Genomic and bioinformatic characterization of mouse mast cells (P815) upon different influenza A virus (H1N1, H5N1, and H7N2) infections. Genomic and bioinformatic characterization of mouse mast cells (P815) upon different influenza A virus (H1N1, H5N1, and H7N2) infections

Background: Influenza A virus (IAV) is a segmented negative-stranded RNA virus that brings a potentially serious threat to public health and animal husbandry. Mast cells play an important role in both the inherent and adaptive immune response. Previous studies have indicated that mast cells support the productive replication of H1N1, H5N1, and H7N2. To date, the distinct molecular mechanism behind the pathogenesis in mast cells among the three different viruses has been poorly understood. Methods: We investigated the genomic profiles in detail and the dynamic change of genomes regulated by different subtypes of IAV in mouse mast cells using microassays in order to the distinct molecular mechanism behind the pathogenesis in mast cells among the three different viruses. Mouse mast cells (P815) were infected at a multiplicity of infection of 1 (infectious viruses/cell) and incubated for 12 hr before collection of total RNA and microarray anlaysis using the Affymetrix platforms. Any two of the three IAV-infected groups were compared to detect genomic distinct. Based on the results of microassays, the further verification was performed by real-time PCR and western blotting. Here, the mRNA levels of 5-HT, PKG and HIF-1 in P815 cells following H1N1, H5N1 and H7N2 infection were determined by real-time PCR. The protein levels of exosomes in P815 cells following H1N1, H5N1 and H7N2 infection were detected by western blotting. Results: Compared with any two of the three IAV-infected groups, much more differentially expressed genes (DEGs), cellular functions and signaling pathways were confirmd in H1N1 or H7N2 group, and H7N2 group showed the highest levels. However, few DEGs were detected as well as various cellular functions and signaling pathways were dramatically suppressed in H5N1 group. With an in-depth study on H1N1 and H7N2 group, we demonstrated the 5-HT signaling pathway and cGMP/PKG signaling pathway terms were enriched in P815 cells infected by H1N1 while HIF-1 signaling pathway terms were enriched in P815 cells infected by H7N2 virus. Furthermore, RT-qPCR results also showed significantly increased mRNA of 5-HT and PKG in H1N1-infected P815 cells as well as HIF-1 in H7N2-infected P815 cells. Besides, exosomes were highly secreted from H1N1-infected or H7N2-infected P815 cells. Conclusions: H1N1 and H7N2 viruses could result in a variety of DEGs in mast cells and activate various cellular functions and signaling pathways while H5N1 virus cause the few DEGs in mast cells as well as suppression of cellular functions and signaling pathways. Furthermore, 5-HT signaling pathway and cGMP/PKG signaling pathway were preferentially activated in P815 cells infected by H1N1 while HIF-1 signaling pathway were preferentially activated in P815 cells infected by H7N2 virus. In addition, exosomes were also preferentially secreted from H1N1-infected or H7N2-infected P815 cells that are potentially pivotal in innate immunity to fight IAV infection. The study provides novel information and insight into the distinct of molecular mechanism of H1N1, H5N1 and H7N2 viruses in mast cells from the perspective of genomic profiles. Overall design: Cells were infected with IAVs at a multiplicty of infection of 1 and cells were incubated for 12 hr at 37 C before RNA extraction and analysis of genomic and bioinformatic characterization.

背景：甲型流感病毒（Influenza A virus, IAV）是一种分节段负链RNA病毒，对公共卫生与畜牧业均构成潜在的严重威胁。肥大细胞（mast cells）在固有免疫与适应性免疫应答中均发挥重要作用。既往研究表明，肥大细胞可支持H1N1、H5N1及H7N2亚型病毒的增殖性复制。截至目前，针对这三种不同亚型病毒在肥大细胞中致病的独特分子机制，相关研究仍较为匮乏。
方法：为阐明这三种病毒在肥大细胞中致病的差异化分子机制，本研究借助微阵列分析技术，详细探究了小鼠肥大细胞中被不同IAV亚型调控的基因组特征及其动态变化。本研究以多感染复数（multiplicity of infection, MOI）为1的剂量感染小鼠肥大细胞株P815，孵育12小时后收集总RNA，采用Affymetrix平台进行微阵列分析。通过两两比较三个IAV感染组以筛选差异表达基因（differentially expressed genes, DEGs）。基于微阵列分析结果，本研究进一步通过实时荧光定量PCR与蛋白质印迹进行验证。本研究通过实时荧光定量PCR检测了H1N1、H5N1及H7N2感染后P815细胞中5-羟色胺（5-HT）、蛋白激酶G（PKG）及缺氧诱导因子-1（HIF-1）的mRNA水平；通过蛋白质印迹法检测了上述三种病毒感染后P815细胞中外泌体（exosomes）的蛋白表达水平。
结果：相较于其余两个感染组，H1N1或H7N2感染组中筛选得到的差异表达基因、细胞功能及信号通路数量显著更多，且H7N2感染组的相关指标水平最高。与之相反，H5N1感染组中仅检测到少量差异表达基因，且多种细胞功能与信号通路均受到显著抑制。通过对H1N1与H7N2感染组进行深入分析，本研究发现：H1N1感染的P815细胞中富集了5-HT信号通路及cGMP/PKG信号通路相关条目，而H7N2感染的P815细胞中则富集了HIF-1信号通路相关条目。此外，实时荧光定量PCR结果显示，H1N1感染的P815细胞中5-HT与PKG的mRNA水平显著上调，H7N2感染的P815细胞中HIF-1的mRNA水平亦显著上调。同时，H1N1或H7N2感染的P815细胞均可大量分泌外泌体。
结论：H1N1与H7N2亚型病毒可在肥大细胞中诱导产生大量差异表达基因，并激活多种细胞功能与信号通路；而H5N1病毒仅能诱导少量差异表达基因，并抑制肥大细胞的细胞功能与信号通路。进一步研究表明，5-HT信号通路与cGMP/PKG信号通路在H1N1感染的P815细胞中优先被激活，而HIF-1信号通路则在H7N2感染的P815细胞中优先被激活。此外，H1N1或H7N2感染的P815细胞可优先分泌外泌体，这一过程在抗IAV感染的固有免疫应答中可能发挥关键作用。本研究从基因组特征角度，为阐明H1N1、H5N1及H7N2三种IAV亚型在肥大细胞中致病的差异化分子机制提供了全新的研究视角与数据支持。
整体实验设计：以多感染复数为1的剂量将细胞暴露于IAV，于37℃孵育12小时后提取RNA，开展基因组特征与生物信息学分析。