manner, and intracellular RSV proteins coprecipitated with alpha-tubulin. NO2Tyr attenuated the RSV-induced release of RANTES, IL-8, and viral particles by 50-90% and decreased alpha-tubulin-associated RSV proteins. 3-chlorotyrosine, another L-tyrosine derivative, had no effects. NO2Tyr also inhibited the RSV-induced shift of the unphosphorylated form I of IRF-3 to the phosphorylated form II. Pre-exposure of the cells to NO(2) (0.15 ppm, 4 h), which produced diffuse protein tyrosine nitration, did not affect RSV-induced release of RANTES, IL-8, or viral particles. NO2Tyr did not affect the potential of viral spreading to the neighboring cells since the RSV titers were not decreased when the uninfected cells were cocultured with the preinfected cells in NO2Tyr-containing medium. These results indicate that NO2Tyr, by replacing the COOH-terminal tyrosine of alpha-tubulin, attenuated RSV infection, and the inhibition appeared to occur at the early stages of RSV infection.     

Incomplete regulation of NF-kappaB by IkappaBalpha during respiratory syncytial virus infection in A549 cells

Respiratory syncytial virus (RSV) infection of airway epithelial cells results in persistent NF-kappaB activation and NF-kappaB-mediated interleukin-8 production. Previous studies in airway epithelial cells demonstrated that tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation is transient due to regulation by IkappaBalpha. However, during RSV infection, IkappaBalpha has only a partial inhibitory effect on NF-kappaB activation. Studies presented here demonstrate that neither increased IkappaBalpha production which occurs as a result of RSV-induced NF-kappaB activation nor inhibition of proteasome-mediated IkappaBalpha degradation results in a reversal of RSV-induced NF-kappaB activation. Thus, while manipulation of IkappaBalpha results in reversal of TNF-alpha-induced NF-kappaB activation, manipulation of IkappaBalpha does not result in a reversal of RSV-induced NF-kappaB activation.