Classical swine fever virus triggers RIG-I and MDA5-dependent signaling pathway to IRF-3 and NF-κB activation to promote secretion of interferon and inflammatory cytokines in porcine alveolar macrophages.
Classical swine fever virus triggers RIG-I and MDA5-dependent signaling pathway to IRF-3 and NF-κB activation to promote secretion of interferon and inflammatory cytokines in porcine alveolar macrophages.
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DOI：
10.1186/1743-422x-10-286
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发表时间：
2013-09-13
期刊：
影响因子：
4.8
通讯作者：
Chen JD
中科院分区：
文献类型：
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作者：
Dong XY;Liu WJ;Zhao MQ;Wang JY;Pei JJ;Luo YW;Ju CM;Chen JD
猪瘟（Classical Swine Fever，CSF）是由猪瘟病毒（CSFV）引起的一种高度接触性传染病。RNA解旋酶视黄酸诱导基因I（RIG-I）和黑色素瘤分化相关基因5（MDA-5）差异性地参与各种RNA病毒的检测。本研究探讨了RIG-I和MDA-5在猪肺泡巨噬细胞（PAM）对猪瘟病毒石门株的抗病毒和炎症反应中的作用。本研究以猪瘟石门株为攻毒病毒，对PAM进行体外培养。免疫荧光染色检测干扰素调节因子（IRF）-3和核因子-κ B（NF-κB）易位，Western Blotting检测RIG-1、MDA 5、干扰素启动子刺激因子1（IPS-1）、IRF-3和NF-κB表达;干扰素β（IFN-β）、IFN-α、白细胞介素-1 β（IL-1β），采用酶联免疫吸附试验（ELISA）检测IL-6和肿瘤坏死因子（TNF-α）的表达，并进行shRNA介导的MDA 5或RIG-I敲低。结果表明，感染CSFV后，细胞内RIG-1和MDA 5的表达增加，IPS-1、IRF-3和NF-κB的活化呈剂量依赖性。对PAM表达的IFN-α、IFN-β、IL-1β、IL-6或TNF-α的评价显示，感染和未感染细胞之间存在显著差异。CSFV感染细胞后24 h内，IFN-α、IFN-β、IL-1β、IL-6和TNF-α的表达呈剂量依赖性增加。同时，CSFV还促进了IRF-3和NF-κB的核转位。我们还直接比较和评估了RIG-I和MDA 5在CSFV感染期间通过shRNA介导的MDA 5或RIG-I的敲低触发先天免疫作用中的作用。我们发现，与对照相比，响应于CSFV感染的IFN-α、IFN-β、IL-1β、IL-6和TNF-α的产生在RIG-I敲除细胞中严重降低，而在MDA 5敲除细胞中中度降低。来源于RIG-I和MDA 5敲低的PAM几乎不能产生IFN和炎性细胞因子。这表明CSFV可以被RIG-I和MDA 5识别，以启动RIG-I信号通路，从而触发针对感染的先天防御。
Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The RNA helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) are differentially involved in the detection of various RNA viruses. In present study, we investigated the roles of RIG-I and MDA-5 in eliciting antiviral and inflammatory responses to CSFV shimen strain in Porcine alveolar macrophages (PAMs). CSFV Shimen strain was used as challenge virus in this study and PAMs were cultured in vitro. Interferon regulatory factor (IRF)-3 and nuclear factor-kappa B (NF-κB) translocation was detected using immunofluorescent staining; RIG-I, MDA5, interferon promoter-stimulating factor 1 (IPS-1), IRF-3 and NF-κB expression was measured by Western Blotting; Interferon beta (IFN-β), IFN-α, interleukin-1beta (IL-1β), IL-6 and tumor necrosis factor (TNF-α) expression was tested by Enzyme-linked immunosorbent assays (ELISA) and shRNA-mediated knockdown of MDA5 or RIG-I was performed. The findings suggested that the initial response to CSFV infection resulted in the higher expression of RIG-I and MDA5 leading to the activation of IPS-1, IRF-3 and NF-κB in a dose-dependent manner. Evaluation of IFN-α, IFN-β, IL-1β, IL-6 or TNF-α expressed by PAMs showed significant differences between infected and uninfected cells. CSFV infected cells induced to express high levels of IFN-α, IFN-β, IL-1β, IL-6 and TNF-α in a dose-dependent way within 24 h post-infection (hpi). At the same time, CSFV improved the nuclear translocation of IRF-3 and NF-κB. We also directly compared and assessed the roles of RIG-I and MDA5 in triggering innate immune actions during CSFV infection through shRNA-mediated knockdown of MDA5 or RIG-I. We found that, compared to the control, the production of IFN-α, IFN-β, IL-1β, IL-6 and TNF-α in response to CSFV infection was heavily reduced in RIG-I knockdown cells while it was moderately decreased in MDA5 knockdown cells. PAMs derived from knockdown of both RIG-I and MDA5 almost failed to produce IFNs and inflammatory cytokines. It indicates that CSFV can be recognized by both RIG-I and MDA5 to initiate the RIG-I signaling pathway to trigger innate defenses against infection.