Single-cell Transcriptomics Analysis Of Lung Immune Cells In Mice With RSV-induced Asthma Exacerbation

Background and ObjectiveAsthma is considered to be one of the most common chronic diseases.It affects 1-18%of the population in different countries.There are approximately 45.7 million asthma patients aged 20 and over in my country,with the asthma prevalence rate of4.2%.Although progress has been made in the treatment of asthma,the acute exacerbation of asthma is still an important problem faced by patients.Asthma exacerbation can occur in patients with different severity of asthma symptoms,leading to the severity of asthma increased,airway remodeling increased,response to bronchodilators and anti-inflammatory treatments decreased.It requiring emergency treatment,hospitalization,or systemic hormone therapy.The medical care system and society have brought huge economic burdens,making people pay more and more attention to the aggravation of asthma.The most common cause of exacerbation of asthma is respiratory virus infection and exposure to environmental allergens,including respiratory syncytial virus(RSV),rhinovirus,house dust mites,cockroaches,pollen,etc.Viral infection has been recognized as the main cause of the worsening of asthma,for patients at risk of asthma or suffering from asthma,respiratory virus infections have an important impact on the occurrence or worsening of asthma symptoms.In most cases,respiratory viral infections do not work alone,but interact with allergic reactions,causing symptoms in asthmatic patients to worsen.House dust mite(HDM)is an allergen closely related to asthma.It can not only activate the adaptive and Th2-derived immune response,but also activate the innate immune response with multiple pathogenic factors and high clinical relevance in asthma,and respiratory syncytial virus is a common virus during asthma exacerbation.Infection with the virus will not only increase the susceptibility to asthma but also increase the body’s responsiveness to allergens.Studies have shown that asthma exacerbation is closely related to the continuous host immune response,which can lead to airway remodeling and airway dysfunction.Airway epithelial cells,as the first line of defense between the host and the external environment,are the main target cells of inhaled allergens and the respiratory tract infection.During the onset of asthma,respiratory viruses,allergens and air pollutants can stimulate airway epithelial cells to secrete alarm hormones(TSLP,IL-33,IL-25)and chemokines to act on dendritic cells,macrophages,neutrophils,and ILC2 to induce innate immune responses,and can also affect the adaptive immune response process involving Th2 and Th17.More and more evidences show that asthma is not just allergic airway inflammation,the intricate molecular network of immune cell infiltration and increased airway resistance during asthma exacerbation is still unclear,and its treatment methods have limitations.We need to understand the interaction between immune cells to reveal the complex pathogenesis of asthma exacerbation.The single-cell RNA sequencing technology(ScRNA-Seq)can effectively understand how the immune system produces a wide range of potential responses to viral infections by studying isolated single cells,and it can also perform unbiased transcriptome analysis on single cells.Revealing the expression of all genes in the whole genome at the single-cell level is conducive to studying the heterogeneity of gene expression between cells,so that we can fully understand the complicated pathogenesis of asthma.Therefore,we use HDM to induce allergic airway inflammation,and then use RSVto stimulate mice to establish an asthma exacerbation model to simulate clinical features,after that we use single-cell transcriptome sequencing technology to analyze the immune cells,gene function and pathway that play an important role in the onset of asthma exacerbation to reveal the complex cellular and molecular mechanisms of asthma exacerbation.Methods1.The RSV virus solution was incubated in a culture flask of Hela cells to observe the pathological changes of Hela cells,and the virus solution was used to infect mice by intranasal,and then the effect of RSV on the airways of mice was observed.2.6 to 8 week BALB/c male mice were randomly divide them into Saline group,HDM/HDM+Vehicle group(H/H+VEH),HDM/HDM+RSV group(H/H+RSV).HDM solution was used to stimulate mice by intranasal to construct a model of allergic airway inflammation,and then RSV were used to infect mice to aggravate asthma symptoms.3.Use the Flexivent device to measure airway reactivity to detect lung function in mice,and obtain mouse bronchoalveolar lavage fluid(BALF),after that leukocytes were classified and counted in BALF.4.The left lobe lung tissue of mouse was fixed and paraffin sectioned,and the lung tissue section was stained with HE stain to evaluate the airway inflammation and mucus secretion of the mouse.Muc5ac gene expression level in mouse lung tissue detected using q RT-PCR to evaluate mucus protein secretion in mice.5.The lung tissue was made into single-cell suspension,and the CD45+immune cells are sorted by a flow cytometer.Single-cell transcriptome sequencing is performed on the sorted cells,and the sequencing results were analyzed by