| Avian H9N2influenza viruses have long been circulating in poultry and were sporadically introduced into humans through close contact. Endothelial cells are believed to play an important role in innate immune cell recruitment and early innate cytokine and chemokine production during virus infection. But little is known about the host responses to H9N2virus infection in endothelial cells.In the present study, we investigated whether endothelial cells could support replication of H9N2viruses and characterized global patterns of gene expression in endothelial cells after inoculation with H9N2virus. In our first experiment, we infected human umbilical vein endothelial cells (HUVECs) with H9N2virus (A/Chicken/Hebei/4/2008), and measured the titers of viruses in the supernatant of HUVECs by plaque-forming units (PFUs). We demonstrated that H9N2virus infection of HUVECs led to a dose-dependent increment in titers, indicating that HUVECs supported replication of H9N2virus.In the second experiment, we inoculated HUVECs with H9N2viruses or inactivated H9N2viral particles inactivated by β-propionolactone at a multiplicity of infection (MOI) of5, and then used microarrays to observe global patterns of gene expression in HUVECs at24hours after inoculation. Microarray analysis showed that H9N2virus infection induced an abundance of differential expressed genes (DEGs;2,091up-expressed genes and4,209down-expressed genes) when compared to its control, exhibiting a transcriptional signature of viral infection. Inactivated H9N2viral particles inoculation only yielded a small amount of DEGs (263DEGs,177up-expressed genes and86down-expressed genes). After this, we mainly focused on three aspects of transcriptional change of genes. First, we observed the expression changes of the genes encoding proteins associated with the structure and function of endothelial cell itself. These genes include apoptosis-related genes, adhesion molecule genes and tight junction protein genes. Our Gene Ontology (GO) function analysis showed that there were317DEGs (up-regulation or down-regulation) enriched in regulation of apoptosis in HUVECs after H9N2virus infection, which hinted that H9N2virus infection might have a profound effect on endothelial cell survival. Adhesion molecules expressed by activated endothelium mediate leukocyte adhesion and transendothelial migration. We found that H9N2virus infection induced the differential expression of some adhesion molecule genes (significantly down-regulated ICAM1gene and significantly up-regulated genes encoding ICAM4and SELE). Alterations in tight junction proteins mediate the endothelial leak. We noted that H9N2virus infection significantly down-regulated the expression of genes encoding tight junction proteins (OCLN, CLDN1, CLDN11and CLDN23). Second, we observed the expression changes of the proinflammatory cytokine genes and chemokine genes. The recruitment of innate immune cells into the lungs combined with excessive proinflammatory cytokine and chemokine production are hallmarks of influenza virus infection. We did not find the significant changes of expressions in proinflammatory cytokine genes (including TNF, IL1A, IL1B, IL6and IL10). But we detected high levels of expressions of chemokine genes (including CCL5, CXCL10and CXCL11) at the transcriptional level following treatment of H9N2viruses and viral particles. Finally, we analyzed the expression changes of the interferon (IFN) genes and interferon-stimulated genes (ISGs). It is believed that production of type â… IFN is the key response to many viral infections, leading to the transcription of various ISGs, some of which encode direct antiviral effectors. Surprisingly, the most significantly up-regulated genes were mainly interferon-stimulated genes (ISGs) after inoculating H9N2viruses and viral particles, although there was no change in interferon gene expression and interferon protein level. These up-regulated ISGs include those encoding interferon-induced protein with tetratricopeptide repeats (IFIT), including IFIT1, IFIT2, IFIT3and IFIT5, interferon-induced transmembrane (IFITM) proteins (IFITM1and IFITM2) and2’-5’oligoadenylate synthetase (OAS) protein (OAS1and OAS2). In addition, we found that inactivated H9N2viral particles were more potent in inducing ISGs expression compared with H9N2viruses. These results suggested that the induction of expression of ISGs were mainly dependent on the interaction between viral particles and endothelial cells, and less associated with viral replication. We speculate that an antiviral pathway may exist unrelated to IFN when endothelial cells are stimulated by H9N2viral particles and virus infection.In our last experiment, we found that baicalin attenuated the titers of viruses in the supernatant of HUVECs. Baicalin treatment following H9N2infection up-and down-regulated the expression of genes related cell biological process and viral replication, and showed no significant change in expression of antiviral genes.In conclusion, our results showed that HUVECs supported the productive replication of H9N2virus, and both H9N2virus infection and viral particle inoculation could significantly up-regulated the transcriptional expression of ISGs and chemokine genes in HUVECs. In addition, H9N2virus infection induces apoptosis related genes expression and significantly down-regulates the expression of genes encoding tight junction proteins. These data might offer further insight into the interaction between endothelial cells and H9N2influenza viruses. |
