| Influenza virus is one of the important pathogens that cause human respiratory tract infections.When the influenza virus infects the body,it induces the host's immune response.Influenza virus invades epithelial cells and induces innate immune response,such as the activation of interferon pathway,which can limit virus replication,and lung resident macrophages can recognize the virus and play a phagocytic role,moreover,dendritic cells can recognize influenza virus to activate T cells.These immune responses induce the production of a large number of cytokines and chemokines,which play the role of virus clearance.Previous studies have found that influenza virus infection causes respiratory and lung damage.However,the lung inflammation and repair caused by influenza virus infection,the dynamic changes of lung cells and how the cells interact with each other are still unclear.This is important that elucidate the key regulatory cells and their functions in lung injury and repair after influenza virus infection,it can provide important datas reference in study the pathogenic mechanism of influenza virus.Firstly,we established a mouse model of repairing lung injury after influenza virus infection in this study.Female C57BL/6J mice aged 6-8 weeks were infected with 20 PFU influenza virus,and the lung tissues were collected at 8h,D1,D4,D9 and D13 after infection.In D4,the viral load of alveolar cavity was the highest,and the recruitment of inflammatory factors and immune cells increased.In order to further analyze the role of lung cells during inflammation,we analyzed lung single-cell sequencing data.Based on the marker genes,there are a total of 75 cell subpopulations were identified including monocyte-macrophage-dendritic cell(17 subgroups),T cell(14 subgroups),B cell(9 subgroups),epithelial cell(12 subgroups),endothelial cell(10 subgroups)and mesenchymal(13 subpopulations)cells.We found that compared with the normal control group,there was no significant difference in the gene transcription level of each cell type at 8h and D1.After infection,D4 and D9 showed two increased expressions of ISGs.All kinds of cells highly expressed interferon-related genes such as Ifit1 and Ifit3 to exert an immune response effect at D4,indicating the antiviral function of the lung cell populations and the existence of efficient microenvironment signal transmission mechanism during the acute phase of infection;macrophages highly expressed chemotactic-related genes such as Ccl7 to promote lymphocyte migration,T cells highly expressed effect-related genes such as Gzmk and Klrdl to promote virus clearance;B cells highly expressed activation-related genes such as Cd86 to exert humoral immune function at D9;T cells and related to proliferation cells were increased related to tissue repair at D13.Macrophages have increased expression of ligands that promote dendritic cells inflammatory and immune responses at D9.Mesenchymal cells(at D9)and endothelial cells(at D13)have increased expression of ligands that promote epithelial cells development and differentiation.In order to analyze whether there are bystander effector cells in lung cells during inflammation,the expression of influenza virus gene fragments in lung cells at different time points was analyzed,and it was found that influenza virus genes can be detected in lung cell at D1/D4 and D9.At D4,the expression of virus genes is highest,and can be detected in cells of all cell types in the lung,and most are detected in epithelial cell and macrophages.We analyzed the transcriptional characteristics of D4 cells.The lung cells of D4 are divided into 15 groups.Among them,the proportion of cells with influenza virus genes detected in interstitial macrophages(58.9%)is the highest,followed by dendritic cells(54.8%)and alveolar epithelial cells(53.4%),respectively.,and influenza virus genes were detected in an average of 41.4%of all cell types.In addition,lung cells were divided into infected cells and bystander cells according to the presence or absence of viral genes expression in D4.By analyzing the difference in gene expression between cells in the normal control group,it was found that the expression of interferon-stimulating genes such as Ifitl,Zbp1,Irf7,and Isg15 showed a gradient expression in the three cells.The normal control group had low expression,and the expression of bystander cells was middle,the infected cells are highly expressed.The results suggest that there is a "bystander" cellular effect in the process of virus infection,which is manifested as the activation of interferon pathways.The influenza virus non-structural protein NS1 has the function of strongly inhibiting the host interferon pathway.By analyzing the distribution characteristics of influenza virus genes in cells,it was found that in cells without NS 1 gene but with other viral genes,the expression of other viral genes was significantly reduced.Among them,the expression of PR8M was reduced by 81.3%,and the expression of PR8NP was reduced by 74.1%.The expression of PR8PB1 was reduced by 92.5%,the expression of PR8PB2 was reduced by 92.9%,and the expression of PR8PA was reduced by 74.7%.Compared with NS1+cells,the high expression of Neurl4,Raver2,Chstl and other genes in NS1-cells indicates that NS1-cells are not virus-sensitive cells.It elucidates that the characteristics of high-expressed genes in this type of cells may explore more host genes related to virus replication.In summary,we analyzed the lung cell composition and its dynamic characteristics through single-cell transcriptome sequencing technology based on the inflammation,injury and repair model of influenza virus-infected mice.The results showed that the viral load of D4 reached its peak and all cell subpopulations were infected.The cells exerted antiviral effects through high expression of interferon-related genes,such as Ifit1 and Ift3;the "bystander" cell effect was present in all cell populations,showing high expression of Ifitl,Zbp1,Irf7 and Isg15 similar to infected cells,the expression level is between normal control cells and infected cells,suggesting the rapid signal transmission network between lung microenvironment during virus infection.In our study,we explained the emergence and function of cell subgroups at different stages of infection,such as the acute and recovery phases after viral infection from the perspective of a single cell subgroup in the lungs,it could provide a more adequate data basis and reference for explaining the cell pathogenic mechanism and treatment strategy in influenza virus infection. |
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