Primary human hepatocytes treated with IFNalpha and IL28B

Recent identification of IL28B gene polymorphisms associated with hepatitis C virus (HCV) clearance suggests a role for type III interferons (IFNs) in hepatitis C infection. The function of type III IFNs in intrinsic antiviral immunity is poorly understood. Here we show that HCV infection of primary human hepatocytes results in a robust induction of type III but not type I IFNs, leading to IFN- stimulated gene (ISG) expression. In addition, HCV infection elicits a much broader range of gene expression alterations in addition to ISG induction. The induction of type III IFNs is mediated by IRF3 and NFkB- dependent pathways. Type III IFN, aside from upregulating ISGs with a different kinetic profile, induces a distinct set of genes from type I IFN, potentially explaining the functional difference between the two types of IFNs. Chimpanzees undergoing experimental HCV infection demonstrated a prompt hepatic induction of IL28, associating with ISG upregulation, but minimal type I IFN induction. Analysis of liver biopsies from HCV-infected patients supported a close correlation among hepatic expression of IL28 and ISGs, but not with type I IFNs. Our study demonstrates that HCV infection results predominantly in type III IFN induction in the liver and the level of induction correlates with hepatic ISG levels, thus providing a mechanistic explanation for the association between IL28, ISG levels and recovery from HCV infection as well as a potential therapeutic strategy for the treatment of non-responders. Microarray analysis of primary human hepatocytes treated samples with IFN, IL28b and T-PolyC after 6 or 24 hours respectively and with recombinant IL28B (25 ng/mL for 24 hours) and JFH1 (MOI of 5 for 36 hours) with three replications.

近期鉴定出与丙型肝炎病毒（hepatitis C virus, HCV）清除相关的IL28B基因多态性，提示III型干扰素（type III interferons, IFNs）在丙型肝炎感染中发挥关键作用，目前学界对III型干扰素在固有抗病毒免疫中的功能尚缺乏深入了解。本研究发现，原代人肝细胞感染HCV后，可有效诱导III型干扰素（而非I型干扰素）的表达，进而激活干扰素刺激基因（IFN-stimulated gene, ISG）的转录；此外，HCV感染除诱导ISG表达外，还会引发更为广泛的基因表达谱改变，且III型干扰素的诱导依赖于IRF3与NFκB依赖的信号通路。与I型干扰素相比，III型干扰素不仅以独特的动力学特征上调ISG表达，还会诱导一套与I型干扰素截然不同的下游基因，这或许可以解释两类干扰素的功能差异。接受实验性HCV感染的黑猩猩，其肝脏中IL28的表达会快速上调，并伴随ISG水平升高，但I型干扰素的诱导水平极低。对HCV感染患者的肝活检组织进行分析后证实，肝脏IL28的表达水平与ISG表达量呈显著正相关，但与I型干扰素的表达无明显关联。本研究表明，HCV感染在肝脏中主要诱导III型干扰素的表达，且其诱导水平与肝脏ISG的表达量密切相关，这为IL28、ISG水平与HCV感染康复之间的关联提供了机制层面的合理解释，同时也为HCV治疗无应答者提供了潜在的干预策略。本研究对经不同处理的原代人肝细胞开展了微阵列分析：分别于处理6小时、24小时后，检测经干扰素、IL28B及T-PolyC处理的样本；同时设置重组IL28B（25 ng/mL，处理24小时）与JFH1（感染复数multiplicity of infection, MOI为5，处理36小时）处理组，每组均设置3次生物学重复。