Cytokine molecular therapy for intervention of virus infection

白介素27(IL-27)是一种异源二聚体细胞因子，由IL-27 p28和EBV诱导蛋白3(EBV-Induced Protein 3，EBV-Induced Protein 3)两个多肽亚基组成。IL-27信号是通过由IL-27R_(WSX-1，TCCR)和gp130组成的异源二聚体受体复合体介导的，表达于多种细胞，包括初始的CD4阳性T细胞。JAK1/STAT1通路的激活在IL-27R复合体下游的信号转导中起着至关重要的作用。IL-27刺激幼稚的CD4+T细胞表达T-bet，随后表达IL-12R62。IL-27依次与IL-12协同作用，诱导Th1免疫应答的启动和维持。IL-27还与IL-12协同诱导干扰素-γ的产生。最近有研究表明，IL-27以STAT1依赖的方式抑制Th17细胞的产生。综上所述，IL-27参与了Th1细胞分化的关键步骤，其中初始的Th前体细胞开始分化为Th1细胞，目前认为IL-27与…有关。还有许多人类疾病，包括恶性疾病、过敏性疾病和炎症性疾病。在这个项目中，我们发现IL-27还参与了一种新的防御病毒感染的机制。将IL-27基因导入感染脑心肌炎病毒(EMCV)的C57BI/6小鼠体内，观察其存活情况。感染病毒但未感染IL-27基因的对照组小鼠在感染后10天内全部死亡，而在此期间转IL-27基因小鼠的存活率保持100%。为了阐明IL-27的抗病毒作用机制，我们还进行了体外实验，强烈表明IL-27直接作用于感染病毒的心肌细胞以抑制病毒复制，而诱导获得性免疫反应不有助于病毒的清除。MDA5途径作为一种抗病毒感染的细胞内固有机制已为人们所熟知，但对MDA5基因表达调控的分子机制知之甚少。检测MDA5是否参与了IL-27介导的病毒抑制作用。从第16天的小鼠胚胎中建立原代心肌细胞，并用IL-27刺激，导致MDA5mRNA的强烈诱导。感染EMCV后，经IL-27处理的细胞在感染后立即显示大量产生干扰素-6，这与未经IL-27处理的细胞产生的干扰素水平较低形成鲜明对比。通过短干扰RNA对MDA5的特异性沉默完全取消了IL-27刺激后在心肌细胞中产生的和病毒效应。这些结果表明，IL-27信号增强了心肌细胞MDA5的表达，导致病毒感染触发的MDA5通路增强，进而在感染早期产生强有力的I型干扰素，从而有效地抑制病毒复制。较少

Interleukin-27 (IL-27) is a heterodimeric cytokine composed of two polypeptide subunits, IL-27 p28 and EBV-induced protein 3 (Ebi3). The IL-27 signal is mediated through the heterodimeric receptor complex that consists of IL-27R_(WSX-1, TCCR)and gp130 and is expressed on various cells including naive CD4 positive T cells. Activation of JAK1/STAT1 pathway is crucially involved in the signal transduction downstream of the IL-27R complex. IL-27 provokes naive CD4 positive T cells to express T-bet and, subsequently, IL-12R62. IL-27 sequentially cooperates with IL-12 to induce initiation and maintenance of Th1 immune responses. IL-27 also synergize with IL-12 to induce interferon (IFN)-y production. Recently it was shown that generation of Th17 cells is suppressed by IL-27 in a STAT1 dependent manner. Taken together, IL-27 is involved in the key step for Th1 commitment where naive Th precursor cells commence differentiation into Th1 cells, and it is now considered that IL-27 is associated w … More ith many human diseases including malignant, allergic and inflammatory disorders. In this project, we found that IL-27 is also involved in a novel defense mechanism against virus infection. C57BI/6 mice that had been infected with Encephalomyocarditis virus (EMCV) were transfected in vivo with IL-27 gene, and their longevity was analyzed. All the control mice that received the virus but not IL-27 gene died within 10 days after the infection, whereas survival rate of the IL-27 gene-transfected mice remained 100% during this period. To clarify the mechanism of the anti-virus activity of IL-27, we also performed in vitro experiments, which strongly suggested that IL-27 directly acted on virus-infected cardiomyocytes to suppress virus replication, whereas induction of acquired immune responses did not contribute to the virus clearance. As an intracellular innate machinery against virus infection, MDA5-mediated pathway is widely known, but little is known on molecular mechanisms to regulate MDA5 expression. Then we assessed whether MDA5 participates in the IL-27-mediated virus suppression. Primary cardiac muscle cells were established form the day 16 mouse embryos and stimulated with IL-27, which led to drastic induction of mRNA for MDA5. Infected with EMCV, the IL-27-treated cells showed massive production of IFN-6 immediately after the infection, which was in sharp contrast to the lower level of IFN production by the cells that were not treated with IL-27. Specific silencing of MDA5 by means of the short interfering RNA completely cancelled the and-virus effect that was otherwise produced in cardiomyocytes after the IL-27 provocation. These findings indicate that IL-27 signaling augments expression of MDA5 in cardiac muscle cells, resulting in a strengthening of MDA5 pathway triggered by virus infection, which in turn generates robust induction of type I IFN at an early phase of infection to effectively suppress virus replication. Less