bioinformatics.Results1.After Hela cells were infected with RSV,the lesions showed cell rounding,the formation of a large number of syncytia and the destruction of the monolayer.Compared with the Saline control group,the total number of cells in BALF and lung tissue increased significantly in the RSV group;The number of neutrophils,macrophages and lymphocytes in BALF,and neutrophils in lung tissue were significantly higher than those in the Saline control group;In the RSV group the expression level of RSV N protein m RNA was higher than in the Saline group.2.RSV can cause the increase of airway resistance in mice with allergic airway inflammation;The results of classification and counting of immune cells in BALF show that the number of total cells,neutrophils,eosinophils,macrophages and lymphocytes in the H/H+RSV group were significantly higher than those of the other two groups.Compared with the Saline control group,the number of various types of cells in the H/H+VEH group was no difference;There are inflammatory cell infiltrations in the lung tissues of the H/H+RSV group and the lung tissues,in the H/H+VEH group,there was also a little inflammatory cell infiltration,and the degree of inflammation in the H/H+RSV group and H/H+VEH group was higher than that in the Saline group,but the difference between the H/H+RSV group and the Saline group was more significant;The expression of Muc5ac m RNA was significantly up-regulated in the H/H+RSV group.3.Single-cell RNA sequencing was performed on CD45~+immune cells in the lung tissue sorted by flow cytometry.The clustered cells were annotated with Single R,and the cell clusters were visualized and expressed with heat map.A total of 40 cell clusters were identified.The types are mainly related to B cells,T cells,dendritic cells,macrophages,neutrophils,ILC2,NK cells,monocytes and basophils.4.After analyzing the cell clusters in lung tissue,we further divided the cells into23 main cell clusters,compared with the Saline group,it was found that the levels of NK cells(NK.DAP10-),NK cells(NK),T cells(T.CD4TESTCJ),Neutrophils(GN.ARTH),Neutrophils(GN.Thio),B cells(B.Fr F),B cells(B1a),Monocytes(MO.6C-Ⅱ-),Monocytes(MO.6C+Ⅱ-),Monocytes(MO.6C-Ⅱ)and DC(DC.PDC.8-)cell clusters were significantly increased in the H/H+VEH group and the H/H+RSV group.Among them,the levels of NK cells(NK.DAP10-),NK cells(NK),B cells(B.Fr F),Neutrophils(GN.Thio),Monocytes(MO.6C+Ⅱ-)and Monocytes(MO.6C-Ⅱ+)cell clusters increased more significantly in the H/H+RSV group,and the levels of Neutrophils(GN.ARTH)and Monocytes(MO.6C-Ⅱ-)cell clusters were higher in the H/H+VEH group.The levels of multiple cell clusters are significantly in the lung tissue of virus-induced asthma exacerbated mice,including T cells(T.4MEM44H62L),T cells(T.8MEM.OTID45LISOVA),Macrophages(MF.103-11B+24-)and Tgd(Tgd.mat.VG2+).5.The GO enrichment analysis results show that,in terms of biological processes,the gene enrichment in the three groups is similar.Most gene enrichment is related to the activation and regulation of T cell,the positive regulation of cytokine production and the adhesion and regulation between cells.In addition,leukocyte proliferation and monocyte proliferation are relatively low in the Saline control group but higher than the experimental group.T cell differentiation is more enriched in the experimental groups than in the Saline control group.In terms of cellular components,genes are mainly enriched in lysosomes,lytic vacuoles and parts related to cell membrane function.The actin cytoskeleton is highly enriched in the H/H+VEH group and is higher than that in the Saline group and H/H+RSV group.In terms of molecular functions,genes are mainly enriched in immune receptors,enzyme activators,transcription coregulator and cytokine receptor activities,ubiquitin and ubiquitin-like protein ligases and GTPase binding functions.The degree of enrichment in transcription coregulator in the Saline group and H/H+VEH group is higher.The feature of DNA-binding transcription factor binding in the H/H+VEH group is significantly higher than the other two groups.The small GTPases and Ras GTPases are combined in the experimental groups.The degree of enrichment in experimental groups was higher than that in the Saline control group,and the degree of enrichment in phospholipid binding in the Saline group and H/H+RSV group was higher than that in the H/H+VEH group.KEGG pathway enrichment analysis shows that genes are enriched in Th1,Th2 and Th17 cell differentiation pathways,T cell receptor signaling pathways,B cell receptor signaling pathways,hematopoietic cell lineage pathways,osteoclast differentiation pathways,coronavirus disease-COVID-19 and apoptosis signaling pathway.Obviously,most of the same enrichment pathways existed in the three groups.ConclusionRSV can cause HDM-induced airway inflammation and enhanced airway hyperresponsiveness.NK cells,monocytes,neutrophils and mature B cells may play an important role in RSV-induced asthma exacerbation.We performed GO gene function enrichment analysis and KEGG pathway enrichment analysis for each group of differentially expressed genes,which are important for us to understand the interaction of innate and adaptive immune responses in the lungs and to seek new targets for the treatment of asthma exacerbation.