| Lung is a vital physiological organ and is composed of a variety of cells that mediate breathing and gas exchange.Lung tissue contains many types of immune cells,such as special alveoli and interstitial macrophages,dendritic cells,granulocytes and lymphocytes,including circulating and tissue-resident memory T cells.The continuous exposure of the lungs to pathogenic and non-pathogenic microorganisms and the environment suggests that kinetic mechanisms are critical to maintaining tissue integrity.Influenza virus(IAV)is a very common respiratory virus,and seasonal flu can cause stress and financial burden on public health every year.Some influenza virus strains will also cause a local epidemic,which will have a huge impact on social stability.The main target cells of the influenza virus are the epithelial cells of the respiratory tract and enter the target cells through the sialic acid receptor.The host’s response to infection results from the combined responses of many individual cells(infected and uninfected).To understand the factors that control host responses at the tissue and organism level,it is crucial to define the mutational patterns of single-cell infection responses.Single-cell sequencing technology refers to a technology that enables high-throughput sequencing of a genome or transcriptome at the level of a single cell and uses it to reveal multiple biological problems.Compared with traditional high-throughput sequencing,single-cell sequencing can analyze the heterogeneity of cells of the same phenotype,and also reveal new cell types and cell development trajectories,which has broad application prospects.To better understand random biological processes,a more accurate understanding of the transcriptome in a single cell is essential to elucidate its role in cell function and to understand how gene expression promotes beneficial or harmful states.This study was based on single-cell transcriptomics to sequence single-cell transcriptomes of alveolar lavage fluid from Day08,Day09,and Day11 after the onset of severe pneumonia in a patient.Five types of cells were found in BALF,including T cells,B cells,monocyte macrophages,DC cells and AT1.In single-cell transcriptome sequencing of T cells,we found that T cells in BALF were significantly reduced at different consecutive time points,and with the change of time points,the cell cycle has a certain effect on cell population,the proportion of CD8+ cytotoxic T cells in G1 was significantly increased,and the expression of cytotoxic related genes and chemokines was higher than that of S or G2M toxic T cells.The proportion of Treg cells is not related to the course of disease,and PDCD1 expression is significantly increased in the later stage of the course of disease.A small amount of mixed NK cells were found in T cells,and it was found that T cells and NK cells cannot be effectively distinguished in single-cell sequencing.Regular changes in gene expression in T cells that are the same as the course of the disease are worth further analysis such as CD48,CCR7,ANXA2,etc.In NK cells we found that the expression of chemokine ligands was abnormally increased at DAY11,such as CCL2,CCL3 and CXCL8.Among B cells,most of the captured B cells were plasma cells expressing CD191oCD38hiCD27hi,and expressed immunoglobulin-related genes IGHG1,IGHG3,and the like.In BSC2,we found a cluster that expresses both B cell marker genes and T cell marker genes.Since there is no mutual transformation between B and T cells,this cluster is likely to be "doublet".In CD 14+M/M cells,the proportion of cells gradually increased over time,indicating the importance of macrophages in lung immunity after influenza infection.CD 14+M/M cells can be divided into seven clusters based on automatic clustering,and the functional enrichment of each subgroup can be found by GO analysis.When the NF-κB pathway was used to characterize the dynamic analysis of monocyte inflammation,most of the gene expression on the signal pathway was decreasing.It could indicate the improvement of the inflammation and the patient’s condition is gradually alleviating while there are no clinical indications.Some of the highly variable significant genes such as IKBKB,MYD88,IRAK1,RIPK1,TNF,and TRADD may be used as marker genes for changes in clinical inflammation indicators.In order to further analyze the immunodynamic changes of lung epithelial cells after influenza virus infection,we performed single-cell transcriptome sequencing of lung tissue at different time points in PR8-infected mice,and we captured fewer cell types,include AT1,AT2,AT1/AT2,ciliated cells.Among them,AT2 can be further typed into ordinary AT2 and Pcolce2hiAT2.In the process of changing epithelial cell ratio,the increase of AT1 in the later period indicates the further reconstruction of the injured lung.Analysis of the expression of ISGs at different time points in the epithelial cells found that(1)the regularity of the change in the expression time of ISGs is determined according to their function;(2)most ISGs,including RNA processing,metabolic regulation,inflammation mediators or regulators,the change of were not obvious,because it is not a direct effector molecule caused by viral infection;(3)ISGs in the predominantly of antiviral family members have more consistent changes in transcription level in different cell types,such as Irf7,Gbp2,Isg15,Ly6e,Lgals3bp,the transcription level has reached its peak on Day4,and part of the transcription level on Day 13 has decreased,indicating that on the natural immunity of Day4 cells have been fully activated,whether it is directly infected by the virus or by the bystander effect between cells(crosstalk).In this paper,we used human single cell transcriptome sequencing technology to analyze the dynamic changes of inflammatory response to influenza infection in humans with pneumonia and mouse PR8 infection models,and provide a reference basis for the dynamic changes of lung immunity and target specific changes. |
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