Zhang Liyong, Xiang Wenpei, Wang Guoliang, Yan Zhengzheng, Zhu Zhaowei, Guo Zhong, Sengupta Rajib, Chen Alex F, Loughran Patricia A, Lu Ben, Wang Qingde, Billiar Timothy R
From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213.
From the Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, the Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
J Biol Chem. 2016 Jul 15;291(29):15093-107. doi: 10.1074/jbc.M116.717942. Epub 2016 May 17.
The sensing of double-stranded RNA (dsRNA) in the liver is important for antiviral defenses but can also contribute to sterile inflammation during liver injury. Hepatocytes are often the target of viral infection and are easily injured by inflammatory insults. Here we sought to establish the pathways involved in the production of type I interferons (IFN-I) in response to extracellular poly(I:C), a dsRNA mimetic, in hepatocytes. This was of interest because hepatocytes are long-lived and, unlike most immune cells that readily die after activation with dsRNA, are not viewed as cells with robust antimicrobial capacity. We found that poly(I:C) leads to rapid up-regulation of inducible nitric oxide synthase (iNOS), double-stranded RNA-dependent protein kinase (PKR), and Src. The production of IFN-β was dependent on iNOS, PKR, and Src and partially dependent on TLR3/Trif. iNOS and Src up-regulation was partially dependent on TLR3/Trif but entirely dependent on PKR. The phosphorylation of TLR3 on tyrosine 759 was shown to increase in parallel to IFN-β production in an iNOS- and Src-dependent manner, and Src was found to directly interact with TLR3 in the endosomal compartment of poly(I:C)-treated cells. Furthermore, we identified a robust NO/cGMP/PKG-dependent feedforward pathway for the amplification of iNOS expression. These data identify iNOS/NO as an integral component of IFN-β production in response to dsRNA in hepatocytes in a pathway that involves the coordinated activities of TLR3/Trif and PKR.
肝脏中双链RNA（dsRNA）的感知对于抗病毒防御很重要，但在肝损伤期间也可能导致无菌性炎症。肝细胞常常是病毒感染的靶标，并且容易受到炎症损伤。在这里，我们试图确定肝细胞对细胞外聚肌苷酸胞苷酸（poly(I:C)，一种dsRNA模拟物）产生I型干扰素（IFN-I）所涉及的途径。这一点很有意思，因为肝细胞寿命长，而且与大多数经dsRNA激活后容易死亡的免疫细胞不同，肝细胞不被视为具有强大抗菌能力的细胞。我们发现，poly(I:C)会导致诱导型一氧化氮合酶（iNOS）、双链RNA依赖性蛋白激酶（PKR）和Src迅速上调。IFN-β的产生依赖于iNOS、PKR和Src，部分依赖于Toll样受体3（TLR3）/TIR结构域衔接蛋白（Trif）。iNOS和Src的上调部分依赖于TLR3/Trif，但完全依赖于PKR。研究表明，TLR3第759位酪氨酸的磷酸化与IFN-β的产生平行增加，且呈iNOS和Src依赖性，并且发现Src在poly(I:C)处理细胞的内体区室中直接与TLR3相互作用。此外，我们确定了一条强大的NO/环磷酸鸟苷（cGMP）/蛋白激酶G（PKG）依赖性前馈途径，用于iNOS表达的放大。这些数据表明，在涉及TLR3/Trif和PKR协同活动的途径中，iNOS/NO是肝细胞对dsRNA产生IFN-β的一个不可或缺的组成部分。
